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.

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.

Assessing Utility of Clinical Exome Sequencing in Diagnosis of Rare Idiopathic Neurodevelopmental Disorders in Indian Population.

BACKGROUND: Neurological diseases are phenotypically and genotypically heterogeneous. Clinical exome sequencing (CES) has been shown to provide a high diagnostic yield for these disorders in the European population but remains to be demonstrated for the Indian population. OBJECTIVE: The study aimed to understand the utility of clinical exome sequencing for the diagnosis of neurodevelopmental disorders. MATERIALS AND METHODS: A cohort of 19 idiopathic patients with neurological phenotypes, primarily intellectual disability and developmental delay, were recruited. CES covering 4620 genes was performed on all patients. Candidate variants were validated by Sanger sequencing. RESULTS: CES in 19 patients provided identified 21 variants across 16 genes which have been associated with different neurological disorders. Fifteen variants were reported previously and 6 variants were novel to our study. Eleven patients were diagnosed with autosomal dominant de novo variants, 7 with autosomal recessive and 1 with X-linked recessive variants. CES provided definitive diagnosis to 10 patients; hence, the diagnostic yield was 53%. CONCLUSION: Our study suggests that the diagnostic yield of CES in the Indian population is comparable to that reported in the European population. CES together with deep phenotyping could be a cost-effective way of diagnosing rare neurological disorders in the Indian population.

Identification and characterization of 30 novel pathogenic variations in 69 unrelated Indian patients with Mucolipidosis Type II and Type III.

Mucolipidosis (ML) (OMIM 607840 & 607838) is a rare autosomal recessive inherited disorder that occurs due to the deficiency of golgi enzyme uridine diphosphate (UDP)- N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) responsible for tagging mannose-6-phosphate for proper trafficking of lysosomal enzymes to lysosomes. Variants in GlcNAc-phosphotransferase (GNPTAB (α, ß subunits) and GNPTG (γ subunits) are known to result in impaired targeting of lysosomal enzymes leading to Mucolipidosis (ML) Type II or Type III. We analyzed 69 Indian families of MLII/III for clinical features and molecular spectrum and performed in silico analysis for novel variants. We identified 38 pathogenic variants in GNPTAB and 5 pathogenic variants in GNPTG genes including missense, frame shift, deletion, duplication and splice site variations. A total of 26 novel variants were identified in GNPTAB and 4 in GNPTG gene. In silico studies using mutation prediction software like SIFT, Polyphen2 and protein structure analysis further confirmed the pathogenic nature of the novel sequence variants detected in our study. Except for a common variant c.3503_3504delTC in early onset MLII, we could not establish any other significant genotype and phenotype correlation. This is one of the largest studies reported till date on Mucolipidosis II/III in order to identify mutation spectrum and any recurrent mutations specific to the Indian ethnic population. The mutational spectrum information in Indian patients will be useful in better genetic counselling, carrier detection and prenatal diagnosis for patients with ML II/III.

Rare cause of Hemophagocytic Lymphohistiocytosis due to mutation in PRF1 and SH2D1A genes in two children - a case report with a review.

BACKGROUND: Hemophagocytic Lymphohistiocytosis (HLH) is a rare, complex, life-threatening hyper-inflammatory condition due to over activation of lymphocytes mediated secretory cytokines in the body. It occurs as a primary HLH due to genetic defect that mostly occurs in the childhood and associated with early neonatal death. Secondary HLH is triggered by secondary to infection and can occur at any age. CASE PRESENTATION: The current report presents two cases of HLH. Case 1, three-months-old boy born to second degree consanguineous parents was clinically suspected with HLH. A pathogenic variant in exon 2 of PRF1 gene [c.386G > C (p.Trp129Ser); FLH-type2] was detected. The parents and the fetus under investigation were shown to be heterozygous carriers, while Case-1 was homozygous for the said variant. Case 2, a one and half-year old male child referred for work-up was born to non-consanguineous young parents. His HLH suspicion was in accordance with HLH-2004 Revised diagnostic guidelines (fulfilling 5/8 criteria). Molecular study revealed hemizygous likely pathogenic variant c.138-3C > G in intron 1 of SH2D1A gene. Both the mother and younger sister were confirmed to be the carrier of the same variant. CONCLUSION: This study has represented two rare cases of HLH carrying missense variant in PRF1 and splice site variant in SH2D1A gene. Detailed molecular analysis has helped the families with precise genetic counselling and prenatal diagnosis during subsequent pregnancy. It is advocated that male patients presenting with EBV-associated HLH may be screened for XLP that may lead to early diagnosis and therapeutic implication if any.

