Laëtitia Michou

Linkage proof for PTPN22, a rheumatoid arthritis susceptibility gene and a human autoimmunity gene

The tyrosine phosphatase PTPN22 allele 1858T has been associated with rheumatoid arthritis (RA) and other autoimmune diseases. RA is the most frequent of those multifactorial diseases. The RA association was usually restricted to serum rheumatoid factor positive disease (RF+). No interaction was shown with HLA-DRB1, the first RA gene. Many case-control studies replicated the RA association, showing an allele frequency increase of ≈5% on average and large variations of population allele frequencies (2.1–15.5%). In multifactorial diseases, the final proof for a new susceptibility allele is provided by departure from Mendels law (50% transmission from heterozygous parents). For PTPN22–1858T allele, convincing linkage proof was available only for type 1 diabetes. We aimed at providing this proof for RA. We analyzed 1,395 West European Caucasian individuals from 465 “trio” families. We replicated evidence for linkage, demonstrating departure from Mendels law in this subset of early RA onset patients. We estimated the overtransmission of the 1858T allele in RF+ families: T = 63%, P < 0.0007. The 1858T allele frequency increased from 11.0% in controls to 17.4% in RF+ RA for the French Caucasian population and the susceptibility genotype (1858T/T or T/C) from 20.2% to 31.6% [odds ratio (OR) = 1.8 (1.2–2.8)]. In conclusion, we provided the linkage proof for the PTPN22–1858T allele and RF+ RA. With diabetes and RA, PTPN22 is therefore a “linkage-proven” autoimmunity gene. PTPN22 accounting for ≈1% of the RA familial aggregation, many new genes could be expected that are as many leads to definitive therapy for autoimmune diseases.

Paget's Disease of Bone in the French Population: Novel SQSTM1 Mutations, Functional Analysis, and Genotype–Phenotype Correlations

Mutation screening of the SQSTM1 gene in 94 French patients with PDB revealed two novel point‐mutations (A381V and L413F) and two new compound heterozygous genotypes (P392L/A381V and P392L/A390X). Functional analysis showed an increased level of SQSTM1/p62 protein in PDB patients and truncated forms of the protein encoded by the A390X allele. Clinical data indicate that PDB patients with SQSTM1 mutation are younger at PDB diagnosis and have more extensive bone lesions.

Rheumatoid arthritis seropositive for the rheumatoid factor is linked to the protein tyrosine phosphatase nonreceptor 22-620W allele

The protein tyrosine phosphatase nonreceptor type 22 (PTPN22) gene encodes for lymphoid tyrosine phosphatase LYP, involved in the negative regulation of early T-cell activation. An association has recently been reported between the PTPN22-620W functional allele and rheumatoid factor-positive (RF+) rheumatoid arthritis (RA), among other autoimmune diseases. Expected linkage proof for consistency cannot be definitely produced by an affected sib-pair (ASP) analysis. Our aim was therefore to search for linkage evidence with the transmission disequilibrium test.DNA from the French Caucasian population was available for two samples of 100 families with one RA patient and both parents, and for 88 RA index cases from RA ASP families. Genotyping was carried out by PCR-restriction fragment length polymorphism. The analysis was performed using the transmission disequilibrium test, genotype relative risk and ASP-based analysis.The transmission disequilibrium test of the PTPN22-620W allele revealed linkage and association for RF+ RA (61% of transmission, P = 0.037). The genotype relative risk showed the risk allele in 34% of RF+ RA patients and in 24% of controls derived from nontransmitted parental chromosomes (P = 0.047, odds ratio = 1.69, 95% confidence interval = 1.03–2.78). The ASP investigation showed no enriched risk allele in RA multiplex families, resulting in a lack of power of ASP analysis, explaining the published negative results.This study is the first to show linkage of PTPN22 to RF+ RA, consistent with PTPN22 as a new RA gene.

Validation of the reshaped shared epitope HLA-DRB1 classification in rheumatoid arthritis

Recently, we proposed a classification of HLA-DRB1 alleles that reshapes the shared epitope hypothesis in rheumatoid arthritis (RA); according to this model, RA is associated with the RAA shared epitope sequence (72–74 positions) and the association is modulated by the amino acids at positions 70 and 71, resulting in six genotypes with different RA risks. This was the first model to take into account the association between the HLA-DRB1 gene and RA, and linkage data for that gene. In the present study we tested this classification for validity in an independent sample. A new sample of the same size and population (100 RA French Caucasian families) was genotyped for the HLA-DRB1 gene. The alleles were grouped as proposed in the new classification: S1 alleles for the sequences A-RAA or E-RAA; S2 for Q or D-K-RAA; S3D for D-R-RAA; S3P for Q or R-R-RAA; and X alleles for no RAA sequence. Transmission of the alleles was investigated. Genotype odds ratio (OR) calculations were performed through conditional logistic regression, and we tested the homogeneity of these ORs with those of the 100 first trio families (one case and both parents) previously reported. As previously observed, the S2 and S3P alleles were significantly over-transmitted and the S1, S3D and X alleles were under-transmitted. The latter were grouped as L alleles, resulting in the same three-allele classification. The risk hierarchy of the six derived genotypes was the same: (by decreasing OR and with L/L being the reference genotype) S2/S3P, S2/S2, S3P/S3P, S2/L and S3P/L. The homogeneity test between the ORs of the initial and the replication samples revealed no significant differences. The new classification was therefore considered validated, and both samples were pooled to provide improved estimates of RA risk genotypes from the highest (S2/S3P [OR 22.2, 95% confidence interval 9.9–49.7]) to the lowest (S3P/L [OR 4.4, 95% confidence interval 2.3–8.4]).

