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.

A novel case of two siblings harbouring homozygous variant in the NEUROG1 gene with autism as an additional phenotype: a case report.

INTRODUCTION: NEUROG1 gene is yet to be associated with a set of human phenotypes in the OMIM database. Three cases have previously been diagnosed with cranial dysinnervation due to biallelic variants in the NEUROG1 gene. This is the fourth and a novel report of a sibling pair harboring a homozygous variant in the NEUROG1 gene with autism as an additional phenotype. A brief review of the literature in conjunction with a genotype-phenotype correlation has been described. A potential hypothesis for the presence of the autistic phenotype in the present case has also been elucidated. CASE PRESENTATION: A female aged 6 years and 9 months born to endogamous and phenotypically healthy parents was diagnosed with global developmental delay, autism spectrum disorder, hearing loss, corneal opacity and no eye blinking. Her MRI of the brain revealed mild peritrigonal white matter hyperintensity, and MRI and CT scan of the temporal bones showed abnormal cranial nerves. The proband's younger sister, aged 4-years, was similarly affected. Whole exome sequencing was performed in the proband, which revealed a novel homozygous, likely pathogenic, truncating frameshift variant, c.228_231dup (p.Thr78ProfsTer122) in exon 1 of the NEUROG1 gene (ENST00000314744.4). Segregation analysis by Sanger sequencing showed the proband and her younger sister to be homozygotes and their parents to be heterozygous carriers. CONCLUSION: This is the fourth report across the globe with a variant identified in the NEUROG1 gene to be associated with cranial dysinnervation phenotype. An additional phenotype of autism in two female siblings was a novel observation. We provide a hypothetical framework which could explain the pleiotropic effect of a dysfunctional NEUROG1 protein leading to autism and posit it as a candidate for diagnosis of autism spectrum disorder with congenital cranial dysinnervation disorder.

Comparative yield of molecular diagnostic algorithms for autism spectrum disorder diagnosis in India: evidence supporting whole exome sequencing as first tier test.

BACKGROUND: Autism spectrum disorder (ASD) affects 1 in 100 children globally with a rapidly increasing prevalence. To the best of our knowledge, no data exists on the genetic architecture of ASD in India. This study aimed to identify the genetic architecture of ASD in India and to assess the use of whole exome sequencing (WES) as a first-tier test instead of chromosomal microarray (CMA) for genetic diagnosis. METHODS: Between 2020 and 2022, 101 patient-parent trios of Indian origin diagnosed with ASD according to the Diagnostic and Statistical Manual, 5th edition, were recruited. All probands underwent a sequential genetic testing pathway consisting of karyotyping, Fragile-X testing (in male probands only), CMA and WES. Candidate variant validation and parental segregation analysis was performed using orthogonal methods. RESULTS: Of 101 trios, no probands were identified with a gross chromosomal anomaly or Fragile-X. Three (2.9%) and 30 (29.7%) trios received a confirmed genetic diagnosis from CMA and WES, respectively. Amongst diagnosis from WES, SNVs were detected in 27 cases (90%) and CNVs in 3 cases (10%), including the 3 CNVs detected from CMA. Segregation analysis showed 66.6% (n = 3 for CNVs and n = 17 for SNVs) and 16.6% (n = 5) of the cases had de novo and recessive variants respectively, which is in concordance with the distribution of variant types and mode of inheritance observed in ASD patients of non-Hispanic white/ European ethnicity. MECP2 gene was the most recurrently mutated gene (n = 6; 20%) in the present cohort. Majority of the affected genes identified in the study cohort are involved in synaptic formation, transcription and its regulation, ubiquitination and chromatin remodeling. CONCLUSIONS: Our study suggests de novo variants as a major cause of ASD in the Indian population, with Rett syndrome as the most commonly detected disorder. Furthermore, we provide evidence of a significant difference in the diagnostic yield between CMA (3%) and WES (30%) which supports the implementation of WES as a first-tier test for genetic diagnosis of ASD in India.

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.

A rare case of a male child with post-zygotic de novo mosaic variant c.538C > T in MECP2 gene: a case report of Rett syndrome.

