Letter Reply: Distribution of TNFA haplotypes in healthy Caucasians: Comment on the articles by Newton et al and Zeggini et al

We thank Ploski and colleagues for their interest in our study. The explanation for the difference in our findings is a typographic error in Table 2 of our article, whereby the alleles for marker TNF ⫺1031 were labeled incorrectly...

Investigation of the role of ANKH in ankylosing spondylitis

Objective The ank/ank mouse develops a phenotype similar to ankylosing spondylitis (AS) in humans. ANKH, the human homolog of the mutated gene in the ank/ank mouse, has been implicated in familial autosomal-dominant chondrocalcinosis and autosomal-dominant craniometaphyseal dysplasia. This study was undertaken to investigate the role of ANKH in susceptibility to and clinical manifestations of AS. Methods Sequence variants were identified by genomic sequencing of the 12 ANKH exons and their flanking splice sites in 48 AS patients; variants were then screened in 233 patients and 478 controls. Linkage to the ANKH locus was assessed in 185 affected-sibling-pair families. Results Five single-nucleotide polymorphisms were identified within the coding region and flanking splice sites. No association between either susceptibility to AS or its clinical manifestations and these novel polymorphisms, or between disease susceptibility and 3 known promoter variants, was seen. No linkage between the ANKH locus and AS was observed. Multipoint exclusion mapping rejected the hypothesis of a locus of a magnitude λ≥1.4 (logarithm of odds score <-2) (equivalent to a genetic contribution of >10% to the AS sibling recurrence risk ratio) within this area contributing to AS. Conclusion These findings indicate that ANKH is not significantly involved in susceptibility to or clinical manifestations of AS.

Association of sporadic chondrocalcinosis with a -4-basepair G-to-A transition in the 5′-untranslated region of ANKH that promotes enhanced expression of ANKH protein and excess generation of extracellular inorganic pyrophosphate

Objective Certain mutations in ANKH, which encodes a multiple-pass transmembrane protein that regulates inorganic pyrophosphate (PPi) transport, are linked to autosomal-dominant familial chondrocalcinosis. This study investigated the potential for ANKH sequence variants to promote sporadic chondrocalcinosis. Methods ANKH variants identified by genomic sequencing were screened for association with chondrocalcinosis in 128 patients with severe sporadic chondrocalcinosis or pseudogout and in ethnically matched healthy controls. The effects of specific variants on expression of common markers were evaluated by in vitro transcription/translation. The function of these variants was studied in transfected human immortalized CH-8 articular chondrocytes. Results Sporadic chondrocalcinosis was associated with a G-to-A transition in the ANKH 5′-untranslated region (5′-UTR) at 4 bp upstream of the start codon (in homozygotes of the minor allele, genotype relative risk 6.0, P = 0.0006; overall genotype association P = 0.02). This -4-bp transition, as well as 2 mutations previously linked with familial and sporadic chondrocalcinosis (+14 bp C-to-T and C-terminal GAG deletion, respectively), but not the French familial chondrocalcinosis kindred 143-bp T-to-C mutation, increased reticulocyte ANKH transcription/ANKH translation in vitro. Transfection of complementary DNA for both the wild-type ANKH and the -4-bp ANKH protein variant promoted increased extracellular PPi in CH-8 cells, but unexpectedly, these ANKH mutants had divergent effects on the expression of extracellular PPi and the chondrocyte hypertrophy marker, type X collagen. Conclusion A subset of sporadic chondrocalcinosis appears to be heritable via a -4-bp G-to-A ANKH 5′-UTR transition that up-regulates expression of ANKH and extracellular PPi in chondrocyte cells. Distinct ANKH mutations associated with heritable chondrocalcinosis may promote disease by divergent effects on extracellular PPi and chondrocyte hypertrophy, which is likely to mediate differences in the clinical phenotypes and severity of the disease.

Progress in spondylarthritis. Progress in studies of the genetics of ankylosing spondylitis

The advent of high-throughput SNP genotyping methods has advanced research into the genetics of common complex genetic diseases such as ankylosing spondylitis (AS) rapidly in recent times. The identification of associations with the genes IL23R and ERAP1 have been robustly replicated, and advances have been made in studies of the major histocompatibility complex genetics of AS, and of KIR gene variants and the disease. The findings are already being translated into increased understanding of the immunological pathways involved in AS, and raising novel potential therapies. The current studies in AS remain underpowered, and no full genomewide association study has yet been reported in AS; such studies are likely to add to the significant advances that have already been made.

