医学遗传与胚胎发育 Chapter 16 临床遗传

1、1Clinical genetics临床遗传2CLINICAL GENETICSDiagnosis of genetic disorders 遗传病的诊断Treatment of genetic disorders 遗传病的治疗Prevention of genetic disorders 遗传病的预防3Diagnosis of genetic disordersSymptomatic diagnosis (临症诊断)Presymptomatic diagnosis (症状前诊断)Prenatal diagnosis (出生前诊断)Preimplantation diagnosis (着床前诊

2、断)4Symptomatic diagnosis (临症诊断)A process of deciding the nature of a diseased condition by examination of the clinical symptoms and the relevant lab tests of a patient.- Clinical examination Physical examination is the basis for clinical diagnosis of all disorders including genetic ones. Particular

3、attention should be directed to characteristic symptoms and principal signs, for example: Gowers sign suggests DMD.5Pedigree analysis plays a central role in clinical genetics. Properly obtained and interpreted, it is one of the most useful tools available to physicians caring for patients with gene

4、tic disorders.Laboratory tests- Chromosomal analysis- Histopathological observations- Biochemical analysis- DNA analysis (gene diagnosis)Pedigree analysis6Presymptomatic diagnosis (症状前诊断)Presymptomatic or preclinical diagnosis is a process of making a diagnostic decision for a person who has not yet

5、 shown clear clinical symptoms. This is to find out the presymptomatic heterozygotes in families with AD genetic disorders that are usually late-onset. To make such a presymptomatic diagnosis, pedigree analysis, very careful clinical examination, and relevant lab tests including DNA analysis if avai

6、lable are needed.7Prenatal diagnosis (出生前诊断)Indications of prenatal diagnosis- One partner of the couple is a carrier of balanced translocation- A pregnant woman with an age of 35 years or older- A couple with normal karyotype have given birth to a child with chromosomal disorder or other congenital

7、 malformation- A pregnant woman who has had recurrent spontaneous abortion（自发性流产） of unknown reason.- A pregnant woman with hydramnios（羊水过多）. A pregnant woman who is the gene carrier of an XR disorder- A couple of consanguineous marriage particularly in kindred (亲戚关系) with genetic disorder.- One par

8、tner of the couple has been exposured to certain teratogen(s)（致畸剂）.8Techniques for prenatal diagnosisInvasive methods（侵袭性方法）- Amniocentesis （羊膜穿刺）: 14-18w- Chorionic villus sampling （绒毛取样）: 7-9w- Cordocentesis （脐穿刺）: 18w- Fetoscopy （胎儿镜）: 15-18w- Amniography （ 羊膜腔造影术 ）: 16wNon-invasive methods （非侵袭性

9、方法）- Ultrasonography （超声） - Radiography （放射线照相）: 16w- Analysis of maternal serum: 12-16w910Preimplantation genetic diagnosis (PGD, 着床前诊断)The biopsy (活检) can be carried out either at 8-cell or blastocyst (胚泡) state. In either case the eggs would be frozen and the diagnosis carried out by PCR. PGD has

10、 raised ethical issues (伦理问题). 11Gene diagnosis定义：通过检测遗传病患者的DNA或mRNA作出诊断。基因诊断的策略： 从基因产物入手作诊断 从基因定位入手作诊断 从比较正常和异常基因的差异入手作诊断12已知基因、已知突变的检测等位基因特异性寡核苷酸杂交分析(allele-specific oligonucleotide, ASO)：探针杂交DNA测序法（DNA Sequencing）：为最基本和最重要的实验手段 PCR-ASO分析MELAS13限制性片段长度多态性(restriction fragment length polymorphism,

11、RFLP) 变性梯度凝胶电泳(denaturing gradient gel electrophoreris, DGGE) 单链构型多态性(single-strand conformational polymorphism, SSCP) 变性高效液相色谱检测(denaturing high performance liquid chromatography, DHPLC) DNA芯片技术（DNA chip） 检测已知基因、未知突变14RFLP分析1516未知基因、未知突变分析候选基因法：选择与遗传病相关联的已知基因作为候选基因，通过家系调查和连锁分析，确定易感/致病基因。全基因组扫描法：利用H

