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1.
S Afr Med J ; 106(6 Suppl 1): S33-7, 2016 May 25.
Article in English | MEDLINE | ID: mdl-27245521

ABSTRACT

Retinal degenerative disorders (RDDs) encompass a group of inherited diseases characterised by vision loss. The genetic and clinical complexity poses a challenge in unravelling the molecular genetic aetiology of this group of disorders. Furthermore, the population diversity in South Africa (SA) presents researchers with a particularly complicated task. Rapid advances in the development of cutting-edge technological platforms over the past two decades, however, have assisted in overcoming some of the challenges. The RDD research team has utilised these escalating technologies, which has facilitated a corresponding increase in molecular diagnoses. A biorepository has been established and comprises ~3 200 patient DNA samples archived with many forms of RDD (including retinitis pigmentosa, macular dystrophies, Stargardt disease, Leber congenital amaurosis, Usher syndrome and Bardet Biedl syndrome). A comprehensive review is presented of the SA journey spanning 25 years, into elucidating the molecular genetic basis of various forms of RDD in SA.


Subject(s)
Biomedical Research/trends , Biomedical Technology/trends , Molecular Biology , Retinal Diseases/physiopathology , Humans , Molecular Diagnostic Techniques , Retinal Diseases/epidemiology , Retinal Diseases/genetics , South Africa/epidemiology
2.
S Afr Med J ; 106(6 Suppl 1): S67-71, 2016 May 25.
Article in English | MEDLINE | ID: mdl-27245531

ABSTRACT

Duchenne muscular dystrophy (DMD) is one of the most common and severe of the inherited dystrophies, with an incidence of 1 in 3 500 live, male births worldwide. Becker muscular dystrophy (BMD) has a lower incidence of 1:14 000 - 18 000 boys and a milder progression and longer life expectancy. Over the last two decades, better understanding of the underlying disease aetiology as well as major advances in medical technology have brought about significantly improved genetic diagnosis and clinical care for B/DMD patients. Exciting developments in the field of gene-based therapies have once again put B/DMD in the limelight, with renewed focus on the importance of comprehensive genetic testing protocols. We present a historical overview of the medical and molecular service for B/DMD offered over the last three decades in South Africa, specifically in the Western Cape, from a clinical as well as a laboratory perspective.


Subject(s)
Genetic Testing/methods , Genetic Therapy/methods , Muscular Dystrophy, Duchenne/therapy , Biomedical Technology , Disease Progression , Humans , Incidence , Life Expectancy , Male , Muscular Dystrophy, Duchenne/diagnosis , Muscular Dystrophy, Duchenne/epidemiology , South Africa/epidemiology
3.
S Afr Med J ; 104(11): 779-84, 2014 Nov.
Article in English | MEDLINE | ID: mdl-26110187

ABSTRACT

BACKGROUND: Duchenne/Becker muscular dystrophy (D/BMD) is an X-linked recessive muscle disorder affecting 1/3 500 live male births worldwide. Up to 70% of all D/BMD cases are caused by exonic deletions or duplications routinely identified in diagnostic laboratories worldwide.The remaining patients harbour other sequence alterations for which testing availability is limited owing to the expense of interrogating the large DMD gene. Genetic screening for D/BMD in South Africa currently includes multiple ligase-dependent probe amplification (MLPA) for exonic deletions and duplications and linkage analysis. No genetic testing for small mutations in the DMD gene is offered, leaving a third of D/BMD families without genetic closure. The advent of potential mutation-specific therapies for DMD necessitates comprehensive testing protocols. OBJECTIVE: To investigate the effectiveness and affordability of high-resolution melting curve analysis (hrMCA) for detection of small/point mutations in the DMD gene, for possible inclusion into the local public health-funded diagnostic service. METHODS: DNA from 24 patients who had previously tested deletion-negative with multiplex polymerase chain reaction (mPCR) was analysed by MLPA and hrMCA. RESULTS: MLPA revealed eight previously undetected exonic rearrangements: five deletions and three duplications. HrMCA of the remaining samples revealed three nonsense, four frameshifts, one splice-site, one missense and one single-base substitution in the Dp427promoter/exon1 of the DMD gene. In addition, 41 polymorphisms and three changes of uncertain significance were detected. CONCLUSION: These findings identify hrMCA as an affordable and effective mutation scanning tool for incorporation into the local diagnostic setting, allowing for better genetic counselling of more DMD families and selection of potential candidates for future therapies.

