The Centromere is a vital chromosomal structure that ensures faithful segregation of replicated chromosomes to their respective daughter cells. With such an important structure, one would expect the underlying centromeric DNA sequence would be highly conserved across all species. It turns out that the underlying centromeric DNA sequences between species ranging from the yeast, fly, mouse to humans are in fact highly diverged suggesting a DNA sequence independent or an epigenetic mechanism of centromere formation. / Neocentromeres are centromeres that form de-novo at genomic locations that are devoid of highly repetitive a-satellite DNA sequences of which normal centromeres are usually comprised from. To date, the 10q25 neocentromere is the most well-characterised, fully functional human centromere that has been used previously to characterise the extent of a number of centromeric protein binding domains and characterise the properties of the underlying DNA sequence. Along with other factors, the existence of neocentromeres has given rise to a hypothesis where centromeres are defined by epigenetic or DNA sequence independent mechanisms. / The putative 10q25 neocentromere domain was recently redefined by high resolution mapping of Centromeric protein A (CENP-A) binding through a chromatin immunoprecipitation and array (CIA) analysis. The underlying DNA sequence was investigated to determine and confirm that the formation of the 10q25 neocentromere was through an epigenetic mechanism. Through a high-density restriction fragment length polymorphism (RFLP) analysis using overlapping PCR amplified DNA derived from genomic DNA representing the 10q25 region before and after neocentromere activation. No sequence polymorphisms, large insertions or deletions were detected and confirmed the epigenetic hypothesis of centromere formation. / DNA methylation is one of many epigenetic factors that are important for cellular differentiation, gene regulation and genomic imprinting. As the mechanisms and functions of DNA methylation have been well characterised, its role at the 10q25 neocentromere was investigated to try and identify the candidate epigenetic mechanism involved in the formation of centromeres. DNA methylation across the neocentromere was assessed using sodium bisulfite PCR and sequencing of selected CpG islands located across the 10q25 neocentromere. Overall, the methylation level of the selected CpG islands demonstrated no difference in DNA methylation before and after neocentromere activation. However, significant hypomethylation upon neocentromere formation was detected close to the protein-binding domain boundaries mapped previously suggesting that this may have a role in demarcating protein binding domains at the neocentromere. / Further analysis of DNA methylation investigated non-CpG island methylation at sites defined as CpG islets and CpG orphans. Interestingly, the DNA methylation level measured at selected CpG islets and CpG orphans across the 10q25 neocentromere were not completely hypermethylated as previously thought, but demonstrated variable methylation that became fully hypermethylated upon neocentromere activation in most sites investigated. These results suggested that a role for DNA methylation existed at the 10q25 neocentromere and that it occurred at sites devoid of CpG islands. / This study has found that DNA methylation at non-CpG island sites was variable contrary to popular belief and, was linked with neocentromere formation through the observation of increased DNA methylation at the 10q25 neocentromere. Inhibition of DNA methylation demonstrated increased neocentromere instability and a decrease in methylation of these CpG islets and CpG orphans confirming the importance of DNA methylation at neocentromeres. This study has characterised a new class of sequences that are involved in the maintenance of chromatin structure through DNA methylation at the 10q25 neocentromere.

Thesis (Ph.D)--Biology, Georgia Institute of Technology, 2009. / Committee Chair: Dr. Soojin Yi; Committee Member: Dr. Eric Vigoda; Committee Member: Dr. James Thomas; Committee Member: Dr. John McDonald; Committee Member: Dr. Kirill Lobachev; Committee Member: Dr. Michael Goodisman. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 149-172).

