The AT2 gene may have a gender-specific effect on kidney function and pulse pressure in type I diabetic patients
2006; Elsevier BV; Volume: 69; Issue: 10 Linguagem: Inglês
10.1038/sj.ki.5000348
ISSN1523-1755
AutoresKim Pettersson-Fernholm, Sara Fröjdö, Johan Fagerudd, Merlin C. Thomas, Carol Forsblom, Maija Wessman, Per‐Henrik Groop,
Tópico(s)Diabetes Treatment and Management
ResumoDiabetic nephropathy shows a higher incidence in male subjects, which may in part be owing to genetic factors. The angiotensin II type 2 receptor (AT2), present in the renal glomerulus, may oppose the deleterious effects of the type I receptor (AT1) through vasodilatation and growth inhibition. We determined whether the functional intronic G1675A or A1818T polymorphism of the X-chromosomal AT2 gene is associated with blood pressure levels or with kidney function. We genotyped 996 (538 female/458 male subjects) Finnish patients with type I diabetes from the FinnDiane-study in a cross-sectional study. DNA samples were amplified using standard polymerase chain reaction protocol and the genotypes were determined by the minisequencing method. Male patients with the AA haplotype had a lower glomerular filtration rate (83±32 vs 94±34 ml min−1 1.73 m−2, P=0.008) and a higher pulse pressure (PP) (62±18 vs 57±15 mm Hg, P=0.002; P<0.05 after adjustment for age) than did those with the GT haplotype. No differences between the genotypes or haplotypes and these variables were evident in females. In males, the G1675A was also an independent variable in a linear regression analysis with PP (r2=0.16, coefficient=3.64, s.e.m.=1.38, P<0.01) as the dependent variable. These data suggest a gender-specific association between the AT2 gene and kidney function and premature aging of the arterial tree in patients with type I diabetes. Diabetic nephropathy shows a higher incidence in male subjects, which may in part be owing to genetic factors. The angiotensin II type 2 receptor (AT2), present in the renal glomerulus, may oppose the deleterious effects of the type I receptor (AT1) through vasodilatation and growth inhibition. We determined whether the functional intronic G1675A or A1818T polymorphism of the X-chromosomal AT2 gene is associated with blood pressure levels or with kidney function. We genotyped 996 (538 female/458 male subjects) Finnish patients with type I diabetes from the FinnDiane-study in a cross-sectional study. DNA samples were amplified using standard polymerase chain reaction protocol and the genotypes were determined by the minisequencing method. Male patients with the AA haplotype had a lower glomerular filtration rate (83±32 vs 94±34 ml min−1 1.73 m−2, P=0.008) and a higher pulse pressure (PP) (62±18 vs 57±15 mm Hg, P=0.002; P<0.05 after adjustment for age) than did those with the GT haplotype. No differences between the genotypes or haplotypes and these variables were evident in females. In males, the G1675A was also an independent variable in a linear regression analysis with PP (r2=0.16, coefficient=3.64, s.e.m.=1.38, P 90 ml min−1 1.73 m−2) than in those with impaired renal function (Table 1). The G allele of the G1675A and the T allele of the A1818T were also associated with a higher absolute GFR value after excluding patients with end-stage renal disease and with a lower pulse pressure (PP) and higher estimated glucose disposal rate (Table 2). However, none of these associations appeared in females. In addition, no association existed between AT2 genotype and albumin excretion rate in either males or females (data not shown).Table 1Allele frequencies and haplotypes by GFR in all patientsGFR>9060≤GFR≤90GFR<60Padf=2.G1675, males G132 (61)53 (46)99 (51) A86 (39)62 (54)94 (49)0.027A1818T, males A149 (68)95 (83)143 (74) T71 (32)20 (17)50 (26)0.013Haplotypes, males GT71 (45)19 (24)50 (35) AA86 (55)61 (76)94 (65)0.004G1675A, females G113 (54)219 (54)144 (55) A97 (46)185 (46)120 (45)NSA1818T, females A155 (74)295 (73)186 (70) T55 (26)109 (27)78 (30)NSHaplotypes, females GT53 (36)97 (36)93 (40) AA94 (64)169 (64)141 (60)NSGFR, glomerular filtration rate; NS, not significant.Data are n (%). All end-stage renal disease patients were classified as GFR<60 ml min−1 1.73 m−2.a df=2. Open table in a new tab Table 2Clinical characteristics according to AT2 polymorphisms in male patientsaData are genotypes and allele frequencies simultaneously owing to X-chromosomal location.G1675AA1818TVariableGAPATPn (%)288 (53.7)244 (46.3)—395 (73.4)143 (26.6)—Age (years)40.7±9.742.7±10.30.02442.0±10.040.3±9.9NSDuration of diabetes (years)27.6±8.628.6±8.7NS28.4±8.826.9±8.2NSHbA1c (%)8.6±1.48.7±1.4NS8.6±1.48.6±1.4NSeGDR (mg kg−1 min−1)5.12±2.264.51±2.200.003bP<0.05 when adjusted for age.4.70±2.245.22±2.250.024cP=NS when adjusted for age.SBP (mm Hg)141±19145±200.019cP=NS when adjusted for age.143±20142±17NSDBP (mm Hg)84±1083±10NS83±1085±110.050cP=NS when adjusted for age.PP (mm Hg)57±1762±18<0.001bP<0.05 when adjusted for age.61±1857±150.028cP=NS when adjusted for age.Albumin excretion rate (mg 24 h−1)dPatients with end-stage renal disease excluded.67 (2–7565)136 (1–5589)NS84± (1–5589)68 (4–7586)NSGFR (ml min−1 1.73 m−2)dPatients with end-stage renal disease excluded.90.4±32.483.4±32.10.027eP=NS (G1675A) and P=0.005 (A1818T) when adjusted for duration of diabetes, use of angiotensin-converting enzyme inhibitors, and urinary albumin excretion rate.85.0±32.093.7±32.80.016eP=NS (G1675A) and P=0.005 (A1818T) when adjusted for duration of diabetes, use of angiotensin-converting enzyme inhibitors, and urinary albumin excretion rate.Diagnosed coronary heart disease22 (8)34 (14)0.021cP=NS when adjusted for age.48 (13)9 (7)0.048cP=NS when adjusted for age.Retinal laser treatment194 (68)164 (67)NS268 (69)92 (65)NSAT2, angiotensin II type 2 receptor; DBP, diastolic blood pressure; eGDR, estimated glucose disposal rate; GFR, glomerular filtration rate; HbA1c, glycosylated hemoglobin; NS, not significant; PP, pulse pressure; SBP, systolic blood pressure.Data are mean±s.d., median (range) or n (%).a Data are genotypes and allele frequencies simultaneously owing to X-chromosomal location.b P<0.05 when adjusted for age.c P=NS when adjusted for age.d Patients with end-stage renal disease excluded.e P=NS (G1675A) and P=0.005 (A1818T) when adjusted for duration of diabetes, use of angiotensin-converting enzyme inhibitors, and urinary albumin excretion rate. Open table in a new tab GFR, glomerular filtration rate; NS, not significant. Data are n (%). All end-stage renal disease patients were classified as GFR<60 ml min−1 1.73 m−2. AT2, angiotensin II type 2 receptor; DBP, diastolic blood pressure; eGDR, estimated glucose disposal rate; GFR, glomerular filtration rate; HbA1c, glycosylated hemoglobin; NS, not significant; PP, pulse pressure; SBP, systolic blood pressure. Data are mean±s.d., median (range) or n (%). As the G allele of the G1675A and the T allele of the A1818T appeared to be protective, when the haplotypes were analyzed as AA against GT, males with the GT haplotype showed similarly a higher GFR, a lower PP, and a higher estimated glucose disposal rate than did those with the AA haplotype (Table 3). This difference remained significant after adjustment for age (Tables 2 and 3). Furthermore, the G1675A was independently associated with PP (r2=0.16, coefficient=3.64, s.e.