Gaucher disease: single gene molecular characterization of one-hundred Indian patients reveals novel variants and the most prevalent mutation.

BACKGROUND: Gaucher disease is a rare pan-ethnic, lysosomal storage disorder resulting due to beta-Glucosidase (GBA1) gene defect. This leads to the glucocerebrosidase enzyme deficiency and an increased accumulation of undegraded glycolipid glucocerebroside inside the cells' lysosomes. To date, nearly 460 mutations have been described in the GBA1 gene. With the aim to determine mutations spectrum and molecular pathology of Gaucher disease in India, the present study investigated one hundred unrelated patients (age range: 1 day to 31 years) having splenomegaly, with or without hepatomegaly, cytopenia and bone abnormality in some of the patients. METHODS: The biochemical investigation for the plasma chitotriosidase enzyme activity and ß-Glucosidase enzyme activity confirmed the Gaucher disease. The mutations were identified by screening the patients' whole GBA gene coding region using bidirectional Sanger sequencing. RESULTS: The biochemical analysis revealed a significant reduction in the ß-Glucosidase activity in all patients. Sanger sequencing established 71 patients with homozygous mutation and 22 patients with compound heterozygous mutation in GBA1 gene. Lack of identification of mutations in three patients suggests the possibility of either large deletion/duplication or deep intronic variations in the GBA1 gene. In four cases, where the proband died due to confirmed Gaucher disease, the parents were found to be a carrier. Overall, the study identified 33 mutations in 100 patients that also covers four missense mutations (p.Ser136Leu, p.Leu279Val, p.Gly383Asp, p.Gly399Arg) not previously reported in Gaucher disease patients. The mutation p.Leu483Pro was identified as the most commonly occurring Gaucher disease mutation in the study (62% patients). The second common mutations identified were p.Arg535Cys (7% patients) and RecNcil (7% patients). Another complex mutation Complex C was identified in a compound heterozygous status (3% patients). The homology modeling of the novel mutations suggested the destabilization of the GBA protein structure due to conformational changes. CONCLUSIONS: The study reports four novel and 29 known mutations identified in the GBA1 gene in one-hundred Gaucher patients. The given study establishes p.Leu483Pro as the most prevalent mutation in the Indian patients with type 1 Gaucher disease that provide new insight into the molecular basis of Gaucher Disease in India.

Batten disease: biochemical and molecular characterization revealing novel PPT1 and TPP1 gene mutations in Indian patients.