12、GP中的MS或SNP作全基因组扫描和连锁分析，初定易感基因的位置。再作精细扫描、测序，克隆出易感基因。外显子组捕获和测序17Exome capture and sequencing外显子组捕获(exome capture and sequencing )技术通过全外显子组扫描，并结合生物信息分析技术，以寻找遗传病患者特有的SNP及单基因病的致病基因。技术平台：罗氏公司推出的Nimblegen外显子捕获平台；安捷伦科技公司推出的SureSelect 平台。采用外显子捕获技术已发现了数十种遗传病的致病基因。 Exome sequencing identified the causing mutat

13、ions of mendelian disorders18Genome-wide association study, GWAS全基因组关联研究(genome-wide association study, GWAS)：指在全基因组层面上，开展多中心、大样本、反复验证的基因（SNP）与疾病的关联研究，全面揭示疾病发生、发展与治疗相关的遗传基因。Klein等于2005年率先在Science上发表了应用GWAS方法证实年龄相关的黄斑变性与补体因子h基因之间存在强烈的关联。2010年，Stahl等在Nat Genet 上发表了应用GWAS荟萃分析法识别7个新的类风湿性关节炎的易感基因，包括IL6ST

14、, SPRED2, RBPJ, CCR6, IRF5, PXK和AFF3。目前已有1350篇采用GWAS方法定位多基因遗传病易感基因的论文发表。192021Treatment of genetic disordersRoutine therapy (常规治疗) of genetic disorders, A combination of the application of the three Rs- Restriction (禁其所忌)：G-6-PD缺乏症- Replacement (补其所缺)：血友病- Removal (去其所余)：各种累积症上海市12种可治性罕见遗传病的患病率及治疗方法

15、疾病患病率治疗方法患者数/年苯丙酮尿症（含四氢生物蝶呤缺乏症）1:1.1万低苯丙氨酸饮食/奶粉，补充BH4，L-多巴、5-羟色氨1016人枫糖尿症1:20万限亮氨酸/异亮氨酸/缬氨酸饮食,补充维生素B10-1人酪氨酸血症1:20万低苯丙氨酸、酪氨酸饮食，补充1,3-环已二酮0-1人甲基丙氨酸血症1:3.5万限亮氨酸/缬氨酸/甲硫氨酸饮食，补充肉碱、维生素B12、甜菜碱4-5人多种羧化酶缺陷病1:6万生物素1-3人高氨血症1:5万饮食治疗，补充精氨酸、瓜氨酸、苯甲酸钠1-2人肝豆状核变性1:5万低铜饮食，青霉胺治疗3-4人先天性耳聋1:1万人工耳蜗，助听器15-20人戈谢病1:20万酶替代治疗，

16、Cerezyme0-1人法布雷病1:20万酶替代治疗，Fabrazyme0-1人粘多糖累积症I型1:10万酶替代治疗，Aldurazyme1-2人糖原累积症II型1:20万酶替代治疗，Myozyme1-2人2223Gene therapy (基因治疗)Definition: Using recombinant DNA technology including gene replacement(替代), gene correction (校正)and gene augmentation(增补) to cure the genetic disorders, cancers and other di

17、seases.24Key procedures of gene augmentation (关键步骤)Transfer of a normal gene Normal gene + vectors (载体) infect defective cellsExpression of normal gene Promoter (启动子) or enhancer (增强子)Safety: No harm to cells and human body, no activation of oncogene and inactivation of tumor suppressor gene.25Gene

18、therapyThe first trials on gene therapy by Blaese in NIH, USA. The first case was a 4-year-old girl who received an intravenous infusion (静脉注射) of her own ADA-gene-corrected T lymphocytes on 14 September 1990.Gene therapy for SCID (Severe Combined Immunodeficiency) caused by ADA (adenosine deaminase

19、 腺苷脱氨酶) deficiency.26Gene therapy for first patientAshanti Desilva who affectedSCID in 1990.27Gene therapy年份项目数各国政府批准的项目类型受试人数肿瘤基因标记遗传病艾滋病心血管病其他19962360-2510-153719972125834348.51.4-2000199938769.26.99.112.6-2.23278各国1996-1999年间基因治疗临床试验项目统计数28基因治疗失败的案例1999年宾夕法尼亚大学的科学家开展了鸟氨酸转氨甲酰酶(ornithine transcarba