=1.38, P<0.01) after adjusting for glycosylated hemoglobin, low-density lipoprotein cholesterol, body mass index, and antihypertensive medication. Again, no association appeared in females.Table 3Overall clinical characteristics between males and females and between the GT and AA haplotypes of the AT2 gene in each sexMalesFemalesMalesFemalesVariableAllAllPGTAAPGTAAPn (%)538 (54.0)458 (46.0)—142 (36.5)247 (63.5)—251 (37.9)412 (62.1)—Age (years)41.6±10.039.5±10.00.00140.2±9.842.6±10.20.02440.0±10.039.5±9.8NSDuration of diabetes (years)28.1±8.727.6±8.2NS26.8±8.128.5±8.7NS27.7±8.327.5±7.8NSHbA1c (%)8.6±1.48.4±1.50.0098.6±1.48.7±1.4NS8.4±1.48.4±1.5NSeGDR (mg kg−1 min−1)4.83±2.256.95±2.48<0.001aP<0.05 when adjusted for age.5.20±2.254.50±2.200.004aP<0.05 when adjusted for age.6.66±2.557.02±2.47NSSBP (mm Hg)143±19138±20<0.001aP<0.05 when adjusted for age.142±17145±20NS138±19138±22NSDBP (mm Hg)83±1080±10<0.001aP<0.05 when adjusted for age.85±1183±10NS79±1080±10NSPP (mm Hg)60±1858±17NS57±1562±180.002aP<0.05 when adjusted for age.58±1758±19NSUAER (mg 24 h−1)bPatients with end-stage renal disease excluded.77 (1–7565)20±(1–6069)<0.00168 (4–7565)132 (1–5589)NS16±(1–4325)19 (1–5259)NSGFR (ml min−1 1.73 m−2)bPatients with end-stage renal disease excluded.87.3±32.476.1±24.0<0.001aP<0.05 when adjusted for age.93.8±32.983.4±32.10.008cP=0.009 when adjusted for duration of diabetes, urinary albumin excretion rate, and use of angiotensin-converting enzyme inhibitors.74.7±24.676.6±24.2NSDiagnosed CHD57 (11)28 (6)0.009dP=NS when adjusted for age.9 (7)34 (14)0.022dP=NS when adjusted for age.17 (7)23 (6)NSRetinal laser treatment360 (68)255 (56)<0.00191 (65)163 (67)NS133 (54)232 (57)NSAT2, angiotensin II type 2 receptor; CHD, coronary heart disease; DBP, diastolic blood pressure; eGDR, estimated glucose disposal rate; GFR, glomerular filtration rate; HbA1c, glycosylated hemoglobin; NS, not significant; PP, pulse pressure; SBP, systolic blood pressure; UAER, urinary albumin excretion rate.Data are mean±s.d., median (range) or n (%).a P<0.05 when adjusted for age.b Patients with end-stage renal disease excluded.c P=0.009 when adjusted for duration of diabetes, urinary albumin excretion rate, and use of angiotensin-converting enzyme inhibitors.d P=NS when adjusted for age. Open table in a new tab AT2, angiotensin II type 2 receptor; CHD, coronary heart disease; DBP, diastolic blood pressure; eGDR, estimated glucose disposal rate; GFR, glomerular filtration rate; HbA1c, glycosylated hemoglobin; NS, not significant; PP, pulse pressure; SBP, systolic blood pressure; UAER, urinary albumin excretion rate. Data are mean±s.d., median (range) or n (%). Despite an association among haplotypes of the AT2 gene, renal function, and blood pressure in men, after adjustment for age, no differences existed in the genotypes or in the haplotypes of the AT2 gene as to prevalence of retinopathy or diagnosed coronary heart disease. Nor were any differences evident in time to onset of DN or end-stage renal disease for either genotype or haplotype in either gender (data not shown). DN is more common and more severe in men.1.Andersen A.R. Christiansen J.S. Andersen J.K. et al.Diabetic nephropathy in Type 1 (insulin-dependent) diabetes: an epidemiological study.Diabetologia. 1983; 25: 496-501Crossref PubMed Scopus (981) Google Scholar, 2.Seliger S.L. Davis C. Stehman-Breen C. Gender and the progression of renal disease.Curr Opin Nephrol Hypertens. 