Up to a third of adults attending the Queen Square UK NHS Specialised Service for Rare Mitochondrial Disease out-patient clinic remain genetically undetermined. This thesis describes research which aimed to establish the molecular basis of mitochondrial disease in these patients and evaluate genotype/phenotype correlations. Both novel clinical syndromes and molecular causes of disease were identified. Furthermore, new insights into the respiratory chain (RC) protein structure were elucidated. Novel clinical phenotypes: Three previously unrecognised mitochondrial disease phenotypes were characterised. First, m.9185T>C in MT-ATP6, encoding subunit 6 of ATP synthase (complex V), was detected in a pedigree exhibiting matrilineal inheritance. Presentation was with axonal Charcot-Marie-Tooth (CMT2) disease. Further screening of 270 patients with genetically unclassified CMT2 demonstrated three additional families harbouring the same mutation, thus proving a causal link between reduced complex V activity and impaired axonal function. Second, a severe distal myopathy was observed in two unrelated patients with de novo dominant POLG mutations. Finally, COX10 mutations were linked to adult cytochrome c oxidase (COX) deficiency. Despite a complex multisystem phenotype comprising short stature, proximal myopathy, fatigue, sensorineural hearing loss, pigmentary maculopathy, renal Fanconi syndrome and premature ovarian failure, the patient’s clinical severity was considerably milder than fatal COX10-related infantile disease. Nuclear gene mutations: Three major experimental strategies were employed to locate mutations in the nuclear genes of adults with clinically and/or biochemically suspected mitochondrial disease. First, a candidate gene approach identified RRM2B mutations in 2/33 patients with multiple mitochondrial DNA deletions. Second, whole-exome sequencing confirmed COX10 mutations can cause adult mitochondrial disease. Finally, combined homozygosity mapping/whole-exome sequencing in a consanguineous family led to the discovery that NDUFA4 mutations cause COX-deficient Leigh syndrome. This example of ‘back-translation’ led to the discovery that NDUFA4, previously considered to be a complex I subunit, is actually an important component of the COX enzyme complex.

Introduction: Epidemiological studies have reported an association between increased bone mineral density (BMD) and osteoarthritis (OA); however, whether increased BMD is a true risk factor for OA remains unclear. To address this question in a novel way, I aimed to determine the prevalence and phenotype of OA in a population of individuals with extremely High Bone Mass (HBM). Methods: HBM cases, defined by dual X-ray absorptiometry (DXA) BMD Zscores, were compared with unaffected family members and general population controls from the Chingford and Hertfordshire cohort studies. The clinical OA phenotype in HBM was established by comparing relevant outcomes such as joint replacement and joint pain in HBM cases versus family controls. The prevalence and phenotype of radiographic OA in HBM was determined through blinded systematic grading of OA features on hip and knee X-rays, with scores compared between the HBM and control groups. Analyses used logistic / linear regression, adjusted for age, gender and body mass index (BMI) with generalised estimating equations to account for within-person clustering, using Stata. Results: HBM cases reported a higher prevalence of joint replacement compared with the family control group (adjusted odds ratio [95% confidence interval] 2.42 [1.06,5.56], p=0.037). An increased prevalence of both radiographic hip (1.52 [1.09,2.11], p=0.013) and knee (1.62 [1.22,2.16], p=O.OOI) OA was seen in HBM cases compared with the combined control group. The radiographic OA phenotype in HBM was predominantly characterised by bone-forming features such as osteophytes and subchondral sclerosis. HBM was also associated with the presence of radiographic pelvic enthesophytes (bony spurs at tendon/ligament insertions) and altered acetabular morphology consistent with a mild skeletal dysplasia. Conclusion: These findings support an increased risk of both clinical and radiographic OA in HBM compared with the general population. Common underlying mechanisms regulating bone formation may underpin both HBM and certain phenotypes of OA.

No description available.

