Office: C303, Teaching Building Gene Mutation, Polymorphism, Gene mapping and identification （From Genotype to Phenotype） 张咸宁 zhangxianning@zju.edu.cn Tel：13105819271; 88208367 Office: C303, Teaching Building 2014/09

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Mutations突变 A mutation is a structural change in genomic DNA sequence due to errors in DNA replication or repair. Mutations may or may not result in an expressed phenotype. Mutations that have no phenotype are called neutral mutations. - Mutations can alter RNA expression, processing and/or stability. - Mutations can also affect protein expression, processing, stability. Mutations can be inherited (genetic/germline mutations) not inherited (somatic mutations)

Structural classification of mutations 1. Point mutations: change in one base pair of DNA. (1) silent mutations: changes in DNA which do not affect protein expression or function. (2) missense mutations: changes in DNA which lead to a change in an amino acid. (3) nonsense mutations: changes in DNA which generate a termination codon and thus stop translation. (4) Regulatory mutations: one which involves the promoter or another regulatory sequence such as an enhancer, silencer, or locus control region. (5) RNA processing mutations: These affect the processing of the primary RNA transcript to form mRNA, either by altering normal RNA splicing or by preventing either normal 5’-capping or 3’-polyadenylation.

Structural classification of mutations 2. Deletions and insertions: small deletions and insertions: If the number of nucleotides deleted or inserted in an exon is not a multiple of three, then the sequence of codons, known as the reading frame, is disrupted. This is referred to as a frame-shift → a truncated protein. large deletions and insertions: These range in size from 20 bp to 10 Mb, beyond which they become visible using a light microscope and are classified as chromosome abnormalities. unequal crossing-over: Crossing-over between misaligned closely adjacent sequences which show close homology results in the formation of a deletion in one chromatid and a duplication in the other. (4) retrotransposition: Transposable elements, SINES and LINES, which have moved from an inert region of the genome to become inserted into an exon elsewhere.

Structural classification of mutations 3. Unstable trinucleotide (‘triplet’) repeat expansions: 某些单基因遗传病是由于脱氧三核苷酸串联重复扩增所引起的，而且这种串联重复的拷贝数可随世代的递增而呈现累加效应，故称这种突变方式为动态突变Dynamic mutation。 Trinucleotide repeats have been identified as the cause of approximately 30 disorders, most of which are extremely rare and involve the central nervous system.

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Mutation Nomenclature Examples c.1444g>a: a mutation at position 1444 in the hexosaminidase A cDNA causing Tay-Sachs disease g.IVS33+2T>A: a mutation substituting an A for T in a splice donor site GT of intron 33 of a gene g.IVS33-2A>T: a mutation substituting a T for an A in the highly conserved AG splice acceptor site in the same intron c.1524_1527delCGTA: a deletion of four nucleotides, numbers 1524 through 1527 in cDNA c.1277_1278insTATC: a four-base insertion between nucleotides 1277 and 1278 in the hexosaminidase A cDNA, a common mutation causing Tay-Sachs disease p.Glu6Val: a missense mutation, glutamic acid to valine at residue 6 in β-globin, that causes sickle cell disease p.Gln39X: a nonsense mutation, glutamine to stop codon (X) at position 39 in β-globin, that causes β0-thalassemia

Heterozygote Advantage杂合子优势 Gene Homozygote Phenotype Heterozygote Advantage Alternative Explanations CFTR Cystic fibrosis Increased resistance to cholera CFTR mutant alleles appeared before Cholera epidemics; increased resistance to typhoid and/or asthma. HLA (MHC) Highly polymorphic Enhancing resistance to infectious disease The loci too polymorphic to be accounted for by heterozygote advantage; may have other benefits. TDS Tay-Sachs mainly in Ashkenazi Jews Resistance to TB The high frequency of disease can be explained by founder effect and genetic drift Gemmell NJ and Slate J. PLoS One. 2006;1:e125.

An epigenetic landscape (Waddington, 1956) Epigenetics表观遗传学 Heritable change in patterns of gene expression mediated by mechanisms other than alterations in primary nucleotide sequence The word Epigenetics was first coined by C. H. Waddington in 1942. An epigenetic landscape (Waddington, 1956) 15

表观遗传(epigenetic inheritance)：通过有 丝分裂或减数分裂来传递非DNA序列信息的现象。 表观遗传学(epigenetics)：研究不涉及DNA序列 改变的基因表达和调控的可遗传变化的学科。 OR 研究从基因演绎为表型的过程和机制的遗传学分支学科。 Epigenome refers to the epigenetic state of a cell.