The role of germline polymorphisms in the T-cell receptor in susceptibility to ankylosing spondylitis

The role of germline polymorphisms of the T-cell receptor A/D and B loci in susceptibility to ankylosing spondylitis was investigated by linkage studies using microsatellite markers in 215 affected sibling pairs. The presence of a significant susceptibility gene (lambda ≤ 1.6) at the TCRA/D locus was excluded (LOD score < -2.0). At the TCRB locus, there was weak evidence of the presence of a susceptibility gene (P = 0.01, LOD score 1.1). Further family studies will be required to determine whether this is a true or false-positive finding. It is unlikely that either the TCRA/D or TCRB loci contain genes responsible for more than a moderate proportion of the non-MHC genetic susceptibility to ankylosing spondylitis.

The effect of LRP5 polymorphisms on bone mineral density is apparent in childhood

Bone mass acquired during childhood is the primary determinant of adult bone mineral density (BMD) and osteoporosis risk. Bone accrual is subject to genetic influences. Activating and inactivating LRP5 gene mutations elicit extreme bone phenotypes, while more common LRP5 polymorphisms are associated with normal variation of BMD. Our aim was to test the hypothesis that LRP5 gene polymorphisms influence bone mass acquisition during childhood. The association between LRP5 gene polymorphisms and bone size and mineralization was examined in 819 unrelated British Caucasian children (n = 429 boys) aged 9 years. Height, weight, pubertal status (where available), total-body and spinal bone area, bone mineral content (BMC), BMD, and area-adjusted BMC (aBMC) were assessed. Dual-energy X-ray absorptiometry (DXA)-gene associations were assessed by linear regression, with adjustment for age, gender, pubertal status, and body size parameters. There were 140, 79, 12, and 2 girls who achieved Tanner stages I-IV, respectively, and 179 and 32 boys who achieved Tanner stages I and II, respectively. The rs2306862 (N740N) coding polymorphism in exon 10 of the LRP5 gene was associated with spinal BMD and aBMC (each P = 0.01) and total-body BMD and aBMC (P = 0.04 and 0.03, respectively). Adjusting for pubertal stage strengthened associations between this polymorphism and spinal BMD and aBMC (P = 0.01 and 0.002, respectively). Individuals homozygous for the T allele had greater spinal BMD and aBMC scores than those homozygous for the C allele. A dose effect was apparent as the mean spinal BMD and aBMC of heterozygous TC individuals were intermediate between those of their TT and CC counterparts. The N740N polymorphism in exon 10 of LRP5 was associated with spinal BMD and aBMC in pre- and early pubertal children. These results indicate that LRP5 influences volumetric bone density in childhood, possibly through effects on trabecular bone formation.

PTHR1 polymorphisms influence BMD variation through effects on the growing skeleton

We investigated whether polymorphisms in PTHR1 are associated with bone mineral density (BMD), to determine whether the association of this gene with BMD was due to effects on attainment of peak bone mass or effects on subsequent bone loss. The PTHR1 gene, including its 14 exons, their exon-intron boundaries, and 1,500 bp of its promoter region, was screened for polymorphisms by denaturing high-performance liquid chromatography (dHPLC) and sequencing in 36 osteoporotic cases. Eleven single-nucleotide polymorphisms (SNPs), one tetranucleotide repeat, and one tetranucleotide deletion were identified. A cohort of 634 families, including 1,236 men (39%) and 1,926 women (61%) ascertained with probands with low BMD (Z< -2.0) and the Children in Focus subset of the Avon Longitudinal Study of Parents and Children (ALSPAC) cohort (785 unrelated individuals, mean age 118 months), were genotyped for the five most informative SNPs (minor allele frequency >5%) and the tetranucleotide repeat. In our osteoporosis families, association was noted between lumbar spine BMD and alleles of a known functional tetranucleotide repeat (U4) in the PTHR1 promoter region (P = 0.042) and between two and three marker haplotypes of PTHR1 polymorphisms with lumbar spine, femoral neck, and total hip BMD (P = 0.021-0.047). This association was restricted to the youngest tertile of the population (age 16-39 years, P = 0.013-0.048). A similar association was found for the ALSPAC cohort: two marker haplotypes of SNPs A48609T and C52813T were associated with height (P = 0.006) and total body less head BMD (P = 0.02), corrected for age and gender, confirming the family findings. These findings suggest a role for PTHR1 variation in determining peak BMD.

