Bi-allelic variants in MYH3 cause recessively-inherited arthrogryposis.

Arthrogryposis is a clinical feature defined by congenital joint contractures in two or more different body areas which occurs in between 1/3000 and 1/5000 live births. Variants in multiple genes have been associated with distal arthrogryposis syndromes. Heterozygous variants in MYH3 have been identified to cause the dominantly-inherited distal arthrogryposis conditions, Freeman-Sheldon syndrome, Sheldon-Hall syndrome, and multiple pterygium syndrome. In contrast, MYH3 variants underlie both dominantly and recessively inherited Contractures, Pterygia, and Spondylocarpotarsal Fusion syndromes (CPSFS) which are characterized by extensive bony abnormalities in addition to congenital contractures. Here we report two affected sibs with distal arthrogryposis born to unaffected, distantly related parents. Sequencing revealed that both sibs were homozygous for two ultra-rare MYH3 variants, c.3445G>A (p.Glu1149Lys) and c.4760T>C (p.Leu1587Pro). Sequencing and deletion/duplication analysis of 169 other arthrogryposis genes yielded no other compelling candidate variants. This is the first report of biallelic variants in MYH3 being implicated in a distal arthrogryposis phenotype without the additional features of CPSFS. Thus, akin to CPSFS, both dominant and recessively inherited distal arthrogryposis can be caused by variants in MYH3.

Development and validation of a French-language cross-cultural adaptation of the Pedi-IKDC Questionnaire.

BACKGROUND: The Pedi-IKDC is an English-language, knee-specific, paediatric questionnaire used by orthopaedic surgeons around the world as a valuable patient-reported outcome measure (PROM). The objective of this study was thus to extend the applicability of the Pedi-IKDC to French-speaking Canadian patients, for both clinical practice and research, by developing a French-language cross-cultural adaptation of the original version. HYPOTHESIS: The French adaptation of the Pedi-IKDC is valid and reliable for evaluating French-speaking children with knee conditions. PATIENTS AND METHODS: The Pedi-IKDC was translated to French by a panel of orthopaedic surgeons then back-translated by a professional translator. The original English version and the back-translation were compared to assess their similarity and confirm the faithfulness of the French translation. The validity of the French version was then tested at a major paediatric hospital in French-speaking Canada, in 203 children, including 163 with knee pain and 40 without knee symptoms. Internal consistency, construct validity, and discriminant capacity of the French version were assessed. RESULTS: Internal consistency of the Pedi-IKDC adaptation was excellent (Cronbach's alpha, 0.934 in the knee-pain group). Construct validity was robust, with all nine hypotheses adapted from the original Pedi-IKDC article demonstrating strong (n=7) or moderate (n=2) correlations (p<0.001). The evaluation of discriminant capacity identified no statistically significant score differences according to most of the respondent characteristics (body mass index, age group, type of diagnosis, and type of treatment). However, scores differed significantly between females and males. DISCUSSION: The French-language cross-cultural adaptation of the Pedi-IKDC obtained using a universally recognized method for translating PROMs demonstrated good performance, with psychometric properties similar to those of the original Pedi-IKDC and of its Danish, Italian, and Russian adaptations. LEVEL OF EVIDENCE: II.

Infected popliteal pseudoaneurysm in a youth basketball player: A case report and brief review of the literature.

INTRODUCTION: An infected popliteal pseudoaneurysm has never been described in the pediatric population. Physicians need to be aware of its presentation and management, in order to diagnose and treat this medical condition adequately. METHODS: We describe the case of a 14-year-old boy who developed myositis and cellulitis centered at the popliteal fossa after playing basketball. A treatment of intravenous cefazolin was started. 5 days later, he experienced a knee pain flare-up, which turned out to be a popliteal pyomyositis with a pseudoaneurysm of the popliteal artery. A saphenous vein graft bypass of the popliteal artery and an excision of the popliteal pseudoaneurysm were performed. Intravenous cefazolin was continued for 6 weeks and prophylactic acetylsalicylic acid for 6 months. RESULTS AND CONCLUSION: This case highlighted the importance of repeating radiologic investigations if a patient suffering from soft tissue infection has persistent pain after several days of appropriate antibiotics. A popliteal pseudoaneurysm can be diagnosed with ultrasound imaging and treated with a popliteal-popliteal bypass. Our patient needed a catheter-guided dilation of the anastomosis at the vein graft 6 months post-surgery, and then evolved favorably and went back to playing basketball 6 months post-dilation.

