DNA Polymorphismsin the Human Genome.Mutations and Genetic

Diseases.

Marie Kopecká 2007/2008 mkopecka@med.muni.cz

• GENOME

• All of the genetic material of a cell or of an individual.

The Human Genome

• Nuclear genome.• Mitochondrial genome.*

Lecture concerns „Nuclear genome”only, for which the term „Human genome” is used.

* 1994

• POLYMORPHISM, GENETIC (Ford, 1940)

• Existence of more than one normal allele at a gene locus

• with the rarest allele exceeding a frequency of 1%.

• A polymorphism may exists at several levels, i.e., variants in DNA sequence,

• amino acid sequence, • chromosomal structure, or • phenotypic traits.

• DNA POLYMORPHISM

• A difference in DNA sequence among individuals, groups, or populations.

• Sources include single nucleotidepolymorphisms (SNPs),

• sequence repeats, • insertions, • deletions and • inversions.

1866 Johan Gregor Mendel: genes (elements)1869 Fritz Miescher: DNA (nuclein)1882 Walther Flemming: chromosomes in mitosis1911 Thomas Hunt Morgan: genes in chromosomes1944 Oswald Avery, Colin MacLeod, Maclyn McCarty:

DNA is carrier of genetic information in bacteria1952 Al Hershey, Martha Chase:

DNA is carrier of genetic information in viruses1953 James Watson, Francis Crick: double-helix of DNA1956 Tjio and Levan: human karyotype1970 Hamilton Smith: restriction endonuclease HindII1977 Fred Sanger/Allan Maxam,Walter Gilbert: DNA sequencing 1983 Kerry Mullis: PCR2001 First „draft” of human genome:

Length of DNA=3.2 x 109 bp. Gene number = 26 383.Nature 409: 860-921, (2001) - 15. 2. 2001 - HUGO, Science 291: 1304, (2001)- 16. 2. 2001 - Celera Genomics2006 Sharp, Cheng, Eichler, ARGHG 7 (2006): human genome 24 652 genes

The Human Genome:First estimation of gene number?

• 1980 Walter Gilbert:

• The human genome has about 3 000 000 000 bp.

• The gene size is about 30 000 bp.• How many genes in human DNA?• 3 000 000 000 : 30 000 = 100 000• The human genome contains about

100 000 genes !

The Human Genome (2001):comparison with other genomes

Yeast 6 200 genesDrosophila 14 000 genesC. elegans 19 000 genesArabidopsis 27 000 genesHomo sapiens ~ 25 000 genes

HUMAN GENOME – Important data:1A. Length of DNA: 2.9 x 109 bp (Campbell, Reece 2005)

Number of genes coding proteins: 25 0001B. Length of DNA 3.2 x 109 pb (Alberts et al. 2004)

Number of genes: 31 0002. DNA (Alberts et al. 2004)A. UNIQUE DNA: (39%)

CODING SEQUENCES 1.5% (Exon regions of genes codingfor proteins, rRNAs and tRNAs)Introns and regulatory sequences (~24%) Unique non-coding DNA (~15%)

B. REPETETIVE DNA (53%)LINEs 21%SINEs 13%Retroviral-like elements 8% DNA- only transposon „fossils” 3% Segmental duplications 3%Simple sequence repeats 5%

C. HETEROCHROMATIN (8%) -

Repetitive sequences at centromeric and telomericregions and intersperesed among the genes.

Examples of repetitive sequences in 7th intron of dystrophin gene

Human genome

• As one commmentator described our genome:„In some ways our genome may resembleyour garage/ bedroom/ refrigerator/ life: highly individualistic, but unkempt; littleevidence of organization; much accumulated„junk“; virtually nothing ever discarded; andthe few patently valuable itemsindiscriminately, apparently carelessly, scattered throughout.”

With the exception of identical twins, no two people have the exact same genome.

When the same region of the genome from two different humans is compared, the nucleotide sequences typically differ by about 0.1 %.

When considering the size of human genome (~3 000 000 000 bp) that amounts ~ 3 000 000genetic differences in each maternal and paternalchromosome set between one person and the next.

Genetic Variations Within the Human Genome

DNA POLYMORPHISM / GENE MUTATIONDNA POLYMORPHISM (D.P.)=a difference in DNA sequence among individuals, groups, or populations.D.P. include SNP, sequence repeats, insertions, deletions, recombination.