Genetics of ankylosing spondylitis and rheumatoid arthritis: Where are we at currently, and how do they compare?

Both ankylosing spondylitis (AS) and rheumatoid arthritis (RA) are common, highly heritable conditions, the pathogenesis of which are incompletely understood. Gene-mapping studies in both conditions have over the last couple of years made major breakthroughs in identifying the mechanisms by which these diseases occur. Considering RA, there is an over-representation of genes involved in TNF signalling and the NFκB pathway that have been shown to influence the disease risk. There is also considerable sharing of susceptibility genes between RA and other autoimmune diseases such as systemic lupus erythematosus, type 1 diabetes, autoimmune thyroid disease and celiac disease, with thus far little overlap with AS. In AS, genes involved in response to IL12/IL23, and in endoplasmic reticulum peptide presentation, have been identified, but a full genomewide association study has not yet been reported.

The role of IL-17-secreting mast cells in inflammatory joint disease

The proinflammatory cytokine IL-17 has an important role in pathogenesis of several inflammatory diseases. In immune-mediated joint diseases, IL-17 can induce secretion of other proinflammatory cytokines such as IL-1, IL-6 and TNF, as well as matrix metalloproteinase enzymes, leading to inflammation, cartilage breakdown, osteoclastogenesis and bone erosion. In animal models of inflammatory arthritis, mice deficient in IL-17 are less susceptible to development of disease. The list of IL-17-secreting cells is rapidly growing, and mast cells have been suggested to be a dominant source of IL-17 in inflammatory joint disease. However, many other innate sources of IL-17 have been described in both inflammatory and autoinflammatory conditions, raising questions as to the role of mast cells in orchestrating joint inflammation. This article will critically assess the contribution of mast cells and other cell types to IL-17 production in the inflammatory milieu associated with inflammatory arthritis, understanding of which could facilitate targeted therapeutic approaches. © 2013 Macmillan Publishers Limited. All rights reserved.

Interaction between ERAP1 and HLA-B27 in ankylosing spondylitis implicates peptide handling in the mechanism for HLA-B27 in disease susceptibility

Ankylosing spondylitis is a common form of inflammatory arthritis predominantly affecting the spine and pelvis that occurs in approximately 5 out of 1,000 adults of European descent. Here we report the identification of three variants in the RUNX3, LTBR-TNFRSF1A and IL12B regions convincingly associated with ankylosing spondylitis (P < 5 × 10-8 in the combined discovery and replication datasets) and a further four loci at PTGER4, TBKBP1, ANTXR2 and CARD9 that show strong association across all our datasets (P < 5 × 10-6 overall, with support in each of the three datasets studied). We also show that polymorphisms of ERAP1, which encodes an endoplasmic reticulum aminopeptidase involved in peptide trimming before HLA class I presentation, only affect ankylosing spondylitis risk in HLA-B27-positive individuals. These findings provide strong evidence that HLA-B27 operates in ankylosing spondylitis through a mechanism involving aberrant processing of antigenic peptides.

Genome-wide association study of CNVs in 16,000 cases of eight common diseases and 3,000 shared controls

Copy number variants (CNVs) account for a major proportion of human genetic polymorphism and have been predicted to have an important role in genetic susceptibility to common disease. To address this we undertook a large, direct genome-wide study of association between CNVs and eight common human diseases. Using a purpose-designed array we typed 19,000 individuals into distinct copy-number classes at 3,432 polymorphic CNVs, including an estimated 50% of all common CNVs larger than 500 base pairs. We identified several biological artefacts that lead to false-positive associations, including systematic CNV differences between DNAs derived from blood and cell lines. Association testing and follow-up replication analyses confirmed three loci where CNVs were associated with diseaseIRGM for Crohns disease, HLA for Crohns disease, rheumatoid arthritis and type 1 diabetes, and TSPAN8 for type 2 diabetesalthough in each case the locus had previously been identified in single nucleotide polymorphism (SNP)-based studies, reflecting our observation that most common CNVs that are well-typed on our array are well tagged by SNPs and so have been indirectly explored through SNP studies. We conclude that common CNVs that can be typed on existing platforms are unlikely to contribute greatly to the genetic basis of common human diseases. © 2010 Macmillan Publishers Limited. All rights reserved.

