shinyseg: a web application for flexible cosegregation and sensitivity analysis.

MOTIVATION: Cosegregation analysis is a powerful tool for identifying pathogenic genetic variants, but its implementation remains challenging. Existing software is either limited in scope or too demanding for many end users. Moreover, current solutions lack methods for assessing the robustness of cosegregation evidence, which is important due to its reliance on uncertain estimates. RESULTS: We present shinyseg, a comprehensive web application for clinical cosegregation analysis. Our app streamlines penetrance specification based on either liability classes or epidemiological data such as risks, hazard ratios, and age of onset distribution. In addition, it incorporates sensitivity analyses to assess the robustness of cosegregation evidence, and offers support in clinical interpretation. AVAILABILITY AND IMPLEMENTATION: The shinyseg app is freely available at https://chrcarrizosa.shinyapps.io/shinyseg, with documentation and complete R source code on https://chrcarrizosa.github.io/shinyseg and https://github.com/chrcarrizosa/shinyseg.

Coexistence of Congenital Adrenal Hyperplasia and Autoimmune Addison's Disease.

Background: Underlying causes of adrenal insufficiency include congenital adrenal hyperplasia (CAH) and autoimmune adrenocortical destruction leading to autoimmune Addison's disease (AAD). Here, we report a patient with a homozygous stop-gain mutation in 3ß-hydroxysteroid dehydrogenase type 2 (3ßHSD2), in addition to impaired steroidogenesis due to AAD. Case Report: Whole exome sequencing revealed an extremely rare homozygous nonsense mutation in exon 2 of the HSD3B2 gene, leading to a premature stop codon (NM_000198.3: c.15C>A, p.Cys5Ter) in a patient with AAD and premature ovarian insufficiency. Scrutiny of old medical records revealed that the patient was initially diagnosed with CAH with hyperandrogenism and severe salt-wasting shortly after birth. However, the current steroid profile show complete adrenal insufficiency including low production of pregnenolone, dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEA-S), without signs of overtreatment with steroids. Conclusion: To the best of our knowledge, this is the first description of autoimmune adrenalitis in a patient with 3ßHSD2 deficiency and suggests a possible association between AAD and inborn errors of the steroidogenesis.

Identification and characterization of rare toll-like receptor 3 variants in patients with autoimmune Addison's disease.

Autoimmune Addison's disease (AAD) is a classic organ-specific autoimmune disease characterized by an immune-mediated attack on the adrenal cortex. As most autoimmune diseases, AAD is believed to be caused by a combination of genetic and environmental factors, and probably interactions between the two. Persistent viral infections have been suggested to play a triggering role, by invoking inflammation and autoimmune destruction. The inability of clearing infections can be due to aberrations in innate immunity, including mutations in genes involved in the recognition of conserved microbial patterns. In a whole exome sequencing study of anonymized AAD patients, we discovered several rare variants predicted to be damaging in the gene encoding Toll-like receptor 3 (TLR3). TLR3 recognizes double stranded RNAs, and is therefore a major factor in antiviral defense. We here report the occurrence and functional characterization of five rare missense variants in TLR3 of patients with AAD. Most of these variants occurred together with a common TLR3 variant that has been associated with a wide range of immunopathologies. The biological implications of these variants on TLR3 function were evaluated in a cell-based assay, revealing a partial loss-of-function effect of three of the rare variants. In addition, rare mutations in other members of the TLR3-interferon (IFN) signaling pathway were detected in the AAD patients. Together, these findings indicate a potential role for TLR3 and downstream signaling proteins in the pathogenesis in a subset of AAD patients.

Childhood lung function and the association with ß2-adrenergic receptor haplotypes.