Example: blue eyes versus brown eyes; straight hair versus curly hair.If a difference in DNA sequence is associated with disease, it is usually

called a genetic mutation.Changes in DNA caused by external agents (mutagens) are also called

„mutations” rather than „polymorphisms”.

GENE MUTATION:A change in the nucleotide sequence of a DNA molecule. Genetic mutations

are a kind of genetic polymorphism.The term „mutation” as opposed to „polymorphism” is generally used to

refer to changes in DNA sequence which are not present in most individuals of a species and either have been associated with disease (or risk of disease) or have resulted from damage inflicted by external agents (viruses, radiation, chemical mutagens.)

• SINGLE NUCLEOTIDE POLYMORPHISM is a source variance in a genome.

• A SNP is a single base variation in DNA. • SNPs are the most simple form and • most common source of genetic polymorphism in the human

genome = 90% of all human DNA polymorphisms. • The bulk of this variation was inherited from our ancestors.

SINGLE NUCLEOTIDE POLYMORPHISM (SNP)

SNPs (cont.)

Frequency:1/1000 base pairs between two equivalent chromosomes.

Distribution:SNPs are not uniformly distributed over the entire human genome,however, they can be found in non-coding and coding regions.

Coding regions:SNPs in a coding region may have two different efects on the resulting protein:Synonymous: the substitution causes no amino acid change = silent mutation.Non-synonymous: the substitution results in an alteration of the encoded aminoacid: -a missense mutation changes the protein by causing a change of codon;-a nonsense mutation results in a misplaced termination codon.

Regulatory regions: SNPs may change the amount of or timing of protein production.

Non-coding regions: In healthy people, DNA polymorphisms probably occurpredominantly in non-coding DNA sequences or in DNA regions that do not influence the function of coded proteins.

These polymorphisms are scattered throughout the genome.More than 90% of all human genes contain at least one SNP.Because SNPs are present at such a high density, they areuseful markers for identification of mutated gene.

SNPs

Most of the SNP and other variations are genetically silent = affect the DNA sequences in noncritical regions of the genome. These SNP have no effect on how we look or how ourcells function.• However, a tiny subset od SNP are presumably responsiblefor nearly all heritable aspects of human individuality. Major task is to learn to recognize those relatively few variationsthat are functionally important against the large backgroundof neutral variation that distinguishes the genomes of anytwo human beings.

Examples:• Single nucleotide polymorphisms (SNPs)

• Variable Number of Tandem Repeats(VNTR)

DNA Polymorphisms in the Human Genome

Single nucleotide polymorphisms (SNPs) [„SNiPS”]

SNP

VNTR

Gene probe

Recognition sitefor restrictionenzyme present

Recognition sitefor restriction enzyme absent

Recognition sites for one restriction endonuclease in homologous chromosomes

Electrophoresis of restriction fragments of DNA and Souhern blot

Variability in a number of repetitive sequence(CACACACA…)

Two types of DNA polymorphism: Single Nucleotide Polymorphism (SNP)(top left) and Variable Number of Tandem Repeats (VNTR) (bottom left), bothresulting in Restriction Fragment Length Polymorphism (RFLP) (right). Polymor-phisms are detectable by Southern blotting,PCR,DNA sequencing,microarrays

• How to use SNP or VNTR ?

• SNP and VNTR change the lenth of restriction fragments of DNA (RFLP).

• It is possible to use RFLP in indirectDNA diagnosis

(see Lab book, p. 114)

Direct DNA diagnosis:Direct identification of mutation in the human

genome by:• DNA sequencing,• Oligonucleotide probes (ASO) in Southern

blotting,• PCR… (see Lab book, p. 113-117)Indirect DNA diagnosis:

If mutation cannot be identified directly, effort is to identify mutation by indirectmethods, f.e. by SNP near mutation by RFLP (see Lab book, 114-117 and the next slides)

Example of linkage of mutated gene causing disease and SNP close on the same chromosome

Dystrophin 2.4 x 106 pb. Gene mutation → Muscle dystrophy

Indirect DNA diagnosis of muscle dystrophy by RFLP

6 year boy

DNA of familymembers + restrictionendonuclease,gel electrophoresis of restriction fragments, Southern blotting