2001; 10: 219-225Crossref PubMed Scopus (109) Google Scholar This is apparently the first study showing a gender-specific association between a gene haplotype/genotype and renal function. The FinnDiane database of more than 4000 patients with type I diabetes (n=4069) includes significantly more males with DN (59 vs 41%) and end-stage renal disease (61 vs 39%) than females (P<0.0001: unpublished observation). The basis of this phenomenon remains to be established. As no effect appeared in females, we hypothesize that the X-chromosomal location of the AT2 gene specifically exposes males with the 1675A genotype or AA haplotype to impaired renal function, perhaps owing to the fact that males have only a single set of alleles or haplotype. The AT2 receptor plays an important role in the developing kidney. The AT2-null mouse exhibits a variety of urological abnormalities, and some studies have demonstrated an association between polymorphisms of the AT2 gene and vesicouretic reflux,10.Nishimura H. Yerkes E. Hohenfellner K. et al.Role of the angiotensin type 2 receptor gene in congenital anomalies of the kidney and urinary tract, CAKUT, of mice and men.Mol Cell. 1999; 3: 1-10Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar another renal disorder with a male preponderance. Recently, it has become clear that following injury to the diabetic kidney, many important developmental genes are expressed, possibly acting in repair and regeneration.11.Dolan V. Hensey C. Brady H.R. Diabetic nephropathy: renal development gone awry?.Pediatr Nephrol. 2003; 18: 75-84PubMed Google Scholar Among these, AT2 is thought to play a key role being linked to inhibition of cell growth, to apoptosis, and to modulation of inflammation and matrix protein accumulation in the kidney. In experimental DN, blockade of the AT2 receptor prevents pathogenic changes in the retina and the kidney.12.Zhang X. Lassila M. Cooper M.E. Cao Z. Retinal expression of vascular endothelial growth factor is mediated by angiotensin type 1 and type 2 receptors.Hypertension. 2004; 43: 276-281Crossref PubMed Scopus (75) Google Scholar, 13.Lee F.T. Cao Z. Long D.M. et al.Interactions between angiotensin II and NF-{kappa}B-dependent pathways in modulating macrophage infiltration in experimental diabetic nephropathy.J Am Soc Nephrol. 2004; 15: 2139-2151Crossref PubMed Scopus (136) Google Scholar It is conceivable that polymorphisms of the AT2 gene may have a similar modulatory effect in type I diabetes. However, regarding cardiovascular remodeling, recent data have also questioned the role of AT2 receptor as having an effect opposite to that of the deleterious AT1, as reviewed by Levy.5.Levy B.I. Can angiotensin II type 2 receptors have deleterious effects in cardiovascular disease? Implications for therapeutic blockade of the renin–angiotensin system.Circulation. 2004; 109: 8-13Crossref PubMed Scopus (152) Google Scholar It is thus controversial whether the AT2 induces arterial hypertrophy and fibrosis, or whether it has an inhibitory effect or both. The renin–angiotensin–aldosterone system also plays an important role in the control of blood pressure, a key risk factor for progressive renal disease in diabetes. We have recently shown that PP increases approximately 20 years earlier in type I diabetic subjects than in non-diabetic subjects.14.Rönnback M. Fagerudd J. Forsblom C. et al.Altered age-related blood pressure pattern in Type 1 diabetes.Circulation. 