Osteoblasts, the bone-forming cells, derive from multipotential bone marrow stromal precursors called colony-forming units-fibroblastic (CFU-F). CFU-F rapidly adhere to plastic upon culture ex vivo, adhesion of such stromal precursors to bone in vivo is likely to be an early event in the anabolic response to bone stimulatory factors. It has been suggested that osteoclasts are involved in the activation of bone formation during bone remodelling. In order to test this, osteoclast conditioned medium was prepared from osteoclasts cultured on either plastic or bone. It was then used as culture medium for bone marrow cells. It was found that the conditioned medium was unable to increase the adherence of bone marrow cells and therefore the number of CFU-F when cultured in 6-well plates. The ability of parathyroid hormone (PTH) to enhance bone formation has recently been exploited in the treatment of osteoporosis. However, the underlying mechanisms are unknown. PTH and other possible osteoblast activating factors were tested for the ability to activate adhesion of CFU-F in vitro. For this, bone marrow cells were incubated in PTH for varying times. Non-adherent cells were then removed, and the adherent cells were incubated in PTH-free medium for 14 days to assess, as colony formation, the number of CFU-F that had adhered in the preceding period. Incubation in PTH caused a substantial increase in the number of CFU-F that adhered within 24 h. This increase was abrogated by peptidic inhibitors of integrins. The increase did not appear to be mediated through a PTHinduced increase in interleukin-6, since interleukin-6 had no effect on CFU-F numbers when substituted for PTH. Similarly, adhesion was unaffected by incubation of bone marrow cells in dibutyryl cyclic AMP, nor by inhibitors or donors of nitric oxide. However, activation of CFU-F in vitro by PTH was strongly inhibited by indomethacin and mimicked by Prostaglandin E2 (PGE2). To test the effects of PTH in vivo, the number of CFU-F that could be extracted from murine bone marrow after administration of an anabolic dose of PTH were measured. A dramatic reduction in the number of CFU-F that could be extracted from their bone marrow commenced within 2 h of treatment. It was also found that indomethacin reversed the PTH mediated reduction of CFU-F that could be extracted from mouse bone marrow. Intermittent PTH administered over a 6 day period increased the dynamic parameters associated with bone formation and there was a concomitant increase in the number of osteoblasts on bone surfaces. These results suggested that PTH rapidly activates adhesion of CFU-F to plastic or bone surfaces. This activation may represent an early event in the anabolic response of bone cells to PTH.

No description available.

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease of elusive origin and characterised by polyclonal B cell hyperactivity and the production of pathogenic antibodies targeting self DNA and nucleoproteins. Clinical manifestations of SLE are highly heterogeneous and include multisystem inflammation of connective tissue and vasculitis of the central nervous system (CNS). SLE is known to have genetic associations, and is widely acknowledged to involve a profound breakdown in immune tolerance. The aim of this project was to identify whether defects in early B cell development that affect the expressed repertoire of immunoglobulin light chains could be observed and to ask whether receptor editing contributes to disease pathogenesis. Chapter 3 describes a high-throughput sequencing analysis of the human immunoglobulin kappa light chain gene repertoire in healthy and SLE mature naive peripheral B cells. We observed that involvement of gene segments as the repertoire develops is equivalent in health and SLE and that a previously described bias towards usage of the gene segment IGKV4-1 in SLE is only observed in the expressed repertoire analysed. Chapter 4 describes an inefficient function of the kappa deleting element (KDE) in SLE, which manifests as reduced frequency of KDE rearrangement status in populations of CD19+ B cells and a failure to inactivate alleles of IGK allowing them to accumulate somatic hypermutations within non-productive IGK rearrangements. Chapter 5 identifies a potential biological outcome of a failure to inactivate rearranged alleles of IGK in SLE; cell surface expression of both kappa and lambda light chains were observed by flow cytometry analyses. Efforts to disprove this were unsuccessful anddetection of both light chain transcripts was confirmed through single cell PCR amplification of cDNA. The data in this thesis suggest that the failure of the KDE to inactivate alleles of IGK efficiently in SLE permits the expression of IGK light chains that have been selected against resulting in allelic inclusion. This mechanism may account for the broader and less stringently regulated antigen-binding B cell repertoire associated with SLE.

Background: Rheumatoid arthritis (RA) is a chronic, systemic, disabling disease which reduces independence, quality of life, and longevity. Upper limb impairment causes considerable disability, contributes to work incapacity, and has substantial monetary and non-monetary, personal and societal consequences. Objectives: The studies in this thesis focus on the development and evaluation of a novel exercise programme for the rehabilitation of global upper limb disability in people with RA. It explores participants’ experiences and the factors influencing their uptake and maintenance of the programme. It evaluates the physical activity (PA) levels of adults with rheumatic diseases against PA guidelines, and assesses the proportion of respondents who report ever receiving PA advice from a healthcare professional (HCP). Methods: Following development of a global upper limb home exercise programme, supplemented by four supervised group education, self-management, and exercise sessions (the EXTRA programme), 108 people with RA of less than 5 years duration were randomly allocated to receive either the EXTRA programme or usual care. Self-reported disability, upper limb functional performance, strength, self-efficacy, quality of life (QOL), and disease activity were assessed at baseline, 12, and 36 weeks. Participants were interviewed to evaluate their experiences of the EXTRA programme. Physical activity participation, recommendation, and preferences were surveyed among 508 adults with a range of rheumatic diseases. Results: Following the EXTRA programme, there were significant improvements to upper limb disability, function, strength, and self-efficacy, but not QOL, and no adverse effects on disease activity or pain. Participants perceived the EXTRA programme to be effective and acceptable. Sixty-one percent of respondents met PA guidelines, although 27% were inactive. Forty-three percent of respondents reported receiving PA advice from a HCP. Walking was the most preferred PA (65%). Conclusions: The EXTRA programme improves upper limb disability, function, strength, and self-efficacy, with no adverse effects on disease activity or pain, in people with RA. Many people with rheumatic diseases are inactive and more than half have never discussed PA with a HCP. Recommending exercise and regular PA should be integral to rheumatic disease management.

