HTR1A a novel type 1 diabetes susceptibility gene on chromosome 5p13-q13

Abstract

Background: We have previously performed a genome-wide linkage study in Scandinavian Type 1 diabetes (T1D) families. In the Swedish families, we detected suggestive linkage (LOD≤2.2) to the chromosome 5p13-q13 region. The aim of our study was to investigate the linked region in search for possible T1D susceptibility genes.

Methodology/principal findings: Microsatellites were genotyped in the Scandinavian families to fine-map the previously linked region. Further, SNPs were genotyped in Swedish and Danish families as well as Swedish sporadic cases. In the Swedish families we detected genome-wide significant linkage to the 5-hydroxytryptamine receptor 1A (HTR1A) gene (LOD 3.98, p<9.8×10(-6)). Markers tagging two separate genes; the ring finger protein 180 (RNF180) and HTR1A showed association to T1D in the Swedish and Danish families (p<0.002, p<0.001 respectively). The association was not confirmed in sporadic cases. Conditional analysis indicates that the primary association was to HTR1A. Quantitative PCR show that transcripts of both HTR1A and RNF180 are present in human islets of Langerhans. Moreover, immunohistochemical analysis confirmed the presence of the 5-HTR1A protein in isolated human islets of Langerhans as well as in sections of human pancreas.

Conclusions: We have identified and confirmed the association of both HTR1A and RFN180, two genes in high linkage disequilibrium (LD) to T1D in two separate family materials. As both HTR1A and RFN180 were expressed at the mRNA level and HTR1A as protein in human islets of Langerhans, we suggest that HTR1A may affect T1D susceptibility by modulating the initial autoimmune attack or either islet regeneration, insulin release, or both.

Figure 1. a–b. Linkage and association analysis in Scandinavian families and case-control cohort.

Linkage analysis of T1D on chromsome 5 in Swedish, -Danish and -Norwegian multiplex families (a). The dashed line represents multipoint linkage from the original scan in the Swedish, Danish and Norwegian families . The thin dark grey line represents the “fine mapping” including 36 microsatellites in all Scandinavian families. A region between D5S407 and D5S428 (at approx 41.5 cM in figure) showed a linkage of LOD 2.16. The dotted line represents the Swedish families using all 40 microsattelites (multipoint). Here, D5S2000 showed strongest linkage (LOD 2.70). The black diamonds represent the “fine mapping” single point analysis in the Swedish families. In the singlepoint analysis for the Swedish families, D5S2048 showed strong linkage (LOD 2.97). The black thick line represents linkage in Swedish families to T1D on chromosome using an extra 4 microsattelites and 61 SNPs, reveiling three linked peaks where the most strongly linked region was the 5p13-q13 (at approx 43 cM on figure) region (LOD 2.7 for rs6295). In the singlepoint analysis using all 61 SNPs and 4 extra microsattelites (white diamonds) rs878567 and rs6295 showed genome-wide significant linkage (LOD 3.9 and LOD 3.65 respectively). Linkage was calculated using the Exponential Equal Weighting model in the Allegro program.SNP association for the Swedish sporadic cases and controls and Swedish and Danish multiplex families (b) was calculated from rs1158292 (63 001 317 bp) to rs6880454 (63 5028 02 bp). Swedish families (diamonds). Danish families (squares), DISS2 (triangles) and BDD (crosses). Association analysis was carried out in the Unphased program. For the BDD cohort, association was calculated using controls included in the Swedish (MS) EIMS study.

Figure 2. LD plot for HTR1A and RNF180.

LD-block of the HTR1A and RNF180 regions was obtained from Haploview using HapMap data. The linkage disequilbrium measure shown is r² and the block definition is solid spine defined by Gabriel et al., . HTR1A and RNF180 are situated in two separate blocks (r² = 0.91 between the two blocks). When LD for the two associated SNPs (rs356570 and rs6880454) is calculated we observe a D́ value of 1 while r² is 0.021. The SNPs in bold were typed to verify the involvement of RNF180 to T1D susceptibility. Rs12697015 tags three other SNPs located in the RNF180 downstream region. rs6880454 tags 17 additional SNPs on either side of RNF180. rs11949052 only tags for itself. Standard settings were used for tagging anlysis in Haplowiev. The bold lines indicate the HTR1A sequenced area.

Figure 3. a-b. Q-PCR expression.

Q-PCR expression for HTR1A (a) and RNF180 (b) from mRNA isolated from a total of 10 human islet donors. Data is presented as means of expression relative to the housekeeping genes cyclophilin A (PPIA), polymerase 2 (POL2A) and hypoxanthine guanine phosphoribosyl transferase (HPRT) +/− SEM.

Figure 4. Human isolated islets and human pancreas triple immunostained for HTR1A, insulin and glucagon.

Human isolated islets (A–D) and human pancreas (E–H) triple immunostained for HTR1A (A, E), insulin (B, F), and glucagon (C,G); merged in D and H. Arrow heads indicate beta cells with HTR1A immunoreactivity, arrows indicate alpha cells with HTR1A immunoreactivity. Scale bar = 20 um in A for upper panel, in E for lower panel.

Figure 5. Human isolated islets and human pancreas with HTR1A antibodies preabsorbed with blocking peptide against HTR1A.

Human isolated islets (A–B) and human pancreas (C–D). A and C immunostained for HTR1A. B and D the same islets as in A and C in consecutive sections immunstained with HTR1A antibodies preabsorbed with blocking peptide against HTR1A, demonstrating complete lack of immunoreaction. Scale bar = 50 um in A for upper panel, in 25 um in C for lower panel.