Michèle Ramsay

Effect of Alcohol Consumption on CpG Methylation in the Differentially Methylated Regions of H19 and IG-DMR in Male Gametes—Implications for Fetal Alcohol Spectrum Disorders

BACKGROUND Exposure to alcohol in utero is the main attributable cause of fetal alcohol spectrum disorders (FASD) which in its most severe form is characterized by irreversible behavioral and cognitive disability. Paternal preconception drinking is not considered to be a significant risk factor, even though animal studies have demonstrated that chronic paternal alcohol consumption has a detrimental effect on the physical and mental development of offspring even in the absence of in utero alcohol exposure. It has been documented that alcohol can reduce the levels and activity of DNA methyltransferases resulting in DNA hypomethylation and that reduced methyltransferase activity can cause activation of normally silenced genes. The aim of this study was to establish a link between alcohol use in men and hypomethylation of paternally imprinted loci in sperm DNA in genomic regions critical for embryonic development, thus providing a mechanism for paternal effects in the aetiology of FASD. METHODS Sperm DNA from male volunteers was bisulfite treated and the methylation patterns of 2 differentially methylated regions (DMRs), H19 and IG-DMR, analyzed following sequencing of individual clones. The methylation patterns were correlated with the alcohol consumption levels of the volunteer males. RESULTS There was a pattern of increased demethylation with alcohol consumption at the 2 imprinted loci with a significant difference observed at the IG-DMR between the nondrinking and heavy alcohol consuming groups. Greater inter-individual variation in average methylation was observed at the H19 DMR and individual clones were more extensively demethylated than those of the IG-DMR. CpG site #4 in the IG-DMR was preferentially demethylated among all individuals and along with the H19 DMR CpG site #7 located within the CTCF binding site 6 showed significant demethylation in the alcohol consuming groups compared with the control group. CONCLUSION This study demonstrates a correlation between chronic alcohol use and demethylation of normally hypermethylated imprinted regions in sperm DNA. We hypothesize that, should these epigenetic changes in imprinted genes be transmitted through fertilization, they would alter the critical gene expression dosages required for normal prenatal development resulting in offspring with features of FASD.

Exposure of Mouse Embryos to Ethanol During Preimplantation Development: Effect on DNA Methylation in the H19 Imprinting Control Region

In the present study, it was hypothesized that disruption of imprinting control in the H19/Igf2 domain may be a mechanism of ethanol-induced growth retardation—a key clinical feature of the fetal alcohol spectrum disorders (FASD). To test this prediction, genomic bisulphite sequencing was carried out on 473 bp of the H19 imprinting control region in DNA obtained from midgestation F(1) hybrid mouse embryos (C57BL/6 × Mus musculus castaneus) exposed to ethanol during preimplantation development. Although ethanol-exposed placentae and embryos were severely growth retarded in comparison with saline-treated controls, DNA methylation at paternal and maternal alleles was unaffected in embryos. However, paternal alleles were significantly less methylated in ethanol-treated placentae in comparison with saline-treated controls. Partial correlations suggested that the relationship between ethanol and placental weight partly depended on DNA methylation at a CCCTC-binding factor site on the paternal allele in placentae, suggesting a novel mechanism of ethanol-induced growth retardation. In contrast, partial correlations suggested that embryo growth retardation was independent of placental growth retardation. Relaxation of allele-specific DNA methylation in control placentae in comparison with control embryos was also observed, consistent with a model of imprinting in which 1) regulation of allele-specific DNA methylation in the placenta depends on a stochastic interplay between silencer and enhancer chromatin assembly factors and 2) imprinting control mechanisms in the embryo are more robust to environmental perturbations.

APOL1 Risk Variants Are Strongly Associated with HIV-Associated Nephropathy in Black South Africans

