第一部分 中国人群新生儿糖尿病（NDM）患者筛查及分子遗传学研究

【研究目的】

  探讨中国人群新生儿糖尿病（NDM）患者的临床特点和分子遗传学特征，研究基因型-临床表型关系，并致力于发现新的NDM致病基因。

【研究方法】

  纳入自2007年8月至2016年8月就诊于北京协和医院的可疑新生儿糖尿病（NDM）患者50例。详细收集临床资料，在知情同意的基础上采集患者及其父母外周血。首先运用Sanger测序技术检测编码K_ATP通道Kir6.2亚单位和SUR1亚单位的KCNJ11和ABCC8基因：对明确由KCNJ11或ABCC8基因突变导致的NDM患者，尝试进行口服格列本脲替代胰岛素转换治疗；如Sanger测序结果为阴性，则运用目标区域捕获测序PANEL检测已知的21个NDM致病基因；如PANEL测序结果仍为阴性，则对患者及其家系成员进行全外显子检测。

【研究结果】

1. 在50例疑似NDM患者中，遗传学确诊K_ATP-NDM患者15例（包括KCNJ11-NDM 10例，ABCC8-NDM 5例），INS-NDM 3例，GCK-NDM 1例，FOXP3-NDM 3例，Wolfram综合征2例。在明确致病基因的24例NDM患者中，仅1例ABCC8-NDM、2例FOXP3-NDM、2例Wolfram综合征为6个月到2岁之间发病，其余19例患者均为出生后6个月以内发病。

2. 发现6个新的突变位点，分别为KCNJ11 p.I182M，ABCC8 p.Q211R、p.I585F、p.R653W、p.A569S和FOXP3 p.P133L。

3. 共13例K_ATP-NDM患者尝试进行口服磺脲类药物替代胰岛素治疗，其中10例患者转换成功（76.9%）。在K_ATP基因同一位点，发生相同或不同氨基酸类型突变，所导致的NDM从临床轻重程度、到对磺脲类药物的治疗反应均存在较大差异。

4. 3例FOXP3-NDM患者均表现为单纯糖尿病，仅1例合并慢性荨麻疹，未观察到其他IPEX综合征的临床组分。

5. 在1例1岁起病的疑似NDM患者中发现CDKAL1基因p.V329I、p.P409L复合杂合突变。

【研究结论】

1. 结合Sanger测序和目标区域捕获测序可以明确82.6%的NDM患者的致病基因。

2. 在中国人群NDM患者中，约60%为编码K_ATP通道的KCNJ11、ABCC8基因突变所致；中国人群K_ATP-NDM具有一系列独特的基因型-临床表型关系；约76.9%的K_ATP-NDM患者可以成功停用胰岛素、转换为磺脲类药物单药治疗；尽早起始磺脲类药物治疗，可有效预防iDEND综合征的发生。

3. 中国人群FOXP3-NDM临床表型与国外文献已报道的FOXP3基因突变临床表型（IPEX综合征）具有显著差异，不除外存在其他基因位点的协同作用、或者与表观遗传修饰有关。

4. CDKAL1可能是中国人群新的单基因糖尿病致病基因。

第二部分 中国人群青少年发病的成人型糖尿病（MODY）患者筛查

及分子遗传学研究

【研究目的】

  探讨中国人群青少年发病的成人型糖尿病（MODY）患者的临床特点和分子遗传学特征。

【研究方法】

  纳入自2013年3月至2017年3月就诊于北京协和医院的可疑MODY家系192个。详细收集临床资料，在知情同意的基础上采集先证者及其家系成员外周血。运用Sanger测序技术对先证者行最常见的MODY致病基因GCK、HNF1α和HNF4α检测。对Sanger测序阴性的先证者，运用目标区域捕获测序PANEL检测已知的14个MODY致病基因。如Sanger测序和PANEL测序均为阴性，则对先证者及其家系成员进行全外显子检测，分析可能的致病基因。

【研究结果】

1. 在符合纳入标准的192个疑似MODY家系（包括372例疑似MODY患者）中，共检测出27个家系（81例患者）携带GCK突变（GCK-MODY，MODY2），7个家系（15例患者）携带HNF1α突变（HNF1α-MODY，MODY3），1个家系（2例患者）携带HNF4α突变（HNF4α-MODY，MODY1）。

