7. Genetic variation

What percentage of the human genome is identical to the human reference genome?

99.6% — approximately 0.4% differs between individuals

How many nucleotides differ between individuals?

Approximately 27,000,000 nucleotides

What are the sources of genomic variation?

Two copies of each nuclear chromosome with different alleles, mitochondrial DNA heteroplasmy, epigenetic variation, and post-zygotic variation

What is neutral genetic variation?

The vast majority of genetic variation that has no effect on phenotype, because the DNA code is redundant and variation does not always occur in exons

How does genetic variation occur?

Through DNA mutations (environmental mutagens, DNA polymerase errors), DNA recombination and meiosis, evolutionary pressure, and DNA repair failures

What are the two main categories of genomic variation by size?

Structural variants (>50bp) and small scale variants (<50bp)

What are examples of structural variants?

Inversions, copy number variants (CNVs), and minisatellites (10-50bp repeats)

What are examples of small scale variants?

Translocations, SNPs/SNVs, indels (small insertions or deletions), and microsatellites (short tandem repeats)

What are SNPs and how frequent are they?

Single nucleotide polymorphisms — occur approximately every 100bp and make up 90% of all DNA sequence variation

How frequent are indels in the genome?

Approximately 1 per kilobase

How many microsatellite sites are in the genome?

Approximately 1,000 sites

What proportion of all DNA sequence variation do SNPs represent?

90%

What was RFLP used for?

Original forensic investigations and identification of sickle cell anaemia

What are the negatives of RFLP?

Limited variation detectable and large amounts of DNA are needed

What is the RFLP process?

1) Digest DNA with restriction endonucleases and separate by size on agarose gel 2) Denature and transfer to membrane 3) Hybridise labelled probe 4) Wash and apply X-ray film 5) Develop film to reveal autoradiograph

Why are short tandem repeats (STRs) preferred over RFLPs?

STRs are more common, have greater discrimination power, and PCR amplification requires less DNA

What are allele specific oligonucleotides (ASOs) used for?

To detect SNPs using DNA primers specific for the SNP sequence under stringent annealing conditions

How do SNP microarrays work?

DNA oligonucleotides are fixed to a plate, fluorescently labelled patient DNA is incubated and washed, then fluorescence is detected to identify SNPs

How many SNPs can a microarray assess simultaneously?

Up to 20,000 SNPs

What can pedigree charts tell us about a disease?

The inheritance pattern — whether it is sex-linked or autosomal, dominant or recessive

What are the confounding factors in mapping genetic disease?

Variable penetrance, non-Mendelian inheritance, de-novo mutations, and missing data

What are the criteria for a good genetic marker?

Clear Mendelian inheritance, easily and cheaply scored, highly polymorphic locus, and part of a panel of hundreds of markers spread across the genome

What is the purpose of genetic markers in disease mapping?

To track where the disease locus is — more markers means higher precision in identifying the disease locus

What are the steps in mapping a genetic disease?

1) Identify affected individuals 2) Pedigree analysis 3) Map genetic markers to disease state 4) Identify disease locus 5) Identify putative disease genes 6) Sequence genes to identify mutations

What was the PARK6 locus study about?

Mapping autosomal recessive juvenile Parkinsonism (ARJP) in a Sicilian family where >50% of cases had no Parkin mutations

What methods were used in the PARK6 study?

DNA extracted from leukocytes, 400 microsatellite markers genotyped across 22 autosomes, amplified by PCR, and separated by gel electrophoresis

What chromosome locus was identified in the PARK6 study?

Chromosome 1p35-36 — the PARK6 locus

What questions must be answered once a sequence variant is found in a locus?

Is it in a gene plausibly linked to the disease? Is it expressed in the affected tissue? Do unrelated affected individuals have mutations in the same gene?

What conditions make locus identification work well?

Diseases caused by loss of function mutations, autosomal dominant diseases, conditions with high penetrance, and a candidate locus that isn't too large

What is a monogenic disease?

A disease following a direct gene → protein → phenotype pathway

What is a complex disease?

A disease where multiple genes and proteins interact with environmental factors to produce a phenotype

What is mitochondrial DNA heteroplasmy?

The presence of more than one type of mitochondrial DNA within a cell, contributing to genomic variation