4.
Eur Respir J ; 39(1): 163-71, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21719487

ABSTRACT

Clinical algorithms for evaluating HIV-infected individuals for tuberculosis (TB) prior to isoniazid preventive therapy (IPT) perform poorly, and interferon-γ release assays (IGRAs) have moderate accuracy for active TB. It is unclear whether, when used as adjunct tests, IGRAs add any clinical discriminatory value for active TB diagnosis in the pre-IPT assessment. 779 sputum smear-negative HIV-infected persons, established on or about to commence combined antiretroviral therapy (ART), were screened for TB prior to IPT. Stepwise multivariable logistic regression was used to develop clinical prediction models. The discriminatory ability was assessed by receiver operator characteristic area under the curve (AUC). QuantiFERON-TB Gold in-tube (QFT-GIT) was evaluated. The prevalence of smear-negative TB by culture was 6.4% (95% CI 4.9-8.4%). Used alone, QFT-GIT and the tuberculin skin test (TST) had comparable performance; the post-test probability of disease based on single negative tests was 3-4%. In a multivariable model, the QFT-GIT test did not improve the ability of a clinical algorithm, which included not taking ART, weight <60 kg, no prior history of TB, any one positive TB symptom/sign (cough ≥ 2 weeks) and CD4+ count <250 cells per mm(3), to discriminate smear-negative culture-positive and -negative TB (72% to 74%; AUC comparison p=0.33). The TST marginally improved the discriminatory ability of the clinical model (to 77%, AUC comparison p=0.04). QFT-GIT does not improve the discriminatory ability of current TB screening clinical algorithms used to evaluate HIV-infected individuals for TB ahead of preventive therapy. Evaluation of new TB diagnostics for clinical relevance should follow a multivariable process that goes beyond test accuracy.


Subject(s)
HIV Infections/diagnosis , Interferons/metabolism , Tuberculosis/therapy , Adult , Algorithms , Area Under Curve , Female , Humans , Infectious Disease Medicine/methods , Interferon-gamma/metabolism , Isoniazid/therapeutic use , Male , Multivariate Analysis , Reproducibility of Results , Sputum/metabolism , Treatment Outcome , Tuberculin Test/methods
5.
Comput Biol Chem ; 32(6): 452-7, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18760969

ABSTRACT

A theoretical analysis is presented with experimental confirmation to conclusively demonstrate the critical role that annealing plays in efficient PCR amplification of GC-rich templates. The analysis is focused on the annealing of primers at alternative binding sites (competitive annealing) and the main result is a quantitative expression of the efficiency (eta) of annealing as a function of temperature (T(A)), annealing period (t(A)), and template composition. The optimal efficiency lies in a narrow region of T(A) and t(A) for GC-rich templates and a much broader region for normal GC templates. To confirm the theoretical findings, the following genes have been PCR amplified from human cDNA template: ARX and HBB (with 78.72% and 52.99% GC, respectively). Theoretical results are in excellent agreement with the experimental findings. Optimum annealing times for GC-rich genes lie in the range of 3-6s and depend on annealing temperature. Annealing times greater than 10s yield smeared PCR amplified products. The non-GC-rich gene did not exhibit this sensitivity to annealing times. Theory and experimental results show that shorter annealing times are not only sufficient but can actually aid in more efficient PCR amplification of GC-rich templates.


Subject(s)
Base Composition , DNA/chemistry , Polymerase Chain Reaction/methods , Models, Theoretical
6.
Clin Genet ; 65(1): 7-10, 2004 Jan.
Article in English | MEDLINE | ID: mdl-15032968

ABSTRACT

Mutations in the RP2 gene account for up to 20% of X-linked recessive retinitis pigmentosa (RP). Arg120stop is to date the most frequently reported mutation found in RP2. Mutation screening was performed during the course of a large screening program of retinal degenerative disorders (RDDs) in South Africa using exon 1 and 2 of RP2 in 20 unrelated families with an X-linked mode of retinal degenerative inheritance. Direct sequencing analysis revealed a C-->T transition at position 358 in the proband in a family of German origin. Subsequent analysis revealed that this Arg120stop mutation cosegregated with the disease in an additional affected family member. The nonsense mutation, Arg120stop, could not however, be detected in the somatic cells of the obligate carrier female. This, the first report of a germ line mutation for a family with RP, has many implications for genetic counseling of retinal degeneration (RD). To avoid inaccurate risk assessment for RP due to epigenetic events, such as the rare occurrence of germ line mosaicism, genetic counseling in families with XLRP should always be guided by molecular testing.