BACKGROUND: Neuronal ceroid lipofuscinoses type I and type II (NCL1 and NCL2) also known as Batten disease are the commonly observed neurodegenerative lysosomal storage disorder caused by mutations in the PPT1 and TPP1 genes respectively. Till date, nearly 76 mutations in PPT1 and approximately 140 mutations, including large deletion/duplications, in TPP1 genes have been reported in the literature. The present study includes 34 unrelated Indian patients (12 females and 22 males) having epilepsy, visual impairment, cerebral atrophy, and cerebellar atrophy. METHODS: The biochemical investigation involved measuring the palmitoyl protein thioesterase 1 and tripeptidy peptidase l enzyme activity from the leukocytes. Based on the biochemical analysis all patients were screened for variations in either PPT1 gene or TPP1 gene using bidirectional Sanger sequencing. In cases where Sanger sequencing results was uninformative Multiplex Ligation-dependent Probe Amplification technique was employed. The online tools performed the protein homology modeling and orthologous conservation of the novel variants. RESULTS: Out of 34 patients analyzed, the biochemical assay confirmed 12 patients with NCL1 and 22 patients with NCL2. Molecular analysis of PPT1 gene in NCL1 patients revealed three known mutations (p.Val181Met, p.Asn110Ser, and p.Trp186Ter) and four novel variants (p.Glu178Asnfs*13, p.Pro238Leu, p.Cys45Arg, and p.Val236Gly). In the case of NCL2 patients, the TPP1 gene analysis identified seven known mutations and eight novel variants. Overall these 15 variants comprised seven missense variants (p.Met345Leu, p.Arg339Trp, p.Arg339Gln, p.Arg206Cys, p.Asn286Ser, p.Arg152Ser, p.Tyr459Ser), four frameshift variants (p.Ser62Argfs*19, p.Ser153Profs*19, p.Phe230Serfs*28, p.Ile484Aspfs*7), three nonsense variants (p.Phe516*, p.Arg208*, p.Tyr157*) and one intronic variant (g.2023_2024insT). No large deletion/duplication was identified in three NCL1 patients where Sanger sequencing study was normal. CONCLUSION: The given study reports 34 patients with Batten disease. In addition, the study contributes four novel variants to the spectrum of PPT1 gene mutations and eight novel variants to the TPP1 gene mutation data. The novel pathogenic variant p.Pro238Leu occurred most commonly in the NCL1 cohort while the occurrence of a known pathogenic mutation p.Arg206Cys dominated in the NCL2 cohort. This study provides an insight into the molecular pathology of NCL1 and NCL2 disease for Indian origin patients.

Biochemical and molecular characterization of adult patients with type I Gaucher disease and carrier frequency analysis of Leu444Pro - a common Gaucher disease mutation in India.

BACKGROUND: Gaucher disease is a rare pan-ethnic disorder which occurs due to an increased accumulation of undegraded glycolipid glucocerebroside inside the cells' lysosomes. A beta-Glucosidase (GBA) gene defect results in glucocerebrosidase enzyme deficiency. Though the disease is mainly diagnosed in childhood, the adult manifestation is often missed or identified late due to the failure to recognize the heterogeneous clinical presentation. The present study includes seven unrelated Indian adult patients (age range: 20-40 years) having splenomegaly, with or without hepatomegaly, cytopenia and bone abnormality. METHODS: The biochemical investigation implicated measuring plasma chitotriosidase enzyme activity followed by confirmatory test of ß-Glucosidase enzyme activity from the leukocytes. The molecular characterization involved patients' initial screening for the common Gaucher mutation (Leu444Pro). Later, all patients were subjected to whole GBA gene coding region study using bidirectional Sanger sequencing. The population screening for common Gaucher disease mutation (Leu444Pro) was executed in 1200 unrelated and healthy Indian subjects by Restriction Fragment Length Polymorphism-Polymerase Chain Reaction technique. The allele frequency was calculated using Hardy-Weinberg formula. RESULTS: The biochemical analysis revealed a significant reduction in the ß-Glucosidase activity in all the patients. Also, an elevated level of plasma Chitotriosidase activity in five patients supported their diagnosis of Gaucher disease. Sanger sequencing established four patients with homozygous variation and three patients with compound heterozygous variation in GBA gene. This study uncovers two missense variants (Ala448Thr and Val17Gly) not previously reported in Gaucher disease patients. Also the known mutations like Leu444Pro, Arg329Cys, Asp315Asn, Ser125Arg, and Arg395Cys were identified in these patients. The homology modeling suggested the destabilization of the protein structure due to novel variants. The Leu444Pro mutation screening in the Indian population spotted two people as a carrier. This emerged the carrier frequency of 1:600 along with wild-type allele frequency 0.97113 and mutant allele frequency 0.02887. CONCLUSIONS: The study reports novel and known variants identified in the GBA gene in seven adult patients. The given study is the first report on the carrier frequency of the Leu444Pro mutant allele in an Indian population which will help understanding the burden and susceptibility of Gaucher disease to affect next generation in India.

A case of Raine syndrome presenting with facial dysmorphy and review of literature.