AIM: To determine associations between ADRB2 polymorphisms and lung function through childhood, and possible modification by gender, pet keeping or tobacco smoke. METHODS: Four ADRB2 single nucleotide polymorphisms (rs1042711, rs1042713, rs1042714 and rs1800888) were genotyped in 953 children from the prospective birth cohort 'Environment and Childhood Asthma' study and analysed for association with flow-volume parameters at birth (tidal breathing) and at 10 years of age (maximally forced), stratified by environmental exposures. RESULTS: The risk of reduced lung function was reduced in 10-year-old children carrying the most common ADRB2 haplotype (CGGC) (OR 0.45 (95% CI 0.25, 0.82)), whereas there was no association between lung function at birth and ADRB2 haplotypes. Tobacco smoke exposure, gender and pet keeping did not significantly interact with the haplotypes in influencing lung function. CONCLUSION: This study demonstrates a possible protective effect by the ADRB2 haplotype I (CGGC) on reduced FEV1 in 10-year-old children, whereas no ADRB2 geno-/haplotypes were significantly associated with neonatal lung function. The ADRB2 gene thus appears to contribute to lung function development in childhood, independently of smoking, pets and gender.

Pet keeping and tobacco exposure influence CD14 methylation in childhood.

BACKGROUND: Several CD14 gene-environment interactions in relation to the development of allergic diseases have been reported, but the underlying biological mechanisms are unclear. We recently showed that CD14 methylation increased during childhood, parallelling a decreased impact of CD14 polymorphisms on soluble CD14 levels. Here, we aim to explore whether environmental stimuli during childhood affects CD14 methylation, thereby providing a biological mechanism through which environment may modulate genetic effect. METHODS: CD14 methylation levels were quantified in 157 children from the prospective Environment and Childhood Asthma birth cohort at ages 2 and 10. Associations between CD14 methylation levels and house dust levels of endotoxin, ß(1,3)-glucans (at 2 yr only), allergens (dog, cat, and house dust mite), pet keeping and tobacco smoke exposure (TSE; questionnaire data) at 2 and 10 yr were explored. RESULTS: Children in homes without pets had larger increases in CD14 methylation through childhood (2-10 yr) compared with children with pets (2.1% increase (p = 0.003) vs. 0.4% decrease (n.s.), global p = 0.04). At 10 yr of age, lower CD14 methylation values were found in children with pets compared with children without pets at both 2 and 10 yr (5.4% vs. 7.5% [p = 0.02]). A similar trend was detected for TSE; children not exposed show larger increases in CD14 methylation, most pronounced in school-age girls exposed vs. not exposed to tobacco (5.5% vs. 7.5% methylation, p = 0.037). CONCLUSION: Pet keeping and TSE appears to limit increase in CD14 methylation from 2 to 10 yr of age. This may partly explain the diverging CD14 allele associations with allergic diseases detected in different environments.

Limitations and possibilities of low cell number ChIP-seq.

BACKGROUND: Chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq) offers high resolution, genome-wide analysis of DNA-protein interactions. However, current standard methods require abundant starting material in the range of 1-20 million cells per immunoprecipitation, and remain a bottleneck to the acquisition of biologically relevant epigenetic data. Using a ChIP-seq protocol optimised for low cell numbers (down to 100,000 cells/IP), we examined the performance of the ChIP-seq technique on a series of decreasing cell numbers. RESULTS: We present an enhanced native ChIP-seq method tailored to low cell numbers that represents a 200-fold reduction in input requirements over existing protocols. The protocol was tested over a range of starting cell numbers covering three orders of magnitude, enabling determination of the lower limit of the technique. At low input cell numbers, increased levels of unmapped and duplicate reads reduce the number of unique reads generated, and can drive up sequencing costs and affect sensitivity if ChIP is attempted from too few cells. CONCLUSIONS: The optimised method presented here considerably reduces the input requirements for performing native ChIP-seq. It extends the applicability of the technique to isolated primary cells and rare cell populations (e.g. biobank samples, stem cells), and in many cases will alleviate the need for cell culture and any associated alteration of epigenetic marks. However, this study highlights a challenge inherent to ChIP-seq from low cell numbers: as cell input numbers fall, levels of unmapped sequence reads and PCR-generated duplicate reads rise. We discuss a number of solutions to overcome the effects of reducing cell number that may aid further improvements to ChIP performance.

