Page of Accepted Preprint first posted on September 0 as Manuscript EJE--0 CYPA polymorphisms in patients with autoimmune Addison s disease, and linkage disequilibrium to HLA risk alleles Ingeborg Brønstad, Beate Skinningsrud, Eirik Bratland, Kristian Løvås,, Dag Undlien,, Eystein Sverre Husebye, & Anette Susanne Bøe Wolff 0 Department of Clinical Science, University of Bergen, 0 Bergen, Norway Department of Medical Genetics, Oslo University Hospital, 00 Oslo, Norway Department of Medicine, Haukeland University Hospital, 0 Bergen, Norway Institute of Medical Genetics, University of Oslo, 0 Oslo, Norway Corresponding author: Ingeborg Brønstad Department of Clinical Science, University of Bergen Laboratory building, th floor 0 Bergen, Norway e-mail: Ingeborg.Bronstad@k.uib.no Telephone: + 0 0 Short title: CYPA in Autoimmune Addison s disease Keywords: CYPA; Autoimmune Addison s disease; Human Leukocyte Antigen; -Hydroxylase Word count: 0 Copyright 0 European Society of Endocrinology.
Page of 0 0 Abstract Objective: Steroid -hydroxylase (OH), encoded by CYPA, is the major autoantigen in autoimmune Addison s disease (AAD). CYPA is located in the region of the human leukocyte antigen (HLA) complex on chromosome p., which harbours several risk alleles for AAD. The objective was to investigate whether CYPA gene variants confer risk of AAD independently of other risk alleles in the HLA loci. Design: DNA samples from Norwegian patients with AAD and 0 healthy controls (HC) previously genotyped for the HLA-A, -B, -DRB, and -DQB and MICA loci were used for genotyping of CYPA. Methods: Genotyping of CYPA was performed by direct sequencing. Linkage of CYPA to the HLA loci was assessed using UNPHASED version.0.0 and PHASE version.. Results: Heterozygotes of the single nucleotide polymorphisms (SNPs) rs, rs, rs, rs and rs were detected significantly more frequently in AAD patients compared to HC (P < 0.00), but all SNPs were in linkage disequilibrium (LD) with high-risk HLA-DRB haplotypes. rsc protected against AAD (Odds ratio = 0., % confidence interval [0.0 0.0], P =. e-0). This SNP was not in LD with HLA loci (P = 0.0), but did not increase protection when considering the effect of HLA-DRB alleles. Mutations causing congenital adrenal hyperplasia were found in heterozygosity in <. % of the cases in both groups. Conclusion: Genotypes of CYPA associated with AAD are in LD with the main autoimmune AAD risk loci HLA-DRB and do not constitute an independent genetic susceptibility locus.
Page of 0 Introduction Autoimmune Addison s disease (AAD) is caused by the destruction of hormone producing cells in the adrenal cortex by autoreactive immunological mechanisms. AAD often occurs together with other organ specific autoimmune disorders, as part of autoimmune polyendocrine syndromes types and (APS- and-, respectively),. APS- is a rare monogenic autosomal recessive disease caused by mutations in the autoimmune regulator (AIRE) gene. Isolated AAD and APS-, however, are thought to be caused by susceptibility variants of multiple genes interacting with environmental triggers. To date, the human leukocyte antigen (HLA) class II haplotypes DRB*0:0 and DRB*0:0 have been shown to be the strongest predisposing genetic factors for AAD with odds ratio (OR) of. and., respectively. Risk is particularly high when these haplotypes are combined (OR = ). The HLA class I genes HLA-A and B (OR =. of HLA-B*0) and MHC-class I related chain A (MICA) also harbour AAD risk alleles -. In addition, several other genes related to the immune system have been associated with AAD, i.e. cytotoxic T lymphocyte antigen- (CTLA-),, protein tyrosine phosphatase non-receptor type (PTPN) 0,, MHC class II transactivator (CIITA), C-type lectin domain family, member A (CLECA), cytochrome P0, family, subfamily B, polypeptide (CYPB ), and programmed death ligand (PD-L). 0 The major autoantigen in AAD is the enzyme steroid -hydroxylase (OH), which is expressed mainly in the adrenal cortex, and circulating autoantibodies (Ab) against OH are present in more than % of patients with AAD in various European cohorts, -. OH is encoded by CYPA, and mutations in this gene are the main cause of congenital adrenal hyperplasia (CAH) 0. Polymorphisms and mutations in CYPA are common,
