Genome annotation of the 1.2MB Region on chromosome 8p22-p23.1 harbouring the gene for Keratolytic Winter Erythema (KWE)

Keratolytic winter erythema (KWE) or Oudtshoorn skin disease is a rare autosomal dominant skin disorder for which the genetic cause remains unknown. The disorder manifests in the form of erythema and hyperkeratosis of the palmar-plantar regions and has been linked to a 1.2Mb region on chromosome 8p22-23.1 between markers D8S1759 and D8S552. A prevalence of 1/7200 has been observed in the South African Afrikaans-speaking white population with a lower unspecified prevalence occurring in the coloured South African population. A number of positional candidate genes within the critical region have been assessed for pathogenic mutations, however to date the causative gene has not been identified. The objective of the current study was to examine the KWE critical region for highly conserved coding and non-coding regions and copy number variants (CNV) and to determine if these regions may play a role in the molecular etiology of the disease. Highly conserved regions were identified based on sequence conservation across a range of evolutionary diverse organisms. These regions were further analysed for possible protein-coding gene structure, regulatory motifs and RNA secondary structure. In addition, a custom CGH tiling array (384K Roche-Nimblegen) was used to identify CNVs across the extended KWE critical region in both affected and unaffected individuals. The multi-species sequence alignment revealed eight regions that showed a high level of conservation above a 70% threshold. Functional analysis of two of the conserved regions led to the identification of a novel protein-coding gene deubiquitinating enzyme 3 (DUB3) within the critical region which presented as a credible functional candidate for KWE. Two of the conserved regions were identified within an open reading frame c8orf13 which has previously been examined and found to contain no pathogenic mutations that segregate with the KWE phenotype. The remaining four highly conserved regions were found within non-coding sequence and computational analysis revealed putative regulatory motifs in the form of transcription factor binding sites. The copy number variation analysis did not show evidence for the presence of any large or small consistent CNV alleles likely to impact on any of the functional candidate genes in the KWE critical region. No common CNV alleles were observed in all of the KWE affected individuals examined and showed absence in unaffected family members. A significant variation in copy number was however observed in affected individuals within a previously defined copy number variable beta-defensin gene cluster which has been associated with psoriasis. Although the exact copy number of the cluster could not be determined in the present study due to the cross hybridization between genes in the family, the CNV observed in affect individuals for the cluster suggests that it may be involved in the modulation of the clinical severity of KWE. The present study has led to the identification of a previously uncharacterised novel gene DUB3 within the KWE critical region which furthermore presented as a plausible functional candidate for the KWE phenotype. In addition, it has revealed that the molecular cause of KWE is unlikely to be exclusively due to copy number variation within the genes in the critical region. The current study has provided valuable insight into the KWE linked critical region and revealed a number of potential regions of interest to be examined in further studies exploring the molecular cause of the disease

Phylogenomic and structure-function relationship studies of proteins involved in EBV associated oncogenesis

