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

http://asia.ensembl.org/

- Mutations may or may not result in an expressed phenotype.

- Mutations can alter RNA expression, processing and/or stability.

- Mutations that have no phenotype are called neutral mutations.

- A mutation is a structural change in genomic DNA sequence due to errors in DNA replication or repair.

- Mutations can also affect protein expression, processing, stability.- Mutations can be inherited (genetic/germline mutations) not inherited (somatic mutations)

Mutations 突变

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: (1) 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.

(2) 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.

(3) 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.

Structural classification of mutations

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

GeneHomozygote Phenotype

Heterozygote Advantage

Alternative Explanations

CFTR Cystic fibrosisIncreased

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.

TDSTay-Sachs mainly in

Ashkenazi JewsResistance 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.

• Heritable change in patterns of gene expression mediated by mechanisms other than alterations in primary nucleotide sequence

Epigenetics 表观遗传学

• 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。

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 (gene expression pattern)

表观遗传学的特点可遗传。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

SAMSAM

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

• 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

非编码 RNA 的调控作用

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。• 1999， RNAi作用机制模型的提出。在线虫、果蝇、拟南芥及斑马鱼等多种生物内发现RNAi 现象。

• 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, PTG

S ）。• 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).

• ‘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).

Polymorphisms 多态性

Uses of Polymorphisms

• 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 Linkage Equilibrium and DisequilibriumDisequilibrium

• Linkage equilibriumLinkage equilibrium– Random association between a marker and a Random association between a marker and a

diseasedisease

• Linkage disequilibriumLinkage disequilibrium （连锁不平衡）（连锁不平衡）– Biased association of a marker (Biased association of a marker (e.ge.g., D or d)., D or d)– May be helpful in identifying ancient May be helpful in identifying ancient

mutations or specific mutant alleles in mutations or specific mutant alleles in genetic isolatesgenetic isolates

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

分子医学研究的策略

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