Bone mineral density (BMD) is a highly heritable trait, indicating that genetic elements are partly responsible for variation in osteoporosis risk. To further understand the genetic variation underlying osteoporosis, I performed genome-wide association (GWA) studies using designs that have largely not been performed in osteoporosis literature to date. Three strategies were used: i) a selected sample of postmenopausal women with high (z ~ 1.5, n = 1,055) or low (z≥1.5, n = 900) hip BMD [as measured by Dual-energy X-ray absorptiometry (DXA)] were used for GWA, followed by replication in an unselected sample of 20,898 adults, ii) a GW A meta-analysis on unselected children from the Avon Longitudinal Study of Parents and Children [ALSPAC (n = 5,330)] and the Generation R study [GEN-R (n = 4,098)], using DXA derived total-body less head BMD (TBLH-BMD) measures, iii) refining total-body BMD measures in children by subregional analysis: i.e. quantifying the genetic and environmental correlation between paediatric BMD measures [ALSPAC (n≥5,299)] of the skull (S-BMD), lower limb (LL-BMD) and upper limb (UL-BMD) using genome-wide complex trait analysis (GCT A) and thereafter performing a GWA meta-analysis on each site using subjects from ALSPAC and GEN-R (n ~ 9,300). The role of bone resorption in bone growth and accrual was investigated via a cross-sectional analysis of 1,130 adolescents from ALSPAC using serum measures of ,β-C-telopeptides of type I collagen (CTX) and quantitative computed tomography (pQCT) measures of the mid-tibia. Two novel BMD associated loci were identified using the selective genotyping strategy: GALNTJ (rs6710518, P = 1.4x lO· 10) and RSP03 (rsI3204965, P = 3.0x lO· 10). Association studies of paediatric TBLH-BMD identified a novel variant in RlN3 (rs754388, P = 3.0x 10.9) and replicated 31 adult BMD associated loci, with six reaching the GWA threshold of association (P < 5.0x 10·R). Sub-regional GCT A analysis indicated that appendicular sites shared a greater proportion of genetic architecture (LL-/UL-BMD rg=0.78, P = 1 x 10.7) when compared to the skull [(UL-/SBMD rg = 0.58, P = 9x l0·7) and (LL-/S-BMD rg = 0.43, P = l x lO'~)]. GWA meta-analysis echoed these findings by identifying twelve known BMD-associated variants that differed in the strength of their association and magnitude of effect with each sub-region. In particular, variants at the WNTl6 and RSP03 showed considerable site-specificity as indicated by strong association with S-BMD and/or UL-BMD, but not with LL-BMD. An investigation into the role of bone resorption in adolescent bone suggested that CTX was positively related to periosteal circumference (PC) [,8 = 0.19 (0 .13, 0.24)] (coefficient = SD change per SD increase in CTX, 95% Cl)], but inversely associated with cortical BMD [,8 = -0.46 (-0.52, -0.40)] and positively related to bone strength as reflected by the strength-strain index (SSI) [,8 = 0.09 (0 .03 , 0.14)]. These relationships were replicated using genetic proxies for bone resorption . . These results suggest that the selective sampling GWA strategy represents an efficient alternative to conventional random sampling designs. However the real world feasibility of selective sampling is questionable, as it requires extensive phenotyping in order to ensure adequate sample size and study power is obtained. BMD measures of children are well suited for GW A, however the replication of adult BMD associated SNPs implies that many of the BMD associated loci identified operate throughout the life course. Whether this strategy enriches for genetic factors involved in bone modelling remains to be seen. BMD at different skeletal sites appears to be influenced by distinct genetic and environmental influences, suggesting that phenotypic refinement of BMD may represent a superior GW A strategy, when compared to using heterogeneous BMO measures (i .e TBLH-BMO). Finally, bone resorption might play an important role in paediatric bone growth, accrual and strength.

