doi: 10.1161/01.RES.0000269420.81524.05
© 2007 American Heart Association, Inc.
Editorials |
The Challenge of Genetic Studies in Hypertension
From the Section on Pharmacology, Division of Intramural Research Programs, National Institute of Mental Health, National Institutes of Health, Department of Health and Human Services, Bethesda, Md.
Correspondence to Juan M. Saavedra M.D., Section on, Pharmacology, DIRP, NIMH, NIH, DHHS, 10 Center Drive, Building 10, Room, 2D-57, Bethesda, MD 20892. E-mail saavedrj{at}mail.nih.gov
See related article, pages 1522–1529
Key Words: endothelin • VEGF • human hypertension • haplotype analysis • salt sensitivity
Essential hypertension is the most common cardiovascular disease,1,2 with genetic elements contributing up to 30% to 50% of the blood pressure variability.3 Identifying contributing genes may allow us to recognize vulnerable individuals, to classify patients in subgroups with definite genetic and pathogenic mechanisms and to achieve better prevention and therapeutics. Using knowledge of pathways involved in cardiovascular regulation, many genes have been implicated in the pathophysiology of hypertension by the candidate gene linkage approach. The approach relies on studies of single nucleotide polymorphic (SNP) markers, in most cases derived from experimental rodent models of the disease. Genes responsible for vasoactive peptides, their receptors, and sodium homeostasis have taken center stage because of the role of these processes in the regulation of fluid volume and vascular reactivity.
However, in spite of intense efforts (11 199 publications for "human hypertension/genetics" listed in PubMed by April 2007) the attempts to replicate association studies have not been encouraging. The question has been raised as to whether high impact journals should even publish these studies.4 The reasons for this problem are clear. Blood pressure is controlled by a complex combination of processes with redundant balancing pressor and depressor mechanisms. Hypertension is a polygenetic condition with multiple interactions of networked genes, strongly influenced by environmental factors. Genes and the environment affect the onset, severity, progression, prognosis and treatment of the disease, and their influence differs among ethnic groups or even population subgroups.1,2 Our knowledge of the mechanisms of blood pressure control and the pathogenesis of hypertension is not complete. Adequate genetic analysis and phenotype definition will not be possible until such mechanisms are better understood. The effect of only one gene is likely to be small and inconsistent across different genetic and environmental backgrounds, a few alleles in a handful of genes are not likely to explain increased blood pressure, and differences among populations are anticipated rather than surprising.5 We should not be surprised that initial findings usually fail when applied to different populations.
In a manuscript published in this issue,6 the authors used SNP-haplotype analysis in a relatively isolated genetic population. They reported the association of the Q276L variant of the 
-1Na+,K+-ATPase (ATP1A1)7,8 and the S44/M74 variant of the dual endothelin-angiotensin II (ET-1-Ang II) (Dear) receptor9,10 with hypertension and also with normal blood pressure. The authors localized and characterized the human Dear receptor by transfection into Cos1 cells, determination of binding affinities, and its immunoreactivity to an anti-human Dear antibody. They concluded that this association, together with the expression of ATP1A1 and Dear in renal tubular cells and vascular endothelium, implicate both variants in the pathogenic mechanisms of hypertension.6
Analysis of this publication requires two major comments. The first relates to the significance of the ATP1A1 and Dear variants. Ruiz-Opazo’s group previously discovered dual receptors for vasoactive peptides.9,11 They proposed these receptors as molecular links to explain the interaction between very important systems linked to hypertension.10,12 In parallel, they reported that variants of the 1 Na+,K+-ATPase, the key enzyme implicated in the development of salt-sensitive hypertension13,14,15 are also linked to the disease in the salt-sensitive/salt-resistant (DS/DR) Dahl rat model of hypertension.7,8,16,17 Based on immunological methods, the authors found that the Q276L variant protein was expressed exclusively in DS rats, and that in transgenic DS rats bearing the DR wild-type 1 Na+,K+-ATPase there was a reduction of the salt-sensitive hypertension and renal disease.7 This strongly implicates the variant not only in hypertension but also in salt sensitivity.7 These discoveries were novel and of great interest, and it is surprising that such a provocative body of work has not been replicated by independent groups. Instead, the proposal that the Na+,K+-ATPase variants may contribute to the pathogenesis of hypertension has been hotly disputed.18,19,20,21
