Evaluation of the X-chromosome inactivation patterns in females with Gabriele-de Vries syndrome and expansion of clinical spectrum

Evaluation of the X-chromosome inactivation patterns in females with Gabriele-de Vries syndrome and expansion of clinical spectrum | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of the X-chromosome inactivation patterns in females with Gabriele-de Vries syndrome and expansion of clinical spectrum Bianca Mie Sato Kurashima, Laura Machado Lara Carvalho, Rafael Mina Piergiorge, and 16 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8846661/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Gabriele-de Vries syndrome (GDVS) is a syndromic form of intellectual disability caused by pathogenic variants in the YY1 gene, which encodes a ubiquitously expressed transcription factor that plays a crucial role in the X-chromosome inactivation (XCI) process. Recent studies have implicated de novo YY1 pathogenic variants in skewed XCI in females with GDVS. Here, we report clinical and molecular features of 11 GDVS patients (ten females), including eight newly identified cases, two being a familial case. Patients exhibited the core phenotype of GDVS but with notable clinical heterogeneity, displaying additional features such as autism spectrum disorder, thyroid dysfunction, hearing impairment, and dysarthria. We also discuss thyroid and other endocrine alterations, autoimmune conditions, and movement disorders in GDVS. Eight YY1 variants were analyzed in silico , exhibiting high pathogenicity scores and predicted structural or functional impact, with most affecting DNA binding or zinc finger domain interactions. Finally, we investigated the XCI patterns of ten female patients, and XCI skewing (moderate or extreme) was detected in blood samples from all of them. Our findings expand the clinical and molecular spectrum of GDVS, in addition to reinforcing a potential involvement of YY1 mutations in modulating XCI patterns in females. Gabriele-de Vries syndrome X-chromosome inactivation YY1 neurodevelopmental disorders variable expressivity Figures Figure 1 Figure 2 Figure 3 1. Introduction Gabriele-de Vries Syndrome (GDVS; OMIM #617557), first described in 2017, is a neurodevelopmental autosomal dominant condition caused by YY1 (OMIM *600013) haploinsufficiency ‌(Gabriele et al. 2017 ). It is characterized by developmental delay (DD), mild to profound intellectual disability (ID), craniofacial dysmorphisms and other highly variable phenotypes, comprising low birth weight, multiple congenital anomalies, feeding difficulties, atypical behavior, among others (Gabriele et al. 2017 ; Nabais Sá et al. 2019 ). GDVS diagnosis relies on genomic testing with the detection of heterozygous pathogenic/likely pathogenic YY1 variants, including missense and loss-of-function (LoF) mutations, and 14q32.2 microdeletions encompassing the YY1 gene (Nabais Sá et al. 2019 ). It is a rare condition, with 48 patients reported so far, 44 for whom a general clinical description is available (Gabriele et al. 2017 ; Morales-Rosado et al. 2018 ; Carminho-Rodrigues et al. 2020 ; Bae et al. 2021 ; Imafidon et al. 2021 ; Balakrishnan and Ranganath 2021 ; Malaquias et al. 2021 ; Tan et al. 2021 ; Zech et al. 2021 ; Zorzi et al. 2021 ; Alali and Vitalone 2022 ; Cherik et al. 2022 ; dos Santos et al. 2022 ; Ferng et al. 2022 ; Khamirani et al. 2022 ; Asato et al. 2023 ; Chaves et al. 2023 ; Chawla et al. 2023 ; Koruga et al. 2023 ; Woo et al. 2023 ; Luo et al. 2024 ; Shin et al. 2024 ; Srour et al. 2024 ; Topa et al. 2024 ; Yang et al. 2024 ; Mudassir et al. 2025 ; Pal et al. 2025 ; Huang et al. 2025 ) (see the Supplementary file – Table S1 ). Gabriele et al. ( 2017 ) also reported other 13 individuals who carried deletions encompassing the YY1 gene. The YY1 gene, located on chromosome 14q32.2, encodes a transcription factor involved in numerous physiological roles, such as DNA replication, cell differentiation, apoptosis, and neurogenesis (Jung et al. 2023 ; Verheul et al. 2020 ). In particular, YY1 plays a central role in the process of X-chromosome inactivation (XCI), a mechanism that evolved in mammals to equalize X-linked gene expression between biological sexes (Jeon and Lee 2011 ; Makhlouf et al. 2014 ; Jégu et al. 2017 ; Sun et al. 2021 ; Dossin and Heard 2022 ). It has been shown that YY1 serves as a transcriptional activator of XIST in both human and mouse contexts (Chapman et al. 2014 ; Makhlouf et al. 2014 ; reviewed in Dossin and Heard 2022 ). Moreover, studies conducted in mice proposed that YY1 protein binding to the Xist locus and its transcript acts as a bridge that allows Xist RNA to coat the future inactive X-chromosome (Xi) (Jeon and Lee 2011 ; Thorvaldsen et al. 2011 ; reviewed in Jégu et al. 2017 and Sun et al. 2021 ). Due to the stochastic nature of XCI, its patterns in healthy females show a continuous distribution, with extreme skewed inactivation (90:10) observed only in a small proportion of them, approximately 2% (Amos-Landgraf et al. 2006 ). Strikingly, female patients with pathogenic YY1 variants for whom XCI patterns have been analysed exhibited marked skewing of XCI in peripheral blood (dos Santos et al. 2022 ; Chaves et al. 2023 ). If confirmed in a larger group of female patients, this phenomenon could have implications on our understanding of YY1 ‘s role in XCI regulation and/or the broader effect of YY1 mutations in affected females. ‌ Herein, we report detailed clinical features and genetic findings of eleven patients with GDVS, including eight previously unreported cases, and present XCI analysis of ten females carrying causal YY1 variants. Furthermore, we performed in silico analysis of the YY1 missense variants identified in the GDVS female patients. 2. Patients and methods Ethics approval for this study was granted by the Institute of Biosciences - University of São Paulo Ethics Committee (CAAE 80921117.5.0000.5464), the Institutional Ethics Committee from the State University of Rio de Janeiro (CAAE 46769315.5.0000.5259), and the French Ethics Committee (French Ministry of Research and Innovation) under approval number DC-2020-4073. Written informed consent was obtained from the patients’ legal guardians. All procedures adhered to the principles outlined in the Declaration of Helsinki, and its subsequent amendments. To ensure confidentiality and data protection, all participant data were anonymized using numerical identification codes, with database access restricted to authorized research team members, in compliance with data protection regulations. We recruited female patients who had previously undergone Whole Exome Sequencing (WES), and were found to carry pathogenic/likely pathogenic YY1 variants. Clinical characterization of the patients was obtained through analysis of previously conducted health tests or reports from healthcare professionals and parents. Peripheral blood samples from probands, their parents and siblings (when applicable) were collected for DNA extraction using standard methods. Sanger sequencing analysis or WES of parents was conducted to perform segregation analysis. For Sanger sequencing, PCR was carried out under standard conditions, and amplicons were sequenced in both directions using BigDye (Thermo Fisher) according to the manufacturer's instructions, with capillary electrophoresis performed on an ABI 3730 DNA Analyzer (Thermo Fisher Scientific Inc.). All variants were re-evaluated and classified according to the American College of Medical Genetics (ACMG) guidelines (Richards et al. 2015 ) and ClinGen updates. Causal variants that had not been previously reported were submitted to ClinVar ( https://www.ncbi.nlm.nih.gov/clinvar/ ). To estimate the proportion of cells carrying an active maternal versus paternal X-chromosome, we employed methylation-sensitive restriction enzyme assays. The XCI patterns of patients P1–P6, P8 and P9 were determined by assessing differential methylation at different X-chromosome loci . One of them is located in a polymorphic region (CAG repeats) of the first exon of the AR gene (mapped to Xq12) (Allen et al. 1992 ); another is mapped to a polymorphic region (GAAA repeats) near the RP2 gene (Xp13.3) (Machado et al. 2014 ); another is mapped to a polymorphic region (CCG repeats) near the HMGB3 gene (Xq28) (Musalkova et al. 2015 ), and the last one is mapped to a polymorphic region (CCG repeats) near the TMEM185A gene (Xq28) (Musalkova et al. 2015 ). Each XCI assay included one male negative control and one female control with a known extremely skewed XCI. Reproducibility of the data was based on the assay of all samples in duplicate. XCI patterns were calculated in agreement with Bittel et al. ( 2008 ), using an average of the duplicates. Ratios ≤ 70:30 were considered random XCI, 71:29–90:10 as moderate skewing, and > 90:10 as extreme skewing, based on criteria reported by Tan et al. ( 2025 ). The six missense variants linked to the YY1 protein were analyzed through AlphaMissense (Cheng et al. 2023 ), and structural modeling based on the AlphaFold 3 database (Varadi et al. 2024). Models incorporating each identified mutation were generated based on the wild-type configuration provided by the 1UBD PDB structure. This approach allowed the exploration of the specific structural perturbations induced by the mutations. Residue interactions with DNA and ions were examined using PDBsum for the wild-type structure (Laskowski et al. 2018 ). Also, the Variant Effect Predictor (VEP) from Ensembl (McLaren et al. 2016 ) was used to assess dosage sensitivity of the YY1 gene. The two frameshift variants leading to premature stop codons were evaluated by LOFTEE through VEP from Ensembl. 3. Results Clinical description Below, the eight newly identified patients GDVS cases are described in more detail, and clinical data for Patients 9–11 are summarized. Complete clinical information can be found in the Supplementary file – Table S2 . Patient 1 Patient 1 (P1) was a Chilean female with a healthy sister and no family history of genetic diseases. She exhibited intrauterine growth restriction and was born prematurely at 36 weeks of gestation, with a birth length of -2.4 SD, and a birth weight of -3.2 SD. Congenital hypotonia and global DD, including delayed motor, speech and language development, were reported. Physical exams at the age of 2 years and 4 months revealed decreased body weight (–4.1 SD), craniofacial asymmetry, large forehead, malar flattening, a pointed chin, low-set and posteriorly rotated ears, periorbital fullness, downslanted palpebral fissures, eyelid ptosis, almond-shaped eyes, telecanthus, convergent strabismus, wide, concave nasal bridge, bulbous nose tip, short columella, downturned corners of mouth, micrognathia, sparse hair, hypertrichosis, marfanoid habitus, big hands and feet, and long fingers with joints hypermobility. Feeding difficulties in consuming solid foods, gastroesophageal reflux, and constipation were reported. She presented with impairment of visual pursuit, atopic dermatitis, bilateral neurosensory hearing impairment, nonverbal autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), self-injurious behavior, sleeping disturbance, unsteady gait and had nasolacrimal duct obstruction, recurrent Moro reflex, poor pincer grasp, and recurrent infections. Brain Magnetic Resonance Imaging (MRI) performed at 3 months revealed a thin corpus callosum . P1 passed away due to sepsis with multiple organ failure and pulmonary hemorrhage, after an infection caused by multiple pathogens, including Streptococcus bacteria, adenovirus and Pseudomonas aeruginosa . Patient 2 Patient 2 (P2) is a Brazilian female born via cesarean section after in vitro fertilization. She has a healthy dizygotic twin sister and no family history of genetic diseases. Her mother developed maternal diabetes during gestation. P2 had intrauterine growth restriction and was born at term (37 weeks) small for gestational age, with a low birth weight (-2.8 SD). Large forehead, downslanted palpebral fissures, eyelid ptosis, thin and sparse hair, micrognathia, and a pointed chin were observed. The patient had congenital torticollis, recurrent Moro reflex and was treated with cranial orthosis between 7 and 10 months of age. She exhibited global DD, including motor and speech development. Hypotonia was noted between 2 to 3 years old, and modest axial ataxia was reported at 18 months, progressing to global ataxia by 3.5 years. MRI revealed mild hydrocephalus. Cranial magnetic resonance imaging at 3 years revealed faint foci of hyperintense signal on T2/FLAIR scattered in the periventricular and deep white matter, potentially related to zones of terminal myelination/gliosis (at 3 years). She is currently 5 years old and has manifested unsteady gait, dysarthria, and sleeping disturbance. She is undergoing assessment for ASD. Patient 3 Patient 3 (P3) is a Brazilian female born from a healthy couple who has two healthy siblings (a younger paternal brother and an older maternal sister). She has a paternal cousin with a mild ID. Her mother displayed maternal hypertension during pregnancy. P3 displayed intrauterine growth restriction and was born prematurely from 35 weeks of gestation by cesarean section. The patient was small for gestational age and was born with a low birth weight (-3.1 SD). She had neonatal jaundice and was treated with phototherapy. P3 exhibited nasolacrimal duct obstruction (treated by surgical intervention), congenital hypotonia, and recurrent emesis. She manifested febrile seizures until 1 year of age. Graves’ disease and Hashimoto’s thyroiditis were diagnosed and treated with Methimazole (later suspended). The patient’s clinical features at 16 years old also include mild ID, reading and writing difficulties, ADHD, and anxiety. Physical exams showed short stature, a large and prominent forehead, receding hairline, epicanthus and telecanthus, proptosis, bilateral ptosis, long philtrum, overfolded helix, simple ears, micrognathia, thick lower lip, pointed chin, arachnodactyly, camptodactyly, tapered phalanx of fingers of the hands, marfanoid habitus, and big hands and feet. She displayed recurrent falls during childhood. Audiometry, echocardiogram, and abdominal ultrasound exams exhibited normal results. Patient 4 Patient 4 (P4) is a French female, one of six siblings from a family originally from the Maghreb. She was born prematurely, had growth delay, speech delay, and academic difficulties with schooling in a medical-educational institute due to mild intellectual disability. She presents with facial dysmorphism including broad forehead, nose with bulbous tip, upslanting palpebral fissures with almond-shaped eyes, enlarged columella, prominent philtrum, pointed chin. She has obesity (1.53 m for 98 kg, BMI 42), convergent strabismus of the left eye, hypermetropia and occasional dizziness. Brain MRI showed normal brain morphology. She had maternal hypertension during her first pregnancy, which happened to be a spontaneous bichorial biamniotic twin pregnancy (mother of patient 5). Patient 5 Patient 5 (P5) is a French male born from a twin bichorial biamniotic pregnancy (son of P4), after an emergency caesarean section at 30 weeks and 3 days, due to severe intrauterine growth restriction. His birth weight was 640 g, length was 31,7 cm and OFC was 25,5 cm. Apgar score was 7 at 5 min, pH at 7,26 and lactate at 3,1. He presented with axial hypotonia, tubulopathy and retinopathy due to prematurity. Cardiac ultrasound showed hypertrophic and hypertrabeculated left ventricle associated to a patent foramen ovale with left-right shunt. Abdominal ultrasound did not identify any visceral malformation but a thickening of the walls of the left pyelon associated with a small ectasia of the caliceal tracts upstream. Prenatal brain MRI and postnatal transfontanellar ultrasounds showed normal brain morphology. Ophthalmologic control showed normal retina but revealed hypermetropia and convergent strabismus. He presented with facial dysmorphism including brachycephaly, broad forehead, nose with bulbous tip, upslanting palpebral fissures, enlarged columella, smooth and prominent philtrum, thin upper lip, pointed chin, faded cupid's bow, low set ears with overfolded helix. His hands had a single transverse palmar crease. He held his head up at three months (adjusted age for prematurity), but is unable to sit or stand without support at 12 months and needs physiotherapy and psychomotricity follow-up. He shows good social interaction. Patient 6 Patient 6 (P6) is a French 6 years old female born from healthy non-consanguineous parents. She has a healthy older brother. Motor delay was noted at 3 months old with hypotonia, sitting was acquired at 12 months and walking at 2 years and a half. Speech delay was noted with only a few words at 3 years. Physical examination revealed multiple hemangiomas and congenital naevus, marfanoid habitus with arachnodactyly, hyperlaxity and pectus excavatum. Broad forehead was noted. She presented some difficulties for feeding. Examination by cardiac echography and cerebral MRI were normal. Ophthalmologic examination was normal. Array-CGH testing showed normal results. Patient 7 Patient 7 (P7) is a girl who was born in Germany after 39 + 5 gestational weeks of an uneventful pregnancy. Her parents and two older siblings are healthy, and there is no history of genetic disease in her family originating from Vietnam. She had a low birth weight (3 P., -1.91 z) and developed recurrent cyanosis after birth. The patient exhibited dystrophy due to severe feeding issues with swallowing difficulties and recurrent emesis. An inefficient motility of the esophagus and gastroesophageal reflux were diagnosed. Her motor development and socio-emotional development were delayed, while speech development was normal. She has recurrent infections intermittently treated with long-term antibiotic medication. Physical exams showed craniofacial