2004; 110: 1076-1082Crossref PubMed Scopus (99) Google Scholar This is thought to reflect premature aging of the arterial tree, whereas duration of diabetes appears to be a strong determinant of premature increase in PP. A small case–control study in non-diabetic subjects has shown an association between polymorphisms in the AT2 gene and prevalence of hypertension only for males.15.Jin J.J. Nakura J. Wu Z. et al.Association of angiotensin II type 2 receptor gene variant with hypertension.Hypertens Res. 2003; 26: 547-552Crossref PubMed Scopus (47) Google Scholar Furthermore, the A allele of the A1675G has been associated specifically with a more pronounced left ventricular hypertrophy, still only in males.8.Herrmann S.M. Nicaud V. Schmidt-Petersen K. et al.Angiotensin II type 2 receptor gene polymorphism and cardiovascular phenotypes: the GLAECO and GLAOLD studies.Eur J Heart Fail. 2002; 4: 707-712Crossref PubMed Scopus (38) Google Scholar, 9.Schmieder R.E. Erdmann J. Delles C. et al.Effect of the angiotensin II type 2-receptor gene (+1675 G/A) on left ventricular structure in humans.J Am Coll Cardiol. 2001; 37: 175-182Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar As the patients in these studies were non-diabetic, they were perhaps too young to display any detectable effect of the AT2 gene on PP. It is thus also possible that the polymorphism can influence the mechanisms leading to left ventricular hypertrophy and left ventricular overload, which could be detected by an elevated PP in our study. The G1675A allele was independently associated with PP in men after adjustment for standard risk factors, although the absolute prevalence of hypertension did not differ significantly between genotypes or haplotypes. This may in part be explained by its association with renal impairment. However, recent studies suggest that the impact of AT2 polymorphisms may be greatest in those patients with hypertension: In one study of non-diabetic men, the AT2 1675A allele was associated with excess risk among those with systolic hypertension, but coronary heart disease risk was independent of AT2 genotype among those normotensive.16.Jones A. Dhamrait S.S. Payne J.R. et al.Genetic variants of angiotensin II receptors and cardiovascular risk in hypertension.Hypertension. 2003; 42: 500-506Crossref PubMed Scopus (84) Google Scholar The fact that AT2 alleles were also associated with the effective glucose disposal rate, a surrogate measure of insulin sensitivity in patients with type I diabetes, was probably driven by the blood pressure indices included in the estimated glucose disposal rate formula, as glycosylated hemoglobin and waist-to-hip ratio did not significantly differ between haplotypes. Nonetheless, a direct effect cannot be discounted, because activation of the AT2 receptor does inhibit insulin-induced extracellular signal-regulated protein kinase-2 activity via a G-protein-mediated pathway.17.Moore S.A. Huang N. Hinthong O. et al.Human angiotensin II type-2 receptor inhibition of insulin-mediated ERK-2 activity via a G-protein coupled signaling pathway.Brain Res Mol Brain Res. 2004; 124: 62-69Crossref PubMed Scopus (6) Google Scholar Data are convincing in support of the functionality of the G1675A polymorphism. First, the G1675A involves a lariat branchpoint motif in intron 1 where the A allele causes a diminished splice efficiency and loss of receptor function.18.Stephens M. Smith N.J. Donnelly P. A new statistical method for haplotype reconstruction from population data.Am J Hum Genet. 2001; 68: 978-989Abstract Full Text Full Text PDF PubMed Scopus (6426) Google Scholar Secondly, the region is adjacent to an intron fragment responsible for directing the gene transcription when the 5′ flanking region of the gene is absent.19.Warnecke C. Willich T. Holzmeister J. et al.Efficient transcription of the human angiotensin II type 2 receptor gene requires intronic sequence elements.Biochem J. 1999; 340: 17-24Crossref PubMed Google Scholar Interestingly, a genome-wide scan in a Finnish population with premature coronary heart disease has pinpointed the locus containing the AT2 gene.20.Pajukanta P. Cargill M. Viitanen L. et al.Two loci on chromosomes 2 and X for premature coronary heart disease identified in early- and late-settlement populations of Finland.Am J Hum Genet. 