APOL1 variants are associated with HIV-associated nephropathy and FSGS in African Americans. The prevalence of these variants in African populations with CKD in HIV-1 infection has not been investigated. We determined the role of APOL1 variants in 120 patients with HIV-associated nephropathy and CKD and 108 controls from a South-African black population. Patients with CKD were selected on the basis of histology. Genotypes were successfully determined for APOL1 G1 and G2 variants and 42 single nucleotide polymorphisms, including 18 ancestry informative markers, for 116 patients with CKD (96.7%; 38 patients with HIV-associated nephropathy, 39 patients with HIV-positive CKD, and 39 patients with HIV-negative CKD), and 108 controls (100%). Overall, 79% of patients with HIV-associated nephropathy and 2% of population controls carried two risk alleles. In a recessive model, individuals carrying any combination of two APOL1 risk alleles had 89-fold higher odds (95% confidence interval, 18 to 912; P<0.001) of developing HIV-associated nephropathy compared with HIV-positive controls. Population allele frequencies were 7.3% for G1 and 11.1% for G2. APOL1 risk alleles were not significantly associated with other forms of CKD. These results indicate HIV-positive, antiretroviral therapy-naïve South-African blacks with two APOL1 risk alleles are at very high risk for developing HIV-associated nephropathy. Further studies are required to determine the effect of APOL1 risk variants on kidney diseases in other regions of sub-Saharan Africa.

The effect of preconception paternal alcohol exposure on epigenetic remodeling of the H19 and Rasgrf1 imprinting control regions in mouse offspring

Imprinted loci play a critical role in fetal development. Their expression is often regulated by CCCTC-binding factor (CTCF) protein binding at imprinting control regions (ICRs). Prenatal alcohol exposure has been shown to reduce global DNA methylation in the developing mouse fetus. This study explored the effect of preconception paternal alcohol exposure on DNA methylation at two paternally methylated ICRs (H19 and Rasgrf1) in the sperm of exposed males and somatic DNA of sired offspring. Significant reductions at the H19 CTCF 1 (p = 0.0027) and CTCF 2 (p = 0.0009) binding sites were observed in the offspring of ethanol-treated sires, which was significantly correlated with reduced weight at postnatal days 35–42 (p < 0.05). As birth weight was unaffected and growth was only delayed during the postnatal weaning period, with subsequent re-convergence, we hypothesize that this may be the result of a mental deficit causing delayed establishment of independent feeding following weaning and would explain why this effect is transient. No difference in DNA methylation was observed in the sperm of alcohol-exposed males, indicating that the transmission of the epigenetic signal at conception is not due to altered methylation, but may be the result of an RNA-mediated mechanism or altered chromatin remodeling.

DNA Polymorphism and Selection at the Melanocortin-1 Receptor Gene in Normally Pigmented Southern African Individuals

Abstract: Skin pigmentation is a polygenic multifactorial trait determined by the cumulative effects of multiple genetic variants and environmental factors. Melanocortin‐1 receptor (MC1R) is one of the genes involved in pigmentation, and has been implicated in the red hair and pale skin phenotype in human Caucasoid individuals. The present study was undertaken to identify variation at the MC1R locus in normally pigmented individuals in two African populations, sub‐Saharan Negroids (22 unrelated individuals) and the San (17 unrelated individuals). The study showed considerable MC1R gene sequence variation with the detection of eight synonymous and three nonsynonymous mutations. This is the first report of nonsynonymous mutations in African individuals in the MC1R gene: L99I was found in a single San individual, S47I was detected in a single Negroid individual, and F196L was detected in five Negroid individuals (5/44; 0.11). The functional significance of these mutations is not known. Three of the eight synonymous mutations found, L106L (CTG → CTA), F300F (TTC → TTT), and T314T (ACA → ACG) (also known as A942G), have been reported previously. T314T was the only variant that showed a significant difference between the Negroid and San populations (0.477 and 0.059, respectively; P= 1.6× 10−5). Its low frequency in the San may be the result of random genetic drift in a population of small size, or selection. Several tests of neutrality of the MC1R coding region in these and other African populations were significant, suggesting that purifying selection (functional constraint) had occurred at this gene locus in Africans. This demonstrates that although some nonsynonymous MC1R mutations are tolerated in individuals with dark skin, this gene has likely played a significant role in the maintenance of dark pigmentation in Africans and normal pigment variation in non‐African populations.

First report of CFTR mutations in black cystic fibrosis patients of southern African origin

Cystic fibrosis (CF) is thought to be rare in the black populations of Africa who have minimal white admixture. Only a few cases have been reported but have not been studied at the molecular level. We report the detection of CFTR mutations in three southern African black patients. One was homozygous for the 3120 + 1G-->A mutation, while the other two were compound heterozygotes each with this mutation on one chromosome. The other mutations were G1249E and a previously unreported in frame 54 bp deletion within exon 17a involving nucleotides 3196-3249 (3196del54). The 3120 + 1G-->A mutation was first described in American black patients and has been shown to be a common mutation in this population (9-14% of CF chromosomes). It was also found in a black CF patient whose father, the 3120 + 1G-->A carrier, is from Cameroon. These data suggest that it is an old mutation which accounts for many of the CFTR mutations in African blacks.