asymmetry, periorbital fullness, malar flattening, protruding ears, and short columella. The patient’s clinical features at 7 years old also include learning disability (IQ 82), attention-deficit disorder (ADD), lack of orientation, Graves’ Disease, decreased body weight (< 1 P., -3.07 z), and IgA deficiency. Patient 8 Patient 8 (P8) is a Brazilian girl born at term. Her birth weight was 2,200 g and her length was 50 cm. She was small for gestational age and did not suck effectively. The patient presented failure to thrive and underwent recurrent hospitalizations due to upper respiratory infections and acute gastroenteritis. Ultrasonography examination at 9.5 years indicated enlarged thyroid gland, with heterogeneous and diffusely hypoechoic texture, diagnosed as hyperthyroidism. At 8.18 years, an electrocardiogram revealed sinus tachycardia and left anterosuperior fascicular block. A physical examination at 11 years and 10 months showed low weight 18.6 kg (z-4), height 141.5 cm, and head circumference 46.5 cm. The observed clinical features were neuropsychomotor developmental delay, intellectual disability, hyperthyroidism, microcephaly, facial dysmorphisms (small face, closed anterior fontanelle, mild midface hypoplasia, proptosis, thick lips), goiter, small nipples, symmetrical limbs, and long, thin fingers with verrucous lesions. Karyotyping was normal. Patients 9, 10 and 11 Patients 9 (P9), 10 (P10) and 11 (P11) had already been described in the literature by Carminho-Rodrigues et al. ( 2020 ), dos Santos et al. ( 2022 ), and Chaves et al. ( 2023 ), respectively. P9 presented with left divergent strabismus, was diagnosed with ADHD, and showed a delay of ~ 2 years in cognitive abilities (Carminho-Rodrigues et al. 2020 ). There were gait and speech difficulties, without cognitive regression. Neurological exams revealed static ataxia with normal muscular tone, kinetic, resting and postural tremor and myoclonic jerks in the left arm. Physical examination at age 21 disclosed facial dysmorphisms (long and asymmetric face, broad forehead, mandibular prognathism, malar flattening and short philtrum), small ears, long nose, full nasal tip, high palate, Gingko leaf-shaped upper lip indentation and thick lower lip. All four members had reduced muscular tone and there was postural and kinetic action tremor in her body. She manifested generalized dystonia, mainly impairing the neck and left hand, and cerebellar features that included gait and stance ataxia. P10 is a Brazilian patient born from a healthy non-consanguineous couple by caesarean delivery at full term (dos Santos et al. 2022 ). Intrauterine growth restriction and hypoactivity were noted. Bilateral hearing loss was detected at 1 year of age, and when she was 3 years old, she failed to thrive and had frequent bilious vomiting. Global developmental delay, hypotonia, low weight, short stature, feeding difficulties, and some pneumonia events were reported, and physical examination revealed flat and triangular face, malar hypoplasia, short palpebral fissures, triangular nose, smooth philtrum, microstomia, arched and narrow palate, thin upper lip, irregular placement of teeth, micrognathia, low-set ears, protruding and malformed auricles, pointed chin, asymmetric thoracic cage, pectus carinatum , small nipples, scoliosis, digital thumb, arachnodactyly, camptodactyly, chorioretinitis, hypoplasia of labia majora and breast, and walking difficulties. Skeletal abnormalities were progressive and included femur and knee dysplasia (surgically treated) and severe scoliosis with osteoporosis. When she was 22, absence of sphincter control, sporadic non-febrile seizures, and feeding issues were still present, she used a wheelchair and exhibited anxiety. P11 is a Brazilian girl who had been described by Chaves et al. ( 2023 ). Here, we update her clinical presentation ( Supplementary file – Table S2 ). Patient 11 exhibited mild ID, delayed speech and language development, ADHD, ASD, self-injurious behavior (trichotillomania), and insomnia. Convergent strabismus was treated surgically by the age of 10. She presented with Hashimoto’s thyroiditis, constipation, recurrent sinus infection (3–8 years), hypotonia, decreased body weight, and atopic dermatitis. Physical signs at 10 years included preauricular pit, periorbital fullness, epicanthus, hypertelorism, almond-shaped eyes, thick lower lip, micrognathia, arachnodactyly, marfanoid habitus, and scoliosis). Genetic findings Table 1 summarizes the molecular characteristics of the YY1 variants detected in the 11 GDVS patients (ten females) described in this study: eight are previously unreported cases and three have already been reported (Carminho-Rodrigues et al. 2020 ; dos Santos et al. 2022 ; Chaves et al. 2023 ). Table 1 Molecular features of YY1 variants and X-chromosome inactivation data of 11 GDVS cases (ten females) Patient 1 Patient 2 Patient 3 Patient 4 (mother of patient 5) Patient 5 Patient 6 Patient 7 Patient 8 Patient 9 Patient 10 Patient 11 YY1 variant (NM_003403.5) c.1102T > C (p.Phe368Leu) c.1147_1151dup (p.Cys385Metfs*18) c.690dupA (p.Asp231Argfs*3) c.690dupA (p.Asp231Argfs*3) c.690dupA (p.Asp231Argfs*3) c.1114A > C (p.Thr372Pro) c.1057T > C (p.Phe353Leu) c.1043C > T (p.Thr348Ile) c.907T > C (p.Cys303Arg) c.1106A > G (p.Asn369Ser) c.1062 + 1G > A (NP_003394.1:p.?) Nationality Chile Brazil Brazil France France France Germany Brazil Swiss Brazil Brazil Age at referral 2 years 5 years 16 years 30 years 6 months 6 years 7 years 11 years 21 years 22 years 8 years 11 months XCI data (blood) AR 71:29 RP2 - NI AR 73:27 RP2 67:33 AR 71:29 RP2 72:28 AR - NI TMEM185A - NI HMGB3 98:2 - (male) AR 54:46 TMEM185A 53:47 HMGB3 24:76 AR 50:50 RP2 27:73 AR 91:9 RP2 57:43 AR 19:81 RP2 87:13 AR - NI RP2 86:14 (data from dos Santos et al., 2022 ) AR 94:6 (data from Chaves et al., 2023 ) ACMG classification likely pathogenic (PM1 moderate, PM2 moderate, PM6 moderate, PP2 supporting) pathogenic (PVS1 strong, PM2 moderate, PM6 moderate, PS4 strong) pathogenic (PVS1 strong, PM2 moderate, PS4 moderate, PM6 moderate) pathogenic (PVS1 very strong, PM2 moderate, PS4 moderate, PM6 moderate) pathogenic (PVS1 very strong, PS4 moderate, PM2 moderate) pathogenic (PS4 strong, PM1 moderate, PP2 supporting, PM2 moderate, PM6 moderate, PP3 supporting) likely pathogenic (PS4 moderate, PM2 moderate, PM1 supporting, PM6 moderate, PP2 supporting) likely pathogenic (PM2 moderate, PM1 supporting, PM6 moderate, PP3 moderate, PP2 supporting) likely pathogenic (PM2 moderate, PM6 moderate, PP3 supporting, PP2 supporting) likely pathogenic (PS4 moderate, PM2 moderate, PM1 moderate, PM6 moderate, PP2 supporting) pathogenic (PVS1 strong, PM2 moderate, PM6 moderate, PS4 strong, PP1 supporting) AlphaMissense 0.9999 0.9999 - - - 0.9938 0.9998 0.989 0.8659 0.9998 NA LOFTEE - low confidence (end_truncated) high confidence high confidence high confidence - - - - - - Biochemical change Phenylalanine is a large, hydrophobic, and aromatic amino acid. Leucine is also hydrophobic but is an aliphatic amino acid due to the lack of an aromatic ring. The change maintains the hydrophobic character but may influence the local folding Cysteine can form disulfide bridges with another cysteine, which improves the protein’s stability and 3D structure. Although methionine also contains sulfur, it does not affect the formation of disulfide bridges. The substitution of cysteine by methionine may compromise the formation of disulfide bridges, affecting the structural integrity of the protein Aspartic acid is a polar amino acid characterized by its negative charge, which enables it to engage in electrostatic interactions and contribute to the formation of salt bridge formation. Arginine is a basic amino acid with a larger, positive charge and the ability to form multiple hydrogen bonds. The substitution represents a charge reversal. It may alter local electrostatic interactions, possibly by disrupting or creating salt bridges Threonine is a polar amino acid with a hydroxyl group (-OH) in its side chain, allowing it to participate in hydrogen bonding and contribute to the stabilization of secondary structures. Proline is unique among amino acids due to its cyclic structure, where the side chain forms a covalent bond with the backbone nitrogen, restricting backbone flexibility and disrupting regular secondary structures. The substitution of threonine by proline may disrupt local secondary structure Phenylalanine is a large, hydrophobic and aromatic amino acid. Leucine is also hydrophobic, but it is an aliphatic amino acid due to the lack of an aromatic ring. The change maintains the hydrophobic character but may influence the local folding Threonine is a polar amino acid, able to interact by hydrogen bonds. Isoleucine is a hydrophobic amino acid, incapable of such bonds Cysteine can form disulfide bonds. Arginine is different in size and charge (positive). The substitution may affect the structure/function of the protein by compromising disulfide bridges formation Asparagine has a polar side chain. Serine has a smaller side chain that contains a hydroxyl group (-OH). This is a conservative substitution, but it may affect local geometry NA NI: not informative; NA: not available; AlphaMissense scores: 0-0.34: likely benign; 0.34–0.564: VUS; 0.564-1: likely pathogenic All patients, except for P5, were found to carry de novo mutations. P5 inherited a YY1 frameshift variant from P4, his affected mother. Variants were reclassified regarding their pathogenicity, following ACMG/ClinGen criteria (Table 1 ) and Clingen updates. Three novel YY1 variants were identified in the present study in P1, P2 and P8. A pathogenic heterozygous YY1 missense variant [chr14:100.277.457 T > C (hg38); YY1 (NM_003403.5):c.1102T > C(p.Phe368Leu)] was identified in P1. A pathogenic heterozygous YY1 frameshift variant was identified in P2 [chr14:100.277.501 C>CTATGT (hg38); YY1 (NM_003403.5):c.1147_1151dup(p.Cys385Metfs*18)]. In P3, P4 and P5, a pathogenic heterozygous YY1 frameshift variant [chr14:100.262.306 G > GA (hg38); YY1 (NM_003403.5):c.690dup(p.Asp231Argfs*3)] was detected. This variant was previously reported in ClinVar (ID 1331550, classified as likely pathogenic) and in the literature (Cherik et al. 2022 ). A heterozygous missense YY1 variant was detected in P6 [chr14:100.277.469 A > C (hg38); YY1 (NM_003403.5):c.1114A > C(p.Thr372Pro)], already described in ClinVar (ID 1703497, as pathogenic). A missense YY1 variant was found in P7 [chr14:100.276.643 T > C (hg38); YY1 (NM_003403.5):c.1057T > C(p.Phe353Leu)] in heterozygosis, which has been previously reported as likely pathogenic in ClinVar (ID 2413123) and in the literature (Topa et al. 2024 ). Lastly, in P8, another heterozygous YY1 missense variant [chr14:100.276.629 C > T (hg38); YY1 (NM_003403.5):c.1043C > T(p.Thr348Ile)], not previously reported, was revealed. Figure 1 s hows the position of each YY1 variant in the YY1 protein. Schematic diagram of YY1 protein, based on dos Santos et al. ( 2022 ). Position and nomenclature of YY1 pathogenic variants of patients whose XCI patterns have been evaluated are represented. Circles, triangles and stars portray missense, frameshift and splicing variants, respectively. Gray and black filled circles/triangles/stars represent moderate (ratios between 71:29 and 90:10) and extreme (> 90:10 ratios) skewing, respectively, in blood DNA samples for the marker that exhibited the highest deviation from a random pattern ( AR , RP2 , TMEM185A or HMGB3 ). Not in scale XCI patterns in blood samples XCI patterns were assessed for 10 patients (P1-P4 and P6-P11). The XCI analysis was performed using DNA samples extracted from peripheral blood. P1 showed moderate skewing for the AR locus at an average ratio of 71:29 and was homozygous for the RP2 locus and hence not informative for this marker. In P2, the AR and RP2 analysis yielded ratios of 73:27 (moderate skewing) and 67:33 (random), respectively. The XCI analysis for P3 revealed moderate skewing for AR and RP2 loci (71:29 and 72:28), respectively). P4 was not informative for AR or TMEM185A , but displayed extreme skewing for HMGB3 (98:2). P6 exhibited random XCI for the AR (54:46) and TMEM185A (53:47) markers, but moderate skewing for HMGB3 (24:76). The XCI analysis using P7’s samples showed a random inactivation ratio for AR (50:50), but moderate skewing for RP2 (27:73). Figure 2 shows these results for three patients, as examples, and the XCI ratios are presented in Table 1 , including those of the three already published GDVS cases (P9–P11). P9 (whose clinical presentation had been previously reported by Carminho-Rodrigues et al. ( 2020 ) exhibited moderate skewing for both the AR (19:81) and RP2 (87:13) loci . P10 was not informative for the AR marker, and the analysis for the RP2 locus indicated moderate skewing (86:14) (data from dos Santos et al. 2022 ). P11’s samples demonstrated extremely skewed XCI (94:6) for the AR locus (data from Chaves et al. 2023 ). Filled circles in the pedigree charts represent females diagnosed with GDVS. Red rectangles highlight moderate XCI skewing. u: undigested; d: digested. The graphics are not in scale. a) P1’s pedigree chart. b) XCI assay using P1’s blood DNA samples for the AR locus (71:29 average ratio). c) P2’s pedigree chart. d) XCI assay using P2’s blood DNA samples for the AR locus (73:27 average ratio). e) P8’s pedigree chart. f) Graphics resulting from XCI assay using P8’s blood DNA samples for the AR locus (91:9 average ratio) In silico analysis of YY1 protein 3D structure YY1 protein contains four zinc-finger domains in its C-terminal region that are essential for DNA binding and transcriptional regulation. Variants located within this region, such as the missense here reported (p.R303Q, p.I348T, p.L353P, p.L368P, p.S369C, and p.M385V), occur within or near the zinc-finger motifs; in terms of structural interactions, the residues Phe368 and Asn369 interact directly with DNA, while Cys385 interacts with zinc. Therefore, all of them are predicted to alter zinc coordination or disrupt the interface with DNA. All the six missense variants listed in Table 1 exhibited high pathogenicity scores according to AlphaMissense, a deep learning method for predicting the pathogenicity of single amino acid substitutions in human proteins based on protein structure and evolutive conservation, with five exceeding 0.9 (AlphaMissense cuttoffs: ≥ .564, likely pathogenic; 0.565 − 0.340, likely neutral; ≤ 0.340, likely benign). These structural disturbances likely reduce YY1’s DNA-binding affinity and impair its transcriptional regulatory function, providing a plausible molecular basis for the associated developmental phenotypes. The changes in the 3D structure of the protein caused by these six missense variants (303R, 348I, 353L, 368L, 369S, and 385M) are represented in Fig. 3 . Although not designed to evaluate LoF variants, Alphamissense can be applied to generated models of truncated proteins, assuming that truncated forms can occur if they escape from the NMD. We evaluated two of the LoF variants: c.690dupA(p.Asp231Argfs*3) and c.1147_1151dup(p.Cys385Metfs*18); the third one can not be evaluated because it is a splice site mutation. The c.690dupA(p.Asp231Argfs*3) variant had a high confidence classification in LOFTEE, suggesting loss of function, whereas the c.1147_1151dup(p.Cys385Metfs*18) variant had a low confidence classification with the truncation site near to the end of the gene. The model for p.Cys385Metfs*18 exhibits C-terminal region loss, extending beyond the α-helical segment, while the experimental structure remains fully resolved throughout the entire length of the molecule (Fig. 3 ). The c.690dupA(p.Asp231Argfs*3) variant, however, could not be depicted in the 3D model, possibly indicating an association with specific protein characteristics. The AlphaFold database shows that the structural model for the YY1 protein (ID: P25490) could indicate that residues 1–295 are situated within a disordered region, a finding that is also supported by DisProt. While the AlphaMissense score for the p.Asp231Argfs*3 variant is lower than that of other variants, it is important to note that this variant is situated within a structurally disordered region. Considering the variants in the study, only p.Asp231Argfs*3 is located outside the zinc finger domain. However, it could play additional functional roles, especially within the Gly-rich region that is linked to HCFC1 interactions. Also, dosage sensitivity data suggest that YY1 is dosage intolerant, as evidenced by high pHaplo and pTriple scores (0.96 and 0.99, respectively). 