This study covers the investigation of evolutionary and structure-function relationship aspects of several cancer related proteins. One part of the study deals with the investigation of a critical protein of Epstein-Barr Virus (EBV) the Nuclear Antigen 1 (EBNA1), and its interactions with different host proteins. One of these host proteins is a member of a large gene family, encoding ubiquitin specific proteases (USP), known as USP7. The second section of the thesis deals with the molecular evolution of the USP gene family. Another set of cellular proteins deregulated during EBV associated oncogenesis are members of the glycoside hydrolase (GH18) family. Their phylogenetic relationships and protein structures were investigated in the third section of this thesis. EBNA1 is the only EBV protein that consistently expressed in all latent forms of the EBV infections. The protein is involved in the genome maintenance and a substantial body of evidence suggests that it has a role in EBV associated oncogenesis. In this study, full length molecular models of the EBNA1 protein were generated using the programmes, I-TASSER, MOE and Modeller. The best models were selected on the basis of plausibility in structural and thermodynamical parameters and from this models of EBNA1 homologues of primates lymphocryptoviruses (LCVs) were generated. The C-terminal DNA binding and homodimerisation domain was predicted to be structurally similar between different LCV EBNA1 homologues, indicative of functional conservation. The central glycine alanine repeat (GAr) domain was predicted to be primarily composed of α helices, while almost all of the protein interaction region was found to be unstructured, irrespective of the prediction approach used and sequence origin. Predicted USP7 and Casein kinase 2 (CK2) binding sites and GAr were observed in the EBNA1 homologues of Old World primate LCVs, but not in the marmoset homologue suggesting the co-evolution of both these sites. Dimer conformations of the EBNA1 monomer models were constructed using SymmDock, where the C-terminal tail was predicted to wrap around the proline rich loop of another monomer, possibly contributing to dimer stability. This feature could be exploited in therapeutic design, hence an inhibitor peptide was designed and a preliminary evaluation was conducted to explore its ability to inhibit EBNA1 function in cell survival. The peptide array libraries of EBNA1 were used to investigate the binding regions and critical contact points between EBNA1 and partner proteins. Human EBP2 and USP7 proteins were expressed in bacteria and probed on the EBNA1 array. The data confirm the previously known binding region for EBNA1-EBP2 and EBNA1-USP7 interactions. In addition further information was gained regarding the critical contact residues and the potential role of phosphorylation of serine residues of EBNA1 in its binding with EBP2 and USP7. The human genome encodes nearly 100 USPs which contribute to regulate the turnover of cellular proteins. These homologues are divided into 16 paralogous groups, all sharing a characteristic peptidase C19 domain. Evolutionary relationships between these homologues were explored by datamining and the phylogenetic reconstruction of peptidase C19 domain sequences. The data reveal an ancient relationship between the genes, with expansion occurring throughout the course of evolution, but particularly at the base of the vertebrates, at the time of the two whole genome duplications. A comparison between the phylogenetic architecture and protein interaction networks suggests the parallel emergence of many molecular pathways and the associated USPs. The GH18 gene family includes chitinases and related non catalytic proteins. Most mammals encode at least three chitinases (CHIT1, CHIA/AMCase and CTBS), as well as several homologues encoding catalytically inactive chitinase-like proteins or chilectins. Phylogenomic analysis shows that the family has undergone extensive expansion, initiating with a duplication event at the root of the vertebrate tree, resulting in the origin of the ancestors of CHIT1 and CHIA. Two further duplications of ancestral CHIA predate the divergence of bony fishes, one leading to a newly identified paralogous group (we have termed CHIO). In tetrapods, additional CHIA duplications predate and postdate the amphibian/mammalian split and relics of some exist as pseudogenes in the human genome. Homology modelling of structurally unresolved GH18 homologues in mouse and human was conducted using Modeller and I-TASSER. All resolved and predicted structures share a TIM barrel (β/α)8 and α+β domain. A central ligand binding cavity was also found in all GH18 homologues. The variation in size and shape of different paralogous proteins, indicate the difference in their ligands specificity and in turn potential functions

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

Using genomics and population genetics to understand genetic variation in Malawi Plasmodium falciparum clinical isolates

The natural selection imposed by host immunity and antimalarial drugs has driven extensive adaptive evolution in Plasmodium falciparum, leading to an ever-changing landscape of genetic variation. We have carried out whole-genome sequencing of 93 P. falciparum clinical isolates from Malawi and used population genetic methods to investigate the genetic diversity and regions under selection. In addition, by computing XP-EHH, PCA and FST we have compared the Malawi isolates to five dispersed others (Kenya, Mali, Burkina Faso, Cambodia and Thailand), and identified genes potentially under positive directional selection. Geographic stratification of genetic diversity in the populations followed continental lines and small population differences were observed within Africa. Positive directional selection signals were identified at or near pfdhps, pfcrt, pfmdr1 and pfgch1 (known drug targets) and in several merozoite invasion ligands (e.g., msp3.8, trap and ama1). We discuss the role of drug selection in promoting fixation of alleles between populations with differing adaptation to local drug pressure. Analysis of copy number variation in Malawi provides a detailed catalogue of new and previously identified gene deletions and duplications with critical roles in cytoadherence, gametocytogenesis, invasion and drug response. This work provides the first genome-wide scan of selection and CNV in Malawi to guide future studies in investigating parasite evolution, changing malaria epidemiology, and monitoring and evaluating impact of malaria interventions as they are deployed