Complication rates following all-epiphyseal ACL reconstructions in skeletally immature patients: A retrospective case series study.

ABSTRACT: The aim was to evaluate the safety of a physeal-sparing anterior cruciate ligament reconstruction technique (ACLR), performed with Orthopediatrics (Warsaw, IN) equipment, by assessing complications.Skeletally immature patients who underwent all-epiphyseal ACLR between 2015 and 2017 with postoperative follow-up were included in this retrospective study. Complications, demographic, clinical, surgical, and imaging data was retrieved from an urban tertiary pediatric hospital database. Physeal status, limb-length discrepancies (LLD), and angular deformities were assessed on preoperative and postoperative radiographs, growth disturbances were reported, and initial and follow-up diameters of tunnels were compared.Nineteen ACLRs were included from 18 patients, 4 females and 14 males, with bone age at surgery of 13.3 ± 1.0 years. At a mean follow-up of 19.2 ±â€Š10.1 months, there were no symptomatic growth disorders requiring intervention. There were: 2 (11.1%) unilateral early physeal closures, 2 (10.5%) new angular deformities (5°-10°), 4 (22.2%) LLD (1-2 cm), 1 (5.6%) contralateral ACLR, 1 (5.6%) femoral screw removal, 2 (10.5%) graft ruptures, and 1 meniscal tear (5.3%). Mean tunnel widening was 1.7 mm and 1.5 mm on the femoral and tibial side, respectively, and no massive osteolysis was recorded at the polyetheretherketone implant site.The complication rates were comparable to those in similar studies, with no growth-related complications at 19.2 months.

Phenotypic and Functional Changes in Peripheral Blood Natural Killer Cells in Crohn Disease Patients.

We investigated activation status, cytotoxic potential, and gut homing ability of the peripheral blood Natural Killer (NK) cells in Crohn disease (CD) patients. For this purpose, we compared the expression of different activating and inhibitory receptors (KIR and non-KIR) and integrins on NK cells as well as their recent degranulation history between the patients and age-matched healthy controls. The study was conducted using freshly obtained peripheral blood samples from the study participants. Multiple color flow cytometry was used for these determinations. Our results show that NK cells from treatment-naïve CD patients expressed higher levels of activating KIR as well as other non-KIR activating receptors vis-à-vis healthy controls. They also showed increased frequencies of the cells expressing these receptors. The expression of several KIR and non-KIR inhibitory receptors tended to decrease compared with the cells from healthy donors. NK cells from the patients also expressed increased levels of different gut-homing integrin molecules and showed a history of increased recent degranulation events both constitutively and in response to their in vitro stimulation. Furthermore, treatment of the patients tended to reverse these NK cell changes. Our results demonstrate unequivocally, for the first time, that peripheral blood NK cells in treatment-naïve CD patients are more activated and are more poised to migrate to the gut compared to their counterpart cells from healthy individuals. Moreover, they show that treatment of the patients tends to normalize their NK cells. The results suggest that NK cells are very likely to play a role in the immunopathogenesis of Crohn disease.

Langerhans Cell Histiocytosis With Vertebral Involvement Diagnosed and Treated Over the Last 15 Years in a Single Canadian Pediatric Academic Institution.

We report 11 children with vertebral lesion of Langerhans cell histiocytosis (LCH) diagnosed and treated between 2000 and 2015. Vertebral lesions were usually present at LCH diagnosis. No child developed neurologic symptoms. Among 29 vertebral lesions, only 2 were unstable. Chemotherapy was used in all children but 3. A LCH recurrence was observed in 6 patients, involving vertebrae in 4 cases. All children were disease-free at their last follow-up. Sequelae were more often radiologic than clinical. Since potential recurrences and incomplete bone regeneration exist, discussion about optimal treatment and long-term follow-up of vertebral lesions are essential.

Altered proteome of high-density lipoproteins from paediatric acute lymphoblastic leukemia survivors.

