D C Smith

Inherited polyglutamine spinocerebellar ataxias in South Africa

OBJECTIVE To determine the frequency and distribution of polyglutamine spinocerebellar ataxias (SCAs) from referrals over a 24-year period to the National Health Laboratory Service (NHLS) in South Africa (SA). METHODS Paper-based clinical reports in the University of Cape Town laboratory and the NHLS electronic patient record database spanning a 24-year period were mined for information regarding the molecular diagnosis, ethnicity and CAG repeat length for individuals referred for molecular genetic testing for the polyglutamine SCAs. RESULTS SCA1 and 7 are the most frequent types of polyglutamine SCA in the SA patient population, followed by SCA2, 3 and 6. SCA1 is the most common type in the coloured, white and Indian populations, whereas the majority of indigenous black African patients are affected with SCA7 and 2. Of individuals tested, 22% were found to be positive for one of the polyglutamine SCAs. CONCLUSION Although trends in the frequency and distribution of the polyglutamine SCAs in SA have not changed significantly since our previous study in 2003, they differ remarkably from those reported elsewhere, and reflect the unique genetic and demographic background of SA. The provision of accurate and complete patient information and family history is crucial to the diagnostic process, to enable comprehensive epidemiological studies and assist in developing therapeutic and patient management strategies.

Allele-specific silencing of mutant Ataxin-7 in SCA7 patient-derived fibroblasts

Polyglutamine (polyQ) disorders are inherited neurodegenerative conditions defined by a common pathogenic CAG repeat expansion leading to a toxic gain-of-function of the mutant protein. Consequences of this toxicity include activation of heat-shock proteins (HSPs), impairment of the ubiquitin-proteasome pathway and transcriptional dysregulation. Several studies in animal models have shown that reducing levels of toxic protein using small RNAs would be an ideal therapeutic approach for such disorders, including spinocerebellar ataxia-7 (SCA7). However, testing such RNA interference (RNAi) effectors in genetically appropriate patient cell lines with a disease-relevant phenotype has yet to be explored. Here, we have used primary adult dermal fibroblasts from SCA7 patients and controls to assess the endogenous allele-specific silencing of ataxin-7 by two distinct siRNAs. We further identified altered expression of two disease-relevant transcripts in SCA7 patient cells: a twofold increase in levels of the HSP DNAJA1 and a twofold decrease in levels of the de-ubiquitinating enzyme, UCHL1. After siRNA treatment, the expression of both genes was restored towards normal levels. To our knowledge, this is the first time that allele-specific silencing of mutant ataxin-7, targeting a common SNP, has been demonstrated in patient cells. These findings highlight the advantage of an allele-specific RNAi-based therapeutic approach, and indicate the value of primary patient-derived cells as useful models for mechanistic studies and for measuring efficacy of RNAi effectors on a patient-to-patient basis in the polyQ diseases.

Evidence for a common founder effect amongst South African and Zambian individuals with Spinocerebellar ataxia type 7

Spinocerebellar ataxia type 7 (SCA7) is an inherited neurodegenerative disease caused by the expansion of a CAG repeat within the ataxin 7 gene, leading to a pathogenic polyglutamine tract within the ataxin 7 protein. SCA7 patients suffer from progressive cerebellar ataxia and macular degeneration. SCA7 is considered to be rare, although founder effects have been reported in South Africa, Scandinavia and Mexico. The South African SCA7-associated haplotype has not been investigated in any other populations, and there have been limited reports of SCA7 patients from other African countries. Here, we describe the first two ethnic Zambian families with confirmed SCA7. Haplotype analysis showed that the South African SCA7 haplotype alleles were significantly associated with the pathogenic expansion in affected Zambian individuals, providing strong evidence for a shared founder effect between South African and Zambian SCA7 patients.

Spinocerebellar ataxia type 7 in South Africa: Epidemiology, pathogenesis and therapy.

Disorders of the nervous system represent a significant proportion of the global burden of non-communicable diseases, due to the trend towards ageing populations. The Department (now Division) of Human Genetics at the University of Cape Town (UCT) has been involved in pioneering research into these diseases since the appointment of Prof. Peter Beighton as Head of Department in 1972. Beightons emphasis on understanding the genetic basis of disease laid the groundwork for investigations into several monogenic neurodegenerative conditions, including Huntingtons disease and the polyglutamine spinocerebellar ataxias (SCAs). In particular, SCA7, which occurs at an unusually high frequency in the South African (SA) population, was identified as a target for further research and therapeutic development. Beginning with early epidemiological surveys, the SCA7 project progressed to molecular genetics-based investigations, leading to the identification of a founder effect in the SA SCA7 patient population in the mid-2000s. Capitalising on the founder haplotype shared by many SCA7 patients, UCT researchers went on to develop the first population-specific gene-silencing approach for the disease. More recently, efforts have shifted to the development of a more accurate model to decipher the precise mechanisms of neurodegeneration, using induced pluripotent stem cells derived from SA SCA7 patients. In many ways, the SA SCA7 journey reflects the legacy and vision of Prof. Peter Beighton, and his efforts to establish world-class, collaborative research into diseases affecting the African continent.

The hereditary ataxias: Where are we now? Four decades of local research

The hereditary ataxias have been studied at the University of Cape Town for more than 40 years, following from initial clinical investigations by Beighton and colleagues in the early 1970s. This group of inherited disorders is characterised by progressive neurodegeneration and associated symptoms, including the inability to coordinate movement. Following initial local and international linkage studies, and the discovery of the genes responsible for the key dominant and recessive inherited ataxias in the 1990s, a local molecular testing service was established at Groote Schuur Hospital. More than 1 600 individuals have been referred through this testing service (now offered by the National Health Laboratory Service), leading to the molecular diagnosis of 253 families with spinocerebellar ataxia types 1, 2, 3, 6 or 7, and 30 families with Friedreichs ataxia. This is likely to be an under-representation of the number of South Africans affected with hereditary ataxia, and future research efforts will focus on increasing the awareness of this group of disorders, both locally and throughout the rest of Africa. Next-generation technologies will be beneficial in identifying additional genes underlying inherited ataxia in indigenous patients to enable more appropriate management and treatment of individuals with molecularly undiagnosed forms of the disease.

