Prader–Willi and PURA Syndromes Concurrently Diagnosed Using High-Throughput Sequencing with Methylation-Specific Multiplex Ligation-Dependent Probe Amplification: A Case Report

Prader–Willi and PURA Syndromes Concurrently Diagnosed Using High-Throughput Sequencing with Methylation-Specific Multiplex Ligation-Dependent Probe Amplification: A Case Report | 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 Case Report Prader–Willi and PURA Syndromes Concurrently Diagnosed Using High-Throughput Sequencing with Methylation-Specific Multiplex Ligation-Dependent Probe Amplification: A Case Report Linmin Zhu, Yujie Han, Qun Xu, Wenmin Hou, Lili Kang, Chen Liu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7941293/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background: A broad and heterogeneous spectrum of disorders present as feeding difficulties and hypotonia during the neonatal period, which are characterized by complex etiologies, diverse clinical manifestations, and considerable symptom overlap, posing diagnostic challenges. The co-occurrence of different genetic disorders in a single patient is rare. We report the a case of Prader–Willi syndrome (PWS) co-occurring with PURA syndrome diagnosed during the neonatal period. Case Presentation: We describe a case of a neonate admitted with feeding difficulties and hypotonia. The infant exhibited persistent feeding problems, hypotonia, a thin upper lip, and immature genitalia. The initial diagnostic workup included high-throughput whole-exome sequencing (WES). The WES results revealed a heterozygous de novo variant, c.764dupA (p.Asn255Lysfs*39), in PURA . This specific variant was absent from all population databases and has not been previously reported in the literature. The variant was classified as pathogenic based on the standards and guidelines of the American College of Medical Genetics and Genomics. This finding was consistent with a diagnosis of PURA syndrome (OMIM #616158). However, PWS could not be ruled out given the clinical phenotype of the patient. Subsequently, methylation-specific multiplex ligation-dependent probe amplification was performed. The paternal methylation imprint at the SNURF-SNRPN locus was completely absent (100%) , confirming the diagnosis of PWS caused by an imprinting center defect. The patient was definitively diagnosed with co-occurring PWS and PURA syndrome. The infant received rehabilitative therapy and growth hormone treatment. The patient showed satisfactory gains in length and weight during follow-up until 9 months of age. However, neurological improvement was poor, and the long-term prognosis remains under observation. Conclusions: We report a case of PWS co-occurring with PURA syndrome diagnosed during the neonatal period. The overlapping clinical presentation challenges the conventional diagnostic paradigm of one disease, one phenotype. We emphasize that the possibility of a dual diagnosis should be considered in infants with complex, rapidly progressive, or atypical features, even after one genetic disorder is identified. A multitiered molecular testing approach should be used to enable precise diagnosis and enabling individualized management, which are fundamental for establishing an accurate prognosis and predicting treatment outcomes. Neonate PWS HTS MLPA dual diagnosis concomitant genetic diseases Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Feeding difficulties and hypotonia in the neonatal period are common clinical presentations that have complex etiologies. Numerous genetic disorders cause these symptoms, which highly overlap in early infancy, hindering differential diagnosis. Clinical diagnosis traditionally adheres to the principle of Occam's razor, indicating that a single underlying disease explains the entire phenotype. Prader–Willi syndrome (PWS) is a genomic imprinting disorder primarily caused by the absence of paternally inherited genes in the 15q11.2-q13 region. The common molecular mechanisms of PWS include paternal deletion, maternal uniparental disomy, imprinting center defects, and chromosomal structural abnormalities [ 1 – 4 ]. PWS is clinically characterized by hypotonia and feeding difficulties in early infancy, followed in childhood by obesity, hypogonadism, and intellectual developmental delay [ 1 ]. PURA syndrome is an autosomal-dominant disorder caused by heterozygous pathogenic variants of the PURA gene (5q31.2). The PURA gene encodes the highly conserved Pur-alpha (Purα) protein (Fig. 1 ), which is crucial for central nervous system development [ 5 – 7 ]. The phenotypic spectrum of PURA syndrome is distinct; the following features are particularly prominent: severe neonatal hypotonia, respiratory problems at birth, feeding difficulties, seizures or epileptic movements, visual issues, moderate-to-severe developmental delays, substantially delayed speech acquisition, and learning disabilities. Additional characteristics include distinctive facial (dysmorphic) features, congenital heart defects, genitourinary malformations, skeletal abnormalities, and endocrine disorders [ 8 – 10 ]. Advancements in genetic testing technologies have led to clinical reports describing the co-occurrence of rare diseases, a phenomenon increasingly recognized as multigenetic inheritance comorbidity or dual genetic diagnosis [ 11 ]. A second diagnosis is prone to being overlooked or misdiagnosed when the two disorders share overlapping symptomatology, compromising clinical management and prognosis. This report describes a case of PWS co-occurring with PURA diagnosed in the neonatal period. Our aim is to alert clinicians that the diagnostic approach must be widened and a multitiered genetic analysis strategy must be adopted when confronted with atypical or progressive clinical presentations to enable precise diagnosis and individualized management. Case Presentation A 9-day-old female infant was admitted to the children's hospital that is affiliated with Shandong University on November 25, 2024, due to feeding difficulties accompanied by generalized hypotonia for 9 days. She was the second child of a second pregnancy, delivered via cesarean section at a gestational age of 37+3 weeks because of a scarred uterus and abnormal fetal heart monitoring. Her birth weight was 2.77 kg. The amniotic fluid was clear, with a volume of 2200 mL, with no history of perinatal asphyxia. She immediately exhibited cyanosis, grunting, a weak cry, and serious hypotonia after birth, leading to transfer to the local neonatal intensive care unit (NICU). The patient received continuous positive airway pressure-assisted ventilation, antibiotics (penicillin + cefotaxime sodium for 3 days), and ambroxol treatment for 9 days. Her respiratory status somewhat improved; however, severe sucking–swallowing dysfunction persisted, with poor oral feeding (approximately 10 mL per feeding), requiring ongoing tube feeding. However, the incidence of hypotonia remained high. The mother experienced gestational diabetes mellitus and reported decreased fetal movements. The family history was unremarkable; both parents were healthy, nonconsanguineous, and had a healthy 3.5-year-old son. The patient had no family history of genetic or congenital disorders. Physical examination upon admission revealed the following: length, 50 cm; head circumference, 33 cm, weight 2.65 kg. She was well-developed and moderately nourished. The findings included a thin upper lip, poor mental response, tachypnea, absent subcostal retraction, and moderate subcutaneous fat. The anterior fontanelle was flat and soft. Lung auscultation revealed coarse breathing sounds without rales. The heart rate was rhythmic at 128 beats/min; the heart sounds were strong, with no murmurs. The abdomen was soft; the liver was palpable 1 cm below the costal margin and soft in consistency. The genitalia appeared immature. Serious hypotonia was observed in all four limbs, and the primitive reflexes were diminished. The infant was admitted to the NCIU and underwent antimicrobial therapy, enteral tube feeding, and feeding skill training. The results of blood tandem mass spectrometry, urine gas chromatography, and blood ammonia and lactate levels returned to normal. Nerve conduction and electromyography studies suggested peripheral neurogenic damage in the limbs, whereas the spinal cord MRI showed no notable abnormalities. The patient experienced seizures 2 weeks after admission The brain MRI performed at this time (2 weeks of age) revealed slightly prolonged T2 signals in some white matter areas and widened extracerebral spaces. the electroencephalogram (EEG) captured during a seizure episode showed delayed background maturity and a discontinuous pattern( Figure 6 ). High-throughput whole-exome sequencing (WES) was performed after