Spinal Muscular Atrophy in the Black South African Population: A Matter of Rearrangement?

Spinal muscular atrophy (SMA) is a neuromuscular disorder, characterized by muscle atrophy and impaired mobility. A homozygous deletion of survival motor neuron 1 (SMN1), exon 7 is the main cause of SMA in ~94% of patients worldwide, but only accounts for 51% of South African (SA) black patients. SMN1 and its highly homologous centromeric copy, survival motor neuron 2 (SMN2), are located in a complex duplicated region. Unusual copy number variations (CNVs) have been reported in black patients, suggesting the presence of complex pathogenic rearrangements. The aim of this study was to further investigate the genetic cause of SMA in the black SA population. Multiplex ligation-dependent probe amplification (MLPA) testing was performed on 197 unrelated black patients referred for SMA testing (75 with a homozygous deletion of SMN1, exon 7; 50 with a homozygous deletion of SMN2, exon 7; and 72 clinically suggestive patients with no homozygous deletions). Furthermore, 122 black negative controls were tested. For comparison, 68 white individuals (30 with a homozygous deletion of SMN1, exon 7; 8 with a homozygous deletion of SMN2, exon 7 and 30 negative controls) were tested. Multiple copies (>2) of SMN1, exon 7 were observed in 50.8% (62/122) of black negative controls which could mask heterozygous SMN1 deletions and potential pathogenic CNVs. MLPA is not a reliable technique for detecting carriers in the black SA population. Large deletions extending into the rest of SMN1 and neighboring genes were more frequently observed in black patients with homozygous SMN1, exon 7 deletions when compared to white patients. Homozygous SMN2, exon 7 deletions were commonly observed in black individuals. No clear pathogenic CNVs were identified in black patients but discordant copy numbers of exons suggest complex rearrangements, which may potentially interrupt the SMN1 gene. Only 8.3% (6/72) of clinically suggestive patients had heterozygous deletions of SMN1, exon 7 (1:0) which is lower than previous SA reports of 69.5%. This study emphasizes the lack of understanding of the architecture of the SMN region as well as the cause of SMA in the black SA population. These factors need to be taken into account when counseling and performing diagnostic testing in black populations.

Fanconi anaemia in black South African patients heterozygous for the FANCG c.637-643delTACCGCC founder mutation.

BACKGROUND: Fanconi anaemia (FA) is an autosomal recessive, genetically heterogeneous disorder, characterised by interstrand crosslink-induced chromosome breaks, congenital abnormalities and predisposition to malignancies. It has a prevalence of about 1/40 000 in black South Africans (SAs). A founder mutation in the FANCG gene occurs in the homozygous state in 77.5% of southern African blacks. OBJECTIVE: To locate additional pathogenic mutations in the FANCG gene of black FA patients who were heterozygous for the founder mutation. Methods. Further mutation analysis of the FANCG gene was undertaken in 7 patients clinically suspected of having FA. The parents of two of the patients were tested for the presence of the founder mutation to determine true heterozygosity in the patients. To clarify whether or not previously unreported variants were pathogenic, 58 random black SA individuals were screened. RESULTS: Three novel single base pair deletions, resulting in frameshift mutations (c.247delA, c.179delT and c.899delT) were identified in 3/7 patients. A fourth patient was found to have a single base substitution resulting in a splice site mutation (c.1636+1G>A). The remaining three patients were not found to harbour any pathogenic mutations. Two non-pathogenic variants were also identified among the seven patients. CONCLUSION: The results of this small sample suggest that a second common mutation in the FANCG gene is unlikely in this population. However, FANCG sequencing should be performed on patients heterozygous for the common founder mutation to attempt to confirm their diagnosis.

Diagnostic, carrier and prenatal genetic testing for fragile X syndrome and other FMR-1-related disorders in Johannesburg, South Africa: a 20-year review.

