Analyses for APC resistance and FV Leiden have made their way into clinical medicine and are now performed routinely all over the world. I have been asked to write a personal historical annotation about the discovery of APC resistance, the early research activities and the rapid progress in this field.
Individuals from families with protein S, protein C, or antithrombin deficiency but also APC resistance had a higher risk for VTE when they inherited combined defects rather than only one defect Koeleman et al, , van Boven et al, Thus, the penetrance of thrombosis increases in these protein C deficient families after introduction of the factor V Leiden allele in the pedigree Brenner et al, Carriers of combinations of defects also presented with thrombosis earlier in life and more frequently.
As a result, thrombophilia was then suggested to be an oligogenetic disease in which inherited predisposition results from 2 or more mutations in genes involved in blood clotting Miletich et al, These findings reaffirm that genetic risk factors do play an important role in the development of VTE. An important question in the field remains whether there are a multitude of genetic risk factors that remain to be identified.
After establishing high levels of heritability in many complex diseases, including VTE, investigators concluded that many genetic risk factors remained to be discovered. New hypotheses were formulated to explain in which part of the genome sequence these missing genetic risk factors were to be found.
The CDCV hypothesis states that several common allelic variants - with appreciable frequency in the population and low penetrance - would account for the genetically determined variance in disease susceptibility of complex diseases like VTE. Other premises of this hypothesis are that the original mutation arose more than , years ago and that the model included absence of selection for or against these variants to make it possible for the variants to persist at a high frequency in the population.
Evolutionary data suggests a proliferation of the human population from a rather small group of founders to 6 billion plus and this would be supplementary evidence for the CDCV theory.
Before the introduction of the CDCV hypothesis, new genetic determinants were investigated by linkage studies. Whole genome linkage studies have been performed in family studies with mini- and microsatellite markers, but later also with single nucleotide polymorphisms SNPs. Linkage analysis assumes that many families share defects in the same locus, while there often is considerable locus heterogeneity in complex diseases, which will dilute linkage signals.
Therefore, association studies of unrelated individuals using genotyping of a large set of single nucleotide polymorphisms SNPs are more appropriate to use for complex diseases. This approach is directly based on the CDCV hypothesis. DNA sequences are inherited in blocks with high linkage disequilibrium. The pattern of SNPs in a block is called a haplotype. Especially the HapMap data has been employed as a source of information about haplotypes in different populations and tagSNPs.
A GWAS study should be designed very carefully to prevent bias and other problems in the subsequent analyses. The study populations most used in GWASs are case-control studies.
Cases and controls should have the same ethnicity and geographical background to avoid false positive results due to population-stratification. For case inclusion, strict criteria should be taken into account to prevent inclusion of phenocopies within the study population. Type I errors, i. In GWAS studies, multiple tests are performed and the significance levels should be corrected for these multiple comparisons.
The Bonferroni correction might be too strict when tested SNPs are in linkage disequilibrium and therefore should not be considered as independent comparisons. Type II errors, false negative results, can be avoided by using large sample sizes. Positive results should be replicated in at least 2 other populations. The effect size and significance of a positive result is often overestimated in the first study.
As a consequence, to replicate a claim, the sample size of the replication studies should be therefore larger than the original GWAS study. The CDCV hypothesis was not accepted by the whole field. Opponents argued that in many complex diseases already a spectrum of disease associated rare variants had become known in direct contradiction of the CDCV hypothesis which states that only a few variants would account for the risk in complex diseases Pritchard, The rare variants would be more important because they were more likely to be functional or have phenotypic effects Gorlov et al, , Pritchard, , Schork et al, Also the observation of familial clustering of complex diseases strengthened the CDRV hypothesis Schork et al, This hypothesis gained increasing support when the genetic variation found with GWAS studies explained collectively only a small fraction of the heritability of any disease in the population.
GWAS studies are not powered for the detection of rare variants. The only strategy available to identify such rare variants is to sequence DNA directly, either in candidate genes or whole genome. To perform large studies with conventional Sanger sequencing is very costly, time consuming, and impossible in practise. With the introduction of next generation sequencing technologies, high-throughput sequencing of many genes became feasible and at a reasonably price. Next generation sequencing can be used for de novo sequencing and re-sequencing purposes.
For humans, re-sequencing is used because the reference sequence is already known from the Human Genome project Collins et al, and will be further improved by the Genomes Project The Genomes Project Consortium, , which just finished the pilot study at the end of Next generation sequencing was first used for targeted re-sequencing of candidate genes in just a few subjects. Nowadays, also whole exome sequencing can be performed for a reasonable price, although the samples sizes in most studies are still limited.
