CM09 - Mutations and Polymorphisms

Biomedical Sciences I

What is a locus?

A DNA segment that occupies a specific position on a chromosome.

What are alleles?

Alternative versions of a DNA sequence at a specific locus. Each locus has two alleles (maternal and paternal).

What is the wild-type/common allele?

The most common allele of a gene in a population.

What are variant or mutant alleles?

All other versions of a gene different from the common allele.

What does it mean if a locus is polymorphic?

The locus has more than two common alleles.

What are “private” alleles?

Rare variants confined to families.

What is zygosity?

The degree of allele similarity at one locus in an organism.

• Homozygous = two copies of the same allele

• Heterozygous = two different alleles

Define genotype.

The genetic information at a locus.

Define phenotype.

The appearance of an organism based on genotype.

What is the role of mutations in genetic diversity?

Mutations are the source of genetic diversity.

How much sequence identity exists among humans?

About 99.9%; there is 0.1% genetically determined variability.

Is there a standard genomic sequence?

No; the most common sequence in a population is used as the reference sequence.

How are mutations classified by size?

• Chromosome mutations (change in chromosome number)

  • Euploidy = multiplication of a chromosome set (e.g., tetraploidy)

  • Aneuploidy = additional chromosomes (trisomy, monosomy)

• Subchromosomal mutations = changes in chromosome portions (copy number variations, structural rearrangements)

• DNA mutations = substitutions, deletions, insertions up to 100 bp

How are mutations classified by function?

From non-functional to lethal.

How are mutations classified by heritability?

Germline (can be passed down to offspring) vs. somatic (can’t be inherited)

What is mutation frequency?

The number of mutations per locus per cell division, dependent on:

• Frequency of spontaneous and induced nucleotide changes

• Probability of repair

• Probability of detection

Do all DNA regions mutate at the same rate?

No, some areas are hotspots.

What is the rate of disease-causing mutations?

It is the incidence of new cases of genetic disease not present in parents and caused by a single mutation.

What causes chromosome mutations?

Chromosome mis-segregation during meiosis; often severe, leading to spontaneously aborted fetuses.

What causes regional mutations?

Homologous recombination between DNA fragments with high homology at different sites, or following repair of double-strand breaks.

What causes gene mutations?

Replication errors (<1 mutation/genome/cell division) or DNA repair errors. Many come from spontaneous mutations that evade repair machinery.

What are the types of nucleotide substitutions?

• Synonymous: nucleotide change that specifies the same amino acid

• Missense: single nucleotide change that specifies a new amino acid (via transitions or transversions)

• Nonsense: point mutation that replaces a codon with a stop codon

• Mutations affecting mRNA processing: abolish/create alternative splice sites → altered splicing pattern

What are dynamic mutations?

Amplifications of simple trinucleotide repeats in coding or untranslated regions.

What are deletions, insertions, and rearrangements?

Changes that may cause frameshift mutations if not in multiples of three → create an altered reading frame and functionally altered protein.

What are the functional consequences of mutations?

• Gain-of-function = overproduction or inappropriate production of protein

• Loss-of-function = reduced protein production (often sufficient if one normal allele remains)

• Haploinsufficiency = 50% of protein is insufficient for function

• Dominant negative = mutated protein inhibits product of normal allele in heterozygosity

• Lethality = mutation results in organism death

What are germline mutations?

Mutations inherited from parents or de novo mutations transmitted to offspring.

• de novo germline mutations are very rare

What are somatic mutations?

Mutations not transmitted to the next generation. They occur in proliferative tissues (e.g., epithelial, hematopoietic) and are frequent in cancers.

Why are somatic mutations typically undetected?

Sequencing usually uses DNA extracted from millions of cells, masking individual mutations.

What is a genetic polymorphism?

A mutation with frequency >1% of all alleles in a population, regardless of type, size, effect, or location.

What are the 4 major types of polymorphisms?

• Single nucleotide polymorphisms (SNPs)

• Insertion-deletion polymorphisms (indels)

• Copy number variants (CNVs)

• Inversion polymorphisms

What are single nucleotide polymorphisms (SNPs)?

• 1 BP is changed

• Average of 1 per 1000 bp (~5–10 million per genome).

• Generally do not affect phenotype

• Most common at CpG sites, with a 25× higher rate (hotspots).

• About 100,000 SNPs are in protein-coding regions

What kinds of SNPs exist in coding regions?

Synonymous and nonsynonymous (leading to protein variants).

What are insertion-deletion polymorphisms (indels)?

