Molecular diagnosis of hereditary hemochromatosis: application of a newly-developed reverse-hybridization assay in the South African population.

A recently developed strip-assay for hemochromatosis provides a rapid method for simultaneous detection of multiple mutations, which among others includes the HFE gene mutations V53M, V59M, H63D, H63H, S65C, Q127H, E168Q, and C282Y, previously detected in the general South African population using gel-based mutation-screening methods. The objective of the study was to determine the frequency of the relatively rare mutations in samples selected for altered iron parameters or a family history of hereditary hemochromatosis (HH) as part of the validation process of the assay for routine diagnostic purposes. The study population consisted of 451 individuals previously screened for mutations C282Y and H63D by restriction enzyme analysis in order to confirm or possibly exclude a diagnosis of HH. These individuals were subjected to mutation screening using the commercially available hemochromatosis strip-assay. Previous positive results for mutations C282Y and H63D in 233 individuals confirmed the accuracy of the reverse-hybridization assay. Mutation S65C was detected in 13 Caucasians, including three compound heterozygotes. These constituted 2% (13/600) of the chromosomes without mutations C282Y or H63D. The African-specific HFE mutation V53M was detected in one out of 11 (9%) African subjects screened. Mutation E168Q was detected in a single Caucasian individual together with mutation H63D. Our data demonstrate the value of the strip-based technology in providing a rapid and reliable comprehensive test for simultaneous analysis of multiple mutations.

Allelic variation in the promoter region of the LDL receptor gene: analysis of an African-specific variant in the FP2 cis-acting regulatory element.

DNA samples of 2303 individuals from nine different population groups were screened for variant -175g-->t in the promoter region of the low-density lipoprotein receptor (LDLR) gene. The -175g-->t variant detected at carrier frequencies of 3-10% in different African population groups was absent in the Caucasian and Asian (Chinese) individuals studied. In contrast to previous findings in Black South Africans where this polymorphism predominated in patients with familial hypercholesterolaemia (FH), it occurred at a significantly lower frequency in hypercholesterolaemics from the recently admixed Coloured population of South Africa compared with population-matched controls (P<0.0001). Haplotype and mutation analysis excluded the likelihood that this finding is due to association with a specific disease-related mutation in FH patients, although reversal of the positive association with FH observed in the Black population may, at least in part, be due to admixture linkage disequilibrium. Transient transfection studies in HepG2 cells demonstrated that the -175t allele is associated with a non-significant decrease ( approximately 7%) of LDLR transcription in the absence of sterols. The data presented in this study raise the possibility that the -175g-->t polymorphism may have subtle effects that become clinically important within certain genetic and/or environmental contexts.

Single nucleotide polymorphisms of the protoporphyrinogen oxidase gene: inter-population heterogeneity of allelic variation.

Five single nucleotide polymorphisms (SNPs) in the protoporphyrinogen oxidase gene (PPOX) were used for inter-population comparisons of six South African populations and two non-South African Caucasian populations. Novel polymorphisms identified in the promoter region and exon 11 of the PPOX gene, as well as three known variants in exon 1 and intron 2, were analysed using single-strand conformation polymorphism (SSCP) and restriction enzyme analyses. Significant population differences were found for four of the five polymorphisms analysed. A G-to-A transition was found at nucleotide position -1081 and is the first polymorphism to be identified in the 5' promoter region of the gene. A novel A-to-C substitution at nucleotide position 3880 in exon 11 was not detected in subjects of European descent. This study represents the first inter-population comparison of allelic variation at the PPOX locus. The significant differences observed between populations demonstrate the importance of population considerations when marker association studies are performed at this locus.

Analysis of the NRAMP1 gene implicated in iron transport: association with multiple sclerosis and age effects.

Multiple sclerosis (MS) is believed to be an autoimmune process occurring in genetically susceptible individuals after an appropriate environmental exposure. We have exploited the homogeneous Afrikaner population of European ancestry to investigate the likelihood that iron dysregulation, in association with infectious and/or autoimmune disease susceptibility, may underlie the MS phenotype in a subgroup of patients. The functional Z-DNA forming repeat polymorphism of the natural resistance-associated macrophage protein-1 (NRAMP1) gene was analyzed in 104 patients diagnosed with MS and 522 Caucasian controls. A family-based control group consisting of 32 parental alleles not transmitted to MS offspring was additionally studied to exclude the likelihood of population substructures. Statistically significant differences in allelic distribution were observed between the patient and control samples drawn from the same population (P < 0.01). Evidence is furthermore provided that alleles considered to be detrimental in relation to autoimmune disease susceptibility may be maintained in the population as a consequence of improved survival to reproductive age following infectious disease challenge. Although it remains to be determined whether the disease phenotype in MS patients with allele 5 of the NRAMP1 promoter polymorphism is directly related to dysregulation of iron or modified susceptibility to viral infection and/or autoimmunity, a combination of these processes most likely underlies the disease phenotype in these patients. In view of the emerging role of polymorphic variants in complex diseases and minimizing of possible confounding factors in this association study, we conclude that allelic variation in the NRAMP1 promoter may contribute significantly to MS susceptibility in the South African Caucasian population.

