Paediatrics (RCH) - Theses
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ItemWhole-genome expression profiling of cord blood mononuclear cells from monozygotic twin pairs( 2010)Substantial variation in gene expression levels exists between individuals, within specific tissues or cell types. With gene expression being the primary mechanism through which genetic information is translated into phenotype, the extent, nature and sources of this variation constitutes an important aspect of human biology. Variation in gene expression levels reflects a complex interplay of genetic and environmental factors. Certain environmental factors and exposures can modify gene expression through epigenetic modifications of DNA and chromatin, thus regulating transcription in a manner largely independent of genetic variation. The sensitivity of epigenetic mechanisms to these factors offers a means through which the environment can modulate expression of the genotype, with effects upon gene expression and ultimately, the phenotype. Studies of variation in gene expression in monozygotic (genetically identical) twins support a substantial environmental contribution to variation in gene expression levels. It is known that the epigenetic and gene expression profiles of monozygotic twins diverge throughout life. Mounting evidence suggests that the period of pre-natal development represents a particularly sensitive one for the occurrence of environmentally induced changes to epigenetic status and gene activity. The current study forms part of a larger research program investigating epigenetic variation in twins and its association with birth weight, maternal nutrition and foetal genotype. The Peri-/Post-natal Epigenetic Twins Study (PETS) builds upon the ‘developmental origins of adult disease’ hypothesis, based on the association between low birth weight and increased risk of cardiovascular and metabolic disease in later life, and focuses on epigenetic changes occurring in utero as the basis of the ‘foetal programming’ phenomenon. The current study is based on the hypothesis that divergence of epigenetic and gene expression profiles occurs from conception in monozygotic twins, in response to differing environments as experienced in utero. This study investigates the gene expression profiles of the cord blood mononuclear cells (CBMCs) of twelve newborn monozygotic twin pairs, including six pairs with birth weight discordance at greater than 15%. Genome-wide expression profiling was performed using the Illumina® Human-6 v2 BeadChip system. Gene expression discordance within twin pairs was assessed using three measures. Of these, the measure of Euclidean distance was considered to be the most systematic and useful. Expression discordance was found to vary substantially across pairs in our sample, with expression discordance being generally lower within twin pairs than between unrelated individuals. A significant correlation was identified between expression discordance and chorionicity, with greater expression discordance in dichorionic pairs compared to monochorionic pairs. The measure of Euclidean distance was also applied to publicly available datasets from genome-wide expression profiling of comparable tissues from adult twin pairs, revealing higher levels of expression discordance within the adult pairs relative to the newborn pairs. All genes surveyed by the microarray analysis were ranked according to the degree of within-pair variation shown across twin pairs. This ranked gene list was subjected to gene ontology analysis to identify gene ontology (GO) terms for which the corresponding ranks were higher than expected. Of the 27 GO terms ranked significantly higher than expected, one third related to immune response or response to other external signals. This data supports our hypothesis that divergence of gene expression profiles occurs from conception in monozygotic twins, and is reflected in differential expression phenotypes detectable at birth. Taken together, these results highlight the role of environment in determining gene expression profiles, and the contribution of environmentally induced changes in gene expression to expression discordance within monozygotic twin pairs. The increased variation observed within dichorionic twin pairs, coupled with the prominence of genes involved in immune/external signal response amongst those showing increased variation across pairs, implies that this variation may arise in response to subtly differing environments experienced by co-twins in utero. Birth weight is a phenotype of particular interest in the Peri-/Post-natal Epigenetic Twins Study (PETS), due to the association of low birth weight with an elevated risk of cardiovascular and metabolic disease in later life. In this study, linear modelling identified 342 genes whose expression levels showed a significant association with birth weight in dichorionic twin pairs. Gene ontology analysis of these genes revealed significant over-representation of GO terms relating to protein dephosphorylation, a process intrinsic to many forms of signal transduction. These data imply a link between the environmental modulation of gene activity via signal response/transduction and the phenotype of birth weight. Due to the myriad number of signal transduction pathways and physiological processes regulated by protein phosphorylation and dephosphorylation, it is not possible to pinpoint with certainty those that may be linked to birth weight or implicated in the association between low birth weight and elevated disease risk from our data. However, these results do provide a basis for further investigation of the specific environmental factors involved in the determination of gene expression variants associated with birth weight. It is to be hoped that future analyses will assist in the identification of the mechanisms underlying the correlation between low birth weight and an elevated risk of cardiovascular and metabolic disease in later life.