DNA methylation (DNAm) is the covalent modification of DNA by addition of a methyl group primarily at the cytosine directly upstream of a guanine. DNAm level plays a central role in transcriptional regulation and is linked to disease. Therefore, understanding genetic and environmental influences on DNAm level in healthy tissue is an important step in the elucidation of trait and disease etiology. However, at present only a minority of easy to access human tissues and ethnicities have been investigated. Therefore, we studied DNAm level measured in five human tissues: cerebellum, frontal cortex, pons, temporal cortex and colon in either North American or South American samples. We applied a novel statistical approach to estimate the heritability attributable to genomic regions (regional heritability, ĥ²/r,g ) for DNAm level at thousands of individual DNAm sites genome-wide. In all five tissues, DNAm level was significantly associated with the local genomic region for more DNAm sites than expected by chance. Moreover, DNAm level could be predicted from the local sequence variants with an accuracy that scaled with the estimated ĥ²/r,g . Our results inform on molecular mechanisms regulating DNAm level and trait etiology in several ways. Firstly, DNAm level at DNAm sites located in genomic risk regions and measured in a tissue relevant to the disease can be influenced by the local genetic variants. Specifically, we found that genetic variation within a region associated with Fluid Intelligence was also associated with local DNAm level at the proline-rich coiled-coil 1 (PRRC1) gene in healthy temporal cortex tissue. Additionally, we replicated the finding of a Colorectal Cancer risk variant (rs4925386) associated with two DNAm sites in healthy colon tissue. More generally, we showed that DNAm sites located within a susceptibility region and measured in a relevant tissue exhibit a similar overall pattern of estimated ĥ²/r,g to DNAm sites outwith a susceptibility region. Secondly, the propensity for DNAm level to be associated with the local sequence variation differs with respect to CpG dinucleotide density and genic location. Most notably, DNAm sites located in CpG dense regions of the genome are less likely to be heritable than DNAm sites located in CpG sparse regions of the genome. Additionally, within both CpG dense and CpG sparse regions of the genome intergenic DNAm sites are more likely to be heritable than intragenic DNAm sites. Overall, our study suggests that variation in DNAm level at some DNAm sites is at least partially controlled by nuclear genetic variation. Moreover, DNAm level in healthy tissue has the potential to act as an intermediary in trait variation and etiology.

Epigenetics refers to heritable changes in phenotype without alterations to the genotype. Epigenetic changes involve two main mechanisms: DNA methylation and histone modification. Methylation of DNA at cytosine bases is the best-studied epigenetic process to date. CpG methylation states are thought to be maintained throughout cell divisions. However, loss of DNA methylation or DNA demethylation has been observed in specific stages of mammalian development. Such prominent examples of developmental DNA demethylation processes occur in developing primordial germ cells and in preimplantation embryos. However, little is known about DNA methylation changes of other tissues in mammalian development. Therefore, the first aim of this PhD study was to investigate changing nuclear distributions and levels of DNA methylation during development in order to discover dynamic variations amongst developing mouse tissues. In addition, a transgenic MBD-GFP mouse was employed to visualise DNA methylation in tissues. Several hypothetical mechanisms for the enzymatic removal of 5mC have been proposed. One of the proposed candidates is Tet-mediated successive oxidation of 5mC to generate 5hmC, 5fC and 5caC. 5hmC has therefore been considered as a transient intermediate in an active cytosine demethylation pathway. Nevertheless, some studies suggest that 5hmC may also function as an epigenetic modification in its own right. Thus, the second aim of this study was to address the research question of how and where 5hmC originates during development. In order to be able to identify tissues undergoing dynamic nuclear changes in DNA methylation and hydroxymethylation states during early mouse development, new working protocols for immunodetection of 5mC and 5hmC on tissue cryosections were required. The protocol optimisation for 5mC immunodetection is discussed in greater detail in Chapter 3. It was found that DNA methylation immunostaining of cryosections required heat-mediated DNA denaturation, which was partly compatible with protein immunostaining. Next, Chapter 4 focuses on identifying tissues undergoing dynamic changes in 5mC and 5hmC patterns during development from E9.5 to E14.5 mouse embryonic stages, using optimised immunohistochemistry protocols. These protocols revealed interesting dynamic observations of 5mC and 5hmC in the developing cerebral neocortex, surface ectoderm, liver, red blood cells, diaphragm and heart. These findings suggested that dynamic changes of 5mC and 5hmC during neocortical and compact myocardial development were in good agreement with a model where the formation of 5hmC may correlate with the loss of old 5mC, but the observations were also consistent with an involvement of de novo methylation in the generation of 5hmC. In other developing tissues, including surface ectoderm, liver, red blood cells, diaphragm and cardiac trabeculae, dynamic changes in 5mC and 5hmC levels were in line with a model where the 5hmC may act as a new epigenetic mark that functions independently. The optimised protocol also confirmed DNA demethylation of the germ cells at E12.5. The presence of three Tet family enzymes (Tet1, Tet2, Tet3) and de novo methyltransferase DNMT3A in mouse E12.5 tissues is reported in the second part of Chapter 4. It was found that Tet1, Tet2, Tet3 and Dnmt3a were present at detectable levels in neocortex, liver, diaphragm and heart. Contrastingly, no apparent signals for Tet1, Tet2, Tet3 and Dnmt3a were observed in red blood cells. This result was expected due to the very low levels of 5hmC staining in E12.5 red blood cells. The third aim of the present study was to investigate the existence of crosstalk between various epigenetic mechanisms. Thus, Chapter 5 focuses on exploring the relationship between 5mC and repressive histone marks, H3K9me3 and H3K27me3. Histone methylation dynamics at H3K9 and H3K27 were observed during mouse fetal development in neocortex and heart. The overall distribution patterns of H3K9me3 and H3K27me3 demonstrated strong association with developmental changes in 5mC, suggesting that these three repressive epigenetic marks work in concert to establish a silenced state of heterochromatin. Chapter 6, on the other hand, focuses on visualising DNA methylation in tissues using mouse transgenic tools. It was found that brain, liver, heart and neural tube expressed high levels of GFP. But no apparent developmental dynamics of GFP was observed. In conclusion, this study will contribute scientific understanding of dynamic DNA methylation and nuclear heterochromatin organisation during mammalian development, and its role in the specification and maintenance of cell lineages forming tissues and organs. This knowledge will provide insight into current barriers to cell fate reprogramming, which will be of benefit to cell regenerative biomedical technologies.