A number of issues should be clarified before the validity and significance of these novel and provocative findings may be properly established. Using the DS/DR model, the authors first reported that the S44/M74 variant in DS cosegregated with high blood pressure in female Dahl rats, whereas in the DR variant (S44P/M74T) the affinity for Ang II was lost.10 Characterization of the Dear receptor in mice22 and humans6 revealed loss of Ang II affinity, and the presence of affinity for the vascular endothelial growth factor signal peptide (VEGFsp).22 The lack of conservation of the Ang II binding domain suggested that the Ang II site is not relevant for the receptor function, calling into question its current nomenclature.22 Dear is a 14 kDa receptor with a single transmembrane domain,9,10 structurally very different from the ET-1 (and Ang II) seven membrane domain, G protein-coupled receptors23 (Figure
). To be considered receptors, proteins should combine the properties of ligand binding, incorporation to specific membrane sites and signal transduction. A full Dear receptor characterization, such as those performed for other receptor candidates in hypertension24 is needed before it may be considered as a major player in hypertension and salt-sensitivity. Signal transduction mechanisms for VEGFsp in the Dear receptor have not been studied, and the novel proposal that VGEFsp is a mimetic ligand for the ET-1 site must be clarified. Characterization should include establishing the relevance of the ET and VEGFsp binding sites, their association with specific signal transduction mechanisms and distribution studies using in situ hybridization to complement immunocytochemical findings. The use of antibodies requires stringent controls to avoid erroneous interpretation of data.25,26 Strict controls must be applied to the use of specific antibodies to detect of the Q276L and Q276 mutants of the subunit isoforms of Na+,K+-ATPase, particularly because the antibodies in question are proposed to differentiate single amino acid substitutions.8 The wild-type Q276 variant is present not only in normotensive DR but also in spontaneously hypertensive rats (SHR).8 This calls into question the generalization of an association of the Q276L variant with hypertension and salt sensitivity, because the SHR are also salt sensitive.27
|
|
The Dear receptor9 was discovered in the rat by application of Blalock’s molecular recognition theory (MRT). The theory is based on hydropathic complementarity, proposing that noncoding, or complementary, strands of genomic DNA encode antisense peptides and that this is the basis for the evolution of peptide receptors.28 Ruiz-Opazo’s group used oligonucleotide probes for each ET-1 and Ang II receptors, sequentially. They independently isolated, from cDNA libraries, identical DNA clones encoding single polypeptide receptors with dual peptide binding sites.9,11 The MRT theory is controversial29,30 and was not confirmed in the case of Ang II, vasopressin and other receptors.30,31 Most evidence for the MRT is based on immunological techniques30 and these methods are subject to question as mentioned above. Moreover, many putative receptors isolated according to MRT theory have not turned out to be receptors.30 This questions the universal applicability of MRT.
The second comment relates to general problems and limitations of association studies in hypertension and other complex heterogeneous disorders, outlined in a recent editorial.32 Glorioso and coworkers6 studied a cohort of essential hypertensive patients, with only 196 female and 237 male hypertensives. The relatively isolated Sardinian population was selected to reduce environmental variability. The phenotype analysis was limited to blood pressure determinations, and normotensives were on average 14 years older than hypertensive subjects. The original studies on DS and DR rats described an association of Dear S44/M74 with gender.10 Association with gender also occurs in the present study.6 Surprisingly, gender associations are opposite; with female gender in DS rats, with male gender in humans.6 It is difficult to explain this discrepancy. The association of the S44/M74 mutant with both hypertension and normal blood pressure6 is confusing and requires further explanation. For these reasons, and because the size of the sample is small, it is too early to determine whether or not the polymorphisms studied in the present communication are associated with hypertension.
