The Diagnostic Odyssey of a Biochemically Confirmed Case of ML II: The First Western Patient With LYSET Deficiency

The Diagnostic Odyssey of a Biochemically Confirmed Case of ML II: The First Western Patient With LYSET Deficiency ABSTRACT The LYSET gene encodes the LYSET transmembrane protein, which regulates lysosome biogenesis by activating the mannose-6-phosphate (M6P) pathway. This is an autosomal recessive, ultrarare, and severe progressive skeletal dysplasia with coarse facies, distended abdomen, short stature, and severe physical disability. In a diagnostic odyssey, we report a female patient, born in 2008, daughter of consanguineous parents, with hand contractures and a typical facial appearance since 5 months old. She was clinically diagnosed at 2 years old with contractures and severe dysplasia. Systolic murmur, thickening of mitral and aortic valves, and tricuspid regurgitation were observed. Nine enzymes showed increased levels in plasma, and seven showed decreased levels in fibroblasts. Abnormal sialic acid profile and GAGs (glycosaminoglycans) were detected in urine. No variants were identified during more than a decade of investigation. A whole-genome analysis identified the homozygous nonsense variant NM_001098621.4:c.112C>T (p.Gln38Ter) in the LYSET gene. The patient had not been diagnosed before due to the recent association of the gene with the lysosomal hydrolase labeling pathway. She died in 2018 from respiratory causes. The discovery of the relationship between the LYSET gene and lysosomal biogenesis was determinative of the diagnostic conclusion. Cases of dysostosis multiplex can be highly challenging due to the rarity of the disease and its clinical similarity to mucopolysaccharidosis (MPS) and mucolipidosis II/III (MLII/III). This is the first western report of a challenging case of an extensive diagnostic odyssey and demonstrates that the LYSET gene must be considered in the differential diagnosis when M6P-labeled lysosomal enzymes are altered. The LYSET gene (TMEM251) codifies the LYSET transmembrane protein located at 14q32.12. This protein regulates lysosome biogenesis by activating the M6P pathway and binding GlcNAc-1-phosphate transferase (GNPTAB) to the membrane-bound transcription factor site-1 protease (MBTPS1), thereby allowing proteolytic activation of GNPTAB. This set of proteins is involved in the regulation of M6P-dependent Golgi-to-lysosome trafficking of lysosomal enzymes [1-4]. Only one article in the literature reports patients with variants in the LYSET gene (two families—six cases), characterized by severe skeletal dysplasia, growth delays, increased lysosomal enzyme levels in dried blood, and normal cognition in the patients [1]. Here, we characterize the first Western patient with ML linked to the LYSET gene, including extensive analysis of skeletal alterations, biochemical characterization, neuropsychomotor developmental delay, and identification of a novel variant. The study was approved by the Hospital de Clínicas de Porto Alegre Research Ethics Committee (FIPE- 2011-0477) and written informed consent had been obtained from the patient's legal guardian. Clinical data were collected in medical records and monitored by a geneticist. A comprehensive biochemical investigation was carried out. Fibroblasts, leukocytes, plasma, and urine were evaluated through enzymatic assays, thin-layer chromatography, dosage of GAGs, and OLS/SOLS chromatography. Genomic DNA was extracted from blood samples and amplification of GNPTAB, GNPTG and MBTPS1 genes was performed by PCR [5], followed by Sanger sequencing using the reference sequences NM_024312.5, NM_032520.5 and NM_003791.4. A Comparative Genome Hybridization Array (aCGH) was performed for the evaluation of duplications, deletions or copy number variations of GNPTAB and GNPTG. An exome sequencing test was conducted when the patient was 5 years old. A whole-genome sequencing was performed on the MGI PCR-free platform and processed with the Sentieon Germline Pipeline v1.0, using the GRCh38 assembly as the reference genome, with a mean coverage of 30×. The female patient was born in 2008, in the Northeast of Brazil, by cesarean section, weighing 2010 g, without prenatal or perinatal complications. Neonatal screening was regular. The parents' ages at birth were 37 years (mother) and 25 