BACKGROUND: Raine syndrome (RS) - an extremely rare autosomal recessive genetic disorder, is caused by a biallelic mutation in the FAM20C gene. Some of the most common clinical features include generalized osteosclerosis with a periosteal bone formation, dysmorphic face, and thoracic hypoplasia. Many cases have also been reported with oro-dental abnormalities, and developmental delay. Most of the cases result in neonatal death. However, a few non-lethal RS cases have been reported where patients survive till adulthood and exhibits a heterogeneous clinical phenotype. Clinical diagnosis of RS has been done through facial appearance and radiological findings, while confirmatory diagnosis has been conducted through a molecular study of the FAM20C gene. CASE PRESENTATION: A 6-year-old girl was born to healthy third degree consanguineous parents. She presented with facial dysmorphy, delayed speech, and delayed cognition. Radiography showed small sclerotic areas in the lower part of the right femur, and an abnormally-shaped skull with minimal sclerosis in the lower occipital region. Computer tomography scan of the brain revealed mild cortical atrophy, and MRI scan of the brain showed corpus callosal dysgenesis with the absence of the rostral area. Chromosome banding at 500 band resolution showed a normal female karyotype. No quantitative genomic imbalance was detected by aCGH. Further study conducted using Clinical Exome Sequencing identified a homozygous missense variation c.1228 T > A (p.Ser410Thr) in the exon 6 of FAM20C gene - a likely pathogenic variant that confirmed the clinical diagnosis of RS. The variant was confirmed in the proband and her parents using Sanger sequencing. Prenatal diagnosis during subsequent pregnancy revealed heterozygous status of the fetus, and a normal carrier child was delivered at term. CONCLUSIONS: The syndrome revealed markedly variable presentations such as facial dysmorphy and developmental delay, and was localized to diffuse bone osteosclerosis. Clinical indications, striking radiological findings and molecular testing of FAM20C gene confirmed the diagnosis of RS. A rarity of the disorder and inconsistent phenotype hindered the establishment of genotype-phenotype correlations in RS. Therefore, reporting more cases and conducting further research would be crucial in defining the variable radiologic and molecular defects of the lethal and non-lethal forms of this syndrome.

Harlequin ichthyosis due to novel splice site mutation in the ABCA12 gene: postnatal to prenatal diagnosis.

BACKGROUND: Harlequin ichthyosis (HI) is a severe genetic disorder caused by the mutation in the ABCA12 gene. Infants born with this condition have markedly thickened, hard stratum corneum skin all over the body. METHODS: A female child born with a thick white plate of skin with deep cracks all over the body was investigated for genes associated with congenital Ichthyosis by Next Generation sequencing. The variant relevant to the clinical indications was identified using Picard and GATK version 3.6. Variant's pathogenicity was predicted by "in silico" tools like Mutation Taster 2, Mutation Assessor and LRT. Bidirectional Sanger sequencing further validated the same variant detected in the proband and confirmed in the parental blood and CVS. RESULTS: A homozygous 5' splice site variation that affects the position at 4 nucleotides downstream to the donor proximal splice site of intron 40 (c.5939+4A>G; ENST00000272895) of the ABCA12 gene was detected in the proband, and the parents were heterozygous for the same variant. This led to the confirmation of diagnosis of Harlequin ichthyosis in the proband. "In silico" prediction of the variant was found to be damaging by MutationTaster2. The CVS sample during subsequent pregnancy was confirmed to be heterozygous for the same variant. CONCLUSIONS: The novel intronic mutation found in the proband confirmed the clinical diagnosis as a severe type of HI and has helped the family in providing precise genetic counseling for further prevention of the disease and carrier screening of other family members.

Novel LINS1 missense mutation in a family with non-syndromic intellectual disability.