obtaining informed parental consent. The results revealed a heterozygous de novo variant of PURA , c.764dupA (p.Asn255Lysfs*39). This variant was classified as pathogenic according to the standards and guidelines of the American College of Medical Genetics and Genomics and was definitively associated with PURA syndrome (OMIM #616158), confirming the diagnosis( Table1 ). However, PWS remained a diagnostic consideration due to the coexisting findings of a thin upper lip and immature genitalia. The subsequent methylation-specific multiplex ligation-dependent probe amplification (MLPA) showed the complete absence (100%) of the paternal methylation imprint (Figure 4). These findings met the molecular diagnostic criteria for PWS caused by either maternal uniparental disomy or an imprinting center defect. The infant was discharged after a 31-day hospitalization. Her breathing was stable, she was able to breastfeed independently, and no further seizure episodes occurred at discharge. The infant was admitted to the Rehabilitation Department of our hospital at 6 months of age due to growth and developmental delay and underwent a comprehensive two-month rehabilitation program. The therapeutic regimens involved kinesiotherapy, sling suspension therapy, vibration therapy, comprehensive upper limb motor training, multisensory training, acupressure, electrobiofeedback therapy, and conductive education. The assessment at admission to the Rehabilitation Department at 6 months of age was as follows: The scores on all subdomains of Gesell’s Developmental Schedules were 15( Table2 ), indicating profound developmental delay (adaptive, gross and fine motor, language, and personal–social skills). The scores on the Peabody Developmental Motor Scales, Second Edition (PDMS-2) were as follows ( Table 3 ) : Gross Motor Quotient (GMQ) = 57 ( very poor ), Fine Motor Quotient (FMQ) = 46 ( very poor ), and Total Motor Quotient (TMQ) = 48 ( very poor ). The EEG was borderline abnormal for infants, with a background rhythm that was mildly slower than that for standard age( Figure 6 ). Regarding motor function, the patient was unable to hold her head steadily, roll over, or sit independently; no active grasping was observed. The brain MRI showed delayed myelination for chronological age (Figure5). Growth hormone therapy was initiated at 6 months of age, starting with a daily dose of 1.3 U (0.15 U/kg/day). This dose was switched at 7 months of age to a long-acting formulation at 3.0 mg/day (0.4 mg/kg/week). The assessment at 9 months of age showed the following: The results on the Gesell Developmental Schedules continued to show profound delay across all subdomains (adaptive, 19; gross motor, 16; fine motor, 19; language, 15; personal–social, 19)( Table2 ). The scores on the PDMS-2 were a GMQ of 53 ( very poor ), an FMQ of 58 ( very poor ), and a TMQ of 51 ( very poor )( Table 3 ). The EEG was persistently borderline abnormal for infants with a background rhythm mildly slower than that for standard age (Figure6). The anthropometric measurements included a head circumference of 48 cm (near the 97th percentile), a length of 72.5 cm (85th–97th percentile), and a weight of 9.3 kg (85th–97th percentile) (Figure7). For motor function, the infant could hold her head steady, roll over with difficulty, elevate her head 90° in the prone position with elbow support, and sit with a forward lean; no active reaching or grasping with the hands was observed. Socially, she smiled responsively and occasionally produced nonspecific vocalizations, but visual tracking was slow. Table 1. Characteristics of the PURA gene of the patient. ACMG, American College of Medical Genetics and Genomics. Gene (Reference Transcript) Chromosomal Location （GRCh37/hg19） Variant Information Genomic Region Zygosity ACMG Classification Population Frequency Associated Disorder Inheritance Pattern Variant Origin PURA（NM-005859.5） Chr5:139494529 C.764dupAp.Asn255Lysfs*39 Exon1 Heterozygous Pathogenic (P) N/A Neurodevelopmental disorder with neonatal respiratory insufficiency, hypotonia, feeding difficulties Autosomal Dominant (AD) De novo Table 2. Patient scores on Gesell ’s Developmental Schedule. Chronological Age (Months) 6 9 Domain Adaptive Gross Motor Language Personal–Social Adaptive Gross Motor Fine Motor Language Personal–Social Developmental Age (Months) 0.9 0.9 0.9 0.9 1.9 1.6 1.9 1.5 1.9 Developmental Quotient (DQ) 15 15 15 15 19 16 19 15 19 Interpretation Profound Developmental Delay Profound Developmental Delay Profound Developmental Delay Profound Developmental Delay Profound Developmental Delay Profound Developmental Delay Profound Developmental Delay Profound Developmental Delay Profound Developmental Delay Table 3. Patient scores on Peabody Developmental Motor Scales, 2nd Edition (PDMS-2). Chronological Age (Months) 6 9 Composite Score Gross Motor (GMQ) RE+ST+LO+OB Fine Motor (FMQ) GR+VI Total Motor (TMQ) RE+ST+LO+OB+GR+VI Gross Motor (GMQ) RE+ST+LO+OB Fine Motor (FMQ) GR+VI Total Motor (TMQ) RE+ST+LO+OB+GR+VI Standard Score 10 2 12 8 6 14 Percentile Rank ＜1 ＜1 ＜1 ＜1 ＜1 ＜1 Developmental Quotient 57 46 48 53 58 51 Qualitative Description Very Poor Very Poor Very Poor Very Poor Very Poor Very Poor Discussion and Conclusions PWS is caused by the loss of expression of paternally inherited genes in a specific region of chromosome 15 (15q11-13) [12]. The global prevalence of PWS is estimated to range from 1 in 10,000 to 1 in 30,000 live births, with reports in the United States ranging from approximately 1 in 10,000 to 1 in 20,000 cases. PWS incidence does not substantially variation across race, sex, or ethnicity [12–14]. PWS is characterized by severe hypotonia and feeding difficulties in infancy, followed by the onset of hyperphagia, behavioral and psychiatric issues, and morbid obesity in childhood. PWS is not a widely known syndrome despite the recognizable phenotype; one-third of affected individuals may remain undiagnosed [13]. PURA syndrome is a developmental encephalopathy and a rare autosomal dominant disorder, with an estimated incidence of <1 in 1,000,000 [9]. PURA syndrome is characterized by neonatal hypotonia, global developmental delay, and a spectrum of neurological symptoms [9]. Advances in clinical awareness and molecular diagnostic technologies are continuously expanding the phenotypic and genotypic spectra of PURA syndrome [15,16]. However, most cases of PURA syndrome that have been reported were diagnosed during infancy or childhood, with relatively few cases identified during the neonatal period. Consequently, many affected individuals and their families attend multiple medical institutions before a diagnosis is established; sometimes diagnosis only occurs after serious developmental delays become apparent. We document a case of a rare co-occurrence of PWS and PURA syndrome that was molecularly diagnosed during the neonatal period. This case challenges Occam's razor as the conventional diagnostic principle and highlights the possibility of multiple genetic disorders underlying complex clinical presentations. This study provides crucial insights for precisely diagnosing neonates presenting with severe hypotonia and feeding difficulties, underscoring the need for a comprehensive genetic evaluation in addition to single genetic explanations. The core features of PURA syndrome include neonatal hypotonia, feeding difficulties, respiratory issues, lethargy, and subsequent serious global developmental delay/intellectual disability [8,9,17]. The patient initially presented with classic severe hypotonia and feeding difficulties. WES identified a de novo pathogenic variant of PURA . These early manifestations substantially overlap those of the PWS phenotype [13,18,19], indicating a high risk of overlooking a diagnosis of PWS if a single genetic etiology is accepted. More specific PWS features were observed in our patient, namely, a thin upper lip and immature genitalia [13,19], which prompted targeted methylation-specific MLPA analysis to ultimately confirm a concurrent PWS diagnosis. This diagnostic experience shows that clinicians must maintain high suspicion of dual diagnoses after one molecular cause has been identified for infants with atypical, overlapping, or progressively inconsistent clinical presentations. A combinatorial diagnostic strategy integrating high-throughput sequencing (such as WES) with targeted assays for specific loci and imprinting disorders (such as MLPA) is crucial for precisely diagnosing such complex cases. The clinical manifestations of the two disorders in the patient in our case likely involved additive effects and interactions, collectively contributing to the exceptionally severe neurodevelopmental outcome s. The follow-up results demonstrated that, despite systematic rehabilitation