2000; 67: 1481-1493Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar Although the AA haplotype of the AT2 gene was associated with a higher PP and renal impairment, no independent association, somewhat surprisingly, was seen with urinary albumin excretion rate. However, not all patients with microalbuminuria will develop overt nephropathy.21.Perkins B.A. Ficociello L.H. Silva K.H. et al.Regression of microalbuminuria in type 1 diabetes.N Engl J Med. 2003; 348: 2285-2293Crossref PubMed Scopus (651) Google Scholar Mechanistically, it is possible that functional polymorphisms in the AT2 gene are more important in progression of renal impairment (i.e., response to injury) than its initial development. This is consistent with family studies suggesting that the genetic predisposition to DN is not specific to diabetes, but is more a risk for progressive renal disease in general. In conclusion, these data suggest a gender-dependent association between the AT2 gene and progressive renal disease and premature aging of the arterial tree in male patients with type I diabetes, although these results require further testing and confirmation in other populations and in prospective studies. All type I diabetic patients who were classifiable according to their urinary albumin excretion rate and who had been entered into the database of the FinnDiane study by August 1999 participated in the study. Consequently, this case–control, cross-sectional, nationwide multicenter study involved 996 patients comprising their ascertained renal status from 20 referral centers between 1994 and 1999. Detailed clinical data and the classification procedure of the patients into four groups according to urinary albumin excretion rate have been described.22.Pettersson-Fernholm K. Karvonen M.K. Kallio J. et al.Leucine 7 to proline 7 polymorphism in the preproneuropeptide Y is associated with proteinuria, coronary heart disease, and glycemic control in type 1 diabetic patients.Diabetes Care. 2004; 27: 503-509Crossref PubMed Scopus (37) Google Scholar The year of renal replacement therapy, year of initiation of microalbuminuria, and year of DN came from medical records. Prevalence of retinopathy, laser treatment, diabetes diagnosis, antihypertensive medication, and diagnosed coronary heart disease were also obtained from medical records. Informed consent was obtained from all subjects participating in the study; the study protocol followed the Declaration of Helsinki Principles and was approved by all local ethics committees. Systolic and diastolic clinically tested office blood pressure, registered as Korotkoff I and V sounds, was measured in each center with a calibrated mercury sphygmomanometer. Two measurements were performed, each after at least 10 min of rest. PP was calculated as the systolic minus the diastolic value. The serum and urine creatinine levels, estimated glucose disposal rate, urinary albumin concentration, and glycosylated hemoglobin were analyzed as described previously.22.Pettersson-Fernholm K. Karvonen M.K. Kallio J. et al.Leucine 7 to proline 7 polymorphism in the preproneuropeptide Y is associated with proteinuria, coronary heart disease, and glycemic control in type 1 diabetic patients.Diabetes Care. 2004; 27: 503-509Crossref PubMed Scopus (37) Google Scholar GFR was calculated with the Cockcroft–Gault formula.23.Cockcroft D.W. Gault M.H. Prediction of creatinine clearance from serum creatinine.Nephron. 