BACKGROUND: Rett syndrome (RTT) is characterized by a normal perinatal period with a normal head size at birth followed by normal development for the first 6 months of life followed by gradual deceleration of head growth, loss of acquired purposeful hand skills, severe expressive and receptive language impairment, severe intellectual disability and gait and truncal apraxia/ ataxia. It is caused due to mutations in the MECP2 gene and follows an X-linked dominant mode of inheritance. It was observed exclusively in females and was believed to be lethal in males. In contrast to this belief, several males were identified with RTT upon genetic analysis, however, most males expired by the age of 2 years due to neonatal encephalopathy. The ones that survived beyond the age of 2 years, were attributed to the presence of an extra X chromosome (co-occurrence of Klinefelter and RTT) or the ones having mosaic cell lines. Only 11 males with somatic mosaicism are known till date. CASE PRESENTATION: This case reports an ultra-rare case of a male affected with RTT surviving beyond the age of 2 years due to post-zygotic de novo somatic mosaicism. He was identified with a known pathogenic variant c.538C > T (p.R180*), which to the best of our knowledge is exclusively seen in females and has never been reported in a male before. CONCLUSION: The present case is the first report of a mosaic male affected with RTT from India. The present report also carried out genotype-phenotype correlations across surviving mosaic males with RTT. We also postulate the effect of variant type, position along the gene and the variant allele fraction in different tissue types to be correlated with disease severity.

Elucidation of the phenotypic spectrum and genetic landscape in primary and secondary microcephaly.

PURPOSE: Microcephaly is a sign of many genetic conditions but has been rarely systematically evaluated. We therefore comprehensively studied the clinical and genetic landscape of an unselected cohort of patients with microcephaly. METHODS: We performed clinical assessment, high-resolution chromosomal microarray analysis, exome sequencing, and functional studies in 62 patients (58% with primary microcephaly [PM], 27% with secondary microcephaly [SM], and 15% of unknown onset). RESULTS: We found severity of developmental delay/intellectual disability correlating with severity of microcephaly in PM, but not SM. We detected causative variants in 48.4% of patients and found divergent inheritance and variant pattern for PM (mainly recessive and likely gene-disrupting [LGD]) versus SM (all dominant de novo and evenly LGD or missense). While centrosome-related pathways were solely identified in PM, transcriptional regulation was the most frequently affected pathway in both SM and PM. Unexpectedly, we found causative variants in different mitochondria-related genes accounting for ~5% of patients, which emphasizes their role even in syndromic PM. Additionally, we delineated novel candidate genes involved in centrosome-related pathway (SPAG5, TEDC1), Wnt signaling (VPS26A, ZNRF3), and RNA trafficking (DDX1). CONCLUSION: Our findings enable improved evaluation and genetic counseling of PM and SM patients and further elucidate microcephaly pathways.

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.

Identification of novel variants in a large cohort of children with Tay-Sachs disease: An initiative of a multicentric task force on lysosomal storage disorders by Government of India.

Tay-Sachs disease (TSD) (OMIM) is a neurodegenerative lysosomal storage disorder caused due to mutations in the HEXA gene. To date, nearly 190 mutations have been reported in HEXA gene. Here, we have characterized 34 enzymatically confirmed TSD families to investigate the presence of novel as well as known variants in HEXA gene. Overall study detected 25 variants belonging to 31 affected TSD patients and 3 carrier couples confirmed by enzyme study. Of these 17 patients harbors 15 novel variants, including seven missense variants [p.V206L, p.Y213H, p.R252C, p.F257S, p.C328G, p.G454R, and p.P475R], four nonsense variant [p.S9X, p.E91X, p.W420X, and p.W482X], two splice site variants [c.347-1G>A and c.460-1G>A], and two small deletion [c.1349delC (p.A450VfsX3) and c.52delG (p.G18Dfs*82)]. While remaining 17 patients harbors 10 previously reported variants that includes six missense variants [p.M1T, p.R170Q, p.D322Y, p.D322N, p.E462V, and p.R499C], one nonsense variant [p.Q106X], two splice site variants [c.1073+1G>A and c.459+4A>G] and one 4 bp insertion [c.1278insTATC (p.Y427IfsX5)]. In conclusion, Indian infantile TSD patients provide newer insight into the molecular heterogeneity of the TSD. Combining present study and our earlier studies, we have observed that 67% genotypes found in Indian TSD patients are novel, which are associated with severe infantile phenotypes, while rest 33% genotypes found in our cohort were previously reported in various populations. In addition, higher frequency of the p.E462V and c.1278insTATC mutations in the present study further support and suggest the prevalence of p.E462V mutation in the Indian population.

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.

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.

Identification of deletion-duplication in HEXA gene in five children with Tay-Sachs disease from India.

BACKGROUND: Tay-Sachs disease (TSD) is a sphingolipid storage disorder caused by mutations in the HEXA gene. To date, nearly 170 mutations of HEXA have been described, including only one 7.6 kb large deletion. METHODS: Multiplex Ligation-dependent Probe Amplification (MLPA) study was carried out in 5 unrelated patients for copy number changes where heterozygous and/or homozygous disease causing mutation/s could not be identified in the coding region by sequencing of HEXA gene. RESULTS: The study has identified the presence of a homozygous deletion of exon-2 and exon-3 in two patients, two patient showed compound heterozygosity with exon 1 deletion combined with missense mutation p.E462V and one patient was identified with duplication of exon-1 with novel variants c.1527-2A > T as a second allele. CONCLUSION: This is the first report of deletion/duplication in HEXA gene providing a new insight into the molecular basis of TSD and use of MLPA assay for detecting large copy number changes in the HEXA gene.