Subject(s)
Codon, Nonsense/genetics , Eye Proteins/genetics , Mosaicism/genetics , Retinitis Pigmentosa/genetics , Adult , Aged , Arginine/genetics , DNA Mutational Analysis , Epigenesis, Genetic , Exons , Female , GTP-Binding Proteins , Humans , Intracellular Signaling Peptides and Proteins , Male , Membrane Proteins , Microtubules , Pedigree , Risk Assessment
8.
Hum Mol Genet ; 10(15): 1555-62, 2001 Jul 15.
Article in English | MEDLINE | ID: mdl-11468273

ABSTRACT

Retinitis pigmentosa (RP) is a genetically heterogeneous disorder characterized by progressive degeneration of the peripheral retina leading to night blindness and loss of visual fields. With an incidence of approximately 1 in 4000, RP can be inherited in X-linked, autosomal dominant or autosomal recessive modes. The RP13 locus for autosomal dominant RP (adRP) was placed on chromosome 17p13.3 by linkage mapping in a large South African adRP family. Using a positional cloning and candidate gene strategy, we have identified seven different missense mutations in the splicing factor gene PRPC8 in adRP families. Three of the mutations cosegregate within three RP13 linked families including the original large South African pedigree, and four additional mutations have been identified in other unrelated adRP families. The seven mutations are clustered within a 14 codon stretch within the last exon of this large 7 kb transcript. The altered amino acid residues at the C-terminus exhibit a high degree of conservation across species as diverse as humans, Arabidopsis and trypanosome, suggesting that some functional significance is associated with this part of the protein. These mutations in this ubiquitous and highly conserved splicing factor offer compelling evidence for a novel pathway to retinal degeneration.


Subject(s)
Genes, Dominant , Mutation , RNA Splicing , Retinitis Pigmentosa/genetics , Animals , Arabidopsis/genetics , Base Sequence , Blotting, Northern , Chromosome Mapping , Chromosomes, Human, Pair 17 , Codon , Conserved Sequence , DNA Mutational Analysis , Exons , Expressed Sequence Tags , Family Health , Female , Genetic Linkage , Humans , Male , Models, Genetic , Molecular Sequence Data , Mutation, Missense , Pedigree , RNA Precursors/metabolism , RNA, Messenger/metabolism , Restriction Mapping , Retina/pathology , Reverse Transcriptase Polymerase Chain Reaction , South Africa , Trypanosoma/genetics
9.
S Afr Med J ; 89(8): 877-8, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10488366

ABSTRACT

Hereditary retinal degenerations may be subdivided into those affecting predominantly the central (macular) or peripheral regions of the retina. Retinitis pigmentosa (RP) affects the photoreceptors; death of the rod cells is followed by a progressive loss of cone cells, resulting in relatively early loss of peripheral vision and progressive constriction of the visual fields. A mutation in the gene encoding the photoreceptor protein, rhodopsin, was the first molecular defect identified as a potential cause of inherited retinal degeneration (RD). In the study reported here, simple tests for rhodopsin involvement in 194 southern African patients with a history of retinal degeneration, including 14 black African patients, were performed. Two RP patients were identified with disease-causing mutations in the rhodopsin gene: one from a black African family in which a codon 347 mutation resulted in a Pro-Leu substitution, and one in a family of Caucasian origin where a codon 58 alteration resulted in a Thr-Arg substitution. This is the first report of a disease-causing rhodopsin mutation in an indigenous black African family with retinitis pigmentosa.


Subject(s)
Black People/genetics , Mutation , Photoreceptor Cells/metabolism , Retinitis Pigmentosa/genetics , Rhodopsin/genetics , Codon , Humans , Polymerase Chain Reaction/methods , Rhodopsin/physiology , South Africa
11.
J Med Genet ; 34(11): 904-6, 1997 Nov.
Article in English | MEDLINE | ID: mdl-9391883

ABSTRACT

The vestibular and ototoxic effects of the aminoglycoside antibiotics (streptomycin, gentamycin, kanamycin, tobramycin, neomycin) are well known; streptomycin, in particular, has been found to cause irreversible, profound, high frequency sensorineural deafness in hypersensitive persons. Aminoglycoside ototoxicity occurs both sporadically and within families and has been associated with a mitochondrial DNA (mtDNA) 1555A to G point mutation in the 12S ribosomal RNA gene. We report on the molecular analysis of a South African family with streptomycin induced sensorineural deafness in which we have found transmission of this same predisposing mutation. It is now possible to identify people who are at risk of hearing loss if treated with aminoglycosides in the future and to counsel them accordingly. In view of the fact that aminoglycoside antibiotics remain in widespread use for the treatment of infections, in particular for tuberculosis, which is currently of epidemic proportions in South Africa, this finding has important implications for the family concerned. In addition, other South African families may potentially be at risk if they carry the same mutation.