1976; 16: 31-41Crossref PubMed Scopus (13100) Google Scholar Impaired renal function was defined as a creatinine clearance of less than 90 ml min−1 1.73 m−2 according to NKF K/DOQI guidelines.24.Levey A.S. Coresh J. Balk E. et al.National Kidney Foundation practice guidelines for chronic kidney disease: evaluation, classification, and stratification.Ann Intern Med. 2003; 139: 137-147Crossref PubMed Scopus (3670) Google Scholar The genotypes of the polymorphisms in this study were determined from DNA samples extracted from peripherally drawn blood samples. Genotyping was performed by standard polymerase chain reaction and a solid-phase minisequencing method. The polymerase chain reaction primers of the G1675A and the A1818T polymorphisms have been described elsewhere.25.Hiraoka M. Taniguchi T. Nakai H. et al.No evidence for AT2R gene derangement in human urinary tract anomalies.Kidney Int. 2001; 59: 1244-1249Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar For these polymorphisms, the minisequencing primers were 5′-CTGTATTTTGCAAAACTCCT-3′ and 5′-TTATGTTAATTTGTTAGGTC-3′, respectively. Genotyping of the G1675A polymorphism failed in two patients owing to polymerase chain reaction failure. Linkage disequilibrium was analyzed with the Linkage Disequilibrium Analyzer 1.026.Ding K. Zhou K. He F. Shen Y. LDA – a java-based linkage disequilibrium analyzer.Bioinformatics. 2003; 19: 2147-2148Crossref PubMed Scopus (116) Google Scholar and the haplotypes were determined by PHASE version 2.0.1.18.Stephens M. Smith N.J. Donnelly P. A new statistical method for haplotype reconstruction from population data.Am J Hum Genet. 2001; 68: 978-989Abstract Full Text Full Text PDF PubMed Scopus (6426) Google Scholar Males and females were analyzed separately in all analyses. This study was supported by grants from Samfundet Folkhälsan, the Folkhälsan Research Foundation, the Finnish Medical Society, the Wilhelm and Else Stockmann Foundation, the Perklén Foundation, and the Liv och Hälsa Foundation. We acknowledge our laboratory technician Ms Tarja Vesisenaho. Finally, we acknowledge the physicians and nurses in each center participating in the collection of the patients: Central Finland Central Hospital: A Halonen, A Koistinen, P Koskiaho, M Laukkanen, J Saltevo, M Tiihonen; Central Hospital of Aland Islands: A-C Blomqvist, M Forsen, H Granlund, B Nyroos; Central Hospital of Kanta-Hame: P Kinnunen, A Orvola, T Salonen, A Vähänen; Central Hospital of Kymenlaakso: R Paldanius, M Riihelä, L Ryysy; Central Hospital of Lansi-Pohja: P Nyländen, A Sademies; Central Ostrobothnian Hospital District: S Anderson, B Asplund, U Byskata, T Virkkala; City of Turku Health Centre: I Hämäläinen, H Virtamo, M Vähätalo; Kainuu Central Hospital: S Jokelainen, P Kemppainen, A-M Mankinen, M Sankari; Lapland Central Hospital: L Hyvärinen, S Severinkangas, T Tulokas; Mikkeli Central Hospital: A Gynther, R Manninen, P Nironen, M Salminen, T Vänttinen; North Karelian Hospital: U-M Henttula, A Rissanen, H Turtola, M Voutilainen; Paijat-Hame Central Hospital: H Haapamäki, A Helanterä, H Miettinen; Satakunta Central Hospital: M Juhola, P Kunelius, M-L Lahdenmäki, P Pääkkönen, M Rautavirta; Savonlinna Central Hospital: T Pulli, P Sallinen, H Valtonen, A Vartia; Seinajoki Central Hospital: E Korpi-Hyövälti, T Latvala, E Leijala; South Karelia Hospital District: T Hotti, R Härkönen, U Nyholm, R Vanamo; Tampere University Hospital: I Ala-Houhala, T Kuningas, P Lampinen, M Määttä, H Oksala, T Oksanen, K Salonen, H Tauriainen, S Tulokas; Turku Health Center: I Hämäläinen, H Virtamo, M Vähätalo; Turku University Central Hospital: M Asola, K Breitholz, R Eskola, K Metsärinne, U Pietilä, P Saarinen, R Tuominen, S Äyräpää; Vasa Central Hospital: S Bergkulla, U Hautamäki, V-A Myllyniemi, I Rusk.
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