DNA methylation and gene expression changes in monozygotic twins discordant for psoriasis: identification of epigenetically dysregulated genes.

Monozygotic (MZ) twins do not show complete concordance for many complex diseases; for example, discordance rates for autoimmune diseases are 20%-80%. MZ discordance indicates a role for epigenetic or environmental factors in disease. We used MZ twins discordant for psoriasis to search for genome-wide differences in DNA methylation and gene expression in CD4(+) and CD8(+) cells using Illumina's HumanMethylation27 and HT-12 expression assays, respectively. Analysis of these data revealed no differentially methylated or expressed genes between co-twins when analyzed separately, although we observed a substantial amount of small differences. However, combined analysis of DNA methylation and gene expression identified genes where differences in DNA methylation between unaffected and affected twins were correlated with differences in gene expression. Several of the top-ranked genes according to significance of the correlation in CD4(+) cells are known to be associated with psoriasis. Further, gene ontology (GO) analysis revealed enrichment of biological processes associated with the immune response and clustering of genes in a biological pathway comprising cytokines and chemokines. These data suggest that DNA methylation is involved in an epigenetic dysregulation of biological pathways involved in the pathogenesis of psoriasis. This is the first study based on data from MZ twins discordant for psoriasis to detect epigenetic alterations that potentially contribute to development of the disease.

A mild form of Mucopolysaccharidosis IIIB diagnosed with targeted next-generation sequencing of linked genomic regions.

Next-generation sequencing (NGS) techniques have already shown their potential in the identification of mutations underlying rare inherited disorders. We report here the application of linkage analysis in combination with targeted DNA capture and NGS to a Norwegian family affected by an undiagnosed mental retardation disorder with an autosomal recessive inheritance pattern. Linkage analysis identified two loci on chromosomes 9 and 17 which were subject to target enrichment by hybridization to a custom microarray. NGS achieved 20-fold or greater sequence coverage of 83% of all protein-coding exons in the target regions. This led to the identification of compound heterozygous mutations in NAGLU, compatible with the diagnosis of Mucopolysaccharidosis IIIB (MPS IIIB or Sanfilippo Syndrome type B). This diagnosis was confirmed by demonstrating elevated levels of heparan sulphate in urine and low activity of α-N-acetyl-glucosaminidase in cultured fibroblasts. Our findings describe a mild form of MPS IIIB and illustrate the diagnostic potential of targeted NGS in Mendelian disease with unknown aetiology.

Alpha-nicotinic acetylcholine receptor and tobacco smoke exposure: effects on bronchial hyperresponsiveness in children.

BACKGROUND: The CHRNA 3 and 5 genes on chromosome 15 encode the alpha subunits of the nicotinic acetylcholine receptor, mediating airway cholinergic activity. Polymorphisms are associated with cigarette smoking, chronic obstructive pulmonary disease, and lung cancer. AIMS: To determine possible associations between CHRNA 3/5 SNP rs8034191 and asthma or lung function in children in one local and one replicate multinational population, and assess if tobacco smoke modified the associations. MATERIALS AND METHODS: The rs8034191 SNP genotyped in 551 children from the environment and childhood asthma (ECA) birth cohort study in Oslo, Norway, and in 516 families from six European centers [the Genetics of Asthma International Network (GAIN) study] was tested for genotypic or allelic associations to current or history of asthma, allergic sensitization (≥ one positive skin prick tests), bronchial hyperresponsiveness (BHR), and lung function (FEV(1%) of predicted and FEV(1) /FVC ratio over/ below the 5th percentile). RESULTS: Although the TT and CT genotypes at SNP rs 8034191 were overall significantly associated with BHR (OR = 3.9, 95% CI 1.5-10.0, p = 0.005), stratified analyses according to exposure to maternal smoking in-utero or indoor smoking at 10 yrs of age showed significant association (OR = 4.4, 95% CI 1.5-12.6, p = 0.006 and OR 5.6, 95% CI 1.7-18.5, p = 0.004, respectively) only in the non-exposed and not in exposed children. The SNP-BHR association was replicated in the non-tobacco-smoke-exposed subjects in one of the GAIN centers (BHR associated with the T allele (p = 0.034)), but not in the collated GAIN populations. Asthma, allergic sensitization, and lung function were not associated with the rs8034191 alleles. CONCLUSION: An interaction between tobacco smoke exposure and a CHRNA3/5 polymorphism was found for BHR in children, but CHRNA3/5 was not associated with asthma or lung function.

