Novel Molecular Mechanisms Leading to Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) is an inherited disorder characterized by increased bone fragility, with severity ranging from very mild to lethal. Mutation in COL1A1 and COL1A2, which code for type I collagen, are identified in the majority (~85%) of patients. Research to identify the cause of OI in individuals without identified mutations has focused on severe and lethal forms of the disease. By focusing on patients not fulfilling the current definition of “severe” adopted by the NHS funded Highly Specialised OI Service for children with severe, complex and atypical OI, we aimed to contribute to the current knowledge of the molecular basis of OI. We employed a number of sequencing strategies to achieve this: targeted exome, Sanger and whole exome analysis. Results in this thesis have established that these approaches, together with appropriate functional analysis, can identify novel causes of OI and bone fragility. We identified four patients with variable presentation and mutations in BMP1 and, importantly, highlight a risk of causing delayed healing, increased stiffness or atypical fractures by anti-resorptive treatment. We report the third occurrence of a c.1178A>G;p.Tyr393Cys P4HB mutation and describe what appears to be an emerging, distinctive radiological phenotype: meta-diaphyseal fractures with metaphyseal sclerosis. We have expanded the clinical spectrum associated with NBAS mutations to include bone fragility that may present as atypical OI. Submission of a UKGTN gene dossier has ensured rapid transition of this research finding into the patient diagnostic pathway. In addition, we have identified a number of novel genes and pathways that warrant further investigation, namely SLC38A10, SRCAP, UGGT1, UBASH3B, SULF2-POSTN, and voltage-gated sodium channel genes. Future work will focus on elucidating the significance of these findings and the development of tools to facilitate analysis and interpretation of genetic data as whole genome sequencing becomes more readily available for OI patient

Novel Molecular Mechanisms Leading to Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) is an inherited disorder characterized by increased bone fragility, with severity ranging from very mild to lethal. Mutation in COL1A1 and COL1A2, which code for type I collagen, are identified in the majority (~85%) of patients. Research to identify the cause of OI in individuals without identified mutations has focused on severe and lethal forms of the disease. By focusing on patients not fulfilling the current definition of “severe” adopted by the NHS funded Highly Specialised OI Service for children with severe, complex and atypical OI, we aimed to contribute to the current knowledge of the molecular basis of OI. We employed a number of sequencing strategies to achieve this: targeted exome, Sanger and whole exome analysis. Results in this thesis have established that these approaches, together with appropriate functional analysis, can identify novel causes of OI and bone fragility. We identified four patients with variable presentation and mutations in BMP1 and, importantly, highlight a risk of causing delayed healing, increased stiffness or atypical fractures by anti-resorptive treatment. We report the third occurrence of a c.1178A>G;p.Tyr393Cys P4HB mutation and describe what appears to be an emerging, distinctive radiological phenotype: meta-diaphyseal fractures with metaphyseal sclerosis. We have expanded the clinical spectrum associated with NBAS mutations to include bone fragility that may present as atypical OI. Submission of a UKGTN gene dossier has ensured rapid transition of this research finding into the patient diagnostic pathway. In addition, we have identified a number of novel genes and pathways that warrant further investigation, namely SLC38A10, SRCAP, UGGT1, UBASH3B, SULF2-POSTN, and voltage-gated sodium channel genes. Future work will focus on elucidating the significance of these findings and the development of tools to facilitate analysis and interpretation of genetic data as whole genome sequencing becomes more readily available for OI patient

Blindness, hearing loss and brown crumbly teeth: determining the molecular basis of Heimler and Heimler plus syndromes and other related conditions.

