Expanding the spectrum of HEXA mutations in Indian patients with Tay-Sachs disease

Abstract

Tay-Sachs disease is an autosomal recessive neurodegenerative disorder occurring due to impaired activity of β-hexosaminidase-A (EC 3.2.1.52), resulting from the mutation in HEXA gene. Very little is known about the molecular pathology of TSD in Indian children except for a few mutations identified by us. The present study is aimed to determine additional mutations leading to Tay-Sachs disease in nine patients confirmed by the deficiency of β-hexosaminidase-A (< 2% of total hexosaminidase activity for infantile patients) in leucocytes. The enzyme activity was assessed by using substrates 4-methylumbelliferyl-N-acetyl-β-d-glucosamine and 4-methylumbelliferyl-N-acetyl-β-d-glucosamine-6-sulfate for total-hexosaminidase and hexosaminidase-A respectively, and heat inactivation method for carrier detection. The exons and exon-intron boundaries of the HEXA gene were bi-directionally sequenced on an automated sequencer. 'In silico' analyses for novel mutations were carried out using SIFT, Polyphen2 and MutationT@ster software programs. The structural study was carried out by UCSF Chimera software using the crystallographic structure of β-hexosaminidase-A (PDB-ID: 2GJX) as the template. Our study identified four novel mutations in three cases. These include a compound heterozygous missense mutation c.524A>C (D175A) and c.805G>C (p.G269R) in one case; and one small 1 bp deletion c.426delT (p.F142LfsX57) and one splice site mutation c.459+4A>C in the other two cases respectively. None of these mutations were detected in 100 chromosomes from healthy individuals of the same ethnic group. Three previously reported missense mutations, (i) c.532C>T (p.R178C), (ii) c.964G>T (p.D322Y), and (iii) c.1385A>T (p.E462V); two nonsense mutations (i) c.709C>T (p.Q237X) and (ii) c.1528C>T (p.R510X), one 4 bp insertion c.1277_1278insTATC (p.Y427IfsX5) and one splice site mutation c.459+5G>A were also identified in six cases. We observe from this study that novel mutations are more frequently observed in Indian patients with Tay-Sachs disease with clustering of ~ 73% of disease causing mutations in exons 5 to 12. This database can be used for a carrier rate screening in the larger population of the country.

Keywords: HEXA gene; Hex-A, β-Hexosaminidase A; LSDs, Lysosomal storage disorders; Lysosomal enzyme; MUG, 4-Methylumbelliferyl-N-acetyl-β-d-glucosamine; MUGS, 4-Methylumbelliferyl-N-acetyl-β-d-glucosamine-6-sulfate; SD, Sandhoff disease; TSD, Tay–Sachs disease; Tay–Sachs disease; β-Hexosaminidase-A.

Fig. 1

Sequence chromatogram of HEXA gene mutations. (a): c.426delT (p.F142LFsX57) (homozygous); (b): c.524A>C (p.D175A) (heterozygous); (c): c.532C>T (p.R178C) (heterozygous); (d): c.709C>T (p.Q237X) (heterozygous); (e): c.805G>C (p.G269R) (heterozygous); (f): c.964G>T (p.D322Y) (homozygous); (g): c.1385A>T (p.E462V) (heterozygous); (h): c.1528C>T (p.R510X); (i): c.459+5G>A (homozygous); (j): c.459+4A>C (heterozygous); (k): c.1277_1278insTATC (p.Y47IfsX5) (homozygous).

Fig. 2

Superimposed native structures (brown) and mutant structure (blue) produced using USCF Chimera software: (a) mutation p.D175A disrupts the β-sheet and (b) p.G269R creates overpacking and backbone disruption.