20、mylase, 简称OTC)缺陷症的基因治疗。该病患者因缺乏OTC基因的正常拷贝，抑制了机体对氨的清除作用。受试者是年仅18岁的JesseGelsinger（来自美国亚利桑那州）的学生。科学家构建了减毒腺病毒和感冒病毒毒株，并以此为载体将正常拷贝的OTC基因导入到患者肝脏。因腺病毒感染导致多器官衰竭(multiorganfailure)而不幸离世。2000年法国发生了SCID-X1（严重复合免疫缺陷）患者接受基因治疗后患上白血病，原因是逆转录病毒载体插入到造血系统原癌基因LMO2启动子附近从而将LMO2激活所致。 近年基因治疗成功案例使用干细胞2930Prevention of genetic

21、 disordersPRIMARY PREVENTION (原发性预防): It is prevent genetic disorders from appearance through offering genetic counseling and guidance to family planning.SECONDARY PREVENTION (继发性预防): It is to prevent genetically defective individuals from expressing their disease phenotype through early diagnosis o

22、f the patients with various screening programs. Genetic screening, Prenatal screening.31Genetic screening(遗传筛查)Maternal serum and amniotic fluid AFT (alpha-fetoprotein) in normal pregnancies and pregnancies affected with neural tube defects (NTD).无创产前基因检测羊水穿刺染色体检查唐氏筛查技术定位唐氏征高准确性的产前筛查法染色体异常诊断金标准广覆盖的产

23、前筛查技术实验技术高通量测序技术羊水细胞培养+染色体核型分析（+FISH技术）生化检测+B超技术（AFP、hCG、E3等）检测疾病唐氏、18、13三体综合征所有染色体数目异常和染色体大片段结构异常唐氏、18三体综合征、开放性神经管缺陷检出率约99%（针对唐氏与18三体征）约100%约70%特异性约99%（针对唐氏与18三体征）针对唐氏综合征等染色体数目异常约为100%约1%2%样本获得孕妇外周血5-10毫升通过羊膜腔穿刺获得羊水孕妇外周血3-5毫升检测时间孕12周-孕24周孕16周-孕24周（以孕20周之前为宜）早孕筛查孕11-13周；中孕筛查孕15-20周报告周期10-15个工作日14-21

24、个天，FISH2-3个天3-10个工作日检测费用2000-3500元1000-4000元约160-200元（不含B超）主要优势不需侵入性操作、检测时间窗较宽诊断金标准、技术可靠、能检测所有染色体异常技术成熟、操作简单、价格具有显著优势主要不足只能检测13、18和21号染色体数目异常、依然只是筛查技术。侵入性操作，孕妇接受度较低，具有潜在流产风险存在较高的漏检率、存在超过5%的假阳性率。3233Genetic screeningNeonatal screening （新生儿筛查）- Screening with newborn blood - Screening with newborn uri

25、ne我国母婴保健法实施办法中规定的新生儿疾病筛查主要包括苯丙酮尿症和先天性甲状腺功能低下症两种。2002年国家卫生部颁布的新生儿疾病筛查管理办法(草案)中规定在新生儿甲低和苯丙酮尿症的检测基础上增加新生儿听力筛查。Guthrie细菌抑制试验检测新生儿血液中苯丙氨酸水平3435Screening for carrier detection（携带者检出）- A carrier of balanced translocation- Heterozygote in AR- Female heterozygote in XR- Heterozygote in AD who does not manife

26、st the disease- Heteroplasmy（杂质） in mitochondrial genetic disorder36Genetic counseling(遗传咨询)Definition: An educational process that identifies and assists couples with a potential risk to their offspring in the decision-making process of family planning. The process seeks to stack the odds as much a

27、s possible in favor of a couples having a normal child. How favorable the odds need be undertake a pregnancy is the personal decision of the couple. 任务是进行婚育指导37Preparation for the genetic counseling sessions- Diagnostic evaluation- Gathering of medical records- Gathering of family history- Estimatin

