Magister Scientiae (Physiotherapy) - MSc(Physio) / Clubfoot is the most common congenital structural deformity that leads to physical impairments in children in many poor developing countries. Inadequately treated or neglected clubfoot has been found to be a common cause of ohysical disability globally among children and young growing adults. Many children are referred to the clinics for treatment but some parents do not comply with the treatment regimen whcih requires attending for consecutive treatment sessions. The purpose of this study was to investigate barriers to treatment attendance parents/caregivers of children with clubfoot encounter in complying with clubfoot treatment during the plaster csting phase in Uganda. / South Africa

Magister Scientiae (Medical Bioscience) - MSc(MBS) / The present study describes molecular aspects of inherited congenital cataract in captive-bred Vervet monkeys. Congenital cataracts are lens opacities that are present at birth or soon after birth and include hereditary cataracts or cataracts caused by infectious agents. The MRC Primate Unit is housing a colony of captive-bred Vervet monkeys in which 7.5% is suffering from congenital cataract. However, the parents of the affected individuals were asymptomatic. Six families within the colony have been identified to be affected by two types of morphologies (Y-sutural and total cataract). Based on the evidence provided above, it was speculated that the colony was affected with autosomal recessive cataract. The main aim of this study was to facilitate a strategy for managing breeding programs by minimizing cataract occurrences in captive-bred Vervet monkeys. Integrated combination of clinical, molecular and bioinformatic strategies were used to identify and assess reciprocal candidate susceptibility genes for cataracts. The genes that are known to be responsible for most human congenital cataract cases were prioritized. The genes include Heat shock transcription factor 4 (HSF4), Crystalline Alpha A (CRYAA), glucosaminyl (N-acetyl) transferase 2 (GCNT2) and Lens intrinsic membrane protein 2 (LIM2). Twenty two subjects were selected based on their morphology (5 carriers, 5 controls and 12 cataracts). 2ml of blood was collected for Deoxyribonucleic acid (DNA) extraction. Coding exons and flanking regions were screened by polymerase chain reaction (PCR) amplification and sequenced. The CLC DNA workbench was used for results analysis. The screening of four genes revealed 20 sequence variants which were not present in the control individuals. Sequencing of HSF4 revealed three mutations: R116R, L245>L and P421>L in exon 5, 10 and 14, respectively. The coding exons for CRYAA showed two sequence variants: S134W and K166N in exon 3. Twelve mutations were identified in exon one of all three GCNT2 transcripts (A, B and C). These mutations include: G212G, H256>H, M258>V, N275>N, V16>I, Y122>F, S15>S, S24>N, S38>S, I118>I, D194>D and Y373>Y which was found in exon three of all transcripts. There were no mutations in LIM2, however, three single nucleotide polymorphisms (SNPs) were identified in exon 2 (P66>P) and 3 (I118>T and A127>T). The above mutations were conserved when aligned with other species. The sequence variations vary among the families and those individuals with the same or different cataract phenotype. Based on these findings, it can be concluded that the four candidate genes harbour mutations that are responsible for both phenotypes. The effect of these mutations in Vervet monkeys is not yet understood, however, their impact will be further investigated. For future studies, it will be of absolute importance to screen the entire family to verify that indeed cataract formation in this colony is inherited in an autosomal recessive manner.

Congenital heart defects (CHD) are structural anomalies affecting the heart, are found in 1% of the population and arise during early stages of embryo development. Without surgical and medical interventions, most of the severe CHD cases would not survive after the first year of life. The improved health care for CHD patients has increased CHD prevalence significantly, and it has been estimated that the population of adults with CHD is growing ~5% per year. Understanding the causes of CHD would greatly help improve our knowledge of the pathophysiology, family counseling and planning and possibly prevention and treatment in the future. The aim of my thesis was to identify novel or known CHD genes enriched for rare coding genetic variants in isolated CHD cases and learn about the relative performance of different study designs. High-throughput next generation sequencing (NGS) was used to sequence all coding genes (whole exome) coupled with various analytical pipelines and tools to identify candidate genes in different family-based study designs. Since there is no general consensus on the underlying genetic model of isolated CHD, I developed a suite of software tools to enable different family-based exome analyses of de novo and inherited variants (chapter 2) and then piloted these tools in several gene discovery projects where the mode of inheritance was already known to identify previously described and novel pathogenic genes, before applying them to an analysis of families with two or more siblings with CHD. Based on the tools developed in chapter 2, I designed a two-stage study to investigate isolated parent-offspring trios with Tetralogy of Fallot (chapter 3). In the first stage, I used whole exome sequence data from 30 trios to identify genes with de novo coding variants. This analysis identified six de novo loss-of-function and 13 de novo missense variants. Only one gene showed recurrent de novo mutations in NOTCH1, a well known CHD gene that has mostly been associated with left ventricle outflow tract malformations (LVOT). Besides NOTCH1, the de novo analysis identified several possibly pathogenic novel genes such as ZMYM2 and ARHGAP35, that harbor de novo loss-of-function variants (frameshift and stop gain, respectively). In the second stage of the study, I designed custom baits to capture 122 candidate genes for additional sequencing using NGS in a larger sample size of 250 parent-offspring trios with isolated Tetralogy of Fallot and identified six de novo variants in four genes, half of them are loss-of-function variants. Both of NOTCH1 and its ligand JAG1 harbor two additional de novo mutations (two stop gains in NOTCH1 and one missense and a splice donor in JAG1). The analysis showed a strongly significant over-representation of de novo loss-of-function variants in NOTCH1 (P=3.8 ×10-9). To assess alternative family-based study design in CHD, I combined the analysis from 13 isolated parent-offspring trios with 112 unrelated index cases of isolated atrioventricular septal defects (AVSD) in chapter 4. Initially, I started with a case/control analysis to test the burden of rare missense variants in cases compared with 5,194 ethnically matching controls and identified the gene NR2F2 (Fisher exact test P=7.7×10-07, odds ratio=54). The de novo analysis in the AVSD trios identified two de novo missense variants in the same gene. NR2F2 encodes a pleiotropic developmental transcription factor, and decreased dosage of NR2F2 in mice has been shown to result in abnormal development of atrioventricular septa. The results from luciferase assays show that all coding sequence variants observed in patients significantly alter the activity of NR2F2 target promoters. My work has identified both known and novel CHD genes enriched for rare coding variants using next-generation sequencing data. I was able to show how using single or combined family-based study designs is an effective approach to study the genetic causes of isolated CHD subtypes. Despite the extreme heterogeneity of CHD, combining NGS data with the proper study design has proved to be an effective approach to identify novel and known CHD genes. Future studies with considerably larger sample sizes are required to yield deeper insights into the genetic causes of isolated CHD.

