Principles BIOC2603 of Molecular Genetics
DNA polymorphisms
Brian Wong [e-mail]
Genetic variations in human
Compare genomic DNA of any two individuals: 99.9% identical (approximately 1 difference per 1 kb) Size of human genome is 3 x 109 bp so 0.1% difference corresponds to about 3 x 106 bp difference These genetic differences result in individual differences in disease susceptibility, responses to drug, etc Finding genetic variations associated with a disease phenotype can help to locate genes responsible for the disease and these variations can also be used for diagnostic purposes Genetic variations can also be used for personal identification and kinship testing
Genetic variations resulting in individual differences
CYP1A2 in liver is responsible for metabolizing caffeine. People with a certain polymorphism in this gene result in a slow metabolizer phenotype and are more likely to suffer heart attack. Those with a fast metabolizer polymorphism have a lower risk.
Cornelis MC et al (2006) JAMA 295:11351141
Discontinuous and continuous variations
Caused by variations in a single gene
Caused by variations in many genes, and interactions between genes and the environment.
From: Griffiths AJF et al (1999) Modern genetics analysis. Freeman.
Polymorphisms
Locus: a defined position in the genome, can be a gene, a stretch of DNA, a restriction enzyme recognition site, or a single nucleotide Alleles: alternative forms of the same gene/locus, caused by mutations Mutation: a change in the gene sequence. Occurs at a low frequency in a population. A locus is polymorphic if the least frequent allele occurs at 1% or greater in a population
Polymorphisms
Phenotypes: color, morphology, etc Protein polymorphism (allozymes)
Differences in size and charge because of amino acid substitutions Identified using native gel electrophoresis
Other hemoglobin variants
Other hemoglobin variants
From: Griffiths AJF et al (1999) Modern genetics analysis. Freeman.
Polymorphisms
DNA polymorphisms
Single-nucleotide polymorphism (SNP) Variable number of tandem repeats (VNTR) polymorphism:
Minisatellite polymorphism: total size 0.5 - 30kb. The unit size of the repeats is variable (15-100bp) but most share a common core sequence (GGGCAGGANG). Minisatellites are also found near telomeres (10-15kb of TTAGGG repeats). Microsatellite polymorphism: the unit size of the repeats is short (e.g. 1 to 6 bp). Also known as short tandem repeats polymorphism or simple sequence repeat polymorphism.
Both SNP and VNTR polymorphism can give rise to restriction fragment length polymorphism (RFLP)
Restriction fragment length polymorphism
Variations in the size of DNA fragments produced after restriction enzyme digestion. Due to:
Single nucleotide polymorphism: single base change abolishes or creates a restriction enzyme recognition site VNTR: a region containing variable number of tandem repeats flanked by two restriction enzyme recognition sites.
To test for RFLP, DNA samples are digested with an appropriate restriction enzyme. The DNA fragments produced are separated by gel electrophoresis and transferred to a nylon membrane (Southern blotting). The fragment length polymorphism is detected by hybridization using a DNA probe near the polymorphic site.
RFLP due to SNP detected by Southern blot hybridization and PCR
300 bp 100 bp
P1, P2 = primers for PCR * = polymorphic site
Allele A Allele B
Southern Blots
PCR products
Gelehrter TD et al (1998) Principles of Medical Genetics. 2nd ed.
RFLP due to variable number of tandem repeat polymorphism detected by Southern blot hybridization
tandem repeat
Probe 1 hybridizes to an unique DNA sequence outside the tandem repeats (locus-specific probe): this detects polymorphisms in a single locus.
Probe 2 hybridizes to the tandem repeats. This detects polymorphisms in a multiple loci.
Gelehrter TD et al (1998) Principles of Medical Genetics. 2nd ed.