Variants up to 1000 bp.

What are simple indels?

One or more DNA base pairs are either added or removed from a DNA sequence.

• Second most frequent form of genetic variation in humans after SNPs

What are microsatellites (short tandem repeat polymorphisms, STRs)?

DNA regions with short sequences (2–4 nucleotides) repeated in tandem. The number of repeats varies between individuals, making them useful for DNA fingerprinting and family testing.

• Often create multiple alleles

How are STRs used in forensics?

To infer familial relationships by DNA fingerprinting (allele study at 13 loci).

What are copy number variants (CNVs)?

Variants in the number of copies of a gene someone has, can be up to thousands of copies. Can cause an altered dosage

What are inversion polymorphisms?

Sequence reversals from a few bp to Mb in size.

What causes inversion polymorphisms?

Sequence homology at edges, leading to homologous recombination.

Are inversions balanced?

Yes, usually no loss or gain of DNA.

What are the steps in SNP detection?

• Discovery → whole genome/exome sequencing and comparison to reference sequence

• Validation → replication assay to exclude sequencing errors; testing in larger populations

• Screening → analyze thousands of SNPs per individual across populations using high-density DNA arrays (SNP arrays)

What is the goal of genetic analysis?

To establish a catalogue of all known human genes and variants and their locations.

How many human variants are catalogued?

Tens of millions, in different populations.

What are the main methods for mapping human disease variants?

Linkage analysis (family-based), association analysis (population-based), and genome sequencing.

What are WGS and WES?

Whole Genome Sequencing and Whole Exome Sequencing.

What is a Genome-Wide Association Study (GWAS)?

A molecular technique analyzing hundreds of thousands to millions of genetic variations simultaneously to determine associations between loci and phenotypes.

What is the strength of candidate gene association studies?

Greater power, but they rely on prior knowledge.

What is the strength of GWAS?

Finds unbiased susceptibility variants for complex traits without prior hypotheses about gene function.

What is the standard study design for GWAS?

Case-control design (matched or unmatched).

What is SNP imputation?

Using genotyped SNPs to infer variants on neighboring SNPs based on reference genomes (e.g., HapMap).

What is linkage disequilibrium?

Non-random association of alleles at linked loci.

Certain genetic variants are inherited together more often than by chance, usually because they are close together on a chromosome (linked)

What are haplotypes?

Sets of closely linked SNPs on the same chromosome that tend to be inherited together.

What is a Manhattan plot?

A scatter plot of association between p-values (y-axis) and chromosomes (x-axis).

Why are traditional p-value thresholds (e.g., 0.05) not suitable for GWAS?

Because GWAS test hundreds of thousands of markers simultaneously.

What threshold is used in GWAS for statistical significance?

p = 5 × 10⁻⁸.

What are the possible meanings if a SNP is significantly associated with a phenotype?

• A causal relationship between SNP and disease

• The SNP is a marker in LD with a causal locus

• A false positive association

What are clinically functional variants?

• Risk variants (increase risk of disease)

• Protective variants (lower risk of disease)

What is the “risk of disease” variant definition?

Having the variant increases likelihood of disease diagnosis.

What is 23andMe?

A direct-to-consumer company testing genome-wide polymorphisms.

What are problems with DTC testing?

Privacy concerns and lack of regulation.

What kinds of traits does 23andMe test for?

Cilantro aversion, hairline, photic sneeze reflex, caffeine metabolism, hair curliness, bitter/sweet taste, newborn hair amount, earlobe type, muscle composition, eye color, dimples.

What carrier statuses does 23andMe test?

Cystic fibrosis, BRCA1, sickle cell anemia, glycogen storage disease, maple syrup urine disease, Sjögren’s syndrome.

What ancestry information does 23andMe provide?

Ancestral composition, maternal/paternal lineage.

What pharmacogenomics does 23andMe test for?

Drug response.

What does the AAO recommend for genetic testing in eye disease? (Don’t need to memorize this)

• Offer testing if causative genes are known

• Use approved labs with peer-reviewed data

• Provide all reports to patients

• Avoid DTC kits

• Order the most specific test based on clinical findings

• Avoid repeat testing unless a clear treatment plan exists

• Do not test asymptomatic minors unless all parents consent or there is justifiable cause

What are limitations of gene-disease associations?

• Not relevant for all patients

• Environmental roles are unknown and hard to predict

• Often only prognostic value (risk can change with confounders)

• Combined risk from multiple SNPs is hard to calculate