28、g of recurrence risk- A group discussion among the members of a genetic program to complete the preparation.Genetic counseling38Estimation of recurrence risk (再显风险) of inherited disorders Recurrence risk of chromosome disorders- For parents who have normal karyotypes: The recurrence risk can be esti

29、mated to be the population incidence of the disorder.- For parents who are balanced translocation carriers or hidden mosaics: The recurrence risk is increased and should be estimated according to concrete situations.Genetic counseling39For example, if the mother is a balanced translocation carrier w

30、ith the karyotype of 45, XX, -14, -21, +t(14q21q). She herself is phenotypically normal, but 6 kinds of eggs will be formed in meiosis, they are as follow: 23,X; 22,X,-14,-21,+t(14q21q) 23,X,-14,+t(14q21q); 22,X,-21 23,X,-21,+t(14q21q); 22,X,-14Genetic counseling40When they fertilized with a normal

31、sperm 23, X then 6 kinds zygote will be formed, they are:46,XX normal45,XX,-14,-21,+t(14q21q) balanced translocation46,XX,-14,+t(14q21q) translocated 21 trisomy45,XX,-21 21 monosomy46,XX,-21,+t(14q21q) translocated 14 trisomy45,XX,-14 14 monosomyGenetic counseling41Recurrence risk of Downs syndrome

32、due to various chromosome aberrationPatient karyotypeFatherMotherRecurrence risk (%)D/G translocationnormalcarrier0.100.15carriernormal0.0521/21 translocationnormalcarrier1.00carriernormal1.0021/22 translocationnormalcarrier0.100.15carriernormal0.0521 trisomynormalnormal0.01Mosaicnormalnormalsmall42

33、Recurrence risk of single-gene disorders Genotype of the couple being disclosedMode of inheritanceMating typeSeg. Freq. among progenyNormalCarrierAffectedADAaaa1/2-1/2AaAa1/4-3/4ARAAaa-1-Aaaa- 1/21/2AaAa1/42/41/443 Segregation frequencies among progeny under X-linked inheritanceMode of inheritanceMa

34、ting typeSeg. freq. among progenyMaleFemaleNormalAffectedNormalAffectedCarrierXDXHYXX1-1-XYXHX1/21/21/21/2-XRXhYXX1-1XYXhXh-1-1XYXhX1/21/21/2-1/2XhYXhX1/21/2-1/21/244There are additional informationThe application of Bayes theorem to calculation of recurrence riskCalculation the risks of recurrence

35、means estimating the probability of being affected for a future pregnancy. The estimate of recurrence risk for a given couple can be made more accurate when information such as age of onset, lab test results, and number of normal children are to be included in the calculation in addition to the segr

36、egation frequencies suggested by the mating type. 45ProceduresWrite down the segregation frequencies, now called prior probabilities for all possible genotypes of the person involved.Consider the conditional probabilities for the person involved for all possible genotypes according to additional inf

37、ormation.Calculate the joint probability, which is the product of the prior and conditional probabilities.Finally calculate the posterior or relative probability, which is the joint probability of being affected (or of being a carrier) divided by the joint probability of not being affected plus the

38、joint probability of being affected.46Estimation of recurrence risk of DMD (XR) for an at-risk couple47ProbabilityII4 is HHII4 is Hh Prior probability1/21/2Condition probability13=1(1/2)3 = 1/8Joint probability8/161/16Posterior probability8/91/9Estimation of recurrence risk of DMD for an at-risk cou

39、pleThe recurrent risk of II-4s son = 1/91/2 =1/1848Estimation of recurrence risk of AR for a first-cousin coupleIII-2为杂合子：2/3III-1为杂合子：1/4III-1与III-2的子代得AR遗传病的风险为：1/42/31/4 = 1/2449Estimation of recurrence risk of Huntington chorea (AD) for a phenotypically normal family memberHuntington chorea 在30岁以前的发病风险为33%50Huntington chorea (AD)ProbabilityII2 is UuII2 is uu Prior probability1/2=0.51/2=0.5Condition probability1-0.33=0.671Joint probability0.330.5Posterior probability0.40.651Ge