A Polymerase Chain Reaction–Based Genotyping Assay for Detecting a Novel Sandhoff Disease–Causing Mutation

Abstract

Sandhoff disease is a rare genetic disorder, however, some northern Saskatchewan communities have a high incidence of the disease (for which the causative mutation has not been described). We discovered a novel mutation causing Sandhoff disease in this community and validated a molecular assay to detect the mutant allele. DNA sequencing was used to search for mutations in the HEXB gene from the most recently affected patient. A polymerase chain reaction (PCR)-based genotyping assay was subsequently designed and validated to detect a novel single-nucleotide deletion using DNA isolated from newborn screening cards. The c.115delG mutation was found in exon 1 of the HEXB gene from 4 patients with clinical presentation of Sandhoff disease. Herein we describe a novel HEXB mutation that is shared among 4 patients with Sandhoff disease, as well as a validated PCR-based genotyping assay that can reliably detect the mutant allele. Because the 4 patients from this community share a common c.115delG mutation in the coding region of the HEXB gene, it may be possible to offer an effective preventive screening program for Sandhoff disease using this assay.

Incidence and carrier frequency of Sandhoff disease in Saskatchewan determined using a novel substrate with detection by tandem mass spectrometry and molecular genetic analysis

“…The assay for detecting the c.115delG variant has been previously described [11] and was used to determine each individual genotype for this particular variant. To summarize, this assay uses real-time PCR to detect the presence of the c.115delG allele in blood eluted from two 3 mm dried blood spot punches.…”

“…Sanger sequencing of the HEXB gene was carried out as previously described [11]. Exons one through fourteen of the HEXB gene and several nucleotides from the flanking intronic regions were amplified by PCR.…”

“…Previously we identified the c.115delG pathogenic variant in the HEXB gene from several Sandhoff disease patients born in this area [11]. No relationship has been identified between Sandhoff disease in northern Saskatchewan where the community is largely Métis (individuals of mixed French/aboriginal descent) and the reports of Sandhoff disease among other French Canadian populations.…”

Advances in Human Biology: Combining Genetics and Molecular Biophysics to Pave the Way for Personalized Diagnostics and Medicine

“…Armed with the abovementioned tools, the ultimate goal is to be able to detect disease-causing DNA defects even before the disease is clinically manifested [184,185]; however, it is equally important to pinpoint the disease-causing effect [66,92,127,128,183] (Figure 2). The last case of investigations is essential for building a library of DNA defects associated with particular diseases, that is, database of genotypes causing particular disease [186].…”