Page of mainly due to the presence of the structurally related pseudogene CYPAP. Variants in CYPA may potentially have an impact on the immunogenicity of OH and thereby influence the chance of an autoreactive response being directed towards it. Examples of such effects are seen in type diabetes where autoantibody frequencies towards the zinc transporter eight (SLC0A) is dependent on which isoform is expressed. Moreover, different isoforms of the thyroid stimulating hormone receptor (TSHR) vary in their immunogenicity,. The expression of CYPA in thymic medullary cells could potentially be dependent on polymorphisms thereby influencing negative selection of OH-specific T cells. 0 We therefore asked if CYPA is a susceptibility gene for AAD. Previously, two studies have addressed CYPA polymorphisms in relation to risk of developing AAD without finding any disease-specific variants,. However, both studies were underpowered; the largest cohort included patients with AAD. In the present study we analysed the much larger well-characterised Norwegian cohort (n = ). Since CYPA is located on chromosome p. in the HLA-III locus, which is in strong linkage disequilibrium (LD) with the HLA-I and II loci, such a large sample size is required to determine whether potential CYPA variants add to the risk of AAD independently of the HLA loci. 0 Subjects and methods Subjects A total of patients with AAD (% females, % males; mean age years) recruited from the National Norwegian Registry of patients with Addison s disease and 0 anonymous Norwegian blood donors controls (HC) were available for genetic analyses. OH
Page of autoantibody index values as determined by radioimmunoassay (RIA) were available for all patients.aad was diagnosed in patients with adrenal insufficiency with either a positive OH-Ab test, or adrenal insufficiency combined with another endocrine autoimmune disease. Of the AAD patients, % were positive for OH-Ab, and % had APS- (Addison s disease plus autoimmune thyroid disease and/or type diabetes), while % had isolated AAD. 0 Ethics All included patients and blood donors signed a written consent form. The study was approved by the Regional Committee for Medical Ethics of Western Norway, and performed according to the Helsinki Declaration. 0 Genetic analyses DNA was isolated from whole blood using standard commercial kits. The sequence of the whole CYPA gene, including 0 exons and introns, were analysed in patients with AAD. Since the antigenicity of OH most likely would be located in the coding regions, coding polymorphisms and rare variants detected in the patients were sought for in a larger material of AAD patients (n = ) and HC (n = 0). The CYPA gene was amplified in two different fragments using primers discriminating the pseudogene from the functional CYPA gene. The primers specific for CYPA were targeted to the base pair (bp) deletion region in exon and the exon cluster region, which are the major markers for distinguishing the pseudogene from CYPA. The first primer pair amplified a fragment starting with the promoter region and ending with the point mutation region in exon (fragment ); the second primer pair amplified the gene from exon spanning the bp
Page of 0 deletion region to 0 bp of the end from exon 0 (fragment ). Expand High Fidelity PCR system (Roche; Basel, Switzerland) was used for amplifying PCR fragment ; the thermal cycling protocol consisted of an initial denaturation step at ºC for minutes, followed by 0 cycles of denaturation at ºC for seconds, annealing at ºC for 0 seconds and elongation at ºC for. minutes. Then the cycle was repeated 0 times with an increment of seconds for each cycle. The PCR was terminated by an elongation step at ºC for minutes. AmpliTaq Gold with Gene Amp PCR system (Life Technologies, Waltham, Massachusetts, USA ) was used for amplifying PCR fragment ; the thermal cycling conditions were initial denaturation at ºC for 0 min, followed by cycles of ºC for 0 sec, ºC for 0 sec and ºC for. min. The final elongation step was run at ºC for min. Variants and single nucleotide polymorphisms (SNP) of CYPA were identified by direct sequencing using standard conditions on a ABI 0 DNA-Analyzer. Primers (purchased from Eurogentec; Seraing, Liège, Belgium) of PCR and sequencing reactions have been described previously -. Sequencing data were aligned to the CYPA gene sequence (http://www.ncbi.nlm.nih.gov/nuccore/m.). As a quality control to check that the pseudogene was not amplified in the PCR product, we checked that no pseudogene variants were present in the sequences. Copy number of the CYPA gene was determined in AAD patients and HC by duplex real time PCR method 0. The genotyping of HLA-A, -B, -DRB and DQB, and MICA was described previously by. 0 Statistical analyses Chi-square and Fisher s exact tests for determination of significant differences between genotype and allele frequencies of patients and HC, grouped according to age at diagnosis, patients with or without type diabetes or thyroid disease and gender; and calculations of OR with % confidence intervals (CI) were performed by IBM SPSS Statistics 0 using
Page of 0 significance level of P < 0.0. Calculation of Hardy-Weinberg equilibrium (p > 0.00 cutoff) and LD map of the CYPA gene SNPs were conducted by Haploview version.. Global association tests with conditional analysis for allele main effects were performed by UNPHASED version.0.0 to determine if associations with CYPA were independent (P < 0.0) of the HLA loci or not. Haplotype frequencies were generated by PHASE version.. D Agostino and Pearson omnibus normality test was used to test whether the values of OH index and age at diagnosis were distributed in a Gaussian manner; and Kruskal-Wallis test with Dunn s multiple comparison was used for testing differences between CYPA/HLA genotypes or haplotypes and OH antibody index values and mean age at diagnosis using the Graph Pad Prism version.0. Results 0 SNPs and haplotypes of CYPA in AAD patients and healthy controls The common exon variants p._0insl/g.0_0insctg (rs), p.k0r/g.a>g (rs), p.ns/g.a>g (rs), the intron variant g.c>a (rs), and the intron variant g. C>T (rs), were frequently detected in both AAD and HC (Table ). Conversely, p.st/g. G>C (rs) appeared rarely (Table ). Several other silent and intron variants were also detected in both AAD patients and HC (listed in Supplementary Table ), but were not further investigated in the large cohort. For the rs, rs and rs SNPs, the heterozygote genotype occurred significantly (P 0.00) more often in patients with AAD than in HC, compared to the
Page of homozygote genotype of the major alleles (Table ). Haplotype reconstruction of CYPA revealed that AA and +GG (in the order rs/rs/rs, from now denoted as the AA and +GG haplotypes) mainly occurred together (Supplementary Table ), with a strong LD (r > 0., D > 0., Fig. a and b). Frequencies of these haplotypes were significantly different between AAD patients and HC (P = 0.00), where. % AAD patients and. % HC had the haplotype AA, and. % AAD patients and. % HC had the haplotype +GG. Other combinations of these SNPs were seen in. % of AAD patients and.% of HC. 0 Regarding the intron variants rs and rs, the heterozygote genotype and the minor alleles were also more common in AAD than in HC (Table ). The frequency of rsc was.% in the AAD group, compared to.% in HC (OR =., [. -.], P = 0.00). The allele frequencies of rst were.% in AAD and.% in HC (OR =. [.0 -.], P = 0.00). The majority of the rst alleles were seen together with the +GG haplotype, but never with the AA haplotype (Supplementary Table ), and was almost in complete LD with all these three coding SNPs (D > 0., Fig. b). 0 The rsc seemed to be a protective variant to AAD, where the allele frequency of the C allele was.% in HC compared to.% in AAD patients (OR = 0., [0.0 0.0], P =. e-0, Table ). The rsc allele was only seen together with the +GG variant of the rs/rs/rs haplotype (Supplementary Table ), which was in complete LD (D =, Fig. b). The rsc allele in the AAD patients was mainly seen ( of 0 alleles) in the haplotype +CGTCG (in order rs/rs/rs/rs/rs/rs). In the control group, the haplotypes +AGTCG and +AGCCG were more frequent ( and of rsc alleles, respectively, Supplementary Table ).