years (father). A widened nasal bridge and thick skin were observed at birth. The onset of clinical manifestations occurred at 5 months of age, with a widened nasal bridge, hands with short, claw-like fingers, and a thoracolumbar hump. Neuropsychomotor and growth delays were observed. The patient presented head support at 3 months, sat unsupported at 8 months, spoke words with at least two syllables at approximately 9 months, crawled at 1 year and 2 months, and walked unsupported at 2 years after physical therapy intervention. Eruption of the first tooth, a lateral incisor, occurred at 1 year of age. Sphincter control began at 2 years and 9 months. The patient had a typical facial appearance, including an elongated skull, bifrontal narrowing, thick hair, ocular proptosis, epicanthus, depressed nasal root, low nasal bridge, anteverted nostrils with bulbous tips, infiltrated face and voluminous lips, the lower inverted and the upper arched. The mouth was kept open, with macroglossia, small teeth, a short neck, a carinated chest and noticeable diffuse joint limitation predominantly in the knees, elbows and hands, as well as genu vagus (Figure 1A,B). Cardiac alterations were observed on the echocardiogram performed at the patient's age of 3 years old, with thickening of the mitral and aortic valve leaflets and mild regurgitation without hemodynamic repercussions. She had functional tricuspid regurgitation with a pulmonary systolic pressure of 27 mmHg. At 2 years and 9 months of age, the patient weighed 10 kg (< 3 W), was 73 cm tall (< 3 W), and had a head circumference of 44.5 cm (< 3 W). She had joint contractures in her hands, elbows, and knees, as well as a thoracolumbar hump. Mongolian spots, a protruding abdomen, and gingival hyperplasia were identified, but teeth were visible. Communication was nonverbal, with partially comprehensible two-syllable words. She could climb stairs. Ophthalmological evaluation was regular. Chest deformities, including asymmetry and pectus carinatum, were noticeable. Severe skeletal dysplasia on radiological examination showed osteopenia, brachycephaly, sella turcica J, hip subluxation, and hypodevelopment of L2 among the main findings (Figure 1C–J). The biochemical diagnosis was performed at 2 years and 4 months and suggested that the patient had ML II alpha/beta, based on increased lysosomal hydrolase activity in plasma and regular activity in leukocytes. In fibroblasts, some values were below the reference values. Abnormal band profile was observed in urine (Table 1). | Biochemical investigation | Sample | Patienta | Reference values | |---|---|---|---| | Age at biochemical investigation (years) | — | 3 | — | | α-Iduronidase | Plasma | 279 | 6.8–13.7 nmol/h/mL | | β-Glucuronidase | Plasma | 832/783/902 | 30–300 nmol/h/mL | | α-N-acetylgalactosaminidase | Plasma | 289/1082 | 11–37/34–162 nmol/h/mL | | α-Mannosidase | Plasma | 2013 | 17–56 nmol/h/mL | | Arylsulfatase A | Plasma | Positive | — | | β-Hexosaminidases A | Plasma | 2606/6599/4599 | 550–1675 nmol/h/mL | | β-Hexosaminidases B | Plasma | 457/12814/15966 | 265–1219 nmol/h/mL | | Total β-Hexosaminidases | Plasma | 3063/19413/22765 | 1000–2857 nmol/h/mL | | % Hexosaminidases A/Total β-Hexosaminidases | Plasma | 34/85/30 | 45%–79% | | Quitotriosidase | Plasma | 78/99/159 | 8.8–132 nmol/h/mL | | Iduronate sulfatase | Plasma | 1221 | 122–463 | | β-Galactosidase | Leukocyte | 118 | 78–280 | | α-Iduronidase | Leukocyte | 44 | 32–56 nmol/h/mg prot | | Arylsulfatase B | Leukocyte | 184 | 72–176 nmol/h/mg prot | | β-Glucosidase | Leukocyte | 10 | 10–45 | | Iduronate sulfatase | Leukocyte | 66 | 31–110 | | β-Hexosaminidase total | Leukocyte | 2936 | 552–1662 nmol/h/mg prot | | %Hex A | Leukocyte | 89 | 48–89 | | β-Galactosidase | Fibroblasts | 11 | 394–1440 nmol/h/mg prot | | β-Glucuronidase | Fibroblasts | 12 | 62–361 nmol/h/mg prot | | Sphingomyelinidase | Fibroblasts | 4 | 49–72 nmol/h/mg prot | | Hex total | Fibroblasts | 876 | 3000–13 490 nmol/h/mg prot | | % Hex A | Fibroblasts | 63 | 46–81 nmol/h/mg prot | | Neuraminidase | Fibroblasts | 2.2 | 30–38 nmol/h/mg prot | | Iduronate sulfatase | Fibroblasts | 66 | 31–110 | | Dosage of GAGs | Urine | 88 | < 2 years: 79–256 mg/L | | Total sialic acid | Urine | 174 | 34–306 μM/mM | | Free sialic acid | Urine | 42 | 21–192 μM/mM | | GAGs chromatography | Urine | Normal | Normal | | OLS/SOLS chromatography | Urine | Anomalous bands | Normal | - Abbreviations: —: not available; CS: chrondoitin sulfate; DS: dermatan sulfate; GAGs: glycosaminoglycans; HS: heparan sulfate; ML: mucolipidosis; OLS: oligosaccharides; SOLS: sialosaccharides. - a When more than one measurement was taken, the different values are presented separated by bars. Sanger sequencing for the GNPTAB, GNPTG, and MBTPS1 genes, aCGH and exome were negative. When the patient was 5 years old, exome analysis was performed with the advent of massively parallel sequencing and no suspicious variants were identified to support the patient's clinical diagnosis. Due to negative molecular analyses, structural variants or variants in regulatory regions were hypothesized. Whole-genome analysis was performed to investigate areas not previously covered by previous studies. The NM_001098621.4:c.112C>T (p.Gln38Ter) variant was identified in the LYSET gene in homozygosity (Figure 1K–M). This is the first report of a Western patient with LYSET deficiency. The patient faced a true odyssey, dying before obtaining a complete diagnosis. Rare genetic Mendelian disorders contribute significantly to pediatric morbidity and mortality, being collectively relevant in frequency, impacting 3.5%–5.9% of the population. There are many challenges related to clinical suspicion, technical and socioeconomic limitations in performing genetic testing and even knowledge gaps about the pathophysiological basis of these diseases when they are ultra-rare, resulting in delayed diagnoses [6-9]. In the only study to date evaluating patients with LYSET deficiency, only one patient had biochemical analyses performed, with results from investigations of hydrolases Iduronate-2 sulfatase, ɑ-Iduronidase, and β-Galactosidase in dried blood showing increased levels. In the present study, biochemical similarities with ML II/III were identified, with increased lysosomal hydrolase activity in plasma and regular activity in leukocytes [1, 10]. Qiao et al. [2] demonstrated the aberrant secretion of lumenal lysosomal proteins and a global loss of cathepsin protease activity in LYSET KO cells, indicating that LYSET deficiency causes a severe defect in M6P-specific lysosomal trafficking. LYSET binds to GNPTAB, which is relocalized to lysosomes and degraded, resulting in M6P tagging failure and explaining why LYSET deficiency is a phenocopy of MLII [2]. The reported consanguinity supports the patient's homozygosity (Figure 1K). The variant p.Gln38Ter is not predicted to undergo nonsense-mediated mRNA decay (NMD); however, the gene was recently discovered and its mechanism of pathogenicity is still being established. The gene has two exons and the variant is located in the second exon. The deduced transmembrane protein is 169 amino acids long with two transmembrane domains and a conserved domain of unknown function (DUF4583). However, with advances in the characterization of LYSET regions, it may be possible to assign new pathogenicity criteria in the future. In the GnomAD database, two alleles were identified among 1 179 620 non-Finnish European individuals, with a frequency of 0.000001695, and no homozygotes were identified [11]. With the case reported in this study, a total of 7 patients with LYSET deficiency were reported. The comparison between the characteristics of the 7 patients is presented in Table 2. Ain et al. [1] described, for the first time, six cases involving five Pakistani sibs and one Iranian girl with homozygous variants in the LYSET gene, resulting in significant skeletal progressive dysplasia, short stature, coarse facial features, and protruding abdomens. Two homozygous variants were identified: c.133C>T; p.(Arg45Trp) and c.215dupA; p.(Tyr72Ter). Functional assays in rats suggested that the disease was associated with chondrocyte differentiation [1]. | Reference | Present work | Ain et al. [1] | Ain et al. [1] | | Number of patients | 1 | 1 | 7a | | Gender | Female | Female | 5 female—2 male | | Origin | Brazil | Iran | Pakistan | | LYSET variants | c.112C>T p.Gln38Ter (homozygous) | c.215dupA p.(Tyr72Ter) (homozygous) | c.133C>T p.