4. Discussion GDVS is caused by YY1 haploinsufficiency due to truncating alterations or missense variants, the latter often mapping to the YY1 zinc-finger motifs (Gabriele et al. 2017 ). Here, we compiled detailed clinical and molecular features of eleven cases ( Supplementary file – Table S2 ), three of them previously reported (Carminho-Rodrigues et al. 2020 ; dos Santos et al. 2022 ; Chaves et al. 2023 ). The patients displayed clinical traits already reported in GDVS, including intrauterine growth restriction, low birth weight, global DD, hypotonia, gait disturbances and craniofacial dysmorphisms, such as broad and prominent forehead, periorbital fullness, eyelid ptosis, micrognathia, pointed chin, and downslanted palpebral fissures (Gabriele et al. 2017 ; Morales-Rosado et al. 2018 ; Carminho-Rodrigues et al. 2020 ; Bae et al. 2021 ; Balakrishnan and Ranganath 2021 ; Malaquias et al. 2021 ; Tan et al. 2021 ; Zorzi et al. 2021 ; Alali and Vitalone 2022 ; Cherik et al. 2022 ; dos Santos et al. 2022 ; Ferng et al. 2022 ; Khamirani et al. 2022 ; Asato et al. 2023 ; Chaves et al. 2023 ; Chawla et al. 2023 ; Koruga et al. 2023 ; Woo et al. 2023 ; Shin et al. 2024 ; Srour et al. 2024 ; Topa et al. 2024 ; Yang et al. 2024 ; Mudassir et al. 2025 ; Pal et al. 2025 ; Huang et al. 2025 ). It is interesting to note that hearing impairment, which was recorded in our series, has been previously described in four patients (Bae et al. 2021 ; dos Santos et al. 2022 ; Cherik et al. 2022 ; Asato et al. 2023 ). Behavioral issues are relatively common in this condition, and were also presented by patients recruited for the present investigation. Attention Deficit Disorder was present in P1 (with Hyperactivity - ADHD), P3 (ADHD), P6, P7, and P11 as well as in several other individuals with GDVS (Gabriele et al. 2017 ; Morales-Rosado et al. 2018 ; Carminho-Rodrigues et al. 2020 ; Alali and Vitalone 2022 ; Cherik et al. 2022 ; Khamirani et al. 2022 ; Chaves et al. 2023 ; Chawla et al. 2023 ). P3, P9 and P10 displayed anxiety, which has also been previously reported in GDVS (Gabriele et al. 2017 ; Cherik et al. 2022 - supplementary material; dos Santos et al. 2022 ; Khamirani et al. 2022 ). P1 exhibited autism and self-injurious behavior, both of which have already been described (Gabriele et al. 2017 ; Cherik et al. 2022 ; Ferng et al. 2022 ; Khamirani et al. 2022 ). P2 is currently being evaluated for ASD. Sleeping disturbances were observed in P1 and P2, similarly to other cases in the literature (Gabriele et al. 2017 ). P1, P3, P7 and P8 had recurrent infections, also documented in individuals with GDVS (Tan et al. 2020; Cherik et al. 2022 - supplementary material; Khamirani et al. 2022 ). Significant clinical heterogeneity was also observed. P2 exhibited dysarthria, which seems to be a rare feature, only reported by Ferng et al. ( 2022 ) and Chawla et al. ( 2023 ). Other uncommon clinical characteristics here described include congenital torticollis (P2), which has only been reported by Cherik et al. ( 2022 ), and abnormalities of the nasolacrimal duct (P3; obstruction), also reported once (Gabriele et al. 2017 ; stenosis). We found an increased frequency of thyroid and autoimmune conditions in this group of patients. P3 and P7 were diagnosed with Graves’ disease; in P3, the condition later evolved into Hashimoto’s thyroiditis. P11 also exhibited Hashimoto’s disease, and P8 was diagnosed with hyperthyroidism. Graves’ disease and Hashimoto’s thyroiditis are both autoimmune thyroid diseases; the Hashimoto’s thyroiditis may produce hyper- or hypothyroidism manifestations, while Graves’ disease causes hyperthyroidism (Liu et al. 2023 ; Davies et al. 2020 ). It is interesting to note that thyroid dysfunction has been previously reported in GDVS, mainly hypothyroidism and thyroid nodules (Gabriele et al. 2017 ; Alali and Vitalone 2022 ; Cherik et al. 2022 ; Asato et al. 2023 ; Pal et al. 2025 ; Huang et al. 2025 ). Remarkably, previous in silico analysis of protein-protein interactions of YY1 protein disclosed enrichment of thyroid hormone signaling, among other biological pathways (dos Santos et al. 2022 ). Other endocrine abnormalities in GDVS include growth hormone deficiency, although rarely described (Gabriele et al. 2017 ). Besides, Morales-Rosado et al. ( 2018 ) published a case of a GDVS patient with the diagnosis of autoimmune myasthenia gravis. In addition, one of the patients (P1) presented with clinical signs that have not been previously reported for GDVS patients to our knowledge, namely hypertrichosis and atopic dermatitis. A more detailed clinical revaluation of P11 – previously described (Chaves et al. 2023 ) – revealed that she also exhibited atopic dermatitis, a finding not noted in the original publication. Atopic dermatitis is a condition that has been associated with an increased risk of developing autoimmune diseases, as studied and discussed by Ahn et al. ( 2024 ). However, it is a frequent manifestation in the normal population, making it hard to ascertain this clinical feature to GDVS. Taken together, these observations hint that thyroid/endocrine disorders (especially hypothyroidism) and autoimmune conditions may represent an important clinical aspect of GDVS, as also proposed by Alali and Vitalone ( 2022 ) and Huang et al. ( 2025 ). These features should be considered in the clinical evaluation and management of GDVS patients. Among the approximately 44 individuals with GDVS clinically described in the literature to date, 20 presented with movement disorders, among whom 12 exhibited dystonia (Gabriele et al. 2017 ; Morales-Rosado et al. 2018 ; Carminho-Rodrigues et al. 2020 ; Balakrishnan and Ranganath 2021 ; Zorzi et al. 2021 ; Malaquias et al. 2021 ; Ferng et al. 2022 ; Khamirani et al. 2022 ; Cherik et al. 2022 ; Chawla et al. 2023 ; Shin et al. 2024 ; Srour et al. 2024 ; Mudassir et al. 2025 ) ( Supplementary file – Table S1 ). The YY1 variant carrier reported by Zech et al. ( 2021 - supporting information) also presented with dystonia; however, since this was the only clinical feature documented for that individual, this case was not included among those with a comprehensive clinical description. Notably, 18 of these 20 clinically characterized cases with dystonia and/or other movement disorders also exhibited neurodevelopmental deficits, such as DD and/or ID (Gabriele et al. 2017 ; Morales-Rosado et al. 2018 ; Carminho-Rodrigues et al. 2020 ; Balakrishnan and Ranganath 2021 ; Zorzi et al. 2021 ; Ferng et al. 2022 ; Khamirani et al. 2022 ; Cherik et al. 2022 ; Shin et al. 2024 ; Srour et al. 2024 ; Mudassir et al. 2025 ). The remaining two cases (Malaquias et al. 2021 ; Chawla et al. 2023 ) did not prominently manifest the core GVDS phenotype, although they did exhibit other nonspecific clinical features commonly reported in the syndrome, including low birth weight and facial dysmorphisms, such as pointed chin and bulbous nose. These observations underscore a phenotypic overlap between YY1 -related movement disorders and neurodevelopmental deficits. Rather than representing distinct diagnostic categories, these phenotypes appear to constitute a single clinical entity associated with YY1 disruption, with a wide and variable manifestation spectrum. This full picture of the GDVS is still evolving; therefore, describing additional cases is a key step to broaden the clinical and molecular spectrum of the condition, with implications for improvement of diagnosis and clinical management. Figure 1 depicts the YY1 protein and the location of the pathogenic variants carried by the affected individuals reported in this study, along with the corresponding XCI pattern observed in the assessed females. P1, P6, P7, P9 (Carminho-Rodrigues et al. 2020 ) and P10 (dos Santos et al. 2022 ) carried missense variants mapped to sequences encoding part of the zinc-finger domain (DNA-binding). The missense variant identified in P8 affects an amino acid located between two zinc finger motifs. The remaining five patients, P2, P3/P4/P5 (frameshift) and P11 (Chaves et al. 2023 ; splice site), carry LoF variants. Interestingly, six out of the eleven variants here reported (variants identified in P1, P2, P6, P7, P9 and P10) impact the zinc-finger DNA binding domains. According to GnomAD, YY1 displays intolerance to missense (z-score = 3.6) and loss-of-function variants (pLI score = 1). Also, the pHaplo (0.96) and pTriple (0.99) values indicate that the YY1 gene is dosage-sensitive; and alterations in its expression may lead to pathological effects. YY1 is a ubiquitously expressed transcription factor involved in the regulation of a wide range of gene promoters, acting either as a repressor or as an activator. Importantly, YY1 is a pivotal gene in the initiation of XCI, since it activates XIST transcription in human and mouse contexts (Chapman et al. 2014 ; Makhlouf et al. 2014 ) and might serve as a docking protein for Xist transcripts to the inactive X-chromosome (Jeon and Lee 2011 ). YY1 interacts with numerous chromatin modifiers (Verheul et al. 2020 ), and is also known to directly and indirectly interact with numerous XCI key regulators (see discussion in dos Santos et al. 2022 ). In Drosophila , YY1 has been shown to participate in the recruitment of polycomb group proteins, which are fundamental for maintaining transcriptional repression of genes (Wilkinson et al. 2006 ). Polycomb group protein recruitment to DNA by YY1 leads to the methylation of histone H3 on lysine 27 (H3K27) (Wilkinson et al. 2006 ). The polycomb complexes PRC1 and PRC2 mediate specific histone modifications, which are involved in the XCI process (Sun et al. 2021 ). Also, YY1 may be implicated in recruiting polycomb complexes to the Xi (Thorvaldsen et al. 2011 ). Additionally, YY1 interacts with histone deacetylases (HDACs) engaged in gene silencing, such as HDAC3, and histone acetyltransferases (HATs) (Verheul et al. 2020 ). Studies point to HDAC3 involvement in XCI, deacetylating histones upon interaction with XIST RNA and helping gene silencing and RNA polymerase II exclusion from the Xi ‌(McHugh et al. 2015 ; Żylicz et al. 2018 ). Moreover, a recent study in mice suggests that the YY1 binding to X-linked genes can act as a barrier to Xist -mediated silencing until XCI late stages (Bowness et al. 2024 ). Given YY1 's prominent involvement in the XCI process, we hypothesized that germline YY1 mutations could drive or impact the XCI pattern in females, leading to skewing (dos Santos et al. 2022 ; Chaves et al. 2023 ). To date, only two published studies have assessed XCI patterns in female patients with YY1 mutations (dos Santos et al. 2022 ; Chaves et al. 2023 ), and both reported XCI skewing. Here, a heterogeneous XCI pattern was observed among the eight cases newly evaluated (P1-P4 and P6-P9). In spite of that, it is noteworthy that in total, seven patients (P1, P2, P3, P4, P7, P9 and P10) exhibited moderate XCI skewing for at least one marker, and three individuals (P4, P8 and P11) showed extreme XCI skewing. These findings may reinforce a potential involvement of YY1 mutations in modulating the XCI pattern in trans , although the mechanistic link is not clear. It is crucial to recognize that XCI skewing can arise due to several mechanisms, such as the stochastic nature of the molecular mechanism, the presence of an X-linked variant unrelated to ID, or even the activity of escapee X-linked genes (Peeters et al. 2023 ). An impressive number of variants in X-linked genes are associated with ID (Migeon 2020 ). Plenge et al. ( 2002 ) first described that female carriers of harmful variants causing X-linked ID (XLID) disorders displayed XCI skewing, with findings suggesting a negative selection against cells harboring the mutation on the active X-chromosome. Notwithstanding the potential protective effect provided by XCI (Migeon 2020 ), female carriers of those XLID pathogenic variants may exhibit skewing favoring the mutant allele, and can still present ID (Vianna et al. 2020 ; Chaves et al. 2023 ). Gabriele et al. ( 2017 ) reported that lymphoblastoid cell lines (LCLs) from two individuals carrying missense mutations [c.1138G > T (p.Asp380Tyr)] and [c.1097T > C (p.Leu366Pro)]) displayed wild-type YY1 levels, after analysis by RNA-seq and Western blot. Upregulation of YY1 was detected in induced pluripotent stem cells (iPSCs) derived from another affected individual with a missense variant (Pereira et al. 2025 ). Jeon and Lee ( 2011 ) proposed that YY1 zinc-fingers interact with both XIST DNA and RNA by binding to different motifs on the DNA and RNA. Therefore, it is plausible to speculate that YY1 isoforms containing missense variants may exhibit reduced affinity to XIST DNA or RNA, resulting in a preferential binding of the wild-type YY1 isoform. In the other way, LoF YY1 mutations, which are known to lead to a strong reduction in the amount of protein in LCLs (Gabriele et al. 2017 ) and iPSCs (Pereira et al. 2025 ) derived from patients with GDVS, would result in a reduced availability of functional YY1. This reduction induces global decrease of YY1 occupancy in the genome (Gabriele et al. 2017 ; Pereira et al. 2025 ); however, if this condition impacts the XCI pattern is a question not yet addressed. Recent findings (Bertin et al. 2026 ) suggested that females may exhibit an altered dynamic utilization of the two X-chromosomal alleles. This would distort the locus - and lineage-specific reactivation of the inactive X, a mechanism proposed to serve as a critical reservoir during differentiation, thereby enhancing the resilience of female neural tissue. Taking into account that YY1 interacts with a myriad of elements involved in XCI and its reduced levels in YY1 -LoF mutations and/or disrupted activity of YY1 -missense mutations, it is tempting to suggest that these harmful YY1 variants could drive a primary XCI skewing, but no obvious explanation can be provided at this point. Alternatively, YY1 depletion caused by germline pathogenic variants could indirectly lead to XCI skewing, by altering progenitor cell development or survival during early development, thereby reducing the available cell pool and increasing the likelihood of biased inactivation patterns. We may also speculate that females with YY1 mutations can develop in the brain a disrupted version of the heterogeneous expression pattern of escapee X-linked genes (Peeters et al. 2023 ). This later hypothesis is interesting when associated with the autoimmune conditions reported here. Autoimmune diseases disproportionately affect women compared to men, with contributing factors such as estrogen, X-linked genes, and microbiota composition; there are several studies addressing how XCI features may drive the sex disparity in autoimmunity of females (reviewed in Mousavi et al. 2020 ). In conclusion, our study expands the clinical and molecular understanding of GDVS. Considering the limited number of reported GDVS cases in the literature, it is of paramount importance to describe new cases along with their clinical and molecular data for improving diagnosis accuracy and exploring potential therapeutic strategies for the syndrome. Moderate XCI skewing was detected in blood samples from seven patients, and extremely skewed XCI patterns were found in three patients, reinforcing that YY1 mutations may impact XCI patterns. Future functional studies to assess the impact of the YY1 mutations on XCI could employ patient-derived cells to evaluate XIST transcription and its interaction with the X-chromosome. Addressing this gap in future research will be crucial to fully elucidate the mechanistic links between YY1 dysfunction and XCI alterations in GDVS. Declarations Acknowledgments We are very grateful to the patients and their families for collaborating in this study. We thank Carlos Augusto Takeuchi for clinically evaluating one of the patients and connecting us with the family. We also thank Dr. Rafael Martins Galupa for his valuable contributions reviewing the manuscript. Ethics approval This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Human Research Ethics Committee of the Institute of Biosciences of the University of São Paulo (CAAE 80921117.5.0000.5464), Institutional Ethics Committee from the State University of Rio de Janeiro (CAAE 46769315.5.0000.5259), University of São Paulo Ethics Committee (CAAE 80921117.5.0000.5464), and the French ethics committee (French Ministry of Research and Innovation, DC-2020-4073. All data presented in this manuscript were fully anonymized, and no identifying images or personal information are included. Consent to participate We obtained written informed consent from the patients’ legal guardians. Consent to publish Written informed consent for publication of clinical and genetic data was obtained from the patients’ legal guardians. Data Availability The reported variants are available in the ClinVar database of genetic variants. ClinVar Variation ID: 2582783, Accession: VCV002582783.1 (P1) ClinVar Variation ID: 3236170, Accession: VCV003236170.1 (P2) ClinVar Variation ID: 1331550, Accession: VCV001331550.4 (P3, P4 and P5; not submitted by us) ClinVar Variation ID: 1703497, Accession: VCV001703497.1 (P6; not submitted by us) ClinVar Variation ID: 2413123, Accession: VCV002413123.6 (P7; not submitted by us) ClinVar Variation ID: 4530027, Accession: VCV004530027.1 (P8) Additional data may be made available by the corresponding author upon reasonable request. Data sharing will be considered in accordance with participant privacy and ethical standards. Author Contributions Ana Cristina Victorino Krepischi and Cíntia Barros Santos-Rebouças conceived and designed the study. Laura Machado Lara Carvalho, Matheus Augusto Araújo Castro, Cíntia Barros Santos-Rebouças, and Ana Cristina Victorino Krepischi established collaborations, recruited patients, and coordinated the collection of clinical data and samples. Bianca Mie Sato Kurashima, Andressa Pereira Gonçalves, and Silvia Souza da Costa performed XCI pattern assays. Bianca Mie Sato Kurashima performed Sanger sequencing, wrote the initial version of the manuscript, and prepared figures 1 and 2 under the guidance of Ana Cristina Victorino Krepischi, Laura Machado Lara Carvalho, and Cíntia Barros Santos-Rebouças. Rafael Mina Piergiorge and Cíntia Barros Santos-Rebouças performed in silico analyses. Rafael Mina Piergiorge prepared figure 3. Lina Quteinheh, Maria Teresa Carminho-Rodrigues, Vincent Michaud, Fanny Morice-Picard, Caroline Rooryck, Matthias Begemann, and Larissa Mattern conducted formal analyses of patients. Francisco Cammarata-Scalisi, Mariana de Carvalho Moreira, Suely Rodrigues dos Santos, and Elis Vanessa de Lima Silva clinically evaluated patients. All authors contributed to Writing – Review & Editing and have read, revised, and approved the final version of the manuscript. Competing Interests The authors declare no conflicts of interest. Funding This work was financed, in part, by the São Paulo Research Foundation (FAPESP), Brazil [Process Numbers #2023/09879-7, #2013/08028-1, #2022/03980-5, #2025/00171-7, #2023/15506-9, and #2025/04380-0], by the Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Brazil [E-26/010.001888/2019; E-26/200.883/2021; E-26/204.043/2024], and by the National Council for Scientific and Technological Development (CNPq), Brazil [#125838/2023-9, #302263/2019-5; #302342/2022-2]. The Article Processing Charge (APC) for this publication was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (ROR identifier: 00x0ma614), Brazil. For the purposes of open access, the authors have applied a Creative Commons CC BY licence to any accepted version of the article. 