Development, validation and application of single molecule molecular inversion probe based novel integrated genetic screening method for 29 common lysosomal storage disorders in India.

BACKGROUND: Current clinical diagnosis pathway for lysosomal storage disorders (LSDs) involves sequential biochemical enzymatic tests followed by DNA sequencing, which is iterative, has low diagnostic yield and is costly due to overlapping clinical presentations. Here, we describe a novel low-cost and high-throughput sequencing assay using single-molecule molecular inversion probes (smMIPs) to screen for causative single nucleotide variants (SNVs) and copy number variants (CNVs) in genes associated with 29 common LSDs in India. RESULTS: 903 smMIPs were designed to target exon and exon-intron boundaries of targeted genes (n = 23; 53.7 kb of the human genome) and were equimolarly pooled to create a sequencing library. After extensive validation in a cohort of 50 patients, we screened 300 patients with either biochemical diagnosis (n = 187) or clinical suspicion (n = 113) of LSDs. A diagnostic yield of 83.4% was observed in patients with prior biochemical diagnosis of LSD. Furthermore, diagnostic yield of 73.9% (n = 54/73) was observed in patients with high clinical suspicion of LSD in contrast with 2.4% (n = 1/40) in patients with low clinical suspicion of LSD. In addition to detecting SNVs, the assay could detect single and multi-exon copy number variants with high confidence. Critically, Niemann-Pick disease type C and neuronal ceroid lipofuscinosis-6 diseases for which biochemical testing is unavailable, could be diagnosed using our assay. Lastly, we observed a non-inferior performance of the assay in DNA extracted from dried blood spots in comparison with whole blood. CONCLUSION: We developed a flexible and scalable assay to reliably detect genetic causes of 29 common LSDs in India. The assay consolidates the detection of multiple variant types in multiple sample types while having improved diagnostic yield at same or lower cost compared to current clinical paradigm.

Lysosomal storage disorders identified in adult population from India: Experience of a tertiary genetic centre and review of literature.

Lysosomal storage disorders (LSDs) in adults have milder phenotype and variable age at presentation. Several studies have described the phenotype, genotype and treatment outcomes for adult-onset LSDs like Gaucher, Fabry, Pompe disease and others. We describe the first systematic study on the occurrence of LSDs in an adult population from India. It describes, the key clinical signs seen in these patients and those from literature review that can aid in early detection. Of 2102 biochemically diagnosed LSDs cases, 32 adult patients were identified with LSDs. Based on the clinical suspicion, screening test and enzyme study was carried out. Twenty-two patients were subjected to a genetic study to identify the causative variant in a respective gene. Of the 32 adult patients, we observed a maximum percentage of 37.5% (n = 12) cases with Gaucher disease, followed by 13% (n = 4) with Fabry disease. We found 10% of cases with MPS IVA and MPS I, and 9% cases with Pompe. Single case of adult mucolipidosis III and two cases each of Type 1 Sialidosis, Niemann-Pick disease B and metachromatic leukodystrophy were identified. We observed two common variants p.Leu483Pro and p.Ala487Thr in the GBA1 gene in 23% of Indian patients with adult Gaucher disease. No common variants were observed in other aforementioned LSDs. Study identified 50% of Fabry patients and 4% of Gaucher patients diagnosed at our centre to be adults. The prevalence of adult Pompe patients was low (3.4%) as compared to 80% reported in the Caucasian population. Adult LSDs such as, MPS III, GM1/GM2 gangliosidosis and Krabbe disease were not identified in our cohort.

Late infantile and adult-onset metachromatic leukodystrophy due to novel missense variants in the PSAP gene: Case report from India.

Metachromatic leukodystrophy (MLD) due to Sap-B deficiency is a rare autosomal recessive disorder caused due to biallelic variants in the PSAP gene. The PSAP gene encodes a precursor protein prosaposin, which is subsequently cleaved to form four active glycoproteins: Sap-A, Sap-B, Sap-C, and Sap-D. In case of deficiency of the sphingolipid activator protein Sap-B, there is a gradual accumulation of cerebroside-3-sulfate in the myelin of the nervous system resulting in progressive demyelination. Only 12 variants have been reported in the PSAP gene causing Sap-B deficiency to date. Here, we report two cases of MLD due to Sap-B deficiency (late-infantile and adult-onset form) harboring two novel missense variants c.688T > G and c.593G > A in the PSAP gene respectively. This study reports the third case of adult-onset MLD due to Sap-B deficiency in the world. The proband, a 3-year-old male child presented with complaints of hypotonia, lower limb tremors and global developmental delay. His MRI showed hyperintense signals in the bilateral cerebellar white matter. Overall, the findings were suggestive of metachromatic leukodystrophy. The second case was a 19-year-old male child with clinical features of regression of speech, gait ataxia and bilateral tremors referred to our clinic. MRI data suggested metachromatic leukodystrophy. Normal enzyme activity of arylsulfatase-A led to a suspicion of saposin B deficiency. For both cases, targeted sequencing was performed. This identified homozygous variant c.688T > G (p.Cys230Gly) and c.593G > A (p.Cys198Tyr) in exon 6 of the PSAP gene, respectively.

