Assessing the severity of the small inframe deletion mutation in the α‐subunit of β‐hexosaminidase A found in the Turkish population by reproducing it in the more stable β‐subunit

Abstract

SummaryGM 2 gangliosidoses are a group of panethnic lysosomal storage diseases in which GM 2 ganglioside accumulates in the lysosome due to a defect in one of three genes, two of which encode the α-or β-subunits of β-N-acetylhexosaminidase (Hex) A. A small inframe deletion mutation in the catalytic domain of the α-subunit of Hex has been found in five Turkish patients with infantile Tay-Sachs disease. To date it has not been detected in other populations and is the only mutation to be found in exon 10. It results in detectable levels of inactive α-protein in its precursor form. Because the α-and β-subunits share 60% sequence identity, the Hex A and Hex B genes are believed to have arisen from a common ancestral gene. Thus the subunits must share very similar three-dimensional structures with conserved functional domains. Hex B, the β-subunit homodimer is more stable than the heterodimeric Hex A, and much more stable than Hex S, the α homodimer. Thus, mutations that completely destabilize the α-subunit can often be partially rescued if expressed in the aligned positions in the β-subunit. To better understand the severity of the Turkish HEXA mutation, we reproduced the 12 bp deletion mutation (1267-1278) in the β-subunit cDNA. Western blot analysis of permanently transfected CHO cells expressing the mutant detected only the pro-form of the β-subunit coupled with a total lack of detectable Hex B activity. These data indicate that the deletion of the four amino acids severely affects the folding of even the more stable β-subunit, causing its retention in the endoplasmic reticulum and ultimate degradation.

Comparison of HCMV IE and EF-1 Promoters for the Stable Expression of β-Subunit of Hexosaminidase in CHO Cell Lines

“…The same lack of detectable β-protein was seen in the pcDNA3.1D/V5-His-TOPO transfected cells. However, extracts from pEFneo-transfected cells contained both a strong pro β-chain band and the processed mature β-chain band, consistent with a high level of β-cDNA expression in these cells (Sinici et al, 2004).…”

“…To lower the PCR mutation frequency, KOD High Fidelity (HiFi) polymerase (Novagen, Japan), which has a 3′ →5′ exonuclease activity, was used. PCR was performed in a total volume of 50 μL as previously described (Sinici et al, 2004). PCR products were separated on a 1% agarose gel and purified by QIAquick gel extraction kit (Qiagen, Canada).…”

“…Western Blot analyses were performed as previously described (Sinici et al, 2004) and protein bands visualized according to the protocol recommended for the Amersham ECL system (Amersham).…”

Molecular analysis of theHEXAgene in Italian patients with infantile and late Onset Tay-Sachs disease: detection of fourteen novel alleles

“…It is interesting to note that the new c.1121A>G (p.Q374R) and c.1123delG (p.R375XfsX6) mutations affected exon 10 in which up to date only an inframe 12 bp deletion was reported in the Turkish population (Ozkara & Navon, 1998;Sinici et al, 2004).…”

“…As shown in Table 2, the mutation profile was characterized by mutations spread over the sequence and consisted of point mutations, small deletions and small duplications causing both missense and nonsense mutations, amino acid deletions, frameshifts and splicing aberrations. It is interesting to note that the new c.1121A>G (p.Q374R) and c.1123delG (p.R375XfsX6) mutations affected exon 10 in which up to date only an inframe 12 bp deletion was reported in the Turkish population (Ozkara & Navon, 1998;Sinici et al, 2004).…”