| Part1:Molecular Genetic Studies in Chinese families with Monogenic ObesityDuring the past decades, obesity has become the main challenge for public health worldwide. Known as a multifactor disease, obesity is influenced both by genetic and environmental factors. It has been estimated that an inherited component to body weight accounts for40-70%. Therefore, study on the molecular mechanism underlying obesity has great significance. Due to the complexity resulting in obesity, studies on the early-onset, extreme, andmonogenic type of obesity have been chosen as the breakthrough for investigating molecular mechanism of obesity. To date, there are more than20genes, in which mutations result in severe monogenic human obesity, have been identified. However, the monogenic causes of obesity identified so far only account for less than5%of severe obesity. Understanding the molecular mechanism of the other95%of obesity is still a great challenge.Human growth is a multi-factorial and complex process, while the height is still predominantly under genetic control. Studies on molecular mechanisms resulting in tall stature and’overgrowth’have perhaps occurred in a less systematic manner, comparing with the study of short stature. Tall stature and overgrowth during childhood can be caused by many reasons, while obesity might be the commonest reason for apparent children tall stature and overgrowth. Therefore, identifying genetic causes of tall stature and overgrowth, weight gain can further our knowledge on the important mechanisms, by which childhood growth and body composition are regulated.In the present study, we have collected one family with autosomal dominant obesity with overgrowth and four nuclear families with early onset obesity. With informed consent of all participating individuals, we performed genetic research in these five families, respectively.We named the first family Obesity with Overgrowth by the fact that besides obesity, two pubertal patients seemed taller than their peers. Firstly, in the proband of this family, we have screened mutations of23genes associated with monogenic forms of obesity in humans or transgenic mice. After that, Affymetrix SNP6.0array was used to detect the copy number variation and performed whole genome linkage analysis in some of the members of the family. Two regions on the short and long arms of Chromosome12were found and they showed the maximal LOD score of2.4. The short arm region located between rs1049168and rs1106753, with a length of1,975,647bp, while the long arm region located between rs17105259and rs10160961, with a length of2,119,818bp. Based on the results of genome linkage analysis,8polymorphic microsatellite markers were selected,4from the linkage region on the short arm and4from the linkage region on the long arm of Chromosome12, and linkage and haplotype analysis was performed to verify the critical region shown by the results from the SNP array data in family1. Genotyping and haplotype analysis results showed that the short arm linkage region were between D12S1050and D12S374, with a length of2,299,067bp, and a maximal LOD score of2.68was obtained for the maker D12S1685at0=0. The long arm linkage region located between D12S313and12q/TG, with a length of1,3913,86bp, and a maximal LOD score of2.68was obtained for the maker12q/TTAT at θ=0.We also performed exome sequencing for the proband. Combining the results of exome sequencing with the results of whole genome linkage analysis, we found a missense mutation in the EFCAB4B gene (R466W) that cosegregated with the patients of the family. We genotyped319unrelated controls for the variant using PCR followed by restriction analysis, and found two of them were heterozygous carriers for this variant. One of the carriers is overweight, while the other one is normal, suggesting the variant we found in this family may not be the pathogenic mutation.Based on the clinical features of the probands of two of the four early onset obese familis, we performed mutation screening of MC4R, LEP and LEPR for both of them. A novel homozygous nonsense mutation in the LEP gene was found in the proband of one family, and both of his parents were carriers of this mutation. To investigate whether the mutations of his parents were inherited from a same ancient, we selected microsatellite markers near the LEP gene and performed haplotype analysis in this family. The results supported our hypothesis. We also found a homozygous nonsense mutation in the MC4R gene in the proband of another family. His parents, his son and daughter were all carriers of this mutation without showing any clinical feature.We performed mutation screening of23genes associated with monogenic forms of obesity in humans or transgenic mice in the probands of two other families, respectively. No mutation had been found in both of them; however, we did find some coding SNPs known to be associated with body weight in the LEPR and SIM1gene of the patient, respectively.In conclusion, we used whole genome linkage analysis combining with exome sequcing to perform mutation screening in a family with autosomal dominant obesity with overgrowth, and found a rare variant that cosegregated with the patients of the family. Though the pathgenicity of this variant is to be determined, our study offers a new method for investigating the molecular mechanism of obesity. We also found two homozygous mutations in LEP and MC4R, in patients with extreme early onset obesity, respectively. The mutation in LEP is also the first LEP gene mutation report in China. Part2:Detection of Pathogenic Mutations of Three Inherited Skin DisordersSkin disease, or dermatosis, is the disease that involves skin. Dermatosis is defined as the pathological changes of skin (including skin and skin appendages, as hair, nail and sweat glands) resulting from internal or external factors. The main causes of dermatosis are infections, allegies and genetics factors. Dermatosis that is caused by changes of genetic material is named genodermatosis. Most of the