2. 发现18个新的突变位点：GCK基因14个，HNF1α基因3个，HNF4α基因1个。

3. 在81例GCK-MODY患者中，平均发现血糖升高的年龄为24.0±16.6岁，最小的为出生后3天，最大的为60岁。高达79.2%的先证者是因常规查体或其他疾病就诊发现血糖升高的。从发现血糖升高到明确诊断为GCK-MODY，平均经过约5年的时间，最短的为2个月，最长的为24年。

4. 本研究中GCK-MODY患者的空腹血糖主要集中分布于6.1～7.6mmol/L，HbA1c主要集中分布于5.7～7.1%。FBG最高值为9.3mmol/L，HbA1c最高值为7.4%。仅2例患者（2.5%）有微量白蛋白尿。

5. 发现了中国人群第一个HNF4α-MODY家系，也是一个MODY和2型糖尿病（T2DM）的混合家系。对10岁的先证者成功停用胰岛素，改为小剂量磺脲类药物治疗，血糖控制良好。

6. 对42个疑似MODY、Sanger测序阴性的先证者进行目标区域捕获测序，阳性率为0，未检测到其他11种已知的MODY致病基因突变。结合Sanger测序+目标区域捕获测序，仍有70%～80%的疑似MODY患者未能找到明确致病基因。

【研究结论】

1. GCK-MODY（MODY2）是中国人群中最常见的MODY类型(约占14-22%)。

2. FBG≤9.3mmol/L，HbA1c≤7.5%或许可作为中国人群MODY2患者的初筛标准。早孕期FBG筛查或许是MODY2与发生在孕中晚期GDM的鉴别关键。

3. 中国人群中存在HNF4α基因突变导致的MODY 1家系。需要重视MODY可能与T2DM混合存在于同一家系的可能性。

4. 及时正确的分子遗传学诊断可为患者提供更加合理的治疗方案，甚至避免不必要的药物治疗。

5. HNF1α-MODY（MODY3）在中国MODY人群中患病率仅约4%。

6. 中国人群具有自己独特的MODY致病基因，70-80%的中国MODY人群仍为MODY-X，需要进行更多的基因学研究。

PART 1 Screening and genetic analysis of neonatal diabetes mellitus (NDM) in Chinese population

Objective

  To investigate the clinical characteristics and molecular genetics of Chinese patients with neonatal diabetes mellitus (NDM), to determine the genotype-phenotype relationships and to identify the underlying novel pathogenic genes of NDM.

Methods

   50 suspected patients with NDM were recruited at Peking Union Medical College Hospital. Detail clinical data were collected. First, the probands were tested for the most common KCNJ11 and ABCC8 genes by Sanger sequencing. Patients with a definite diagnosis of K_ATP-NDM were switched from insulin injection to oral glibenclamide. Second, targeted next-generation sequencing with 21 known NDM causative genes were done for those patients with negative results of Sanger sequencing. Third, whole exome sequencing was performed for those patients with negative results from the aforementioned previous sequencing steps, as well as their family members.

Results  

1. Of the 50 patients with suspected NDM, 15 patients with K_ATP-NDM (including 10 KCNJ11-NDM and 5 ABCC8-NDM), 3 with INS-NDM, 1 with GCK-NDM, 3 with FOXP3-NDM and 2 with Wolfram syndrome were identified. Of the 24 patients with definite genetic diagnosis, 19 had an onset within 6 months of birth.

2. 6 new mutation sites in 3 known pathogenic genes were identified as follows: KCNJ11 p.I182M, ABCC8 p.Q211R, p.I585F, p.R653W, p.A569S, and FOXP3 p.P133L.

3. Switching from insulin to glibenclamide monotherapy was successful in 76.9% (10/13) of the patients. Different clinical severity and treatment respond to sulfonylureas were observed even among patients with the same mutation or with different amino acids changes at the same mutation site.

4. All 3 patients with FOXP3-NDM presented with simple diabetes and only 1 with chronic urticaria. No other clinical components of IPEX syndrome were observed.

