Published ahead of print on August 5, 2004, doi:10.1164/rccm.200404-481OC
© 2004 American Thoracic Society doi: 10.1164/rccm.200404-481OC
The Clara Cell10 Adenine38Guanine Polymorphism and Sarcoidosis Susceptibility in Dutch and Japanese SubjectsHeart Lung Center Utrecht, Utrecht; Departments of Pulmonology and Clinical Chemistry, St. Antonius Hospital, Nieuwegein, The Netherlands; Clinical Genomics Group, Royal Brompton Hospital and National Heart and Lung Institute, Imperial College of Science, Technology and Medicine, London, United Kingdom; and Department of Respiratory Medicine, Central Clinic Kyoto, Graduate School of Medicine, Kyoto University, Kyoto, Japan Correspondence and requests for reprints should be addressed to Jules M. M. van den Bosch, M.D., Ph.D., Department of Pulmonology, St. Antonius Hospital, Koekoekslaan 1, Nieuwegein, 3435 CM The Netherlands. E-mail: j.vandenbosch{at}antonius.net
CC10 (CC16, uteroglobin) is a pulmonary protein postulated to play a counter regulatory role in sarcoidosis pathogenesis. The adenine38guanine (A38G) polymorphism of the encoding CC10 gene (SCGB1A1) is functional. Recently, an association between the low CC10 producing 38A allele and sarcoidosis susceptibility has been reported in Japanese patients from Hokkaido. The aim of the present study was to confirm this association in a clinically well characterized population of Dutch white and Kyoto Japanese patients with sarcoidosis and control subjects. No difference in genotype or allele frequency was found between patients with sarcoidosis and control subjects in either ethnic population. Remarkably, however, a significant difference was found between the control subjects from Kyoto and Hokkaido, but not between the Japanese groups of patients with sarcoidosis. Furthermore, review of previously published A38G genotyping results showed a consistent difference in CC10 A38G allele frequencies between whites and Japanese subjects. We conclude that the CC10 A38G polymorphism does not influence sarcoidosis susceptibility in Dutch whites or in Japanese subjects from Kyoto. This stresses the importance of studying the influence of polymorphisms on disease susceptibility in multiple ethnically and geographically distinct disease and control populations before reaching conclusions.
Key Words: Clara cell secretory protein 10 • uteroglobin • polymorphism • sarcoidosis
Clara cell secretory protein 10 (CC10) is a protein with a molecular weight of approximately 10 kD produced by nonciliated bronchiolar Clara cells (1). Human CC10 is homologous to Clara cell secretory protein 16 (CC16) and uteroglobin (UGB) (1). Although its exact role has yet to be determined, there are arguments for believing that CC10 serves as an immunosuppressive mediator. CC10 interferes with IFN- Sarcoidosis is a complex granulomatous disease thought to be caused by an unknown antigenic stimulus from the environment in combination with genetic susceptibility (5, 6). Higher values of serum CC10 levels were found in patients presenting with pulmonary infiltrates on chest radiograph compared with those without parenchymal involvement (7, 8). Interestingly, high bronchoalveolar lavage fluid and serum CC10 levels were found to be associated with a favorable prognosis in this disease (9). There is growing evidence for the contribution of genetic polymorphisms to interindividual differences in the regulatory mechanisms of protein production. Therefore, certain cytokine genotypes might increase disease susceptibility. Laing and colleagues were the first to describe an adenine (A) to guanine (G) substitution in the promoter region of the gene encoding CC10 (UGB A38G; current gene symbol: SCGB1A1) (10). Compared with G homozygotes (38GG), those homozygous and heterozygous for the polymorphic variant (38AA and 38AG, respectively) had an increased risk of developing asthma (10). Furthermore, the 38AA genotype was associated with significantly reduced plasma CC16 levels in both individuals with and without asthma (11). Also, in our patients with sarcoidosis, 38A-allele carriers showed significantly reduced levels compared with non–A-allele carriers (12). Therefore, it is believed that the CC10 A38G polymorphism is functional and plays a role in regulating pulmonary inflammation. Recently, Ohchi and colleagues investigated the A38G variant in Japanese patients with sarcoidosis (13). They found an increased carriership of the 38A allele in Hokkaido patients compared with control subjects from the same region in northern Japan, suggesting a contribution in the genetics underlying sarcoidosis susceptibility (13). The aim of the present study was to validate this association in clinically well defined groups of patients with sarcoidosis from The Netherlands and from Japan. Some of the results of this study have previously been reported in the form of an abstract (12).
