A molecular investigation of a mixed ancestry family displaying dementia and movement disorders

[摘要] A South African family of Mixed Ancestry presented with a rapidly progressive dementia and amovement disorder which affected a number of individuals across three generations. The initialsymptoms included personality changes and tremors that escalated to severe dementia andeventually a completely bedridden state. It was determined that the mean age at onset was in thethird decade of life and affected individuals died within 10-15 years after the onset of symptoms.The aim of the present study was to elucidate the genetic cause of the disorder in this family and tofurther investigate the patho-biology of the disease.Mutations that could possibly cause the observed phenotype in this family were screened for. Theseincluded loci implicated in Huntington's disease, Parkinson's disease, Dentatorubral-PallidoluysianAtrophy, Spinocerebellar ataxias (types 1, 2, 3, 6, and 7), Huntington's disease-like 2 (HDL2) andseveral mitochondrial disorders. Single-strand Conformation Polymorphism (SSCP) analysis anddirect sequencing were used to detect possible mutations while genotyping on an ABI geneticanalyser was used to detect disorders caused by repeat expansions. Haplogroup and Short TandemRepeats (STRs) analyses of the Y-chromosome and mitochondrial DNA of one affected familymember was used to determine the family's genetic ancestry. Reverse transcriptase polymerasechain reaction (RT- PCR) and complementary DNA (cDNA) analyses of the Junctophlin-3 (JPH3)gene was performed to provide information on the expression profile of this gene.After the exclusion of several genetic loci it was shown that this family had HDL2. This is a raredisease caused by a CAG/CTG repeat expansion in an alternatively spliced version of the JPH3gene. HDL2 occurs almost exclusively in individuals of Black African ancestry. The genetic ancestrydata suggested that the family member was most likely of South African Mixed Ancestry making thisthe first reported family of South African Mixed Ancestry with HDL2. A pilot study investigated therepeat distribution amongst three South African sub-populations in order to determine whether therewas a bias in the repeat distribution that possibly predisposes Black Africans to develop the disease.The results showed a statistically significant difference (P= 0.0014) in the distribution of the repeatsbetween the Black African and Caucasian cohorts. However, no conclusions could be drawn as towhether Black Africans harboured larger repeats that predisposes them to developing HDL2.The expanded repeat is located in an alternatively spliced version of the JPH3 mRNA. Interestingly,this repeat is not present in the mouse homologue of the gene although the rest of the genomicsequence is highly conserved across the human, mouse and chimpanzee genomes. Using foetalbrain cDNA and PCR primers designed to be specific for different JPH3 isoforms, independentconfirmation of the presence of two JPH3 mRNA transcripts (the full length and a shorter alternativelyspliced version) was provided. In the absence of brain tissue from an HDL2-affected individual, it wasinvestigated whether both JPH3 mRNA transcripts could be detected in lymphocytes. Using RNAisolated from the transformed lymphocytes of two HDL2-affected family members, real-time PCRwas attempted. These experiments produced inconclusive results and required further optimisation.Further RT-PCR experiments for JHP3 expression in different tissues (brain and other) obtainedfrom HDL2-affected individuals would be of interest.The present study identified the first Mixed Ancestry family with HDL2. This family will now be ableto request genetic counselling and pre-symptomatic testing for all at-risk family members. Aspects ofthis study provided independent confirmation of characteristics of the mutated gene. More researchon HDL2 will be crucial in understanding the pathogenesis of this disease.

[发布机构] Stellenbosch University