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Parkinsonism Relat Disord 124:106995. https://doi.org/10.1016/j.parkreldis.2024.106995 Srour L, Baroudi K, Saleh A, Shaker R, Mokbel R, Alam CA (2024) The 30th Case of Autosomal Dominant Gabriele-de Vries Syndrome: Diagnosis and Management in a 16-Month-Old Lebanese Boy. Acta Sci Clin Case Rep 5:04–07 Sun Z, Fan J, Zhao Y (2021) trans-Acting Factors and cis Elements Involved in the Human Inactive X Chromosome Organization and Compaction. Genet Res 2021:1–7. https://doi.org/10.1155/2021/6683460 Tan L, Li Y, Liu F, Huang Y, Luo S, Zhao P, Gu W, Lin J, Zhou A, He X (2021) A 9-month-old Chinese patient with Gabriele-de Vries syndrome due to novel germline mutation in the YY1 gene. Mol Genet Genomic Med 9:e1582. https://doi.org/10.1002/mgg3.1582 Tan X, Yang Y, Wu X, Zhu J, Wang T, Jiang H, Chen S, Lou S (2025) An investigation of a hemophilia A female with heterozygous intron 22 inversion and skewed X chromosome inactivation. 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Front Cell Dev Biol 8:592164. https://doi.org/10.3389/fcell.2020.592164 Vianna EQ, Piergiorge RM, Gonçalves AP, dos Santos JM, Calassara V, Rosenberg C, Krepischi ACV, Boy da Silva RT, dos Santos SR, Ribeiro MG et al (2020) Understanding the Landscape of X-linked Variants Causing Intellectual Disability in Females Through Extreme X Chromosome Inactivation Skewing. Mol Neurobiol 57:3671–3684. https://doi.org/10.1007/s12035-020-01981-8 Wilkinson FH, Park K, Atchison ML (2006) Polycomb recruitment to DNA in vivo by the YY1 REPO domain. Proc Natl Acad Sci U S A 103:19296–19301. https://doi.org/10.1073/pnas.0603564103 Woo H, Kim WS, Kim JS (2023) A rare epilepsy phenotype in Gabriele-de Vries syndrome: A new case and literature review. Neurol Asia 28:1063–1067. https://doi.org/10.54029/2023eat Yang J, Yu C, Lyn N, Liu L, Li D, Shang Q (2024) Clinical analysis of Gabriele-de Vries caused by YY1 mutations and literature review. Mol Genet Genomic Med 12:e2281. https://doi.org/10.1002/mgg3.2281 Zech M, Jech R, Boesch S, Škorvánek M, Necpál J, Švantnerová J, Wagner M, Sadr-Nabavi A, Distelmaier F, Krenn M et al (2021) Scoring Algorithm‐Based Genomic Testing in Dystonia: A Prospective Validation Study. Mov Disord 36:1959–1964. https://doi.org/10.1002/mds.28614 Zorzi G, Juan I, Danti FR, Bustos BI, Invernizzi F, Panteghini C, Reale C, Garavaglia B, Chiapparini L, Lubbe SJ et al (2021) YY1-Related Dystonia: Clinical Aspects and Long‐Term Response to Deep Brain Stimulation. Mov Disord 36:1461–1462. https://doi.org/10.1002/mds.28547 Żylicz JJ, Bousard A, Žumer K, Dossin F, Mohammad E, da Rocha ST, Schwalb B, Syx L, Dingli F, Loew D et al (2018) The Implication of Early Chromatin Changes in X Chromosome Inactivation. Cell 176:182–197e23. https://doi.org/10.1016/j.cell.2018.11.041 6.Statements & Declarations Additional Declarations No competing interests reported. Supplementary Files ESM1.pdf Table S1 Summary of published cases of Gabriele-de Vries syndrome (GDVS). The table includes publication of reference, number of novel cases reported, identification of individual patients when more than one case is reported, variant nomenclature, variant type and reported classification of pathogenicity, inheritance pattern, the presence or absence of a clinical description, age of referral or examination and sex of the affected individuals, the presence or absence of neurodevelopmental disorders, movement abnormalities, hormone/thyroid dysfunction and/or autoimmune conditions, and additional observations (Available in the Supplementary file) Table S2 Summary of the clinical traits of the eleven patients with heterozygous pathogenic/likely pathogenic variants in YY1 (three of them previously reported) who were evaluated in this study (Available in the Supplementary file) Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 18 May, 2026 Reviewers invited by journal 18 Feb, 2026 Editor assigned by journal 11 Feb, 2026 Submission checks completed at journal 11 Feb, 2026 First submitted to journal 10 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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03:38:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8846661/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8846661/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103177595,"identity":"71866cd2-ad91-4542-9932-0a8480fdfad9","added_by":"auto","created_at":"2026-02-22 16:52:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":95497,"visible":true,"origin":"","legend":"\u003cp\u003eYY1 protein structure and position of pathogenic variants identified in the patients\u003c/p\u003e\n\u003cp\u003eSchematic diagram of YY1 protein, based on dos Santos et al. (2022). Position and nomenclature of \u003cem\u003eYY1 \u003c/em\u003epathogenic variants of patients whose XCI patterns have been evaluated are represented. Circles, triangles and stars portray missense, frameshift and splicing variants, respectively. Gray and black filled circles/triangles/stars represent moderate (ratios between 71:29 and 90:10) and extreme (\u0026gt;90:10 ratios) skewing, respectively, in blood DNA samples for the marker that exhibited the highest deviation from a random pattern (\u003cem\u003eAR\u003c/em\u003e, \u003cem\u003eRP2\u003c/em\u003e, \u003cem\u003eTMEM185A \u003c/em\u003eor \u003cem\u003eHMGB3\u003c/em\u003e). Not in scale\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8846661/v1/84271e90afb657821e3344cf.png"},{"id":103504630,"identity":"a2c671bd-53dc-44af-bb61-079b1211ff37","added_by":"auto","created_at":"2026-02-26 13:20:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":97243,"visible":true,"origin":"","legend":"\u003cp\u003ePedigree charts of three female patients exemplifying the XCI study\u003c/p\u003e\n\u003cp\u003eFilled circles in the pedigree charts represent females diagnosed with GDVS. Red rectangles highlight moderate XCI skewing. u: undigested; d: digested. The graphics are not in scale. \u003cstrong\u003ea) \u003c/strong\u003eP1’s pedigree chart. \u003cstrong\u003eb)\u003c/strong\u003e XCI assay using P1’s blood DNA samples for the \u003cem\u003eAR locus \u003c/em\u003e(71:29 average ratio). \u003cstrong\u003ec)\u003c/strong\u003e P2’s pedigree chart. \u003cstrong\u003ed)\u003c/strong\u003e XCI assay using P2’s blood DNA samples for the \u003cem\u003eAR locus \u003c/em\u003e(73:27 average ratio). \u003cstrong\u003ee)\u003c/strong\u003e P8’s pedigree chart. \u003cstrong\u003ef)\u003c/strong\u003e Graphics resulting from XCI assay using P8’s blood DNA samples for the \u003cem\u003eAR locus \u003c/em\u003e(91:9 average ratio)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8846661/v1/e4b98838e380e55501f80f65.png"},{"id":103177596,"identity":"51bc1552-45ab-489d-b08c-49f21beb5dd6","added_by":"auto","created_at":"2026-02-22 16:52:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":262943,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn silico\u003c/em\u003eanalysis of the missense variants and p.Cys385Metfs*18 included in this study\u003cstrong\u003e\u003cbr\u003e\na) \u003c/strong\u003eThree-dimensional model of the YY1 protein complexed with DNA, obtained from the PDB database (ID: 1UBD). The regions highlighted in pink indicate the amino acid positions examined in this study. \u003cstrong\u003eb–h)\u003c/strong\u003e Structural overlap between the structural models generated by AlphaFold 3 and the crystallographic model. The altered residues are identified, and the side chains are highlighted. The variant p.Cys385Metfs*18\u003cstrong\u003e \u003c/strong\u003e(h) introduces a premature stop codon and is therefore represented as a truncated protein model. The model (yellow) exhibits a loss of the C-terminal region extending beyond the α-helical segment, while the experimental structure (beige) remains fully resolved throughout the entire length of the molecule. Since the 231R mutation did not cover the crystallographic model, it was not represented in this figure. The text presented in red highlights the missense changes depicted in images b to h\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8846661/v1/9b05ea06ec348c7fe56a579b.png"},{"id":103509369,"identity":"902c8c45-8354-4408-8f52-7e67c8aa6d27","added_by":"auto","created_at":"2026-02-26 13:58:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1521277,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8846661/v1/90dc77cb-f696-49e8-b44c-beb42e2c315d.pdf"},{"id":103504721,"identity":"9544135a-8d57-4107-b53c-d049dd030fcd","added_by":"auto","created_at":"2026-02-26 13:21:06","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":856894,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S1 \u003c/strong\u003eSummary of published cases of Gabriele-de Vries syndrome (GDVS). The table includes publication of reference, number of novel cases reported, identification of individual patients when more than one case is reported, variant nomenclature, variant type and reported classification of pathogenicity, inheritance pattern, the presence or absence of a clinical description, age of referral or examination and sex of the affected individuals, the presence or absence of neurodevelopmental disorders, movement abnormalities, hormone/thyroid dysfunction and/or autoimmune conditions, and additional observations\u003c/p\u003e\n\u003cp\u003e(Available in the \u003cstrong\u003eSupplementary file\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S2 \u003c/strong\u003eSummary of the clinical traits of the eleven patients with heterozygous pathogenic/likely pathogenic variants in \u003cem\u003eYY1\u003c/em\u003e (three of them previously reported) who were evaluated in this study\u003c/p\u003e\n\u003cp\u003e(Available in the \u003cstrong\u003eSupplementary file\u003c/strong\u003e)\u003c/p\u003e","description":"","filename":"ESM1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8846661/v1/0e91111041de4de7a96734d8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of the X-chromosome inactivation patterns in females with Gabriele-de Vries syndrome and expansion of clinical spectrum","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGabriele-de Vries Syndrome (GDVS; OMIM #617557), first described in 2017, is a neurodevelopmental autosomal dominant condition caused by \u003cem\u003eYY1\u003c/em\u003e (OMIM *600013) haploinsufficiency \u0026zwnj;(Gabriele et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). It is characterized by developmental delay (DD), mild to profound intellectual disability (ID), craniofacial dysmorphisms and other highly variable phenotypes, comprising low birth weight, multiple congenital anomalies, feeding difficulties, atypical behavior, among others (Gabriele et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Nabais S\u0026aacute; et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGDVS diagnosis relies on genomic testing with the detection of heterozygous pathogenic/likely pathogenic \u003cem\u003eYY1\u003c/em\u003e variants, including missense and loss-of-function (LoF) mutations, and 14q32.2 microdeletions encompassing the \u003cem\u003eYY1\u003c/em\u003e gene (Nabais S\u0026aacute; et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It is a rare condition, with 48 patients reported so far, 44 for whom a general clinical description is available (Gabriele et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Morales-Rosado et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Carminho-Rodrigues et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Bae et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Imafidon et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Balakrishnan and Ranganath \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Malaquias et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Tan et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zech et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zorzi et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Alali and Vitalone \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Cherik et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; dos Santos et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ferng et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Khamirani et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Asato et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Chaves et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Chawla et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Koruga et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Woo et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Luo et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Shin et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Srour et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Topa et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Mudassir et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Pal et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) (see the \u003cb\u003eSupplementary file \u0026ndash; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Gabriele et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) also reported other 13 individuals who carried deletions encompassing the \u003cem\u003eYY1\u003c/em\u003e gene.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eYY1\u003c/em\u003e gene, located on chromosome 14q32.2, encodes a transcription factor involved in numerous physiological roles, such as DNA replication, cell differentiation, apoptosis, and neurogenesis (Jung et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Verheul et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In particular, \u003cem\u003eYY1\u003c/em\u003e plays a central role in the process of X-chromosome inactivation (XCI), a mechanism that evolved in mammals to equalize X-linked gene expression between biological sexes (Jeon and Lee \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Makhlouf et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; J\u0026eacute;gu et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Dossin and Heard \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). It has been shown that YY1 serves as a transcriptional activator of \u003cem\u003eXIST\u003c/em\u003e in both human and mouse contexts (Chapman et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Makhlouf et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; reviewed in Dossin and Heard \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Moreover, studies conducted in mice proposed that YY1 protein binding to the \u003cem\u003eXist locus\u003c/em\u003e and its transcript acts as a bridge that allows \u003cem\u003eXist\u003c/em\u003e RNA to coat the future inactive X-chromosome (Xi) (Jeon and Lee \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Thorvaldsen et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; reviewed in J\u0026eacute;gu et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e and Sun et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Due to the stochastic nature of XCI, its patterns in healthy females show a continuous distribution, with extreme skewed inactivation (\u0026lt;\u0026thinsp;10:90/\u0026gt;90:10) observed only in a small proportion of them, approximately 2% (Amos-Landgraf et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Strikingly, female patients with pathogenic \u003cem\u003eYY1\u003c/em\u003e variants for whom XCI patterns have been analysed exhibited marked skewing of XCI in peripheral blood (dos Santos et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Chaves et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). If confirmed in a larger group of female patients, this phenomenon could have implications on our understanding of \u003cem\u003eYY1\u003c/em\u003e\u0026lsquo;s role in XCI regulation and/or the broader effect of \u003cem\u003eYY1\u003c/em\u003e mutations in affected females.\u003c/p\u003e \u003cp\u003e\u0026zwnj; Herein, we report detailed clinical features and genetic findings of eleven patients with GDVS, including eight previously unreported cases, and present XCI analysis of ten females carrying causal \u003cem\u003eYY1\u003c/em\u003e variants. Furthermore, we performed \u003cem\u003ein silico\u003c/em\u003e analysis of the \u003cem\u003eYY1\u003c/em\u003e missense variants identified in the GDVS female patients.\u003c/p\u003e"},{"header":"2. Patients and methods","content":"\u003cp\u003e \u003cstrong\u003eEthics approval\u003c/strong\u003e \u003cp\u003efor this study was granted by the Institute of Biosciences - University of S\u0026atilde;o Paulo Ethics Committee (CAAE 80921117.5.0000.5464), the Institutional Ethics Committee from the State University of Rio de Janeiro (CAAE 46769315.5.0000.5259), and the French Ethics Committee (French Ministry of Research and Innovation) under approval number DC-2020-4073. Written informed consent was obtained from the patients\u0026rsquo; legal guardians. All procedures adhered to the principles outlined in the Declaration of Helsinki, and its subsequent amendments. To ensure confidentiality and data protection, all participant data were anonymized using numerical identification codes, with database access restricted to authorized research team members, in compliance with data protection regulations.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eWe recruited female patients who had previously undergone Whole Exome Sequencing (WES), and were found to carry pathogenic/likely pathogenic \u003cem\u003eYY1\u003c/em\u003e variants. Clinical characterization of the patients was obtained through analysis of previously conducted health tests or reports from healthcare professionals and parents.