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Stroke. 2004;35:2752-2753

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(Stroke. 2004;35:2752.)
© 2004 American Heart Association, Inc.

Letters to the Editor

ACE and Subarachnoid Hemorrhage: Strategies for Genetics of Stroke

Mohammad Saeed, MD

Department of Biological and Biomedical Sciences, Aga Khan University, Karachi, Pakistan

To the Editor:

I thank Slowik et al for their article investigating the angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism in subarachnoid hemorrhage (SAH).1 I wish to comment on several aspects of this article related to the design of genetic association studies for complex disorders such as SAH, which are important in interpreting their findings. It is impressive to note the high odds ratios achieved, however this study was less than half the size of the one it aimed to confirm.2 The populations of the 2 studies were of European origin and the allele frequencies were also similar. Using calculations previously described3 and the data from Keramatipour et al,2 an estimated sample size of 600 subjects is required to show the presence of an association of ACE I/D with SAH with 80% power. It is surprising that Slowik et al achieved twice as high an odds ratio with half the sample size. The small sample size of this study could have resulted in a type 1 error.

ACE I/D polymorphism has been extensively studied in several disorders with conflicting results. Interestingly, Zhu et al showed that the I/D polymorphism is not functional and its association with elevated plasma ACE levels is secondary to a linkage disequilibrium (LD) effect.4 We recently showed this to be the case for the association of the I/D polymorphism with essential hypertension as well.5,6 To verify the effect of a gene it is important to genotype several single nucleotide polymorphisms (SNPs) across the gene, study the structure of the haplotype block and look for associations with the haplotypes, which considerably increases the power of a case-control study. Moreover, improvised study designs such as sib pair and TDT-trios are formidable tools for teasing out the genetic architecture of complex disorders. Elaborate algorithms exploring the combinatorial effects of SNPs in several candidate genes across the genome have recently become available and can add power to the analysis.7 Using novel study designs entire pathways, such as the renin-angiotensin system (RAS), can be explored in complex disorders such as SAH. This is important as some components of RAS increase angiotensin II formation whereas others decrease it.8 It is also worthy to collect details of disease characteristics which may serve as covariates in subsequent allele, genotype and haplotype analysis. The study design can be enhanced by including intermediate phenotypes in the study.9 Recently, genome-wide SNP analysis using microarrays has brought linkage analysis for complex disorders within the realm of reality,10 provided suitable pedigrees are available.

Without such elaborate efforts it will be difficult to meaningfully interpret results of genetic association studies for complex disorders. Studies of human polymorphisms had a modest presence in 1980 with just over 100 publications. After the explosive entry of ACE I/D in 199211 and ApoE {epsilon}-4 in 199312 in the complex disease arena, there was an exponential rise in the studies of human polymorphisms, most being association studies. Approximately 3500 such studies are now published annually in indexed journals. Making sense of such a large number of studies, several presenting contradictory findings, is a colossal task. Unless well-determined guidelines are followed, and robust study designs and appropriate sample sizes are used, many resources will go to waste. Also, a majority of the genome will remain unexplored in most disorders, and only a few loci will be investigated repeatedly with no additional information gained. It is important that we realize the significance of well-planned genetic association studies aimed at stroke and associated complex disorders. In light of this, the National Institutes of Health funded Siblings with Ischemic Stroke Study is much appreciated.13 Additionally, a Stroke Consortium could be made which would allow the use of DNA and other resources to investigators on approval of a proposal in a similar fashion as the Framingham Heart Study.14 We must act before it is too late.

