常染色体显性多囊肾病患者表现型与基因型分析及遗传干预研究

发布时间：2018-06-15 07:19

本文选题：常染色体显性多囊肾病 + 临床表现　；参考：《第二军医大学》2016年博士论文

【摘要】：常染色体显性多囊肾病(autosomal dominant polycystic kidney disease,ADPKD)(以下简称多囊肾病)是人类最常见的单基因遗传性肾病,发病率在0.1~0.25%,全世界约有1250万患者,是导致终末期肾病(end-stage renal diseases,ESRD)的第四位病因,占5%~10%。其临床特征突出表现为双侧肾脏发生无数进行性增大的液性囊泡,损害肾脏的正常结构和功能,50%以上患者在60岁时进展至ESRD,需行透析或肾移植治疗。该病除累及肾脏外,还可导致肝脏、胰腺、脾脏、泌尿生殖系统囊肿,心瓣膜病,结肠憩室和颅内动脉瘤等肾外病变。是一种严重危害人类健康且多系统受累的遗传性疾病。ADPKD遗传特点为代代发病,与性别无关,子代发病机率均为50%,致病基因已被确认为PKD1基因与PKD2基因。其中约85%患者因PKD1基因突变致病,约15%患者因PKD2基因突变致病;基因突变类型与疾病临床表现密切相关,PKD1基因致病患者其临床表现及疾病进展速度显著重于PKD2基因突变患者。受基因检测成本及技术检出率限制,临床诊断ADPKD主要依靠家族遗传史+临床表现+影像学检查“三联”法来确诊。但已有临床调查研究证实,约10%~20%的临床表现符合ADPKD患者无明确家族史。在这种情况下,单纯依靠临床表现进行ADPKD诊断是否可靠?随着新一代测序技术(next generation sequencing,NGS)的不断发展,PKD1与PKD2基因突变的检出能力不断提高,但现有的各类基因诊断数据库内容均十分匮乏,几乎没有国内人群检测数据。既往研究显示,ADPKD基因突变不存在突变热点,再联系疾病的发病机制中的“二次打击”“三次打击”学说,我们推测:在有家族史与无家族史(自发突变致病)的ADPKD患者间是否存在基因突变位点(和/或)类型上的差异,并伴随有临床表现差别。为了验证这一假设,我们选择2009年6月至2015年12月间在长征医院肾内科长期随访、临床诊断明确的ADPKD患者,经询问病史、查看既往病例与影像学检查结果、父母等直系亲属行超声检查确认等方式,共筛选出有明确家族史ADPKD患者348例,确认无家族史患者119例。统计结果显示:无明确家族史ADPKD患者在多囊肝的发生率上显著少于有明确家族史患者(p0.01),疾病诊断年龄平均较有明确家族史患者推迟2年,但没有统计学差异。无家族史ADPKD患者在脑血管事件发生率上略低于有家族史患者(0.84%VS 1.44%),合并糖尿病发生率(1.68%VS 1.43%)相当,均无统计学差异。无家族史患者中两人分别罹患胃癌及前列腺癌,两组间在性别组成、高血压发生率、治疗情况及疾病进展等其他方面均没有统计学差异,仅发现≤18岁年龄组中无家族史患者估算肾小球滤过率(egfr)下降速度快于有家族史的患者(p0.05),41~50岁年龄组无家族史患者肾脏起始体积显著小于有家族史患者(p0.05)。采用cox回归分析显示,患者所处年龄段(p=0.033)、诊断adpkd年龄(p=0.032)及有无家族史(p=0.026)是预测肾体积增长率快速进展的危险因素。为进一步比较两组患者在疾病基因突变位点上的差异,我们从以上研究队列中选取有或无明确家族史的患者各30例,进行pkd1/2基因突变位点检测。其中有家族史患者检出致病突变位点28例,2例检测结果为阴性,整体检出率93.3%;无家族史adpkd患者pkd1/2基因突变位点检测结果阳性者仅有20例(4例为可疑致病突变),在剩余未检出pkd1/2基因致病突变的10例患者中额外实施了tsc、pkhd和hnf-1β三个可引起类似adpkd临床及肾脏影像表现的基因突变检测,发现其中5例患者存在有pkhd1基因杂合突变,均为18岁以上成年人,未出现肝功能异常及肝硬化表现,提示有复合杂合子遗传等复杂遗传模式参与了无家族史患者的疾病遗传。仍有5例患者上述基因致病突变检测结果呈阴性,基因致病突变整体检出率为83.3%。在所有检出的pkd1/2基因突变位点中,约47.9%为未见报道的新发突变,其中有家族史患者组发现11个新突变位点,无家族史患者组检出12个,其中4例为可疑致病突变,因其属于错义突变,且没有家族史可供验证。所有检出突变并未提示有突变热点存在,基因检测结果进一步证实了临床诊断在无家族史adpkd患者中的不确定性。基于检出的明确基因致病突变,结合已有的技术条件,在这60例患者中,我们征得了其中6例育龄期患者及其配偶(5例有明确家族史,1例无明确家族史)的知情同意,实施胚胎植入前遗传诊断(preimplantationgeneticdiagnosis,pgd)技术干预致病基因遗传。通过药物促排卵、体外卵胞浆内单精子注射(intracytoplasmicsperminjectionicsi)授精,发育形成65个囊胚期胚胎,经pgd检测筛选出8个不携带致病基因、染色体正常的健康胚胎,为4例患者进行了4个胚胎移植,最终有1例胚胎成功妊娠存活,发育至18周,胎儿宫内发育正常,羊水穿刺结果确证为不携带致病基因遗传胚胎。结果显示,pgd技术安全可靠,对无明确家族史的adpkd夫妇而言,卵巢促排卵效果将直接决定pgd技术实施的可靠性及成功率。综上所述,本研究发现有或无明确家族史、临床诊断常染色体显性多囊肾病患者在多囊肝、预测疾病进展状态方面存在显著差异,在此基础上,抽取患者进行pkd1/2基因检测,整体pkd1/2基因检出率为80%,其中,有家族史患者检出率为93.3%,新突变位点占39.3%;无家族史患者检出率为66.7%,新突变位点占60%,其中检出16.7%患者为非PKD基因突变致病。检测结果极大的丰富了国内人群ADPKD基因检测信息数据库,长片段PCR+NGS基因检测技术可靠、检出率理想,是临床诊断的有效武器。另一方面,无家族史临床诊断ADPKD患者的PKD基因突变检出率显著降低(P=0.023),且检出明确非PKD基因致病突变,这说明单纯依靠临床诊断ADPKD患者准确性堪忧,有必要进行PKD基因检测以确诊,并应额外检测可能导致出现与ADPKD类似临床表现的基因突变。临床表现的差异可能与ADPKD的诊断偏差有关。本研究最后应用PGD操作使1对夫妇成功妊娠不携带致病基因突变的胚胎,证实了PGD技术在干预ADPKD疾病遗传中的可操作性,为将来开展多中心干预研究及推广应用打下了良好的基础,为阻断ADPKD遗传、生育健康下一代提供了可靠的技术手段,必将取得巨大的社会效益。
[Abstract]:Autosomal dominant polycystic kidney disease (autosomal dominant polycystic kidney disease, ADPKD) (hereinafter referred to as polycystic kidney disease) is the most common monogenic hereditary nephropathy in humans. The incidence is in 0.1~0.25%, about 12 million 500 thousand patients all over the world. It is the fourth cause of end-stage renal disease (end-stage renal diseases, ESRD), which accounts for the clinical manifestation of 5%~10%.. It is characterized by a myriad of progressive fluid vesicles in the bilateral kidneys which damage the normal structure and function of the kidneys. More than 50% of the patients progressed to ESRD at the age of 60, requiring dialysis or renal transplantation. The disease could lead to the liver, pancreas, spleen, urogenital cysts, valvular disease, colonic diverticulum and cranium except for the kidney. The hereditary disease, such as internal aneurysm, is a hereditary disease.ADPKD hereditary disease which is serious harm to human health and multisystem. It is not related to sex, and the incidence of subgeneration is 50%. The pathogenic gene has been identified as PKD1 