\u003c/p\u003e \u003cp\u003ePeripheral blood samples from probands, their parents and siblings (when applicable) were collected for DNA extraction using standard methods. Sanger sequencing analysis or WES of parents was conducted to perform segregation analysis. For Sanger sequencing, PCR was carried out under standard conditions, and amplicons were sequenced in both directions using BigDye (Thermo Fisher) according to the manufacturer's instructions, with capillary electrophoresis performed on an ABI 3730 DNA Analyzer (Thermo Fisher Scientific Inc.).\u003c/p\u003e \u003cp\u003eAll variants were re-evaluated and classified according to the American College of Medical Genetics (ACMG) guidelines (Richards et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and ClinGen updates. Causal variants that had not been previously reported were submitted to ClinVar (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/clinvar/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/clinvar/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo estimate the proportion of cells carrying an active maternal \u003cem\u003eversus\u003c/em\u003e paternal X-chromosome, we employed methylation-sensitive restriction enzyme assays. The XCI patterns of patients P1\u0026ndash;P6, P8 and P9 were determined by assessing differential methylation at different X-chromosome \u003cem\u003eloci\u003c/em\u003e. One of them is located in a polymorphic region (CAG repeats) of the first exon of the \u003cem\u003eAR\u003c/em\u003e gene (mapped to Xq12) (Allen et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1992\u003c/span\u003e); another is mapped to a polymorphic region (GAAA repeats) near the \u003cem\u003eRP2\u003c/em\u003e gene (Xp13.3) (Machado et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e); another is mapped to a polymorphic region (CCG repeats) near the \u003cem\u003eHMGB3\u003c/em\u003e gene (Xq28) (Musalkova et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), and the last one is mapped to a polymorphic region (CCG repeats) near the \u003cem\u003eTMEM185A\u003c/em\u003e gene (Xq28) (Musalkova et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEach XCI assay included one male negative control and one female control with a known extremely skewed XCI. Reproducibility of the data was based on the assay of all samples in duplicate. XCI patterns were calculated in agreement with Bittel et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), using an average of the duplicates. Ratios\u0026thinsp;\u0026le;\u0026thinsp;70:30 were considered random XCI, 71:29\u0026ndash;90:10 as moderate skewing, and \u0026gt;\u0026thinsp;90:10 as extreme skewing, based on criteria reported by Tan et al. (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe six missense variants linked to the YY1 protein were analyzed through AlphaMissense (Cheng et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and structural modeling based on the AlphaFold 3 database (Varadi et al. 2024). Models incorporating each identified mutation were generated based on the wild-type configuration provided by the 1UBD PDB structure. This approach allowed the exploration of the specific structural perturbations induced by the mutations. Residue interactions with DNA and ions were examined using PDBsum for the wild-type structure (Laskowski et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Also, the Variant Effect Predictor (VEP) from Ensembl (McLaren et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) was used to assess dosage sensitivity of the \u003cem\u003eYY1\u003c/em\u003e gene. The two frameshift variants leading to premature stop codons were evaluated by LOFTEE through VEP from Ensembl.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e \u003cb\u003eClinical description\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBelow, the eight newly identified patients GDVS cases are described in more detail, and clinical data for Patients 9\u0026ndash;11 are summarized. Complete clinical information can be found in the \u003cb\u003eSupplementary file \u0026ndash; Table S2\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePatient 1\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePatient 1 (P1) was a Chilean female with a healthy sister and no family history of genetic diseases. She exhibited intrauterine growth restriction and was born prematurely at 36 weeks of gestation, with a birth length of -2.4 SD, and a birth weight of -3.2 SD. Congenital hypotonia and global DD, including delayed motor, speech and language development, were reported. Physical exams at the age of 2 years and 4 months revealed decreased body weight (\u0026ndash;4.1 SD), craniofacial asymmetry, large forehead, malar flattening, a pointed chin, low-set and posteriorly rotated ears, periorbital fullness, downslanted palpebral fissures, eyelid ptosis, almond-shaped eyes, telecanthus, convergent strabismus, wide, concave nasal bridge, bulbous nose tip, short columella, downturned corners of mouth, micrognathia, sparse hair, hypertrichosis, marfanoid habitus, big hands and feet, and long fingers with joints hypermobility. Feeding difficulties in consuming solid foods, gastroesophageal reflux, and constipation were reported. She presented with impairment of visual pursuit, atopic dermatitis, bilateral neurosensory hearing impairment, nonverbal autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), self-injurious behavior, sleeping disturbance, unsteady gait and had nasolacrimal duct obstruction, recurrent Moro reflex, poor pincer grasp, and recurrent infections. Brain Magnetic Resonance Imaging (MRI) performed at 3 months revealed a thin \u003cem\u003ecorpus callosum\u003c/em\u003e. P1 passed away due to sepsis with multiple organ failure and pulmonary hemorrhage, after an infection caused by multiple pathogens, including \u003cem\u003eStreptococcus\u003c/em\u003e bacteria, adenovirus and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePatient 2\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePatient 2 (P2) is a Brazilian female born via cesarean section after \u003cem\u003ein vitro\u003c/em\u003e fertilization. She has a healthy dizygotic twin sister and no family history of genetic diseases. Her mother developed maternal diabetes during gestation. P2 had intrauterine growth restriction and was born at term (37 weeks) small for gestational age, with a low birth weight (-2.8 SD). Large forehead, downslanted palpebral fissures, eyelid ptosis, thin and sparse hair, micrognathia, and a pointed chin were observed. The patient had congenital torticollis, recurrent Moro reflex and was treated with cranial orthosis between 7 and 10 months of age. She exhibited global DD, including motor and speech development. Hypotonia was noted between 2 to 3 years old, and modest axial ataxia was reported at 18 months, progressing to global ataxia by 3.5 years. MRI revealed mild hydrocephalus. Cranial magnetic resonance imaging at 3 years revealed faint foci of hyperintense signal on T2/FLAIR scattered in the periventricular and deep white matter, potentially related to zones of terminal myelination/gliosis (at 3 years). She is currently 5 years old and has manifested unsteady gait, dysarthria, and sleeping disturbance. She is undergoing assessment for ASD.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePatient 3\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePatient 3 (P3) is a Brazilian female born from a healthy couple who has two healthy siblings (a younger paternal brother and an older maternal sister). She has a paternal cousin with a mild ID. Her mother displayed maternal hypertension during pregnancy. P3 displayed intrauterine growth restriction and was born prematurely from 35 weeks of gestation by cesarean section. The patient was small for gestational age and was born with a low birth weight (-3.1 SD). She had neonatal jaundice and was treated with phototherapy. P3 exhibited nasolacrimal duct obstruction (treated by surgical intervention), congenital hypotonia, and recurrent emesis. She manifested febrile seizures until 1 year of age. Graves\u0026rsquo; disease and Hashimoto\u0026rsquo;s thyroiditis were diagnosed and treated with Methimazole (later suspended). The patient\u0026rsquo;s clinical features at 16 years old also include mild ID, reading and writing difficulties, ADHD, and anxiety. Physical exams showed short stature, a large and prominent forehead, receding hairline, epicanthus and telecanthus, proptosis, bilateral ptosis, long philtrum, overfolded helix, simple ears, micrognathia, thick lower lip, pointed chin, arachnodactyly, camptodactyly, tapered phalanx of fingers of the hands, marfanoid habitus, and big hands and feet. She displayed recurrent falls during childhood. Audiometry, echocardiogram, and abdominal ultrasound exams exhibited normal results.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePatient 4\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePatient 4 (P4) is a French female, one of six siblings from a family originally from the Maghreb. She was born prematurely, had growth delay, speech delay, and academic difficulties with schooling in a medical-educational institute due to mild intellectual disability. She presents with facial dysmorphism including broad forehead, nose with bulbous tip, upslanting palpebral fissures with almond-shaped eyes, enlarged columella, prominent philtrum, pointed chin. She has obesity (1.53 m for 98 kg, BMI 42), convergent strabismus of the left eye, hypermetropia and occasional dizziness. Brain MRI showed normal brain morphology. She had maternal hypertension during her first pregnancy, which happened to be a spontaneous bichorial biamniotic twin pregnancy (mother of patient 5).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePatient 5\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePatient 5 (P5) is a French male born from a twin bichorial biamniotic pregnancy (son of P4), after an emergency caesarean section at 30 weeks and 3 days, due to severe intrauterine growth restriction. His birth weight was 640 g, length was 31,7 cm and OFC was 25,5 cm. Apgar score was 7 at 5 min, pH at 7,26 and lactate at 3,1. He presented with axial hypotonia, tubulopathy and retinopathy due to prematurity. Cardiac ultrasound showed hypertrophic and hypertrabeculated left ventricle associated to a patent foramen ovale with left-right shunt. Abdominal ultrasound did not identify any visceral malformation but a thickening of the walls of the left pyelon associated with a small ectasia of the caliceal tracts upstream. Prenatal brain MRI and postnatal transfontanellar ultrasounds showed normal brain morphology. Ophthalmologic control showed normal retina but revealed hypermetropia and convergent strabismus. He presented with facial dysmorphism including brachycephaly, broad forehead, nose with bulbous tip, upslanting palpebral fissures, enlarged columella, smooth and prominent philtrum, thin upper lip, pointed chin, faded cupid's bow, low set ears with overfolded helix. His hands had a single transverse palmar crease. He held his head up at three months (adjusted age for prematurity), but is unable to sit or stand without support at 12 months and needs physiotherapy and psychomotricity follow-up. He shows good social interaction.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePatient 6\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePatient 6 (P6) is a French 6 years old female born from healthy non-consanguineous parents. She has a healthy older brother. Motor delay was noted at 3 months old with hypotonia, sitting was acquired at 12 months and walking at 2 years and a half. Speech delay was noted with only a few words at 3 years. Physical examination revealed multiple hemangiomas and congenital naevus, marfanoid habitus with arachnodactyly, hyperlaxity and pectus excavatum. Broad forehead was noted. She presented some difficulties for feeding. Examination by cardiac echography and cerebral MRI were normal. Ophthalmologic examination was normal. Array-CGH testing showed normal results.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePatient 7\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePatient 7 (P7) is a girl who was born in Germany after 39\u0026thinsp;+\u0026thinsp;5 gestational weeks of an uneventful pregnancy. Her parents and two older siblings are healthy, and there is no history of genetic disease in her family originating from Vietnam. She had a low birth weight (3 P., -1.91 z) and developed recurrent cyanosis after birth. The patient exhibited dystrophy due to severe feeding issues with swallowing difficulties and recurrent emesis. An inefficient motility of the esophagus and gastroesophageal reflux were diagnosed. Her motor development and socio-emotional development were delayed, while speech development was normal. She has recurrent infections intermittently treated with long-term antibiotic medication. Physical exams showed craniofacial asymmetry, periorbital fullness, malar flattening, protruding ears, and short columella. The patient\u0026rsquo;s clinical features at 7 years old also include learning disability (IQ 82), attention-deficit disorder (ADD), lack of orientation, Graves\u0026rsquo; Disease, decreased body weight (\u0026lt;\u0026thinsp;1 P., -3.07 z), and IgA deficiency.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePatient 8\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePatient 8 (P8) is a Brazilian girl born at term. Her birth weight was 2,200 g and her length was 50 cm. She was small for gestational age and did not suck effectively. The patient presented failure to thrive and underwent recurrent hospitalizations due to upper respiratory infections and acute gastroenteritis. Ultrasonography examination at 9.5 years indicated enlarged thyroid gland, with heterogeneous and diffusely hypoechoic texture, diagnosed as hyperthyroidism. At 8.18 years, an electrocardiogram revealed sinus tachycardia and left anterosuperior fascicular block. A physical examination at 11 years and 10 months showed low weight 18.6 kg (z-4), height 141.5 cm, and head circumference 46.5 cm. The observed clinical features were neuropsychomotor developmental delay, intellectual disability, hyperthyroidism, microcephaly, facial dysmorphisms (small face, closed anterior fontanelle, mild midface hypoplasia, proptosis, thick lips), goiter, small nipples, symmetrical limbs, and long, thin fingers with verrucous lesions. Karyotyping was normal.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePatients 9, 10 and 11\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePatients 9 (P9), 10 (P10) and 11 (P11) had already been described in the literature by Carminho-Rodrigues et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), dos Santos et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and Chaves et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), respectively.\u003c/p\u003e \u003cp\u003eP9 presented with left divergent strabismus, was diagnosed with ADHD, and showed a delay of ~\u0026thinsp;2 years in cognitive abilities (Carminho-Rodrigues et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). There were gait and speech difficulties, without cognitive regression. Neurological exams revealed static ataxia with normal muscular tone, kinetic, resting and postural tremor and myoclonic jerks in the left arm. Physical examination at age 21 disclosed facial dysmorphisms (long and asymmetric face, broad forehead, mandibular prognathism, malar flattening and short philtrum), small ears, long nose, full nasal tip, high palate, Gingko leaf-shaped upper lip indentation and thick lower lip. All four members had reduced muscular tone and there was postural and kinetic action tremor in her body. She manifested generalized dystonia, mainly impairing the neck and left hand, and cerebellar features that included gait and stance ataxia.\u003c/p\u003e \u003cp\u003eP10 is a Brazilian patient born from a healthy non-consanguineous couple by caesarean delivery at full term (dos Santos et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Intrauterine growth restriction and hypoactivity were noted. Bilateral hearing loss was detected at 1 year of age, and when she was 3 years old, she failed to thrive and had frequent bilious vomiting. Global developmental delay, hypotonia, low weight, short stature, feeding difficulties, and some pneumonia events were reported, and physical examination revealed flat and triangular face, malar hypoplasia, short palpebral fissures, triangular nose, smooth philtrum, microstomia, arched and narrow palate, thin upper lip, irregular placement of teeth, micrognathia, low-set ears, protruding and malformed auricles, pointed chin, asymmetric thoracic cage, \u003cem\u003epectus carinatum\u003c/em\u003e, small nipples, scoliosis, digital thumb, arachnodactyly, camptodactyly, chorioretinitis, hypoplasia of labia majora and breast, and walking difficulties. Skeletal abnormalities were progressive and included femur and knee dysplasia (surgically treated) and severe scoliosis with osteoporosis. When she was 22, absence of sphincter control, sporadic non-febrile seizures, and feeding issues were still present, she used a wheelchair and exhibited anxiety.\u003c/p\u003e \u003cp\u003eP11 is a Brazilian girl who had been described by Chaves et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Here, we update her clinical presentation (\u003cb\u003eSupplementary file \u0026ndash; Table S2\u003c/b\u003e). Patient 11 exhibited mild ID, delayed speech and language development, ADHD, ASD, self-injurious behavior (trichotillomania), and insomnia. Convergent strabismus was treated surgically by the age of 10. She presented with Hashimoto\u0026rsquo;s thyroiditis, constipation, recurrent sinus infection (3\u0026ndash;8 years), hypotonia, decreased body weight, and atopic dermatitis. Physical signs at 10 years included preauricular pit, periorbital fullness, epicanthus, hypertelorism, almond-shaped eyes, thick lower lip, micrognathia, arachnodactyly, marfanoid habitus, and scoliosis).