Enamel is the body’s hardest, most mineralised tissue and protects the underlying tissues of the tooth from the forces exerted during mastication and from bacterial attack. Amelogenesis imperfecta (AI) is a heterogeneous group of genetic conditions that result in defective dental enamel formation. AI can present as either isolated disease or as part of a syndrome. This thesis documents the identification of the genes involved in Heimler syndrome (HS) and in hypomineralised AI. HS is an autosomal recessively inherited combination of sensorineural hearing loss, AI and variable nail abnormalities, with or without visual defects. Biallelic mutations in the peroxisomal biogenesis factor genes, PEX1 and PEX6 were identified through whole exome sequencing (WES) of ten HS patients from seven families. Both proteins were found to be present throughout the retina. Mutations in PEX1 and PEX6 were already known to cause the Zellweger syndrome spectrum disorders (ZSSD), a group of conditions of varying severity. HS represents a mild ZSSD and results from combinations of mutations that include at least one hypomorphic variant, leaving patients with some residual peroxisomal function. For two additional families, mutations in the Usher syndrome (USH) gene, USH2A, were identified, highlighting the phenotypic overlap of HS with the more common USH. One family with autosomal dominantly inherited hypomineralised AI was recruited and DNA from three affected individuals was subjected to WES. DNA copy number variant analysis identified a heterozygous in-frame deletion of exons 3-6 of amelotin. Exfoliated primary teeth from an affected family member had enamel that was of a lower mineral density compared to control enamel and exhibited structural defects. Some of this appeared to be associated with organic material as evidenced by elemental analysis. Both cases in this study highlight the heterogeneity of AI and the importance of a genetic diagnosis for the clinical management of patients

Whole exome sequencing: a customised approach to exploring the genetic basis of musculoskeletal soft tissue injuries