Single nucleotide polymorphism (SNP)
Most abundant form of variations in human, occurs about 1 in 1000 bp Single base change
Transition (pyrimidine pyrimidine; purine purine)
C T; G A
Transversion (pyrimidine purine)
C A; C G; T A; T G
Most are biallelic polymorphism; tri- and tetra-allelic SNPs are very rare. May result in creation or abolishment of restriction enzyme recognition site, give rise to restriction fragment length polymorphism
Single nucleotide polymorphism (SNP)
SNPs can be found in
Coding regions (cSNPs, very few are found)
Synonymous: different SNP alleles code for the same amino acid Non-synonymous: different SNP alleles code for different amino acid
Non-coding regions (non-coding SNPs)
Promoters 5 or 3 untranslated regions Splice sites Introns Intergenic region
Shastry BS (2002) SNP alleles in human disease and evolution. J Hum Genet 47:561566
Microsatellite polymorphism
Most human microsatellite loci are identified from randomly cloned genomic DNA fragments:
Cloned genomic fragments that hybridized to labeled simple repetitive sequence (e.g. CATTCATTCATT) are sequenced to determine the unique DNA sequences flanking the simple repeats. These unique flanking sequences are used as primers for the polymerase chain reaction (PCR) to detect the microsatellite locus. Variation in the number of simple sequence repeats will appear as polymorphism in the sizes of the PCR products after gel electrophoresis.
Microsatellites are highly polymorphic, some can have nine or more alleles.
9 repeats 8 repeats 6 repeats 5 repeats
Microsatellite in the tyrosine hydroxylase (TH) gene on human chromosome 11p15.5. PCR using primers flanking the CATT repeats amplified alleles whose sizes differ by multiples of 4 bp.
(9/5) (9/6) (5/6) (9/9) (9/5) (9/8) (5/6)
From: Hearne CM et al (1992) Microsatellites for linkage analysis of genetic traits. Trends in Genetics 8:288294
Sources of DNA variation in a population
Mutation
Chemical modifications in DNA Errors in DNA replication and repair Strand slippage due to mispairing mediated by direct repeats in DNA sequences during replication give rise to VNTR polymorphism
Recombination
Independent assortment and crossing-over Unequal crossing-over at nonallelic site of sister or nonsister chromatids due to misalignment of nonallelic repetitive DNA may also give rise to VNTR
Migration
Introduction of genes from one population into another
Normal replication
Backward slippage causes insertion
Forward slippage causes deletion
Strand slippage during replication causes variation in number of short tandem repeats.
From: Strachan T and Read AP (1999) Human Molecular Genetics 2. Fig 9.5
Generation of minisatellite polymorphisms: (A) Unequal crossing-over and (B) unequal sister chromatid exchange result in duplication or deletion of repeats.
From: Strachan T and Read AP (1999) Human Molecular Genetics 2. Fig 9.7
How is polymorphism maintained?
Neutral theory of evolution: most mutations are neutral (no advantageous nor disadvantageous phenotypic effects) and the allele frequency changed by genetic drift Balancing selection: mutation creates variations and natural selection favored different alleles at different times, in different places, in different combinations
heterozygote advantage individual heterozygous for a locus is more favourable e.g. in sickle-cell anaemia, heterozygous individuals are resistant to malaria while individuals homozygous for the locus are susceptible to malaria
Applications of DNA polymorphisms Serve as markers
Population genetics and evolutionary studies. E.g. inferring demographic history Molecular diagnosis of diseases Mapping
e.g. genetic maps of the human genome e.g. identification of disease susceptibility genes
DNA fingerprinting
e.g. forensics and paternity tests
DNA sequence variations used in phylogenetic studies
Mitochondrial DNA from 182 individuals of diverse geographic origin, including 121 native Africans, Europeans, Papua New Guineans, Asians, a native Australian, African-Americans were compared. There was a greater sequence variations among Africans than among Asian or Europeans. Since mutations accumulated over time, more mutations mean longer time to evolve variations so concluded that modern human originated in Africa.
Wilson AC and Cann RL (1992) Scientific American 266(4):22-27.