Although structurally unrelated, the human killer cell immunoglobulin-like (KIR) and the rodent lectin-like Ly49 receptors serve similar functional roles in natural killer (NK) cells. Moreover, both gene families display variegated and mostly mono-allelic expression patterns established at the transcriptional level. DNA methylation, but not histone modifications, has recently been shown to play an important role in maintenance of the expression patterns of KIR genes but the potential role of DNA methylation in the expression of Ly49 genes was unknown. My thesis focuses on the role of epigenetic modifications, especially DNA methylation, in the maintenance of mouse Ly49 gene expression. I show that hypomethylation of the region encompassing the main promoter of Ly49a and Ly49c in primary C57BL/6 (B6) mouse NK cells correlates with expression of these genes. Using B6 x BALB/c Fl hybrid mice, I demonstrate that the expressed allele of Ly49a is hypomethylated while the non-expressed allele is heavily methylated, indicating a role for epigenetics in maintaining mono-allelic Ly49 gene expression. Furthermore, the Ly49a promoter region is heavily methylated in fetal NK cells but variably methylated in non-lymphoid tissues. In apparent contrast to the KIR genes, I show that histone acetylation state of the promoter region strictly correlate with Ly49A and Ly49G expression status. Also, the instability of Ly49G expression on some lymphoid cell lines is at least in part due to changes in the level of histone acetylation of the promoter region. As for the activating Ly49 receptors, it seems that although DNA methylation levels of the promoter regions do correlate with the state of expression of these receptors, the pattern of DNA methylation is different from that of the inhibitory Ly49a and c genes. In conclusion, my results support a role for epigenetic mechanisms in the maintenance of Ly49 expression. Moreover, these epigenetic mechanisms appear to vary among the Ly49 genes and also differ from those governing KIR expression. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

DNA methylation is one of the principal epigenetic modifications that plays an essential role in transcriptional regulation. After fertilization, mammalian embryos undergo dynamic changes in genome-wide DNA methylation patterns and the changes are essential for normal embryo development. Ten-eleven translocation (TET) methylcytosine dioxygenases are implicated in DNA demethylation by catalyzing the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). The three members of TET protein family, TET1, TET2, and TET3, are highly expressed in preimplantation embryos in a stage-specific manner. Previous studies demonstrated that TET proteins are involved in diverse biological processes such as gene regulation, pluripotency maintenance, and cell differentiation by mediating 5mC oxidation. My dissertation research was conducted to elucidate the mechanistic roles of TET proteins in epigenetic reprogramming of mammalian embryos using porcine embryos as a model. The first set of studies focused on the relationship between TET proteins and pluripotency. To understand the role of TET proteins in establishing pluripotency in preimplantation embryos, CRISPR/Cas9 technology and TET-specific inhibitors were applied. TET1 depletion unexpectedly resulted in an increased expression of NANOG and ESRRB genes in blastocysts, although the DNA methylation levels of NANOG promoter were not changed. Interestingly, transcript abundance of TET3 was increased in blastocysts carrying inactivated TET1, which might have had an effect on the increase of NANOG and ESRRB. When the activity of TET enzymes was inhibited to eliminate the compensatory increase of TET3 under the absence of functional TET1, the expression levels of NANOG and ESRRB were decreased and methylation level of NANOG promoter was increased. In addition, ICM specification was impaired by the inhibition of TET enzymes. These results suggest that the TET family is a critical component of the pluripotency network of porcine embryos by regulating expression of genes involved in pluripotency and early lineage specification. In the next set of studies, the presence of TET3 isoforms in porcine oocytes and cumulus cells was investigated to dissect the gene structure of TET3 that could assist in understanding mechanistic actions of TET3 in the DNA demethylation process. Among the three TET3 isoforms identified in cumulus cells, only the pTET3L isoform, which contains CXXC domain that carry DNA binding property, was verified in mature porcine oocytes. Expression level of the pTET3L isoform was much higher in mature oocytes compared to that in somatic cells and tissues. In addition, the transcript level of this isoform was significantly increased during oocyte maturation. These results suggest that pTET3L isoform is predominantly present in mature porcine oocytes and that CXXC domain may play an important role in DNA demethylation in zygotes. In a follow-up study, the