References

  1. Slowik A, Borratynska A, Pera J, Betlej M, Dziedzic T, Krzyszkowski T, Czepko R, Figlewicz DA, Szczudlik A. II genotype of the angiotensin-converting enzyme gene increases the risk for subarachnoid hemorrhage from ruptured aneurysm. Stroke. 2004; 35: 1594–1597.[Abstract/Free Full Text]
  2. Keramatipour M, McConnell RS, Kirkpatrick P, Tebbs S, Furlong RA, Rubinsztein DC. The ACE I allele is associated with increased risk for ruptured intracranial aneurysms. J Med Genet. 2000; 37: 498–500.[Abstract/Free Full Text]
  3. Risch N, Merikangas K. The future of genetic studies of complex human diseases. Science. 1996; 273: 1516–1517.[Medline] [Order article via Infotrieve]
  4. Zhu X, Bouzekri N, Southam L, Cooper RS, Adeyemo A, McKenzie CA, Luke A, Chen G, Elston RC, Ward R. Linkage and association analysis of angiotensin I-converting enzyme (ACE)-gene polymorphisms with ACE concentration and blood pressure. Am J Hum Genet. 2001; 68: 1139–1148.[CrossRef][Medline] [Order article via Infotrieve]
  5. Saeed Mahmood M, Saboohi K, Osman Ali S, Bokhari AM, Frossard PM. Association of the angiotensin-converting enzyme (ACE) gene G2350A dimorphism with essential hypertension. J Hum Hypertens. 2003; 17: 719–723.[CrossRef][Medline] [Order article via Infotrieve]
  6. Saeed M, Naseer A, Siddiqui S, Saboohi K, Osman Ali S, Frossard PM. Association of the ACE gene heplotype with essential hypertension. J Hum Hypertens. 2004 Aug 5. [Epub ahead of print].
  7. Yamada R, Tanaka T, Unoki M, Nagai T, Sawada T, Ohnishi Y, Tsunoda T, Yukioka M, Maeda A, Suzuki K, Tateishi H, Ochi T, Nakamura Y, Yamamoto K. Association between a single-nucleotide polymorphism in the promoter of the human interleukin-3 gene and rheumatoid arthritis in Japanese patients, and maximum-likelihood estimation of combinatorial effect that two genetic loci have on susceptibility to the disease. Am J Hum Genet. 2001; 68: 674–685.[CrossRef][Medline] [Order article via Infotrieve]
  8. De Mello WC, Danser AH. Angiotensin II and the heart: on the intracrine renin-angiotensin system. Hypertension. 2000; 35: 1183–1188.[Abstract/Free Full Text]
  9. Saeed M. Editorial comment–Unraveling the pagodian architecture of stroke as a complex disorder. Stroke. 2004 Apr;35: 824–825.
  10. John S, Shephard N, Liu G, Zeggini E, Cao M, Chen W, Vasavda N, Mills T, Barton A, Hinks A, Eyre S, Jones KW, Ollier W, Silman A, Gibson N, Worthington J, Kennedy GC. Whole-genome scan, in a complex disease, using 11 245 single-nucleotide polymorphisms: comparison with microsatellites. Am J Hum Genet. 2004; 75: 54–64.[CrossRef][Medline] [Order article via Infotrieve]
  11. Cambien F, Poirier O, Lecerf L, Evans A, Cambou JP, Arveiler D, Luc G, Bard JM, Bara L, Ricard S. Deletion polymorphism in the gene for angiotensin-converting enzyme is a potent risk factor for myocardial infarction. Nature. 1992; 359: 641–644.[CrossRef][Medline] [Order article via Infotrieve]
  12. Corder EH, Saunders AM, Strittmatter WJ, Schmechel DE, Gaskell PC, Small GW, Roses AD, Haines JL, Pericak-Vance MA. Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer’s disease in late onset families. Science. 1993; 261: 921–923.[Abstract/Free Full Text]
  13. Meschia JF, Brown RD Jr, Brott TG, Chukwudelunzu FE, Hardy J, Rich SS. The Siblings With Ischemic Stroke Study (SWISS) protocol. BMC Med Genet. 2002; 3: 1.[CrossRef][Medline] [Order article via Infotrieve]
  14. National Heart, Lung and Blood Institute. Framingham Heart Study: Biological Materials (DNA) and Genetic Data Index. Available at: http://www.nhlbi.nih.gov/about/framingham/policies/index.htm. Accessed November 1, 2004.

Response:

Agnieszka Slowik, MD; Anna Borratynska, MD; Tomasz Dziedzic, MD Andrzej Szczudlik, MD

Department of Neurology, Jagiellonian University, Krakow, Poland

Agnieszka Kieltyka, MSc

Department of Epidemiology and Preventive Medicine, Jagiellonian University, Krakow, Poland