Lysosomal storage disorders: from biology to the clinic with reference to India.

Lysosomal storage disorders (LSDs) are a group of seventy different metabolic storage diseases due to accumulation of substrate mainly in the form of carbohydrate, lipids, proteins, and cellular debris. They occur due to variant in different genes that regulate lysosomal enzymes synthesis, transport, and secretion. In recent years, due to an increased availability of various therapies to treat these disorders, and increased diagnostic tools, there has been an escalated awareness of LSDs. Due to heterogeneous population and various social reasons, India is likely to have a high frequency of LSDs. Therefore, to understand the burden of various LSDs, its molecular spectrum, and understanding the phenotype-genotype correlation, Indian Council of Medical Research (ICMR) and Department of Health Research (DHR), Government of India had set up a task force in the year 2015. It has resulted in identifying common LSDs, and founder variant for some of the storage disorders and molecular spectrum of various LSDs across the country. This review describes in detail the spectrum of LSDs, its molecular epidemiology and prevention in context to Indian population.

Late infantile form of multiple sulfatase deficiency with a novel missense variant in the SUMF1 gene: case report and review.

BACKGROUND: Multiple sulfatase deficiency (MSD) is a rare lysosomal storage disorder caused due to pathogenic variants in the SUMF1 gene. The SUMF1 gene encodes for formylglycine generating enzyme (FGE) that is involved in the catalytic activation of the family of sulfatases. The affected patients present with a wide spectrum of clinical features including multi-organ involvement. To date, almost 140 cases of MSD have been reported worldwide, with only four cases reported from India. The present study describes two cases of late infantile form of MSD from India and the identification of a novel missense variant in the SUMF1 gene. CASE PRESENTATION: In case 1, a male child presented to us at the age of 6 years. The remarkable presenting features included ichthyosis, presence of irritability, poor social response, thinning of corpus callosum on MRI and, speech regression. Clinical suspicion of MSD was confirmed by enzyme analysis of two sulfatase enzymes followed by gene sequencing. We identified a novel missense variant c.860A > T (p.Asn287Ile) in exon 7 of the SUMF1 gene. In case 2, a two and a half years male child presented with ichthyosis, leukodystrophy and facial dysmorphism. We performed an enzyme assay for two sulfatases, which showed significantly reduced activities thereby confirming MSD diagnosis. CONCLUSION: Overall, present study has added to the existing data on MSD from India. Based on the computational analysis, the novel variant c.860A > T identified in this study is likely to be associated with a milder phenotype and prolonged survival.

The GALNS p.P77R variant is a probable Gujarati-Indian founder mutation causing Mucopolysaccharidosis IVA syndrome.

BACKGROUND: Mucopolysaccharidosis IVA (Morquio syndrome A, MPS IVA) is an autosomal recessive lysosomal storage disorder caused due to biallelic variants in the N-acetylgalactoseamine-6-sulfate sulfatase (GALNS) gene. The mutation spectrum in this condition is determined amongst sub-populations belonging to the north, south and east India geography, however, sub-populations of west Indian origin, especially Gujarati-Indians, are yet to be studied. We aimed to analyse the variants present in the GLANS gene amongst the population of Gujarat by sequencing all exons and exon-intron boundaries of the GALNS gene in patients from 23 unrelated families. RESULTS: We report 11 variants that include eight missense variants: (p.L36R, p.D39G, p.P77R, p.C79R, pP125L, p.P151L, p.G255A and p.L350P), one splice site variant: (c.121-7C > G), one small insertion: (c.1241_1242insA, p.I416HfsTer2) and one small deletion: (c.839_841delACA). Of these, three missense variants (p.D39G, p.G255A and p.L350P), one splice site and the two indels mentioned above are novel. Interestingly, we observed a higher than anticipated prevalence of p.P77R variant in our cohort (n = 14/25, 56%). Haplotype analysis in cases with p.P77R variant and 63 ethnicity matched healthy population controls suggested a 4 SNP haplotype block present in cases compared to controls (likelihood ratio test p-value = 1.16 × 10-13), thereby suggesting p.P77R variant as a founder variant in the Gujarati-Indian population. Furthermore, age of mutation analysis suggested the variant to have arisen approximately 450 years ago in the population. CONCLUSION: p.P77R variant in the GLANS gene is likely to be a founder variant in MPS IVA patients of Gujarati-Indian ancestry and appeared approximately 450 years ago in the population. To our knowledge, this is the first variant to be posited as a founder variant in the GLANS gene in patients with MPS IVA syndrome.

