Silja S. Amundsen

INFLORESCENCE DEFICIENT IN ABSCISSION Controls Floral Organ Abscission in Arabidopsis and Identifies a Novel Family of Putative Ligands in Plants

Abscission is an active process that enables plants to shed unwanted organs. Because the purpose of the flower is to facilitate pollination, it often is abscised after fertilization. We have identified an Arabidopsis ethylene-sensitive mutant, inflorescence deficient in abscission (ida), in which floral organs remain attached to the plant body after the shedding of mature seeds, even though a floral abscission zone develops. The IDA gene, positioned in the genomic DNA flanking the single T-DNA present in the ida line, was identified by complementation. The gene encodes a small protein with an N-terminal signal peptide, suggesting that the IDA protein is the ligand of an unknown receptor involved in the developmental control of floral abscission. We have identified Arabidopsis genes, and cDNAs from a variety of plant species, that encode similar proteins, which are distinct from known ligands. IDA and the IDA-like proteins may represent a new class of ligands in plants.

Association analysis of the 1858C>T polymorphism in the PTPN22 gene in juvenile idiopathic arthritis and other autoimmune diseases.

A functional single nucleotide polymorphism, 1858C>T, in the PTPN22 gene, encoding a tyrosine phosphatase, has been reported to be associated with type I diabetes and some other autoimmune diseases. To further investigate whether this polymorphism may be a general susceptibility factor for autoimmunity, we performed an association study in five different autoimmune diseases, three previously not tested. We found an association with juvenile idiopathic arthritis (OR=1.41; P=0.04), not previously reported, and a tendency for an association with coeliac disease (OR=1.35; P=0.08). In primary sclerosing cholangitis, no association was observed (OR=0.95; P=0.8). Furthermore, we confirmed the increased risk in rheumatoid arthritis (OR=1.58; P=0.001), but could not find support for an association with systemic lupus erythematosus (OR=0.94; P=0.8). Altogether, we have provided further evidence of an association between autoimmune diseases and the 1858C>T polymorphism in PTPN22.

Association study of IL2/IL21 and FcgRIIa: significant association with the IL2/IL21 region in Scandinavian coeliac disease families

The first genome-wide association study performed in a UK coeliac disease (CD) case-control cohort revealed association with a linkage disequilibrium block containing the KIAA1109/Tenr/IL2/IL21 genes. Also recently, an association with a non-synonymous polymorphism in FcγRIIa (CD32a) was reported in CD with an unusually strong P-value. We aimed to replicate the reported associations with the single nucleotide polymorphisms rs13119723 A>G and rs6822844 G>T in the KIAA1109/Tenr/IL2/IL21 region and rs1801274 G>A in the FcγRIIa gene in a family sample consisting of 325 Swedish/Norwegian families using the robust transmission disequilibrium test. The family sample used in this study included 100 families with two or more children affected by CD and 225 families with one affected child. We could confirm significant association between the polymorphisms rs13119723 A>G and rs6822844 G>T located in the KIAA1109/Tenr/IL2/IL21 region and CD (P-value 0.001 and 0.002, respectively). However, we found no association with the FcγRIIa rs1801274 G>A polymorphism (P-value=0.3). In conclusion, our results support the KIAA1109/Tenr/IL2/IL21 region as a true CD susceptibility region.

Interferon regulatory factor 5 gene polymorphism confers risk to several rheumatic diseases and correlates with expression of alternative thymic transcripts

OBJECTIVES Polymorphisms in genes related to the IFN pathway were investigated for susceptibility to rheumatic diseases and correlation with gene expression in thymus. METHODS Forty-five polymorphisms were genotyped in Norwegian patients with RA (n = 518), JIA (n = 440), SLE (n = 154) and healthy controls (n = 756). Forty-two thymic samples were used for gene expression analysis. Six hundred and fifty SLE patients and 737 healthy controls from Spain were available for replication. RESULTS We found a novel association between interferon regulatory factor 5 (IRF5), rs2004640 and JIA, in particular with the polyarthritis RF-negative patients [odds ratio (OR) = 1.60; 95% confidence interval (CI) 1.17, 2.20; P = 0.003]. Also, we confirmed the associations between rs2004640 and SLE (OR = 1.95; 95% CI 1.50, 2.53; P = 3.75 × 10(-7)), which was further strengthened in a meta-analysis (OR = 1.44; 95% CI 1.36, 1.52; P = 2.11 × 10(-37)). Suggestive evidence of association between rs2004640 and RA was found in the Norwegian discovery cohort (OR = 1.19; 95% CI 1.02, 1.40; P = 0.029) and strengthened in a meta-analysis (OR = 1.11; 95% CI 1.05, 1.18; P = 0.00028). Expression levels of exon 1B IRF5 transcripts were dependent on the presence of the rs2004640 T risk allele in thymic tissue, while exon 1A transcript levels correlated with IRF5 promoter CGGGG-indel variants. CONCLUSION The IFN pathway gene, IRF5, is a common susceptibility factor for several rheumatic and autoimmune diseases, and risk variants are correlated with expression of alternative IRF5 transcripts in thymus implying a regulatory role.

