Noah Fram

Biology 303 (Genetics) term paper

November 3, 2007

Association between environmental and genetic causes of cerebral hemorrhaging

There are a variety of diseases commonly ascribed to environmental causes, including such commonly known ones as obesity and hypertension, which are in fact largely the responsibility of genetic mutations. Cerebral hemorrhaging, or the rupture of capillaries supplying the brain with oxygen, is among the most deadly of these (3rd highest cause of death in the United States). The most commonly known form of it, stroke, often causes mental retardation and occasionally can result in severe mental impairment (inability to feed oneself, inability to use language, undeveloped or nonexistent motor control, etc.) Until the genomic era, the only method for stroke prevention was relative abstinence from alcohol, or at least refraining from over-indulgence.

While alcohol is still accepted as a major risk factor for stroke (8), twin studies and family aggregation analysis pointing to a genetic component soon led many biologists to start seeking a genetic mutation which would cause lesions in cranial blood vessels. They tended to focus on a specific type of cerebral hemorrhage, cerebral cavernous malformation (CCM), a known autosomal dominant Mendelian disorder, and soon pinpointed three genes keying for CCM: KRIT1 on 7q (7), MGC4607 on 7p (3), and PDCD10 on 3q (4). The KRIT1 region was detected first because most of the studies focused on Hispanics, who demonstrated a high expression of the CCM trait, and the vast majority of Hispanics with CCM have the KRIT1 mutation.

One of the chief problems with studying cerebral hemorrhaging is its all-too-frequent lethality. Because of this lethality, there have been a number of studies investigating certain mutations in model organisms, frequently zebrafish. Studying fish embryos is much simpler than mammalian ones because even in vitro, fish are free-swimming. Of course, fish and mammals have adapted to different environments, so their systems do not show as much conservation of human processes as, for example, mice. What makes zebrafish so common in studies of vascular development and integrity is that their genetic makeup in this area is remarkably similar to that of mammals (1).

David Buchner and his team conducted an experiment to determine which gene was causing one of a variety of types of cerebral hemorrhaging in zebrafish (1). While gene pathways such as Notch, TGFβ, and VEGF had already been identified as causing cerebral hemorrhaging (mutants mush for brains, bubblehead, migraine, gridlock, and leaky heart), no pathway had been labeled as causing the redhead strain.

The rhd (redhead) gene was mapped to a specific section of zebrafish chromosome 2 using bulked segregant analysis. After identifying the rhd nonrecombinant interval as lying between the D2Umi14 and D2Umi17 markers, they decided to focus on four of the seven genes present in this interval. Among these genes was pdcd10-like, which they chose for special investigation because its human ortholog, PDCD10, plays a major role in CCM. Direct sequence comparison between mutant and wild type embryos demonstrated that while pdcd10-like did not play any obvious role in the rhd phenotype, that two other candidate genes, golph4-like and pak2a both could be important. They finally pinpointed pak2a as the mutant gene through RT-PCR (real-time polymerase chain reaction), which when combined with pak2a’s unique position as within the refined nonrecombinant region between markers D2Umi17 and D2Umi18 clearly identifies pak2a as the gene responsible for rhd.

Pak2a’s human ortholog, PAK2, is also responsible for in vitro cerebral hemorrhaging. However, there is very little experimental research about the human PAK2 gene because in vitro hemorrhaging is lethal in humans. This is not necessarily true for zebrafish embryos, however. For this reason, most population-oriented experiments with cerebral hemorrhaging are done with model organisms. In fact, CCM is such a popular topic with stroke researchers just because it tends to strike in vivo, and is not universally lethal. Understanding mutations in the KRIT1 gene or its relations is not nearly as daunting a task as understanding a mutant which causes humans to die in embryo.

