The identification and characterisation of the causative gene mutation for keratolytic winter erythema (KWE) in South African families

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment for the degree of Doctor of Philosophy Johannesburg, 2017Keratolytic winter erythema (KWE) is a rare autosomal dominant skin disorder characterized by recurrent episodes of palmoplantar erythema and epidermal peeling, and symptoms worsen in winter. KWE is relatively common in South African (SA) Afrikaners and was mapped to 8p23.1-p22 through a common haplotype in SA families. The aim of this study was to identify and characterize the causal mutation for KWE in SA families. Targeted resequencing of 8p23.1-22 was performed in three families and seven unrelated controls. Reads were aligned to the reference genome using BWA. GATK and Pindel were used to call small and large structural variants, respectively. A 7.67 kb tandem duplication was identified upstream of the CTSB gene and encompassing an enhancer element that is active in a keratinocytes (based on H3K27ac data). The tandem duplication segregated completely with the KWE. The tandem duplication overlaps with a 15.93 kb tandem duplication identified in two Norwegian families at a 2.62 kb region encompassing the active enhancer suggesting that the duplication of the enhancer leads to the KWE phenotype. Existing chromatin structure, CTCF binding and chromatin interaction data from several cell lines, including keratinocytes were analysed and three potential topological subdomains were identified, all containing the enhancer and CTSB, or CTSB and FDFT1 or both genes and NEIL2. Additionally, we showed that the enhancer’s activity correlated with CTSB expression, but not with FDFT1 and NEIL2 expression in differentiating keratinocytes and other cell lines. RNA polymerase II ChIA-PET interaction data in cancer cell lines showed that the enhancer interacts with CTSB but not FDFT1 or NEIL2. These data suggest that the enhancer normally regulates CTSB expression. Relative gene expression and immunohistochemistry from palmar biopsies from South African and Norwegian participants (7 Affected and 7 Controls) showed a significantly higher expression of CTSB, but not FDFT1 and NEIL2, in affected individuals compared to the controls and that CTSB was significantly more abundant in the granular layer of affected individuals compared to controls. We conclude that the enhancer duplication causes KWE by upregulating CTSB expression and causing an overabundance of CTSB in the granular layer of the epidermis.MT201

Exploring the role of genetic variation at the leptin and leptin receptor genes (LEP and LEPR) in obesity and hypertension in a black South African cohort

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand , Johannesburg, in partial fulfillment of the requirements for the degree of Master of Science (Medicine) in Human Genetics,2013Obesity and hypertension often occur together and are risk factors for cardio-metabolic disorders. Single nucleotide polymorphisms (SNPs) in the leptin (LEP) and leptin receptor (LEPR) genes have been shown to be associated with obesity and hypertension, but have not been well explored in African populations. The aims of this study were to determine the heritability estimates of anthropometric and blood pressure (BP) measures and leptin levels; to identify additional informative SNPs in and around the LEP and LEPR genes; and to examine the potential relationships between these SNPs and measures of obesity, hypertension and leptin levels in a black South African cohort. Participants from the African Programme on Genes in Hypertension (APOGH) with various anthropometric and BP measurements were genotyped for LEP and LEPR SNPs using the BeadXpress platform. Heritability estimates were determined using Statistical Analysis for Genetic Epidemiology (S.A.G.E.) software and relationships between LEP or LEPR SNPs and obesity, leptin levels and hypertension were assessed using SAS 9.3 and gPLINK vs2.050, taking into account family relationships, various confounders and correcting for multiple testing. The Bonferroni method was used to correct for multiple testing and P≤0.00076 was considered as statistically significant for SNP association tests. Seven-hundred-and-thirteen individuals were successfully genotyped and there were more women (66%) than men. The prevalence of obesity (42%) and hypertension (46%) were high in the sample. Significant heritability (h2 %, P<0.05) was noted for body weight (38%), body mass index (26%), waist (35%) and hip circumference (42%), waist-to-hip ratio (46%), skinfold thickness (44%), systolic (34%), diastolic (27%) and central systolic (33%) BP; but leptin levels were not significantly heritable (h2 %=15%, P=0.228). LEP rs17151914 (P=0.0002) and LEPR rs6690661 (P=0.0007) were significantly associated with leptin levels and diastolic BP, respectively, in women. The LEP rs17151913T-rs6956510G haplotype was associated with an increase in central systolic BP in women (P=0.012 with Bonferroni correction) whereas the LEPR rs2154381C-rs1171261T haplotype was associated with lower systolic BP in men (P=0.0359 with Bonferroni correction). LEP gene variants were significantly correlated with effects on leptin levels in women and the LEPR gene variants were significantly correlated with effects on diastolic BP also in women. These results indicate that further exploration of the role of genetic variation in the LEP and LEPR genes in obesity and hypertension in individuals of African ancestry is warranted

Duplicated Enhancer Region Increases Expression of CTSB and Segregates with Keratolytic Winter Erythema in South African and Norwegian Families

Contains fulltext : 174194.pdf (publisher's version ) (Closed access)Keratolytic winter erythema (KWE) is a rare autosomal-dominant skin disorder characterized by recurrent episodes of palmoplantar erythema and epidermal peeling. KWE was previously mapped to 8p23.1-p22 (KWE critical region) in South African families. Using targeted resequencing of the KWE critical region in five South African families and SNP array and whole-genome sequencing in two Norwegian families, we identified two overlapping tandem duplications of 7.67 kb (South Africans) and 15.93 kb (Norwegians). The duplications segregated with the disease and were located upstream of CTSB, a gene encoding cathepsin B, a cysteine protease involved in keratinocyte homeostasis. Included in the 2.62 kb overlapping region of these duplications is an enhancer element that is active in epidermal keratinocytes. The activity of this enhancer correlated with CTSB expression in normal differentiating keratinocytes and other cell lines, but not with FDFT1 or NEIL2 expression. Gene expression (qPCR) analysis and immunohistochemistry of the palmar epidermis demonstrated significantly increased expression of CTSB, as well as stronger staining of cathepsin B in the stratum granulosum of affected individuals than in that of control individuals. Analysis of higher-order chromatin structure data and RNA polymerase II ChIA-PET data from MCF-7 cells did not suggest remote effects of the enhancer. In conclusion, KWE in South African and Norwegian families is caused by tandem duplications in a non-coding genomic region containing an active enhancer element for CTSB, resulting in upregulation of this gene in affected individuals