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.

Novel mutations in the glucocerebrosidase gene of Indian patients with Gaucher disease.

Gaucher disease (GD) is the most common glycolipid storage disorder resulting from glucocerebrosidase deficiency due to mutations in the GBA gene. Study was performed in 33 unrelated patients with low ß-glucosidase activity in leukocytes and/or fibroblasts. The exons and exon-intron boundaries of the GBA gene were bidirectionally sequenced using an automated sequencer. Mutations were confirmed in parents and were looked up in public databases, and in silico analysis was carried for novel mutations. We identified two novel missense mutations G289A (c.866G>C) and I466S (c.1397T>G) in exons 7 and 10, respectively, in two (6.06%) patients that destabilize the protein structure. L444P (c.1448T>C) was the most common mutation identified in 20/33 (60.60%) non-neuronopathic and 1/33 (3.03%) sub-acute neuronopathic form based on clinical presentation at the time of investigation. Other nine rare mutations were: R463C (c.1504C>T), R395C (c.1300C>T), R359Q (c.1193G>A), G355D (c.1181G>A), V352M (c.1171G>A) and S356F (c.1184C>T) found in each patient (18.18%). Compound heterozygous mutation L444P (c.1448T>C)/R496C (c.1603C>T) in exon 10/11 and L444P (c.1448T>C)/R329C (c.1102C>T) were observed in exon 10/8 in one each patient (6.06%). Two patients (6.06%) from Sri Lanka showed E326K (c.1093G>A) mutation in exon 8. We conclude that L444P is the most common mutant allele with exons 8 and 10 as the hot spot region of GBA gene observed in Indian GD patients.

Burden of lysosomal storage disorders in India: experience of 387 affected children from a single diagnostic facility.

Lysosomal storage disorders (LSDs) are considered to be a rare metabolic disease for the national health forum, clinicians, and scientists. This study aimed to know the prevalence of different LSDs, their geographical variation, and burden on the society. It included 1,110 children from January 2002 to December 2012, having coarse facial features, hepatomegaly or hepatosplenomegaly, skeletal dysplasia, neuroregression, leukodystrophy, developmental delay, cerebral-cerebellar atrophy, and abnormal ophthalmic findings. All subjects were screened for I-cell disease, glycolipid storage disorders (Niemann-Pick disease A/B, Gaucher), and mucopolysaccharide disorders followed by confirmatory lysosomal enzymes study from leucocytes and/or fibroblasts. Niemann-Pick disease-C (NPC) was confirmed by fibroblasts study using filipin stain. Various storage disorders were detected in 387 children (34.8 %) with highest prevalence of glycolipid storage disorders in 48 %, followed by mucopolysaccharide disorders in 22 % and defective sulfatide degradation in 14 % of the children. Less common defects were glycogen degradation defect and protein degradation defect in 5 % each, lysosomal trafficking protein defect in 4 %, and transport defect in 3 % of the patients. This study demonstrates higher incidence of Gaucher disease (16 %) followed by GM2 gangliosidosis that includes Tay-Sachs disease (10 %) and Sandhoff disease (7.8 %) and mucopolysaccharide disorders among all LSDs. Nearly 30 % of the affected children were born to consanguineous parents and this was higher (72 %) in children with Batten disease. Our study also demonstrates two common mutations c.1277_1278insTATC in 14.28 % (4/28) and c.964G>T (p.D322Y) in 10.7 % (3/28) for Tay-Sachs disease in addition to the earlier reported c.1385A>T (p.E462V) mutation in 21.42 % (6/28).

Identification of Novel Mutations in FAH Gene and Prenatal Diagnosis of Tyrosinemia in Indian Family.

Carrier of tyrosinemia type I was diagnosed by sequencing FAH (fumarylacetoacetate hydrolase) gene. It leads to the identification of heterozygous status for both c.648C>G (p.Ile216Met) and c.1159G>A (p.Gly387Arg) mutations in exons 8 and 13, respectively, in the parents. The experimental program PolyPhen, SIFT, and MT predicts former missense point mutation as "benign" that creates a potential donor splice site and later one as "probably damaging" which disrupts secondary structure of protein.