Mendelian-inherited heart disease: a gateway to understanding mechanisms in heart disease Update on work done at the University of Stellenbosch.

The presence of founder effects in South Africa for many single-gene diseases, which include heart diseases such as progressive familial heart block types I and II, hypertrophic cardiomyopathy and the long QT syndromes, afforded us the opportunity to identify causal genes and associated mutations through genetic mapping and positional cloning. From finding the genes, the emphasis has shifted to elucidating how primary defects cause disease and recognising factors that could explain the often pronounced phenotypic variability seen in persons carrying the same inherited defect. In some of these diseases, sudden unexpected death has been a frequent occurrence in young, apparently healthy individuals who had not been aware that they had inherited an underlying risk. Herein, we review progress in identifying genes, mutations and risk factors associated with the diseases mentioned.

Progressive familial heart block type II (PFHBII): a clinical profile from 1977 to 2003.

An evaluation of a 38-year-old Caucasian woman, who was referred to Tygerberg Hospital (Western Cape Province, RSA) with Wenckebach second-degree or possibly complete atrioventricular (AV) block that had progressed from first-degree AV block, identified a family history of the cardiac conduction system disorder progressive familial heart block type II (PFHBII). This prompted a retrospective clinical review of the subjects described in the original study, as well as additional family members who had not been examined in the original study. Progression of clinical features was observed, but more importantly, PFHBII was clinically redefined as an AV nodal disorder, which may progress to dilated cardiomyopathy (DCM).

Violence in male patients with schizophrenia: risk markers in a South African population.

OBJECTIVE: We investigate the role of functional variants in the catecholamine-O-methyl transferase gene (COMT) and the monoamine oxidase-A gene (MOA-A), as well as previously identified non-genetic risk factors in the manifestation of violent behaviour in South African male schizophrenia patients. METHOD: A cohort of 70 acutely relapsed male schizophrenia patients was stratified into violent and non-violent subsets, based on the presence or absence of previous or current violent behaviour. Standardized violence rating scales were also applied and the COMT/NlaIII and MAO-A promoter region variable number of tandem repeats (VNTR) polymorphisms were genotyped. RESULTS: A multiple logistic regression model based on the clinical, genetic and socio-demographic variables indicated that delusions of control (OR = 3.7, 95% CI = 1.21-11.61) and the combined use of cannabis and alcohol (OR = 6.89, 95% CI = 1.28-37.05) were two significant predictors of violent behaviour in this schizophrenia population. No association was found between the tested polymorphisms and violent behaviour. CONCLUSIONS: Although the sample size may have limited power to exclude a minor role for these specific gene variants, such a small contribution would have limited clinical relevance given the strong significance of the non-genetic markers. These findings suggest that currently proactive management of violent behaviour in this schizophrenia population should continue to be based on clinical predictors of violence.

Mutations of the light meromyosin domain of the beta-myosin heavy chain rod in hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (HCM) is caused by mutations in 9 sarcomeric protein genes. The most commonly affected is beta-myosin heavy chain (MYH7), where missense mutations cluster in the head and neck regions and directly affect motor function. Comparable mutations have not been described in the light meromyosin (LMM) region of the myosin rod, nor would these be expected to directly affect motor function. We studied 82 probands with HCM in whom no mutations had been found in MYH7 exons encoding the head and neck regions of myosin nor in the other frequently implicated disease genes. Primers were designed to amplify exons 24 to 40 of MYH7. These amplimers were subjected to temperature modulated heteroduplex analysis by denaturing high-performance liquid chromatography. An Ala1379Thr missense mutation in exon 30 segregated with disease in three families and was not present in 200 normal chromosomes. The mutation occurred on two haplotypes, indicating that it was not a polymorphism linked with another disease-causing mutation. The position of this residue within the LMM region of myosin suggests that it may be important for thick filament assembly or for accessory protein binding. A further missense mutation in exon 37, Ser1776Gly, segregated with disease in a single family and was absent from 400 population-matched control chromosomes. Because the Ser1776 residue occupies a core position in the myosin rod at which the substitution of glycine is extremely energetically unfavorable, it is likely to disrupt the coiled-coil structure. We conclude that mutation of the LMM can cause HCM and that such mutations may act through novel mechanisms of disease pathogenesis involving myosin filament assembly or interaction with thick filament binding proteins.

Obsessive-compulsive disorder and a novel polymorphism adjacent to the oestrogen response element (ERE 6) upstream from the COMT gene.

