基于全外显子测序技术的中国新生儿糖尿病分子遗传学研究

Neonatal diabetes mellitus(NDM) occurs in approximately 1 out of every 100,000 live births. It can be either permanent (PNDM)or transient(TNDM), and recent studies indicate that is likely to have an underlying genetic cause, particularly when diagnosed before 6 months of age.. It was found that permanent NDM (PNDM) can result from at least 12 kinds of genes mutations mainly associated with insulin secretion and pancreatic development. . Most commonly, PNDM is due to activating mutations in either of the genes encoding the two subunits of the ATP-sensitive potassium channel. In most of these patients, switching from insulin to oral sulfonylurea therapy leads to improved metabolic control as well as possible amelioration of occasional associated neurodevelopmental disabilities.. The diagnosis and treatment of neonatal diabetes of that type is a great success of molecular genetics and represents a model for personalized medicine. Despite great effort and progress on the molecular genetics of NDM, approximately 40% of NDM diagnosed before 6 months of age continues to have no currently identifiable cause and the most appropriate treatment for them remains unknown..In 2009,our team reported the first Chinese NDM case with mutations in KCNJ11 (R201H)which successfully switched from insulin to oral sulfonylurea therapy( Hormone and Metabolic Research 2009；41：451-453)..Till now, 40 cases of neonatal diabetes have been followed up by us for five years, which is the nationwide largest cohort of NDM. Among them,10 cases with mutations in KCNJ11 or ABCC8 have been switched from insulin to oral sulfonylurea therapy and have shown improvement of metabolic control, as well as amelioration of associated neurodevelopmental disabilities. In our cohort, 23 cases have not been found to carry those known mutations..Recently, whole-exome sequencing approach have been developed, which has made breakthrough in the genetic basis of rare Mendelian disorders in small numbers of affected individuals.. Hereby, we propose this molecular genetic study of neonatal diabetes in our largest chinese cohort using next-generation sequencing of the whole exome to determine novel gene mutations of NDM and followed by function study. .The result will facilitate our understanding of the pathogenesis of NDM ,as well as that of common forms of diabetes ,such as type 1 and type 2 diabetes.

近年来新生儿糖尿病（neonatal diabetes mellitus，NDM）已成为糖尿病分子遗传学研究的热点领域之一，已发现至少12种与胰岛素分泌和胰腺发育异常有关的NDM致病基因，其中由KCNJ11和ABCC8基因突变致病的绝大多数NDM可由胰岛素注射治疗成功转换为磺脲类口服治疗。但仍有相当多的NDM病例未找到明确的致病基因突变。该领域国内研究甚少。本课题组于2009年报道了中国首例KCNJ11基因突变NDM并成功转换为磺脲类口服治疗。目前共收集40例NDM，是全国单中心最大的队列；17例已明确致病基因，10例治疗转换成功；余尚未发现致病基因突变。全外显子测序与GWAS技术相比，精确度和准确性更高，尤其在检测稀有突变中能发挥更重要的作用。本研究拟应用全外显子测序技术在中国NDM患者中进行分子遗传学研究，旨在发现新的NDM致病基因并进行功能研究，为基于分子遗传学的个体化治疗奠定基础。

近年来新生儿糖尿病（neonatal diabetes mellitus，NDM）已成为糖尿病分子遗传学研究的热点领域之一，已发现至少12种与胰岛素分泌和胰腺发育异常有关的NDM致病基因，其中由KCNJ11和ABCC8基因突变致病的绝大多数NDM可由胰岛素注射治疗成功转换为磺脲类口服治疗。此类疾病少见，但对患者本人及其家族危害大；属于疑难疾病，常不能得到正确诊治。建立这类疾病的分子遗传学诊断平台并据此给予个体化治疗，具有重要的社会意义及临床应用价值。探寻此类疾病的新致病基因并鉴定其生物学功能有益于加深对胰岛素分泌的分子调控网络和葡萄糖代谢调控机制的认识，有助于为与胰腺功能和葡萄糖代谢有关的疾病提供新的诊治靶点和策略，具有深远的理论研究价值。本项目主要完成了以下三方面工作：I. 建立了NDM的电子数据库、生物标本库以及临床诊治路径并进行长期随访。目前共有临床诊断54例NDM进入数据库。其中随访时间较长的30例患者的临床特点及治疗反应相关资料已在中华糖尿病杂志发表。II. 应用二代靶向测序技术，建立覆盖全部已知可能致病基因的高通量临床遗传学检测平台，明确分子诊断。为提高检出率，建立了覆盖单基因糖代谢异常疾病已知的58个致病基因的高通量检测平台。对38例新生儿糖尿病进行分子遗传学诊断和分型，并予遗传咨询及个体化治疗。同时，也对新生儿糖尿病以外的其他单基因糖代谢异常疾病进行了检测，包括22例青少年的成人起病型糖尿病（MODY）、4例先天性高胰岛素血症（CHI）、4例脂肪萎缩性糖尿病及3例不典型1型糖尿病患者。其中部分资料已发表在中华糖尿病杂志。III. 应用全外显子组测序技术探索新致病基因。对4例新生儿糖尿病患者及其一级亲属共14人进行了全外显子组测序，但未发现新的致病基因。在项目执行中，因全外显子组测序费用较高，完成检测的患者数量有限。同期，国际上其他研究中心在更大规模样本中应用全外显子组测序技术也未发现和报道新的致病基因，检测出的皆为已知致病基因。基于目前研究结果，对于新生儿糖尿病的新致病基因的进一步探索应依托的是全基因组测序，同时也需要关注表观遗传学在其中的参与作用。此外，单基因糖代谢异常疾病的致病机制常有紧密联系，对这一类疾病作为整体研究能够提供更多信息。本项目结果已在国内核心期刊发表论文4篇；2次在美国糖尿病学会学术年会的中国专场口头发言；共培养4名临床医学博士。