The Genetic And Clinical Characteristics Of SHP Gene Mutations In Chinese Overweight And Obese Population

Obesity is defined as a state of increased fat mass of sufficient magnitude to produce adverse health consequences. Because obesity is associated with substantially increased morbidity from type 2 diabetes, metabolic syndrome, hypertension, stroke, and some forms of cancer, it is clear that obesity represents a major public health problem. By the World Health Organization (WHO) definition, currently at least 50% of the population of most Western countries are either overweight or obese. Because of changes to the traditional diet, reduced levels of physical avtivity, and increased sedentary lifestyles, which led to rapid increase of overweight or obesity in China. Although the prevalence of obesity in China is relatively low compared with Western countries such as the United States, where over half of adults are either overweight or obese, it is the rapid increase of the condition, especially among children, that is particularly alarming.Human obesity is a complex trait influenced by interacting environmental and genetic factors. Although the increasing prevalence of obesity in most developed and developing countries is clearly driven by secular changes in physical activity and easy availability of high-energy foods, it is also clear that inherited factors play a major role in determining adiposity. Obesity is complex disease, the complex disease caused by the interaction of polygenic determinants and environmental factors, currently the identification of the polygenic determinants of adiposity across the general human population has been challenging and has had limited success thus far. However, the study of extreme human phenotypes with Mendelian patterns of inheritance has been illuminating in a number of ways.More recently, several monogenic disorders that results from disruption of the leptin-melanocortin pathway have been identified. There are the mutations in the genes for leptin, the leptin receptor, prohormone convertase 1, pro-opiomelanocortin and melanocortin-4 receptor. The proteins encoded by these genes are functionally related to regulate/participate in central melanocortin pathway,thus influence energy homeostasis. However, the obesity caused by these genes usually identified in Caucasian,and in addition to the prevalence of MC4R gene mutation was high, the prevalence of other gene mutations was very low, which only have been identified sporadically in Caucasian.Several loss-of-function mutations in the small heterodimer partner (SHP; also called NR0B2 for nuclear receptor subfamily 0, group B, member 2 ) gene have recently been shown to cosegregate with a mild form of an early-onset type of obesity among Japanese(carrier prevalence about 6%), which demonstrated that mutations in SHP gene are associated a dominantly inherited form of early-onset mild obesity. It is tempting to speculate that SHP gene mutations are a component of the genetic background of obesity in Japanese, although the lack of the significance of such mutations in the development of adult-onset obesity in Japanese population. But, subsequently studies among the Caucasian obesity population demonstrated that functional SHP gene mutations associated with obesity is considerably lower among Caucasian, compared to the prevalence in the previously reported Japanese population.The human SHP gene, located on chromosome 1p36.1 consists of two exons separated by an introns of 1.8 kb, the protein encoded by this gene is a 257-amino acid orphan nuclear receptor. Because of the absence of a DNA-binding domain, which makes SHP belongs to a heterogeneous group of NR-related proteins that are substantially distinct from conventional NRs in both structure and function. To date, SHP is implicated in the regulation of more than 40 diverse target genes involved in cholesterol/bile acid metabolism, lipogenesis, gluconeogenesis, xenobiotic drug metabolism and steroidogenesis. In this study, firstly, to investigate the prevalence of SHP gene mutations in Chinese overweight or obesity population by direct sequencing. Subsequently, was to determine whether the mutations, which identified in this study, affected the function of SHP with Dual-Luciferase Reporter Assay System. Finally, was to determine the clinical pathophysiology of mutation carriers. The aim of our study was to investigate the genetic and clinical characteristics of SHP gene mutations in Chinese overweight or obesity population, in order to provide theoretical guidance for early clinical intervention to SHP gene mutation carriers. The whole study is composed of three parts.PartⅠMutation analysis of small heterodimer partner (SHP) gene in Chinese