Family and twin studies have consistently provided evidence for involvement of genetic mechanisms in obsessive-compulsive disorder (OCD). This has given rise to association studies involving several candidate genes in an endeavour to identify susceptibility factors. One of the more promising candidate genes appears to be the catecol-O-methyltransferase (COMT) gene. Recent association studies in North American and Afrikaner populations have reported a likely association between a functional polymorphism of COMT (linked with COMT enzyme activity levels) and OCD. COMT expression has been demonstrated to be regulated by oestrogen through the oestrogen-response elements (EREs) in the promoter region of the gene. In the light of this association, the authors tested for an association between a novel polymorphism (C --> T transition) adjacent to ERE 6 in the promoter area of COMT and OCD in 48 Afrikaners and 48 ethnically matched controls. The C --> T transition was not significantly associated with OCD (P = 0.93) or gender (P = 0.67). These findings, although limited by a small sample size, suggest that the novel polymorphism adjacent to ERE 6 in the promoter area of COMT does not play a major role in the genetic predisposition to OCD.

Association between a catechol-o-methyltransferase polymorphism and obsessive-compulsive disorder in the Afrikaner population.

BACKGROUND: It has been proposed that the catechol-o-methyl transferase gene (COMT) may play a role in the pathogenesis of obsessive-compulsive disorder (OCD). Whereas studies in a North American population showed that the low activity (L) allele of a functional polymorphism in COMT was associated with OCD in male patients, this result was not supported by studies in a Japanese population. The present association study assessed the risk for OCD conferred by this COMT polymorphism in a geographically different patient group, namely, the relatively genetically homogeneous Afrikaner population of South Africa. METHODS: Fifty-four unrelated OCD patients and fifty-four sex-matched controls were recruited from the same Afrikaner community. Patients and controls were phenotyped (DSM-IV) and genotyped for a NlaIII polymorphism with H (high activity) or L (low activity) alleles in the COMT gene. RESULTS: The H/L genotype was significantly more common than expected in the OCD patient group (P = 0.0017). LIMITATIONS: Replication studies with related individuals may be useful in discovering factors underpinning the H/L genotype abundance in the Afrikaner population. CONCLUSIONS: These results emphasise the need for further studies in genetically homogeneous populations to help define the complex etiology of this disease.

The origins of hypertrophic cardiomyopathy-causing mutations in two South African subpopulations: a unique profile of both independent and founder events.

Hypertrophic cardiomyopathy (HCM) is an autosomal dominantly inherited disease of the cardiac sarcomere, caused by numerous mutations in genes encoding protein components of this structure. Mutation carriers are at risk of sudden cardiac death, mostly as adolescents or young adults. The reproductive disadvantage incurred may explain both the global occurrence of diverse independent HCM-associated mutations and the rare reports of founder effects within populations. We have investigated whether this holds true for two South African subpopulations, one of mixed ancestry and one of northern-European descent. Previously, we had detected three novel mutations-Ala797Thr in the beta-myosin heavy-chain gene (betaMHC), Arg92Trp in the cardiac troponin T gene (cTnT), and Arg645His in the myosin-binding protein C gene (MyBPC)-and two documented betaMHC mutations (Arg403Trp and Arg249Gln). Here we report three additional novel mutations-Gln499Lys in betaMHC and Val896Met and Deltac756 in MyBPC-and the documented betaMHC Arg719Gln mutation. Seven of the nine HCM-causing mutations arose independently; no conclusions can be drawn for the remaining two. However, the betaMHC Arg403Trp and Ala797Thr and cTnT Arg92Trp mutations were detected in another one, eight, and four probands, respectively, and haplotype analysis in families carrying these recurring mutations inferred their origin from three common ancestors. The milder phenotype of the betaMHC mutations may account for the presence of these founder effects, whereas population dynamics alone may have overridden the reproductive disadvantage incurred by the more lethal, cTnT Arg92Trp mutation.

Identification of a new missense mutation in MyBP-C associated with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy is a primary cardiac disease, characterised by idiopathic myocardial hypertrophy, and is caused by defects in sarcomeric protein encoding genes. One of these genes is cardiac myosin binding protein C (MyBP-C), in which a number of splice site and duplication mutations causing HCM have been described. During mutation screening of a South African HCM population by PCR-SSCP, a missense mutation, Arg654His, was detected in one proband. Although the mutation was present in his three adult children, only the proband himself was markedly affected. This is the first report of a disease associated missense mutation in MyBP-C which does not affect the myosin or titin binding domains.

Sudden death due to troponin T mutations.