The best, non-hypothesis driven, method would be whole genome sequencing, but this is still quite expensive especially when using large sample sizes. Targeted re-sequencing of candidate genes was initially executed by first amplifying the target sequences by PCR and then sequence these PCR products with a next generation sequencer.
The PCR steps are very time consuming and to accelerate the whole sample preparation process, a new method was developed: target enrichment. This method uses predesigned probes to enrich the DNA for the selected target genes and wash away remaining non-selected DNA sequences.
Next generation sequencers are improving constantly and are generating more and more reads with increasing read length. As a consequence, the total data output from one sequencing run is increasing and all these data need to be analyzed. The data analysis of the sequencing reactions remains a challenge.
Especially the distinction of sequencing errors from real mutations is difficult and is best served by using a high coverage level, i. However, PCR errors that originated in the sample preparation phase cannot always be distinguished from real mutations and this problem is not solved by using higher coverage levels. Therefore, findings from next generation sequencing still have to be confirmed with Sanger sequencing. In the next three sections, some genome wide linkage analysis studies, GWAS studies, and high-throughput sequencing studies in the field of thrombophilia will be discussed.
High-throughput sequencing results for VTE are not available and therefore we will discuss some results from other complex diseases. Several genome wide linkage studies have been performed for venous thromboembolism. Twelve of these families were selected through probands with idiopathic thrombophilia. The other 9 families were selected irrespective of any phenotype.
Several genome scans were performed in the GAIT study. For the first scan microsatellite markers were genotyped Soria et al, while microsatellite markers were used in the second scan Lopez et al, The investigators focussed on associations between genetic markers and intermediate phenotypes of VTE, like lipoprotein a levels Lopez et al, , factor XII levels Soria et al, , total plasma homocysteine Malarstig et al, , and C4BP plasma levels Buil et al, He published his work in Norway in , but his results were not widely known until after the war, when he was able to publish in The Lancet.
However, its deficit is described in by Olav Egeberg. Armand Quick. This test is used to help detect and diagnose a bleeding disorder or excessive clotting disorder. The doctor Karl Link and collaborators identify the molecule responsible in the clover: dicoumarol, the antidote for which is vitamin K.
The anticoagulant isolated independently by Maurice Doyon in also proved to be heparin. In this paper, Morawitz described four coagulation factors: fibrinogen, prothrombin, thrombokinase, and calcium.
Morawitz was a pioneer in the study of coagulation, and his landmark paper is still regarded as a springboard for further study of the physiology of blood; He also pioneered blood transfusion, initially without the benefit of blood typing, and studied angina and the use of quinidine as an antiarrhythmic.
He wrote about the wife of a pharmacist who, after a difficult delivery, developed swelling and pain of the right leg, extending from the knee to the hip, with no inflammation and discoloring of the skin.
Wiseman surmised that thrombus formation was caused by a systemic alteration in circulating blood, thereby pioneering the concept of hyper-coagulability. He also he established a symptomatic treatment of thrombosis with the use of a lace stocking as a means of compression.
It describes the case of a young man from Normandy named Raoul who, at the age of 20, developed unilateral oedema in the right ankle that subsequently extended up to the thigh. The case does not give enough details to know for sure. The mutation is less common in other populations. A particular mutation in the F5 gene causes factor V Leiden thrombophilia. The F5 gene provides instructions for making a protein called coagulation factor V. This protein plays a critical role in the coagulation system, which is a series of chemical reactions that forms blood clots in response to injury.
The coagulation system is controlled by several proteins, including a protein called activated protein C APC. APC normally inactivates coagulation factor V, which slows down the clotting process and prevents clots from growing too large.
As a result, the clotting process remains active longer than usual, increasing the chance of developing abnormal blood clots. Other factors also increase the risk of developing blood clots in people with factor V Leiden thrombophilia.
These factors include increasing age, obesity, injury, surgery, smoking, pregnancy, and the use of oral contraceptives birth control pills or hormone replacement therapy.
The risk of abnormal clots is also much higher in people who have a combination of the factor V Leiden mutation and another mutation in the F5 gene. Additionally, the risk is increased in people who have the factor V Leiden mutation together with a mutation in another gene involved in the coagulation system.
The chance of developing an abnormal blood clot depends on whether a person has one or two copies of the factor V Leiden mutation in each cell. People who inherit two copies of the mutation, one from each parent, have a higher risk of developing a clot than people who inherit one copy of the mutation.
Considering that about 1 in 1, people per year in the general population will develop an abnormal blood clot, the presence of one copy of the factor V Leiden mutation increases that risk to 3 to 8 in 1,, and having two copies of the mutation may raise the risk to as high as 80 in 1, Genetics Home Reference has merged with MedlinePlus.
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