DNA polymorphisms and personalized medicine
Identify polymorphisms (e.g. SNP) that correlate with drug response
custom design a drug treatment plan for a person based on the predicted drug response
Identify polymorphisms (e.g. SNP) that associated with a disease
develop screening method to identify individuals susceptible to the help disease to find gene responsible for the disease
different alleles of the SNP loci
Roses A.D. (2000) Pharmacogenetics and the practice of medicine. Nature 405:857865
Polymorphism and drug response
CYP450:
CYP2D6 and CYP2C19 genes are important in the metabolism of ~25% of all prescription drugs. based on genotypes of these genes, patients can be classified as poor, intermediate, extensive, or ultra rapid metabolizer knowing the genotypes of a person at these genes may help physicians in individualizing treatment selection and dosing for drugs metabolized through these genes, e.g. antidepressants, antipsychotics, beta-blockers, and some chemotherapy drugs diagnostic test available for genotyping of CYP2D6 and CYP2C19
Use of DNA polymorphism in disease diagnosis
The restriction site for MstII is destroyed in S globin allele
The restriction site for MstII is present in the normal A globin allele
RFLPs as diagnostic markers in diseases: The sickle cell mutation destroys an Mst II site and generates a disease-specific RFLP.
Uses of DNA polymorphisms in gene mapping
Serve as markers that correlate with phenotypic variations (e.g. drug response, disease susceptibility) because of its proximity to the actual underlying genetic factor, i.e. the genetic marker is close to the genetic factor such that there is little recombination between them
DNA polymorphism can be used to detect linkage.
A,B,C: alleles of a polymorphic marker normal disease
DNA polymorphism can be used to detect linkage.
A,B,C: alleles of a polymorphic marker normal disease
No correspondence between the inheritance of alleles and inheritance of the disease no linkage between marker and disease gene. They segregate independently.
H: dominant disease allele h: normal allele
Correspondence between the inheritance of alleles and inheritance of the disease those inherited allele C always inherited the disease. The marker and disease gene are linked.
Pierce BA (2005) Genetics 2nd ed.
A microsatellite locus can show linkage to a disease gene
M: M: M: M:
3 repeats 5 repeats 2 repeats 8 repeats
Griffiths et al (2005) Introduction to Genetic analysis 8th ed.
A microsatellite locus can show linkage to a disease gene
8 repeats 5 repeats 3 repeats 2 repeats
All affected individuals have M allele. M allele is linked to the disease allele P.
An RFLP linked to a disease gene
Griffiths et al (2005) Introduction to Genetic analysis 8th ed.
An RFLP linked to a disease gene
The normal allele, d, segregates with RFLP allele 2. The disease allele D, segregates with RFLP allele 1 (except in child 8). So the gene responsible for the disease is on the same chromosome as the RFLP marker.
D: dominant disease allele
normal disease
Griffiths et al (2005) Introduction to Genetic analysis 8th ed.
Population association studies
Linkage disequilibrium (LD): non-random association of alleles of two adjacent loci, i.e. two loci do not segregate independently. LD is high when two loci are physically close to each other (5-20kb). LD decreases as distance between 2 loci increases.
Haplotype
Haplotype = haploid genotype Haplotype refers to a set alleles on the same chromosomal region. Each individual has two haplotypes for any given genomic region, one from paternal chromosome, one from maternal chromosome. SNP represents a single nucleotide variation. Haplotype represents a much longer sequence of nucleotide that tends to be inherited together.
Individual 1 Individual 2 Individual 3 Individual 4
(http://www.hapmap.org/)
Linkage disequilibrium
Ancestor
Current generation
A1 to J1 are alleles of genetic loci A to J (can be SNP, RFLP, mini-satellite, etc) surrounding a gene locus (N) in an ancestor. Different ancestors may have different combinations of alleles. The normal gene (N) was mutated to a disease gene (D) in this ancestor. In subsequent generations, recombination (crossing-over) between homologous chromosomes lead to exchange of alleles such that D was linked to alleles other than those that originally presented. After many generations, only the alleles of the closest loci that surround the disease gene are retained. The disease gene is in linkage disequilibrium with F1 and E1. To find the disease gene in the current generation, we identify alleles of marker loci (e.g. F1 and E1) that are more commonly found in persons with the disease phenotype. The disease gene should be close to, i.e. in linkage disequilibrium with these marker loci. The chromosome around the marker loci are then screened for genes.