Associations between genetic factors, tobacco smoking and autoantibodies in familial and sporadic rheumatoid arthritis

Objectives: The objective of this study was to investigate the association between genes (HLA-DRB1 and PTPN22) and tobacco smoking, separately as well as combined, and serological markers of rheumatoid arthritis (RA) in a French population with RA. Methods: 274 patients with RA with half of them belonging to RA multicase families, were genotyped for HLA-DRB1 allele and for PTPN22-1858 polymorphism. IgM rheumatoid factor and anti-cyclic citrullinated peptide (anti-CCP) antibodies were determined by ELISA method. The search for association relied on χ2 test and odds ratio with 95% confidence interval calculation. The interaction study relied on the departure-from-additivity-based method. Results: The presence of at least one shared epitope (SE) allele was associated with anti-CCP antibodies presence (82.5% vs. 68.4%, p = 0.02), particularly with HLA-DRB1*0401 allele (28.0% vs. 16.4%, p = 0.01). Tobacco exposure was associated with anti-CCP antibodies, but only in presence of SE. A tendency toward an interaction was found between tobacco, the presence of at least one HLA-DRB1*0401 allele and anti-CCP antibodies (attributable proportion due to interaction = +0.24 (−0.21+0.76)). The cumulative dose of cigarette smoking was correlated with anti-CCP antibody titres (r = 0.19, p = 0.04). The presence of both SE and 1858T alleles was associated with a higher, but not significantly different, risk for anti-CCP antibodies presence than for each separately. No association was found between PTPN22-1858T allele and tobacco smoking for autoantibody positivity. Conclusions: Our findings suggest an association between SE alleles and tobacco smoking for anti-CCP positivity and a tendency toward an interaction between the HLA-DRB1*0401 allele and smoking for anti-CCP positivity in this sample of RA.

IRF5 rs2004640‐T allele, the new genetic factor for systemic lupus erythematosus, is not associated with rheumatoid arthritis

Background: Recently, a new genetic factor within the interferon regulatory factor 5 (IRF5) gene was demonstrated for systemic lupus erythematosus (SLE) through linkage and association: the rs2004640-T allele. IRF5 is involved in the production of rheumatoid arthritis (RA) cytokines, and SLE already shares with RA one genetic factor within the tyrosine phosphatase PTPN22 gene. Aim: To test the hypothesis that the SLE IRF5 genetic factor could also be shared with RA. Patients and methods: 100 French Caucasian trio families with RA were genotyped and analysed with the transmission disequilibrium test, the frequency comparison of the transmitted and untransmitted alleles, and the genotype relative risk. 97% power was available to detect at least a trend in favour of a factor similar to that reported for SLE. Results: The analysis showed the absence of linkage and association globally and in “autoimmune” RA subsets, with a weak non-significant trend against the IRF5rs20046470-T allele. Given the robustness of familial-based analysis, this slight negative trend provided strong evidence against even a weaker factor than that reported for SLE. Conclusion: Our results exclude the IRF5rs2004640-T allele as a major genetic factor for RA in this French Caucasian population.

Change in fracture risk and fracture pattern after bariatric surgery: nested case-control study

Objective To investigate whether bariatric surgery increases the risk of fracture. Design Retrospective nested case-control study. Setting Patients who underwent bariatric surgery in the province of Quebec, Canada, between 2001 and 2014, selected using healthcare administrative databases. Participants 12 676 patients who underwent bariatric surgery, age and sex matched with 38 028 obese and 126 760 non-obese controls. Main outcome measures Incidence and sites of fracture in patients who had undergone bariatric surgery compared with obese and non-obese controls. Fracture risk was also compared before and after surgery (index date) within each group and by type of surgery from 2006 to 2014. Multivariate conditional Poisson regression models were adjusted for fracture history, number of comorbidities, sociomaterial deprivation, and area of residence. Results Before surgery, patients undergoing bariatric surgery (9169 (72.3%) women; mean age 42 (SD 11) years) were more likely to fracture (1326; 10.5%) than were obese (3065; 8.1%) or non-obese (8329; 6.6%) controls. A mean of 4.4 years after surgery, bariatric patients were more susceptible to fracture (514; 4.1%) than were obese (1013; 2.7%) and non-obese (3008; 2.4%) controls. Postoperative adjusted fracture risk was higher in the bariatric group than in the obese (relative risk 1.38, 95% confidence interval 1.23 to 1.55) and non-obese (1.44, 1.29 to 1.59) groups. Before surgery, the risk of distal lower limb fracture was higher, upper limb fracture risk was lower, and risk of clinical spine, hip, femur, or pelvic fractures was similar in the bariatric and obese groups compared with the non-obese group. After surgery, risk of distal lower limb fracture decreased (relative risk 0.66, 0.56 to 0.78), whereas risk of upper limb (1.64, 1.40 to 1.93), clinical spine (1.78, 1.08 to 2.93), pelvic, hip, or femur (2.52, 1.78 to 3.59) fractures increased. The increase in risk of fracture reached significance only for biliopancreatic diversion. Conclusions Patients undergoing bariatric surgery were more likely to have fractures than were obese or non-obese controls, and this risk remained higher after surgery. Fracture risk was site specific, changing from a pattern associated with obesity to a pattern typical of osteoporosis after surgery. Only biliopancreatic diversion was clearly associated with fracture risk; however, results for Roux-en-Y gastric bypass and sleeve gastrectomy remain inconclusive. Fracture risk assessment and management should be part of bariatric care.