Association between SNPs and phenotypes does not necessarily prove functional causality of disease. None of the candidate genes identified by linkage studies using SNPs has shown strong linkage with hypertension, and the results have been conflicting, with few replications between populations.2,32,33,34 Several steps can be taken to prevent further confusion and lack of replication. It is important to use rodent models of genetic hypertension critically for translational studies between rat and human, taking advantage of developments in genome analysis. It is now possible to conduct genome-wide studies in rat models to identify quantitative trait loci (QTLs) of interest.1,35,36 The use of genome-wide scans using techniques to detect microsatellite polymorphisms and multiple SNPs, and analysis of the complete human genome sequence data37 may complement the hypothesis-driven candidate gene approach to discover novel genes that have not yet been implicated in the pathogenesis of hypertension.38,39 To determine candidate genes that merit translational studies in human populations, the strongest evidence is the transgenic rescue experiment, the rescue of the mutant animal from the disease phenotype following genomic incorporation of a DNA clone containing the normal allele.40 Successful complementation provides direct functional evidence for the role of the gene in question, and there are recent successful examples of this approach.41,42
Because negative evidence in replication studies is expected from statistical theory,5 high quality studies should include large sample sizes with high power statistical analyses. Insufficient numbers may prevent us from determining whether a particular SNP is associated with hypertension at all. The complete studies should include extensive phenotype characterization, report associations that make biological sense, contain an initial study as well as an independent replication, and systematically investigate the interaction between genes and the environment.43,44 Important phenotype characteristics should be carefully defined in terms of present age, age of onset of hypertension, gender, disease severity and body size, with consideration of disease progression from the early to the later stages of the disease. Replication studies should use similar inclusion and exclusion criteria and include a population of similar extraction and ethnic background, exposed to similar environmental factors, including differences in lifestyle and protein and salt intake.2,5 Subjects on antihypertensive treatments should be included because familial components of blood pressure variance are important. This information is lost when treated patients are excluded, reducing the evidence of linkage.45 Family-based analysis is more powerful than population-based studies for detecting linkage and/or linkage disequilibrium.34,46 However, even when isolated founder populations are studied by extensive phenotyping and whole genome-linkage scans, many loci are revealed. These loci probably interact in a nonlinear fashion,2,47 indicating that the genetic variations in founder populations may not be relevant to other populations.
Better study designs require a collaborative multidisciplinary approach. These study designs should include sharing and combining, before publication, the data resources and the results obtained from different but similar studies and between scientific teams32,48 and they should be supported by the editorial boards of high impact journals. Mondry33 suggested that all association studies should be registered in a public access electronic database and should be supported by a decision from editorial boards not to consider inappropriately designed studies for the review process, similar to that required for clinical trials.49 A similar process should be applied to grant reviews, because increased funding and further molecular and statistical sophistication may not be sufficient and may very well be adding to the confusion. We need more stringent requirements to guide future studies in the right direction, eliminating any positive publication bias and the tendency to over-interpret marginal results. Recognition of the complexity of hypertension and the difficulties ahead, improved study design, data sharing and collaborative studies, coupled with stringent criteria for acceptance in high impact journals, will permit us to advance our understanding of a disease of such immense importance for clinical practice and public health.
 |
Acknowledgments |
|---|
The author wishes to thank Dr Julius Benicky for the preparation of the figure and his helpful comments, Drs Ignacio Larrayoz-Roldán and Jin Zhou for their helpful comments and Marcia Sloger for her assistance in the preparation of the manuscript.
Sources of Funding
This study was supported by the Division of Intramural Research Programs, National Institute of Mental Health, NIH, DHHS, USA.
Disclosures
None.
|
Footnotes |
|---|
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
|
References |
|---|
 Top References |
|---|
1. McBride MW, Graham D, Delles C, Dominiczak AF. Functional genomics in hypertension. Curr Opin Nephrol Hypert. 2006; 15: 145–151.[Medline] [Order article via Infotrieve]
2. Cowley AW. The genetic dissection of essential hypertension. Nat Rev Genet. 2006; 7: 829–840.[Medline] [Order article via Infotrieve]
3. Garcia EA, Newhouse S, Caulfield MJ, Munroe PB. Genes and hypertension. Curr Pharmaceut Design. 2003; 9: 1679–1689.[CrossRef][Medline] [Order article via Infotrieve]
4. Anonymous. Freely associating. Nat Genet. 1999; 22: 1–2.[CrossRef][Medline] [Order article via Infotrieve]
5. Lalouel J-M, Rohrwasser A. Power and replication in case-control studies. Am J Hypertens. 2002; 15: 201–205.[CrossRef][Medline] [Order article via Infotrieve]
6. Glorioso N, Herrera VLM, Bagamasbad P, Filigheddu F, Troffa C, Argiolas G, Bulla E, Decano JL, Ruiz-Opazo N. Association of ATP1A1 and Dear SNP-haplotypes with essential hypertension with gender-specific and haplotype-specific effects. Circ Res. 2007; 100: 1522–1529.
