Lecture Notes
基因突变、多态性及疾病基因的定位和鉴定
张咸宁（细胞生物学与医学遗传学系）
2013/09
I. 人类基因组
A. 核基因组
1. ~3.2 × 109 bp
2. ~20,000–25,000 genes
3. Less than 10% of these encode proteins. Approximately 50% of the genome
consists of different forms of repetitive DNA widely distributed across the genome.
4. Most genes contain several exons, which are expressed, separated by
intervening sequences known as introns, which are spliced out of mature mRNA.
B. 线粒体基因组
1. Each human cell contains several hundred or more mitochondria in its
cytoplasm. The mitochondria, which produce ATP, have their own unique DNA.
Mitochondrial DNA (mtDNA) is maternally inherited and has a high mutation rate.
2. mtDNA: 16,568 bp. The mitochondrial genome encodes 2 rRNAs, 22 tRNAs,
and 13 polypeptides involved in oxidative phosphorylation.
II. 突变
A. 变异的类型
SNPs
CNVs
In/dels
Inversions
Segmental duplications
B. 多态性
1. 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.
2. It has been estimated that there are some 10–15 million common sequence
variants that are of sufficient frequency (minor allele frequency >5%) in one or more
populations to be considered polymorphic in our species.
C. 突变
1. A mutation is a structural change in genomic DNA sequence due to errors in
DNA replication or repair.
2. Mutations can be inherited (genetic/germline mutations) or not inherited
(somatic mutations).
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3. Structural classification of mutations
(1) Point mutations: change in one base pair of DNA.
1) silent mutations:
2) missense mutations:
3) nonsense mutations:
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.
(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.
(3) Unstable trinucleotide (‘triplet’) repeat expansions (动态突变):
Trinucleotide repeats have been identified as the cause of approximately 30
disorders, most of which are extremely rare and involve the central nervous system.
4. 突变的蛋白质效应
1) Loss of Pr function (the great majority): is seen in (1)recessive
diseases;(2)diseases involving haploinsufficiency, in which 50% of the gene product
is insufficient for normal function; and (3)dominant negative mutations, in which the
abnormal protein product interferes with the normal protein product.
2) Gain of function: are sometimes seen in dominant diseases.
3) Novel property: infrequent.
4) The expression of a gene at the wrong time (Heterochronic expression), or in
the wrong place (Ectopic expression), or both. (uncommon, except in cancer).
D. 表观遗传学
The newly emerging field of epigenetics is providing us with a basis for
understanding how heritable changes other than those in DNA sequence can influence
phenotypic variation.
An epigenetic trait is a stable, mitotically and meiotically heritable phenotype
that results from changes in gene expression without alterations in the DNA sequence.
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Epigenetics is the study of the ways in which these changes alter cell- and
tissue-specific patterns of gene expression. Epigenetic regulation of gene expression
uses reversible modifications of DNA and chromatin structure to mediate the
interaction of the genome with a variety of environmental factors and to generate
changes in the patterns of gene expression in response to these factors. The
epigenome refers to the epigenetic state of a cell.
There are 3 major epigenetic mechanisms: (1) reversible modification of DNA by
the addition or removal of methyl groups; (2) modification of histones by the addition
or removal of chemical groups; and (3) regulation of gene expression by small,
noncoding RNA molecules.
III. 分离和鉴定人类疾病基因
A. 一般策略：
B. 连锁不平衡: Random association between a genetic marker and a disease.
LOD score（LODs 值）: A statistical method that tests genetic marker data in
families to determine whether two loci are linked. The lod score is the logarithm of the
odds in favor of linkage. By convention, a LOD score of 3 (odds of 1000:1 in favor) is
accepted as proof of linkage and a LOD score of -2 (100:1 against) as proof that the loci
are unlinked.
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C. 全外显子组测序（whole exome sequencing，WES） makes medical
genomics a reality
Exome（外显子组）: the collection of all exons in the human genome.
D. 疾病动物模型的构建
The need for animal models of human disease
 Many genes have been isolated based upon biochemical properties, sequence
similarities to known genes or patterns of expression; Their true biological
function is unknown

Knockout mice provide keys to the in vivo function of a gene product/s

Introduction of mutated/altered genes can similarly provide insight about the
role of the gene in a disease process

Developmental processes can be examined by altering the timing of gene
expression.
Transgenic Mice（转基因小鼠）


Random integration of DNA into the genome of mouse cells
A transgene consists of a cDNA encoding the protein of interest driven by a
promoter that regulates timing and location of expression.

transgenic mice are used to model various types of cancer
Gene Targeting（基因打靶）

In vitro mutagenesis can be used to generate alterations in genes

DNA is taken up by cells and integrated into the genome

Homologous recombination can be used to replace normal genes with those
carrying mutations

generation of chimeric mice
o Mutant alleles are introduced into embryonic stem cell by homologous
recombination
o ES cells with the mutation are introduced into the mouse embryo creating
chimeric animals
o Animals are mated to determine if the mutant gene is present in the germ
line

Generation of heterozygous and homozygous offspring
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