Mapping Mendelian Disease

Question 1

What are the different types of genetic disease?

Answer 1

• Monogenic/Mendelian - Disease caused by the effect of a single gene with little impact from the environment
• Polygenic/Non-mendelian - Disease caused by the effects of multiple gene with each individual gene having a small contribution to the development of the disease
• Multifactorial - Disease caused by the effect of multiple genes as well as environmental factors

Question 2

What are the 3 methods of gene mapping that can be used when trying to identify a genetic variant of a candidate gene that causes a particular mendelian disease?

Answer 2

• Homozygosity mapping (Autozygosity mapping)
• Linkage analysis
• GWAS (genome-wide association studies)

Question 3

What is Homozygosity mapping (Autozygosity mapping)?

Answer 3

• A method used on consanguineous families (families who descend from a common ancestor) to find the location of a particular disease-causing gene within the genome.
• By looking for regions of homozygosity within the genome of affected family members that aren’t present within unaffected family member genomes you can locate areas where the disease-causing gene loci is.

Question 4

Explain the concept of “Autozygosity”

Answer 4

Autozygosity occurs when individuals are homozygous at a particular gene loci due to inheritance from a common ancestor

Question 5

What is linkage analysis?

Answer 5

• A method used to used to map the location of a particular disease-causing gene within the genome.
• It does this by comparing the co-inheritance/co-segregation of genetic markers with a disease gene between affected family members and unaffected family members

Question 6

What is genetic linkage?

Answer 6

The tendency of alleles located at gene loci very close to each other on a chromosome to be inherited together

Question 7

Why is it that genes located on adjacent loci are more likely to be inherited together compared with genes on loci that are far apart?

Answer 7

• Genes that are on adjacent loci are a lot more likely to be on the same sister chromatid that gets exchnaged during homologous chromosomes during recombination
• They get inherited togetehr because they’re still together just on the the other homologous chromosome to the one they originated from

Question 8

What is a haplotype?

Answer 8

A haplotype is an area of a chromosome in which multiple alleles at multiple gene loci are linked to each other

Question 9

How is a haplotype used to identify the location of a disease causing gene within the genome?

Answer 9

• Haplotypes can be produced for every family member within a pedigree by producing genotypes for marker genes across the genome for every family member
• For each marker gene genotype for every individual you identify the maternal and paternal alleles
• This allows you to identify multiple alleles, chromosomal segments, that every affected family member has inherited
• The disease gene therefore is likely to be within one of the chromomsomal segments identified

Question 10

What is a genetic marker?

Answer 10

• A gene within a known location on a chromosome that is used to identify the location of a disease-causing gene
• Two types of genetic marker used are:
  
  • Microsatellite marker
  • SNP marker

Question 11

How are genetic markers used to to identify the location of disease-causing genes?

Answer 11

• Look at inheritance patterns of particular marker genes within the genome of individuals affected by disease and compare them with the inheritance patterns of the same markers of family members without the disease
• If a genetic marker is linked to a disease gene it will be more likely that the gentic marker is co-inherited with the disease gene in all affected individuals

Question 12

Briefly explain the process of linkage analysis?

Answer 12

1. Produce a pedigree diagram for the families that you’re studying
2. Take a blood sample from each family member within the pedigree and perform a SNP microarray on each sample - this is used to sequence the entire genome and produces data based on the location of every known SNP within each chromosome.
3. Generate a file with your pedigree information plus the genotyping data from the microarray
4. Put all of the information within the file into a linkage programme, e.g. merlin which will then produce a LOD score for every single marker gene on every single chromosome within the genome

Question 13

What are the 2 types of linkage analysis that can be used to identify a disease-causing gene?

Answer 13

• Non-parametric linkage analysis
• Parametric linkage analysis

Question 14

What is non-parametric linkage analysis?

Answer 14

• Technique used to study the probability that particular alleles inherited by affected individuals within a family are identical by descent (IUD)
• Areas where alleles of the affected individuals are identical by descent are areas where the disease-causing gene is likely to be located.

Question 15

What does it mean when it’s said that an allele/gene is “identical by descent?”