Four novel coeliac disease regions replicated in an association study of a Swedish-Norwegian family cohort.

Recent genome-wide association studies have identified 1q31 (RGS1), 2q11–12 (IL18RAP), 3p21 (CCR1/CCR3/CCR2), 3q25–26 (IL12A/SCHIP1), 3q28 (LPP), 4q27 (IL2/IL21), 6q25 (TAGAP) and 12q24 (SH2B3) as susceptibility regions for coeliac disease (CD). We have earlier replicated association with the IL2/IL21 region. This study aimed at replicating the remaining regions in a family cohort using the transmission disequilibrium test, which is not prone to population stratification as a source of false-positive results. Nine single nucleotide polymorphisms (SNPs) within these regions were genotyped in 325 Swedish–Norwegian CD families. We found significant associations with the same alleles in the regions 1q31 (rs2816316; Pnc=0.0060), 3p21 (rs6441961; Pnc=0.0006), 3q25–26 (rs17810564; Pnc=0.0316 and rs9811792; Pnc=0.0434) and 3q28 (rs1464510; Pnc=0.0037). Borderline, but non-significant, associations were found for rs917997 (IL18RAP), whereas no evidence for association could be obtained for rs13015714 (IL18RAP) or rs1738074 (TAGAP). The lack of replication of the latter SNPs could be because of limited power. rs3184504 (SH2B3) was not analysed because of assay failure. The most significantly associated region, 3p21 (CCR1/CCR3/CCR2), was further analysed by typing of 30 SNPs, with the aim of identifying the causal variant responsible for the initial association. Several SNPs showed association with CD, but none displayed associations stronger than rs6441961, nor did any of them add to the effect initially marked by rs6441961 in a conditional analysis. However, differential effects of rs6441961*C carrying haplotypes were indicated, and we thus cannot exclude the possibility that our inability to obtain evidence for multiple independent effects in the CCR1/CCR3/CCR2 gene region was related to a power issue.

A comprehensive screen for SNP associations on chromosome region 5q31-33 in Swedish/Norwegian celiac disease families.

Celiac disease (CD) is a gluten-induced enteropathy, which results from the interplay between environmental and genetic factors. There is a strong human leukocyte antigen (HLA) association with the disease, and HLA-DQ alleles represent a major genetic risk factor. In addition to HLA-DQ, non-HLA genes appear to be crucial for CD development. Chromosomal region 5q31–33 has demonstrated linkage with CD in several genome-wide studies, including in our Swedish/Norwegian cohort. In a European meta-analysis 5q31–33 was the only region that reached a genome-wide level of significance except for the HLA region. To identify the genetic variant(s) responsible for this linkage signal, we performed a comprehensive single nucleotide polymorphism (SNP) association screen in 97 Swedish/Norwegian multiplex families who demonstrate linkage to the region. We selected tag SNPs from a 16 Mb region representing the 95% confidence interval of the linkage peak. A total of 1404 SNPs were used for the association analysis. We identified several regions with SNPs demonstrating moderate single- or multipoint associations. However, the isolated association signals appeared insufficient to account for the linkage signal seen in our cohort. Collective effects of multiple risk genes within the region, incomplete genetic coverage or effects related to copy number variation are possible explanations for our findings.

Coeliac disease-associated polymorphisms influence thymic gene expression

Significant associations between coeliac disease (CD) and single nucleotide polymorphisms (SNPs) distributed over 40 genetic regions have been established. The majority of these SNPs are non-coding and 20 SNPs were, by expression quantitative trait loci (eQTL) analysis, found to harbour cis regulatory potential in peripheral blood mononuclear cells (PBMC). Almost all regions contain genes with an immunological relevant function, of which many act in the same biological pathways. One such pathway is T-cell development in the thymus, a pathway previously not explored in CD pathogenesis. The aim of our study was to explore the regulatory potential of the CD-associated SNPs (n=50) by eQTL analysis in thymic tissue from 42 subjects. In total, 43 nominal significant (P<0.05) eQTLs were found within 24 CD-associated chromosomal regions, corresponding to 27 expression-altering SNPs (eSNPs) and 40 probes (eProbes) that represents 39 unique genes (eGenes). Nine significant probe–SNP pairs (corresponding to 8 eSNPs and 7 eGenes) overlapped with previous findings in PBMC (rs12727642-PARK7, rs296547-DDX59, rs917997-IL18RAP, rs842647-AHSA2, rs13003464-AHSA2, rs6974491-ELMO1, rs2074404-NSF (two independent probes) and rs2298428-UBE2L3). When compared across more tissues, we found that 14 eQTLs could represent potentially novel thymus-specific eQTLs. This implies that CD risk polymorphisms could affect gene regulation in thymus.