Genetics of bone diseases: Paget's disease, fibrous dysplasia, osteopetrosis, and osteogenesis imperfecta

Over the last few years, research into the genetics of bone diseases has produced new insights into the pathophysiology of bone remodeling. The identification of SQSTM1 mutations in Pagets disease of bone established that osteoclast activation involved both binding to ubiquitin and the proteasome pathway. However, murine models fail to replicate the full phenotype, and somatic SQSTM1 mutations have been identified, suggesting a role for complex mechanisms. In patients with fibrous dysplasia of bone, postzygotic somatic mutations in the GNAS gene are now well documented. Technological advances have improved the detection of somatic mutations in peripheral blood cells. Osteopetrosis is characterized by increased bone density due to deficient osteoclastic bone resorption. Most of the genes involved in the various clinical patterns of osteopetrosis have been identified. The identification of LRP5 gain-of-function mutations in autosomal dominant osteopetrosis type I prompted a revision of the classification scheme, and this form is now being included among the high-bone-mass diseases. Osteogenesis imperfecta is characterized by an inherited abnormality in bone formation that manifests as osteopenia with increased bone fragility. Mutations in the COL1A1 and COL1A2 genes are found in over 90% of patients. The recent identification of mutations in the CRTAP, LEPRE1, and PPIB genes in recessive forms has radically changed the classification of osteogenesis imperfecta and generated new pathophysiological hypotheses.

Novel SQSTM1 mutations in patients with Paget's disease of bone in an unrelated multiethnic American population

More than 20 mutations of the Sequestosome 1 (SQSTM1) gene have been reported in patients of European descent affected by Pagets disease of bone (PDB). In this investigation, a systematic screening for SQSTM1 mutations was conducted in consecutively evaluated unrelated patients with phenotypical PDB living in the New York City area (NY, United States). Seventy unrelated PDB patients with a multiethnic background, mostly of Jewish, Italian American, and Western European ancestries, were recruited. Sequencing of exons 7 and 8 was performed on DNA samples isolated from peripheral blood. Seven patients (10%) had SQSTM1 mutations, of which three had a family history of PDB. Four patients carried the C1215T (P392L) mutation, and three patients carried novel SQSTM1 missense mutations: T1085A (S349T), C1209T (A390V), and T1290A (L417Q) mutations. All PDB patients with SQSTM1 mutations had polyostotic involvement, and the mean number of affected bones was significantly higher in pagetic patient carriers of a SQSTM1 mutation when compared to non-mutated PDB patients (4.0 vs. 2.0, respectively; P = 0.003). Haplotype analysis in patient carriers of the P392L mutation revealed that all P392L mutations were carried by haplotype 2. The SQSTM1 mutation rate in unrelated American patients described in the present study was similar to that reported in European populations.

Genetics of Dupuytren’s disease

Dupuytrens disease (DD) is a progressive fibrosis of the palmar fascia characterized by the formation of a nodule, which evolves into a cord. DD is the most common hereditary disease of the connective tissue preferentially affecting Caucasoids originating from Northern Europe. Some environmental factors are associated with DD, namely alcohol consumption, tobacco exposure and, possibly, manual activities. Diabetes and epilepsy are the most frequently reported DD-associated diseases. The genetic mode of inheritance is not well understood, but seems to be heterogeneous: most frequently, autosomal dominant with variable penetrance, and rarely recessive autosomal or maternal (matrilinear), suggesting a mitochondrial heredity. Initially, a suggestion of linkage with the DUPC1 locus at 16q was proposed. Then, among the genomic variations observed in DD, alterations in the copy number of genes in chromosomal regions 10q22, 16p12.1 and 17p12, associations with the HLA-DRB1*15 allele and a mutation in the rRNA 16s identified in forms with a matrilinear heredity, were reported. Finally, a genome-wide study has shown a genetic association of DD with 6, 11 and 16 chromosomes. Pathophysiology of DD involves mainly myofibroblasts and the extracellular matrix of collagen. Gene and protein expression studies have confirmed the central role of the β catenin of the TGFβ pathways in the pathogenesis of DD.