An ultra-rare case of immunoskeletal dysplasia with neurodevelopmental abnormalities in an Indian patient with homozygous c.953C > T variant in EXTL3 gene: a case report.

BACKGROUND: Immunoskeletal dysplasia with neurodevelopmental abnormalities (ISDNA) is an ultra-rare genetic condition that belongs to the group of spondyloepimetaphyseal dysplasias. It is caused due to presence of biallelic variants in the EXTL3 gene. The encoded exostosin like glycosyltransferase 3 (EXTL3) protein plays a key role in heparan sulfate synthesis. The skeletal and nervous systems are prominently affected in ISDNA with variability in immunological manifestations. Here, we report the 15th case of ISDNA (third patient of an Indian ancestry) in the world, along with a review of literature. CASE PRESENTATION: A 15-month-old female child with clinical indications of global developmental delay, short stature, coarse facial features, and hypotonia was referred to our clinic. Spondyloepimetaphyseal dysplasias associated with extra-skeletal manifestations was suspected based on clinic-radiological correlation. Whole exome sequencing analysis revealed the presence of a homozygous known pathogenic variant c.953C > T (p. Pro318Leu) in exon 3 of the EXTL3 gene, thereby confirming diagnosis of ISDNA. CONCLUSION: We present an ultra-rare case of ISDNA- third patient of Indian ancestry and only the 15th reported case in the literature. On review of all cases in the literature, we find that the affected individuals show abnormalities primarily in three systems namely- skeletal, nervous and immune system. Notably, patients harbouring the same variant in EXTL3 gene show phenotypic variability especially with respect to presence or absence of immunological manifestations, suggesting a role of unknown modifiers. Hence, it is currently not possible to correlate the variant position in the EXTL3 gene with disease severity.

Case Report: Recurrent Variant c.298 TA in CCN6 Gene Found in Progressive Pseudorheumatoid Dysplasia Patients From Patni Community of Gujarat: A Report of Three Cases.

Biallelic mutations in the CCN6 gene are known to cause a rare genetic disorder-progressive pseudorheumatoid dysplasia (PPD). PPD is characterized by distinct joint deformities of interphalangeal joints, stiffness, gait disturbance, abnormal posture, and absence of inflammation, resulting in significant morbidity. The largest case series of PPD from India suggests c.233G>A and c.1010G>A to be the most common mutations in the CCN6 gene, although the distribution of these variants among endogamous communities in India has not been carried out. We here report three cases of PPD from three independent families belonging to the Patni community of Gujarat, a community known to practice endogamy. All three cases had short stature, gait disturbance, scoliosis, and interphalangeal joint deformities. Analysis by whole-exome sequencing in the first case showed the presence of a previously known, homozygous, missense variant c.298T>A (p.Cys100Ser) in exon 3 of the CCN6 gene in all cases. Due to all three families belonging to the same community, analysis by Sanger sequencing in the remaining two cases for the variant mentioned earlier showed both cases to be of homozygous mutant genotype. Unaffected family members, i.e., parents and siblings, were either heterozygous carriers or wildtype for the said variant. The present case series is the first report of a recurrent variant occurring across multiple PPD-affected individuals from unrelated families belonging to the same community from India.

Treatment for Lysosomal Storage Disorders.

Lysosomal storage disorders comprise a group of approximately 70 types of inherited diseases resulting due to lysosomal gene defects. The outcome of the defect is a deficiency in either of the three: namely, lysosomal enzymes, activator protein, or transmembrane protein, as a result of which there is an unwanted accumulation of biomolecules inside the lysosomes. The pathophysiology of these conditions is complex affecting several organ systems and nervous system involvement in a majority of cases. Several research studies have well elucidated the mechanism underlying the disease condition leading to the development in devising the treatment strategies for the same. Currently, these approaches aim to reduce the severity of symptoms or delay the disease progression but do not provide a complete cure. The main treatment methods include Enzyme replacement therapy, Bone marrow transplantation, Substrate reduction therapy, use of molecular chaperones, and Gene therapy. This review article presents an elaborate description of these strategies and discusses the ongoing studies for the same.