AtMBD8 is involved in control of flowering time in the C24 ecotype of Arabidopsis thaliana

The Arabidopsis thaliana accession C24 is a vernalization-responsive, moderately late flowering ecotype. We report that a mutation in AtMBD8, which encodes a protein with a putative Methyl-CpG-Binding Domain (MBD), in C24 background, results in a delay in flowering time during both long and short days. The atmbd8-1 mutant responded to vernalization as wild type (wt) plants. Consistent with a role in modulation of flowering time, an AtMBD8::GUS-reporter construct was expressed in the shoot meristem region and developing leaves. Full-genome transcriptional profiling revealed very few changes in gene expression between atmbd8-1 and wt plants. The expression level of FLC, the major repressor of transition to flowering, was unchanged in atmbd8-1, and in accordance with that, genes upstream of FLC were unaffected by the mutation. The expression level of CONSTANS, involved in photoperiodic control of flowering, was very similar in atmbd8-1 and wt plants. In contrast, the major promoters of flowering, FT and SOC1, were both downregulated. As FT is a regulator of SOC1, we conclude that AtMBD8 is a novel promoter of flowering that acts upstream of FT in the C24 accession. In contrast to atmbd8-1, the Colombia (Col) SALK T-DNA insertion line, atmbd8-2, did not display a delayed transition to flowering. Transcriptional profiling revealed that a substantial number of genes were differentially expressed between C24 and Col wt seedlings. Several of these genes are also differentially expressed in late flowering mutants. We suggest that these differences contribute to the contrasting effect of a mutation in AtMBD8 in the two ecotypes.

Clinical and molecular characteristics in three families with biallelic mutations in IGHMBP2

Biallelic mutations in IGHMBP2 cause spinal muscular atrophy with respiratory distress type 1 (SMARD1) or Charcot-Marie-Tooth type 2S (CMT2S). We report three families variably affected by IGHMBP2 mutations. Patient 1, an 8-year-old boy with two homozygous variants: c.2T>C and c.861C>G, was wheelchair bound due to sensorimotor axonal neuropathy and chronic respiratory failure. Patient 2 and his younger sister, Patient 3, had compound heterozygous variants: c.983_987delAAGAA and c.1478C>T. However, clinical phenotypes differed markedly as the elder with sensorimotor axonal neuropathy had still unaffected respiratory function at 4.5 years, whereas the younger presented as infantile spinal muscular atrophy and died from relentless respiratory failure at 11 months. Patient 4, a 6-year-old girl homozygous for IGHMBP2 c.449+1G>T documented to result in two aberrant transcripts, was wheelchair dependent due to axonal polyneuropathy. The clinical presentation in Patients 1 and 3 were consistent with SMARD1, whereas Patients 2 and 4 were in agreement with CMT2S.

ASSOCIATION BETWEEN GENOTYPES AND PHENOTYPES IN COELIAC DISEASE

Background: Coeliac disease (CD) is a genetically driven immunological intolerance to dietary gluten with a wide range of clinical presentations. The aim of this study was to investigate the heritability of the phenotype in CD and the influence on the phenotype of different genes associated with the disease. Patients and Methods: One hundred and seven families with at least 2 siblings with CD were collected. The patients were grouped in symptom grades on the basis of the clinical presentation, the age at diagnosis, and sex. Stratification analyses of the human leucocyte antigen-DQA1 and human leucocyte antigen-DQB1 genotypes, the CTLA4 +49A/G polymorphism, the CTLA4 haplotype MH30*G:−1147*T:+49*A:CT60*G:CT61*A, and the 5q31-33 loci were done. Results: The heritability of the phenotype was estimated to be 0.45. Significant association and linkage was found between the clinical presentation and the CTLA4 +49A/G polymorphism but not for the other genotypes. No correlation was found between genotypes and age at diagnosis or sex. Conclusions: Our results indicate that the heritability is determiner of the phenotype in CD. The CTLA4 +49A/G polymorphism is correlated to the clinical presentation: the AA genotype is associated with clinically silent disease.