Extensive variation and low heritability of DNA methylation identified in a twin study.

Disturbance of DNA methylation leading to aberrant gene expression has been implicated in the etiology of many diseases. Whereas variation at the genetic level has been studied extensively, less is known about the extent and function of epigenetic variation. To explore variation and heritability of DNA methylation, we performed bisulfite sequencing of 1760 CpG sites in 186 regions in the human major histocompatibility complex (MHC) in CD4+ lymphocytes from 49 monozygotic (MZ) and 40 dizygotic (DZ) twin pairs. Individuals show extensive variation in DNA methylation both between and within regions. In addition, many regions also have a complex pattern of variation. Globally, there appears to be a bimodal distribution of DNA methylation in the regions, but a significant fraction of the CpG sites are also heterogeneously methylated. Classification of regions into CpG islands (intragenic and intergenic), 5' end of genes not associated with a defined CpG island, conserved noncoding regions, and random CpG sites shows region-type differences in variation and heritability. Analyses revealed slightly lower intra-pair differences among MZ than among DZ pairs, suggesting some genetic influences on DNA methylation variation, with most of the variance attributed to nongenetic factors. Overall, heritability estimates of DNA methylation were low. Our heritability estimates are, however, somewhat deflated due to the presence of batch effects that artificially inflate the estimates of shared environment.

Multiple loci in the HLA complex are associated with Addison's disease.

CONTEXT: A strong association between autoimmune Addison's disease (AAD) and major histocompatibility complex class II-encoded HLA-DRB1-DQA1-DQB1 haplotypes is well known. Recent evidence from other autoimmune diseases has suggested that class I-encoded HLA-A and HLA-B gene variants confer HLA-DRB1-DQA1-DQB1-independent effects on disease. OBJECTIVE: We aimed to explore AAD predisposing effects of HLA-A and -B and further investigate the role of MICA and HLA-DRB1-DQA1-DQB1 in a much larger material than has previously been studied. DESIGN: HLA-A, -B, -DRB1, and -DQB1 and a microsatellite in MICA were genotyped in 414 AAD patients and 684 controls of Norwegian origin. RESULTS: The strongest association was observed for the DRB1 locus, in which the DRB1*03:01 and DRB1*04:04 conferred increased risk of AAD, particularly in a heterozygous combination [odds ratio 22.13; 95% confidence interval (11.39-43.98); P = 6 × 10(-20)]. After conditioning on DRB1, association with AAD was still present for HLA-B and MICA, suggesting the presence of additional risk factors. CONCLUSIONS: The major histocompatibility complex harbors multiple risk loci for AAD, in which DRB1 appears to represent the main risk factor.

Does the relative risk for type 1 diabetes conferred by HLA-DQ, INS, and PTPN22 polymorphisms vary with maternal age, birth weight, or cesarean section?