role of the TET3 CXXC domain in controlling post-fertilization demethylation in porcine embryos was investigated by injecting TET3 GFP-CXXC into mature porcine oocytes. The injected CXXC was exclusively localized in the pronuclei, indicating that the CXXC domain may localize TET3 to the nucleus. The CXXC overexpression reduced the 5mC level in zygotes and enhanced the DNA demethylation of the NANOG promoter in 2-cell stage embryos. Furthermore, the transcript abundance of NANOG and ESRRB was increased in blastocysts derived from GFP-CXXC overexpressing zygotes. These results provide an evidence that the CXXC domain of TET3 is critical for post-fertilization demethylation of porcine embryos and proper expression of pluripotency related genes in blastocysts. In the last set of studies, the impact of MBD proteins on porcine embryo development was examined under the hypothesis that competitive binding of MBD and TET proteins to 5mC contributes to the proper maintenance of DNA methylation levels in embryos. Cloning of porcine MBD1, MBD3, and MBD4 from mature oocytes indicates that the genes are highly conserved among different species, implying the involvement of porcine MBD proteins in the maintenance of DNA methylation. MBD1 overexpression in oocytes impaired preimplantation development of porcine embryos, suggesting that the MBD1 overexpression may have negatively affected porcine embryo development because proper DNA methylation levels were not preserved under the high level of MBD1. Collectively, the studies in my dissertation demonstrate that TET family proteins are important epigenetic players involved in the regulation of pluripotency and reprogramming of DNA methylation, and are thus crucial for normal embryo development. The findings in the dissertation will improve our understanding of epigenetic events occurring in mammalian embryos, and have the potential to overcome epigenetic defects that are observed in pluripotent stem cells and in-vitro derived embryos. / Doctor of Philosophy / Epigenetic modifications are heritable changes affecting the level of gene expression without changing the sequence of the genome. DNA methylation, one of the biggest epigenetic marks in mammalian genome, is often correlated to gene repression. In mammals, DNA methylation patterns are dramatically changed during preimplantation development to acquire embryonic developmental potential. Understanding of the epigenetic changes occurring in preimplantation embryos is necessary for producing healthy domestic animals in agriculture and for developing strategies for the treatment of epigenetic defects in human. Ten-eleven translocation (TET) family enzymes, TET1, TET2, and TET3, are known to function as a DNA methylation modifier by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). My dissertation research was performed to elucidate the role of TET family during preimplantation development using porcine embryos as a model. Pluripotency refers to the ability of cells to differentiate into all cell types of a mature organism. Pluripotent cells emerge in embryos as embryonic cells acquire lineage-specific characteristics. The first set of studies focused on the role of TET enzymes in regulating the pluripotency of porcine embryos. The impacts of inhibited activities of TET enzymes on the expression of pluripotency related genes were examined. We found that the inhibition of all TET enzymes leads to a decreased expression of pluripotency related genes, an altered DNA methylation level on a gene segment controlling pluripotency, and the impaired formation of pluripotent cell lineage in porcine embryos. This study demonstrates that the TET family is critical for the acquisition of pluripotency in porcine embryos. In the following sets of studies, the function of TET3 protein in the demethylation process occurring in preimplantation embryos was investigated. Fertilized mammalian embryos undergo genome-wide demethylation process to reset germ cell specific epigenetic marks into the embryonic epigenome. Previous studies indicate that TET3 is responsible for the demethylation process in mammalian embryos, although detailed mechanistic action of TET3 is still elusive. Here, we identified a predominant expression of a specific TET3 gene in porcine oocytes. The TET3 gene contained a CXXC domain, a potential DNA binding module, suggesting that TET3 may mediate DNA demethylation through its DNA binding property. To examine the function of the CXXC domain in TET3-mediated DNA demethylation, isolated CXXC domain was injected into porcine oocytes. The injection of CXXC domain facilitated DNA demethylation in embryos, demonstrating that the DNA binding property of TET3 is important for its functionality. In the last study, we investigated the importance of genes known to interact with TET enzymes in porcine embryos. Methyl-CpG-binding domain proteins (MBDs) have the ability to bind methylated region on the genome and play a critical role in mediating DNA methylation and gene repression. Our