| Summary: | Thesis (MScMedSc (Biomedical Sciences. Molecular Biology and Human Genetics))--Stellenbosch University, 2008. === A South African family of Mixed Ancestry presented with a rapidly progressive dementia and a
movement disorder which affected a number of individuals across three generations. The initial
symptoms included personality changes and tremors that escalated to severe dementia and
eventually a completely bedridden state. It was determined that the mean age at onset was in the
third 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 to
further investigate the patho-biology of the disease.
Mutations that could possibly cause the observed phenotype in this family were screened for. These
included loci implicated in Huntington’s disease, Parkinson’s disease, Dentatorubral-Pallidoluysian
Atrophy, Spinocerebellar ataxias (types 1, 2, 3, 6, and 7), Huntington’s disease-like 2 (HDL2) and
several mitochondrial disorders. Single-strand Conformation Polymorphism (SSCP) analysis and
direct sequencing were used to detect possible mutations while genotyping on an ABI genetic
analyser was used to detect disorders caused by repeat expansions. Haplogroup and Short Tandem
Repeats (STRs) analyses of the Y-chromosome and mitochondrial DNA of one affected family
member was used to determine the family’s genetic ancestry. Reverse transcriptase polymerase
chain 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 rare
disease caused by a CAG/CTG repeat expansion in an alternatively spliced version of the JPH3
gene. HDL2 occurs almost exclusively in individuals of Black African ancestry. The genetic ancestry
data suggested that the family member was most likely of South African Mixed Ancestry making this
the first reported family of South African Mixed Ancestry with HDL2. A pilot study investigated the
repeat distribution amongst three South African sub-populations in order to determine whether there
was 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 repeats
between the Black African and Caucasian cohorts. However, no conclusions could be drawn as to
whether 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 genomic
sequence is highly conserved across the human, mouse and chimpanzee genomes. Using foetal
brain cDNA and PCR primers designed to be specific for different JPH3 isoforms, independent
confirmation of the presence of two JPH3 mRNA transcripts (the full length and a shorter alternatively
spliced version) was provided. In the absence of brain tissue from an HDL2-affected individual, it was
investigated whether both JPH3 mRNA transcripts could be detected in lymphocytes. Using RNA
isolated from the transformed lymphocytes of two HDL2-affected family members, real-time PCR
was attempted. These experiments produced inconclusive results and required further optimisation.
Further RT-PCR experiments for JHP3 expression in different tissues (brain and other) obtained
from HDL2-affected individuals would be of interest.
The present study identified the first Mixed Ancestry family with HDL2. This family will now be able
to request genetic counselling and pre-symptomatic testing for all at-risk family members. Aspects of
this study provided independent confirmation of characteristics of the mutated gene. More research
on HDL2 will be crucial in understanding the pathogenesis of this disease.
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