Subject(s)
Anti-Bacterial Agents/adverse effects , Deafness/chemically induced , Deafness/genetics , Mitochondria , Streptomycin/adverse effects , Female , Humans , Male , Pedigree , South Africa
12.
J Med Genet ; 33(6): 511-4, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8782054

ABSTRACT

Cleidocranial dysplasia (CCD) is an autosomal dominant disorder, features of which include a patient anterior fontanelle, a bulging calvarium, hypoplasia or aplasia of the clavicles, a wide public symphysis, dental anomalies, vertebral malformation, and short stature. The Cape Town kindred which is under our genetic management was originally described more than four decades ago and now consists of more than 1000 people. Following reports of rearrangements on chromosomes 6 and 8 in people with CCD, we have carried out linkage analyses between highly information microsatellite dinucleotide repeat markers in the rearranged regions and the disorder in a branch of this South African CCD kindred, consisting of 38 subjects, 18 of whom are affected. Maximum lod scores (at theta = 0.00) of 7.14 (for marker D6S459), 4.32 (TCTE), 4.99 (D6S452), 5.97 (D6S269), and 3.95 (D6S465) confirm linkage of the disorder to the short arm of chromosome 6. Our data indicate that the CCD gene is located within a minimal region of approximately 10 cM flanked by the marker D6S451 distally and D6S466 proximally. This information is vital towards isolating and characterising the gene for CCD, and is being used to construct a physical map of 6p21.1-6p21.3. More importantly, mapping of the locus in the South African kindred of mixed ancestry, in which the "founder" of the disorder was of Chinese origin, suggests that a single locus is responsible for classic CCD.


Subject(s)
Chromosomes, Human, Pair 6 , Cleidocranial Dysplasia/genetics , Child , Chromosome Mapping , Female , Genetic Heterogeneity , Genetic Linkage , Humans , Male , Pedigree , South Africa
15.
Hum Mol Genet ; 4(8): 1459-62, 1995 Aug.
Article in English | MEDLINE | ID: mdl-7581389

ABSTRACT

Retinitis pigmentosa is one of the most common causes of severe visual handicap in middle to late life. Prior to this report, seven loci had previously been mapped for the autosomal dominant form of this disorder (adRP). We now report the identification of a novel adRP locus on chromosome 17q. To map the new locus, we performed linkage analysis with microsatellite markers in a large South African kindred. After exclusion of 13 RP candidate gene loci (including rhodopsin and peripherin-RDS), we obtained significant positive lod scores at zero recombination fraction (theta = 0) for D17S808 (Z = 4.63) and D17S807 (Z = 5.69). Multipoint analysis gave a maximum lod score of 8.28 between these two markers. From haplotype analysis, the disease locus lies in the interval between markers D17S809 and D17S942. Three candidate genes for retinal dystrophies map to this chromosomal region and these genes are currently being investigated for possible involvement with adRP in this family.


Subject(s)
Chromosomes, Human, Pair 17/genetics , Genes, Dominant , Genetic Linkage , Retinitis Pigmentosa/genetics , Adult , Chromosome Mapping , Female , Haplotypes , Humans , Lod Score , Male , Microsatellite Repeats , Pedigree
16.
Hum Mol Genet ; 3(6): 915-8, 1994 Jun.
Article in English | MEDLINE | ID: mdl-7951236

ABSTRACT

Retinitis pigmentosa (RP) is a group of genetically and clinically heterogeneous retinopathies, some of which have been shown to result from mutations in two different known retinal genes, rhodopsin (3q) and peripherin-rds (6p). Three additional anonymous loci at 7p, 7q and pericentric 8 have been implicated by linkage studies. There are still, however, a few families in which all known loci have been excluded. In this report we present data indicating a location, on the short arm of chromosome 17, for the autosomal dominant RP (ADRP) locus in a large South African (SA) family of British ancestry. Positive two-point lod scores have been obtained for nine markers (D17S938, Z = 5.43; D17S796, Z = 4.82; D17S849, Z = 3.6; D17S786, Z = 3.55; TP53, Z = 3.55; D17S578, Z = 3.29; D17S960, Z = 3.16; D17S926, Z = 1.51; D17S804, Z = 0.47 all at theta = 0.10 except D17S804 and D17S926, theta = 0.20). These data provide definitive evidence for the localization of an ADRP gene on chromosome 17p. The human recoverin gene has been localized to 17p13.1 and was consequently a prime candidate for ADRP in the family studied. However, mutation screening of the three exons of this gene failed to produce any evidence of recoverin being the gene involved in the pathogenesis of ADRP in this SA family.


Subject(s)
Chromosomes, Human, Pair 17 , Eye Proteins , Genes, Dominant , Lipoproteins , Membrane Glycoproteins , Nerve Tissue Proteins , Retinitis Pigmentosa/genetics , Calcium-Binding Proteins/genetics , Chromosome Mapping , DNA/blood , DNA/genetics , Exons , Female , Genetic Linkage , Genetic Markers , Hippocalcin , Humans , Intermediate Filament Proteins/genetics , Lod Score , Male , Pedigree , Peripherins , Recoverin , Retinaldehyde/genetics , Retinitis Pigmentosa/blood , Rhodopsin/genetics , South Africa
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