gene and PKD2 gene. About 85% of the patients are caused by the mutation of the PKD1 gene and about 15% of the patients are due to the PKD2 gene process. The type of gene mutation is closely related to the clinical manifestation of the disease. The clinical manifestation and disease progression of the patients with PKD1 gene are significantly higher than that of the PKD2 gene mutation patients. The cost of gene detection and the detection rate are limited. The clinical diagnosis ADPKD is mainly confirmed by the "triple" method of family genetic history + temporary bed performance + imaging examination. However, clinical studies have confirmed that the clinical manifestations of about 10%~20% are in line with the undefined family history of ADPKD patients. In this case, the reliability of ADPKD diagnosis by simply relying on clinical manifestations? With the continuous development of a new generation sequencing technology (next generation sequencing, NGS), the detection ability of PKD1 and PKD2 gene mutations is increasing, but The existing genetic diagnosis database is very scarce, and there are almost no domestic population detection data. Previous studies have shown that the ADPKD gene mutation does not have mutation hot spots, and then the "two hit" "three strikes" theory in the pathogenesis of the disease, we speculate that there is a family history and a family history of A (spontaneous mutation). In order to verify this hypothesis, we chose the long-term follow-up of the nephrology department of the Changzheng Hospital from June 2009 to December 2015, and the clinical diagnosis of ADPKD patients in the Long March Hospital from June 2009 to December 2015 to check the results of previous and imaging examinations, in order to verify this hypothesis. A total of 348 patients with a clear family history of ADPKD were selected and 119 cases were confirmed without family history. The statistical results showed that the incidence of polycystic liver disease in ADPKD patients without a clear family history was significantly less than that of patients with a clear family history (P0.01), and the average age of the diagnosis of the disease was more than that of a family history. It was delayed for 2 years, but there was no statistical difference. The incidence of cerebrovascular events in ADPKD patients without family history was slightly lower than that of patients with family history (0.84%VS 1.44%), and the incidence of diabetes (1.68%VS 1.43%) was similar. No statistical difference was found in the incidence of diabetes mellitus (1.43%). Among the patients without family history, two were respectively suffering from gastric cancer and prostate cancer, and the two groups were in sex composition and hypertension. There was no statistical difference in the incidence, treatment and disease progression. It was found that the estimated glomerular filtration rate (EGFR) decreased faster than the family history (P0.05) in the age group with no family history in the age group of the age of 18 (P0.05), and the initial volume of the renal viscera in the 41~50 age group was significantly smaller than that of the family history (P0.05). Ox regression analysis showed that the patient's age (p=0.033), the diagnosis of the ADPKD age (p=0.032) and the family history (p=0.026) were the risk factors for predicting the rapid progress of the renal volume growth rate. In order to further compare the difference between the two groups of patients at the mutation site of the disease, we choose a patient with or without a clear family history from the above cohort. The pkd1/2 gene mutation loci were detected in 30 cases, of which 28 cases were detected by family history, 2 cases were negative, and the overall detection rate was 93.3%. Only 20 cases (4 cases of suspected mutagenesis) were positive for pkd1/2 gene mutation and no pkd1/2 gene mutation was detected in the rest of the family history patients. In 10 patients, three additional mutations in TSC, pkhd and HNF-1 beta that could cause similar mutations in the clinical and renal imaging features of ADPKD were detected. 