Caution Regarding the Interpretation of Homoallelism in Polyglutamine Multiplex Assays: A Recommendation for Confirmatory Testing of Homozygous Alleles

Spinocerebellar ataxia type 7 (SCA7) is an inherited dominant neurodegenerative disease caused by the expansion of a CAG repeat within the ATXN7 gene. Standard molecular diagnostic testing for SCA7 involves amplification of the region surrounding the CAG repeat via end-labeled PCR and subsequent capillary electrophoresis. In addition, multiplex methods exist that may be used to test for multiple polyglutamine spinocerebellar ataxias in a single assay. Herein, we used a SCA7 singleplex method to screen 111 individuals for whom the multiplex method detected a single normal allele. A total of six retested individuals (5.4%) were shown to have a pathogenic expansion at the ATXN7 locus. An additional triplet-primed PCR method was used to test the same cohort, and revealed no further disease-causing alleles. This study demonstrates the importance of using complementary methods to rule out apparent homoallelism during molecular testing for polyglutamine diseases.

Stem cells on South African shores: proposed guidelines for comprehensive informed consent.

In October 2012, Shinya Yamanaka and Sir John Gurdon were awarded the Nobel Prize in Physiology or Medicine for their pioneering research into the reprogramming of mature cells into a pluripotent state. The technology developed by Yamanaka and colleagues allows researchers to turn terminally differentiated somatic cells back into a stem-cell state. These induced pluripotent stem cells (iPSCs) can be cultured indefinitely in the laboratory, and can undergo directed differentiation into any cell type of interest. This is highly beneficial for disease-modelling studies, since researchers are able to culture cells that are not normally obtainable, such as neurons or retinal cells. Furthermore, the differentiated cells can be used to test potential therapies in patient-derived cells, and may even be used for future therapeutic cell transplantation.

Transcriptional and electrophysiological aberrations in an induced pluripotent stem cell-derived model of spinocerebellar ataxia type 7

Spinocerebellar ataxia type 7 (SCA7) is an inherited neurodegenerative disease caused by a polyglutamine repeat expansion in the ATXN7 gene. Patients with this disease suffer from a degeneration of their cerebellar Purkinje neurons and retinal photoreceptors that result in a progressive ataxia and loss of vision. As with many neurodegenerative diseases, studies of pathogenesis have been hindered by a lack of disease-relevant models. To this end, we have generated induced pluripotent stem cells (iPSCs) from a cohort of SCA7 patients in South Africa. First, we differentiated the SCA7 affected iPSCs into neurons which showed evidence of a transcriptional phenotype affecting components of STAGA (ATXN7 and KAT2A) and the heat shock protein pathway (DNAJA1 and HSP70). We then performed electrophysiology on the SCA7 iPSC-derived neurons and found that these cells show features of functional aberrations. Lastly, we were able to differentiate the SCA7 iPSCs into retinal photoreceptors that also showed similar transcriptional aberrations to the SCA7 neurons. Our findings demonstrate that iPSC-derived neurons and photoreceptors from SCA7 patients express molecular and electrophysiological differences that are indicative of impaired neuronal health. We hope that these findings will contribute towards the ongoing efforts to establish the cell-derived models of neurodegenerative diseases that are needed to develop patient-specific treatments.Spinocerebellar ataxia type 7 (SCA7) is an inherited neurodegenerative disease that is characterised by ataxia and visual loss. It results from a degeneration of cerebellar Purkinje neurons and retinal photoreceptors caused by a polyglutamine repeat expansion in the ATXN7 gene, a component of the STAGA transcription co-activator complex. As with many neurodegenerative diseases, studies of pathogenesis have been hindered by a lack of disease-relevant models. To this end, we have generated the first induced pluripotent stem cells (iPSCs) from South African SCA7 patients, where the disease occurs at an unusually high frequency as a result of a founder effect. These iPSCs were capable of differentiation into neural and retinal cells, and showed evidence of a transcriptional phenotype affecting components of STAGA (ATXN7 and KAT2A) and the heat shock protein pathway (DNAJA1 and HSP70). Functionally, SCA7 iPSC-derived neurons exhibited more negative resting membrane potentials and increased input resistance compared to controls, suggesting reduced excitability in response to synaptic input. These results provide the first evidence of a disease phenotype in SCA7 iPSC-derived cells, establishing a valuable model for the study of neurodegenerative diseases and the development of population-specific therapies.

Towards guidelines for informed consent for prospective stem cell research

Stem cell science is advancing at an unprecedented rate, with thousands of research papers being published every year and many clinical trials for a wide range of conditions underway as registered on ClinicalTrials.gov. This rapidly expanding and alluring field has brought with it ever more complex and multifaceted ethical issues, many of which require new guidelines, consent protocols and even change in legislation, since they do not fit comfortably in the existing bioethical regulations and protocols. Keeping up with the ethical implications of stem cell research is daunting to the expert and non-expert. We review the various types of stem cells and then focus on multipotent and pluripotent cell types, since it is these cell types that bring with them the greatest research and therapeutic potential, while concurrently delivering novel ethical conundrums. Certain key considerations are currently lacking and what is needed is how to obtain permission from individuals who donate their biological material for both scientific inquiry and eventually, for their potential therapeutic utility.