5. Compound heterozygous mutations p.V329I and p.P409L of CDKAL1 were identified in a 1-year onset proband.

Conclusions

1. 82.6% of the causative genes in NDM patients could be identified by Sanger sequencing and targeted next-generation sequencing.

2. About 60% Chinese NDM patients were caused by KCNJ11 or ABCC8 gene mutations. K_ATP-NDM had their unique genotype- phenotype relationships in Chinese population. 76.9% of the K_ATP-NDM patients could be successfully transferred from insulin to glibenclamide monotherapy. Early initiation of sulfonylureas is of great importance in the prevention of neurological abnormalities of DEND/iDEND syndrome.

3. The clinical phenotypes of FOXP3-NDM in Chinese population are significantly different from that reported in other populations, which could not be excluded from the synergistic effects of other loci or with epigenetic modifications.

4. Compound heterozygous mutations in CDKAL1 were firstly found and related to early-onset monogenic diabetes. CDKAL1 may be a new discovered pathogenic gene of monogenic diabetes in Chinese population.

PART 2  Screening and genetic analysis of maturity onset diabetes of the young (MODY) in Chinese population

Objective

  To investigate the clinical characteristics and molecular genetics of Chinese patients with maturity onset diabetes of the young (MODY).

Methods

  Suspected MODY probands were recruited at Peking Union Medical College Hospital. Detail clinical data were collected. First, the probands were tested for the most common GCK, HNF1α and HNF4α genes by Sanger sequencing. Second, targeted next-generation sequencing with 14 known MODY causative genes were done for those patients with negative results of Sanger sequencing. Third, whole exome sequencing was performed for those patients with negative results from the aforementioned sequencing steps, as well as their family members.

Results

1. 192 suspected MODY families including 372 suspected MODY patients were recruited. We totally identified 27 GCK-MODY families (including 81 GCK-MODY patients), 7 HNF1α--MODY families (including 15 HNF1α-MODY patients) and 1 HNF4α--MODY families (including 2 HNF4α-MODY patients) among them.

2. 18 new mutation sites were identified, including 14 in GCK, 3 in HNF1α and 1 in HNF4α.

3. In 81 patients with GCK-MODY, the average age to find elevated blood glucose was 24.0±16.6 years, with a minimum of 3 days after birth and a maximum of 60 years. Up to 79.2% of the probands found that due to routine physical examination or other medical treatment. The average time from finding blood glucose elevated to a definite genetic diagnosis of GCK-MODY was about 5 years. The shortest is 2 months, and the longest is 24 years.

4. The fasting blood glucose (FBG) in GCK-MODY patients is mainly distributed from 6.1 to 7.6mmol/L, and HbA1c is mainly distributed from 5.7% to 7.1%. The maximum value of FBG is 9.3mmol/L, and the maximum value of HbA1c is 7.4%. Only 2 patients (2.5%) had microalbuminuria.

5. We identified the first HNF4α-MODY family in Chinese population as well as a hybrid family of MODY and T2DM. We successfully transferred with a small dose of sulfonylurea instead of insulin therapy for the 10-year-old proband.

6. Targeted next-generation sequencing were done for 42 suspected MODY patients with negative results of Sanger sequencing, and the positive rate was 0. No other 11 known MODY mutations were detected. There are still 70%～80% of suspected MODY patients failed to find a clear causative gene combining with Sanger sequencing and targeted next-generation sequencing.

Conclusions

1. GCK-MODY (MODY2) is the most common type of MODY in Chinese population (14-22%).

2. FBG≤9.3mmol/L, HbA1c≤7.5% may be taken as the initial screening standard for MODY2 patients in Chinese population. Early pregnancy FBG screening may be the key to identify MODY2 from those GDM in late pregnancy.

3. There are MODY 1 pedigrees in Chinese population. MODY may co-exist with type 2 diabetes in a same pedigree, which should be paid more attention.

4. Timely molecular genetic diagnosis can provide more reasonable treatment, and even to avoid unnecessary treatment.

5. The prevalence of HNF1α-MODY in Chinese MODY population is only about 4%

6. The Chinese population has its own unique MODY pathogenic genes. 70-80% of Chinese MODY are still MODY-X.