Background: Several variants have been associated with the risk of musculoskeletal soft tissue injuries, suggesting a role for genetics in the aetiology of chronic Achilles tendinopathy (AT) and anterior cruciate ligament (ACL) ruptures. Genetic risk modifiers have primarily been identified using a hypothesis driven candidate gene approach. However, the ability to identify all risk-conferring variants using this approach alone is limited. Therefore, the primary aim of this thesis was to further define the molecular signatures of musculoskeletal soft tissue injuries mapping to specific genes. The genes encoding the tenascin-C glycoprotein (TNC, chromosome 9), the α1 chain of type XXVII collagen (COL27A1, chromosome 9), matrix metalloproteinase 3 (MMP3, chromosome 11) and the α1 chain of type I collagen (COL1A1, chromosome 17) were previously associated with the risk of injury and were therefore prioritised for further interrogation. Variants within these regions, which had either been previously associated with injury risk or prioritised from the list of new candidates identified by whole exome sequencing (WES) through the application of a customised tiered filtering strategy, were genotyped in several self-identified white AT and ACL rupture cohorts. The second aim of this study was to determine whether the observed risk-associated variants in the self-identified white cohorts were similar to those underpinning injury in the ancestrally admixed South African Coloured cohort, using ACL ruptures as the phenotypic model. The specific objectives of this thesis were: • To select well-phenotyped participants to be sequenced using an extended whole exome sequencing platform • The development and application of a reusable bioinformatics analyses pipeline involving a customised, tiered filtering strategy to select candidates for interrogation from the list of variants identified by WES. The TNC, COL27A1, MMP3 and COL1A1 genes were prioritised for further interrogation using this approach. • To test the association between the selected candidate variants and the risk of chronic AT and ACL ruptures using a case-control genetic association study design. The candidates selected from the list of variants identified by WES included: TNC rs1061494 (T>C), rs2104772 (T>A) and rs1061495 (T>C) and COL27A1 rs2567706 (A>G), rs2241671 (G>A) and rs2567705 (A>T). In addition, several variants previously associated with the risk of injury including TNC rs1138545 (C>T), MMP3 rs3025058 (5A>6A), rs679620 (G>A), rs591058 (C>T) and rs650108 (G>A) and COL1A1 rs1107946 (G>T) and rs1800012 (G>T), were also prioritised for investigation in additional injury cohorts. • To functionally annotate the prioritised variants using a host of in silico bioinformatic analyses tools. Methods Whole exome sequencing and data processing: Ten asymptomatic controls and ten chronic AT cases were selected for sequencing. Controls were older than 47 years of age, were physically active and had not reported any previous tendon or ligament injuries. Cases were younger than 35 years of age, had suffered chronic, bilateral Achilles tendinopathy and/or reported several Achilles tendon injuries. Paired-end WES was performed on the Illumina HiSeq 2000/2500 platform at 30X coverage. A customised, tiered filtering strategy was developed to screen for candidate variants. All candidate variants were confirmed using Sanger Sequencing and genotyped, together with the other prioritised variants in the larger injury cohorts. Case-control genetic association studies Achilles tendon injury cohorts: Three cohorts were independently recruited from South Africa (SA), Australia (AUS) and the United Kingdom (UK). The South African White (SAW)- Achilles tendon injury cohort consisted of 165 controls (SAW-CONAT), 123 cases with chronic Achilles tendinopathy (SAW-TEN) and 47 cases with acute Achilles tendon ruptures (SAWRUP). The UK-Achilles tendon injury cohort consisted of 130 controls (UK-CON), 87 cases with chronic Achilles tendinopathy (UK-TEN) and 35 cases with acute Achilles tendon rupture (UKRUP). The AUS-Achilles tendon injury cohort included 210 controls (AUS-CON) and 85 cases with chronic Achilles tendinopathy (AUS-TEN). Anterior cruciate ligament rupture cohorts: The first ACL rupture cohort consisted of 232 control participants (SAW-CONACL) and 234 cases with surgically diagnosed ACL ruptures (SAW-ACL), of which 135 were reportedly non-contact in mechanism (SAW-NON). All participants in this group were self-identified to be of South African White ancestry. The participants in the second South African ACL rupture cohort were of mixed ancestry and self-identified as being South African Coloured (SAC). This group consisted of 100 controls (SAC-CON) and 97 participants with surgically diagnosed ACL ruptures (SAC-ACL), of which 50 were reportedly non-contact in mechanism (SAC-NON). The TNC and COL27A1 genomic intervals were explored through the TNC rs1061494, rs1138545, rs2104772 and rs1061495 variants and the COL27A1 rs2567706, rs2241671 and rs2567705 variants in the SAW- and UK-Achilles tendon injury cohorts, in addition to the SAWand SAC-ACL rupture cohorts. The MMP3 locus was explored using the previous riskassociated rs3025058, rs679620, rs591058 and rs650108 variants in the AUS-Achilles tendon injury and SAW-ACL rupture cohorts. Chapter 5 explored the COL1A1 locus using the previous risk-associated COL1A1 rs1107946 and rs1800012 variants in the SAW- and UK-Achilles tendon injury cohorts, in addition to the SAW-ACL rupture and SAC-ACL rupture cohorts. Statistical analyses were performed using the R programming environment, with statistical significance set at PC) variant is predicted to overlap the sequence motifs of the muscle initiator nuclear protein, members of the myogenic family of transcription factors and RNA polymerase II subunit A. Furthermore, the rs1061494 variant demonstrated marked differences in its predicted pre-mRNA structure. The other TNC variant associated with injury risk, rs2104772 (T>A), was predicted to be deleterious by two independent annotation tools. The investigated COL27A1 variants were suggested to interact with several predicted enhancers of cellular function. However, this gene is still relatively uncharacterised in musculoskeletal soft tissue injuries, and therefore, these variants will require further interrogation. The 8kb MMP3 genomic interval demonstrated high levels of linkage disequilibrium. Furthermore, MMP3 was predicted to interact with the MMP12 gene mediated by chromatin looping. The COL1A1 rs1800012 (G>T) variant overlaps the recognition sequence of the Sp1 transcription factor in intron 1. This variant is also proposed to interact with the functional promoter variants, rs1107946 (G>T) and rs11327935 (indel/T). This interaction is suggested to be mediated by chromatin looping. Furthermore, the rare rs1800012 T allele is predicted to result in a looser mRNA conformation immediately surrounding the variant within the pre-mRNA sequence compared to the ancestral G allele. Conclusion: These results provide proof of concept for the use of WES and a customised tiered filtering strategy to identify and prioritise variants for further interrogation using traditional molecular techniques. This approach, utilising previous research to guide a targeted analysis of a WES dataset has highlighted the potential risk modifying effects of several new variants in the TNC and COL27A1 genes. Furthermore, haplotype analysis has implicated several signatures encompassing variants previously associated with risk of injury in the four investigated genes. Although no new candidate variants within the MMP3 and COL1A1 genes were independently associated with risk of injury, unique allele combinations were observed to co-segregate with an altered injury risk profile. Therefore, this study has genetically characterised several previously implicated loci and highlighted new sequence signatures, which may potentially contribute to the susceptibility of musculoskeletal soft tissue injuries. The next step would be to explore the functional significance of these sequence signatures in vitro; a process that would help further characterise the biological mechanisms underpinning the observed risk associations