OBJECTIVES: This study was designed to verify initial observations of the clinical and prognostic features of hypertrophic cardiomyopathy caused by cardiac tropnin T gene mutations. BACKGROUND: The most common cause of sudden cardiac death in the young is hypertrophic cardiomyopathy, which is usually familial. Mutations causing familial hypertrophic cardiomyopathy have been identified in a number of contractile protein genes, raising the possibility of genetic screening for subjects at risk. A previous report suggested that mutations in the cardiac troponin T gene were notable because they were associated with a particularly poor prognosis but only mild hypertrophy. Given the variability of some genotype:phenotype correlations, further analysis of cardiac troponin T mutations has been a priority. METHODS: Deoxyribonucleic acid from subjects with hypertrophic cardiomyopathy was screened for cardiac troponin T mutations using a ribonuclease protection assay. Polymerase chain reaction-based detection of a novel mutation was used to genotype members of two affected pedigrees. Gene carriers were examined by echocardiography and electrocardiology, and a family history was obtained. RESULTS: A novel cardiac troponin T gene mutation, arginine 92 tryptophan, was identified in 19 of 48 members of two affected pedigrees. The clinical phenotype was characterized by minimal hypertrophy (mean [+/-SD] maximal ventricular wall thickness 11.3 +/- 5.4 mm) and low disease penetrance by clinical criteria (40% by echocardiography) but a high incidence of sudden cardiac death (mean age 17 +/- 9 years). CONCLUSIONS: These data support the observation that apparently diverse cardiac troponin T gene mutations produce a consistent disease phenotype. Because this is one of poor prognosis, despite deceptively mild or undetectable hypertrophy, genotyping at this locus may be particularly informative in patient management and counselling.

Evidence of a long QT founder gene with varying phenotypic expression in South African families.

We report five South African families of northern European descent (pedigrees 161, 162, 163, 164, and 166) in whom Romano-Ward long QT syndrome (LQT) segregates. The disease mapped to a group of linked markers on chromosome 11p15.5, with maximum combined two point lod scores, all generated at theta = 0, of 15.43 for the D11S922, 10.51 for the D11S1318, and 14.29 for the tyrosine hydroxylase (TH) loci. Recent studies have shown that LQT is caused by an Ala212Val mutation in a potassium channel gene (KVLQT1) in pedigrees 161 to 164. We report that the same mutation is responsible for the disease in pedigree 166. Haplotype construction showed that all the families shared a common haplotype, suggesting a founder gene effect. DNA based identification of gene carriers allowed assessment of the clinical spectrum of LQT. The QTc interval was significantly shorter in both carriers and non-carriers in pedigree 161 (0.48 s and 0.39 s, respectively) than the same two groups in pedigree 161 (0.52 s and 0.42 s, respectively). The spectrum of clinical symptoms appeared more severe in pedigree 162. The possible influence of modulating genetic factors, such as HLA status and sex of family members, on the expression of an LQT founder gene is discussed.

Clinical and prognostic evaluation of familial hypertrophic cardiomyopathy in two South African families with different cardiac beta myosin heavy chain gene mutations.

BACKGROUND: Familial hypertrophic cardiomyopathy is the most common inherited cardiac disorder, with sudden cardiac death at a young age the most frequent cause of death in affected individuals. Some cases of familial hypertrophic cardiomyopathy are caused by missense mutations of the beta myosin heavy chain (beta MHC) gene on chromosome 14 and at least 17 such mutations have been described. Recent reports suggest that a correlation exists between a specific beta MHC gene mutation and prognosis in familial hypertrophic cardiomyopathy. This premise is currently being used as a basis to provide counselling for affected families. This mutation/prognosis association, however, has not been widely assessed as yet. The clinical and prognostic features of two South African families of mixed racial descent, in which different beta MHC gene mutations were segregating, were studied to evaluate this correlation. The results were compared with those of previously published reports of European families carrying the same mutations. METHODS: The beta MHC gene missense mutations in two affected families were identified by single strand conformation polymorphism analysis and sequencing (pedigree 106: Arg403Trp; pedigree 108: Arg249Gln). All family members were subjected to genotypic analysis using polymerase chain reaction amplification and restriction enzyme based mutation detection techniques. Clinical, electrocardiographic, and echocardiographic studies were performed on genotypically affected individuals in these two kindreds. RESULTS: The number of individuals identified in pedigree 106 with the Arg403Trp mutation was 32.10 individuals bore the Arg249Gln mutation in pedigree 108. The penetrance rate in adults (equal to or greater than 16 years), using the strict echocardiographic criterion of maximum left ventricular wall thickness > or = 13 mm, was 25% for pedigree 106 and 33% for pedigree 108. Familial hypertrophic cardiomyopathy compatible electrocardiographic and echocardiographic abnormalities were seen in 60% of genotypically positive individuals aged > or = 16 years in pedigree 106 and 80% in pedigree 108. The prognosis was uniformly benign in the two families. For pedigree 106 this corresponded to a report of no early sudden cardiac deaths in a French family with the Arg403Trp mutation. For pedigree 108 the absence of such deaths was in apparent contrast to the four cases reported in 24 genotypically affected individuals in a study of a kindred of European ancestry bearing the Arg249Gln mutation. CONCLUSION: This study of a large South African kindred confirmed the benign nature of the Arg403Trp mutation suggested in a previous report. The number and the relatively young age of affected individuals in a second South African family must be considered when comparing the absence of familial hypertrophic cardiomyopathy associated deaths with the intermediate survival reported for the Arg249Gln mutation in a European family. This investigation lends support to current evidence relating specific beta MHC gene mutations to prognosis, which may be used as a basis to provide counselling for affected families.

