Lecture Notes: Gene Mutation, Polymorphism, Gene mapping and
identification
ZHANG Xian-Ning, PhD
(Department of Biochemistry and Genetics, Zhejiang University School of Medicine)
Sep., 2011
I. Human Genome
A. Nuclear genome
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
(Table 1).
4. Most genes contain several exons, which are expressed, separated by
intervening sequences known as introns, which are spliced out of mature mRNA
(Figure 1).
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B. Mitochondrial genome
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: 16,569 bp. The mitochondrial genome encodes 2 rRNAs, 22 tRNAs,
and 13 polypeptides involved in oxidative phosphorylation.
II. Human Mutations
A. Types of variation (Table 2)
B. Polymorphisms
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.
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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. Mutations
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).
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 (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.
4. Functional classification of mutations
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.
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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. Epigenetic
The term that refers to any factor that can affect gene function without change in
the genotype. Some typical epigenetic factors involve alterations in DNA methylation,
chromatin structure, histone modifications, and transcription factor binding that
change genome structure and affect gene expression without changing the primary
DNA sequence.
III. Identify Human Disease Gene and Mutations
A. General strategy (Figure 2)
B. Linkage equilibrium: Random association between a marker and a disease.
LOD score: 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.
C. Whole Exome Sequencing (WES) makes medical genomics a reality
Exome: the collection of all exons in the human genome.
D. Construction of a disease animal model
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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
 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
 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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