HUGE Lecture 6 09/10/26

What was used to discover disease genes before the HGP

linkage analysis in large disease-affected families

What was used to discover disease genes after the HGP

exome/genome sequencing and association mapping across populations

What is the goal of meiosis

Make haploid gametes

Mendel’s laws of inheritence

Law of dominance, segregation, and independent assortment

Segregation

during gamete formation, paired alleles for a trait segregate such that each gamete receives only one allele (meiosis 1 and 2)

Independent assortment

during gamete formation, the alleles of two (or more) different genes get sorted independently of one another; traits are inherited independently

Gene link

Genes/loci that are located on the same chromosome are not always free to participate in independent assortment

Complete linkage

two genes/loci are located so close together on the same chromosome that they are practically never separated by crossing over (always inherited together)

Incomplete linkage

genes/loci are on the same chromosome but are far enough apart that crossing over can sometimes happen between them, leading to the possibility of different allele combinations in offspring

Homologous recombination

Genetic recombination (crossing over) in which nucleotide sequences are exchanged between two similar or identical molecules of DNA (homologous chromosomes)

When does homologous recombination occur

prophase 1 of meiosis 1

What is the expected recombination frequency between two genes if they are considered unlinked

50%

Expected recombination frequency of independent assortment

50%

What recombination frequency indicates linkage between the genes

below 50%

Centimorgan

unit of measurement used to express genetic distance and linkage in chromosomes

How do centimorgans work

pairs of genes/loci that recombine 1% of the time are 1cM apart

Are genetic distance and physical distance the same and why

no, due to recombination hotspot that leads 2 genes that are physically close to look far

How to calculate gene distance

recombinants/total * 100 = centimorgans

Linkage disequilibrium (LD)

Measure of how often alleles (DNA markers) occur together in a population (more often than would be expected by chance)

What does LD describe

non-random association between alleles of genetic loci that lie in proximity to each other

LOD score (logarithm of odds)

statistical estimate of whether two genetic loci are physically near enough to each other on a particular chromosome that they are likely to be inherited together

What LOD score indicated two genes are located close to one another

3 or higher (1000:1 of the two genes are linked)

What was necessary for mapping human disease genes without a genome reference sequence

genetic markers

What size family is needed to use linkage analysis to identify human genes

large family

Steps in linkage analysis to identify human disease genes

1. Disease phenotype segregates in a family

2. Identify known common genetic marker linked to disease phenotype expression

3. Given LD, disease causing variant is likely to reside near the genetic marker

What does RFLP stand for

Restriction Fragment Length Polymorphism

What is an RFLP

DNA variation that causes DNA fragments of different lengths after being cut by a restriction enzyme

What did RFLPs allow researchers to do

Allowed researchers to identify the general location of potential disease genes on chromosomes

How do RFLPs work for locus identification

1. Restriction enzymes cut DNA at specific sequences

2. Gel electrophoresis

3. Southern blotting with probe

4. Assess banding pattern

Chromosome walking

technique used to find a specific gene when you know approximately where it is on a chromosome, but don't know its exact location

Chromosome walking steps

Walking across the chromosomes in overlapping chunks of sequences to identify variants that may be deleterious

What are contributors to successful linkage analysis

• Lack of genetic heterogeneity (phenotype caused by one genetic locus)

• Phenotype is specific

• Families well phenotyped

• High penetrance

When is genome/exome sequencing employed

when a specific gene, or set of genes, cannot be implicated based on clinical presentation

Exome sequencing

sequencing just protein coding regions

Gene panel

sequencing specific genes based on phenotypic expression

Autosomal dominant inheritance

Single variant- one copy of a mutated gene is needed to cause the condition

Autosomal recessive inheritance

compound heterozygous or homozygous- person must inherit two non working copies of a gene (one from each parent)

Autozygosity mapping

genetic technique used to find the genes responsible for autosomal recessive disorders by locating identical DNA segments inherited from a common ancestor

How is autozygosity mapping conducted

Using whole exome, whole genome, or chromosomal microarray data

What types of diseases are prevalent in consanguineous families

recessive diseases

How can you confirm gene is associated with disease (patients)

Substantial phenotype overlap between affected patients

How can you confirm gene is associated with disease (gene specificity)

Consider phenotype specificity (potential for other genes to be associated with similar phenotypes?)

How can you confirm gene is associated with disease (variation)

Gene intolerant of genetic variation (especially for dominant disease)

How can you confirm gene is associated with disease (frequency)

Population frequency data (disease associated variants are often rare)

What does GWAS stand for

genome wide association study

What is GWAS

commonly used approach to identify genes associated with a range of human traits, including most common diseases

What do GWAS studies require

Requires large cohorts of study participants (affected and unaffected individuals)

SNP array

thousand to over a million genetic markers