单基因遗传病高级遗传.ppt

单基因遗传病,Single gene disorders Monogenic disorders,Etiology of diseases. For any condition the overall balance of genetic and environmental determinants can be represented by a point somewhere within the triangle.,Classification of Genetic Disorders,Single gene disorders are caused by defects in one particular gene, and often have simple and predictable inheritance patterns. They affect about 1 per cent of the population as a whole.,Classification of genetic disorders,Multifactorial Variants in genes causing alteration of function Single gene Mutations in single genes (often causing loss of function Chromosomal Chromosomal imbalance causes alteration in gene dosage Mitochondrial Somatic mutations (cancer),Male,Recessive Homozygotes with two copies of the altered gene are affected,Dominant Heterozygotes with one copy of the altered gene are affected,X-linked recessive Males with one copy of the altered gene on the X-chromosome are affected,Multifactorial (common) - “Environmental” influences act on a genetic predisposition to produce a liability to a disease. - One or more organ system affected. - Person affected if liability above a threshold. Single gene (1% liveborn) - Dominant/recessive pedigree patterns (Mendelian inheritance). - Can affect structural proteins, enzymes, receptors, transcription factors. Chromosomal (0.6% liveborn) - Thousands of genes may be involved. - Multiple organ systems affected at multiple stages in gestation. - Usually de novo (trisomies, deletions, duplications) but can be inherited (translocations).,Genetic disorders,Single gene disorders: disorders in which inheritance is due to a single mutant gene,Mendelian inheritance Genes are units of heredity, based in DNA Phenotype (physical or functional abnormalities) Genotype (DNA change) 4. Autosomal vs X-linked determined by whether the responsible gene is carried on one of the autosomal chromosomes or on the X chromosome 5. Dominant vs Recessive , based on phenotypic expression,Single gene disorders,- High risks to relatives - Dominant/recessive pedigree patterns - Some isolated cases due to new dominant mutations - Structural proteins, enzymes, receptors, transcription factors,Characteristics of single gene inheritance,Autosomal Dominant vertical (successive), risk of affected offspring 50% (both sex) Autosomal Recessive horizontal, multiple sibs affected, usually one generation, consanguinity (+) risk of affected offspring 25%, carrier 50% X-linked Dominant daughters of affected males (+), sons of affected males (-), affected females transmit the disorder to offspring of both sexes, risk of affected offspring 50%, but twice as many affected females as affected males (no male to male) X-linked Recessive males through carrier women, males affected almost exclusively, females affected only when affected father and carrier mother or with skewed X-inactivation Y-linked males affected,Characteristics of Autosomal Dominant inheritance 1. The phenotype usually appears in every generation, each affected person having an affected parent Exceptions : (1)fresh mutation (2)the disorder is not expressed or is expressed only subtly in a person who has inherited the responsible gene. 2. Any child of an affected parent has a 50 percent risk of inheriting the trait 3. both males and females are affected in a 1 : 1 ratio,Autosomal dominance inheritance (AD),Pedigree showing typical inheritance of a form of progressive sensorineural deafness (DFNA1) inherited as an autosomal dominant trait,Characteristics of Autosomal Recessive Inheritance 1. An autosomal Recessive phenotype, typically is seen only in the sibship of the proband, not in parents, offspring, or other relatives. 2. both sexes are affected with equal frequency at a ratio of 1:1 3. Parents of an affected child are asymptomatic carriers of mutant alleles. heterozygous parents have a risk of 25% of affected offspring 4. The parents of the affected person may in some cases be consanguineous. This is especially likely if the mutant gene is rare in the population.,Autosomal Recessive Inheritance (AR),Characteristics of X-linked Dominant Inheritance 1. The incidence of the trait is much higher in females than in males (about twice) ,but affected females typically have milder (variable) expression of the phenotype. 