Netzwerkgestützte Analyse von chronischen Entzündungserkrankungen

An investigation of the (partially shared) genetic factors of the diseases Ankylosing Spondylitis, Crohn's disease, primary sclerosing cholangitis, psorisis and ulcerative colitis using network methods

The Shared Genetic Architecture of Modifiable Risk for Dementia and its Influence on Brain Health

Targeting modifiable risk factors for dementia may prevent or delay dementia. However, the mechanisms by which risk factors influence dementia remain unclear and current research often ignores commonality between risk factors. Therefore, my thesis aimed to model the shared genetic architecture of modifiable risk for dementia and explored how these shared pathways may influence dementia and brain health. I used linkage disequilibrium score regression and genomic structural equation modelling (SEM) to create a multivariate model of the shared genetics between Alzheimer’s disease (AD) and its modifiable risk factors. Although AD was genetically distinct, there was widespread genetic overlap between most of its risk factors. This genetic overlap formed an overarching Common Factor of general modifiable dementia risk, in addition to 3 subclusters of distinct sets of risk factors. Next, I performed two multivariate genome-wide association studies (GWASs) to identify the risk variants that underpinned the Common Factor and the 3 subclusters of risk factors. Together, these uncovered 590 genome-wide significant loci for the four latent factors, 34 of which were novel findings. Using post-GWAS analyses I found evidence that the shared genetics between risk factors influence a range of neuronal functions, which were highly expressed in brain regions that degenerate in dementia. Pathway analysis indicated that shared genetics between risk factors may impact dementia pathogenesis directly at specific loci. Finally, I used Mendelian randomisation to test whether the shared genetic pathways between modifiable dementia risk factors were causal for AD. I found evidence of a causal effect of the Common Factor on AD risk. Taken together, my thesis provides new insights into how modifiable risk factors for dementia interrelate on a genetic level. Although the shared genetics between modifiable risk factors for dementia seem to be distinct from dementia pathways on a genome-wide level, I provide evidence that they influence general brain health, and so they may increase dementia risk indirectly by altering cognitive reserve. However, I also found that shared genetics risk between risk factors in certain genomic regions may directly influence dementia pathogenesis, which should be explored in future work to determine whether these regions represent targets to prevent dementia

Investigation of the Molecular Basis of Three New Disorders of Brain Growth and Development Identified Amongst the Amish