Mutation analysis in a small cohort of New Zealand patients originating from the United Kingdom demonstrates genetic heterogeneity in familial hypercholesterolemia.

Familial hypercholesterolemia (FH) and familial defective apolipoprotein B-100 (FDB) are relatively common lipid disorders caused by mutations in the low-density lipoprotein receptor (LDLR) and apolipoprotein B (apo B) genes, respectively. Molecular analysis at these loci was performed in eight New Zealand subjects with clinical features of heterozygous FH. Utilization of an in vitro lymphocyte receptor assay demonstrated normal receptor function in four patients, three of whom screened positive for the founder-type apo B mutation, R3500Q, causing FDB. Four patients with reduced LDLR function, consistent with heterozygous FH, revealed three previously documented mutations in exons 3 (W66X), 6 (C292Y) and 7 (G322S) of the LDLR gene and, a novel 2-bp deletion (TC or CT) after nucleotide 1204 (or 1205) in exon 9. The remaining patient was found to be FH/FDB negative after extensive mutation screening using both denaturing gradient gel electrophoresis and heteroduplex-single strand conformation polymorphism analysis. Haplotype analysis at the LDLR and apo B loci finally excluded the likelihood that mutations in these two genes underlie the FH phenotype in the molecularly uncharacterized New Zealand family originating from the United Kingdom. This family represents a valuable source of material for future genetic dissection of autosomal dominant hypercholesterolemia (ADH), shown to be a heterogeneous disease through molecular analysis.

A reverse-hybridization assay for the rapid and simultaneous detection of nine HFE gene mutations.

Hereditary hemochromatosis (HH) is a very common autosomal recessive disorder of iron metabolism and frequently associated with mutations in the HFE gene. Molecular genetic testing for HFE mutations is considered valuable for carrier identification, as well as for early diagnosis of the disease, allowing simple treatment by phlebotomy and normal survival of patients. We have developed a reverse-hybridization assay for the routine diagnosis of eight previously described and one novel (E168Q) HFE point mutations. The test is based on multiplex DNA amplification and ready-to-use membrane teststrips, which contain oligonucleotide probes for each wild-type and mutated allele immobilized as an array of parallel lines. The procedure is rapid and accessible to automation on commercially available equipment, and by adding new probes the teststrip can easily be adapted to cover an increasing number of mutations.

Predominance of a 6 bp deletion in exon 2 of the LDL receptor gene in Africans with familial hypercholesterolaemia.

In South Africa, the high prevalence of familial hypercholesterolaemia (FH) among Afrikaners, Jews, and Indians as a result of founder genes is in striking contrast to its reported virtual absence in the black population in general. In this study, the molecular basis of primary hypercholesterolaemia was studied in 16 Africans diagnosed with FH. DNA analysis using three screening methods resulted in the identification of seven different mutations in the coding region of the low density lipoprotein (LDLR) gene in 10 of the patients analysed. These included a 6 bp deletion (GCGATG) accounting for 28% of defective alleles, and six point mutations (D151H, R232W, R385Q, E387K, P678L, and R793Q) detected in single families. The Sotho patient with missense mutation R232W was also heterozygous for a de novo splicing defect 313+1G-->A. Several silent mutations/polymorphisms were detected in the LDLR and apolipoprotein B genes, including a base change (g-->t) at nucleotide position -175 in the FP2 LDLR regulatory element. This promoter variant was detected at a significantly higher (p<0.05) frequency in FH patients compared to controls and occurred in cis with mutation E387K in one family. Analysis of four intragenic LDLR gene polymorphisms showed that the same chromosomal background was identified at this locus in the four FH patients with the 6 bp deletion. Detection of the 6 bp deletion in Xhosa, Pedi, and Tswana FH patients suggests that it is an ancient mutation predating tribal separation approximately 3000 years ago.