2. Affected males with normal mates have normal sons and Affected daughters. 3. Both male and female offspring of Affected female have a 50 percent risk of inheriting the phenotype. 4.The pedigree pattern is the same as autosomal dominant inheritance.,X-linked Dominant Inheritance (XD),Characteristics of X-Linked Recessive Inheritance The incidence of the trait is much higher in males than in females. The gene is ordinarily never transmitted directly from father to son (male-to-male), but it is transmitted by an affected male to all his daughters . A carrier Female for an X-chromosomal mutation has a risk of 50% For an affected son. The gene may be transmitted through a series of carrier females; affected males inherit the mutant allele from the mother only Heterozygous females are usually unaffected, but some may express the condition with variable severity as determined by the pattern of X inactivation,X-linked Recessive Inheritance (XR),Y-linked inheritance,Gene : YA （ mutant allele ） Ya Genotype : XYA XYa holandric inheritance (全男性遗传) male-to-male,Y-linked inheritance,Single gene disorders Huntington Disease Myotonic Dystrophy Hereditary Motor Sensory Neuropathy (HMSN) Neurofibromatosis Marfan syndrome Cystic Fibrosis Spinal Muscular Atrophy (SMA) Duchenne Muscular Dystrophy Hemophilia,如何确定所研究的疾病是单基因病？ 确认方法主要有以下两种： 1) 参考OMIM(Online Mendelian Inheritance in Man)数据库，根据疾病的表型或者临床症状等确定是否属于OMIM收录的单基因病。 OMIM除了简略描述各种疾病的临床特征、诊断、鉴别诊断、治疗与预防外，还提供已知相关致病基因的连锁关系、染色体定位、组成结构和功能、动物模型等资料，并附有经缜密筛选的相关参考文献。 网址为： 2) 绘制疾病的遗传系谱图，通过系谱图分析其遗传方式来判断是否属于孟德尔遗传病。 系谱分析法是研究人类遗传规律的重要方法。在临床上，常用系谱分析法来判断某种疾病的遗传方式。系谱图就是从先证者（proband）（家系中第一个被医生确诊的某遗传病的患者，或者具有某种性状的成员）入手，追溯调查其所有家庭成员的数目、亲属关系、某种遗传病（或性状）的分布等资料，按照一定格式绘制而成的图解。,单基因病研究举例及进展,Rieger 综合征(#MIM 180500)致病基因PITX2的研究,Rieger 综合征是Axenfeld-Rieger症候群中最为严重的一型 典型临床表现： 眼前节发育不良，继发青光眼 口颌发育异常： 先天多数牙缺失，过小牙，畸形牙 面中部发育不足，下颌前突等 脐残断回缩异常 遗传方式：常染色体显性遗传，发生率约为1:200,000,临床资料：,家系I 先证者,Rieger综合征相关基因和染色体区域,PITX2 4q25 FOXC1 6p25 PAX6 11p13 13q14,All these Rieger-syndrome-associated genesencode transcription factors and have been shown to play important roles in embryonic development,测 序 结 果,Wild type,1-III:4 cloned,1-III:4 uncloned,家系中的每位患者均存在PITX2基因第5外显子第717-720位ACTT四个碱基的杂合缺失,导致该基因的读码框移位,蛋白质大段功能域缺失,而家系中正常人不存在此突变。,PITX2基因的特征,1996年定位克隆得到同源盒(homeobox) 基因PITX2，编码一种转录因子，属于paired-bicoid基因家族，在发育中高度保守，cDNA编码区与小鼠的同源基因91相同，蛋白质的homeobox区100相同。 Paired-bicoid 的标志是在同源结构域(homeodomain) 第3螺旋50位有一赖氨酸残基，这一残基识别TAAT盒后的CC序列。 小鼠Pitx2参与牙胚的定位，在牙齿发育的较早阶段表达于口腔上皮组织。Pitx2-/- 的小鼠牙胚的发育停止在蕾状期。Pitx2还是心脏形态，上下颌骨的前突，垂体发育所必需的。,PITX2 基因结构图,PITX2的重要功能域,PITX2基因的各种变异剪切体均含相同的homeobox结构域(HD)和C末端，但是N端由不同的外显子组成。 对PITX2分子C端功能的研究提示C端能抑制DNA的结合从而为PITX2与协同因子Pit-1作用创造条件。,PITX2突变谱的总结,15/23的突变发生在HD，7/23的突变发生在HD的3编码区。,图中矩形表示PITX2基因的外显子，标出了翻译的起始（ATG）及终止（TGA）位点。矩形中黑色的区域表示基因的Homeobox结构域。图中的红色星形、三角形、椭圆、圆形、箭头依次表示不同的突变类型：剪切位点突变、缺失突变、点突变、无义突变及插入突变。,PITX2突变功能研究,T68P位于HD第2个螺旋，该突变不改变蛋白质对DNA结合功能，但是使之失去调节基因转录的功能； L54Q位于HD的第1个螺旋，该突变使蛋白质的稳定性丧失； K88E恰改变HD的第50位赖氨酸，不仅使蛋白质丧失功能，并且抑制野生型蛋白正常功能的行使； V45L位于HD的第1个螺旋，该突变仅轻微降低DNA结合活性，而能够大幅上调报告基因的表达。,PITX2突变类型与临床表型的关系,不同类型的突变在临床表型上存在差异。Kozlowski等针对与不同临床表型相关的5种PITX2突变的功能研究表明，当突变蛋白还拥有部分功能时临床的表型也较轻。 Espinoza等的实验也证实没有牙齿异常表型的突变R84W与PITX2调控基因Dlx2启动子的结合能力与野生型相似，而含有所有临床表型的突变T68P则无法调节Dlx2的表达。,本研究中PITX2基因突变示意图,1 38 98 228 248 271,N,C,OAR,HD,野生型,突变型,1 44 82,N,C,ACTT Del,突变位于HD的起始部位，移除大段重要功能域，所引起的临床表型，特别是牙齿的缺失是目前所有报道中最严重的,.,Mutations in 5 regulatory region,Selective Tooth Agenesis (STHAG), is the common generalized term used to describe congenitally missing teeth and is one of the most frequent developmental anomalies in humans. Genetic linkage studies on non-syndromic hypodontia have so far identified four genes underlying this condition: MSX1 ,PAX9,WNT10A and AXIN2 STHAG3 (OMIM #604625) is caused by heterozygous mutation in the PAX9 gene on chr14q12-13,which codes a transcription factor, and essential for the switch in odontogenic potential from the epithelium to the mesenchyme, are expressed in the dental mesenchyme at the initiation stage of tooth development,A family with Hypodontia,Pedigree of family DEN29 with haplotypes for a SNP within (rs28933972) and microsatellite markers (D14SA1462,D14S1463, D14S1464) near the PAX9 locus. The shaded haplotype is that segregating with the hypodontia phenotype.,A novel g.