One hundred thirty-eight unrelated and randomly selected Dutch white patients with sarcoidosis (78 men, 60 women; mean age ± SD: 37 ± 10 years) were included in the study. In 97 patients, the diagnosis of sarcoidosis was established when clinical findings were supported by histologic evidence, and after exclusion of other known causes of granulomatosis. Forty-one patients presented with the classic Löfgren's syndrome of fever, erythema nodosum, bilateral hilar lymphadenopathy, and joint symptoms. The diagnosis in these patients was mostly made without biopsy proof (14). Verbal and written patient consent was obtained from all subjects, and authorization was given by the Ethics Committee of the St. Antonius Hospital, Nieuwegein (Utrecht region). The Dutch control subjects comprised 114 white donors from the Blood Transfusion Service in Utrecht, which takes donors mainly from the Utrecht region. All donors were routinely checked for health before donation and gave their written consent. One hundred unrelated Japanese patients with sarcoidosis from the Kyoto region seen at Central Clinic Kyoto were randomly included in the study. In all patients the diagnosis of sarcoidosis was established in keeping with the diagnostic criteria used for the Dutch patients. One hundred seventeen healthy individuals from the Kyoto region were included as control subjects. All subjects gave their informed consent. The CC10 A38G polymorphism was determined using sequence-specific primers and polymerase chain reaction (PCR) that utilizes SSPs with 3'-end mismatches and identifies the presence of specific allelic variants through PCR amplification. We used the sequence-specific forward primers 5'-CAG AGA CGG AAC CAG AGA CA and 5'-AGA GAC GGA ACC AGA GAC G in combination with the consensus reverse primer 5'-TCC TGA GAG TTC CTA AGT CC at a final concentration of 30 ng/µl, with an expected PCR product size of 150 and 151 base pairs, respectively. In the primer mix, we included the control primers 5'-TGC CAA GTG GAG CAC CCA A and 5'-GCA TCT TGC TCT GTG CAG AT at a final concentration of 2 ng/µl. PCR reactions were run as previously described at a final volume of 13 µl overlaid with 10 µl of mineral oil (15). The presence of an allele-specific band of the expected size, in conjunction with a control band, was considered to be positive evidence for each particular allele. The absence of an allele-specific band and the presence of a control band were considered to be evidence for the absence of an allele.
The genotype frequencies and the frequency of an allele in the chromosomal pool of each population (allele frequency) were determined by direct counting for both control and sarcoidosis groups. All genotype frequencies were tested for Hardy-Weinberg equilibrium. A classical
Allele frequencies of the CC10 A38G promoter polymorphism in the Dutch and Kyoto Japanese patients with sarcoidosis and the control subjects are summarized in Table 1. Both populations were in Hardy-Weinberg equilibrium for all genotypes. No differences in genotype and allele frequencies were observed between Dutch patients and control subjects or between patients and control subjects from Kyoto. Furthermore, subgroup analysis within the Dutch patients with sarcoidosis did not reveal significant differences in allele frequencies between Löfgren and non-Löfgren cases.
The difference in the Dutch cohort for the CC10 A38G A allele was 0.330–0.338 = –0.008, and the 95% confidence interval of that small difference was 0.074 to –0.091. In the Kyoto cohort this difference was –0.012 with a 95% confidence interval of 0.08 to –0.104; and in the Hokkaido cohort this difference was 0.102 with a 95% confidence interval of 0.16 to –0.04. Equivalence in the width of these intervals, i.e., similar accuracy of the estimates in the three cohorts, indicated sufficient study power to detect differences.