Gene for progressive familial heart block type I maps to chromosome 19q13.

BACKGROUND: Progressive familial heart block type I (PF-HBI) is a dominantly inherited cardiac bundle-branch conduction disorder that has been traced through nine generations of a large South African kindred. Similar conduction disorders have been reported elsewhere; however, the cause of these diseases is unknown. The aim of the present study was to determine by linkage analysis the approximate chromosomal position of the gene causing PFHBI, thereby allowing family-based diagnosis and the development of positional cloning strategies to identify the causative gene. METHODS AND RESULTS: Eighty-six members of three pedigrees, 39 members of which were affected with PFHBI, were genotyped at four linked polymorphic marker loci mapped to chromosome 19, bands q13.2-q13.3 (chromosome 19q13.2-13.3). Maximum two-point logarithm of the odds scores (which represent the logarithm of the odds ratio of detecting linkage versus nonlinkage) generated were 6.49 (theta = 0) for the kallikrein locus, 5.72 (theta = 0.01) for the myotonic dystrophy locus, 3.44 (theta = 0) for the creatine kinase muscle-type locus and 4.51 (theta = 0.10) for the apolipoprotein C2 locus. The maximum multipoint logarithm of the odds score was 11.6, with the 90% support interval positioning the PFHBI locus within a 10 cM distance centering on the kallikrein 1 locus. CONCLUSIONS: The gene for PFHBI maps to an area of approximately 10 cM on chromosome 19q13.2-13.3. There are several candidate genes in this interval; although a recombination event ruled out the myotonic dystrophy locus from direct involvement with PFHBI, the proximity of these two loci may be relevant to the observed cardiac abnormalities of myotonic dystrophy. The results provide a means of DNA-based diagnosis in the families studied and a foundation for cloning studies to identify the causative gene.

Polymerase chain reaction-based detection of MN blood group-specific sequences in the human genome.

The MN blood group antigens have traditionally been detected by serotyping; however, development of a DNA-based method offers flexibility in the determination of this highly polymorphic system. Genotyping the MN blood group antigens was performed by polymerase chain reaction amplification of the specific alleles (PASA) in the human genome. In separate paired reactions, M or N allele-specific oligonucleotide primers were amplified with a common distal primer. Only in the presence of the homologous template was a 781-base pair polymerase chain reaction amplification product visible after agarose gel electrophoresis and ethidium bromide staining. This method of genotyping could be performed using either 1 microgram of extracted DNA or 0.5 microL of whole blood, and the results showed 100-percent correlation with those obtained by serotyping. PASA-based genotyping of MN blood group antigens, which requires a small amount of starting material, has application in linkage and population studies and in forensic medicine.

Synthesis of a wild-type and three mutant Cucurbita maxima trypsin inhibitor-encoding genes by a single-strand approach.

A single-strand approach to gene assembly, based on a modification of an in vitro complementary oligodeoxyribonucleotide template-directed ligation of the desired sequence to a linearized vector [Chen et al., Nucleic Acids Res. 18 (1990) 871-878], is described. The gene coding for the wild-type Cucurbita maxima trypsin inhibitor of 29 amino acid residues [Bode et al., FEBS Lett. 242 (1989) 285-292], as well as three mutant forms of the gene, in which two of the three disulfide bonds have been replaced singly or as a pair, have been synthesized in a single synthesis run with minimal manual intervention. Subsequent to ligation to pUC9 and in vivo gapped duplex repair by Escherichia coli, their sequences have been verified.

A modified protoplast-regeneration protocol facilitating the detection of cloned exoenzyme genes in Bacillus subtilis.

Incorporation of starch or casein into protoplast-regeneration medium facilitated shotgun cloning of alpha-amylase and neutral protease genes from an unidentified Bacillus sp. in Bacillus subtilis by polyethylene glycol-induced protoplast transformation. This modification and the use of the plasmid vector pPL603b enabled us to simultaneously select for promoter-bearing recombinant plasmids that expressed amylase or protease activity. The inserts were found to be 4 and 4.6 kb, respectively. Although protease activity directed by the cloned gene was only 2- to 4-fold higher than for the donor strain, that of alpha-amylase was 28-fold higher.