Newer sequencing technologies decipher molecular variations and increase the knowledge of pathogenesis of complex diseases like intellectual disability (ID), affecting 2-3% of the population. We report a novel family with a missense mutation in LINS1 as a cause for non-syndromic ID. Clinical exome sequencing for ID related genes carried out for a male with dysmorphism, mutism, and cognitive delay was uninformative. Subsequently, "pathogenic" and "likely pathogenic" variants associated with other inherited disorders were searched for as secondary findings. Further, PCR-RFLP carried out in other family members confirmed the result. A novel missense variant (c.937G>A) in exon 5 of LINS1 was detected in the proband. His affected elder brother was homozygous and the parents were heterozygous respectively, for the mutation. No mutation was observed in his unaffected sister. Mutations in LINS1 were suspected in this non-syndromic ID case with mutism. LINS1 alterations affect ELAV1 expression and result in reduction in the commissural axonal growth, thus affecting peripheral and central neuronal function. LINS1 acts in association with ß-catenin to influence WNT1 signaling. It is hypothesized that mutations in LINS1 may alter HuR expression during neural differentiation, leading to ID in humans. © 2017 Wiley Periodicals, Inc.

GM2 gangliosidosis AB variant: novel mutation from India - a case report with a review.

BACKGROUND: GM2 gangliosidosis-AB variants a rare autosomal recessive neurodegenerative disorder occurring due to deficiency of GM2 activator protein resulting from the mutation in GM2A gene. Only seven mutations in nine cases have been reported from different population except India. CASE PRESENTATION: Present case is a one year old male born to 3rd degree consanguineous Indian parents from Maharashtra. He was presented with global developmental delay, hypotonia and sensitive to hyperacusis. Horizontal nystagmus and cherry red spot was detected during ophthalmic examination. MRI of brain revealed putaminal hyperintensity and thalamic hypointensity with some unmyelinated white matter in T2/T1 weighted images. Initially he was suspected having Tay-Sachs disease and finally diagnosed as GM2 gangliosidosis, AB variant due to truncated protein caused by nonsense mutation c.472 G > T (p.E158X) in GM2Agene. CONCLUSION: Children with phenotypic presentation as GM2 gangliosidosis (Tay-Sachs or Sandhoff disease) and normal enzyme activity of ß-hexosaminidase-A and -B in leucocytes need to be investigated for GM2 activator protein deficiency.

Lysosomal Storage Disorders in Indian Children with Neuroregression Attending a Genetic Center.

OBJECTIVE: To study the etiology of neuroregression in children having deficiency of the lysosomal enzymes. DESIGN: Review of medical records. SETTING: Specialized Genetic Center. PARTICIPANTS: 432 children aged 3 mo-18 y having regression in a learned skill, selected from 1453 patients referred for diagnostic workup of various Lysosomal storage disorders (LSDs). METHODS: Plasma chitotriosidase, quantitative and qualitative glycosaminoglycans, and mucolipidosis-II/II screening followed by confirmatory enzyme study using specific substrate was carried out; Niemann-Pick disease Type-C was studied by fillipin stain method on skin fibroblasts. RESULTS: Total 309 children (71.5%) were diagnosed with different lysosomal storage disorders as the underlying cause of neuroregression. Plasma chitotriosidase was raised in 82 of 135; 64 (78%) of these had various LSDs. 69 out of 90 cases showed high excretion of glycoaminoglycans, and 67 (97.1%) of these were confirmed to have enzyme deficiency for various mucoplysaccharide disorders. While 3 of 90 children with positive Icell screening had confirmed mucolipidosis II/III disease. Among all, glycolipid storage disorders were the most common (50.2%) followed by mucopolysaccharidosis (MPS) (21.7%) and sulphatide degradation defect (17.5%). Neuronal ceroid lipofucinosis1 and 2 (7.4%), mucolipidosis-II/III (1%), Sialic acid storage disorder (1%), Niemann-Pick disease type-C (1%) and Fucosidosis (0.3%) were observed with less frequency. Most common phenotypes in all subjects were cherry red spot (18.5%), hepatosplenomegaly (17.9%), coarse facies (15%), seizures (13.1%) and skeletal abnormalities (12.14%). CONCLUSIONS: Lysosomal storage disorders are considered to be one of the common causes in children with regression in learned skill, dysmorphic features and cherry red spot. Among these, glycolipid storage disorders are the most common, followed by mucopolysaccharidosis.