Genomics and proteomics have played a major role in making cerebral hemorrhaging easier to study without having to rely on organisms such as zebrafish for experimental data (5). For example, an Icelandic project involving 476 stroke patients used a whole-genome scan to identify linkage to genes on chromosomes 5q12 and 13q12-13 which have been named STRK1 and STRK2. Also, mutations in the genes for factor V Leiden and prothrombin apparently are potential risk factors for stroke, especially in women taking certain types of oral contraceptives. Some genetic factors affect different types of stroke in different ways: elevated serum cholesterol is positively correlated with ischemic (embolic) stroke risk but negatively with hemorrhagic stroke. Finally, some genes play double duty, as the same genes which can cause diabetes and hypertension also increase risk of stroke.

One of the most interesting points about the Hispanic-American population of diagnosed CCM patients is that it displays a rather pronounced founder effect. In other words, there is a high statistical probability that all or most of those cases are descended from a single, ancient mutation, in a fashion similar to Huntington’s disease. However, this does not hold true for other populations. In 1999, Sophie Laberge et al published an article in the European Journal of Human Genetics which described a genetic analysis of a group of 36 French families (6). Haplotype analysis of the families with the conditional linkage probability >.95 (shown at left) showed that none of them shared the Hispanic-American haplotype. Comparative analysis also demonstrated no evidence of a founder effect within this French population.

Examined from an historical perspective, this observation could lead to a variety of very important conclusions in understanding CCM. For example, given the extensive history of genetic mixing between the Spanish conquistadores and the South American natives (whose progeny now dominate the Hispanic-American population), a founder effect could imply that the CCM1 mutation was not present in the native population but was brought across the Atlantic by the Spaniards. Taking this one step further, perhaps that particular mutation was more common among Spaniards than the South Americans; this would not only explain, to some extent, the founder effect among Hispanic-Americans but also the lack of one among the French.

Essentially, genomic/proteomic analysis of potential risks for cerebral hemorrhaging and other disorders like it is a relatively new development, but one with a great deal of promise. Stroke itself does not require a genetic component but, like obesity, understanding the hereditary aspects of stroke could very well help scientists develop more effective preventative medications. In at least one case, research into risk factors associated with metabolizing stroke medication (CYP450 enzymes in particular can cause varied sensitivity to a stroke medication called warfarin) (5) could help doctors determine which drug in what dosage would be most appropriate for that specific patient.

BIBLIOGRAPHY

  1. Buchner, D. A.; et al. (2007) Pak2a mutations cause cerebral hemorrhage in redhead zebrafish. PNAS, 104(35), 13996-14001. National Academy of Sciences of the USA.
  2. Craig, HD; et al. (1998) Multilocus linkage identifies two new loci for a mendelian form of stroke, cerebral cavernous malformation, at 7p15-13 and 3q25.2-27. Human Molecular Genetics, 7, 1851-8. Oxford University Press.
  3. Denier, C; et al. (2004) Mutations within the MGC4607 Gene Cause Cerebral Cavernous Malformations. American Journal of Human Genetics, 74, 326-37. American Society of Human Genetics.
  4. Guclu, Bulent; et al. (2005) Mutations in apoptosis-related gene, PDCD10, cause cerebral cavernous mutation 3. Neurosurgery, 57(5), 1008-13. Congress of Neurological Surgeons.
  5. Gwinn-Hardy, Katrina; Dawson, Valina. (2004) Genomic-Proteomics and Stroke: Introduction. Stroke, 35(suppl 1), 2731-4. American Heart Association, Inc.
  6. Laberge, S; et al. (1999) Genetic heterogeneity and absence of founder effect in a series of 36 French cerebral cavernous angiomas families. European Journal of Human Genetics, 7(4), 499-504.
  7. Laberge, S; et al. (1999) Truncating mutations in CCM1, encoding KRIT1, cause hereditary cavernous angiomas. Nature Genetics, 23, 189-93. Nature America, Inc.

8.  Liu, Jin; et al. (2007) A βPix–Pak2a signaling pathway regulates cerebral vascular stability in zebrafish. PNAS, 104(35), 13990-5. National Academy of Sciences of the USA.

  1. Woo, Daniel; et al. (2002) Genetic and environmental risk factors for intracerebral hemorrhage: preliminary results of a population-based study. Stroke, 33(5), 1190-7. American Heart Association, Inc.