Molecular Genetic Analysis of Non-Catalytic Pol IV and V Subunits

Among eukaryotes, plants have the distinction of encoding multisubunit RNA polymerases used exclusively for RNA directed DNA Methylation: RdDM) in addition to Pol I, II, and III. In Arabidopsis thaliana, Pol IV is required for the biogenesis of 24nt siRNAs whereas Pol V transcription is needed for cytosine methylation of the DNA sequences corresponding to these siRNAs. The ancestry of Pol IV and V can be traced back to Pol II, and Pol II, IV and V still utilize multiple non-catalytic subunits encoded by the same genes. Genetic analysis of non-catalytic subunits that are highly similar reveals that these subunits are not necessarily redundant. For instance, NRPB9b but not its 97% similar paralog, NRPB9a is required for RdDM. Likewise, Pol IV and Pol V-specific 7th largest subunits are very similar yet have different involvements in RdDM. In some of the non-catalytic subunit mutants of Pol IV, 24nt siRNA accumulation is not dramatically reduced, yet RNA silencing is disrupted. This contrasts with Pol IV catalytic subunit mutants in which siRNA biogenesis and RdDM are coordinately disrupted. Taken together, these results suggest that Pol IV might possess functions in RdDM that are in addition to, and separable from siRNA biogenesis. Differences in Pol V subunit composition based on the use of alternative non-catalytic subunit variants might also have functional consequences for RdDM. The evidence suggests that alternative non-catalytic subunits in Pol IV and V are likely to influence interactions with other proteins needed for RdDM

Dissecting the genotype to phenotype relationships of genomic disorders

Over the last decade, major advances in the development and application of microarray-based comparative genomic hybridisation (aCGH) technology have significantly contributed to our understanding of Genomic Disorders. My aims here were to provide insight into the genotype to phenotype relationships of three Genomic Disorders; CUL4B-deleted X-Linked Mental Retardation (XLMR), Wolf-Hirschhorn Syndrome (WHS) and 16p11.2 Copy Number Variant Disorder. CUL4B encodes a structural component of the Cullin-RING-ligase 4-containing class of E3 ubiquitin ligases. CUL4B-deleted XLMR represents a syndromal form of mental retardation whereby patients exhibit other clinical features aside from the MR, such as seizures, growth retardation and disrupted sexual development. I used CUL4B-deleted patient-derived cell lines to investigate the impacts of CUL4B loss on mitochondrial function. I have shown that loss of CUL4B is associated with a distinct set of mitochondrial phenotypes, identifying CUL4B-deleted XLMR as a disorder associated with mitochondrial dysfunction. Furthermore, I have uncovered a reciprocal relationship between CUL4B and Cereblon, providing evidence of a potential role for the CUL4-CRBN E3 ligase complex in maintaining mitochondrial function. Deletion or duplication of the 16p11.2 region is associated with macro-/microcephaly respectively. Here, I have evaluated the cellular consequences of 16p11.2 CNV, specifically with regards KCTD13 expression, DNA replication and checkpoint activation. WHS is typically caused by a small hemizygous telomeric deletion of the 4p16.1 region. Haploinsufficiency of 4p16.1 is associated with microcephaly, growth retardation and complex developmental abnormalities. I investigated the impacts of LETM1 copy number change in WHS patient-derived cells. Here, I have shown that copy number change of LETM1 specifically segregates with mitochondrial dysfunction, likely underlying the seizure phenotype exhibited by the large subgroup of WHS patients whose deletions incorporate LETM1 as well as the rarer instances of the reciprocal duplication. In this thesis I use patient-derived cell lines from three Genomic Disorders as a fundamental tool providing new pathomechanistic insight into the clinical presentation of these conditions