BACKGROUND AND OBJECTIVE: Maternal age at birth, birth weight, and cesarean section has been associated with a weak but significant increase in risk of type 1 diabetes. The objective was to assess whether the relative risk for type 1 diabetes conferred by established susceptibility loci human leukocyte antigen (HLA)-DQ, INS, and PTPN22 differed depending on these perinatal factors. METHODS: We employed a case-control study with 456 cases of type 1 diabetes diagnosed before 15 yr of age and 1377 population-based control children. HLA genotypes were divided into high to moderate risk (DQ8/DQ2, DQ8/DQ8, DQ8/X, DQ2/DQ2) vs. all other genotypes. Case-only analysis using logistic regression was used to test for significant interaction. RESULTS: There was no significant difference in the relative risks conferred by HLA-DQ, INS, or PTPN22 by maternal age, birth weight, or mode of delivery, except the relative risk conferred by PTPN22 which was 2.11 [95% confidence interval (CI): 1.64-2.72] for those born vaginally and 0.99 (95% CI: 0.50-1.99) for those born by cesarean section [p(interaction) = 0.028]. CONCLUSION: The relative risks conferred by the three established susceptibility genes investigated here were independent of the perinatal factors, apart from a possible interaction between PTPN22 and mode of delivery.

A CLEC16A variant confers risk for juvenile idiopathic arthritis and anti-cyclic citrullinated peptide antibody negative rheumatoid arthritis.

OBJECTIVE: Variants in CLEC16A have conferred susceptibility to autoimmune diseases in genome-wide association studies. The present work aimed to investigate the locus' involvements in juvenile idiopathic arthritis (JIA) and further explore the association with rheumatoid arthritis (RA), type 1 diabetes (T1D) and Addison's disease (AD) in the Norwegian population. METHODS: Three single nucleotide polymorphisms (SNPs) were genotyped in patients with RA (n=809), JIA (n=509), T1D (n=1211) and AD (n=414) and in healthy controls (n=2149). RESULTS: All diseases were associated with CLEC16A, but with different SNPs. The intron 22 SNP, rs6498169, was associated with RA (p=0.006) and JIA (p=0.016) and the intron 19 SNPs, rs12708716/rs12917716, with T1D (p=1x10-5) and AD (p=2x10-4). The RA association was confined to the anti-cyclic citrullinated peptide antibody (anti-CCP) negative subgroup (p=2x10-4). CONCLUSION: This is the first report of a CLEC16A association with JIA and a split of the RA association according to anti-CCP status. Different causative variants underlie the rheumatic versus the organ specific diseases.

An inverse association between history of childhood eczema and subsequent risk of type 1 diabetes that is not likely to be explained by HLA-DQ, PTPN22, or CTLA4 polymorphisms.

BACKGROUND: Established genetic susceptibility loci for type 1 diabetes are important in immune regulation and may play a role also in atopic disorders, potentially explaining the inverse association between childhood eczema and subsequent risk for type 1 diabetes previously reported. OBJECTIVE: We aimed to directly assess whether HLA-DQ, CTLA4, and PTPN22 genes could explain the putative association between childhood eczema and lower subsequent risk of type 1 diabetes observed in several case-control studies. METHODS: We designed a case-control study with 339 incident cases of type 1 diabetes identified in the Norwegian childhood diabetes registry, and 985 population-based control children. DNA was collected, and physician-diagnosed childhood eczema was ascertained by a questionnaire administered to the parents of children with and without type 1 diabetes. RESULTS: The previously reported association between childhood eczema and lower risk of type 1 diabetes was confirmed (odds ratio,OR, 0.61, 95% confidence interval, CI, 0.40-0.95] and this was consistent in subgroups defined by HLA-DQ, CTLA4, and PTPN22 genotypes. The OR was essentially not influenced by adjustment for genetic variation at these loci (OR simultaneously adjusted for the three genetic loci: 0.55, 95% CI: 0.32-0.92). The ratio of the unadjusted to adjusted OR was 1.12, with a corresponding 95% CI from 0.84 to 1.50. CONCLUSION: In this first study of its kind, we demonstrated directly that the observed inverse association between childhood eczema and type 1 diabetes is not likely to be explained by the established diabetes susceptibility genes HLA-DQ, CTLA4, or PTPN22.

T cell responses to steroid cytochrome P450 21-hydroxylase in patients with autoimmune primary adrenal insufficiency.