5 of them had PKHD1 gene heterozygous mutations, all of which were over 18 years old, without abnormal liver function and liver cirrhosis, suggesting complex heterozygote inheritance and other complex genetic patterns. There were 5 patients with no family history. 5 cases were still negative. The overall detection rate of the gene mutation was 83.3%. in all pkd1/2 mutation sites, and about 47.9% of the new mutations were unreported. Among them, the family history group found 11 new mutation sites, without family history. 12 cases were detected in the patient group, of which 4 were suspected to be pathogenic mutations because they were missense mutations, and there was no family Shi Ke for verification. All the mutations did not indicate the existence of mutation hot spots. The results of the gene detection further confirmed the inaccuracy of the clinical diagnosis in the ADPKD patients without family history. In these 60 patients, 6 of the 60 patients with childbearing age and their spouses (5 with a clear family history, 1 without a clear family history) were accepted, and the genetic diagnosis of preimplantation genetic diagnosis (preimplantationgeneticdiagnosis, PGD) was used to interfere with the genetic inheritance of the pathogenetic gene. 65 blastocysts were developed by insemination (intracytoplasmicsperminjectionicsi) insemination, and 8 healthy embryos that did not carry pathogenic genes and normal chromosomes were screened by PGD, and 4 embryos were transplanted for 4 patients. In the end, 1 embryos were successfully pregnant and developed to 18 weeks, fetal intrauterine development was normal, amniotic fluid was worn. The results showed that the PGD technology was safe and reliable. For ADPKD couples without a clear family history, the ovulation effect of the ovaries would directly determine the reliability and success rate of the implementation of PGD technology. In summary, this study found or had no definite family history and the clinical diagnosis of autosomal dominant polycystic kidney. On the basis of pkd1/2 gene detection, the overall pkd1/2 gene detection rate was 80%, among which, the detection rate of family history was 93.3%, the new mutation site was 39.3%, the detection rate of the patients without family history was 66.7% and the new mutation site was 60%, of which 16.7 was detected. % of the patients were non PKD gene mutations. The detection results greatly enriched the information database of ADPKD gene detection in domestic population, the long segment PCR+NGS gene detection technology was reliable, the detection rate was ideal, and it was an effective weapon in clinical diagnosis. On the other hand, the detection rate of PKD gene mutation in ADPKD patients without family history diagnosis was significantly lower (P=0.023). A clear non PKD gene mutation was detected. This indicates that the accuracy of the clinical diagnosis of ADPKD patients simply depends on the accuracy of the clinical diagnosis. It is necessary to make a diagnosis of the PKD gene, and the additional detection may lead to the mutation of the gene similar to the clinical manifestation of the ADPKD. The difference in clinical manifestation may be related to the diagnostic deviation of ADPKD. The final application of this study is to apply PGD exercise. In order to make the 1 couples successfully pregnant without the mutation of the pathogenic gene, the feasibility of the PGD technology in the intervention of the heredity of ADPKD disease has been confirmed, which has laid a good foundation for the future research and application of multi center intervention. It provides a reliable technical means for blocking the inheritance of ADPKD and the next generation of reproductive health. Social results.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R692

【相似文献】