Normal allele for dystrophin

Deletion mutation in the dystrophin alelle

Conclusion:Mother´s sister does not have mutated allele

(GR)

Examples of gene mutations: substitution, insertion (addition),deletion…

Consequences of the gene mutation for the function of mutated protein product

deletion mutation

Indirect DNA diagnosis of phenylketonuria(AR)by means of RFLP

Conclusion:II. 2 will behealthyheterozygote

Another example: mutated enzyme

DNA offamilymembers+ restrictionendonu-clease, gelelectro-phoresisandSouthernblotting

It is easier than to look for single SNPs to identify the wholehaplotype* block and to identify SNP inside of this block!

Haplotype* = combination of alelles and futher DNA markers in linear block of DNA that is not interrupted by recombination and that is transferred for many generations.

• The investigation of the human genomecontinues.

• The aim is to understand the molecularnature of mutation of genes responsible for human diseases.

• Identification of DNA polymorphisms of individuals and of human haplotype map mayhelp to identify

• pre-dispositions to diseases, • the diagnosis and the course of diseases and• their potential therapy.

Specific terms• ALLELE The alternative forms of a genetic character found at a given locus on a chromosome.• DIPLOID The chromosome complement consists of two copies (homologues) of each chromosome.

In humans, the each chromosome pair is from a different origin (mother, father).• GENE (Johannsen, 1909) A hereditary factor that constitutes a single unit of hereditary material. It

corresponds to a segment of DNA that codes for the synthesis of a single polypeptide chain.• GENE LOCUS (Morgan, 1915) The position of a gene on a chromosome.• GENE MAP The position of gene loci on chromosomes. Physical map – the absolute position of gene

loci, their distance from each other being expressed by the number of base pairs between them. Genetic map – expresses the distance of genetically linked loci by their frequency of recombinations.

• GENETIC MARKER A polymorphic genetic property that can be used to distinguish the parental origin of alleles.

• GENETIC POLYMORPHISM A difference in DNA sequence among individuals, groups, or populations. Sources include SNPs, sequence repeats, insertions, deletions and recombination.

• GENOME All of the genetic material of a cell or of an individual. • GENOMICS The scientific field dealing with the structure and function of the entire genome.• GENOTYPE An exact description of the genetic constitution of an individual, with respect to a single

trait or a larger set of traits. The genetic constitution of an organism as revealed by genetic or molecular analysis, i.e. the complete set of genes, both dominant and recessive, possessed by a particular cell or organism.

• GENOTYPING Genotyping is normally defined as detecting the genotypes of individual SNPs.• HAPLOTYPE (haploid genotype). A combination of alleles of closely linked loci that are found in a

single chromosome and tend to be inherited together. The linear, ordered arrangement of alleles in a chromosome. A particular pattern of sequential SNPs (or alleles) found on a single chromosome. These SNPs tend to be inherited together over time and can serve as disease- gene markers.

• HOMOZYGOUS A diploid organism having identical alleles of a given gene on both homologous chromosomes.

• HETEROZYGOUS A diploid organism having different alleles of a given gene on both homologous chromosomes.

• LINES Long interspersed nuclear elements= long interspersed repetitive DNA sequence. POLYMORPHISM, GENETIC (Ford, 1940) Existence of more than one normal allele at a gene locus with the rarest allele exceeding a frequency of 1%. A polymorphism may exists at several levels, i.e., variants in DNA sequence, amino acid sequence, chromosomal structure, or phenotypic traits.

• PHENOTYPE (Johannsen, 1909) The observable effect of one or more genes on an individual or a cell.The observable properties of an individual as they have developed under the combined influences of the individual´s genotype and the effects of environmental factors.

• PROTEOM The complete set of all protein encoding genes or all proteins produced by them.• PSEUDOGENE Nucleotide sequence of DNA similar to functional gene, but containing many mutations

that prevent expression.• SINE Short interspersed nuclear element=short repetitive DNA sequences.• SINGLE NUCLEOTIDE POLYMORPHISM (SNP) SNP is a source variance in a genome. A SNP is a

single base variation in DNA. SNPs are the most simple form and most common source of geneticpolymorphism in the human genome (90% of all human DNA polymorphisms).