Extremes of brain growth have frequently been associated with impaired neurodevelopment and cognition. A significant contribution to our understanding of the processes involved in brain development has been made by the study of single gene disorders which are rare in the general population, but occur with increased frequency in certain endogamous populations. This project stems from findings of a long-running clinical-genetic program, called ‘Windows of Hope’, based amongst the Amish. The project aims were to clinically characterise three new autosomal recessive disorders of brain growth and development, to define the genes and mutations responsible for each, and to investigate the function of the molecules identified. A final further objective of the project in keeping with the wider aims of the Windows of Hope study was to translate the findings of this research into direct clinical benefits to the families and community involved. A combination of clinical phenotyping, autozygosity and linkage mapping, and functional studies, were used to investigate each disorder, which enabled the identification of the novel disease genes in each case. Chapter three describes the identification of a hypomorphic mutation in PCNA responsible for a novel DNA repair disorder. Thus, although it was considered by many that mutations in PCNA would not be compatible with life due to its crucial role in genomic stability and cell division, this study disproves this notion and describes a molecular ‘missing piece’ in DNA repair spectrum disorders. While the relationship between pervasive developmental disorders and megalencephaly is well described, very few single gene disorders associated with this clinical relationship have been identified. Chapter four documents the discovery of two founder mutations in the KPTN gene associated with such a phenotype. The functional data shows that the encoded wild type molecule (kaptin) associates with dynamic actin cytoskeletal structures in cultured neurons and that the causative mutations result in loss of function perturbing this interaction, defining kaptin as a new molecule which is crucial for normal human brain development and function. Chapter five details the investigation of the eponymously named “Hershberger syndrome”, a disorder originally described by McKusick in the Ohio Amish in 1967. Clinical and genetic studies of affected individuals revealed that the syndrome was comprised of two distinct disorders; Aicardi Goutières syndrome due to mutation in SAMHD1, and a new condition characterised by profound neurological impairment, cerebellar involvement and nephrosis. This condition, renamed “nephrocerebellar syndrome” was found to be caused by homozygous mutations in two closely linked genes, WHAMM and WDR73. The genetic and functional data supported the involvement of both molecules in the disease, suggesting that this is a composite phenotype. The identification and functional characterisation of three new genes responsible for abnormalities in brain growth provides an invaluable insight into disease pathogenesis and also identifies molecular pathways important for normal brain development. This is turn enables clinicians to provide a much needed diagnosis for affected individuals and their families as well as the wider community. Medical Research CouncilNewlife Foundation for Disabled Childre

Optimizing Exposome-wide Assessments in Cardiometabolic Risk

This thesis is focused on cardiovascular disease (CVD) and type 2 diabetes mellitus (T2D), two concomitant conditions that appear with growing concern. In our work, we aim to improve the identification of individuals at-risk of cardiometabolic disease through the characterization of complex environmental exposures (i.e. diet, physical activity), that temporally vary, and the health effects on cardiometabolic traits and disease. Our projects were based upon the Västerbotten Health Survey (VHU) and the Malmö Diet and Cancer (MDCS) studies, which included extensive data on lifestyle, biological intermediates, and clinical outcomes. In Paper I, we utilized the so-called environmental-wide association approach (EWAS), using longitudinal data from > 31,000 adults in VHU study. Under generalized linear models, from ~ 300 candidate exposures, 11 modifiable variables were associated with most of the cardiometabolic traits; the prioritised variables belonged to smoking, coffee intake, physical activity, alcohol intake, and context-specific lifestyle domains. In Paper II, we implemented a machine learning-based model to identify individuals with variable susceptibility to lifestyle risk factors for T2D and CVD. Individuals with sensitivity to blood lipids, and blood pressure associated predictors were at higher risk to develop cardiometabolic disease. Furthermore, when pooling across sensitive groups from the two cohorts, the findings suggest a particular vulnerable subpopulation with different risk profile. In Paper III, a series of causal-inference experiments from VHU and publicly available genome-wide association study (GWAS) summary statistics were used to triangulate evidence of the direct and mediated effects by adiposity and physical activity, of macronutrient intake (fat, carbohydrates, protein and sugar) and cardiometabolic disease. Using structural equation modelling, the mediation analyses enhanced with Mendelian randomization analysis, showed a likely causal putative association between carbohydrate intake and T2D. In addition, the integrative genomic analyses suggested a candidate causal variant localized to the established T2D gene TCF7L2. In Paper IV, we conducted a systematic review and metanalysis of observational studies, complemented by Mendelian randomization analysis using GWAS summary statistics, investigating causal associations of individuals with high, yet normal, glycaemia associated with cardiovascular complications. Prediabetes was likely causally associated with coronary heart disease; suggesting higher, but not diabetic levels of blood glucose confer a risk, thus, effective preventive strategies may prove successful in prediabetes