Toll-like receptor 4 and CD14 polymorphisms in ankylosing spondylitis: Evidence of a weak association in finns

Objective To investigate the association of CD14 and Toll-like receptor (TLR4) with ankylosing spondylitis (AS). Methods A promoter variant in CD14 and 2 coding polymorphisms in TLR4 were investigated in UK and Finnish families with AS and in a UK case-control study. A metaanalysis of published TLR4 and CD14 studies was performed. Results In the Finnish study the CD74-260bp T variant showed an association (p = 0.006), and the common 2-marker TLR4 haplotype showed a weak association (global p = 0.03), with AS. No associations were seen in the UK based studies or in the metaanalyses. Conclusion CD14 and TLR4 showed an association with AS in the Finns only.

Estrogen receptor α regulates area-adjusted bone mineral content in late pubertal girls

Context: Whether the action of estrogen in skeletal development depends on estrogen receptor α as encoded by the ESR1 gene is unknown. Objectives: The aim of this study was to establish whether the gain in area-adjusted bone mineral content (ABMC) in girls occurs in late puberty and to examine whether the magnitude of this gain is related to ESR1 polymorphisms. Design: We conducted a cross-sectional analysis. Setting: The study involved the Avon Longitudinal Study of Parents and Children (ALSPAC), a population-based prospective study. Participants: Participants included 3097 11-yr-olds with DNA samples, dual x-ray absorptiometry measurements, and pubertal stage information. Outcomes: Outcome measures included separate prespecified analyses in boys and girls of the relationship between ABMC derived from total body dual x-ray absorptiometry scans and Tanner stage and of the interaction between ABMC, Tanner stage, and ESR1 polymorphisms. Results: Total body less head and spinal ABMC were higher in girls in Tanner stages 4 and 5, compared with those in Tanner stages 1, 2, and 3. In contrast, height increased throughout puberty. No differences were observed in ABMC according to Tanner stage in boys. For rs2234693 (PvuII) and rs9340799 (XbaI) polymorphisms, differences in spinal ABMC in late puberty were 2-fold greater in girls who were homozygous for the C and G alleles, respectively (P = 0.001). For rs7757956, the difference in total body less head ABMC in late puberty was 50% less in individuals homozygous or heterozygous for the A allele (P = 0.006). Conclusions: Gains in ABMC in late pubertal girls are strongly associated with ESR1 polymorphisms, suggesting that estrogen contributes to this process via an estrogen receptor α-dependent pathway.

Mutation of perinatal myosin heavy chain

Veugelers et al. (July 29 issue)1 report on patients with the trismus–pseudocamptodactyly syndrome as having a “Carney complex variant.” Among more than 500 patients with the Carney complex in our database, there are none with the trismus–pseudocamptodactyly syndrome.2,3...

A novel human leucocyte antigen-DRB1 genotyping method based on multiplex primer extension reactions

We have developed and validated a semi-automated fluorescent method of genotyping human leucocyte antigen (HLA)-DRB1 alleles, HLA-DRB1*01-16, by multiplex primer extension reactions. This method is based on the extension of a primer that anneals immediately adjacent to the single-nucleotide polymorphism with fluorescent dideoxynucleotide triphosphates (minisequencing), followed by analysis on an ABI Prism 3700 capillary electrophoresis instrument. The validity of the method was confirmed by genotyping 261 individuals using both this method and polymerase chain reaction with sequence-specific primer (PCR-SSP) or sequencing and by demonstrating Mendelian inheritance of HLA-DRB1 alleles in families. Our method provides a rapid means of performing high-throughput HLA-DRB1 genotyping using only two PCR reactions followed by four multiplex primer extension reactions and PCR-SSP for some allele groups. In this article, we describe the method and discuss its advantages and limitations.