-1258GA mutation in a conserved putative regulatory element of PAX9 is associated with autosomal dominant molar hypodontia Clin Genet 2011: 80: 26572,Multiple-species comparison of a 60 bp segment bearing the g.-1258GA variant identified by the arrow,Repeat expansion disease,a set of genetic disorders caused by trinucleotide repeat expansion, a kind of mutation where trinucleotide repeats in certain genes exceeding the normal, stable, threshold, which differs per gene.,Nature Reviews Genetics 2010 Vol 11 786-99,Huntington Disease (HD),Clinical Classification Movement/Cognitive/Psychiatric disorder Mean onset age 35-55 years. Prevalence Incidence 1 in 10,000. Genetic Testing Diagnostic Presymptomatic counselling protocol.,Huntington Disease (HD),Physical features: - involuntary movements - weight loss - abnormal gait - speech & swallowing difficulties. Psychiatric Manifestations: - personality changes - depression - aggression - early onset dementia.,Genetic aspects of Huntington disease,Inheritance AD Chromosome locus 4p16.3 Trinucleotide repeat CAG in 5 translated region Repeat sizes Normal 26 Mutable 27-35 Reduced penetrance 36-39 Fully penetrant 40 Protein product Huntingtin Early onset form Juvenile Paternally transmitted,Dynamic mutations (动态突变): Mutations in some disorders involve amplification of trinucleotide repeat sequences during gametogenesis. Become mutated through a two-step process. The first mutation, called the premutation, doesnt cause any clinical symptoms. A second mutation was required to convert the premutation into a full mutation capable of causing the clinical symptoms,Structure of the Huntington disease gene. Short vertical bars represent exons.,Huntington disease - a triplet repeat disease,CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG . CAG,11-35 CAG triplet repeats are normal: encodes a run of 11-35 glutamine amino acid residues in the protein.,A run of 35 glutamine residues causes the protein to aggregate in the brain cells and cause progressive cell death.,Runs of 35 CAG repeats in the HD gene expand further (particularly during male meiosis) causing earlier age of onset in children of men who have the gene anticipation.,normal brain HD patient brain,Mechanisms of expansion,Mechanism of TNR instability during DNA replication and/or repair processes and the possible roles of DNA repair proteins. DNA repair proteins could either be involved in the formation of slipped-DNAs, the intermediates of TNR instability, or else they could be involved in the processing of these intermediates by mediating correct, escaped, or error-prone repair. Correct repair restores the parental repeat size. Escaped repair involves sealing of the nick resulting in either excess or fewer repeatsrepair proteins may bind to the slipped-DNA preventing repair. Error-prone repair results from incomplete excision of excess repeats leading to a variety of expanded repeat sizes.,Mechanisms of pathogenesis for satellite instability disorders.,Cont. Mechanisms of pathogenesis for satellite instability disorders. DNA Repair Vol 7 (2008) 113554,The RASopathies: developmental syndromes of Ras/MAPK pathway dysregulation,Current Opinion in Genetics & Development 2009, 19:230236,Ras/mitogen-activated protein kinase (MAPK) pathway,Plays an essential role in the control of the cell cycle and differentiation Its dysregulation has profound developmental consequences.,Each RASopathies each exhibit unique phenotypic features Many share characteristic overlapping features Craniofacial dysmorphology Cardiac malformations Cutaneous, musculoskeletal and ocular abnormalities, Varying degrees of neurocognitive impairment In some syndromes, an increased risk of developing cancer.,Outcomes of mutations,The vast majority of these mutations result in increased signal transduction down the Ras/MAPK pathway Usually to a lesser extent than somatic mutations associated with oncogenesis. It is likely that the strongly activating oncogenic mutations cannot be tolerated as germline mutations. The most common oncogenic BRAF mutation, V600E, does not occur in CFC syndrome The specific KRAS mutations associated with NS are not the same as the known somatic mutations associated with cancer.,Kinasopathy .,A clinical phenotype that is caused by germline mutations in the kinase domain of functional proteins that lead to a loss-of-function or gain-of-function of the protein,Kinase mutations