hypothesis was that a competitive binding of MBD and TET proteins to methylated regions was critical for proper DNA methylation levels in embryos. We identified that porcine MBD sequences were very similar to other species in terms of gene structure, indicating that the genes could also possess gene repressing activity by competing with TET enzymes during porcine embryo development. Injection of MBD1 mRNA to oocytes impaired normal embryo development, suggesting that the injected MBD1 may have negatively affected early embryo development in pigs by disrupting the proper maintenance of DNA methylation levels. My dissertation researches demonstrate that maintaining proper DNA methylation levels through the TET family is critical for normal embryo development in pigs. This research assists in improving our understating of epigenetic dynamics occurring in mammalian embryos and offers a potential solution to the epigenetic defects frequently observed in mammalian embryos produced through artificial reproductive technologies and pluripotent stem cells reprogrammed from somatic cells.

Hypothalamus and adipose tissue are essential central and peripheral sites regulating energy homeostasis. Disruption of energy homeostasis can lead to diseases like anorexia and obesity in humans and reduced productivity in animals. Therefore, integrating knowledge in hypothalamic appetite regulation and adipose tissue metabolism is essential to maintain homeostasis. The aim of this dissertation was to elucidate molecular and cellular mechanisms of energy homeostasis in birds. We determined adipose tissue physiological changes during the first two weeks post-hatch in chickens from lines selected for low (LWS) and high (HWS) body weight. LWS was more dependent on yolk and subcutaneous fat mobilization for growth from hatch to day 4 post-hatch, with hyperplasia-predominated replenishment of the reservoir. In contrast, HWS was more dependent on feed for growth and maintained depot mass through hyperplasia and hypertrophy. From day 4 to 14 post-hatch, compared to maintenance of depot weight and adipocyte size in LWS, HWS accumulated clavicular and abdominal fat with minimal lipolysis. There was greater expression of precursor and proliferation markers in LWS with more apoptotic cells in the abdominal stromal vascular fraction on day 14 post-hatch, suggesting that apoptosis contributed to lower adipogenic potential and lack of abdominal fat in LWS. Exposure to thermal and nutritional stressors at hatch impaired growth by reducing yolk utilization and lowering body weight, lean and fat masses in LWS. Stress exposure resulted in increased global DNA methylation and DNA methyltransferase activity in the arcuate nucleus of the hypothalamus in LWS. Moreover, there was decreased binding to methyl-CpG-binding domain protein 2 in the promoter of corticotropin-releasing factor (CRF) because of hypomethylation in one CpG site at its core binding site in stressed LWS, which explains the increased CRF expression in the paraventricular nucleus of the hypothalamus. We next determined effects of nutritional status on adipose tissue physiology in Japanese quail, a less-intensively selected avian species. Six-hour fasting promoted lipolysis and gene expression changes in 7-day old quail with some changes restored to original levels within 1 hour of refeeding. Overall, our results reveal novel cellular and molecular mechanisms regulating appetite and adiposity in birds early post-hatch. / Doctor of Philosophy / Hypothalamus and adipose tissue are essential for regulating energy homeostasis in central and peripheral body sites, respectively. Disruption of energy homeostasis can lead to diseases like anorexia and obesity in humans and reduced productivity in animals. Therefore, integrating knowledge in hypothalamic appetite regulation and adipose tissue metabolism is essential to maintain energy homeostasis in both humans and animals. The aim of this dissertation was to elucidate molecular and cellular mechanisms of energy homeostasis in birds. We first determined adipose tissue physiological changes in chickens during the first two weeks post-hatch from lines selected for low (LWS) and high (HWS) body weight. These chickens have been selected for juvenile body weight for over 60 generations. The LWS are lean and anorexic, while HWS eat compulsively and develop obesity and metabolic syndrome. Such characteristics make the body weight line chickens good animal models to study physiological changes under anorexia and obesity. We found that LWS was more dependent on yolk reserves and subcutaneous fat mobilization for growth from hatch to day 4 post-hatch, with replenishment of the fat reservoir by increases in cell number. By contrast, HWS was more dependent on feed for growth and maintained depot mass through increased cell number and cell size. From day 4 to 14 post-hatch, HWS accumulated fat throughout the body, with less fat breakdown as compared to LWS. There was greater expression of cellular precursor and