Answer 15

It means that two or more members of the same family have inherited the allele from a common ancestor without recombination occuring.

Question 16

Why doesn’t non-parametric linkage analysis make any assumptions about the family it is used on?

Answer 16

• Because it isn’t based on a genetic model.
• You don’t produce a pedigree for the family so you don’t know the pattern of inheritance of the disease gene within the family

Question 17

What is parametric linkage analysis?

Answer 17

Technique used to study the probability that a disease-causing gene is linked to a particular genetic marker

Question 18

What are the differences between parametric and non-parametric linkage analysis?

Answer 18

• Parametric linkage analysis does apply assumptions because you know the inheritance pattern of the family
• During parametric linkage analysis you do produce a pedigree of the family that you’re studying
• Results from parametric linkage analysis also highlight regions where the genotypes follow the imposed inheritance pattern rather than just highlighting the LOD scores for the marker genes across the genome

Question 19

What is a LOD score?

Answer 19

• A LOD (logarithm of the odds) score is a statistical estimate of whether 2 genes or a gene and a disease-causing gene are linked.

Question 20

What LOD score is generally considered to show evidence of significant linkage and what LOD score is generally considered to show evidence against linkage?

Answer 20

• LOD scores > 3.0 are taken as significant evidence for linkage
• LOD scores < -2.0 show significant non-linkage
• LOD scores between -2 and 3 are inconclusive

Question 21

What exactly does getting a high LOD score indicate?

Answer 21

• Indicates that alleles within that particular position on the chromosome for affected individuals are the same, but different to unaffected individuals.
• Fact that alleles for affected individuals are the same means that affected individuals more likely to inherit these alleles as well as the disease-causing gene
• In otehr words these alleles are inherited with, or linked to, the disease-causing gene

Question 22

Once you have identified an area on the chromosome that is significantly linked to the disease gene what steps do you then take to identify one candidate gene that may be disease-causing?

Answer 22

• You use whole exome sequencing to sequence every single gene within the region of linkage of an affected patient
• Whole exome sequencing will identify previously unidentified variants within a particular genes within the linakge that may be disease-causing
• This will narrow down the no. of candidate genes to about 1 or 2

Question 23

Apart from whole exome sequencing what other sequencing techniques can be used to reduce the no. of candidate genes within an area of linkage?

Answer 23

• Sanger sequencing (barely used today)
• Whole genome sequencing (more time consuming than whole exome sequencing)

Question 24

After using sequencing to lower the no. of candidate genes that contain variants that may be disease-causing down, how can you validate that these variants do indeed cause disease?

Answer 24

• You use in vivo and in vitro methods e.g. cell culture or producing disease models using animals

Question 25

Give an example of an in vivo experiment that was used to validate whether or not a variant within a gene was disease-causing after being identified using linkage analysis

Answer 25

• Parametric linkage analysis of the family of a girl with primary lymphoedema showed linkage to a set of genes in chromosome 18
• Sequencing then identified variants in CCBE1 gene that may be causing disease.
• To validate this study was done into a mutant zebrafish which was full of fluid
• Study showed reason why this mutated Zebrafish was full of fluid was because it had a mutation within the CCBE1 gene
• This in vivo experiment proved that the variant (mutation) within the CCBE1 gene does indeed cause lymphoedema.

Question 26

What is primary lymphoedema?

Answer 26

• A chronic oedema caused by a developmental abnormality (birth defect) of the lymphatic system

Question 27

Patients with primary lymphoedema are separated into 5 different groups based on their clinical features. In one of these groups there’s a disease called Generalised lymphatic dysplasia (Hennekam syndrome), What are some of the clinical features of this disease?

Answer 27

• Peripheral lymphoedema - arms, legs, face
• Mild developmental delay
• Intestinal lymphangiectasia - dilatation of the intestinal lymphatics and loss of lymph fluid into the gastrointestinal tract

Question 28

In another one of the 5 primary lymphodema groups there’s a disease called 4-limb lymphoedema. What are some of the clinical features of 4-limb lymphoedema?

Answer 28

• Swelling in the legs and arms
• Autosomal dominant
• Pubertal/adult onset