基因表达模式在细胞世代之间的可遗传性并不依赖细胞内DNA的序列信息。 (gene expression pattern) 决定细胞类型（>200）的不是基因本身，而是基因表达模式，通过细胞分裂来传递和稳定地维持具有组织和细胞特异性的基因表达模式对整个机体的结构和功能协调至关重要。 基因表达模式在细胞世代之间的可遗传性并不依赖细胞内DNA的序列信息。

表观遗传学的特点 可逆性（reversible）的基因表达调节。 （基因活性或功能的改变）； 可遗传。即通过有丝分裂或减数分裂，能在细 胞或个体世代间遗传； 可逆性（reversible）的基因表达调节。 （基因活性或功能的改变）； 没有DNA序列的改变或不能用DNA序列变化 来解释。

表观遗传现象/修饰 DNA 甲基化（DNA methylation） 组蛋白修饰（histone modification） 染色质重塑（chromatin remodeling） 基因组印记（genomic imprinting） X染色体失活（X chromosome inactivation） RNA相关沉默（RNA interference等） 副突变（paramutation） 位置效应斑（position effect variegation） 组蛋白密码（histone code） RNA 编辑（RNA editing） ……

DNA甲基化 DNA甲基化(DNA methylation)是研究得最清楚、也是最重要的表观遗传修饰形式，主要是基因组DNA上的胞嘧啶第5位碳原子和甲基间的共价结合，胞嘧啶（C）由此被修饰为5甲基胞嘧啶(5-methylcytosine，5mC)。 DNMT1 SAM C 5mC

DNA甲基化 哺乳类基因组中5mC占胞嘧啶总量的2%~7%，约 70%的5mC存在于CpG二核苷酸。 在结构基因的5’端调控区域, CpG二核苷酸常常 以成簇串联形式排列，这种富含CpG的区域称为 CpG岛(CpG islands)，其大小为500~1000 bp， 约56%的编码基因含CpG岛。 基因调控元件(如启动子)所含CpG岛中的5mC会阻 碍转录因子复合体与DNA的结合。 DNA甲基化一般与基因沉默相关联； 非甲基化一般与基因的活化相关联； 去甲基化往往与一个沉默基因的重新激活相关 联。

组蛋白修饰的类型 乙酰化：一般与活化的染色质构型相关联，乙酰化 修饰大多发生在H3、H4的 Lys 残基上。 甲基化：发生在H3、H4的 Lys 和 Arg残基上，可 以与基因抑制有关，也可以与基因的激活相关，这 往往取决于被修饰的位置和程度。 磷酸化：发生与 Ser 残基，一般与基因活化相 关。 泛素化：一般是C端Lys修饰，启动基因表达。 SUMO（一种类泛素蛋白）化：可稳定异染色质。 其他修饰（如ADP的核糖基化）

组蛋白修饰 组蛋白中被修饰氨基酸的种类、位置和修饰类型 称为组蛋白密码（histone code），遗传密码的表 观遗传学延伸，决定了基因表达调控的状态，并 且可遗传。 The sum of the complex patterns and interactions of histone modifications that change chromatin organization and gene expression is called the histone code.

遗传学与表观遗传学

非编码RNA的调控作用 Short interfering RNA (siRNA) Micro RNA (mirRNA) Double-stranded RNA (dsRNA) Short heterochromatic RNA (shRNA) Transcripts from repeated sequences (ALU, LTR) Ribosomal and transfer RNAs 27

RNA干扰（RNAi）现象 1995，RNAi（RNA干扰）现象首次在线虫中 发现。 1998，RNAi概念的首次提出。 2001，RNAi技术成功诱导培养的哺乳动物细 胞基因沉默现象。RNAi 技术被《Science》评 为2001年度的十大科技进展之一。

RNA干扰（RNAi）现象 RNAi作用是生物体内的一种通过双链RNA分子在mRNA水平上诱导特异性序列基因沉默的过程。 由于RNAi发生在转录后水平，故又称为转录后基因沉默（post-transcriptional gene silencing, PTGS）。 RNAi是一种重要而普遍表观遗传的现象。 Two types of short RNA molecules are involved in RNA induced gene silencing: The small interfering RNAs (siRNAs) and the microRNAs (miRNAs. >1048).

Polymorphisms多态性 ‘Mutations’ that are propagated and maintained in the population at relatively high frequencies are called polymorphisms. Polymorphism is defined as the existence of two or more alleles, where the rare allele appears with a frequency greater than 1% in the population.指同一群体中存在有2种或以上可变基因型的现 象，每种类型的比例应大于1﹪。 Most mutations are quickly lost from population due to deleterious effects (natural selection) or genetic drift (random fluctuations). Mutations may become polymorphisms due to selective advantage (heterozygotes for hemoglobin sickle cell mutation are more resistant to malaria) or genetic drift遗传漂变 (founder effect, small group of individuals found a new population).

Uses of Polymorphisms studies in families and populations. Polymorphisms are used as ‘genetic markers’ for studies in families and populations. + Majority of human genome variance is represented within rather than between populations. Mapping of genes that cause inherited diseases (linkage analysis).

Linkage Equilibrium and Disequilibrium Random association between a marker and a disease Linkage disequilibrium（连锁不平衡） Biased association of a marker (e.g., D or d) May be helpful in identifying ancient mutations or specific mutant alleles in genetic isolates 35

分子医学研究的策略 确定疾病的表型 →收集疾病家系、患病同胞对家系、隔离人群 → 连锁分析、关联研究、外显子组捕获测序！ → 物理作图、构建重叠群（contig） → 分离转录物 → 分析候选基因、突变检测 → 基因测序 → 确定候选基因以及基因突变与所观察到的表 型的关系 → 疾病基因和蛋白质的功能研究 → 疾病的分子诊断和治疗研究

Biesecker LG. Exome sequencing makes medical genomics a reality Nature Genetics 2010;42: 13–14.