proliferation markers in LWS, with more dying cells in their abdominal fat on day 14 post-hatch, suggesting that programmed cell death is responsible for the lack of fat cell development in LWS. Exposure to thermal and nutritional stressors at hatch impaired growth by reducing yolk utilization and lowering body weight, lean and fat masses in LWS. There were many molecular changes in the hypothalamus, including changes in DNA that led to increased activation of corticotropin-releasing factor (CRF), a signaling molecule that is known to regulate the body's stress and appetite responses. Stress exposure increased global DNA methylation and DNA methyltransferase activity in the arcuate nucleus of the hypothalamus in LWS. Moreover, there was less methylation at the core binding site of methyl-CpG-binding domain protein 2 (MBD2), a protein that binds to methylated DNA to repress gene expression, in the CRF gene, in stressed LWS. In response to stress, there was decreased binding of MBD2 to the promoter region of CRF, which may explain increased expression of CRF in the paraventricular nucleus of LWS. These results demonstrate that early-life stressful events can cause epigenetic changes (like DNA methylation) that lead to alterations in physiology and behavior that persist to later in life. We next determined effects of nutritional status on adipose tissue physiology in Japanese quail, which have undergone less artificial selection than chickens and are more representative of a wilder-type bird. Six-hour fasting promoted lipolysis and gene expression changes in 7-day old quail with some changes restored to original levels within 1 hour of refeeding. Overall, our results provide novel perspectives on cellular and molecular mechanisms regulating appetite and adiposity in birds during early post-hatch development.

DNA methylation is an epigenetic mechanism with roles that range from the fine tuning of transcription to genome wide dynamic acclimation to changing environments and regulation of developmental processes. While recent work has confirmed the presence and regulatory functions of DNA methylation in non-bilaterians, its role and distribution in Porifera has never been addressed. In this study, we performed whole genome bisulfite sequencing of the demosponge Amphimedon queenslandica and show that DNA methylation occurs mostly in CpG dinucleotides of coding regions. While high levels of gene-body methylation correlate positively with high expression and co-occur with the histone modification H3K36me3, they are not associated with amelioration of spurious transcription as found in other metazoans; nonetheless, per-exon methylation levels are predictive for exon retention suggesting a role in mRNA splicing. Additionally, analyses of Amphimedon and other sponges genomic data consistently revealed biased dinucleotide frequencies that suggest a long history of methylation-driven CpG conversion. Despite a genome wide loss of CpG dinucleotides, these are positively selected in exons and in methylated genes. These results indicate DNA methylation as a component of early metazoans regulome and challenge hypothesis on CpG methylation acting as a means for codon usage optimization.

A research report submitted to the Faculty of Medicine, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the Degree of Master of Science in Medicine. Johannesburg October, 1997 / Fragile X syndrome is, second to Down syndrome, the commonest form of genetic mental retardation. The aim of this research project was to investigate the impact of having a child with this syndrome on the family relationships. The subjects were 21 mothers and 9 fathers of affected children. The data were collected by means of specially constructed questionnaires in interviews with 19 mothers and 8 fathers and completed by post in three cases. A control group of parents with a normal child, matched for sex and age of the affected child, family size and ethnic groups, was interviewed. The data were computerised and analyzed. The results showed that more experimental parents than controls enjoyed their child’s nature, but disliked the behavioural problems. About half of the experimental parents tended not to reward good behaviour physically. However, although most of the affected children were accepted by their siblings, they had fewer friends and more problems with their peers. Some parents thought that their relationship with their spouse had improved and others thought that it had deteriorated after the affected child’s birth. Most parents in both study groups would request prenatal diagnosis in subsequent pregnancies and significantly more experimental parents than controls would request a termination of pregnancy for an affected fetus. Most parents were satisfied with the health service they received. These results show that family dynamics are disturbed by the presence of a child with FMR. Counsellors and therapists working with these families should be aware of the effects of the syndrome on the family / IT2017