7. Herrera VL, Xie HX, Lopez LV, Schork NJ, Ruiz-Opazo N. The 1 Na,K-ATPase gene is a susceptibility hypertension gene in the Dahl salt-sensitiveHSD rat. J Clin Invest. 1998; 102: 1102–1111.[Medline]
[Order article via Infotrieve]
8. Kaneko Y, Cloix J-F, Herrera VLM, Ruiz-Opazo N. Corroboration of Dahl SQ276L 1 Na,K-ATPase protein sequence: impact on affinities for ligands and on E1 conformation. J Hypert. 2005; 23: 745–752.[Medline]
[Order article via Infotrieve]
9. Ruiz-Opazo N, Hirayama K, Akimoto K, Herrera VLM. Molecular characterization of a dual endothelin-1/Angiotensin II receptor. Mol Med. 1998; 4: 96–108.[Medline] [Order article via Infotrieve]
10. Kaneko Y, Herrera VLM, Didishvili T, Ruiz-Opazo N. Sex-specific effects of dual ET-1/Ang II receptor (Dear) variants in Dahl SALT-sensitive/resistant hypertension rat model. Physiol Genom. 2005; 20: 157–164.
11. Ruiz-Opazo N, Akimoto K, Herrera VL. Identification of a novel dual angiotensin II/vasopressin receptor on the basis of molecular recognition theory. Nat Med. 1995; 1: 1074–1081.[CrossRef][Medline] [Order article via Infotrieve]
12. Ruiz-Opazo N, Lopez LV, Herrera VL. The dual Ang II/AVP receptor gene N119S/C163R variant exhibits sodium-induced dysfunction and cosegregates with salt-sensitive hypertension in the Dahl salt-sensitive hypertensive rat model. Mol Med. 2002; 8: 24–32.[CrossRef][Medline] [Order article via Infotrieve]
13. Mobasheri A, Avila J, Cózar-Castellano, Brownleader MD, Trevan M, Francis MJO, Lamb JF, Martin-Vasallo P. Na+,K+-ATPase isozyme diversity: comparative biochemistry and physiological implications of novel functional interactions. Biosci Rep. 2000; 20: 51–91.[CrossRef][Medline] [Order article via Infotrieve]
14. Iwamoto T, Kita S. Hypertension, Na+/Ca2+ exchanger, and Na+, K+-ATPase. Kidney Int. 2006; 69: 2148–2154.[CrossRef][Medline] [Order article via Infotrieve]
15. Dostanic-Larson I, Lorenz JN, Van Huysse JW, Neumann JC, Moseley AE, Lingrel JB. Physiological role of the 1- and 2-isoforms of the Na+-K-ATPase and biological significance of their cardiac glycoside binding site. Am J Physiol Regul Integr Comp Physiol. 2006; 290: R524–R528.
16. Herrera VL, Emanuel JR, Ruiz-Opazo N, Levenson R, Nadal-Ginard B. Three differentially expressed Na,K-ATPase subunit isoforms: structural and functional implications. J Cell Biol. 1987; 105: 1855–1865.
17. Ruiz-Opazo N, Barany F, Hirayama K, Herrera VL. Confirmation of mutant 1 Na,K-ATPase gene and transcript in Dahl salt-sensitive/JR rats. Hypertension. 1994; 24: 260–270.
18. Simonet L, St. Lezin E, Kurtz TW. Sequence analysis of the 1 Na+,K+-ATPase gene in the Dahl salt-sensitive rat. Hypertension. 1991; 18: 689–693.
19. Rapp JP, Dene H. Failure of alleles at the Na+,K(+)-ATPase 1 locus to cosegregate with blood pressure in Dahl rats. J Hypertens. 1990; 8: 457–462.[CrossRef][Medline]
[Order article via Infotrieve]
20. Harris EL, Barnard R. A1079T transversion in the gene of the 1 isophorm of the Na+/K+ ATPase in the Dahl S rat. J Hypertens. 2006; 24: 1209–1210.[Medline]
[Order article via Infotrieve]
21. Harris E, Barnard R. Reply to Herrera and Ruiz-Opazo’s letter published in the June 2006 issue. J. Hypertens. 2006; 24: 2312–2313.[Medline] [Order article via Infotrieve]
22. Herrera VLM, Ponce LRB, Bagamasbad PD, YanPelt BD, Didishvili T, Ruiz-Opazo N. Embryonic lethality in Dear gene-deficient mice: new player in angiogenesis. Physiol Genom. 2005; 23: 257–268.