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ItemA prospective study of the psychological impact, understanding and disclosure of gene test results for hypertrophic cardiomyopathy and long QT syndrome.( 2010)Hypertrophic cardiomyopathy (HCM) and Long QT syndrome (LQTS) are inherited cardiovascular conditions for which genetic testing has become more common in clinical practice. The most concerning feature of these conditions is sudden death which can be prevented if those at risk are identified. Research into the clinical and molecular aspects of these conditions has advanced rapidly but research on the psychosocial implications of genetic testing for these conditions has lagged behind. The present study aimed to add to the limited body of research about the impact of genetic testing for HCM and LQTS to guide genetic health professionals working with families who are considering genetic testing. A multi-centre prospective questionnaire-based study was conducted to examine the impact of diagnostic and predictive testing for HCM and LQTS. Understanding of test results, risk perception, motivations for and concerns about testing and psychological impact of result disclosure were examined as well as disclosure of gene test result to relatives. Participants were recruited from four Australian and one British site. Questionnaires were completed before testing and at two weeks and three months post-disclosure. Data from participants who underwent diagnostic testing (n=46, 15-76 years old, 40 (80%) tested positive) were analysed separately to those who had predictive testing (n=77, 14-67 years old, 29 (38%) tested positive). A high proportion of participants from both the diagnostic and predictive gene testing groups were pleased they had testing, recalled their result accurately and in the diagnostic group, understood the implications of their result for their own health and their relatives. In those who had predictive testing, perceptions of the likelihood of developing disease, level of worry, and the number of concerns about LQTS and HCM reported were consistent with gene test result. More than 90% of participants in both groups disclosed their gene test results to first‐degree relatives. Concerns about sudden death were evident in participants from both the diagnostic and predictive group, confirming anecdotal reports from clinical practice. Concerns about the possibility of at-risk relatives inheriting either LQTS or HCM and the unpredictable natural history of both conditions were also common concerns. For the predictive testing group, multivariable linear regression analysis adjusting for baseline psychological scores and potential confounders, and accounting for family clusters demonstrated a higher mean anxiety (p=0.005) and distress (p=0.003) score in gene positive compared to gene negative participants at 2 weeks, but these differences were less apparent at 3 months. There was no difference in depression scores at any time point in those who underwent predictive testing. The same analysis was performed in the diagnostic testing group and there were no statistically significant differences in adjusted mean anxiety, distress and depression scores when comparing participants with gene positive and gene negative results at any time point. Overall, high proportions of participants who underwent diagnostic and predictive testing were pleased to have undergone testing, understood the implications of their result and disclosed their result to relatives. There was no evidence for significant negative psychological sequelae three months after receipt of test result in participants who had either diagnostic or predictive testing. Implications for practice are presented.
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ItemRespiratory function tracking in survivors of prematurity and low birth weight( 2009)INTRODUCTION: To investigate longitudinal ‘tracking’ in lung growth, decline or stability of adult preterm/very low birth weight survivors over the study period. To investigate whether those who have clinically abnormal lung function fall further behind their contemporaries or demonstrate evidence of ‘catch-up’ growth. METHODS: 210 babies born ≤34 weeks’ gestation and with birth weights ≤ 1500g in 1977-1982 were followed up at 8, 11, 14, 18 and 25 years of age. Respiratory outcome was evaluated using Spirometry. Spirometry was performed at each of the follow-up periods. Mixed model regression was used to assess longitudinal lung function trajectories and the influence of Bronchopulmonary dysplasia, being small for gestational age and abnormal lung function. Restricted maximum likelihood modelling was used for longitudinal FEV1 z-score as it allows for analysis of data from different time points that are not necessarily evenly spaced, without being affected by missing data. RESULTS: 137 VLBW children completed lung function testing at 8 years of age. Twenty VLBW children (14.6%) had abnormal FEV1 z-scores and 26 (21.0%) [Defined as >2 standard deviation’s below the mean]. VLBW survivors showed minimal ‘catch-up’ in FEV1 z-score over the 13 years of the study; those without BPD (Bronchopulmonary dysplasia) FEV1 improved 0.034 (p=0.014) z-scores, those with BPD FEV1 improved 0.033 (p=0.039) z-scores. VLBW with BPD survivors did not return to within normal limits; those with abnormal FEF25-75 z-scores did not show significant ‘catch-up’ growth (p=0.41) and remained abnormal. CONCLUSIONS: Mixed models do not reveal any significant effects from being born with VLBW and its interaction with age; it does lead to a reduction in FEV1/FVC mean z-score. Although those VLBW individuals who were small for gestational age had significantly reduced FEV1, FVC and FEV1/FVC mean z-scores and these small for gestational age individuals show evidence of ‘catch-up’ growth in terms of lung function, with the exception of FEV1/FVC z-score which falls further from their contemporaries. This effect on FEV1 and FVC z-scores may suggest some protective element or compensatory mechanism of being born small for gestational age. Reassuringly, Bronchopulmonary dysplasia as a neonate did not significantly affect lung function variables as these individuals as they age. Those with clinically abnormal lung function show a progressive failure to achieve optimal lung function whether or not they had Bronchopulmonary dysplasia.
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ItemChildhood pneumonia and hypoxaemia in an urban diarrhoeal hospital, Dhaka, Bangladesh( 2010)The aim of this prospective cohort study was to evaluate clinical and socio-demographic predictors of pneumonia, deaths from pneumonia, and hypoxaemia in children. All under-five children who were admitted to the special care ward of ICDDR,B during September 2007-December 2007 were enrolled. Children sleeping in a bare bed and those having parents/caregivers with poor knowledge were at risk of pneumonia. Children with severe malnutrition, hypoxaemia, or severe sepsis were at higher risk of death. Chest wall-indrawing was the best predictor of hypoxaemia.