Congenital heart defects (CHD) are the largest class of birth defects in humans and are a major cause of infant mortality and morbidity. Deciphering the molecular and genetic etiologies central for heart development and the pathogenesis of congenital heart diseases (CHD) is a challenging puzzle. We have previously demonstrated that the zinc-finger kruppel-like transcription factor KLF13, expressed predominantly in the atria, binds evolutionarily conserved regulatory elements known as CACC-boxes and transcriptionally activates several cardiac promoters. KLF13 loss of function in Xenopus embryos was associated with cardiac developmental defects underscoring its critical role in the heart. In the current study, using in vivo and in vitro approaches, we examined KLF13’s mechanisms of action and its interaction with other cardiac regulators. To test the evolutionary conserved role in the mammalian heart, we deleted the Klf13 gene in transgenic mice using homologous recombination. Mice with homozygote deletion of Klf13 were born at reduced frequency owing to severe heart defects. We also report the existence of a novel isoform of KLF13, referred to here as KLF13b. Furthermore, we report that KLF13 interacts biochemically and genetically with the T-box transcription factor TBX5 which is a key regulator of heart development. Our data provide novel insight into the role of KLF13 in cardiac transcription and suggest that KLF13 maybe a genetic modifier of congenital heart disease. Furthering our knowledge of protein-protein interactions and gene transcription will enhance genotype-phenotype correlation and contribute to better understanding of the etiology of CHD.

The peripheral and highly lipid-exposed M4 α-helix, although distant from the agonist binding site, channel gate, and other important gating structures, is involved in modulating function of the nicotinic acetylcholine receptor. M4 "senses" changes in the surrounding lipid environment and may consequently affect receptor function by altering specific interactions between the M4 C-terminus and the Cys-loop. An example of this lipid sensing ability is demonstrated by a lipid-facing Cys418 to Trp substitution on αM4 (αM4 C418W) of the muscle-type receptor, which subtly alters protein-lipid interactions and potentiates channel function 16-fold, leading to a slow-channel congenital myasthenic syndrome. Through the use of mutational studies and mutant cycle analysis, I determine that, contrary to previous studies, M4–Cys-loop interactions are not critical to wild-type muscle-type receptor function, nor are they involved in C418W-induced potentiation. Instead, C418W potentiates channel activity by enhancing local M4-M1 interactions mediated by three polar side-chains, which are absolutely critical to potentiation. I show that altered M4-M1 interactions are ultimately translated to two important gating structures, which work in tandem to stabilize the open conformation of the receptor. These studies highlight how altered protein-lipid interactions can affect channel function and contribute to our understanding of the underlying gating mechanism of the muscle-type receptor.