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.

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.

Genetic and phenotypic dissection of 1q43q44 microdeletion syndrome and neurodevelopmental phenotypes associated with mutations in ZBTB18 and HNRNPU.

Subtelomeric 1q43q44 microdeletions cause a syndrome associating intellectual disability, microcephaly, seizures and anomalies of the corpus callosum. Despite several previous studies assessing genotype-phenotype correlations, the contribution of genes located in this region to the specific features of this syndrome remains uncertain. Among those, three genes, AKT3, HNRNPU and ZBTB18 are highly expressed in the brain and point mutations in these genes have been recently identified in children with neurodevelopmental phenotypes. In this study, we report the clinical and molecular data from 17 patients with 1q43q44 microdeletions, four with ZBTB18 mutations and seven with HNRNPU mutations, and review additional data from 37 previously published patients with 1q43q44 microdeletions. We compare clinical data of patients with 1q43q44 microdeletions with those of patients with point mutations in HNRNPU and ZBTB18 to assess the contribution of each gene as well as the possibility of epistasis between genes. Our study demonstrates that AKT3 haploinsufficiency is the main driver for microcephaly, whereas HNRNPU alteration mostly drives epilepsy and determines the degree of intellectual disability. ZBTB18 deletions or mutations are associated with variable corpus callosum anomalies with an incomplete penetrance. ZBTB18 may also contribute to microcephaly and HNRNPU to thin corpus callosum, but with a lower penetrance. Co-deletion of contiguous genes has additive effects. Our results confirm and refine the complex genotype-phenotype correlations existing in the 1qter microdeletion syndrome and define more precisely the neurodevelopmental phenotypes associated with genetic alterations of AKT3, ZBTB18 and HNRNPU in humans.

Pulmonary manifestations in Niemann-Pick type C disease with mutations in NPC2 gene: case report and review of literature.

BACKGROUND: Niemann-Pick disease type C (NPC) is an inherited metabolic disorder; due to defect in cellular cholesterol trafficking. It is clinically a heterogeneous disease with variable age of onset with multiple organ systems being involved. NPC1 gene is involved in 95% cases where as remaining ~5% cases are linked with NPC2 gene. CASE PRESENTATION: Case-1, a 14-months-old female presented with recurrent respiratory distress, failure to thrive and hepatosplenomegaly. Lung biopsy was suggestive of alveolar proteinosis and liver biopsy confirmed foamy macrophages. Molecular analysis revealed homozygous mutation c.141C > A in exon 2 of NPC2 gene. Case-2, a 3-year-old male presented with dyspnoea and hepatomegaly noticed at 1 year of age. HRCT-scan of thoracic region showed consolidation with mediastinal lymphadenopathy. Broncho-alveolar lavage revealed moderate amount of foamy macrophages and bone marrow examination detected foam cells. Homozygous T > C transition in intron 1 of the NPC2 gene was identified. CONCLUSION: Our study demonstrates that NPC2 can present in early years of life with pulmonary complications like alveolar proteinosis and hepatosplenomegaly or hepatomegaly due to mutation in NPC2 gene. An early suspicion will help clinicians to clinch its diagnosis, management and genetic counselling.

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.

Does vitamin D play a significant role in type 2 diabetes?

BACKGROUND: Vitamin D deficiency reportedly is associated with type 2 diabetes (T2DM). We aim to examine whether 25-hydroxyvitamin D (25OHD) has clinically significant influence on hemoglobin glycation (HbA1c) and insulin resistance (HOMA-IR) in T2DM subjects. METHODS: Present study was carried out in 912 subjects (429 T2DM cases and 483 non-diabetic controls) from Western India. The enrolled study subjects were investigated for biochemical parameters like FBS, PPBS, HbA1c, FI, HOMA-IR and 25OHD levels in blood. RESULTS: Vitamin D deficiency was seen in 91.4% and 93.0% of T2DM cases and control subjects respectively. There was no association of serum 25OHD deficiency on HbA1c or HOMA-IR in T2DM cases (p = 0.057 & p = 0.257 respectively) and in control subjects (p = 0.675 & p = 0.647 respectively). CONCLUSION: Our findings suggests that though vitamin D deficiency is prevalent in T2DM and non-diabetic subjects, its role in hemoglobin glycation and insulin resistance could not be established.