Article Question Responses

1. step 1. splitting participants into groups

- the article did this by splitting participants into 3 populations samples based on their descent: Afro-Caribbean group, Afro-German group, and a healthy European expatriates, they were then screened for sickle cell disease and beta-thalassaemia by hemoglobin electrophoresis. There was then a 4th group called HbSS which were affected British individuals with sickle cell anemia

- in the QTL paper, mice were collected and tested for their PPI responses and then split into groups based on high or low response levels

step 2. phenotyping

- in the QTL paper, they kind of put steps 1 and 2 together by splitting participants into groups based on their phenotyping results which was their PPI responses

- in the article the participants were phenotyped based on their levels of fetal hemoglobin, and the F cell levels were determined by flow cytometry. The HbSS patients were routinely measured for HbF levels in the clinic

step 3. genotyping

- in the QTL paper, they collected DNA markers called "microsatellites"  which are short stretches of repeat DNA sequences with high mutation rates. this was another way to assign individuals to different groups based on what microsatellites were present in their DNA. used computational model to detect QTL signals across whole genome to identify potential contributions to different PPI responses

- in the article, geneotyping was performed by PCR/restiction enzyme analysis and was used for all HMIP-2 markers. This genotyping was used to find different HMIP-2 markers and haplotypes related to these different ethnic groups.

step 4. statistical analysis

- in the QLT paper, computational models were used to extract more detailed information on possible genetic contributions to PPI responses. Once the gene was identified, they tested the transcription levels for that gene in the different groups of mice. The researchers then performed a complementation test to provide evidence that their gene was one of the genes responsible for the PPI-QTL signal on chromosome 10

- in the sickle cell article, they ran statistical analysis on the association of FC and HbF traits with HMIP-2 marker alleles and investigated the linkage disequilibrium between markers and marker haplotypes. They also calculated the effective number of haplotypes.They then tested the haplotypes (11 SNPs) to see which ones had a strong association in all ethnic types in the study. By showing an influence of HMIP-2 locus to healthy and affected individuals of African origin, they believe this can lead to more powerful approaches for identifying the loci involved in the determination of HbF persistence. 

2. The article states that, "the number of HbF carrying cells, referred to as F cells...shows 89% heritability". This leads us to believe that VG would contribute more to the variance of the HbF and F cell phenotype. Since the variation is due to inheritance and genetics, if we were to draw a linear regression scatterplot for this trait, the slope of the line would be closer to 1. The offspring phenotype is going to more closely reflect the phenotypes of their parents. This leaves only about 11% of the variation of this phenotype up to environmental factors. This is understandable since sickle cell is an inherited disease.

3. There are 4 general causes of linkage disequilibrium (LD). They are physical linkage, selection, genetic drift, and population admixture. Physical linkage is when the loci are located on the same chromosome and near enough to each other such that cross-over between the loci rarely occurs. They are so close that they basically can't be separated through crossing-over. Selection is probably the most common cause of linkage disequilibrium, and most likely plays a role in our paper. This is when alleles at one locus affect the phenotype at a second locus. This means that one locus will affect the frequency of a second locus. If the first locus is advantageous, it will be selected for, and no matter if the second allele is advantageous or disadvantageous, it will also be selected for. Genetic drift leading to LD is when sampling error a mutation at one locus creates coupling of the new, mutated allele with specific-alleles at physically-linked loci. So this cause plays back into the first cause, which was physical linkage. This just makes new alleles linked because one of the alleles is mutated. The final cause of LD is population admixture, which also plays a role in our paper. This is when the combination of two populations, each of which are in linkage equilibrium (like the African population and the other ethnic populations), can create a single population that is in linkage disequilibrium. 

If we are trying to guess what happened in our article to make HMIP-2 locus in LD, I would say in the African population, a mutated HMIP-2 locus probably became advantageous. This is the locus that influences your number of F cells, your F cells are what carry HbF which is what sickle cell patients tend to have higher levels of. So the African population was probably in linkage equilibrium with a higher frequency of HMIP-2 mutated allele and then population admixture probably occurred with Europeans or people in the Caribbean, where HMIP-2 is at linkage equilibrium for a normal allele and then population admixture occurred. 

4. Our focal disease, malaria, is a disease which infects red blood cells and that is the site were they multiply within the body. After multiplying the disease can then destroy the red blood cells and lead to anemia. These also results in large amounts of free hemoglobin being released into the bloodstream. Malaria is a prominent disease in many African countries. At some point, there was probably a mutation in the SNPs at the HMIP-2 locus that lead to mutant haplotypes that led to sickle cell anemia. This turned into an advantageous trait because the malaria parasite cannot affect sickle shaped cells. This would lead to either a cure for the disease or at least a less severe form of the disease. Since less blood cells would be affected, less red blood cells would be destroyed and would lead to less free hemoglobin in the blood and better circulation of oxygen to the body. 

Bonus: The functional difference between HbF and HbA is that HbF binds less tightly to 2,3-BPG than does HbA. 2,3-BPG is a molecule that binds to hemoglobin and lowers its affinity for oxygen and promotes oxygen release. Higher levels of 2,3-DPG in the blood allows the delivery of more oxygen to tissues even under low oxygen tension (high altitiudes). This difference between fetal and adult hemoglobin is what accounts for the leftward shift of the oxygen saturation curve of fetal Hb compared to adult Hb.