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ItemAttitudes towards newborn screening for Pompe disease among affected adults, family members and parents of ‘healthy’ children( 2009)Pompe disease is a rare autosomal recessive condition caused by a deficiency in lysosomal alpha glucosidase. It is a progressive and often fatal muscular disease with wide variation in clinical presentation. Two broad clinical categories of Pompe disease have been identified; infantile- and late- onset. In the past decade, enzyme replacement therapy has shown promising results in treating the underlying pathology, resulting in improved clinical outcome. Clinical trials indicating that initiation of treatment at an earlier disease stage leads to a higher chance of preventing permanent damage have led to the proposition of introducing newborn screening for Pompe disease. All forms of Pompe disease are caused by the same pathology, and thus newborn screening has the potential to identify those affected with the more severe infantile-onset form as well as those with late-onset disease who may not present with symptoms until late in life. The aim of this study was to investigate attitudes towards newborn screening for Pompe disease among affected adults, their family members and parents of ‘healthy’ children. Affected adults were recruited through support groups in Australia, the United Kingdom and United States; family members of affected adults were recruited from Australia; and parents of ‘healthy’ children were recruited through maternal child health clinics in Victoria, Australia. Participants completed questionnaires exploring their experiences of Pompe disease and/or newborn screening and their attitudes towards newborn screening for Pompe disease. Support for newborn screening for Pompe disease was high among adults with Pompe disease (85.4%), parents of ‘healthy’ children (93.9%) and all three family members of affected adults who participated in this study. However, when offered a theoretical screening test that would only identify infantile-onset Pompe disease, 42.1% of adults with Pompe disease and 53.1% of parents of ‘healthy’ children preferred this screen, indicating that these stakeholders have some concerns regarding detection of late-onset disease in infancy. Factors influencing attitudes were investigated and support for newborn screening in affected adults was highly correlated with age of onset of disease; a preference to have been diagnosed in infancy; a belief that an earlier diagnosis would have made symptoms easier to cope with; and a stronger confidence in the efficacy of enzyme replacement therapy. Potential benefits of diagnosis of late-onset disease in infancy were identified as being able to avoid the diagnosis odyssey, access enzyme replacement therapy at the optimal time, and allow individuals to make appropriate life choices. Participants identified increased anxiety in parents and the potential for over-protectiveness, in addition to possible discrimination, as harms of newborn screening for Pompe disease. Families in which an infant is identified with the potential for late-onset Pompe disease will need assistance to adapt to and manage this diagnosis, so that anxiety is minimised and unnecessary limitations are not placed on the child. Whilst potential medical and psychosocial benefits can result from newborn screening, it is important to carefully consider the potential for harm and the resources required to appropriately manage these so that ultimately benefit outweighs harm.
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ItemExploring the experiences of people who have consented to tumour testing for a hereditary disposition to cancer( 2009)Due to the costly and technically challenging nature of genetic testing, methods have been developed to target more specifically those who are at increased risk of carrying the Hereditary Non-Polyposis Colorectal Cancer (HNPCC) mutation. HNPCC is an inherited colorectal cancer syndrome. Testing of tumour material (which has previously been removed during surgery) for features of HNPCC has been found to be an effective and economic method of identifying those at higher risk of having a mutation. Only those at higher risk of having a mutation will undergo genetic testing. This practice of “tumour testing” has become widespread. There is currently no clarity about requirements for consent prior to testing of stored tumour tissue. The person giving consent to tumour testing does not always have an appointment with a genetics service prior to giving consent. This can be contrasted to genetic testing on blood samples where laws and guidelines state that informed consent is required prior to genetic testing and that comprehensive genetic counselling and support should be provided as part of this process. Protocols for genetic testing have been developed as a result of extensive research around the impact and implications of genetic testing. Consumer opinion and participation through research is an important aspect of health policy and guideline development. Accordingly the purpose of this study was to contribute to such development by gaining insight into the experiences, understandings, decision making processes and opinions of those who had given consent to have their own or their relatives tumour tested. Seventeen people who had given consent for tumour testing either for themselves, or on behalf of a deceased relative were recruited through a Familial Cancer Centre and in-depth interviews conducted. The interviews were transcribed and analysed using thematic analysis. Some participants had no memory of consenting to tumour testing. Others remembered basic concepts. Negative implications of testing were unknown or viewed as unimportant. Participants did not understand the difference between tumour testing and germline testing. Despite lack of memory or understanding participants did not want additional or more detailed pre-test information although they did want more follow-up and support after receipt of results. The decision to consent to testing was made as soon as participants were informed of the availability of tumour testing - the major reason being to provide information for the family that would aid in cancer prevention. Participants were more concerned with accessibility to testing than pre test information and counselling. Findings in this study indicated participants made decisions heuristically rather than systematically and this as well as participants’ opinions and other decision-making research has implications for the traditional view of informed consent around genetic related decisions. This in turn has implications for policy and guidelines in the area. Implications for current practise as a result of findings from this study include ensuring participants understand negative implications of testing and follow up and support of those with negative as well as positive results to tumour testing.