interventions and growth hormone therapy, Gesell’s Developmental Quotients indicated profound developmental delay at 9 months of age across all domains (adaptive, gross motor, fine motor, language, and personal–social). All quotients from the Peabody Developmental Motor Scales-2 similarly remained very poor. Growth hormone therapy considerably improved physical growth (length and weight); however, the beneficial effects on the neurological prognosis were limited. These findings suggest that the severe impact of the PURA gene defect on the central nervous system and the neurodevelopmental impairments associated with PWS were synergistic [13,20]. These synergistic impacts resulted in more profound developmental delays than would be expected from either condition alone. This observation is valuable when assessing the long-term prognosis of patients with rare comorbidities and formulating realistic management strategies. This study underscores the critical need to provide early counseling in these cases, even during aggressive therapy, to note the potential for a guarded neurodevelopmental prognosis. The molecular diagnosis methods indicated two distinct genetic mechanisms in our case. First, WES identified a de novo heterozygous frameshift variant, PURA c.764dupA (p.Asn255Lysfs*39). This previously unreported variant is a frameshift mutation predicted to severely disrupt protein function and structure, consistent with the autosomal dominant inheritance pattern of PURA. Subsequently, methylation-specific MLPA analysis revealed a methylation pattern at chromosomal region 15q11.2-q13, which is typical of paternal (maternal) uniparental disomy, providing definitive molecular evidence for a diagnosis PWS. The diagnosis in this case underscores the pivotal role and effectiveness of the combination of WES and MLPA in clinical genetics. WES serves as a broad-spectrum tool for discovering unknown genetic variants, the primary advantage of which is a hypothesis-free, high-throughput capacity to detect single nucleotide variants and small insertions/deletions (indels) across the exome. The identification of the de novo PURA variant exemplifies the powerful discovery capability of WES [21,22]. However, WES has limitations in sensitivity in detecting large copy number variations such as deletions or duplications of entire exons or genes [23]. MLPA fills this technical gap. MLPA is a targeted assay that provides gold-standard sensitivity and specificity in analyzing copy number and methylation status at specific loci, such as the PWS critical imprinted region at 15q11.2-q13 [24,25]. This tiered diagnostic strategy of using WES for initial screening followed by targeted validation efficiently considers the complete spectrum of pathogenic variations from small sequence alterations to large structural variants. This strategy provides a critical paradigm and practical pathway for precisely diagnosing complex and heterogeneous genetic disorders. In summary, this case provides a documented instance of the co-occurrence of PWS and PURA. This case serves to alert clinicians that the diagnostic mindset of Occam's razor should be eschewed when confronted with complex phenotypes; an open diagnostic approach should be maintained toward potential concomitant genetic disorders. The combined application of broad-screening technologies, such as WES, and targeted, in-depth assays, such as MLPA, is pivotal for precisely diagnosing such rare diseases. An early and accurate diagnosis must be established for providing genetic counseling, monitoring complications, and formulating individualized rehabilitation management plans. Future efforts will require the accumulation and reporting of more cases and long-term follow-ups to deepen our understanding of the additive effects of multigenetic disorders on phenotypes as well as the natural histories of multigenetic disorders, enabling treatment strategies to be continuously refined and patient outcomes to be improved. Abbreviations PWS Prader–Willi syndrome WES Whole-exome sequencing NCIU Neonatal intensive care unit EEG Electroencephalogram MLPA Multiplex ligation-dependent probe amplification PDMS-2 Peabody Developmental Motor Scales, Second Edition GMQ Gross Motor Quotient FMQ Fine Motor Quotient TMQ Total Motor Quotient ACMG American College of Medical Genetics and Genomics UPD Uniparental disomy Declarations Ethics approval and consent to participate The studies involving humans were approved by The ResearchEthics Boards of Children’s Hospital Affiliated to Shandong University(Approval No. SDFE-IRB/T-2025128). The studies were conducted in accordance with thelocal legislation and institutional requirements. Written informed consent for participation in this study was provided by theparticipants’ legal guardians/next of kin. Written informed consent was obtained from the individual(s), and minor(s)’ legalguardian/next of kin, for the publication of any potentially identifiable images or data included in this article. Clinical Trial Registration Number: Not applicable Consent for publication All the images and patient material presented in this study have consented for publication and they are available to see upon request. Written informed consent was obtained from all subjects and/or their legal guardian(s) for all the images and patient material presented in this study. Availability of data and materials The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author. Competing interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding This work was supported by the Jinan High-Level Talent Fund for the Healthcare Industry [Grant number: 202412]. Availability of data and materials The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2025) in National Genomics Data Center (Nucleic Acids Res 2025), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA-Human: HRA014603) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa-human. Authors' contributions LMZ and CL were responsible for physical examinations, collection of clinical data and family history, and drafting the initial manuscript. QX and YJH assisted in data interpretation and language polishing. WMH was responsible for collecting clinical cases. LLK and XYL secured research funding, provided guidance in patient management, and participated in the overall guidance of manuscript drafting and revision. All authors have read and approved the final manuscript. Acknowledgements We thank the patient and her parents for agreeing to the publication of this report. We also thank the people who have contributed to the development and execution of this study References Butler MG, Hartin SN, Hossain WA, et al. Molecular genetic classification in Prader-Willi syndrome: A multisite cohort study. J Med Genet. 2019;56(3):149-153. doi:10.1136/jmedgenet-2018-105301. Epub 2018 May 5. Strom SP, Hossain WA, Grigorian M, et al. A Streamlined approach to Prader-Willi and Angelman syndrome molecular diagnostics. Front Genet. 2021;12:608889. doi: 10.3389/fgene.2021.608889. eCollection 2021. Duis J, Butler MG. Syndromic and nonsyndromic obesity: underlying genetic causes in humans. Adv Biol (Weinh). 2022;6(10):e2101154. doi:10.1002/adbi.202101154. Epub 2022 Jun 9. Butler MG, Duis J. Chromosome 15 imprinting disorders: genetic laboratory methodology and approaches. 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15:34:46","extension":"xml","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":78089,"visible":true,"origin":"","legend":"","description":"","filename":"5c8f0e54fe33439a9213af42a241cc661structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7941293/v1/12eb9c8d267cb628b6fd3962.xml"},{"id":97720349,"identity":"db41ee93-45ed-43d4-96ce-fb7b98195ea4","added_by":"auto","created_at":"2025-12-08 15:34:45","extension":"html","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":86502,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7941293/v1/79a4144b75dee35792a21df9.html"},{"id":97720337,"identity":"2a48738c-c88d-4b3e-a6c3-d7209b122462","added_by":"auto","created_at":"2025-12-08 15:34:45","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":535089,"visible":true,"origin":"","legend":"\u003cp\u003edisplays the Sanger sequencing of the PURA gene in the affected child (A), the father (B), and the mother (C). The child was heterozygous for a \u003cem\u003ede novo\u003c/em\u003e c.764dupA (p.Asn255Lysfs*39) variant in the PURA gene.