4. mutations + polymorphisms

Locus (loci)

DNA segment that occupies a specific position on a chromosome

Alleles

alternative versions of DNA sequence at a specific locus

2 alleles for each locus: maternal & paternal

wild-type/common allele vs variants/mutants

Most genes have one prevailing allele = wild-type/common allele

All other versions = variants/mutants

polymorphic

A locus with >2 common alleles 

“private” alleles

Rare variants confined to families

Zygosity

degree of allele similarity at one locus in an organism

Homozygous

2 copies of same allele at one locus

Heterozygous

2 different alleles at same locus

Genotype

genetic information at a locus

Phenotype

appearance of an organism based on genotype

source of genetic diversity

Mutations

how much sequence identity among individuals

Approx. 99.9% sequence identity among individuals

0.1% = genetically determined variability

reference sequence

NO standard genomic sequence!

The most common sequence in a population

Mutation Classifications

Classification by:

size (chromosome, subchromosomal + DNA mutations)

function (from non-functional to lethal)

heritability (germline vs somatic)

Chromosome mutations

intact structure, change in chromosome number

Euploidy + Aneuploidy

Euploidy

multiplication of a chromosome set (tetraploidy)

Aneuploidy

additional chromosomes (trisomy, monosomy)

Subchromosomal mutations

changes in portions of the chromosomes

copy number variations, structural rearrangements

DNA mutations

substitutions, deletions, insertions up to 100bp

Mutation frequency

number of mutations/locus/cell division

what is mutation frequency dependent on?

Frequency of spontaneous and induced nucleotide changes

Probability of repair

Probability of detection

mutation rates very by: 

species + genes

areas of DNA mutate differently (hot spots) 

Rate of disease-causing mutations

incidence of new cases of genetic disease NOT present in parents and caused by a single mutation

Chromosome mutations

Result of chromosome mis-segregation during meiosis

Generally severe, resulting in spontaneously aborted fetuses

Regional mutations

Result of homologous recombination between fragments with high homology at different sites or following repair of double-strand breaks

Gene mutations

Replication errors <1 mutation/genome/cell division

DNA repair errors

• Frequently from spontaneous mutations that evade the repair machinery

nucleotide substitutions

• synonymous

• missense

• nonsense

• mutation affecting mRNA processing

• dynamic mutations

Synonymous mutations

nucleotide change that specifies the same amino acid (AA)

Missense mutations

single nucleotide change that specifies a new AA

Transitions + Transversions

transition

purine → purine

pyrimidine → pyrimidine

transversion

purine → pyrimidine

pyrimidine → purine

Nonsense mutation

point mutations resulting in replacement of coding codon by a “stop” codon

Mutation affecting mRNA processing

mutations that abolish or create alternative intron-exon junctions

causes alterations of the splicing pattern

Dynamic mutations

amplifications of simple trinucleotide repeats in the coding region or untranslated regions

deletion, insertions, rearrangement mutations (frameshift)

rearrangement of small nr. of nucleotides (not a multiple of 3) resulting in altered reading frame → functionally altered protein

consequences of mutations

gain/ lose function, lethality, domitive negative

gain of function

Overproduction/ inappropriate production of protein

loss of function

Reduced production of protein

The product of the normal allele is generally sufficient for function

haploinsufficiency

50% of protein is insufficient for function

dominant negative 

a mutated protein interferes with the function of the normal protein, so even though a healthy allele is present, the overall function is disrupted.

heredity of mutations: germline mutations

Mutations inherited from parents or de novo (spontaneous, not from parent) mutations that are transmitted to offspring

De novo mutations are very rare

heredity of mutations: somatic mutations

Are not transmitted to the next generations

Genomic heterogeneity specific to highly proliferative tissues (eg., epithelial,
hematopoietic cells)

Typically undetected

what mutation type is frequent in cancers? 

Somatic mutations

Genetic polymorphisms

Mutations with a frequency exceeding 1% of all alleles in a population

(regardless of the type, size, effect, or location of mutation)

1. Single nucleotide polymorphisms (SNPs)

change of 1bp

Average 1 for every 1000bp (approx. 5-10 million SNPs/genome)

Generally, do not produce phenotypic differences

where are SNP hotspots? whats the rate?

25x higher rate at adjacent CG (CpG)

how many SNPs in protein-coding genes?

approx 100,000

nonsynonymous → protein variants

2. Insertion-deletion polymorphisms (indels)

up to 1000bp

Simple or Microsatellites

Simple Indels

presence or absence of a short fragment → 2 alleles

Microsatellites (short tandem repeat polymorphisms STR) 

variable number of repeated short segments (2, 3, or 4 nt) → multiple alleles

Infer familial relationships by DNA fingerprinting (study of alleles at 13 loci)

3. Copy number variants (CNVs) 

up to hundreds of kb

Can include dozens of genes → altered gene dosage

4. Inversion polymorphisms

few bp – mb 

Sequence homology at edges (homologous recombination)

Balanced – no loss/gain of DNA

detection of mutations process

1. discovery

2. validation

3. screening 

1. Discovery
   –

Initial identification

Whole genome/exome sequencing + comparison to reference sequence

2. Validation
   

Replication assay to exclude sequencing errors

Larger population to get statistical occurrence

3. Screening
   

Sequencing/analysis of thousands of SNPs from same individual and multiple individuals

High-density DNA arrays (SNP arrays)

Genetic analysis

Establish a catalogue of all known human genes and variants and their location

On-going list of tens of millions of variants in different populations

Methods for mapping human disease variants

Linkage analysis (family-based)

Association analysis (population-based)

Genome sequencing

genome-wide association studies

Molecular technique that analyses simultaneously hundreds of thousands of variations in genomic DNA to determine if a genetic locus is associated with a certain phenotype

Candidate gene associations have greater power, but rely on previous knowledge

GWAS find unbiased susceptibility variants for complex traits, without a prior hypothesis of gene function

Study design

Standard case-control (matched or unmatched)

SNP imputation

Genotyped SNPs allow for determining possible variants on neighboring SNPs based on reference genomes (HapMap)

Linkage disequilibrium

non-random association of alleles at linked loci

Measure of tendency of some alleles to be inherited together as haplotypes

Haplotypes

sets of closely linked SNPs present on the same chromosome, which tend to be inherited together

Manhattan plot

scatter plot of association between statistical significance as p-value on the y-axis against chromosomes on the x-axis

Association of hundreds of thousands of markers → traditional statistical significance thresholds (eg. p<0.05) not appropriate; association
considered statistically significant if p=5x10-8

If a SNP is significantly associated with the phenotype

Causal relationship between SNP and disease

Marker in linkage disequilibrium with causal locus

False positive

Clinically functional variants: Risk variants

increase risk of disease

Clinically functional variants: Protective variants

lower risk of disease

Clinically functional variants: Risk of disease

diagnosis of disease

23andme

testing of genome-wide polymorphisms

Problems : privacy, lack of regulation

Traits: cilantro aversion, hairline, photic sneeze reflex, caffeine metabolism, hair curliness, bitter taste, newborn hair amount, earlobe type, muscle composition, eye color, dimples, sweet taste preference

Carrier status: cystic fibrosis, BRCA1, sickle cell anemia, glycogen storage disease, maple syrup urine disease, Sjorgen’s syndrome

Ancestry: ancestral composition, maternal/paternal lineage

Drug response

Clinical utility/ limitations

Gene-disease associations not relevant for all patients

Environmental role unknown and hard to predict and estimate

Relevance only for prognosis (confounding factors can change risk)

Combined risk from multiple SNPs is hard to calculate