A new point mutation (G412 to A) at the last nucleotide of exon 3 of hexosaminidase α-subunit gene affects splicing

doi:10.1016/S0387-7604(02)00219-X

Brain and Development

Volume 25, Issue 3, April 2003, Pages 203-206

https://doi.org/10.1016/S0387-7604(02)00219-X Get rights and content

Abstract

We report the sixth mutation associated with the infantile form of Tay-Sachs disease in the Turkish population. The mutation is a single nucleotide transition (G to A) at the last nucleotide of exon 3 of hexosaminidase A (HEX A) α-subunit gene. The 14 exons and their flanking sequences of the HEX A gene were amplified and analyzed by polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP). Sequencing of exon 3 showed a homozygous mutation. Cultured patient’s fibroblasts produced no detectable mRNA for HEX A α-subunit gene by Northern blot analysis. We speculate that abnormal mRNA was rapidly degraded following transcription. Our data are consistent with the idea that the severe infantile form of Tay-Sachs disease is associated with a total lack of Hex A activity in the patient. A similar mutation (G to T) had been observed at the 5′-donor splice site of exon 3. It resulted in abnormal splicing and skipping of exon 3. The other acceptor and donor splice site mutations described in the HEX A gene ablate normal mRNA splicing. Identification of multiple mutant HEX A alleles shows molecular heterogeneity of infantile Tay-Sachs disease in our population.

Introduction

Tay-Sachs disease is an autosomal recessive sphingolipid storage disease due to a deficiency of the hexosaminidase A (hex A) enzyme. It is a catastrophic disorder and is characterized by accumulation of GM2 ganglioside in the neurons of the cerebral cortex. Clinically, the result is a spectrum of disease states ranging from severe and fatal to mild and chronic. Affected infants do not attain early milestones such as sitting or crawling and lose most cognitive functions by the second year. Mutations at the α-subunit gene of the lysosomal enzyme hex A are responsible for the enzyme deficiency and Tay-Sachs disease [1]. To date 20 splice site mutations out of 87 mutations have been reviewed at the HEX A gene [2]. Previously, five Tay-Sachs mutations that cause infantile type of the disease have been detected in the Turkish population: two point mutations that yield stop codons, one splice site and one deletion mutations [3], [4]. Three of these five mutations are unique to the Turkish population.

In the present study, we report a new mutation at the last nucleotide of exon 3 of hex α-chain gene in a Turkish infant with infantile type of Tay-Sachs disease and its effect on gene expression.

Section snippets

Clinical summary of the patients

Two patients with infantile Tay-Sachs disease were included in this study. The mutation of patient 1 was not detected by single stranded conformational polymorphism (SSCP) analysis.

Identification of the mutation

PCR amplification products of all HEX A exons and flanking sequences were subjected to SSCP analysis. All amplification products produced a pattern of bands that was identical for normal control DNA except for band patterns of exon 3-containing fragments (Fig. 1).

Detection of the mutation in genomic DNA

DNA sequence analysis of exon 3 revealed a transition mutation G412 to A at the last nucleotide of exon 3 (Fig. 2). The patient was homozygote for the mutation.

The effect of the G to A transition mutation at the splice site of exon 3

Discussion

A novel HEX A mutation has been identified in a Turkish infant with acute infantile type Tay-Sachs disease. The exon 3 donor splice site mutation is the second splice site and sixth mutation found in the Turkish population. The other splice site mutation was at the IVS5+1 position of the HEX A gene. In this work, our data indicated that this novel mutation results in a lack of HEX A mRNA by defective splicing. The lack of detectable α-subunit is most likely because of the instability of the

Acknowledgements

This work was supported by Scientific and Technical Research Council of Turkey (TUBITAK) and Deutsche Forschung Gemeinschaft. We thank Dr R. Proia for providing the cDNA clones of human Hex, and Dr Christina Schütte and Dr Julia Bär for their technical assistance.

References (8)

D.J Mahuran
Biochemical consequences of mutations causing the GM2 gangliosidoses
Biochim Biophys Acta
(1999)
H.A Özkara et al.
At least six different mutations in Hex A gene cause Tay-Sachs disease among the Turkish population
Mol Genet Metab
(1998)
R.A Gravel et al.
The GM2 gangliosidoses
H.A Özkara et al.
Donor splice site mutation in intron 5 of the Hex A gene in a Turkish infant with Tay-Sachs disease
Hum Mutat
(1995)

There are more references available in the full text version of this article.

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Original articleA new point mutation (G412 to A) at the last nucleotide of exon 3 of hexosaminidase α-subunit gene affects splicing

Abstract

Introduction

Section snippets

Clinical summary of the patients

Identification of the mutation

Detection of the mutation in genomic DNA

Discussion

Acknowledgements

Biochim Biophys Acta

Mol Genet Metab

The GM2 gangliosidoses

Donor splice site mutation in intron 5 of the Hex A gene in a Turkish infant with Tay-Sachs disease

Hum Mutat

Original article
A new point mutation (G412 to A) at the last nucleotide of exon 3 of hexosaminidase α-subunit gene affects splicing