Using haplotypes to deduce gene position
SNPs 4 and 5 are in linkage disequilibrium with the disease gene patients tend to have SNP 4 and 5. The disease gene is around SNP 4 and 5.
Principle of multi-locus DNA fingerprinting.
A, B, C are minisatellite loci sharing a similar core sequence in the tandem repeats (e.g. 1kb in length). A, B, and C can be on the same or different chromosomes and each locus have different number of repeats on each homologous chromosome. DNA is digested with an restriction enzyme that does not cut within the repeat region. This generate a set of DNA fragments with different sizes corresponding to variation in the number of repeats. The DNA fragments are analyzed by Southern Blotting using a probe that hybridizes to the repeat regions. The probe therefore identifies polymorphism at many loci at a time. The resulting hybridization pattern is known as DNA fingerprint.
Use of DNA fingerprinting
(A): paternity test M (mother), C (Child) F1, F2 (two possible fathers) Child has minisatellite repeat lengths inherited from either the mother or F1 forensics The minisatellite repeat lengths in the specimen match those of suspect 1
(B):
Note: current DNA fingerprinting methods use multiple singlelocus probes (e.g. short tandem repeat polymorphisms).
From: Strachan T and Read AP (1999) Human Molecular Genetics 2. Fig 17.19
Use of DNA polymorphism in forensic investigation
Background:
Tsar Nicholas II, Tsarina Alexandra (wife), and 4 daughters, 1 son, three servants, 1 doctor were killed on July 16, 1918 during the Russian revolution bodies were buried together in July 1991, Russian government recovered 1000 bone fragments from the burial site and assembled into 5 females and 4 males believed to be the remains of the Tsar family. analysis of the amelogenin gene allowed the determination of sex of the bones analysis of five short tandem repeat polymorphic loci in the nine skeletons suggested that 5 skeletons belonged to a single family (Tsar, Tsarina, and three children) how to confirm whether the remains were those of the Russian royal family?
Use of DNA polymorphism in forensic investigation
Extract mitochondrial DNA (mtDNA) from bone recovered from burial site Extract mitochondrial DNA from Prince Philip who is a maternal relative (grand nephew) of Tsarina Alexandra Comparison of the mtDNA sequences reveal the sibling status of the children and the identification of the mother The mtDNA sequence of the putative Tsar was compared to that of two relatives of maternal descent The two relatives have the same mtDNA sequence of the putative Tsar, except at position 16169: Tsar is heteroplasmic (C/T) while the relative is homoplasmic (T) Could the bone really be the remains of Tsar Nicholas II?
ALEXEI
Lineage of Tsarina Alexandra, showing relationship to Prince Philip
Lineage of Tsar Nicholas II, showing relationship to two maternal relatives tested
(from Gill P. et al (1994) Nature Genetics 6: 130135)
To further confirm heteroplasmy in the Tsar's lineage, the mtDNA was extracted from the bone of Georgij Romanov, Tsar's brother. The mtDNA sequence of Georgij Romanov matched that of the putative Tsar and was also heterplasmic (C/T) at position 16169. This confirmed heteroplasmy in Tsar's lineage. The mother of Tsar Nicholas must also be heteroplasmic, and that the heteroplasmy segregated to homoplasmy in later generations.
72% C, 28% T
38% C, 62% T
Lineage of Tsar Nicholas II, black symbols indicate maternal lineage
Sequence of mtDNA around position 16169 from bones of putative Tsar Nicholas II, the bone of Georgij Romanov and blood of Xenia Cheremeteff-Stfiri
From: Ivanov P.L. et al (1996) Nature Genetics 12:417420
References
Griffiths AJF et al (2000) Introduction to Genetic Analysis. 7th ed. Freeman and Company, New York. Chapters 5, 13, and 14 or Griffiths AJF et al (1999) Modern Genetic Analysis. Freeman and Company, New York. Chapters 11 and 12. Hartwell LH (2004) Genetics: From Genes to Genomes. 2nd ed. McGraw-Hill. Chapter 11. Strachan T and Read AP (2004) Human Molecular Genetics 3. Garland Science. Chapter 7.