Computational selection and prioritization of candidate genes for Fetal Alcohol Syndrome

BackgroundFetal alcohol syndrome (FAS) is a serious global health problem and is observed at high frequencies in certain South African communities. Although in utero alcohol exposure is the primary trigger, there is evidence for genetic- and other susceptibility factors in FAS development. No genome-wide association or linkage studies have been performed for FAS, making computational selection and -prioritization of candidate disease genes an attractive approach.Results10174 Candidate genes were initially selected from the whole genome using a previously described method, which selects candidate genes according to their expression in disease-affected tissues. Hereafter candidates were prioritized for experimental investigation by investigating criteria pertinent to FAS and binary filtering. 29 Criteria were assessed by mining various database sources to populate criteria-specific gene lists. Candidate genes were then prioritized for experimental investigation using a binary system that assessed the criteria gene lists against the candidate list, and candidate genes were scored accordingly. A group of 87 genes was prioritized as candidates and for future experimental validation. The validity of the binary prioritization method was assessed by investigating the protein-protein interactions, functional enrichment and common promoter element binding sites of the top-ranked genes.ConclusionThis analysis highlighted a list of strong candidate genes from the TGF-β, MAPK and Hedgehog signalling pathways, which are all integral to fetal development and potential targets for alcohols teratogenic effect. We conclude that this novel bioinformatics approach effectively prioritizes credible candidate genes for further experimental analysis.

The H3Africa policy framework: negotiating fairness in genomics

Human Heredity and Health in Africa (H3Africa) research seeks to promote fair collaboration between scientists in Africa and those from elsewhere. Here, we outline how concerns over inequality and exploitation led to a policy framework that places a firm focus on African leadership and capacity building as guiding principles for African genomics research.

Absence of GJB2 gene mutations, the GJB6 deletion (GJB6-D13S1830) and four common mitochondrial mutations in nonsyndromic genetic hearing loss in a South African population.

OBJECTIVE The purpose of this study was to determine the prevalence of mutations in the GJB2 gene, the GJB6-D13S1830 deletion and the four common mitochondrial mutations (A1555G, A3243G, A7511C and A7445G) in a South African population. METHODS Using single-strand conformation polymorphism and direct sequencing for screening GJB2 mutation; Multiplex PCR Amplification for GJB6-D13S1830 deletion and Restriction Fragment-Length Polymorphism (PCR-RFLP) analysis for the four common mtDNA mutations. We screened 182 hearing impaired students to determine the frequency of these mutations in the population. RESULTS None of the reported disease causing mutations in GJB2 nor any novel pathogenic mutations in the coding region were detected, in contrast to the findings among Caucasians. The GJB6-D13S1830 deletion and the mitochondrial mutations were not observed in this group. CONCLUSION These results suggest that GJB2 may not be a significant deafness gene among sub-Saharan Africans, pointing to other unidentified genes as responsible for nonsyndromic hearing loss in these populations.

Africa: Continent of genome contrasts with implications for biomedical research and health

The genomic architecture of African populations is poorly understood and there is considerable variation between ethno‐linguistic groups. Genome‐wide approaches have been extensively applied to search for genetic associations to complex traits in Europeans, but rarely in Africans. This is largely attributed to lower levels of funding, poor infrastructure and public health systems, and to the small pool of trained scientists. High levels of genetic variation and underlying population structure in Africans present significant challenges, but lower levels of linkage disequilibrium provide an opportunity for more effective localisation of causal variants. High throughput technologies, including dense genotyping arrays, genome sequencing and epigenome studies, together with plummeting costs, are making research more affordable, even for African scientists. Understanding the interactions between genome structure and environmental influences is essential to interpreting their contributions to the increase in infectious diseases and non‐communicable diseases, exacerbated by adverse environments and lifestyle choices. The unique genome dynamics in African populations have an important role to play in understanding human health and susceptibility to disease.