CONTEXT: Autoimmune Addison's disease is thought to result from T cell mediated autoimmunity. Autoantibodies against the steroidogenic cytochrome P450 enzyme 21-hydroxylase (21OH) are found in most patients, and 21OH is therefore a likely target for antigen-specific T cells. OBJECTIVE: The aim was to study cellular immunity to 21OH and its associations with 21OH autoantibodies and human leukocyte antigen alleles in autoimmune Addison's disease. DESIGN/PATIENTS: Peripheral blood mononuclear cells were collected from 33 patients with autoimmune Addison's disease and 21 controls. Cellular proliferation and production of cytokines in response to stimulation with 21OH or 21OH-derived peptides were tested. RESULTS: Cellular proliferation (P = 0.0009) and secretion of interferon-gamma (P < 0.0001) in response to 21OH was significantly higher in patients compared to healthy controls and associated with the presence of 21OH autoantibodies (P = 0.0052). Furthermore, the 21OH-specific production of interferon-gamma was enhanced in the presence of 21OH autoantibodies. This effect was partially inhibited by antibodies against the Fc receptor for IgG, CD32. Moreover, mature dendritic cells proved superior to the other antigen-presenting cells in invoking cellular responses to 21OH. An association between cellular immunity to 21OH and the high-risk HLA genotype for Addison's disease, DRB1*0301-DQ2/DRB1*0404-DQ8, was observed (P = 0.0089). Finally, a significant association between the DRB1*0404-DQ8 haplotype and cellular responses to a 21OH-derived peptide predicted to bind to DRB1*0404 was detected (P = 0.0055). CONCLUSION: Patients with autoimmune Addison's disease have circulating 21OH-specific T cells, with amino acids 342-361 of 21OH possibly constituting a disease-specific epitope presented by HLA-DRB1*0404.

Clinical, immunological, and genetic features of autoimmune primary adrenal insufficiency: observations from a Norwegian registry.

OBJECTIVE: Primary adrenal insufficiency [Addison's disease (AD)] is rare, and systematic studies are few, mostly conducted on small patient samples. We aimed to determine the clinical, immunological, and genetic features of a national registry-based cohort. DESIGN: Patients with AD identified through a nationwide search of diagnosis registries were invited to participate in a survey of clinical features, health-related quality of life (HRQoL), autoantibody assays, and human leukocyte antigen (HLA) class II typing. RESULTS: Of 664 registered patients, 64% participated in the study. The prevalence of autoimmune or idiopathic AD in Norway was 144 per million, and the incidence was 0.44 per 100,000 per year (1993-2007). Familial disease was reported by 10% and autoimmune comorbidity by 66%. Thyroid disease was most common (47%), followed by type 1 diabetes (12%), vitiligo (11%), vitamin B12 deficiency (10%), and premature ovarian insufficiency (6.6% of women). The mean daily treatment for AD was 40.5 mg cortisone acetate and 0.1 mg fludrocortisone. The mean Short Form 36 vitality scores were significantly diminished from the norm (51 vs. 60), especially among those with diabetes. Concomitant thyroid autoimmunity did not lower scores. Anti-21-hydroxylase antibodies were found in 86%. Particularly strong susceptibility for AD was found for the DR3-DQ2/ DRB1*0404-DQ8 genotype (odds ratio, 32; P = 4 x 10(-17)), which predicted early onset. CONCLUSIONS: AD is almost exclusively autoimmune, with high autoimmune comorbidity. Both anti-21-hydroxylase antibodies and HLA class II can be clinically relevant predictors of AD. HRQoL is reduced, especially among diabetes patients, whereas thyroid disease did not have an impact on HRQoL. Treatment modalities that improve HRQoL are needed.

X-linked congenital adrenal hypoplasia with hypogonadotropic hypogonadism caused by an inversion disrupting a conserved noncoding element upstream of the NR0B1 (DAX1) gene.