Page of 0 Are the CYPA associations independent of HLA I and II loci? To this end a global regression analysis was performed to determine if AAD associations of CYPA were independent (P < 0.0) of high risk HLA loci. Table shows that all the CYPA SNPs associations, except rs (P = 0.0), were lost when HLA-DRB was set as conditional marker, suggesting that the HLA-type determines the AAD-risk. The SNPs rs, rs and rs showed independence from the HLA-DQB locus (P = 0.0, P = 0.000 and P = 0.0, respectively), while the rs, rs and rs SNPs were in LD with HLA-DQB (Table ). All the exon variants of CYPA further showed independency to the HLA-A, HLA-B and MICA while the two intron variants were both in LD with HLA-A and MICA. The rs was in LD with HLA-B, whereas rs was not (P = 0.0) (Table ). 0 We next wanted to investigate how CYPA alleles are linked to the HLA-DRB risk alleles. Supplementary Table shows the major CYPA haplotypes (the SNPs rs/rs/rs/rs with haplotype AGA and +GGG) in combination with the most common HLA-DRB haplotypes generated by PHASE. The HLA-DRB*00 mainly occurred together with the +GGG haplotype (i.e.. % and. % of AAD and HC, respectively), while the HLA-DRB*0:0 haplotype was most commonly seen with the AGA variant (. % and. % of AAD and HC cases, respectively (Supplementary Table ). The HLA-DRB* and *0 haplotypes also occurred most frequently together with the +GGG CYPA haplotype, while the HLA-DRB*0:0 was most frequently seen together with AGA (Supplementary Table ). Furthermore, the individuals with heterozygous HLA- DRB*0:0/*0:0 high-risk genotype, were also heterozygote for CYPA AA/+GG in. % AAD patients (n = ) and. % HC (n = ), respectively.
Page 0 of For rs, the HLA-DRB*0:0 haplotype was most frequently reconstructed together with the minor C allele, while the majority of HLA-DRB*0:0, -* and -*0 haplotypes were seen together with the A allele. For HLA-DRB*0:0 both rs alleles were more equally distributed (Supplementary Table ). For rs SNP, the minor T allele was mainly seen together with the HLA-DRB*0:0 haplotype (Supplementary Table ). 0 The majority of the rsc alleles in AAD ( of 0) were reconstructed by PHASE together with the high-risk HLA-DRB*0:0, while the rest with HLA-DRB*0:0 ( of 0) and HLA-DRB*:0 ( of 0). For the HC group, the rsc allele was mainly seen together with the haplotypes HLA-DRB*:0 ( of ),-*0 ( of ) and -*0:0 ( of ). The protective effect of rsc was lost (p > 0.0) when considering the effect of the HLA-DRB haplotypes (Table ). 0 CYPA variants related to biochemical and clinical parameters Although all of the patients in the present study were diagnosed with AAD, % lacked OH-Ab at the time of testing. Our analyses revealed that the frequencies of heterozygous variants of CYPA were higher in the OH-Ab positive group (Supplementary Table ). The mean OH-Ab levels at diagnosis were also higher in patients with the AAD associated genotypes of CYPA, although not statistically significant (Supplementary Table ). Furthermore, the HLA-DRB*0:0 allele was found in. % and. % of OH-Ab positives and negatives, respectively. Likewise, for HLA-DRB*0:0, the allele frequency was. % and. % for OH-Ab positives and negatives. HLA-DRB*0:0 and -* were more common in the OH-Ab negative group at % and 0. %, respectively, compared to. % and. % in the OH-Ab positive group. Finally, the HLA- 0
Page of DRB*0:0/*0:0 genotype was found only in OH-Ab positives. The mean OH-Ab levels at diagnosis were significantly higher in patients with the HLA-DRB*0:0 and - *0:0 haplotypes compared to other haplotypes, and similarly for the HLA- DRB*0:0/*0:0 genotype (p < 0.000). Patients with the HLA-B*0 had significantly higher OH Ab indices at diagnoses compared to patients with other HLA-B haplotypes (p = 0.0), while the OH Ab indices were significantly higher in patients with the HLA-A*0 compared to HLA-A*0 (p = 0.00). The results are summarized in Supplementary Table. 