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7941293/v1/233c007cdff9d9e3bfd8e212.jpeg"},{"id":97720330,"identity":"89a0a03d-9f64-40ef-95bb-15827c8e92a3","added_by":"auto","created_at":"2025-12-08 15:34:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":10527,"visible":true,"origin":"","legend":"\u003cp\u003eshows the pedigree chart of the proband's family\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7941293/v1/9384f80d3b913d75de7209b7.png"},{"id":97894120,"identity":"17d9bae1-62f2-43d6-932e-e80bfe357edb","added_by":"auto","created_at":"2025-12-10 15:31:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":323558,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMutated site in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePURA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e is located within a highly conserved sequence across diverse species, such as mice, chickens, \u003c/strong\u003e\u003cem\u003eXenopus\u003c/em\u003e\u003cstrong\u003e, zebrafish, fruit flies, and \u003c/strong\u003eC. elegans\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7941293/v1/441afe7e9704bf7e51d5ed1d.png"},{"id":97894768,"identity":"0b96f145-322b-46c0-9288-bbd0b5ba1fc1","added_by":"auto","created_at":"2025-12-10 15:33:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1019266,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMLPA results for patient. Note:\u003c/strong\u003e Probes SLC9A2-2 and ESCO2-1 served as nonmethylation controls; UBE3A-1 was predominantly nonmethylated in the blood samples. Probe ME028-1 detects copy number variation; probe ME028-2 assesses methylation status.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7941293/v1/bed3caf763a05549f002c9a2.png"},{"id":97720331,"identity":"31c5961f-ec55-4928-b57e-41a4beee78b2","added_by":"auto","created_at":"2025-12-08 15:34:45","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":186597,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBrain MRI at 6 months of age. \u003c/strong\u003eT1-weighted image demonstrating \u003cstrong\u003e(A)\u003c/strong\u003e poorly defined, high signal intensity in the genu of the corpus callosum (arrow); \u003cbr\u003e\n \u003cstrong\u003e(B)\u003c/strong\u003e indistinct high signal intensity in the central white matter (arrow).\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7941293/v1/171ceaed0763e73c4e4e376a.jpeg"},{"id":97720343,"identity":"8c5e845c-c5ce-4f58-b6df-5ae803f87ba5","added_by":"auto","created_at":"2025-12-08 15:34:45","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":782406,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSerial electroencephalogram (EEG) findings\u003c/strong\u003e. \u003cbr\u003e\n \u003cstrong\u003e(1)\u003c/strong\u003e EEG at 2 weeks of age showed delayed background maturity and a discontinuous pattern. \u003cstrong\u003e(2)\u003c/strong\u003e EEG at 6 months of age demonstrates a background rhythm that is mildly slower than the age-appropriate standard. \u003cstrong\u003e(3)\u003c/strong\u003e EEG at 9 months of age continued to show a background rhythm mildly slower than the standard for the age.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7941293/v1/952c989ea0dc6e59e9dd1b82.jpeg"},{"id":97720339,"identity":"c2d092bc-0bb0-4811-a80a-3868846f6c1d","added_by":"auto","created_at":"2025-12-08 15:34:45","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":628825,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGrowth parameters on standard growth curves at 9 months of age.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7941293/v1/aedadb7b51a7f20a5048de16.jpeg"},{"id":97902673,"identity":"9fc6c9cf-57cf-41bc-95c1-9fb9cbc85697","added_by":"auto","created_at":"2025-12-10 15:53:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5750817,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7941293/v1/251f4ab8-3b18-4daf-ad7e-0c6d0c79d559.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Prader–Willi and PURA Syndromes Concurrently Diagnosed Using High-Throughput Sequencing with Methylation-Specific Multiplex Ligation-Dependent Probe Amplification: A Case Report","fulltext":[{"header":"Background","content":"\u003cp\u003eFeeding difficulties and hypotonia in the neonatal period are common clinical presentations that have complex etiologies. Numerous genetic disorders cause these symptoms, which highly overlap in early infancy, hindering differential diagnosis. Clinical diagnosis traditionally adheres to the principle of Occam's razor, indicating that a single underlying disease explains the entire phenotype. Prader\u0026ndash;Willi syndrome (PWS) is a genomic imprinting disorder primarily caused by the absence of paternally inherited genes in the 15q11.2-q13 region. The common molecular mechanisms of PWS include paternal deletion, maternal uniparental disomy, imprinting center defects, and chromosomal structural abnormalities [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. PWS is clinically characterized by hypotonia and feeding difficulties in early infancy, followed in childhood by obesity, hypogonadism, and intellectual developmental delay [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. PURA syndrome is an autosomal-dominant disorder caused by heterozygous pathogenic variants of the \u003cem\u003ePURA\u003c/em\u003e gene (5q31.2). The \u003cem\u003ePURA\u003c/em\u003e gene encodes the highly conserved Pur-alpha (Purα) protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), which is crucial for central nervous system development [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The phenotypic spectrum of PURA syndrome is distinct; the following features are particularly prominent: severe neonatal hypotonia, respiratory problems at birth, feeding difficulties, seizures or epileptic movements, visual issues, moderate-to-severe developmental delays, substantially delayed speech acquisition, and learning disabilities. Additional characteristics include distinctive facial (dysmorphic) features, congenital heart defects, genitourinary malformations, skeletal abnormalities, and endocrine disorders [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Advancements in genetic testing technologies have led to clinical reports describing the co-occurrence of rare diseases, a phenomenon increasingly recognized as multigenetic inheritance comorbidity or dual genetic diagnosis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. A second diagnosis is prone to being overlooked or misdiagnosed when the two disorders share overlapping symptomatology, compromising clinical management and prognosis.\u003c/p\u003e\u003cp\u003eThis report describes a case of PWS co-occurring with PURA diagnosed in the neonatal period. Our aim is to alert clinicians that the diagnostic approach must be widened and a multitiered genetic analysis strategy must be adopted when confronted with atypical or progressive clinical presentations to enable precise diagnosis and individualized management.\u003c/p\u003e"},{"header":"Case Presentation","content":"\u003cp\u003eA 9-day-old female infant was admitted to the children\u0026apos;s hospital that is affiliated with Shandong University on November 25, 2024, due to feeding difficulties accompanied by generalized hypotonia for 9 days. She was the second child of a second pregnancy, delivered via cesarean section at a gestational age of 37+3 weeks because of\u0026nbsp;a scarred uterus and abnormal fetal heart monitoring. Her birth weight was 2.77 kg. The amniotic fluid was clear, with a volume of 2200 mL, with no history of perinatal asphyxia. She immediately exhibited cyanosis, grunting, a weak cry, and serious hypotonia after birth, leading to transfer to the local neonatal intensive care unit (NICU). The patient received continuous positive airway pressure-assisted ventilation, antibiotics (penicillin + cefotaxime sodium for 3 days), and ambroxol treatment for 9 days. Her respiratory status somewhat improved; however, severe sucking\u0026ndash;swallowing dysfunction persisted, with poor oral feeding (approximately 10 mL per feeding), requiring ongoing tube feeding. However, the incidence of hypotonia remained high.\u0026nbsp;The mother experienced gestational diabetes mellitus and reported decreased fetal movements. The family history was unremarkable; both parents were healthy, nonconsanguineous, and had a healthy 3.5-year-old son. The patient had no family history of genetic or congenital disorders. Physical examination\u0026nbsp;upon admission revealed the following: length, 50 cm; head circumference, 33 cm, weight 2.65 kg. She was well-developed and moderately nourished. The findings included a thin upper lip, poor mental response, tachypnea, absent subcostal retraction, and moderate subcutaneous fat. The anterior fontanelle was flat and soft. Lung auscultation revealed coarse breathing sounds without rales. The heart rate was rhythmic at 128 beats/min; the heart sounds were strong, with no murmurs. The abdomen was soft; the liver was palpable 1 cm below the costal margin and soft in consistency. The genitalia appeared immature. Serious hypotonia was observed in all four limbs, and the primitive reflexes were diminished.