Lens growth and development is a life-long process in which epithelial cells from the perimeter of the lens migrate towards the centre of the lens and follow a dynamic programme of differentiation and structured DNA and organelle degradation resulting in the optical clarity of the lens. Myotonic dystrophy type 1 (DM1) is a genetic disease resulting in multiple symptoms including skeletal muscle wasting, cardiac conduction defects, myotonia and endocrine system malfunction. DM1 is caused by an expanded CTG repeat in the 3' untranslated region (UTR) of the myotonic dystrophy protein kinase (DMPK) gene. Mutant DMPK RNA has been demonstrated to form abnormal foci within the cell nucleus in muscle cells from DM1 patients. The link between these foci and the multiple symptoms of DM1 is not fully understood. The alternative pre-mRNA splicing factor muscle blind-like 1 (MBNL1) is found in the foci and sequestration of MBNL1 is a leading hypothesis for pathogenesis. Results shown in this thesis demonstrate that only a small percentage of nuclear MBNL1 is sequestrated into foci. Furthermore MBNL1 is shown to co-localise with splicing speckles in a transcriptionally dependent manner. Interestingly eye lens histological samples suggest a change in MBNL1 distribution during lens growth. The data presented in this thesis shows a strong relationship between MBNL1 and CUGexp pre-mRNA foci and presents data which highlights the sensitivity of lens epitheial cells to changes in MBNL1.

PAR-2 is a GPCR activated in response to proteolytic cleavage of the N terminus. Previous studies have revealed a critical role for PAR-2 in a murine arthritic model, reporting PAR-2 KO mice are resistant to disease. Further studies have identified PAR-2 on rheumatoid synovial CD68+ macrophages. Also, ex vivo studies revealed blockade of PAR-2 using a specific antagonist results in substantial reduction of many pro-inflammatory cytokines, known to contribute to rheumatoid pathology including IL-1β, IL-6 and TNFα. Although these studies clearly identify PAR-2 as having a role in disease, the cellular mechanisms involved require elucidation. This doctoral project therefore focuses on identifying the effects of PAR-2 activation/blockade in immunological responses of innate and adaptive cells which are thought to play a pivotal role in inflammatory disease pathology. In order to assess the role of PAR-2 in innate and adaptive immune systems, studies were carried out to determine the key differences between PAR-2 WT and KO macrophage and T cell populations. Cellular markers, cytokine release and populations of T cells in peripheral lymphoid organs were assessed. The PAR-2 murine studies also comprised an in vivo component to assess and compare innate cell recruitment in an innate model of peritoneal inflammation induced by thioglycollate. The later stages of the thesis focused on human studies by examination of buffy coat derived innate and adaptive cells. Characterisation of PAR-2 expression in these cell types and effect of PAR-2 activation/blockade on innate responses was determined by analysis of cytokine production, cell surface markers and morphological studies. Key results from these investigations identified an important role for PAR-2 in innate cell recruitment with significantly reduced macrophage numbers in PAR-2 KO in the thioglycollate model. PAR-2 KO derived macrophages also had significantly reduced expression of co-stimulatory markers, whilst the responses and proliferation of murine ova specific T cells (OT2) were significantly reduced in PAR-2 KO mice. Human studies identified PAR-2 expression in human macrophages, B and T cells. PAR-2 antagonism during monocyte to macrophage maturation resulted in impaired cellular maturation as determined by significantly decreased cell area in both M1 and M2 type macrophages. In addition to this, PAR-2 activation in M1 macrophages significantly upregulated HLA-DR, an antigen presenting molecule, haplotypes of which have been highly associated with increased risk of rheumatoid disease. Stimulations of these cells with a panel of TLR agonists revealed significant differences in TNFα when stimulated with the addition of PAR-2 agonist or antagonist. Human B and T cells were also analysed for PAR-2 expression, which was dramatically upregulated in response to pro-inflammatory stimuli. These data add to the existing literature supporting a role for PAR-2 in innate cell development and activation. This doctoral project highlights a role for PAR-2 in macrophage development, and recruitment of inflammatory cells, clearly identifying an important role for PAR-2 in innate immunology. In addition to this, the data generated is strongly supportive of a role for PAR-2 in adaptive cell responses, not only by indirect effects on innate cells but also direct effects on the adaptive cells themselves. This data is also relevant to studies of autoimmune disease, by investigating the potential pathological role of PAR-2 in arthritis. The data suggests that the PAR-2 mediated mechanism, responsible for the amelioration of arthritis in KO mice, may involve reduced macrophage activation, recruitment and pro-inflammatory cytokine release. The data also provides evidence for a role for PAR-2 in T cell specific responses, thought to be critical in RA disease.