(Arg45Trp) (homozygous)b | | Consanguinity | + | + | + | | Onset of symptoms | 5 m | 6 m | 1 yo | | Age at death | 10 | 5 | ~20 | | Short stature | + | + | + | | Dysostosis multiplex | + | + | + | | Coarse facial features | + | + | + | | Protruding abdomen | + | + | + | | Speech | 2–3 words | 2–3 words | + | | GAGs in urine | − | − | − | | Cognition | Affected | NA | Normal | | Lysosomal enzymes | Altered in plasma, fibroblasts and leukocytes | Altered in dried blood spots | NA | - Abbreviations: +, present; −, absent; GAG, glycosaminoglycans; m, months; NA, not assessed; yo, years old. - a Data was presented in the article for five patients, but for several parameters, different individuals did not have data analyzed. - b Five patients analyzed genetically were homozygous for the variant. The five Pakistani patients with p.(Arg45Trp) homozygous variants presented increasingly coarse facial features, protruding abdomens, progressive skeletal changes, and the two older patients died in their twenties. The Iranian girl with p.(Tyr72Ter) homozygous variants had short stature, craniosynostosis, kyphoscoliosis, hip-joint subluxation, and died at the age of 5 years. We believe that the severity of the symptoms can be justified in the cases of the Brazilian and Iranian girls with more similar clinical presentation and earlier deaths by the type of variant identified, a nonsense variant, which results in truncated or non-functional shortened proteins or undergo to mRNA decay, while the patients with greater survival presented a missense variant, although the protein domains are not yet well characterized [12, 13]. The clinical characterization of bone disease showed widespread osteopenia. In the cases reported until now, LYSET deficiency causes severe skeletal dysplasia and extreme short stature like MLII, showing the impacts that defects in the lysosomal hydrolase labeling pathway have on bone resorption. Common major features of the MLII diseases are severe skeletal defects such as short stature, bowing of limbs, progressive joint contractures, thoracic deformity, and kyphoscoliosis. Pathogenic variants in GNPTAB but not GNPTG result in increased bone resorption in MLII and III alpha/beta, but not in MLIII gamma, showing that the diseases are not only genetically but also phenotypically different in the pathophysiology of bone disease. In this context, LYSET deficiency appears to resemble MLII [14]. The discovery of the relationship between the LYSET gene and lysosomal biogenesis was determinative for the diagnostic conclusion. Cases of dysostosis multiplex can be highly challenging due to the rarity of the disease and clinical similarity to MPS and ML. This is the first western report of a complex case in an extensive diagnostic odyssey and demonstrates that the LYSET gene must be considered in the differential diagnosis when M6P-labeled lysosomal enzymes are altered. Author Contributions Writing: Fernanda Sperb-Ludwig. Data collection, organization, analysis, and tabulation: Fernanda Sperb-Ludwig, Taciane Alegra, Leonardo Martinello da Rosa, Nataniel Floriano Ludwig, and Renata Voltolini Velho. Article review and obtained funding: Fernanda Sperb-Ludwig and Ida Vanessa Doederlein Schwartz. Acknowledgments This work was supported by FAPERGS 08/2023-24/2551-0000591-9, CAPES—Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, and FIPE/HCPA (2011-0477;2022-0206). The Article Processing Charge for the publication of this research was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) (ROR identifier: 00x0ma614). Funding This work was supported by FAPERGS - Fundo de Amparo à Pesquisa do Rio Grande do Sul (24/2551-0000591-9), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Fundo de Incentivo à Pesquisa (FIPE- HCPA: 2011-0477; 2022-0206). We thank the Inborn Errors of Metabolism Laboratory of the Medical Genetics Service HCPA. Conflicts of Interest The authors declare no conflicts of interest. Data Availability Statement The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Peer Review For transparency, the peer review documents associated with this article are available at https://doi.org/10.1111/cge.70165. | Reviews Round 4 | | | Editor Decision Letter | 2026/03/10 | | Reviews Round 3 | | | Editor Decision Letter | 2026/02/05 | | Reviewer 1 Report | 2026/02/05 | | Reviewer 1 Report Attachment 1 | 2026/02/05 | | Reviews Round 2 | | | Editor Decision Letter | 2026/01/30 | | Reviews Round 1 | | | Editor Decision Letter | 2026/01/09 |