\u003c/p\u003e\n\u003cp\u003eThe infant was admitted to the NCIU and underwent antimicrobial therapy, enteral tube feeding, and feeding skill training. The results of blood tandem mass spectrometry, urine gas chromatography,\u0026nbsp;and blood ammonia and lactate levels returned to normal. Nerve conduction and electromyography studies suggested peripheral neurogenic damage in the limbs, whereas the spinal cord MRI showed no notable abnormalities. The patient experienced seizures 2 weeks after admission The brain MRI performed at this time (2 weeks of age) revealed slightly prolonged T2 signals in some white matter areas and widened extracerebral spaces. the electroencephalogram (EEG) captured during a seizure episode showed delayed background maturity and a discontinuous pattern(\u003cstrong\u003eFigure\u003c/strong\u003e\u003cstrong\u003e6\u003c/strong\u003e). High-throughput whole-exome sequencing (WES) was performed after obtaining informed parental consent. The results revealed a heterozygous \u003cem\u003ede novo\u003c/em\u003e variant of \u003cem\u003ePURA\u003c/em\u003e, c.764dupA (p.Asn255Lysfs*39). This variant was classified as pathogenic according to the standards and guidelines of the American College of Medical Genetics and Genomics and was definitively associated with PURA syndrome (OMIM #616158), confirming the diagnosis(\u003cstrong\u003eTable1\u003c/strong\u003e).\u0026nbsp;However, PWS remained a diagnostic consideration due to the coexisting findings of a thin upper lip and immature genitalia. The subsequent methylation-specific multiplex ligation-dependent probe amplification (MLPA) showed the complete absence (100%) of the paternal methylation imprint (Figure\u0026nbsp;4). These findings met the molecular diagnostic criteria for PWS caused by either maternal uniparental disomy or an imprinting center defect.\u003c/p\u003e\n\u003cp\u003eThe infant was discharged after a 31-day hospitalization. Her breathing was stable, she was able to breastfeed independently, and no further seizure episodes occurred at discharge.\u003c/p\u003e\n\u003cp\u003eThe infant was admitted to the Rehabilitation Department of our hospital at 6 months of age due to growth and developmental delay and underwent a comprehensive two-month rehabilitation program. The therapeutic regimens involved kinesiotherapy, sling suspension therapy, vibration therapy, comprehensive upper limb motor training, multisensory training, acupressure, electrobiofeedback therapy, and conductive education. \u003cstrong\u003eThe assessment at admission to\u0026nbsp;\u003c/strong\u003ethe Rehabilitation Department at\u003cstrong\u003e\u0026nbsp;6 months of age was as follows:\u003c/strong\u003e The scores on all subdomains of \u003cstrong\u003eGesell\u0026rsquo;s Developmental Schedules were\u003c/strong\u003e 15(\u003cstrong\u003eTable2\u003c/strong\u003e), indicating \u003cem\u003eprofound developmental delay\u003c/em\u003e (adaptive, gross and fine motor, language, and personal\u0026ndash;social skills). The scores on the \u003cstrong\u003ePeabody Developmental Motor Scales, Second Edition (PDMS-2) were as follows\u003c/strong\u003e(\u003cstrong\u003eTable\u003c/strong\u003e\u003cstrong\u003e3\u003c/strong\u003e)\u003cstrong\u003e:\u003c/strong\u003e Gross Motor Quotient (GMQ) = 57 (\u003cem\u003every poor\u003c/em\u003e), Fine Motor Quotient (FMQ) = 46 (\u003cem\u003every poor\u003c/em\u003e), and Total Motor Quotient (TMQ) = 48 (\u003cem\u003every poor\u003c/em\u003e). \u003cstrong\u003eThe EEG was\u003c/strong\u003e borderline abnormal for infants, with a background rhythm that was mildly slower than that for standard age(\u003cstrong\u003eFigure\u003c/strong\u003e\u003cstrong\u003e6\u003c/strong\u003e). \u003cstrong\u003eRegarding motor function, the patient was\u003c/strong\u003e unable to hold her head steadily, roll over, or sit independently; no active grasping was observed. \u003cstrong\u003eThe brain MRI\u003c/strong\u003e showed delayed myelination for chronological age (Figure5). Growth hormone therapy was initiated at 6 months of age, starting with a daily dose of 1.3 U (0.15 U/kg/day). This dose was switched at 7 months of age to a long-acting formulation at 3.0 mg/day (0.4 mg/kg/week).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe assessment at 9 months of age showed the following:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;The results on the\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eGesell Developmental Schedules continued to show\u003c/strong\u003e \u003cem\u003eprofound delay\u003c/em\u003e across all subdomains (adaptive, 19; gross motor, 16; fine motor, 19; language, 15; personal\u0026ndash;social, 19)(\u003cstrong\u003eTable2\u003c/strong\u003e). The scores on the \u003cstrong\u003ePDMS-2 were a\u003c/strong\u003e GMQ of 53 (\u003cem\u003every poor\u003c/em\u003e), an FMQ of 58 (\u003cem\u003every poor\u003c/em\u003e), and a TMQ of 51 (\u003cem\u003every poor\u003c/em\u003e)(\u003cstrong\u003eTable\u003c/strong\u003e\u003cstrong\u003e3\u003c/strong\u003e). \u003cstrong\u003eThe EEG was persistently\u003c/strong\u003e borderline abnormal for infants with a background rhythm mildly slower than that for standard age (Figure6). \u003cstrong\u003eThe anthropometric measurements included a\u003c/strong\u003e head circumference of 48 cm (near the 97th percentile), a length of 72.5 cm (85th\u0026ndash;97th percentile), and a weight of 9.3 kg (85th\u0026ndash;97th percentile) (Figure7). \u003cstrong\u003eFor motor function, the infant\u003c/strong\u003e could hold her head steady, roll over with difficulty, elevate her head 90\u0026deg; in the prone position with elbow support, and sit with a forward lean; no active reaching or grasping with the hands was observed. Socially, she smiled responsively and occasionally produced nonspecific vocalizations, but visual tracking was slow.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. Characteristics of the \u003cem\u003ePURA\u003c/em\u003e gene of the patient. ACMG,\u0026nbsp;\u003c/strong\u003eAmerican College of Medical Genetics and Genomics.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"609\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eGene (Reference Transcript)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eChromosomal Location\u003c/strong\u003e（GRCh37/hg19）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVariant Information\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 49px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGenomic Region\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 43px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eZygosity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eACMG Classification\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePopulation Frequency\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 91px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAssociated Disorder\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInheritance Pattern\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVariant Origin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003ePURA（NM-005859.5）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003eChr5:139494529\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003eC.764dupAp.Asn255Lysfs*39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 49px;\"\u003e\n \u003cp\u003eExon1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 43px;\"\u003e\n \u003cp\u003eHeterozygous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003ePathogenic (P)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 91px;\"\u003e\n \u003cp\u003eNeurodevelopmental disorder with neonatal respiratory insufficiency, hypotonia, feeding difficulties\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003eAutosomal Dominant (AD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e\u003cem\u003eDe novo\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Patient scores on Gesell\u003c/strong\u003e\u003cstrong\u003e\u0026rsquo;s Developmental Schedule.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eChronological Age (Months)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 306px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eDomain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eAdaptive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eGross Motor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eLanguage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003ePersonal\u0026ndash;Social\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003eAdaptive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003eGross Motor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003eFine Motor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003eLanguage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003ePersonal\u0026ndash;Social\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eDevelopmental Age (Months)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e1.