BACKGROUND: Fragile X syndrome (FXS), the most common inherited cause of intellectual disability (ID) worldwide, is caused by the expansion of a CGG repeat in the fragile X mental retardation gene (FMR-1) gene. OBJECTIVES; To review, retrospectively, the genetic services for FXS and other FMR-1-related disorders - including fragile X-associated tremor/ataxia syndrome (FXTAS) and FMR-1-related primary ovarian insufficiency (POI) - at the Division of Human Genetics, Johannesburg, for diagnostic, carrier and prenatal genetic testing.Methods. The records of 2 690 patients with ID and suspected FXS (ID/?FXS) who had genetic testing for FMR-1 between 1992 and 2012 were reviewed. Of these, 2 239 had diagnostic testing, 430 carrier or cascade testing and 17 prenatal testing for FXS. Four had FXTAS or POI testing. Polymerase chain reaction (PCR) and/or Southern blotting techniques were used to test the patients' samples for FMR-1 and FMR-2 expansions. RESULTS; Of the 2 239 patients who had diagnostic testing, 128 (5.7%) had a full mutation, 12 (0.5%) had a premutation and 43 (1.9%) an intermediate allele. In 17 prenatal tests, eight fetuses tested positive for FXS. FMR-1 CGG repeat distribution analysis in 1 532 males negative for the FMR-1 expansion showed that 29 and 30 CGG repeats were the most common (61.1%), but distribution was significantly different in the black and white populations.CONCLUSION; The findings support the presence of FXS, as the most common cause of ID, in all local populations. The FMR-1 CGG repeat distribution varied from that found in other studies. The number of family members tested was relatively low suggesting that many at-risk individuals are not being referred.

Testing for haemoglobinopathies in Johannesburg, South Africa: a 30-year review.

BACKGROUND: Haemoglobinopathies are seen mostly in regions where malaria occurs or has occurred, but population migration has resulted in affected individuals being identified in many countries globally. The first molecular genetics services for diagnostic testing and prenatal diagnosis were established, both worldwide and in South Africa (SA), for haemoglobinopathies. OBJECTIVE: To analyse the diagnostic service offered by the Division of Human Genetics, National Health Laboratory Service and University of the Witwatersrand, from 1983 to 2012. METHODS: A retrospective file analysis (N=1 249) was performed for all individuals who had molecular genetic testing for α-thalassaemia, ß-thalassaemia and sickle cell anaemia to examine indications for testing, population origins of patients and molecular genetics findings. RESULTS: The α-thalassaemia testing was requested predominantly to explain microcytic hypochromic haematological indices. Five α-globin deletions were identified, the most common being the -α3.7, in individuals of different ethnicities. For ß-thalassaemia and sickle cell anaemia, most testing was performed for prenatal diagnosis purposes. For sickle cell anaemia, most prenatal tests were requested by African families. The ß-thalassaemia families were mostly of Indian or Mediterranean origin. The most common mutation identified in all Indian groups was IVS1 nt5 (G>C) (c.92+5G>C) and in individuals from the Mediterranean, IVS1 nt110 (G>A) (c.93-21G>A). CONCLUSION: The molecular genetics service for haemoglobinopathies in SA is comprehensive and specific to the needs of local ethnic groups. Clinically significant haemoglobinopathies occur at significant frequencies in specific high-risk ethnic groups. Appropriate screening programmes should be initiated so that genetic counselling and reproductive options can be offered.

Genetic testing for Duchenne/Becker muscular dystrophy in Johannesburg, South Africa.

BACKGROUND: Genetic testing for Duchenne/Becker muscular dystrophy (DMD/BMD) mutations initially involved multiplex polymerase chain reaction (mPCR), which targeted two mutation hotspots in the gene and detected deletions in affected males. A newer technology, multiplex ligation-dependent probe amplification (MLPA), was introduced for diagnostic testing in 2007. OBJECTIVES: To evaluate MLPA relative to mPCR as a technique for DMD/BMD diagnostic testing and to establish whether the mutation profile in affected individuals differs between different South African ethnic groups. METHODS; From January 2000 - May 2007, genetic diagnostic testing for DMD/BMD was undertaken in 128 male patients using mPCR. From May 2007 onwards, MLPA replaced this technique and 261 males were investigated. MLPA is a kit-based technology available from MRC-Holland.Results. Of the 128 and 261 probands tested using mPCR and MLPA, respectively, 31% and 34% were found to carry a deletion mutation. Further, MLPA could detect duplication mutations (11.5%), complex rearrangements (1.5%) and small mutations (1.5%). In black patients, deletion mutations were found to cluster in the 3' region of the gene. No population-specific pathogenic mutations were found. CONCLUSIONS: The mutation detection rate for mPCR and MLPA is similar for deletion mutations, but MLPA proved to be a better diagnostic approach as it could detect other types of mutations as well, including duplications, complex rearrangements and small mutations. MLPA could also diagnose mutation status in at-risk female relatives, which is not possible with mPCR.