23. Marasciulo FL, Montagnani M, Potenza MA. Endothelin-1: the Yin and Yang on vascular function. Current Med Chem. 2006; 13: 1655–1665.[CrossRef]
24. Felder RA, Jose PA. Mechanisms of disease: the role of GRK4 in the etiology of essential hypertension and salt sensitivity. Nat Clin Pract Nephrol. 2006; 2: 637–650.[CrossRef][Medline] [Order article via Infotrieve]
25. Burry RW. Perspectives: Specificity Controls for Immunocytochemical Methods. J Histochem Cytochem. 2000; 48: 163–165.
26. Saper CB. An open letter to our readers on the use of antibodies. J Comp Neurol. 2005; 493: 477–478.[CrossRef][Medline] [Order article via Infotrieve]
27. Kubo T, Hagiwara Y. Enhanced central hypertonic saline-induced activation of angiotensin II-sensitive neurons in the anterior hypothalamic area of spontaneously hypertensive and Dahl S rats. Brain Res Bull. 2006; 68: 335–340.[CrossRef][Medline] [Order article via Infotrieve]
28. Blalock JE. Genetic origins of protein shape and interaction rules. Nat Med. 1995; 1: 876–878.[CrossRef][Medline] [Order article via Infotrieve]
29. Houen G. Evolution of the genetic code: the nonsense, antisense, and antinonsense codes make no sense. Bio System. 1999; 54: 39–46.
30. Root-Bernstein RS. Peptide self-aggregation and peptide complementarity as bases for the evolution of peptide receptors: a view. J Mol Recognit. 2005; 18: 4049.
31. De Gasparo M, Whitebread S, Einsle K, Heusser C. Are the antibodies to a peptide complementary to angiotensin II useful to isolate the angiotensin II receptor? Biochem J Let. 1989; 261: 310–311.
32. Saavedra JM. Studies on genes and hypertension. A daunting task. J Hypertens. 2005; 23: 929–932.[Medline] [Order article via Infotrieve]
33. Mondry A, Loh M, Liu P, Zhu A-L, Nagel M. Polymorphisms of the insertion/deletion ACE and M235 AGT genes and hypertension: surprising new findings and meta-analysis of data. BMC Nephrol. 2005; 6: 1–11.
34. Luft FC. Geneticism of essential hypertension. Hypertension. 2004; 43: 1–5.
35. Dutil J, Eliopoulos V, Tremblay J. Hamet P, Charron S, Deng AY. Multiple quantitative trait loci for blood pressure interacting epistatically and additively on Dahl chromosome 2. Hypertension. 2005; 45: 557–564.
36. Grondin M, Eliopoulos V, Lambert R. Deng Y, Ariyarajah A, Moujahidine M, Dutil J, Charron S, Deng AY. Complete and overlapping congenics proving the existence of a quantitative trait locus for blood pressure on Dahl rat chromosome 17 Physiol Genomics. 2005; 21: 112–116.
37. International Human Genome Sequencing Consortium. Finishing the euchromatic sequence of the human genome. Nature. 2004; 431: 931–945.[CrossRef][Medline] [Order article via Infotrieve]
38. Samani NJ. Genome scans for hypertension and blood pressure regulation. Am J Hypertens. 2003; 16: 167–171.[CrossRef][Medline] [Order article via Infotrieve]
39. Dominiczak AF, Graham D, McBride MW, Brain NJ, Lee WK, Charchar FJ, Tomaszewski M, Delles C, Hamilton CA. Corcoran Lecture. Cardiovascular genomics and oxidative stress. Hypertension. 2005; 45: 636–642.
40. Michalkiewicz M, Michalkiewicz T, Ettinger RA, Rutledge EA, Fuller JM, Moralejo DH, Van Yserloo B, MacMurray AJ, Kwitek AE, Jacob HJ, Lander ES, Lernmark A. Transgenic rescue demonstrates involvement of the lan5 gene in T cell development in the rat. Physiol Genomics. 2004; 19: 228–232.