CONTEXT: X-linked congenital adrenal hypoplasia with hypogonadotropic hypogonadism (AHCH) is known to be caused by coding mutations in the nuclear receptor subfamily 0, group B, member 1 (NR0B1) gene, encoding the transcriptional repressor dosage-sensitive sex-reversal adrenal hypoplasia critical region on the X chromosome protein 1 (DAX1). OBJECTIVE/PATIENTS: Four males in a family were affected by AHCH. Our aim was to locate the genetic cause of their disease, knowing that they had no mutation in the obvious candidate gene, NR0B1. DESIGN: Linkage analysis of the X chromosome and mutational screening of conserved noncoding regions upstream of NR0B1 were performed. To functionally characterize the genetic defect, studies of transcription and expression of DAX1 and steroidogenic factor 1 (SF-1) were done. RESULTS: A 60 Mb inversion on the X chromosome with one of the inversion breakpoints located in a conserved noncoding region 4 kb upstream of NR0B1 was detected. The inversion causes relocation of a putative SF-1 binding site implicated in murine gonadal development. A reporter construct lacking this enhancer element upstream of NR0B1 was unresponsive to SF-1 transcriptional activation. Immunohistochemistry suggested that the inversion leads to SF-1 silencing in the patients' testes both in childhood and in adult life. CONCLUSION: We report a noncoding mutation causing AHCH, an inversion resulting in a phenotype similar to what is caused by intragenic NR0B1 null mutations. The inversion seems to disrupt and/or relocate regulatory sites crucial in DAX1 expression.

Genome-wide linkage analysis with clustered SNP markers.

Single nucleotide polymorphisms (SNPs) have recently replaced microsatellites as the genetic markers of choice in linkage analysis, primarily because they are more abundant and the genotypes more amenable for automatic calling. One of the most recently launched linkage mapping sets (LMS) is the Applied Biosystems Human LMS 4K, which is a genome-wide linkage set based on the SNPlex technology and the use of clustered SNPs. In this article the authors report on their experience with this set and the associated genotyping software GeneMapper version 4.0, which they have used for linkage analyses in 17 moderate to large families with assumed monogenic disease. For comparison of methods, they also performed a genome-wide linkage analysis in 1 of the 17 families using the Affymetrix GeneChip Human Mapping 10K 2.0 array. The conclusion is that both methods performed technically well, with high call rates and comparable and low rates of Mendelian inconsistencies. However, genotyping is less automated in GeneMapper version 4.0 than in the Affymetrix software and thus more time consuming.

Polymorphisms in CLEC16A and CIITA at 16p13 are associated with primary adrenal insufficiency.

CONTEXT/OBJECTIVES: It is known that different autoimmune diseases often share the same susceptibility genes. In this study we aimed to investigate if loci found associated with common autoimmune diseases in recent genome-wide association studies also could be susceptibility loci for autoimmune Addison's disease (primary adrenal insufficiency). DESIGN/PATIENTS: A total of 139 tagging single nucleotide polymorphisms (SNPs) in 11 candidate genes (IL2, IL21, IL2RA, CLEC2D, CD69, ERBB3, PTPN11, SH2B3, CLEC16A, CIITA, and PTPN2) were genotyped in a case/control study design consisting of Norwegian Addison's disease patients (n = 332) and Norwegian healthy control individuals (n = 1029). Five SNPs were subsequently selected for analysis in a United Kingdom sample set consisting of Addison's disease patients (n = 210) and controls (n = 191). RESULTS: Polymorphisms in CLEC16A and CIITA remained significantly associated with Addison's disease in the Norwegian sample set at the 0.05 level, even after correction for multiple testing. CLEC16A and CIITA are both located at 16p13, but linkage disequilibrium patterns and logistical regression analyses suggest that SNPs in these two genes are independently associated with Addison's disease. We were not able to confirm these associations in the United Kingdom material, however, this may well be due to the limited sample size and lack of statistical power. CONCLUSION: Two alleles at 16p13 are independently associated with the risk of Addison's disease in the Norwegian population, suggesting this chromosomal region to harbor common autoimmunity gene(s), CLEC16A and CIITA being possible independent candidates.