0 The AAD associated CYPA variants were more frequent in patients with onset of the disease before 0 years age than in those with later onset. In addition, the protective variant rsc was not detected in patients with disease onset before 0 years (Supplementary Table ). There was no association between autoimmune thyroid disease of the APS - patients and the CYPA variants. The rst allele was, however, more frequent in APS- patients with type diabetes than in AAD patients without type diabetes (OR =., [.0 -.0], P = 0.0), but there was no such association to the other CYPA variants. There was no association between gender and CYPA variants. 0 Rare variants of CYPA in AAD patients and healthy controls A rare heterozygous coding variant, p. AT/g.G>A, was found in /0 patients with AAD and in /0 HC. All four were also heterozygous for the rare haplotype HLA-B*0 which was absent in other patients and controls. Known disease causing mutations frequently reported in patients with CAH, were also found as heterozygous in both AAD patients and HC: The I splice variant (rsg) was detected in /0 AAD patients and / HC. The
Page of base pair deletion in exon (rs00) was found in one AAD patient and in one HC. The non-classical CAH variant p.vl (rs) was found in /0 AAD patients and / HC, while p.ps (rs) was found in / AAD patients and / HC. No copy number variations were detected in AAD patients or HC; they all had two copies of CYPA. 0 Discussion It is not known why certain proteins, such as OH become autoantigens in organ-specific autoimmune diseases. However, common features are that autoantigens are intracellular enzymes with restricted tissue expression. One possibility is that only altered or modified selfproteins can induce autoreactive immune responses, and that genetic polymorphisms may represent such alterations. By direct sequencing of CYPA in AAD patients we did not find any disease specific risk alleles, but heterozygous genotypes of common CYPA variants were significantly more frequent in AAD patients than in HC. However, we found that all the CYPA SNPs associations, except for rs, were dependent on the HLA-DRB locus, which is the strongest single genetic risk factor of AAD,,,. Therefore, the higher heterozygous genotype frequency of the two main haplotypes of CYPA alleles, AA and +GG, merely reflects the high risk genotype of HLA-DRB*0:0/*0:0. The protective rsc association could also be explained by the linkage to HLA protective variants. 0 The non-coding AAD-associated CYPA variants rs and rs were also dependent of MICA and HLA-A or B. The rst allele was further associated with type diabetes of the APS- patients included in the study. Interestingly, the rsc and
Page of rst alleles have both shown to be in LD with components of the. ancestral haplotype. 0 Our data showed that all of our patients with the DRB*0:0/*0:0 genotype were positive for OH Ab and had the highest OH Ab indices compared to other genotypes. This is consistent of HLA-DRB*0:0/*0:0 genotype association with expression of OH Ab. The corresponding CYPA genotype did not add to the high OH Ab association of HLA- DRB*0:0/*0:0, which underlines the strength of HLA-DRB risk factors. Similarly, we here found a trend for association betweencypa genotypes and age of disease onset. This is in agreement with previous findings of a significant lower mean age of onset in patients who carries the HLA-DRB*0:0/*0:0 genotype. 0 A hypothesis proposes that low protein expression due to genetic polymorphisms might increase the probability of autoreactive T cells to escape from the thymus during negative selection. This has been suggested for the variable number of tandem repeats polymorphism in the gene encoding insulin and its association to production of Ab and autoreactive T cells to insulin in type diabetes, 0. Although the HLA-DRB locus proves to be the highest predisposing factor of AAD, the significance of CYPA heterozygote genotypes is not clear since it is not known if the gene is inherited co-dominantly like the HLA genes. In conclusion, sequencing of the exons and introns of CYPA did not reveal any disease specific risk variants associated with AAD. Instead, CYPA polymorphisms are in LD with the high risk haplotypes of the HLA-DRB loci, and do not independently add to increased risk or protection, but merely reflects the effect of HLA-DRB. Declaration of interest: The authors declare no conflict of interest.