Dissection of the genetic background of childhood onset progressive myoclonic epilepsies

The progressive myoclonic epilepsies (PMEs) are a clinically and etiologically heterogeneous group of symptomatic epilepsies characterized by myoclonus, tonic-clonic seizures, psychomotor regression and ataxia. Different disorders have been classified as PMEs. Of these, the group of neuronal ceroid lipofuscinoses (NCLs) comprise an entity that has onset in childhood, being the most common cause of neurodegeneration in children. The primary aim of this thesis was to dissect the molecular genetic background of patients with childhood onset PME by studying candidate genes and attempting to identify novel PME-associated genes. Another specific aim was to study the primary protein properties of the most recently identified member of the NCL-causing proteins, MFSD8. To dissect the genetic background of a cohort of Turkish patients with childhood onset PME, a screen of the NCL-associated genes PPT1, TPP1, CLN3, CLN5, CLN6, MFSD8, CLN8 and CTSD was performed. Altogether 49 novel mutations were identified, which together with 56 mutations found by collaborators raised the total number of known NCL mutations to 364. Fourteen of the novel mutations affect the recently identified MFSD8 gene, which had originally been identified in a subset of mainly Turkish patients as the underlying cause of CLN7 disease. To investigate the distribution of MFSD8 defects, a total of 211 patients of different ethnic origins were evaluated for mutations in the gene. Altogether 45 patients from nine different countries were provided with a CLN7 molecular diagnosis, denoting the wide geographical occurrence of MFSD8 defects. The mutations are private with only one having been established by a founder-effect in the Roma population from the former Czechoslovakia. All mutations identified except one are associated with the typical clinical picture of variant late-infantile NCL. To address the trafficking properties of MFSD8, lysosomal targeting of the protein was confirmed in both neuronal and non-neuronal cells. The major determinant for this lysosomal sorting was identified to be an N-terminal dileucine based signal (9-EQEPLL-14), recognized by heterotetrameric AP-1 adaptor proteins, suggesting that MFSD8 takes the direct trafficking pathway en route to the lysosomes. Expression studies revealed the neurons as the primary cell-type and the hippocampus and cerebellar granular cell layer as the predominant regions in which MFSD8 is expressed. To identify novel genes associated with childhood onset PME, a single nucleotide polymorphism (SNP) genomewide scan was performed in three small families and 18 sporadic patients followed by homozygosity mapping to determine the candidate loci. One of the families and a sporadic patient were positive for mutations in PLA2G6, a gene that had previously been shown to cause infantile neuroaxonal dystrophy. Application of next-generation sequencing of candidate regions in the remaining two families led to identification of a homozygous missense mutation in USP19 for the first and TXNDC6 for the second family. Analysis of the 18 sporadic cases mapped the best candidate interval in a 1.5 Mb region on chromosome 7q21. Screening of the positional candidate KCTD7 revealed six mutations in seven unrelated families. All patients with mutations in KCTD7 were reported to have early onset PME, rapid disease progression leading to dementia and no pathologic hallmarks. The identification of KCTD7 mutations in nine patients and the clinical delineation of their phenotype establish KCTD7 as a gene for early onset PME. The findings presented in this thesis denote MFSD8 and KCTD7 as genes commonly associated with childhood onset symptomatic epilepsy. The disease-associated role of TXNDC6 awaits verification through identification of additional mutations in patients with similar phenotypes. Completion of the genetic spectrum underlying childhood onset PMEs and understanding of the gene products functions will comprise important steps towards understanding the underlying pathogenetic mechanisms, and will possibly shed light on the general processes of neurodegeneration and nervous system regulation, facilitating the diagnosis, classification and possibly treatment of the affected cases.Ei saatavill