Denise A. Figlewicz, PhD

Department of Neurology, University of Michigan, Ann Arbor, Michigan

We thank Dr Saeed for his letter emphasizing a very important aspect concerning the design of genetic studies, ie the calculation of the power of a study and the number of patients included in the control and case groups needed to provide replicable results. Interestingly, while analyzing the studies that assess the significance of different polymorphisms in a variety of stroke etiologies, which have been published in Stroke during the last five years, we found that this aspect of methodology was addressed only in a few cases. In general, the previous authors published in Stroke calculated the required number of cases and controls to achieve the power of 80% (P=0.05). This was based on allele or genotype frequencies in the populations to achieve a given minimum odds ratios,1 relative risk by allele,2 or the proportion of the studied alleles in cases and controls.3 Other journals, such as Neurology4 and Human Genetics5 have already published the guidelines for genetic association studies in humans. We feel it would be reasonable to prepare such guidelines for publication in Stroke. The information regarding the power of a study should be provided, in a uniform fashion, within the methodology section of all articles related to this topic.

A very important aspect in the design of genetic association studies (or any clinical study, for that matter) is the ability to generate significant and replicable results while keeping within budget constraints. It is well known that the population frequency of the II genotype, and not I allele, of the angiotensin-converting enzyme (ACE) gene is approximately 25% (23.7% in English patients6 and 23.4% in our controls7). Bearing this in mind, we calculated that having 90 patients with aneurysmal subarachnoid hemorrhage and 128 controls, we would be able to obtain statistically significant differences between the groups if the odds ratio was >2.5, assuming the study’s achievable power to be 80%, and P=0.05. In our study, type I error (probability of rejecting true H0 hypothesis) was 0.00001, which is very low.

It is commonly known that ACE gene insertion/deletion polymorphism, because it is an intronic marker, may be functionally neutral, but may be in linkage disequilibrium with other (functional) mutations within ACE or another gene.8 The genetic association studies, such as we performed, generally answer the question as to whether the target allele or genotype remain risk factors for the disease, but they do not answer the question, does the causal relationship exist between them? We can only speculate therefore whether such a causal relationship exists. We have already planned to expand our studies of the ACE gene, examining several single nucleotide polymorphisms across the gene, and calculating association with haplotype(s). We agree with Dr Saeed that introducing genome-wide single nucleotide polymorphism analysis using microarrays is an approach that will ultimately enable study of complex disease mechanisms in greater detail.

References

  1. Jannes J, Hamilton-Bruce MA, Pilotto L, Smith BJ, Mullighan CG, Bardy PG, Koblar SA. Tissue plasminogen activator –7351 C/T enhancer polymorphism is a risk factor for lacunar stroke. Stroke. 2004; 35: 1090–1094.[Abstract/Free Full Text]
  2. Zhang B, Dhillon S, Geary I, Howell WM, Iannotti F, Day IN, Ye S. Polymorphisms in matrix metalloproteinase-1, -3, -9, and -12 genes in relation to subarachnoid hemorrhage. Stroke. 2001; 32: 2198–2202.[Abstract/Free Full Text]
  3. Onda H, Kasuya H, Yoneyama T, Hori T, Nakajima T, Inoue I. Endoglin is not a major susceptibility gene for intracranial aneurysm among Japanese. Stroke. 2003; 34: 1640–1644.[Abstract/Free Full Text]
  4. Bird TD, Jarvik GP, Wood NW. Genetic association studies: genes in search of diseases. Neurology. 2001; 57: 1153–1154.[Free Full Text]
  5. Cooper DN, Nussbaum RL, Krawczak M. Proposed guidelines for papers describing DNA polymorphism-disease associations. Hum Genet. 2002; 110: 207–208.[CrossRef][Medline] [Order article via Infotrieve]
  6. Keramatipour M, McConnell RS, Kirkpatrick P, Tebbs S, Furlong RA. Rubinsztein DC. The ACE I allele is associated with increased risk for ruptured intracranial aneurysms. J Med Genet. 2000; 37: 498–500.[Abstract/Free Full Text]
  7. Slowik A, Borratynska A, Pera J, Betlej M, Dziedzic T, Krzyszkowski T, Czepko R, Figlewicz DA, Szczudlik A. II genotype of the angiotensin-converting enzyme gene increases the risk for subarachnoid hemorrhage from ruptured aneurysm. Stroke. 2004; 35: 1594–1597.[Abstract/Free Full Text]
  8. Niu T, Chen X, Xu X. Angiotensin converting enzyme gene insertion/deletion polymorphism and cardiovarscular disease: therapeutic implication. Drugs. 2002; 62: 977–993.[CrossRef][Medline] [Order article via Infotrieve]




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