mapping mandelian disease

Question 1

What are the classification of mendelian disease?

Answer 1

1. monogeninc- caused by single gene with no impact from environment- eg. PKD
2. non- mendelian/polygenic: disease or traits caused by many different genes, each having small individual impacts- eg. psoriasis
3. multifactorial: caused by interaction of multiple genes and multiple environmental factor- E.g heart disease.

Question 2

How do we study genetic disease?

Answer 2

1. gene identification by gene mapping :
   - homozygosity mapping
   - linkage analysis
   - GWAS
2. Find disease-causing mutations: sequencing
3. Prove they cause disease: using in silico, in vitro and in vivo tools.

Question 3

What is the principle of genetic linkage?

Answer 3

• genetic linkage is the tendency for alleles at neighbouring loci to segregate together at meiosis
• therefore to be linked, two loci must lie very close together
• A haplotype defines the multiple alleles at linked loci. They mark chromosomal segments, which can be tracked through pedigrees and populations.
• cross overs (recombination events) during meiosis are more likely to occur between loci separated by some distance than those close together.

Question 4

How can we use information about linked alleles to identify disease causing genes?

Answer 4

• if an allele is linked to a disease locus, the same allele will be inherited by two affected relatives more often than expected by chance.
• if the allele and the disease locus are linked all the affected individuals in a family are more likely to inherit this haplotype block.
• if the alleles and disease and locus are unlinked to the disease locus, the affected individuals in the family are less likely to inherit the marker alleles.

Question 5

what is the goal of linkage analysis?

Answer 5

• find genomic regions linked to disease
• gene mapping tool
• using observed loci(alleles) to draw inferences about an unobserved locus (disease gene)
• family based design (from few large families to many small nuclear or sibling pairs)

Question 6

What is the method of linkage analysis?

Answer 6

1. take a pedigree and get as many DNA sample as possible
2. generate a genotyping data for pedigree
3. physical and genetic distribution of markers on a genotyping array (600SNP markers distributed uniformly across human genome)
4. generate file using genotyping data from microarray (how maternal/paternal marker alleles are inherited - homozygous/heterozygous) and pedigree information (one family, gender, etc)
5. run a linkage process
6. Can apply assumptions using parametric analysis (homozygous recessive, etc- imposes rules about inheritance and disease frequency) or without assumption using NPL(non- parametric linkage testing looking for identity by descent -IBD)

Question 7

What do parametric test results show?

Answer 7

• highlights regions with high LOD scores where all affected are equal but different to those unaffected and all genotype follow imposed inheritance pattern.
• LOD score > 3.0 are taken for significant evidence of linkage. Disease gene likely to be found between two markers found in the linkage peak of graph.
• below -2 show significant non-linkage. -Between -2 and 3 are inconclusive.

Question 8

What is primary lymphodema?

Answer 8

• chronic oedema caused by developmental abnormality in lymphatic system
• progressive disease
• phenotypes vary(age of onset, site, inheritance patterns, associated features, genetic causes)

Question 9

Why is it important to study primary lymphodema using traditional linkage analysis and sanger sequencing?

Answer 9

• no cure for it
• it helps identify cause of autosomal recessive form of primary lymphoedma
• treatment is manual lymph drainage using massage, bandaging and compression stocking.
• more similar the clinical symptoms the easier it is to find genetic marker between patients. so flow chart of symptoms can lead to a possible disease causing gene.

Question 10

What is an example of generalised lymphatic dysplasia?

Answer 10

=> Hennekam syndrome (HS)

• antenatal hydrops with asites and pleural effusions
• odematous at birth
• intestinal lymphangiectasia
• peripheral lymphoedema; arms , legs, face
• mild developmental delay

Question 11

What did study of family pedigree of lymphatic dysplasis find?

Answer 11

• assumed autosomal recessive as parents were unaffected
• genotype data generated
• maximum LOD was 2.1 so not significant but still 2 regions identified on graph.
• peak linked to chromosome 18
• recombinant found in 3 affected children (which were AA for mutant allele, the unaffected were TA or TT)
• 130 candidate genes in region. Sanger sequencing identified mutated gene(CCBE1) that was homozygous in patient but heterozygous in parents .
• proof of pathology found in same mutated gene in zebrafish caused absence of trunk lymphatic vessels.

Question 12

What is 4 limb lymphoedema?

Answer 12

• autosomal dominant
• pubertal/adult onset
• associated with venous incompetence
• no other abnormalities.

Question 13

What did 4-limb lymphoedema of 2 families study find?

Answer 13

• genotype generate, linkage analysis linked disease to markers in one region with 173 candidate genes. NGS of whole exome showed 2 gene mutations in the region. Sanger sequencing showed only one gene co-segregated with disease status.
• whole exome sequencing(WES) was carried out on DNA from 1 affected individual from family 1, which gave 2 hetrozygous unreported changes in the linkage region of chromosome 1.