>Magister Scientiae - MSc / Molecular genetics: strategies to indentify congenital cataract genes in captive-bred Vervet monkeys Zandisiwe Emilia Magwebu MSc thesis, Department of Medical Biosciences, University of the Western Cape The present study describes molecular aspects of inherited congenital cataract in captive-bred Vervet monkeys. Congenital cataracts are lens opacities that are present at birth or soon after birth and include hereditary cataracts or cataracts caused by infectious agents. The MRC Primate Unit is housing a colony of captive-bred Vervet monkeys in which 7.5% is suffering from congenital cataract. However, the parents of the affected individuals were asymptomatic. Six families within the colony have been identified to be affected by two types of morphologies (Ysutural and total cataract). Based on the evidence provided above, it was speculated that the colony was affected with autosomal recessive cataract. The main aim of this study was to facilitate a strategy for managing breeding programs by minimizing cataract occurrences in captive-bred Vervet monkeys. Integrated combination of clinical, molecular and bioinformatic strategies were used to identify and assess reciprocal candidate susceptibility genes for cataracts. The genes that are known to be responsible for most human congenital cataract cases were prioritized. The genes include Heat shock transcription factor 4 (HSF4), Crystalline Alpha A (CRYAA), glucosaminyl (N-acetyl) transferase 2 (GCNT2) and Lens intrinsic membrane protein 2 (LIM2). Twenty two subjects were selected based on their morphology (5 carriers, 5 controls and 12 cataracts). 2ml of blood was collected for Deoxyribonucleic acid (DNA) extraction. Coding exons and flanking regions were screened by polymerase chain reaction (PCR) amplification and sequenced. The CLC DNA workbench was used for results analysis. The screening of four genes revealed 20 sequence variants which were not present in the control individuals. Sequencing of HSF4 revealed three mutations: R116R, L245>L and P421>L in exon 5, 10 and 14, respectively. The coding exons for CRYAA showed two sequence variants: S134W and K166N in exon 3. Twelve mutations were identified in exon one of all three GCNT2 transcripts (A, B and C). These mutations include: G212G, H256>H, M258>V, N275>N, V16>I, Y122>F, S15>S, S24>N, S38>S, I118>I, D194>D and Y373>Y which was found in exon three of all transcripts. There were no mutations in LIM2, however, three single nucleotide polymorphisms (SNPs) were identified in exon 2 (P66>P) and 3 (I118>T and A127>T). The above mutations were conserved when aligned with other species. The sequence variations vary among the families and those individuals with the same or different cataract phenotype. Based on these findings, it can be concluded that the four candidate genes harbour mutations that are responsible for both phenotypes. The effect of these mutations in Vervet monkeys is not yet understood, however, their impact will be further investigated. For future studies, it will be of absolute importance to screen the entire family to verify that indeed cataract formation in this colony is inherited in an autosomal recessive manner.

INTRODUCTION: Congenital Heart Defects are the most common congenital defects in the United States. They affect a significant proportion of all births, and many babies with CCHDs are not expected to survive their first year. While diagnostic and surgical interventions have drastically improved mortality rates, a growing population of adolescent and adult CHD patients continue to face unique developmental, psychological and QoL issues. METHODS: Medical journal articles were utilized to determine the prevalence, mortality rates, survival rates, adverse psychological outcomes, and follow-up rates for CHD patients as they transitioned through adolescence. Most articles came from Pediatrics, The Journal of Pediatrics, and Circulation. RESULTS: From 1980-2005, the prevalence of CHDs in the United States increased, while CHD mortality decreased by nearly half. Over a similar time period, CHD patients were more likely to have poor psychological, behavioral, and QoL outcomes than their healthy peers. Specifically, CHD patients were likely to have developmental disorders, lower QoL, and loss to follow-up when transitioning to adult care providers. CHD patients also demonstrated a poor understanding of their condition, especially with respect to need for follow-up, identifying symptoms of deteriorating heart condition, and the negative effects of smoking, drugs, and alcohol. DISCUSSION: Diagnostic and surgical interventions for CHD patients have led to increased survival. However, many of these interventions occur in the early stages of life, leaving a gap in medical management of CHD patients as they transition through adolescence. This is represented by high attrition rates for those following-up with adult care providers, adverse psychosocial outcomes, and patients’ lack of knowledge about their condition. CHD patients may benefit from more comprehensive, coordinated and formal transition programs that incorporate good social support systems, self-efficacy, and education about their condition.

Background: Naviculectomy was originally described for resistant congenital vertical talus deformity but was later expanded to use in rigid cavus deformity. This study reviews the operative outcomes of complete excision of the navicular for recurrent deformity in the talipes equinovarus (TEV) population. Methods: After institutional review board approval, all patients undergoing naviculectomy at a single institution were identified. Clinical, radiographic, and pedobarographic data (minimum 2 years’ follow-up) were reviewed. Results: Twelve patients (14 feet) with TEV from 1984 to 2019 were included. All feet had minimum 1 prior operative intervention on the affected foot (mean age = 4.0 years, range 0.2-14.5), with 8/14 having at least 3 prior operative procedures. Complete navicular excision with concomitant procedures was performed in all patients (mean age = 11.7 years, range 5.5-16.1). Mean clinical follow-up from naviculectomy was 5.1 years (range, 2.2-11.2). During follow-up, 6 patients required subsequent surgery, most often secondary to pain and progressive deformity. One patient underwent elective below-knee amputation of the affected extremity. Of the remaining 11 patients, 7 of 11 reported continued pain and 8 of 11 maintained adequate range of motion at the ankle at the most recent follow-up. Conclusion: Clinical follow-up demonstrated deteriorating results in a large percentage of patients. The high rate of additional procedures and continued pain in the current series suggests that even as a salvage procedure, naviculectomy may not provide adequate results for patients. Level of Evidence: Level IV, case series.

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