Acute lymphoblastic leukemia (ALL) is the most frequent malignancy in children. With the use of more modern, efficient treatments, 5-year survival has reached more than 90% in this population. However, this achievement comes with many secondary and long-term effects since more than 65% of the survivors experience at least one severe complication, including the metabolic syndrome and cardiovascular diseases. The main objective of the present work was to characterize the composition of HDL particles isolated from pediatric ALL survivors. HDLs from 8 metabolically healthy ALL survivors, 8 metabolically unhealthy ALL survivors and 8 age- and gender-matched controls were analyzed. The HDL fraction from the survivors contained less cholesterol than the controls. In addition, proteomic analyses revealed an enrichment of pro-thrombotic (e.g., fibrinogen) and pro-inflammatory (e.g., amyloid A) proteins in the HDLs deriving from metabolically unhealthy survivors. These results indicate an alteration in the composition of lipid and protein content of HDL from childhood ALL survivors with metabolic disorders. Although more work is needed to validate the functionality of these HDLs, the data seem relevant for survivor health given the detection of potential biomarkers related to HDL metabolism and functionality in cancer.

Predicting lowest hemoglobin level and risk of blood transfusion in spinal fusion surgery for adolescent idiopathic scoliosis.

PURPOSE: The aim of this study was to evaluate the factors associated with timing of lowest hemoglobin (Hb) level and the need for postoperative blood transfusion in posterior spinal fusion for adolescent idiopathic scoliosis. METHODS: We conducted a retrospective review of all adolescent scoliosis patients undergoing posterior spinal fusion at our institution, 2002-2014. Surgery consisted of segmental pedicle screw fixation using multi-level pedicle screws. Blood-saving techniques were used in all patients. Data included Cobb angle, pre- and postoperative Hb levels, preoperative autologous blood donation (PABD), surgery duration, and allogeneic or autologous transfusion. We used linear and logistic regressions for statistical analysis. RESULTS: There were 456 patients (402 female, 54 male), mean age 16 ± 5 years. Lowest Hb was observed on postoperative Days 2 (32.2%) and 3 (33.3%); 45.1% of postoperative transfusions occurred on Day 2. One hundred and eighty-eight (41%) patients who provided PABD had significantly lower preoperative Hb and received more transfusions intraoperatively (22.6% vs. 5.2%) and postoperatively (20% vs. 6.3%) than others. Probability of transfusion increased 49.6 (95% CI 17.40-141.37) times with preoperative Hb < 11 g/dL as compared to preoperative Hb > 14 g/dL. Probability of transfusion increased 4.3- and 9.8-fold when surgery duration exceeded 5 and 6 h, respectively. Probability of transfusion increased 3.3- and 5.3-fold with Cobb angle > 70° and 80°, respectively. CONCLUSIONS: We identified clear patient-specific perioperative parameters that affect risk of perioperative blood transfusion, including Cobb angle, PABD and preoperative Hb. Hb measurement beyond postoperative Day 3 is considered unnecessary unless clinically indicated. These slides can be retrieved under Electronic Supplementary Material.

Validation of the French version of the KOOS-child questionnaire.

PURPOSE: The Knee Injury Osteoarthritis Outcome Score (KOOS) questionnaire is one of the frequently used outcome scores in pediatric studies. However, a recent study demonstrated that the pediatric population had a limited understanding of some of its questions. Therefore, the KOOS-Child questionnaire was developed specifically for this population. Our team produced a French adaptation based on the English version. The objective of the current study was to validate the French adaptation of the KOOS-Child questionnaire. METHODS: After ethic board approval, the questionnaire was translated from English to French by two French speaking orthopedic surgeons. Following consensus, the translated version was retranslated to English by a professional translator. A group of experts compared the original and back translated version and decided on a final adapted questionnaire version. Ninety-nine 8-16 year-old patients were prospectively recruited from our pediatric orthopedic surgery clinic. Twenty-one control participants and 78 patients suffering from knee pain were recruited. The participants were asked to answer the translated French version of the KOOS-Child questionnaire and two validated French pediatric quality of life surveys. RESULTS: Statistical analysis demonstrated no statistically significant demographic difference between the control population and the patients suffering from a knee pathology. The mean for the five different domains of the KOOS-Child questionnaire showed statistical differences (p < 0.001) between the two groups. Construct validity was demonstrated through testing of previously validated hypothesis of correlation. Internal consistency was also confirmed in injured patients. CONCLUSIONS: In conclusion, the current study results demonstrate good to excellent internal consistency, good construct validity and inconclusive discriminant capacity of the French adaptation of the KOOS-Child questionnaire. LEVEL OF EVIDENCE: II.