41. Pravenec M, Landa V, Zidek V, Musilova A, Kazdova L, Qi N, Wang J, St Lezin E, Kurtz TW. Transgenic expression of CD36 in the spontaneously hypertensive rat is associated with amelioration of metabolic disturbances but has no effect on hypertension. Physiol Res. 2003; 52: 681–688.[Medline] [Order article via Infotrieve]
42. H
bner N, Wallace CA, Zimdahl H, Petretto E, Schulz H, Maciver F, Mueller M, Hummel O, Monti J, Zidek V, Musilova A, Kren V, Causton H, Game L, Born G, Schmidt S, Muller A, Cook SA, Kurtz TW, Whittaker J, Pravenec M, Aitman TJ. Integrated transcriptional profiling and linkage analysis for identification of genes underlying disease. Nat Genet. 2005; 37: 243–253.[CrossRef][Medline]
[Order article via Infotrieve]
43. Kardia SLR. Context-dependent genetic effects in hypertension. Curr Hypertens Reports. 2000; 2: 32–38.[Medline] [Order article via Infotrieve]
44. Farahani P, Dolovich L, Levine M. Exploring design-related bias in clinical studies on receptor genetic polymorphism of hypertension. J Clin Epidemiol. 2007; 60: 1–7.[CrossRef][Medline] [Order article via Infotrieve]
45. Cui JS, Hopper JL, Harrap SB. Antihypertensive treatments obscure familial contributions to blood pressure variation. Hypertension. 2003; 41: 207–210.
46. Terwilliger JD, Haghighi F, Hiekkalinna TS, Göring HHH. A biased assessment of the use of SNPs in human complex traits. Curr Opin Genet Dev. 2002; 12: 726–734.[CrossRef][Medline] [Order article via Infotrieve]
47. Hamet P Merlo E, Seda O, Broeckel U, Tremblay J, Kaldunski M, Gaudet D, Bouchard G, Deslauriers B, Gagnon F, Antoniol G, Pausova Z, Labuda M, Jomphe M, Gossard F, Tremblay G, Kirova R, Tonellato P, Orlov SN, Pintos J, Platko J, Hudson TJ, Rioux JD, Kotchen TA, Cowley AW Jr. Quantitative founder-effect analysis of French Canadian families identifies specific loci contributing to metabolic phenotypes of hypertension. Am J Hem Genet. 2005; 76: 815–832.[CrossRef]
48. Boerwinkle E. All for one and one for all: introduction to a coordinated analysis of the Gly 460-Trp a-adducin polymorphism. Am J Hypertens. 2000; 3: 734–735.
49. Couser WG, Drueke T, Halloran P, Kasiske B, Klahr S, Morris P. Uniform clinical trial registration policy for journals of kidney diseases, dialysis, and transplantation. J Am Soc Nephrol. 2005; 16: 837.
50. Conchon S, Clauser E. AT1 receptor molecular aspects. In: Unger T, Scholkens BA, eds. Angiotensin Vol I. New York: Springer-Verlag Berlin Heidelberg; 2004: 269–295.
51. Breu V, Hashido K, Broger C, Miyamoto C, Furuichi Y, Hayes A, Kalina B, Loffler BM, Ramuz H, Clozel M. Separable binding sites for the natural agonist endothelin-1 and the non-peptide antagonist bosentan on human endothelin-A receptors. Eur J Biochem. 1995; 231: 266–270.[Medline] [Order article via Infotrieve]
52. Adachi M, Hashido K, Trzeciak A, Watanabe T, Furuichi Y, Miyamoto C. Functional domains of human endothelin receptor. J Cardiovasc Pharmacol. 1993; 22 (Suppl 8): S121–S124.[CrossRef][Medline] [Order article via Infotrieve]
53. Krystek SR Jr., Patel PS, Rose PM, Fisher SM, Kienzle BK, Lach DA, Liu EC, Lynch JS, Novotny J, Webb ML. Mutation of peptide binding site in transmembrane region of a G protein-coupled receptor accounts for endothelin receptor subtype selectivity. J Biol Chem. 1994; 269: 12383–12386.
Related Article:
Circ. Res. 2007 100: 1522-1529.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||