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e1.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e1.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eDevelopmental Quotient (DQ)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eInterpretation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eProfound Developmental Delay\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eProfound Developmental Delay\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eProfound Developmental Delay\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003eProfound Developmental Delay\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003eProfound Developmental Delay\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003eProfound Developmental Delay\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003eProfound Developmental Delay\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003eProfound Developmental Delay\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003eProfound Developmental Delay\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. Patient scores on Peabody Developmental Motor Scales, 2nd Edition (PDMS-2).\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"616\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eChronological Age (Months)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 248px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eComposite Score\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 576px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGross Motor (GMQ)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003eRE+ST+LO+OB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFine Motor (FMQ)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eGR+VI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal Motor (TMQ)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eRE+ST+LO+OB+GR+VI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGross Motor (GMQ)\u0026nbsp;\u003c/strong\u003eRE+ST+LO+OB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFine Motor (FMQ)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eGR+VI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eMotor (TMQ)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eRE+ST+LO+OB+GR+VI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStandard Score\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePercentile Rank\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e＜1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e＜1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e＜1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e＜1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e＜1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e＜1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDevelopmental Quotient\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e51\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eQualitative Description\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003eVery Poor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003eVery Poor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003eVery Poor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003eVery Poor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eVery Poor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003eVery Poor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Discussion and Conclusions","content":"\u003cp\u003ePWS is caused by the loss of expression of paternally inherited genes in a specific region of chromosome 15 (15q11-13) [12]. The global prevalence of PWS is estimated to range from 1 in 10,000 to 1 in 30,000 live births, with reports in the United States ranging from approximately 1 in 10,000 to 1 in 20,000 cases. PWS incidence does not substantially variation across race, sex, or ethnicity [12\u0026ndash;14]. PWS is characterized by severe hypotonia and feeding difficulties in infancy, followed by the onset of hyperphagia, behavioral and psychiatric issues, and morbid obesity in childhood. PWS is not a widely known syndrome despite the recognizable phenotype; one-third of affected individuals may remain undiagnosed [13]. PURA syndrome is a developmental encephalopathy and a rare autosomal dominant disorder, with an estimated incidence of \u0026lt;1 in 1,000,000 [9]. PURA syndrome is characterized by neonatal hypotonia, global developmental delay, and a spectrum of neurological symptoms [9]. Advances in clinical awareness and molecular diagnostic technologies are continuously expanding the phenotypic and genotypic spectra of PURA syndrome [15,16]. However, most cases of PURA syndrome that have been reported were diagnosed\u0026nbsp;during infancy or childhood, with relatively few cases identified during the neonatal period. Consequently, many affected individuals and their families attend multiple medical institutions before a diagnosis is established; sometimes diagnosis only occurs after serious developmental delays become apparent.\u0026nbsp;We document a case of a rare co-occurrence of PWS and PURA syndrome that was molecularly diagnosed\u0026nbsp;during the neonatal period. This case challenges Occam\u0026apos;s razor as the conventional diagnostic principle and highlights the possibility of multiple genetic disorders underlying complex clinical presentations. This study provides crucial insights for precisely diagnosing neonates presenting with severe hypotonia and feeding difficulties, underscoring the need for a comprehensive genetic evaluation in addition to single genetic explanations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe core features of PURA syndrome include neonatal hypotonia, feeding difficulties, respiratory issues, lethargy, and subsequent serious global developmental delay/intellectual disability\u0026nbsp;\u003c/strong\u003e[8,9,17].\u0026nbsp;\u003cstrong\u003eThe patient initially presented with classic severe hypotonia and feeding difficulties. WES identified a\u0026nbsp;\u003c/strong\u003e\u003cem\u003ede novo\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;pathogenic variant of \u003cem\u003ePURA\u003c/em\u003e.\u003c/strong\u003e These early manifestations substantially overlap those of the PWS phenotype [13,18,19], indicating a high risk of overlooking a diagnosis\u0026nbsp;of PWS if a single genetic etiology is accepted.\u0026nbsp;\u003cstrong\u003eMore specific PWS\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003efeatures were observed in our patient, namely, a thin upper lip and immature genitalia\u003c/strong\u003e [13,19],\u003cstrong\u003e\u0026nbsp;which prompted targeted methylation-specific MLPA analysis to ultimately confirm a concurrent PWS diagnosis.\u003c/strong\u003e This diagnostic experience shows that clinicians must maintain high suspicion of dual diagnoses after one molecular cause has been identified for infants with atypical, overlapping, or progressively inconsistent clinical presentations.\u0026nbsp;\u003cstrong\u003eA combinatorial diagnostic strategy integrating high-throughput sequencing (such as WES) with targeted assays for specific loci and imprinting disorders (such as MLPA) is crucial for precisely diagnosing such complex cases.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe clinical manifestations of the two disorders in the patient in our case likely involved additive effects and interactions, collectively contributing to the exceptionally severe neurodevelopmental outcome\u003c/strong\u003e\u003cstrong\u003es.\u003c/strong\u003e The follow-up results demonstrated that, despite systematic rehabilitation interventions and growth hormone therapy, Gesell\u0026rsquo;s Developmental Quotients indicated profound developmental delay at 9 months of age across all domains (adaptive, gross motor, fine motor, language, and personal\u0026ndash;social). All quotients from the Peabody Developmental Motor Scales-2 similarly remained very poor.\u0026nbsp;\u003cstrong\u003eGrowth hormone therapy considerably improved physical growth (length and weight); however, the beneficial effects on the neurological prognosis were limited.\u003c/strong\u003e These findings suggest that the severe impact of the \u003cem\u003ePURA\u003c/em\u003e gene defect on the central nervous system and the neurodevelopmental impairments associated with PWS were synergistic [13,20]. These synergistic impacts resulted in more profound developmental delays than would be expected from either condition alone.