overweight or obese populationObjective: To investigate the prevalence of mutations and sequence variants of SHP gene in Chinese overweight or obese population, and clinical phenotype of mutation carriers, mode of inheritance and the penetrance of the SHP gene mutation. Methods: We screened the whole coding region and intron/exon boundaries for SHP in 500 unrelated Chinese overweight or obese subjects and 272 unrelated nondiabetic and nonobese control subjects by direct sequencing of the amplified polymerase chain reaction products. Results: Screening the 500 unrelated subjects with overweight or obesuty resulted in the identification of a total of five different mutations in coding region, all in a heterozygous state, in eleven subjects. In overweight group, two novel missense mutations (G171A and G189E) were found in two different unrelated subjects. Two of the previously reported mutatioms (R34X and H53fsdel10) were identified in this group, in which the nonsense mutation R34X that was found in one subject, and frameshift mutation H53fsdel10 that was found in five unrelated subjects. One novel silence mutation P10P, which located in the N-terminal end of the protein, also was identified in one subject. Furthermore, we also identified an overweight subject who was heterozygous substitution (C→T) at the position 10 bp downstream from the 3'-end of exon1. In obesity group, the frameshift mutation H53fsdel10 that was found in two unrelated subjects (2.6%). None of the above variants were found in 272 healthy nonobese and nondiabetic Chinese controls. Furthermore, in extended family studies that these mutations did not co-segregate with overweight or obesity within family in several pedigrees in our study, the penetrance of SHP gene mutations(R34X, H53fsdel10, G174A and G192E)is only 62.5% (15/24) in our study. Conclusion: Genetic variants in the SHP gene may be a key genetic factor responsible for overweight or obesity, increased insulin level in Chinese.PartⅡIn vitro functional analysis of mutant SHP proteinObjective: To determine whether the mutations identified in Chinese overweight or obese subjects alter the function of the SHP protein. Methods: The wild-type and heterozygotic mutant SHP gene were cloned into eukaryotic expression plasmid vector, the liver-derived HepG2 and insulin producing MIN-m9 cells were transfected with pcDNA-HNF-4α, pGL3-HNF-1α, test DNA and pRL-SV40, respectively. The effect of the wild-type and mutant protein on HNF-4α-mediated transactivation of HNF-1αwas examined by using a Dual-Luciferase Reporter Assay System. Results: The expression of wild-type efficiently decreased the HNF-4αdependent transactivation activity in both cell lines. When compared the effects of mutant proteins on HNF-4α-mediated transactivation of HNF-1αgene promoter with that of wild-type in both HepG2 and MIN6-m9 cells. The significant reductions of the suppression activity were observed, while the R34X, H53fsdel10 and G189E mutant proteins were expressed. Moreover, the G171A protein showed a weak suppression in MIN-m6 cells when five times more of the mutant protein was expressed(25 ng of HNF-4αplasmid and 125 ng of G171A plasmid in the transfections). Conclision: The R34X, H53fsdel10, G171A and G189E mutation undermine the ability of transcriptional suppression of SHP. It may be the possible reason responsible for overweight or obesity in Chinese.PartⅢClinical pathophysiology study on SHP gene mutation carriersObjective: To further investigate clinical pathophysiology feature of SHP gene mutation carriers and evaluate the in vivo effects of the SHP gene mutation to glucose metabolism, lipid metabolism and other endocrine function. Methods: We determine some endocrine hormone level of SHP gene mutation carriers to analyze whether SHP gene mutation accompanied by endocrine abnormalities; assay blood lipid profile level to analyze whether SHP gene mutation affect blood lipid metabolism; and evaluate theβcell function of SHP gene mutation carriers by oral glucose tolerance test and nonglucose secretagogue arginine test. Results: The subjects with SHP gene mutations exhibited no endocrine abnormalities. All SHP gene mutations carriers with normal liver and renal function, serum lipid concentrations were within normal ranges. All subjects with SHP deficiency had hyperinsulinema and showed insulin resistance. The acute insulin response to nonglucose secretagogue arginine was significantly reduced in SHP deficiency subjects compared with nondiabetic and nonobese healthy subjects, overweight or obese subjects, and obese type 2 diabetes. Conclusion: SHP gene mutation is unlikely to accomply by endocrine abnormalities and dislipidema, but, which may lead to deterioration ofβ-cell function.