\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenetic findings\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e summarizes the molecular characteristics of the \u003cem\u003eYY1\u003c/em\u003e variants detected in the 11 GDVS patients (ten females) described in this study: eight are previously unreported cases and three have already been reported (Carminho-Rodrigues et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; dos Santos et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Chaves et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMolecular features of \u003cem\u003eYY1\u003c/em\u003e variants and X-chromosome inactivation data of 11 GDVS cases (ten females)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePatient 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePatient 2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePatient 3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePatient 4\u003c/p\u003e \u003cp\u003e(mother of patient 5)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePatient 5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePatient 6\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003ePatient 7\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003ePatient 8\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003ePatient 9\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePatient 10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003ePatient 11\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eYY1\u003c/b\u003e \u003cb\u003evariant\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(NM_003403.5)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec.1102T\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e \u003cp\u003e(p.Phe368Leu)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec.1147_1151dup\u003c/p\u003e \u003cp\u003e(p.Cys385Metfs*18)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ec.690dupA\u003c/p\u003e \u003cp\u003e(p.Asp231Argfs*3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ec.690dupA\u003c/p\u003e \u003cp\u003e(p.Asp231Argfs*3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ec.690dupA\u003c/p\u003e \u003cp\u003e(p.Asp231Argfs*3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ec.1114A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e \u003cp\u003e(p.Thr372Pro)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003ec.1057T\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e \u003cp\u003e(p.Phe353Leu)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003ec.1043C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003cp\u003e(p.Thr348Ile)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003ec.907T\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e \u003cp\u003e(p.Cys303Arg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ec.1106A\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e \u003cp\u003e(p.Asn369Ser)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003ec.1062\u0026thinsp;+\u0026thinsp;1G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e \u003cp\u003e(NP_003394.1:p.?)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNationality\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChile\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBrazil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBrazil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFrance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFrance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eFrance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eGermany\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eBrazil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eSwiss\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eBrazil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eBrazil\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge at referral\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e30 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e7 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e11 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e21 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e22 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e8 years 11 months\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eXCI data\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(blood)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAR\u003c/em\u003e 71:29\u003c/p\u003e \u003cp\u003e\u003cem\u003eRP2\u003c/em\u003e - NI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eAR\u003c/em\u003e 73:27\u003c/p\u003e \u003cp\u003e\u003cem\u003eRP2\u003c/em\u003e 67:33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eAR\u003c/em\u003e 71:29\u003c/p\u003e \u003cp\u003e\u003cem\u003eRP2\u003c/em\u003e 72:28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eAR\u003c/em\u003e - NI\u003c/p\u003e \u003cp\u003e\u003cem\u003eTMEM185A -\u003c/em\u003e NI\u003c/p\u003e \u003cp\u003e\u003cem\u003eHMGB3\u003c/em\u003e 98:2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003cp\u003e(male)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eAR\u003c/em\u003e 54:46\u003c/p\u003e \u003cp\u003e\u003cem\u003eTMEM185A\u003c/em\u003e 53:47 \u003cem\u003eHMGB3\u003c/em\u003e 24:76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eAR\u003c/em\u003e 50:50\u003c/p\u003e \u003cp\u003e\u003cem\u003eRP2\u003c/em\u003e 27:73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cem\u003eAR\u003c/em\u003e 91:9\u003c/p\u003e \u003cp\u003e\u003cem\u003eRP2\u003c/em\u003e 57:43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cem\u003eAR\u003c/em\u003e 19:81\u003c/p\u003e \u003cp\u003e\u003cem\u003eRP2\u003c/em\u003e 87:13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cem\u003eAR\u003c/em\u003e - NI\u003c/p\u003e \u003cp\u003e\u003cem\u003eRP2\u003c/em\u003e 86:14\u003c/p\u003e \u003cp\u003e(data from dos Santos et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cem\u003eAR\u003c/em\u003e 94:6\u003c/p\u003e \u003cp\u003e(data from Chaves et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eACMG classification\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003elikely pathogenic\u003c/p\u003e \u003cp\u003e(PM1 moderate, PM2 moderate, PM6 moderate, PP2 supporting)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epathogenic\u003c/p\u003e \u003cp\u003e(PVS1 strong, PM2 moderate, PM6 moderate,\u003c/p\u003e \u003cp\u003ePS4 strong)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003epathogenic\u003c/p\u003e \u003cp\u003e(PVS1 strong,\u003c/p\u003e \u003cp\u003ePM2 moderate, PS4 moderate, PM6 moderate)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epathogenic\u003c/p\u003e \u003cp\u003e(PVS1 very strong,\u003c/p\u003e \u003cp\u003ePM2 moderate, PS4 moderate, PM6 moderate)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003epathogenic\u003c/p\u003e \u003cp\u003e(PVS1 very strong, PS4 moderate,\u003c/p\u003e \u003cp\u003ePM2 moderate)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003epathogenic\u003c/p\u003e \u003cp\u003e(PS4 strong, PM1 moderate, PP2 supporting, PM2 moderate, PM6 moderate, PP3 supporting)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003elikely pathogenic\u003c/p\u003e \u003cp\u003e(PS4 moderate, PM2 moderate, PM1 supporting, PM6 moderate, PP2 supporting)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003elikely pathogenic\u003c/p\u003e \u003cp\u003e(PM2 moderate, PM1 supporting, PM6 moderate, PP3 moderate, PP2 supporting)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003elikely pathogenic\u003c/p\u003e \u003cp\u003e(PM2 moderate, PM6 moderate, PP3 supporting, PP2 supporting)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003elikely pathogenic\u003c/p\u003e \u003cp\u003e(PS4 moderate, PM2 moderate, PM1 moderate, PM6 moderate, PP2 supporting)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003epathogenic\u003c/p\u003e \u003cp\u003e(PVS1 strong, PM2 moderate, PM6 moderate,\u003c/p\u003e \u003cp\u003ePS4 strong, PP1 supporting)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAlphaMissense\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.9999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.9999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.9938\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.9998\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.989\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.8659\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.9998\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLOFTEE\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003elow confidence (end_truncated)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ehigh confidence\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ehigh confidence\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ehigh confidence\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBiochemical change\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhenylalanine is a large, hydrophobic, and aromatic amino acid. Leucine is also hydrophobic but is an aliphatic amino acid due to the lack of an aromatic ring. The change maintains the hydrophobic character but may influence the local folding\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCysteine can form disulfide bridges with another cysteine, which improves the protein\u0026rsquo;s stability and 3D structure. Although methionine also contains sulfur, it does not affect the formation of disulfide bridges. The substitution of cysteine by methionine may compromise the formation of disulfide bridges, affecting the structural integrity of the protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003eAspartic acid is a polar amino acid characterized by its negative charge, which enables it to engage in electrostatic interactions and contribute to the formation of salt bridge formation. Arginine is a basic amino acid with a larger, positive charge and the ability to form multiple hydrogen bonds. The substitution represents a charge reversal. It may alter local electrostatic interactions, possibly by disrupting or creating salt bridges\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThreonine is a polar amino acid with a hydroxyl group (-OH) in its side chain, allowing it to participate in hydrogen bonding and contribute to the stabilization of secondary structures. Proline is unique among amino acids due to its cyclic structure, where the side chain forms a covalent bond with the backbone nitrogen, restricting backbone flexibility and disrupting regular secondary structures. The substitution of threonine by proline may disrupt local secondary structure\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003ePhenylalanine is a large, hydrophobic and aromatic amino acid. Leucine is also hydrophobic, but it is an aliphatic amino acid due to the lack of an aromatic ring. The change maintains the hydrophobic character but may influence the local folding\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eThreonine is a polar amino acid, able to interact by hydrogen bonds. Isoleucine is a hydrophobic amino acid, incapable of such bonds\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCysteine can form disulfide bonds. Arginine is different in size and charge (positive). The substitution may affect the structure/function of the protein by compromising disulfide bridges formation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eAsparagine has a polar side chain. Serine has a smaller side chain that contains a hydroxyl group (-OH). This is a conservative substitution, but it may affect local geometry\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"12\"\u003eNI: not informative; NA: not available; AlphaMissense scores: 0-0.34: likely benign; 0.34\u0026ndash;0.564: VUS; 0.564-1: likely pathogenic\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAll patients, except for P5, were found to carry \u003cem\u003ede novo\u003c/em\u003e mutations. P5 inherited a \u003cem\u003eYY1\u003c/em\u003e frameshift variant from P4, his affected mother. Variants were reclassified regarding their pathogenicity, following ACMG/ClinGen criteria (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and Clingen updates. Three novel \u003cem\u003eYY1\u003c/em\u003e variants were identified in the present study in P1, P2 and P8.\u003c/p\u003e \u003cp\u003eA pathogenic heterozygous \u003cem\u003eYY1\u003c/em\u003e missense variant [chr14:100.277.457 T\u0026thinsp;\u0026gt;\u0026thinsp;C (hg38); \u003cem\u003eYY1\u003c/em\u003e(NM_003403.5):c.1102T\u0026thinsp;\u0026gt;\u0026thinsp;C(p.Phe368Leu)] was identified in P1. A pathogenic heterozygous \u003cem\u003eYY1\u003c/em\u003e frameshift variant was identified in P2 [chr14:100.277.501 C\u0026gt;CTATGT (hg38); \u003cem\u003eYY1\u003c/em\u003e (NM_003403.5):c.1147_1151dup(p.Cys385Metfs*18)]. In P3, P4 and P5, a pathogenic heterozygous \u003cem\u003eYY1\u003c/em\u003e frameshift variant [chr14:100.262.306 G\u0026thinsp;\u0026gt;\u0026thinsp;GA (hg38); \u003cem\u003eYY1\u003c/em\u003e(NM_003403.5):c.690dup(p.Asp231Argfs*3)] was detected. This variant was previously reported in ClinVar (ID 1331550, classified as likely pathogenic) and in the literature (Cherik et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). A heterozygous missense \u003cem\u003eYY1\u003c/em\u003e variant was detected in P6 [chr14:100.277.469 A\u0026thinsp;\u0026gt;\u0026thinsp;C (hg38); \u003cem\u003eYY1\u003c/em\u003e(NM_003403.5):c.1114A\u0026thinsp;\u0026gt;\u0026thinsp;C(p.Thr372Pro)], already described in ClinVar (ID 1703497, as pathogenic). A missense \u003cem\u003eYY1\u003c/em\u003e variant was found in P7 [chr14:100.276.643 T\u0026thinsp;\u0026gt;\u0026thinsp;C (hg38); \u003cem\u003eYY1\u003c/em\u003e(NM_003403.5):c.1057T\u0026thinsp;\u0026gt;\u0026thinsp;C(p.Phe353Leu)] in heterozygosis, which has been previously reported as likely pathogenic in ClinVar (ID 2413123) and in the literature (Topa et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Lastly, in P8, another heterozygous \u003cem\u003eYY1\u003c/em\u003e missense variant [chr14:100.276.629 C\u0026thinsp;\u0026gt;\u0026thinsp;T (hg38); \u003cem\u003eYY1\u003c/em\u003e(NM_003403.5):c.1043C\u0026thinsp;\u0026gt;\u0026thinsp;T(p.Thr348Ile)], not previously reported, was revealed. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u003cb\u003es\u003c/b\u003ehows the position of each \u003cem\u003eYY1\u003c/em\u003e variant in the YY1 protein.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSchematic diagram of YY1 protein, based on dos Santos et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Position and nomenclature of \u003cem\u003eYY1\u003c/em\u003e pathogenic variants of patients whose XCI patterns have been evaluated are represented. Circles, triangles and stars portray missense, frameshift and splicing variants, respectively. Gray and black filled circles/triangles/stars represent moderate (ratios between 71:29 and 90:10) and extreme (\u0026gt;\u0026thinsp;90:10 ratios) skewing, respectively, in blood DNA samples for the marker that exhibited the highest deviation from a random pattern (\u003cem\u003eAR\u003c/em\u003e, \u003cem\u003eRP2\u003c/em\u003e, \u003cem\u003eTMEM185A\u003c/em\u003e or \u003cem\u003eHMGB3\u003c/em\u003e). Not in scale\u003c/p\u003e \u003cp\u003e \u003cb\u003eXCI patterns in blood samples\u003c/b\u003e \u003c/p\u003e \u003cp\u003eXCI patterns were assessed for 10 patients (P1-P4 and P6-P11). The XCI analysis was performed using DNA samples extracted from peripheral blood. P1 showed moderate skewing for the \u003cem\u003eAR locus at an average\u003c/em\u003e ratio of 71:29 and was homozygous for the \u003cem\u003eRP2 locus\u003c/em\u003e and hence not informative for this marker. In P2, the \u003cem\u003eAR\u003c/em\u003e and \u003cem\u003eRP2\u003c/em\u003e analysis yielded ratios of 73:27 (moderate skewing) and 67:33 (random), respectively. The XCI analysis for P3 revealed moderate skewing for \u003cem\u003eAR\u003c/em\u003e and \u003cem\u003eRP2\u003c/em\u003e loci (71:29 and 72:28), respectively). P4 was not informative for \u003cem\u003eAR\u003c/em\u003e or \u003cem\u003eTMEM185A\u003c/em\u003e, but displayed extreme skewing for \u003cem\u003eHMGB3\u003c/em\u003e (98:2). P6 exhibited random XCI for the \u003cem\u003eAR\u003c/em\u003e (54:46) and \u003cem\u003eTMEM185A\u003c/em\u003e (53:47) markers, but moderate skewing for \u003cem\u003eHMGB3\u003c/em\u003e (24:76). The XCI analysis using P7\u0026rsquo;s samples showed a random inactivation ratio for \u003cem\u003eAR\u003c/em\u003e (50:50), but moderate skewing for \u003cem\u003eRP2\u003c/em\u003e (27:73). Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows these results for three patients, as examples, and the XCI ratios are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, including those of the three already published GDVS cases (P9\u0026ndash;P11). P9 (whose clinical presentation had been previously reported by Carminho-Rodrigues et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) exhibited moderate skewing for both the \u003cem\u003eAR\u003c/em\u003e (19:81) and \u003cem\u003eRP2\u003c/em\u003e (87:13) \u003cem\u003eloci\u003c/em\u003e. P10 was not informative for the \u003cem\u003eAR\u003c/em\u003e marker, and the analysis for the \u003cem\u003eRP2 locus\u003c/em\u003e indicated moderate skewing (86:14) (data from dos Santos et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). P11\u0026rsquo;s samples demonstrated extremely skewed XCI (94:6) for the \u003cem\u003eAR locus\u003c/em\u003e (data from Chaves et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFilled circles in the pedigree charts represent females diagnosed with GDVS. Red rectangles highlight moderate XCI skewing. u: undigested; d: digested. The graphics are not in scale. \u003cb\u003ea)\u003c/b\u003e P1\u0026rsquo;s pedigree chart. \u003cb\u003eb)\u003c/b\u003e XCI assay using P1\u0026rsquo;s blood DNA samples for the \u003cem\u003eAR locus\u003c/em\u003e (71:29 average ratio). \u003cb\u003ec)\u003c/b\u003e P2\u0026rsquo;s pedigree chart. \u003cb\u003ed)\u003c/b\u003e XCI assay using P2\u0026rsquo;s blood DNA samples for the \u003cem\u003eAR locus\u003c/em\u003e (73:27 average ratio). \u003cb\u003ee)\u003c/b\u003e P8\u0026rsquo;s pedigree chart. \u003cb\u003ef)\u003c/b\u003e Graphics resulting from XCI assay using P8\u0026rsquo;s blood DNA samples for the \u003cem\u003eAR locus\u003c/em\u003e (91:9 average ratio)\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn silico\u003c/b\u003e \u003cb\u003eanalysis of YY1 protein 3D structure\u003c/b\u003e\u003c/p\u003e \u003cp\u003eYY1 protein contains four zinc-finger domains in its C-terminal region that are essential for DNA binding and transcriptional regulation. Variants located within this region, such as the missense here reported (p.R303Q, p.I348T, p.L353P, p.L368P, p.S369C, and p.M385V), occur within or near the zinc-finger motifs; in terms of structural interactions, the residues Phe368 and Asn369 interact directly with DNA, while Cys385 interacts with zinc. Therefore, all of them are predicted to alter zinc coordination or disrupt the interface with DNA. All the six missense variants listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e exhibited high pathogenicity scores according to AlphaMissense, a deep learning method for predicting the pathogenicity of single amino acid substitutions in human proteins based on protein structure and evolutive conservation, with five exceeding 0.9 (AlphaMissense cuttoffs: \u0026ge; .564, likely pathogenic; 0.565\u0026thinsp;\u0026minus;\u0026thinsp;0.340, likely neutral; \u0026le; 0.340, likely benign). These structural disturbances likely reduce YY1\u0026rsquo;s DNA-binding affinity and impair its transcriptional regulatory function, providing a plausible molecular basis for the associated developmental phenotypes. The changes in the 3D structure of the protein caused by these six missense variants (303R, 348I, 353L, 368L, 369S, and 385M) are represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Although not designed to evaluate LoF variants, Alphamissense can be applied to generated models of truncated proteins, assuming that truncated forms can occur if they escape from the NMD. We evaluated two of the LoF variants: c.690dupA(p.Asp231Argfs*3) and c.1147_1151dup(p.Cys385Metfs*18); the third one can not be evaluated because it is a splice site mutation. The c.690dupA(p.Asp231Argfs*3) variant had a high confidence classification in LOFTEE, suggesting loss of function, whereas the c.1147_1151dup(p.Cys385Metfs*18) variant had a low confidence classification with the truncation site near to the end of the gene. The model for p.Cys385Metfs*18 exhibits C-terminal region loss, extending beyond the α-helical segment, while the experimental structure remains fully resolved throughout the entire length of the molecule (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The c.690dupA(p.Asp231Argfs*3) variant, however, could not be depicted in the 3D model, possibly indicating an association with specific protein characteristics. The AlphaFold database shows that the structural model for the YY1 protein (ID: P25490) could indicate that residues 1\u0026ndash;295 are situated within a disordered region, a finding that is also supported by DisProt. While the AlphaMissense score for the p.Asp231Argfs*3 variant is lower than that of other variants, it is important to note that this variant is situated within a structurally disordered region. Considering the variants in the study, only p.Asp231Argfs*3 is located outside the zinc finger domain. However, it could play additional functional roles, especially within the Gly-rich region that is linked to HCFC1 interactions. Also, dosage sensitivity data suggest that \u003cem\u003eYY1\u003c/em\u003e is dosage intolerant, as evidenced by high pHaplo and pTriple scores (0.96 and 0.99, respectively).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eGDVS is caused by \u003cem\u003eYY1\u003c/em\u003e haploinsufficiency due to truncating alterations or missense variants, the latter often mapping to the \u003cem\u003eYY1\u003c/em\u003e zinc-finger motifs (Gabriele et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Here, we compiled detailed clinical and molecular features of eleven cases (\u003cb\u003eSupplementary file \u0026ndash; Table S2\u003c/b\u003e), three of them previously reported (Carminho-Rodrigues et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; dos Santos et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Chaves et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe patients displayed clinical traits already reported in GDVS, including intrauterine growth restriction, low birth weight, global DD, hypotonia, gait disturbances and craniofacial dysmorphisms, such as broad and prominent forehead, periorbital fullness, eyelid ptosis, micrognathia, pointed chin, and downslanted palpebral fissures (Gabriele et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Morales-Rosado et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Carminho-Rodrigues et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Bae et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Balakrishnan and Ranganath \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Malaquias et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Tan et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zorzi et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Alali and Vitalone \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Cherik et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; dos Santos et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ferng et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Khamirani et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Asato et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Chaves et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Chawla et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Koruga et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Woo et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Shin et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Srour et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Topa et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Mudassir et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Pal et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). It is interesting to note that hearing impairment, which was recorded in our series, has been previously described in four patients (Bae et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; dos Santos et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Cherik et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Asato et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Behavioral issues are relatively common in this condition, and were also presented by patients recruited for the present investigation. Attention Deficit Disorder was present in P1 (with Hyperactivity - ADHD), P3 (ADHD), P6, P7, and P11 as well as in several other individuals with GDVS (Gabriele et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Morales-Rosado et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Carminho-Rodrigues et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Alali and Vitalone \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Cherik et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Khamirani et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Chaves et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Chawla et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). P3, P9 and P10 displayed anxiety, which has also been previously reported in GDVS (Gabriele et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Cherik et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e - supplementary material; dos Santos et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Khamirani et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). P1 exhibited autism and self-injurious behavior, both of which have already been described (Gabriele et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Cherik et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ferng et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Khamirani et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). P2 is currently being evaluated for ASD. Sleeping disturbances were observed in P1 and P2, similarly to other cases in the literature (Gabriele et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). P1, P3, P7 and P8 had recurrent infections, also documented in individuals with GDVS (Tan et al. 2020; Cherik et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e - supplementary material; Khamirani et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSignificant clinical heterogeneity was also observed. P2 exhibited dysarthria, which seems to be a rare feature, only reported by Ferng et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and Chawla et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Other uncommon clinical characteristics here described include congenital torticollis (P2), which has only been reported by Cherik et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and abnormalities of the nasolacrimal duct (P3; obstruction), also reported once (Gabriele et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; stenosis).\u003c/p\u003e \u003cp\u003eWe found an increased frequency of thyroid and autoimmune conditions in this group of patients. P3 and P7 were diagnosed with Graves\u0026rsquo; disease; in P3, the condition later evolved into Hashimoto\u0026rsquo;s thyroiditis. P11 also exhibited Hashimoto\u0026rsquo;s disease, and P8 was diagnosed with hyperthyroidism. Graves\u0026rsquo; disease and Hashimoto\u0026rsquo;s thyroiditis are both autoimmune thyroid diseases; the Hashimoto\u0026rsquo;s thyroiditis may produce hyper- or hypothyroidism manifestations, while Graves\u0026rsquo; disease causes hyperthyroidism (Liu et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Davies et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It is interesting to note that thyroid dysfunction has been previously reported in GDVS, mainly hypothyroidism and thyroid nodules (Gabriele et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Alali and Vitalone \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Cherik et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Asato et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Pal et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Remarkably, previous \u003cem\u003ein silico\u003c/em\u003e analysis of protein-protein interactions of YY1 protein disclosed enrichment of thyroid hormone signaling, among other biological pathways (dos Santos et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOther endocrine abnormalities in GDVS include growth hormone deficiency, although rarely described (Gabriele et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Besides, Morales-Rosado et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) published a case of a GDVS patient with the diagnosis of autoimmune myasthenia gravis. In addition, one of the patients (P1) presented with clinical signs that have not been previously reported for GDVS patients to our knowledge, namely hypertrichosis and atopic dermatitis. A more detailed clinical revaluation of P11 \u0026ndash; previously described (Chaves et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) \u0026ndash; revealed that she also exhibited atopic dermatitis, a finding not noted in the original publication. Atopic dermatitis is a condition that has been associated with an increased risk of developing autoimmune diseases, as studied and discussed by Ahn et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, it is a frequent manifestation in the normal population, making it hard to ascertain this clinical feature to GDVS. Taken together, these observations hint that thyroid/endocrine disorders (especially hypothyroidism) and autoimmune conditions may represent an important clinical aspect of GDVS, as also proposed by Alali and Vitalone (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and Huang et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). These features should be considered in the clinical evaluation and management of GDVS patients.\u003c/p\u003e \u003cp\u003eAmong the approximately 44 individuals with GDVS clinically described in the literature to date, 20 presented with movement disorders, among whom 12 exhibited dystonia (Gabriele et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Morales-Rosado et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Carminho-Rodrigues et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Balakrishnan and Ranganath \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zorzi et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Malaquias et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ferng et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Khamirani et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Cherik et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Chawla et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Shin et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Srour et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Mudassir et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) (\u003cb\u003eSupplementary file \u0026ndash; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). The \u003cem\u003eYY1\u003c/em\u003e variant carrier reported by Zech et al. (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2021\u003c/span\u003e - supporting information) also presented with dystonia; however, since this was the only clinical feature documented for that individual, this case was not included among those with a comprehensive clinical description. Notably, 18 of these 20 clinically characterized cases with dystonia and/or other movement disorders also exhibited neurodevelopmental deficits, such as DD and/or ID (Gabriele et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Morales-Rosado et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Carminho-Rodrigues et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Balakrishnan and Ranganath \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zorzi et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ferng et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Khamirani et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Cherik et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Shin et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Srour et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Mudassir et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The remaining two cases (Malaquias et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Chawla et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) did not prominently manifest the core GVDS phenotype, although they did exhibit other nonspecific clinical features commonly reported in the syndrome, including low birth weight and facial dysmorphisms, such as pointed chin and bulbous nose. These observations underscore a phenotypic overlap between \u003cem\u003eYY1\u003c/em\u003e-related movement disorders and neurodevelopmental deficits. Rather than representing distinct diagnostic categories, these phenotypes appear to constitute a single clinical entity associated with \u003cem\u003eYY1\u003c/em\u003e disruption, with a wide and variable manifestation spectrum. This full picture of the GDVS is still evolving; therefore, describing additional cases is a key step to broaden the clinical and molecular spectrum of the condition, with implications for improvement of diagnosis and clinical management.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e depicts the YY1 protein and the location of the pathogenic variants carried by the affected individuals reported in this study, along with the corresponding XCI pattern observed in the assessed females. P1, P6, P7, P9 (Carminho-Rodrigues et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and P10 (dos Santos et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) carried missense variants mapped to sequences encoding part of the zinc-finger domain (DNA-binding). The missense variant identified in P8 affects an amino acid located between two zinc finger motifs. The remaining five patients, P2, P3/P4/P5 (frameshift) and P11 (Chaves et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; splice site), carry LoF variants. Interestingly, six out of the eleven variants here reported (variants identified in P1, P2, P6, P7, P9 and P10) impact the zinc-finger DNA binding domains. According to GnomAD, \u003cem\u003eYY1\u003c/em\u003e displays intolerance to missense (z-score\u0026thinsp;=\u0026thinsp;3.6) and loss-of-function variants (pLI score\u0026thinsp;=\u0026thinsp;1). Also, the pHaplo (0.96) and pTriple (0.99) values indicate that the \u003cem\u003eYY1\u003c/em\u003e gene is dosage-sensitive; and alterations in its expression may lead to pathological effects.