\u0026nbsp;\u003cstrong\u003eThis observation is valuable when assessing the long-term prognosis of patients with rare comorbidities and formulating realistic management strategies. This study underscores the critical need to provide early counseling in these cases, even during aggressive therapy, to note the potential for a guarded neurodevelopmental prognosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;molecular diagnosis methods indicated two distinct genetic mechanisms in our case.\u003c/strong\u003e First, WES identified a \u003cem\u003ede novo\u003c/em\u003e heterozygous frameshift variant, \u003cem\u003ePURA\u003c/em\u003e c.764dupA (p.Asn255Lysfs*39). This previously unreported variant is a frameshift mutation predicted to severely disrupt protein function and structure, consistent with the autosomal dominant inheritance pattern of PURA. Subsequently, methylation-specific MLPA analysis revealed a methylation pattern at chromosomal region 15q11.2-q13, which is typical of paternal (maternal) uniparental disomy, providing definitive molecular evidence for a diagnosis PWS.\u0026nbsp;\u003cstrong\u003eThe diagnosis in this case underscores the pivotal role and effectiveness of the combination of WES and MLPA in clinical genetics.\u003c/strong\u003e WES serves as a broad-spectrum tool for discovering unknown genetic variants, the primary advantage of which is a hypothesis-free, high-throughput capacity to detect single nucleotide variants and small insertions/deletions (indels) across the exome. The identification of the \u003cem\u003ede novo\u003c/em\u003e \u003cem\u003ePURA\u003c/em\u003e variant exemplifies the powerful discovery capability of WES [21,22]. However, WES has limitations in\u0026nbsp;sensitivity in detecting large copy number variations such as deletions or duplications of entire exons or genes [23].\u0026nbsp;\u003cstrong\u003eMLPA fills this technical gap.\u003c/strong\u003e MLPA is a targeted assay that provides gold-standard sensitivity and specificity in analyzing copy number and methylation status at specific loci, such as the PWS critical imprinted region at 15q11.2-q13 [24,25].\u0026nbsp;\u003cstrong\u003eThis tiered diagnostic strategy of using WES for initial screening followed by targeted validation efficiently considers the complete spectrum of pathogenic variations from small sequence alterations to large structural variants.\u003c/strong\u003e This strategy provides a critical paradigm and practical pathway for precisely diagnosing complex and heterogeneous genetic disorders.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn summary, this case provides a documented instance of the co-occurrence of PWS and PURA.\u003c/strong\u003e This case serves to alert clinicians that the diagnostic mindset of Occam\u0026apos;s razor should be eschewed when confronted with complex phenotypes; an open diagnostic approach should be maintained toward potential concomitant genetic disorders.\u0026nbsp;\u003cstrong\u003eThe combined application of broad-screening technologies, such as WES, and targeted, in-depth assays, such as MLPA, is pivotal for precisely diagnosing\u0026nbsp;such rare diseases.\u003c/strong\u003e \u003cstrong\u003eAn early and accurate diagnosis must be established for providing genetic counseling, monitoring complications, and formulating individualized rehabilitation management plans.\u003c/strong\u003e Future efforts will require the accumulation and reporting of more cases and long-term follow-ups to deepen our understanding of the additive effects of multigenetic disorders on phenotypes as well as the natural histories of multigenetic disorders, enabling treatment strategies to be continuously refined and patient outcomes to be improved.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ePWS Prader\u0026ndash;Willi syndrome\u003c/p\u003e\n\u003cp\u003eWES Whole-exome sequencing\u003c/p\u003e\n\u003cp\u003eNCIU Neonatal intensive care unit\u003c/p\u003e\n\u003cp\u003eEEG Electroencephalogram\u003c/p\u003e\n\u003cp\u003eMLPA Multiplex ligation-dependent probe amplification\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePDMS-2 Peabody Developmental Motor Scales, Second Edition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGMQ Gross Motor Quotient\u003c/p\u003e\n\u003cp\u003eFMQ Fine Motor Quotient\u003c/p\u003e\n\u003cp\u003eTMQ Total Motor Quotient\u003c/p\u003e\n\u003cp\u003eACMG American College of Medical Genetics and Genomics\u003c/p\u003e\n\u003cp\u003eUPD Uniparental disomy\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThe studies involving humans were approved by The ResearchEthics Boards of Children\u0026rsquo;s Hospital Affiliated to Shandong University(Approval No. SDFE-IRB/T-2025128). The studies were conducted in accordance with thelocal legislation and institutional requirements. Written informed consent for participation in this study was provided by theparticipants\u0026rsquo; legal guardians/next of kin. Written informed consent was obtained from the individual(s), and minor(s)\u0026rsquo; legalguardian/next of kin, for the publication of any potentially identifiable images or data included in this article.\u003c/p\u003e\n\u003cp\u003eClinical Trial Registration Number: Not applicable\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eAll the images and patient material presented in this study have consented for publication and they are available to see upon request. Written informed consent was obtained from all subjects and/or their legal guardian(s) for all the images and patient material presented in this study.\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Jinan High-Level Talent Fund for the Healthcare Industry [Grant number: 202412].\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eThe raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (Genomics, Proteomics \u0026amp; Bioinformatics 2025) in National Genomics Data Center (Nucleic Acids Res 2025), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA-Human: HRA014603) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa-human.\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; contributions\u003c/p\u003e\n\u003cp\u003eLMZ and CL were responsible for physical examinations, collection of clinical data and family history, and drafting the initial manuscript. QX and YJH assisted in data interpretation and language polishing. WMH was responsible for collecting clinical cases. LLK and XYL secured research funding, provided guidance in patient management, and participated in the overall guidance of manuscript drafting and revision. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eWe thank the patient and her parents for agreeing to the publication of this report. We also thank the people who have contributed to the development and execution of this study\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eButler MG, Hartin SN, Hossain WA, et al. Molecular genetic classification in Prader-Willi syndrome: A multisite cohort study. J Med Genet. 2019;56(3):149-153. doi:10.1136/jmedgenet-2018-105301. Epub 2018 May 5.\u003c/li\u003e\n\u003cli\u003eStrom SP, Hossain WA, Grigorian M, et al. A Streamlined approach to Prader-Willi and Angelman syndrome molecular diagnostics. Front Genet. 2021;12:608889. doi: 10.3389/fgene.2021.608889. eCollection 2021.\u003c/li\u003e\n\u003cli\u003eDuis J, Butler MG. Syndromic and nonsyndromic obesity: underlying genetic causes in humans. Adv Biol (Weinh). 2022;6(10):e2101154. doi:10.1002/adbi.202101154. Epub 2022 Jun 9.\u003c/li\u003e\n\u003cli\u003eButler MG, Duis J. Chromosome 15 imprinting disorders: genetic laboratory methodology and approaches. Front Pediatr. 2020;8:154. doi:10.3389/fped.2020.00154. eCollection 2020.\u003c/li\u003e\n\u003cli\u003eGeer LY, Marchler-Bauer A, Geer RC, et al. The NCBI BioSystems database. Nucleic Acids Res. 2010;38(Database issue):D492-6. doi:10.1093/nar/gkp858. Epub 2009 Oct 23.\u003c/li\u003e\n\u003cli\u003eTanaka AJ, Bai R, Cho MT, et al. De novo mutations in PURA are associated with hypotonia and developmental delay. Cold Spring Harb Mol Case Stud. 2015;1(1):a000356. doi:10.1101/mcs.a000356.\u003c/li\u003e\n\u003cli\u003eHokkanen S, Feldmann HM, Ding H, et al. Lack of Pur-alpha alters postnatal brain development and causes megalencephaly. Hum Mol Genet. 2012;21(3):473-84. doi:10.1093/hmg/ddr476. Epub 2011 Oct 18.\u003c/li\u003e\n\u003cli\u003eJohannesen KM, Gardella E, Gjerulfsen CE, et al. The PURA study group (2021). PURA-related developmental and epileptic encephalopathy: Phenotypic and genotypic spectrum. [J].Neurol Genet. 2021;7(6):e613. doi:10.1212/NXG.0000000000000613. eCollection 2021 Dec.\u003c/li\u003e\n\u003cli\u003eLee BH, Reijnders MRF, Abubakare O, et al. Expanding the neurodevelopmental phenotype of PURA syndrome. Am J Med Genet A. 2018;176(1):56-67. doi:10.1002/ajmg.a.38521. Epub 2017 Nov 17.