The 38A allele frequency in our Kyoto control subjects was significantly higher compared with the frequency in Hokkaido control subjects reported by Ohchi and colleagues (
A significant difference was observed in allele frequency distribution between Dutch and Japanese patients with sarcoidosis from Hokkaido ( A review of the literature on the CC10 A38G polymorphism in white control subjects (from Australia, the UK, and Germany) showed results similar to those of the Dutch, further supporting the observation of an essential racial difference in A38G allele frequencies between whites and Japanese subjects (Table 1) (10, 18, 19).
CC10, the predominant product of Clara cells, lines the bronchiolar epithelium and is thought to protect the respiratory tract from local inflammation through its immunosuppressive actions (1). The A38G promoter is potentially of great immunogenetic interest, as there is in vivo evidence that it accounts for interindividual variations in CC10 production (11). Recently, a study from Ohchi and colleagues demonstrated that the 38A allele was associated with sarcoidosis susceptibility in Hokkaido Japanese (13). The present study, however, in clinically well defined sarcoidosis patients from The Netherlands and the Kyoto region in Japan, could not confirm these findings. Notably, the 38A-allele frequency in Hokkaido control subjects was significantly lower compared with Kyoto control subjects, whereas the 38A-allele frequency in both populations with sarcoidosis was similar. Studies on the same polymorphism in other Japanese populations (from Niigata and Kitakyushu) showed genotyping results similar to those of our Kyoto controls. Niigata, Kitakyushu, and Kyoto are all located in the central and southern part of Japan, and Hokkaido is an island in northern Japan, which may suggest a geographically determined genetic difference within Japan. Alternatively, as already indicated by Iannuzzi, gene environment interactions may be different in northern and southern parts of Japan (20). But if this is true, then the results found by Ohchi and colleagues, and interpreted in the context of the results presented in this paper, indicate that the absence of the 38A allele actually protects against sarcoidosis in northern Japanese (13). Another important finding in this study is the remarkably consistent A38G allele distribution in four different white populations from The Netherlands, Germany, the UK and Australia. This suggests strong genetic homogeneity for CC10 across northern Europe (and Australia, where ancestry is from the same part of Europe). Furthermore, comparing these results with those from the Kyoto control subjects and the Japanese control subjects from Niigata and Kitakyushu showed a clear racial difference in the A38G distribution between whites and Japanese. This information is crucial for geneticists interested in the unraveling of common complex inflammatory pulmonary conditions, in which counter regulation by CC10 might be important. Our study highlights one of the pivotal aspects of genetic association studies, i.e., replication of a reported genetic association between a candidate gene and a disease like sarcoidosis. Subsequent testing of the reported findings in one or, even better, more ethnically distinct populations is still an essential strategy in this respect. A thorough review of the various problems of reporting genetic associations with complex diseases, e.g., replication of genetic associations before declaration of evidence as convincing, was recently given by Colhoun and colleagues (21). We conclude that the CC10 A38G polymorphism does not influence disease susceptibility in Dutch whites or Japanese subjects with sarcoidosis from Kyoto. We show significant differences in the allele frequencies of this polymorphism between Japanese and whites. The fact that no association between the CC10 38A allele and sarcoidosis susceptibility was found, stresses that polymorphisms need to be studied in multiple ethnical and geographic distinct populations before reaching conclusions.
The authors thank Natalie Pot and Adrian Kruit for their technical support.
Supported by a grant from AstraZeneca (to R.J.). Conflict of Interest Statement: R.J. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; H.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; J.C.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; H.J.T.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; R.M.d.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; R.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; M.Y. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; S.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; T.I. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; K.I.W. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; S.N. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; J.M.M.v.d.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this article. Received in original form April 9, 2004; accepted in final form July 29, 2004
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TOP
ABSTRACT
METHODS
and tumor necrosis factor-
–mediated actions and diminishes their biological activity (
2 test was used for a 2 x 2 table, which delivers (R – 1) (C – 1) = 1 degree of freedom (df) (SPSS for Windows; SPSS Inc., Chicago, IL). Fisher exact test was used when the underlying criteria for the