\u003c/p\u003e \u003cp\u003eYY1 is a ubiquitously expressed transcription factor involved in the regulation of a wide range of gene promoters, acting either as a repressor or as an activator. Importantly, \u003cem\u003eYY1\u003c/em\u003e is a pivotal gene in the initiation of XCI, since it activates \u003cem\u003eXIST\u003c/em\u003e transcription in human and mouse contexts (Chapman et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Makhlouf et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and might serve as a docking protein for \u003cem\u003eXist\u003c/em\u003e transcripts to the inactive X-chromosome (Jeon and Lee \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). YY1 interacts with numerous chromatin modifiers (Verheul et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and is also known to directly and indirectly interact with numerous XCI key regulators (see discussion in dos Santos et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In \u003cem\u003eDrosophila\u003c/em\u003e, YY1 has been shown to participate in the recruitment of polycomb group proteins, which are fundamental for maintaining transcriptional repression of genes (Wilkinson et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Polycomb group protein recruitment to DNA by YY1 leads to the methylation of histone H3 on lysine 27 (H3K27) (Wilkinson et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The polycomb complexes PRC1 and PRC2 mediate specific histone modifications, which are involved in the XCI process (Sun et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Also, YY1 may be implicated in recruiting polycomb complexes to the Xi (Thorvaldsen et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Additionally, \u003cem\u003eYY1\u003c/em\u003e interacts with histone deacetylases (HDACs) engaged in gene silencing, such as HDAC3, and histone acetyltransferases (HATs) (Verheul et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Studies point to HDAC3 involvement in XCI, deacetylating histones upon interaction with \u003cem\u003eXIST\u003c/em\u003e RNA and helping gene silencing and RNA polymerase II exclusion from the Xi \u0026zwnj;(McHugh et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Żylicz et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Moreover, a recent study in mice suggests that the YY1 binding to X-linked genes can act as a barrier to \u003cem\u003eXist\u003c/em\u003e-mediated silencing until XCI late stages (Bowness et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGiven \u003cem\u003eYY1\u003c/em\u003e's prominent involvement in the XCI process, we hypothesized that germline \u003cem\u003eYY1\u003c/em\u003e mutations could drive or impact the XCI pattern in females, leading to skewing (dos Santos et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Chaves et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). To date, only two published studies have assessed XCI patterns in female patients with \u003cem\u003eYY1\u003c/em\u003e mutations (dos Santos et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Chaves et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and both reported XCI skewing. Here, a heterogeneous XCI pattern was observed among the eight cases newly evaluated (P1-P4 and P6-P9). In spite of that, it is noteworthy that in total, seven patients (P1, P2, P3, P4, P7, P9 and P10) exhibited moderate XCI skewing for at least one marker, and three individuals (P4, P8 and P11) showed extreme XCI skewing. These findings may reinforce a potential involvement of \u003cem\u003eYY1\u003c/em\u003e mutations in modulating the XCI pattern \u003cem\u003ein trans\u003c/em\u003e, although the mechanistic link is not clear.\u003c/p\u003e \u003cp\u003eIt is crucial to recognize that XCI skewing can arise due to several mechanisms, such as the stochastic nature of the molecular mechanism, the presence of an X-linked variant unrelated to ID, or even the activity of escapee X-linked genes (Peeters et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). An impressive number of variants in X-linked genes are associated with ID (Migeon \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Plenge et al. (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) first described that female carriers of harmful variants causing X-linked ID (XLID) disorders displayed XCI skewing, with findings suggesting a negative selection against cells harboring the mutation on the active X-chromosome. Notwithstanding the potential protective effect provided by XCI (Migeon \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), female carriers of those XLID pathogenic variants may exhibit skewing favoring the mutant allele, and can still present ID (Vianna et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Chaves et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGabriele et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) reported that lymphoblastoid cell lines (LCLs) from two individuals carrying missense mutations [c.1138G\u0026thinsp;\u0026gt;\u0026thinsp;T (p.Asp380Tyr)] and [c.1097T\u0026thinsp;\u0026gt;\u0026thinsp;C (p.Leu366Pro)]) displayed wild-type YY1 levels, after analysis by RNA-seq and Western blot. Upregulation of \u003cem\u003eYY1\u003c/em\u003e was detected in induced pluripotent stem cells (iPSCs) derived from another affected individual with a missense variant (Pereira et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Jeon and Lee (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) proposed that YY1 zinc-fingers interact with both \u003cem\u003eXIST\u003c/em\u003e DNA and RNA by binding to different motifs on the DNA and RNA. Therefore, it is plausible to speculate that YY1 isoforms containing missense variants may exhibit reduced affinity to \u003cem\u003eXIST\u003c/em\u003e DNA or RNA, resulting in a preferential binding of the wild-type YY1 isoform. In the other way, LoF \u003cem\u003eYY1\u003c/em\u003e mutations, which are known to lead to a strong reduction in the amount of protein in LCLs (Gabriele et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and iPSCs (Pereira et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) derived from patients with GDVS, would result in a reduced availability of functional YY1. This reduction induces global decrease of YY1 occupancy in the genome (Gabriele et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Pereira et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2025\u003c/span\u003e); however, if this condition impacts the XCI pattern is a question not yet addressed.\u003c/p\u003e \u003cp\u003eRecent findings (Bertin et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2026\u003c/span\u003e) suggested that females may exhibit an altered dynamic utilization of the two X-chromosomal alleles. This would distort the \u003cem\u003elocus\u003c/em\u003e- and lineage-specific reactivation of the inactive X, a mechanism proposed to serve as a critical reservoir during differentiation, thereby enhancing the resilience of female neural tissue. Taking into account that YY1 interacts with a myriad of elements involved in XCI and its reduced levels in \u003cem\u003eYY1\u003c/em\u003e-LoF mutations and/or disrupted activity of \u003cem\u003eYY1\u003c/em\u003e-missense mutations, it is tempting to suggest that these harmful \u003cem\u003eYY1\u003c/em\u003e variants could drive a primary XCI skewing, but no obvious explanation can be provided at this point. Alternatively, YY1 depletion caused by germline pathogenic variants could indirectly lead to XCI skewing, by altering progenitor cell development or survival during early development, thereby reducing the available cell pool and increasing the likelihood of biased inactivation patterns. We may also speculate that females with \u003cem\u003eYY1\u003c/em\u003e mutations can develop in the brain a disrupted version of the heterogeneous expression pattern of escapee X-linked genes (Peeters et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This later hypothesis is interesting when associated with the autoimmune conditions reported here. Autoimmune diseases disproportionately affect women compared to men, with contributing factors such as estrogen, X-linked genes, and microbiota composition; there are several studies addressing how XCI features may drive the sex disparity in autoimmunity of females (reviewed in Mousavi et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn conclusion, our study expands the clinical and molecular understanding of GDVS. Considering the limited number of reported GDVS cases in the literature, it is of paramount importance to describe new cases along with their clinical and molecular data for improving diagnosis accuracy and exploring potential therapeutic strategies for the syndrome. Moderate XCI skewing was detected in blood samples from seven patients, and extremely skewed XCI patterns were found in three patients, reinforcing that \u003cem\u003eYY1\u003c/em\u003e mutations may impact XCI patterns. Future functional studies to assess the impact of the \u003cem\u003eYY1\u003c/em\u003e mutations on XCI could employ patient-derived cells to evaluate \u003cem\u003eXIST\u003c/em\u003e transcription and its interaction with the X-chromosome. Addressing this gap in future research will be crucial to fully elucidate the mechanistic links between \u003cem\u003eYY1\u003c/em\u003e dysfunction and XCI alterations in GDVS.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eWe are very grateful to the patients and their families for collaborating in this study. We thank Carlos Augusto Takeuchi for clinically evaluating one of the patients and connecting us with the family. We also thank Dr. Rafael Martins Galupa for his valuable contributions reviewing the manuscript.\u003c/p\u003e\n\u003ch2\u003eEthics approval\u003c/h2\u003e\n\u003cp\u003eThis study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Human Research Ethics Committee of the Institute of Biosciences of the University of S\u0026atilde;o Paulo (CAAE 80921117.5.0000.5464), Institutional Ethics Committee from the State University of Rio de Janeiro (CAAE 46769315.5.0000.5259), University of S\u0026atilde;o Paulo Ethics Committee (CAAE 80921117.5.0000.5464), and the French ethics committee (French Ministry of Research and Innovation, DC-2020-4073. All data presented in this manuscript were fully anonymized, and no identifying images or personal information are included.\u003c/p\u003e\n\u003ch2\u003eConsent to participate\u003c/h2\u003e\n\u003cp\u003eWe obtained written informed consent from the patients\u0026rsquo; legal guardians.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eConsent to publish\u003c/h2\u003e\n\u003cp\u003eWritten informed consent for publication of clinical and genetic data was obtained from the patients\u0026rsquo; legal guardians.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe reported variants are available in the ClinVar database of genetic variants.\u003c/p\u003e\n\u003cp\u003eClinVar Variation ID: 2582783, Accession: VCV002582783.1 (P1)\u003c/p\u003e\n\u003cp\u003eClinVar Variation ID: 3236170, Accession: VCV003236170.1 (P2)\u003c/p\u003e\n\u003cp\u003eClinVar Variation ID: 1331550, Accession: VCV001331550.4 (P3, P4 and P5; not submitted by us)\u003c/p\u003e\n\u003cp\u003eClinVar Variation ID: 1703497, Accession: VCV001703497.1 (P6; not submitted by us)\u003c/p\u003e\n\u003cp\u003eClinVar Variation ID: 2413123, Accession: VCV002413123.6 (P7; not submitted by us)\u003c/p\u003e\n\u003cp\u003eClinVar Variation ID: 4530027, Accession: VCV004530027.1 (P8)\u003c/p\u003e\n\u003cp\u003eAdditional data may be made available by the corresponding author upon reasonable request. Data sharing will be considered in accordance with participant privacy and ethical standards.\u003c/p\u003e\n\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\n\u003cp\u003eAna Cristina Victorino Krepischi and C\u0026iacute;ntia Barros Santos-Rebou\u0026ccedil;as conceived and designed the study. Laura Machado Lara Carvalho, Matheus Augusto Ara\u0026uacute;jo Castro, C\u0026iacute;ntia Barros Santos-Rebou\u0026ccedil;as, and Ana Cristina Victorino Krepischi established collaborations, recruited patients, and coordinated the collection of clinical data and samples. Bianca Mie Sato Kurashima, Andressa Pereira Gon\u0026ccedil;alves, and Silvia Souza da Costa performed XCI pattern assays. Bianca Mie Sato Kurashima performed Sanger sequencing, wrote the initial version of the manuscript, and prepared figures 1 and 2 under the guidance of Ana Cristina Victorino Krepischi, Laura Machado Lara Carvalho, and C\u0026iacute;ntia Barros Santos-Rebou\u0026ccedil;as. Rafael Mina Piergiorge and C\u0026iacute;ntia Barros Santos-Rebou\u0026ccedil;as performed\u003cem\u003e\u0026nbsp;in silico\u003c/em\u003e analyses. Rafael Mina Piergiorge prepared figure 3. Lina Quteinheh, Maria Teresa Carminho-Rodrigues, Vincent Michaud, Fanny Morice-Picard, Caroline Rooryck, Matthias Begemann, and Larissa Mattern conducted formal analyses of patients. Francisco Cammarata-Scalisi, Mariana de Carvalho Moreira, Suely Rodrigues dos Santos, and Elis Vanessa de Lima Silva clinically evaluated patients. All authors contributed to Writing \u0026ndash; Review \u0026amp; Editing and have read, revised, and approved the final version of the manuscript.\u003c/p\u003e\n\u003ch2\u003eCompeting Interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was financed, in part, by the S\u0026atilde;o Paulo Research Foundation (FAPESP), Brazil [Process Numbers #2023/09879-7, #2013/08028-1, #2022/03980-5, #2025/00171-7, #2023/15506-9, and #2025/04380-0], by the Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado do Rio de Janeiro (FAPERJ), Brazil [E-26/010.001888/2019; E-26/200.883/2021; E-26/204.043/2024], and by the National Council for Scientific and Technological Development (CNPq), Brazil [#125838/2023-9, #302263/2019-5; #302342/2022-2]. The Article Processing Charge (APC) for this publication was funded by the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior (CAPES) (ROR identifier: 00x0ma614), Brazil. For the purposes of open access, the authors have applied a Creative Commons CC BY licence to any accepted version of the article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAhn J, Shin S, Lee GC, Han BE, Lee E, Ha EK, Shin J, Lee WS, Kim JH, Han MY (2024) Unraveling the link between atopic dermatitis and autoimmune diseases in children: Insights from a large-scale cohort study with 15-year follow-up and shared gene ontology analysis. 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Cell 176:182\u0026ndash;197e23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cell.2018.11.041\u003c/span\u003e\u003cspan address=\"10.1016/j.cell.2018.11.041\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e6.Statements \u0026amp; Declarations\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"human-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"huge","sideBox":"Learn more about [Human Genetics](https://www.springer.com/journal/439)","snPcode":"439","submissionUrl":"https://submission.nature.com/new-submission/439/3","title":"Human Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Gabriele-de Vries syndrome, X-chromosome inactivation, YY1, neurodevelopmental disorders, variable expressivity","lastPublishedDoi":"10.21203/rs.3.rs-8846661/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8846661/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGabriele-de Vries syndrome (GDVS) is a syndromic form of intellectual disability caused by pathogenic variants in the \u003cem\u003eYY1\u003c/em\u003e gene, which encodes a ubiquitously expressed transcription factor that plays a crucial role in the X-chromosome inactivation (XCI) process. Recent studies have implicated \u003cem\u003ede novo YY1\u003c/em\u003e pathogenic variants in skewed XCI in females with GDVS. Here, we report clinical and molecular features of 11 GDVS patients (ten females), including eight newly identified cases, two being a familial case. Patients exhibited the core phenotype of GDVS but with notable clinical heterogeneity, displaying additional features such as autism spectrum disorder, thyroid dysfunction, hearing impairment, and dysarthria. We also discuss thyroid and other endocrine alterations, autoimmune conditions, and movement disorders in GDVS. Eight \u003cem\u003eYY1\u003c/em\u003e variants were analyzed \u003cem\u003ein silico\u003c/em\u003e, exhibiting high pathogenicity scores and predicted structural or functional impact, with most affecting DNA binding or zinc finger domain interactions. Finally, we investigated the XCI patterns of ten female patients, and XCI skewing (moderate or extreme) was detected in blood samples from all of them. Our findings expand the clinical and molecular spectrum of GDVS, in addition to reinforcing a potential involvement of \u003cem\u003eYY1\u003c/em\u003e mutations in modulating XCI patterns in females.\u003c/p\u003e","manuscriptTitle":"Evaluation of the X-chromosome inactivation patterns in females with Gabriele-de Vries syndrome and expansion of clinical spectrum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-22 16:52:32","doi":"10.21203/rs.3.rs-8846661/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"314723463198182842352803382902611184072","date":"2026-05-18T09:50:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-18T05:35:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-12T01:54:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-12T01:53:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Human Genetics","date":"2026-02-11T03:19:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"human-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"huge","sideBox":"Learn more about [Human Genetics](https://www.springer.com/journal/439)","snPcode":"439","submissionUrl":"https://submission.nature.com/new-submission/439/3","title":"Human Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"08a426fe-473e-4e12-992f-9a0504e88ee3","owner":[],"postedDate":"February 22nd, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"314723463198182842352803382902611184072","date":"2026-05-18T09:50:33+00:00","index":79,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-22T16:52:32+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-22 16:52:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8846661","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8846661","identity":"rs-8846661","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}