\u003c/li\u003e\n\u003cli\u003eReijnders MRF, Janowski R, Alvi M, et al. PURA syndrome: Clinical delineation and genotype-phenotype study in 32 individuals with review of published literature. [J]. Med Genet. 2018;55(2):104-113. doi:10.1136/jmedgenet-2017-104946. Epub 2017 Nov 2.\u003c/li\u003e\n\u003cli\u003ePosey JE, Harel T, Liu P, et al., Resolution of disease phenotypes resulting from multilocus genomic variation, N Engl J Med. 2017;376(1):21-31. doi:10.1056/NEJMoa1516767. Epub 2016 Dec 7.\u003c/li\u003e\n\u003cli\u003eCassidy SB, Schwartz S, Miller JL, et al. Prader-Willi syndrome. Genet Med. 2012;14(1):10-26. doi:10.1038/gim.0b013e31822bead0. Epub 2011 Sep 26.\u003c/li\u003e\n\u003cli\u003eLionti T, Reid SM, White SM, et al. A population-based profile of 160 Australians with Prader-Willi syndrome: trends in diagnosis, birth prevalence and birth characteristics.Am J Med Genet A. 2015;167A(2):371-8. doi:10.1002/ajmg.a.36845. Epub 2014 Nov 25.\u003c/li\u003e\n\u003cli\u003eBohonowych J, Miller J, McCandless SE, et al. The global Prader-Willi syndrome registry: development, launch, and early demographics. Genes (Basel). 2019;10(9):713. doi: 10.3390/genes10090713.\u003c/li\u003e\n\u003cli\u003eBoczek NJ, Macke EL, Kemppainen J, et al. Expansion of PURA-related phenotypes and discovery of a novel PURA variant: a case report. [J]. Child Neurol Open. 2020:7:2329048X20955003. doi: 10.1177/2329048X20955003. eCollection 2020 Jan-Dec.\u003c/li\u003e\n\u003cli\u003eTrau SP, Pizoli CE. PURA syndrome and myotonia. [J]. Pediatr Neurol. 2020;104:62-63. doi: 10.1016/j.pediatrneurol.2019.09.008. Epub 2019 Oct 19.\u003c/li\u003e\n\u003cli\u003eMargot R F Reijnders，Robert Janowski，Mohsan Alvi，et al. PURA syndrome: Clinical delineation and genotype-phenotype study in 32 individuals with review of published literature[J]. J Med Genet. 2018;55(2):104-113. DOI: 10.1136/jmedgenet-2017-104946.\u003c/li\u003e\n\u003cli\u003eGrootjen LN, Uyl NEM, van Beijsterveldt IALP, et al. Prenatal and neonatal characteristics of children with Prader-Willi syndrome. J Clin Med. 2022;11(3):679. doi: 10.3390/jcm11030679.\u003c/li\u003e\n\u003cli\u003eButler MG, Miller JL, Forster JL. Prader-Willi syndrome\u0026mdash;clinical genetics, diagnosis and treatment approaches: an update. Curr Pediatr Rev. 2019;15(4):207-244. doi: 10.2174/1573396315666190716120925.\u003c/li\u003e\n\u003cli\u003eKuo CJ， Lee KH， Huang CC，et al. Pur\u0026alpha; regulates the induction of Znf179 transcription during neuronal differentiation. [J]. Biochem Biophys Res Commun. 2020;533(4):1477-1483. DOI: 10.1016/j.bbrc.2020.10.047\u003c/li\u003e\n\u003cli\u003eBamshad MJ, Ng SB, Bigham AW, et al.Exome sequencing as a tool for Mendelian disease gene discovery.Nat Rev Genet. 2011;12(11):745-55. doi: 10.1038/nrg3031.\u003c/li\u003e\n\u003cli\u003eYang Y, Muzny DM, Reid JG, et al. Clinical whole-exome sequencing for the diagnosis of mendelian disorders.N Engl J Med. 2013.\u003c/li\u003e\n\u003cli\u003ede Ligt J, Willemsen MH, van Bon BW, et al. Diagnostic exome sequencing in persons with severe intellectual disability.N Engl J Med. 2012;367(20):1921-9. doi: 10.1056/NEJMoa1206524. Epub 2012 Oct 3.\u003c/li\u003e\n\u003cli\u003eSchouten, JP, McElgunn CJ, Waaijer R, et al. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res. 2002;30(12):e57. doi: 10.1093/nar/gnf056.\u003c/li\u003e\n\u003cli\u003eStuppia L, Antonucci I, Palka J, et al. Use of the MLPA assay in the molecular diagnosis of gene copy number alterations in human genetic diseases. Int J Mol Sci. 2012;13(3):3245-3276. doi: 10.3390/ijms13033245. Epub 2012 Mar 8.\u003cstrong\u003e\u003c/strong\u003e\u003c/li\u003e\n\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":"bmc-pediatrics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bped","sideBox":"Learn more about [BMC Pediatrics](http://bmcpediatr.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bped/default.aspx","title":"BMC Pediatrics","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Neonate, PWS, HTS, MLPA, dual diagnosis, concomitant genetic diseases","lastPublishedDoi":"10.21203/rs.3.rs-7941293/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7941293/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e A broad and heterogeneous spectrum of disorders present as feeding difficulties and hypotonia during the neonatal period, which are characterized by complex etiologies, diverse clinical manifestations, and considerable symptom overlap, posing diagnostic challenges. The co-occurrence of different genetic disorders in a single patient is rare. We report the a case of Prader–Willi syndrome (PWS) co-occurring with PURA syndrome diagnosed during the neonatal period.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCase Presentation: \u003c/strong\u003eWe describe a case of a neonate admitted with feeding difficulties and hypotonia. The infant exhibited persistent feeding problems, hypotonia, a thin upper lip, and immature genitalia. The initial diagnostic workup included high-throughput whole-exome sequencing (WES). The WES results revealed a heterozygous \u003cem\u003ede novo\u003c/em\u003e variant, c.764dupA (p.Asn255Lysfs*39), in \u003cem\u003ePURA\u003c/em\u003e. This specific variant was absent from all population databases and has not been previously reported in the literature. The variant was classified as pathogenic based on the standards and guidelines of the American College of Medical Genetics and Genomics. This finding was consistent with a diagnosis of PURA syndrome (OMIM #616158). However, PWS could not be ruled out given the clinical phenotype of the patient. Subsequently, methylation-specific multiplex ligation-dependent probe amplification was performed. The paternal methylation imprint at the SNURF-SNRPN locus was completely absent (100%) , confirming the diagnosis of PWS caused by an imprinting center defect. The patient was definitively diagnosed with co-occurring PWS and PURA syndrome. The infant received rehabilitative therapy and growth hormone treatment. The patient showed satisfactory gains in length and weight during follow-up until 9 months of age. However, neurological improvement was poor, and the long-term prognosis remains under observation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eWe report a case of PWS co-occurring with PURA syndrome diagnosed during the neonatal period. The overlapping clinical presentation challenges the conventional diagnostic paradigm of one disease, one phenotype. We emphasize that the possibility of a dual diagnosis should be considered in infants with complex, rapidly progressive, or atypical features, even after one genetic disorder is identified. A multitiered molecular testing approach should be used to enable precise diagnosis and enabling individualized management, which are fundamental for establishing an accurate prognosis and predicting treatment outcomes.\u003c/p\u003e","manuscriptTitle":"Prader–Willi and PURA Syndromes Concurrently Diagnosed Using High-Throughput Sequencing with Methylation-Specific Multiplex Ligation-Dependent Probe Amplification: A Case Report","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-08 15:34:41","doi":"10.21203/rs.3.rs-7941293/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2025-12-18T21:42:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"75684843323921793654337706125262105326","date":"2025-12-13T13:32:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"263234266216434114727256449734380662081","date":"2025-12-13T10:44:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-06T13:29:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"200078003898083243551594361971132222445","date":"2025-12-06T12:33:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-04T12:28:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-01T20:29:55+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-12T13:01:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-12T11:49:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Pediatrics","date":"2025-11-12T11:45:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-pediatrics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bped","sideBox":"Learn more about [BMC Pediatrics](http://bmcpediatr.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bped/default.aspx","title":"BMC Pediatrics","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"37041594-fec6-4c8a-98ca-698ead464171","owner":[],"postedDate":"December 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-12-08T15:34:41+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-08 15:34:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7941293","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7941293","identity":"rs-7941293","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}