Earlier age of onset of Alzheimer's disease in patients with both the transferrin C2 and apolipoprotein E-epsilon 4 alleles.

The etiology of Alzheimer's disease is now known to be multifactorial. The genetic factors transferrin C2 (TfC2) and apolipoprotein E epsilon 4 (ApoE-epsilon 4) have both been associated with Alzheimer's disease (AD). Transferrin is the carrier protein for iron in the blood, while ApoE is involved with the transport and redistribution of lipids. In the present study, the polymerase chain reaction (PCR) method was used to determine the frequency of both TfC2 and ApoE-epsilon 4 in 27 AD patients, 9 vascular dementia (VaD) patients, and 27 controls. Patients were diagnosed according to the criteria as set out in the 4th edition of the Diagnostic and Statistical Manual of the American Psychiatric Association (DSM-IV). The frequency of the TfC2 allele for the AD patients was 24%, while for the VaD patients it was 12.5%, which was not significantly different from the controls at 13%. The frequency of ApoE-epsilon 4 for the AD patients was 44%, for the VaD patients 22%, and controls 17%. Of the 27 AD patients, 8 had both TfC2 and ApoE-epsilon 4. The age of onset of the disease in these 8 patients (51-67 years, mean 60.25) was significantly earlier (p < 0.02) than in the remaining AD patients (49-76 years, mean 66.9). None of the VaD patients had both the TfC2 and the ApoE-epsilon 4 alleles.

Significance of linkage disequilibrium between mutation C282Y and a MseI polymorphism in population screening and DNA diagnosis of hemochromatosis.

An increasing number of studies demonstrate a lack of phenotypic expression in subjects found to be homozygous for the common hereditary hemochromatosis (HH) mutation, C282Y. In this study the impact of possible overestimation of C282Y homozygosity, as a consequence of a MseI polymorphism identified in intron 4 of the HFE gene, was investigated in South African subjects. Utilization of a modified polymerase chain reaction (PCR)-based assay highlighted the potential implications with respect to genotype/phenotype correlation studies, particularly in the general population. Mistyping rather than lack of disease association provides a plausible explanation for the phenomenon of C282Y homozygosity without iron overload. Reassessment of C282Y mutation status in such cases may result in justified population screening in HH.

Mutation -59c-->t in repeat 2 of the LDL receptor promoter: reduction in transcriptional activity and possible allelic interaction in a South African family with familial hypercholesterolaemia.

The low-density lipoprotein receptor (LDLR) plays a major role in cholesterol homeostasis. Mutations in the regulatory region of the LDLR gene, although rare, have been shown to alter transcriptional activity of the gene and can cause familial hypercholesterolaemia (FH). In this study, a transition (c-->t) was identified at nucleotide position -59 within repeat 2 of the LDLR promoter in a South African FH patient of mixed ancestry. By screening 17 family members of the index case for this promoter mutation, two additional single base changes (-124c-->t and-175g-->t) were identified, located at recently described cis- acting regulatory sequences of the LDLR promoter. Both the-59c-->t and the-124c-->t transitions were identified in the normocholesterolaemic son of the index patient. Reporter plasmids containing the normal and mutant promoter fragments were constructed by directional cloning. Transcription studies using a luciferase reporter system demonstrated that the-59c-->t mutation significantly reduces promoter activity in both the presence and absence of sterols ( approximately 40% of normal activity), while the-124c-->t variant increases transcription ( approximately 160%) of the LDLR gene. The intra-familial phenotypic variability observed amongst individuals with the-59c-->t mutation can probably be ascribed to allelic interaction, suggesting that variation in the LDLR promoter region may contribute significantly to the phenotypic expression of FH-related mutations in populations where these mutations prevail.

Intronic mutations at splice junctions in the low-density lipoprotein receptor gene.

Most of the low-density lipoprotein receptor (LDLR) gene mutations causing familial hypercholesterolemia (FH) have been identified in the coding region of the gene. We have screened 180 patients for disease-related gene defects and report the identification of three previously described (IVS3+1G-->A, IVS9-1G-->A and IVS16-2A-->G) and two novel mutations (IVS2+1G-->A and IVS14+1G-->T) at splice junctions. Approximately 9% (38/404) of LDLR gene point mutations identified to date in FH patients occur in introns and may affect splicing. The severe consequences of these mutations make them an important target for the molecular analysis of FH.

Founder mutations in the LDL receptor gene contribute significantly to the familial hypercholesterolemia phenotype in the indigenous South African population of mixed ancestry.