Page of Funding The study was supported by grants from the Regional Health Authorities of Western Norway, Bergen Medical Research Foundation, The Norwegian Research council (project no 0) and the EU FP project Euradrenal (grant number 0). I.B is a PhD student supported by University of Bergen. 0 Acknowledgements We thank Hajirah Muneer Elin Theodorsen and Elisabeth Tombra Halvorsen for excellent organizing of DNA sampling and antibody analyses. We also thank all the patients and their medical doctors who have contribute to the National Norwegian Registry of patients with Addison s disease (ROAS).
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Page of Legend to figures: Figure a) r linkage plot of common variants of CYPA. b) D linkage plot of common variants of CYPA.
Page of Table Differences in genotype frequencies (%) of the most common variants in CYPA detected in patients with autoimmune Addison s disease (AAD) and healthy controls (HC). P values were calculated by Chi square test for x tables and Fisher s exact test on x tables. CYPA SNP Genotype P - value OR % CI P - value rs +/+ +/- -/- +/+ vs +/- AAD () 0 (.) (.) 0.00... 0.000 HC (.) (.) 0 (.0) rs AA AC CC AA vs AC AAD (.) () (.) 0.000.. -. 0.000 HC 0 (.) (.) (.0) rs GG GA AA GG vs GA AAD 0 (.) 0 (.) (.) 0.00... 0.00 HC (.0) (.) (.) rs CC CT TT CC vs CT AAD (.0) (.) 0 (0.) 0.0000.0... e- HC (.) (0.) (.) rs GG GC CC G vs C AAD 0 (.) 0 (.) 0 (0). e- 0. 0.0 0.0. e-0 HC (.0) (.) (.) rs GG GA AA GG vs GA AAD (.) (.) (.0) 0.00... 0.000 HC (.) (.0) (.)
Fisher s exact test Page of
Page of Table Global regression analysis of the HLA and CYPA loci for determination of independent associations in autoimmune Addison s disease calculated by UNPHASED. Test locus Analysis for conditional locus (P - values) A B MICA DRB DQB rs rs rs rs rs rs HLA-A 0.0 0.00 0. 0.0. e- 0.00. e- 0.00. e-.0 e- HLA-B. e-. e- 0.0. e-. e-.0 e-0. e-. e-.0 e-0. e- MICA.0 e- 0.000 0.00. e-. e-. e-. e-.0. e-. e-. e- HLA-DRB. e-. e-. e-. e-. e-. e-. e-0. e-. e-. e- HLA-DQB. e-0. e-. e- 0.0. e-. e-. e-. e-.0 e-0. e- rs. e-. e-. e- 0.0 0.0 0.00 0.0. e- 0.0 0. rs 0.0 0. 0. 0. 0.0 0.000 0.000 0.0 0.00 0.000 rs 0.000. e- 0.000 0. 0.0 0. 0.00. e- 0. 0. rs 0. 0.0 0. 0. 0.. e- 0.. e- 0.000. e- rs.0 e-. e-. e- 0.0 0.000.0 e-. e-. e-. e-. e- rs.0 e-.0 e-. e- 0. 0.0 0. 0.00 0.. e- 0.0
Page of Table Haplotype reconstruction of protective and high risk alleles of HLA-DRB and rs. P values were calculated by Fisher s exact test. HLA-DRB rs AAD HC Odds Ratio % Confidence Interval P-Value *0 C *0 G *00 C *00 G *0 C 0 *0 G *00 G 0 *00 C 0 0.0 0.0. 0. 0.0 0...0 0. 0.00.0 0.0 0. 0.0. 0.
Page of xmm ( x DPI)