in human disease: interpreting genotypephenotype relationships Nature Reviews Genetics 2010 Vol 11 60-74,The characters of mutational hotspots associated kinase structures,Common neutral mutations tend to occupy the C-terminal regions of the catalytic core and substrate-binding or catalytic residues are avoided. Inherited germline disease causing mutations, most of which result in loss-of function developmental and/or metabolic disorders, tend to cluster in regions of the catalytic core involved in regulation and substrate binding, especially residues that participate in proteinprotein and allosteric interactions. Acquired somatic mutations that cause or contribute to cancer tend to populate ATP binding and catalytic residues.,Unlocking Mendelian disease using exome sequencing Gilissen et al. Genome Biology 2011, 12:228,Exome sequencing,also known as targeted exome capture， is an efficient strategy to selectively sequence the coding regions of the human genome to identify novel genes associated with rare and common disorders. It is estimated that the protein coding regions of the human genome constitute about 85% of the disease-causing mutations.,Work flow of exome sequencing,Ex.Identified that DHODH is the gene responsible to Miller syndrome (MIM 263750),A timeline illustrating technological breakthroughs and hallmark publications for Mendelian disease gene identification. (a) The main historical events leading up to the introduction of whole exome sequencing (WES).,A timeline illustrating technological breakthroughs and hallmark publications for Mendelian disease gene identification. (cont)(b) The main exome sequencing events and landmark publications. More than 30 Mendelian disease genes have been identified by exome sequencing so far.,Table 2. Mendelian disease gene identifications by exome or genome sequencing,Contributions of exome sequencing to Mendelian disease studies,Enrich, extend, and possibly even complete our search for the heritable basis of Mendelian disease. Improving clinical diagnosis, greatly increase our understanding of the most basic causes of disease.,Limitations of exome sequencing,Fails to identify causative variants in regulatory regions spread across the genome (transcription binding sites, enhancers, and so on).,microRNA and single gene disorders,Original causal mechanisms miRNA as a candidate gene of the disease Mutation or SNP (also referred to as miRSNP) is located in the miRNA-binding site, or nearby Succesive effects of mutated proteins,Mechanisms involving microRNA in inherited diseases,Clin Genet 2010: 77: 306313,#613074 DEAFNESS, AUTOSOMAL DOMINANT 50; DFNA50 mapped the phenotype to chromosome 7q32, caused by mutations in a microRNA, miR-96 This was the first study to implicate a miRNA in a mendelian disorder,Mutations in the seed region of human miR-96 are responsible for nonsyndromic progressive hearing loss. Nature Genet. 41: 609-613, 2009,miRNA as a candidate gene of the disease,Sequence analysis excluded UBE2H, SMO, ATP6V1F,CALU, CCDC136, TSPAN33, KLHDC10, C7ORF68, FLNC, IMPDH1 and MIR129-1. A set of three genes encoding miRNAs (MIR96, MIR182 and MIR183) was annotated within the interval. They are transcribed as a single polycistronic transcript and were reported to be expressed in the inner ear,.,Mutations in the seed region of MIR96 cause DFNA50 hearing loss. (a) Pedigrees of the Spanish families (family 1, top; 2, bottom) segregating DFNA50 hearing loss. (b) Electropherograms depict the 23-nt mature sequence of human miR-96. The nucleotides corresponding to the seed region are boxed.,Predicted secondary structure and processing of the wild-type and mutant forms of miR96.,Downregulation of predicted primary targets is impaired by the miR-96 (+13GA) and (+14CA) mutations.,Lewis MA, Quint E, Glazier AM et al. An ENU-induced mutation of miR-96 associated with progressive hearing loss in mice. Nat Genet 2009: 41: 614618.,A mouse mutant presented a phenotype similar to the human disease, which carries a (AT) base substitution in the seed sequence of miR-96.,Sequence Variants in SLITRK1 Are Associated with Tourettes Syndrome Science 2005：310, 317-320,The single-base change maps to the 3UTR of the SLITRK1 transcript and corresponds to a highly conserved nucleotide within the predicted binding site for the human miRNA hs