The South African population harbors genes that are derived from varying degrees of admixture between indigenous groups and immigrants from Europe and the East. This study represents the first direct mutation-based attempt to determine the impact of admixture from other gene pools on the familial hypercholesterolemia (FH) phenotype in the recently founded Coloured population of South Africa, a people of mixed ancestry. A cohort of 236 apparently unrelated patients with clinical features of FH was screened for a common mutation causing familial defective apolipoprotein B-100 (FDB) and seven low-density lipoprotein receptor (LDLR) gene defects known to be relatively common in South Africans with FH. Six founder-type 'South African mutations' were responsible for FH in approximately 20% of the study population, while only 1 patient tested positive for the familial defective apolipoprotein B-100 mutation R3500Q. The detection of multiple founder-type LDLR gene mutations originating from European, Indian and Jewish populations provides direct genetic evidence that Caucasoid admixture contributes significantly to the apparently high prevalence of FH in South African patients of mixed ancestry. This study contributes to our knowledge of the biological history of this unique population and illustrates the potential consequences of recent admixture in populations with different disease risks.

Spectrum of mutations in the HFE gene implicated in haemochromatosis and porphyria.

Mutation analysis was performed on DNA samples of 965 individuals from four different ethnic groups in South Africa, in an attempt to determine the spectrum of sequence variants in the haemochromatosis ( HFE ) gene. This population screening approach, utilizing a combined heteroduplex and single-strand conformation polymorphism (HEX-SSCP) method, revealed three previously described and four novel missense mutations. Novel variants V53M and V59M were identified in exon 2, Q127H in exon 3 and R330M in exon 5. The exon 5 variant was identified in one of 13 patients referred for a molecular diagnosis of hereditary haemochromatosis (HH), who tested negative for the known C282Y and H63D mutations. Mutation Q127H was detected in exon 3 of the HFE gene together with mutation H63D in an apparently severely affected patient previously shown to carry the protoporphyrinogen oxidase ( PPOX ) gene mutation R59W, which accounts for dominantly inherited variegate porphyria (VP) in >80% of affected South Africans. The mutant allele frequency of the C282Y mutation was found to be significantly lower in 73 apparently unrelated VP patients with the R59W mutation than in 102 controls drawn from the same population ( P = 0.005). The population screening approach used in this study revealed considerable genotypic variation in the HFE gene and supports previous data on the involvement of this gene in the porphyria phenotype.

High prevalence of the Cys282Tyr HFE mutation facilitates an improved diagnostic service for hereditary haemochromatosis in South Africa.

OBJECTIVE: The aim of the study was to investigate the molecular basis of hereditary haemochromatosis (HH) in South Africa in order to establish a reliable, cost-effective molecular diagnostic service for this potentially lethal disorder. DESIGN: DNA samples of patient and control groups were screened for two common haemochromatosis (HFE) gene mutations. The local frequencies of mutations C282Y and H63D were determined and the DNA results correlated with biochemical parameters. SETTING: Patients were referred from private practitioners, health workers and pathologists for a molecular diagnosis of HH at the University of Stellenbosch Medical School. Twenty-two of the 244 referrals were clinically diagnosed with HH, while the remaining patients were family members of the probands or unrelated subjects referred solely on the basis of an abnormal iron profile. RESULTS: Seventeen of the 22 patient referrals (77%) diagnosed with HH were homozygous for the C282Y mutation, 3 (14%) were compound heterozygotes for mutations C282Y and H63D, and 2 patients (9%) did not exhibit either mutation. Screening of 458 control individuals from the general South African population demonstrated a carrier frequency of approximately 17% for the C282Y mutation among whites, implying that up to 1 out of every 115 South Africans of European descent may be homozygous for this founder-type mutation. Among 64 healthy blood donors of mixed ancestry, we detected 2 individuals heterozygous and 1 homozygous for the C282Y mutation. CONCLUSIONS: The detection of mutations C282Y and H63D at a high frequency in the majority of affected South African patients facilitates accurate pre-clinical and confirmatory diagnosis of HH in South Africa. Early detection by DNA screening and subsequent treatment by repeated phlebotomy can prevent disease onset in affected individuals. DNA diagnosis is particularly applicable to a common genetic disease such as HH, which is underdiagnosed and potentially lethal, but treatable.

Molecular analysis reveals a high mutation frequency in the first untranslated exon of the PPOX gene and largely excludes variegate porphyria in a subset of clinically affected Afrikaner families.