genodermatosis, such as vitiligo, psoriasis, et al, are polygenic diseases; however, there are also nearly300monogenic genodermatosis. Herein this thesis, we perform molecular genetics study on three monogenic genodermatosis, Bazex-Dupre-Christol syndrome, X-linked Hypohidrotic Ectodermal Dysplasia, and Congenital insensitivity to pain with anhidrosis, to investigate or detect mutations resulting in these diseases.Bazex-Dupre-Christol syndrome (BDCS, MIM301845) is a rare genodermatosis that was first recognized by Bazex and colleagues in1964. Affected individuals can manifest a broad range of cutaneous symptoms, while frequently and regularly observed clinical signs comprise the triad of hypotrichosis, follicular atrophoderma, and early onset of basal cell carcinomas (BCCs). Further symptoms include milia, hypohidrosis, hyperpigmentation of the face, trichoepitheliomas and hair shaft abnormalities. BDCS shows an X-linked dominant inheritance mode, the gene for BDCS has been mapped to an11.4Mb interval on chromosome Xq25-q27.1(ChrX:128,242,493-139,631,728) in2011. However, no mutation has been found yet. In the present study, we have collected two BDCS families from Europe. Whole exome sequencing and Affymetrix SNP6.0array was performed to investigate the pathogenic mutation of BDCS. After we received results of exome sequencing, we design primers to validate the variants found in the reported linkage region; however, no potential causative variants were found. Then the raw data of the exome sequencing within the linkage region were reanalyzed. The exons of genes with low or null coverage were picked up and were sequenced; moreover, the genes within the linkage interval that were not shown in the raw data were sequenced, too. Unfortunately, we still did not find any potential causative variants. Using Affymetrix SNP6.0array for copy number variantion detection, we have found duplication on chromosome Xq26.1-q26.2in all of the patients we had. However, the duplication located in a region that had overlap with known CNVs, therefore the pathgenicity of the duplication still need to be further confirmed. Ectodermal Dysplasia (ED) is not a single disease, but a class of syndromes characterized by abnormal development of two or more of ectodermal-derived structures, such as hair, teeth, and nails and so on. Of all these syndromes, X-linked Hypohidrotic Ectodermal Dysplasia (XLHED, MIM305100) is the most common form. Male patients present the triad of hypohidrosis, hypotrichosis and anodontia. Female carriers, however, show considerably variability in the severity of the disease, and about30%of them do not present obvious manifestations. XLHED is caused by mutations in EDA, which is located at Xq12-13. To date, there are nearly180pathogenic mutations that have been found in the EDA gene. More than98%of these mutations in EDA result in XLHED. However, there are several mutations found to cause another X-linked disease:X-linked dominant incisor hypodontia (MIM300606). To detect mutations of EDA gene that resulted in XLHED in the six families we collected, PCR amplification and automatic sequencing of EDA gene coding region with flanking regions were performed to detect mutations in EDA. We identified six mutations in6Chinese families with XLHED, including two novel mutations, a missense mutation and a deletion. The pathogenicity of novel missense mutation was determined by restriction analysis in family members and in52unrelated female controls. To confirm the deletion, we performed qPCR assays to validate the relative copy number (CNV) of the proband and his mother. Moreover, to explore the extent of the deletion, PCR assays were performed with primer pairs located between exon7and exon8and downstream of exon9. Finally, the deletion size was determined to be10kb by using Gap-PCR. This deletion we found also affected a gene AWAT2, downstream of EDA. Based on the results of mutation analysis, we also performed prenatal diagnosis for one female carrier, who got a male without EDA mutation at the end.Congenital insensitivity to pain with anhidrosis (CIPA, MIM256800), which is classified as hereditary sensory and autonomic neuropathy type IV (HSAN-IV), is a rare autosomal recessive disorder. CIPA is characterized by absence of reaction to noxious stimuli, anhidrosis (inability to sweat), recurrent episodic fever, self-mutilating behavior, and often mental retardation. Mutations in the human neurotrophic tyrosine kinase receptor type1(NTRK1) are responsible for this disease [3]. NTRK1, which is also designated as TRKA, is located on chromosome1q21-22, and encodes a receptor tyrosine kinase TRKA that contains either790or796amino acids, and is a high-affinity receptor for nerve growth factor (NGF). In two Chinese Han families with CIPA, we found three novel missense mutations (p.P397L, p.R692C, p.R771C) in the proband of family1; his mother carried two of them (p.P397L and p.R771C); while his father didn’t carry the third mutation, suggesting p.R692C was de novo or his father was chimera for this mutation. We found homozygous one base pair duplication in the proband of family2. Both of his parents were carriers of this mutation.In conclusion, we chose several methods to perform pathogenic mutation analysis for these three genodermatosis. Based on the mutation screening within linkage region of BDCS, we have basically ruled out the possibility that BDCS was caused by mutation in the exome of the reported linkage region. We also indentified duplication on chromosome Xq26.1-q26.2in all of the four patients we had, however, the pathgenicity of the duplication still need to be further confirmed. Through mutation detection performed on XLHED and CIPA families, we found pathnogenic mutations in all of the families, including5novel mutations. Our finding should be helpful for genetics counseling and prenatal diagnosis for these families, and the novel mutations also expand the EDA and NTRK1gene mutation spectrum. |
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