A subset of probands from 11 South African families with clinical and/or biochemical features of variegate porphyria (VP), but without the known protoporphyrinogen oxidase (PPOX) gene defects identified previously in the South African population, were subjected to mutation analysis. Disease-related mutation(s) could not be identified after screening virtually the entire PPOX gene by heteroduplex single-strand conformation polymorphism analysis (HEX-SSCP), although three new sequence variants were detected in exon 1 of the gene in three normal controls. The presence of these single base changes at nucleotide positions 22 (C/G), 27 (C/A) and 127 (C/A), in addition to the known exon 1 polymorphisms I-26 and I-150, indicates that this untranslated region of the PPOX gene is particularly mutation-prone. Furthermore, microsatellite markers flanking the PPOX and alpha-1 antitrypsin (PI) gene, on chromosomes 1 and 14, respectively, were used to assess the probability of involvement of these loci in disease presentation. Common alleles transmitted from affected parent to affected child were determined where possible in the mutation-negative index cases. Allelic frequencies of these <> alleles were compared to findings in the normal population, but no predominant disease-associated allele could be identified. Co-segregation of a specific haplotype with the disease phenotype could also not be demonstrated in a large Afrikaner family. It is concluded that further studies are warranted to determine the genetic factor(s) underlying the autosomal dominant pattern of inheritance in molecularly uncharacterized cases showing clinical symptoms of an acute porphyria.

Intensive short course chemotherapy in the management of tuberculous meningitis.

SETTING: Short course chemotherapy for tuberculous meningitis (TBM) is advocated by several groups, but relatively few children have been so treated and followed up. METHODS: A prospective, observational study of isoniazid (INH), rifampicin (RMP) and ethionamide (ETH) in a dosage of 20 mg/kg, and pyrazinamide (PZA) 40 mg/kg, all given once daily in hospital for 6 months. Surviving children were followed up for a year after discharge. RESULTS: Ninety five children, 39 (41%) at stage III, 52 (55%) at stage II and 4 (4%) at stage I TBM were studied. Ten (26%) at stage III and 3 (6%) at stage II died before completion of therapy. Five surviving children (6%) moved on discharge and were untraceable; seven children (9%) were lost during follow up and three were inadvertently restarted on antituberculosis therapy. Two children with severe stage III disease died after discharge. One child experienced a probable disease recrudescence 1 month after discharge. Eighteen children (20%) developed a mildly elevated serum bilirubin concentration during the first month of treatment. In five of these children INH, RMP, ETH and PZA were stopped and streptomycin (SM) and ethambutol substituted. In all cases the original treatment was restarted without incident. One child developed overt jaundice after 5 months of treatment due to hepatitis A infection. CONCLUSIONS: Our experience suggests that young children with TBM can be safely treated for 6 months with high doses of antituberculosis agents without overt hepatotoxicity and with a low risk of relapse.

Familial hypercholesterolemia: potential diagnostic value of mutation screening in a pediatric population of South Africa.

Three founder-related low-density lipoprotein receptor (LDLR) gene mutations, D154N, D206E and V408M, cause familial hypercholesterolemia (FH) in approximately 90% of South African Afrikaners. Two hundred and twenty-one South African children, from 85 affected families, were screened for the specific mutation identified previously in the index case. Sixty boys and 56 girls were heterozygous for mutation D154N (FH3), D206E (FH1) or V408M (FH2). Total and LDL cholesterol (LDLC) levels were similar among the children heterozygous for the three founder mutations, and mean values were significantly higher compared to those without a known mutation (p < 0.0001). Plasma cholesterol levels overlapped considerably between the different groups, suggesting that modifiable lifestyle factors remain important in children with FH. This study demonstrates the potential diagnostic value of mutation screening in a pediatric population with an enrichment of particular gene mutations.

Mutation analysis in familial hypercholesterolemia patients of different ancestries: identification of three novel LDLR gene mutations.

Twelve familial hypercholesterolemia (FH) patients of different ancestries living in South Africa were subjected to mutation analysis of the low-density lipoprotein receptor (LDLR) gene. Nine different mutations were identified in 10 patients. Six of these, including the founder-related mutation C660X identified in two Lebanese patients, have previously been described in other FH patients with compatible genetic backgrounds, and/or in patients originating from countries where admixture is not uncommon. Characterization of an abnormal electrophoresis pattern detected in exon 4 of the LDLR gene by heteroduplex single-strand conformation polymorphism (HEX-SSCP) analysis, revealed a novel G deletion at codon 185 (617delG) which resulted in a downstream stop codon. Two of the new mutations identified resulted in amino acid substitutions and were designated R57C and Q357P.