Insight from mitochondrial functions and proteomics to understand cardiometabolic disorders in survivors of acute lymphoblastic leukemia.

BACKGROUND: Childhood acute lymphoblastic leukemia (cALL) is the most prevalent form of cancer in children. Due to advances in treatment and therapy, young cALL subjects now achieve a 90% survival rate. However, this tremendous advance does not come without consequence since ~2/3 of cALL survivors are affected by long-term and late, severe complications. Although the metabolic syndrome is a very serious sequel of cALL, the mechanisms remain undefined. It is also surprising to note that the mitochondrion, a central organelle in metabolic functions and the main cellular energy generator, have not yet been explored. OBJECTIVES: To determine whether cALL survivors exhibit impairments in their mitochondrial functions and proteomic profiling in relationship with metabolic disorders in cALL survivors compared to healthy controls. METHODS AND RESULTS: Anthropometric measures, metabolic characteristics and lipid profiles were assessed, mitochondria isolated from peripheral blood mononuclear cells, and proteomic analyzed. Our data demonstrated that metabolically. Unhealthy survivors exhibited several metabolic syndrome components (e.g. overweight, insulin resistance, dyslipidemia, inflammation) whereas Healthy cALL survivors resemble the Controls. In line with these abnormalities, functional experiments in these subjects revealed a significant decrease in the protein expression of mitochondrial antioxidant superoxide dismutase, PGC1-α transcription factor (a key modulator of mitochondrion biogenesis), and an increase in pro-apoptotic cytochrome c. Proteomic analysis of mitochondria by mass spectrometry revealed changes in the regulation of proteins related to inflammation, apoptosis, energy production, redox and antioxidant activity, fatty acid ß-oxidation, protein transport and metabolism, and signalling pathways between groups. CONCLUSIONS: Through the use of proteomic analysis, our work demonstrated a number of significant alterations in protein expression in mitochondria of cALL survivors, especially the metabolically Unhealthy survivor group. Further investigation of these proteins may help delineate the mechanisms by which mitochondrial dysfunctions exert cardiometabolic derangements in cALL survivors.

Lipid and lipoprotein abnormalities in acute lymphoblastic leukemia survivors.

Survivors of acute lymphoblastic leukemia (ALL), the most common cancer in children, are at increased risk of developing late cardiometabolic conditions. However, the mechanisms are not fully understood. This study aimed to characterize the plasma lipid profile, Apo distribution, and lipoprotein composition of 80 childhood ALL survivors compared with 22 healthy controls. Our results show that, despite their young age, 50% of the ALL survivors displayed dyslipidemia, characterized by increased plasma triglyceride (TG) and LDL-cholesterol, as well as decreased HDL-cholesterol. ALL survivors exhibited lower plasma Apo A-I and higher Apo B-100 and C-II levels, along with elevated Apo C-II/C-III and B-100/A-I ratios. VLDL fractions of dyslipidemic ALL survivors contained more TG, free cholesterol, and phospholipid moieties, but less protein. Differences in Apo content were found between ALL survivors and controls for all lipoprotein fractions except HDL3 HDL2, especially, showed reduced Apo A-I and raised Apo A-II, leading to a depressed Apo A-I/A-II ratio. Analysis of VLDL-Apo Cs disclosed a trend for higher Apo C-III1 content in dyslipidemic ALL survivors. In conclusion, this thorough investigation demonstrates a high prevalence of dyslipidemia in ALL survivors, while highlighting significant abnormalities in their plasma lipid profile and lipoprotein composition. Special attention must, therefore, be paid to these subjects given the atherosclerotic potency of lipid and lipoprotein disorders.

Static and dynamic compression application and removal on the intervertebral discs of growing rats.

Fusionless implants are used to correct pediatric progressive spinal deformities, most of them spanning the intervertebral disc. This study aimed at investigating the effects of in vivo static versus dynamic compression application and removal on discs of growing rats. A microloading device applied compression. 48 immature rats (28 d.o.) were divided into two groups (43d, 53d). Each group included four subgroups: control (no surgery), sham (device installed without loading), static (0.2 MPa) and dynamic compressions (0.2 MPa ± 30% with 0.1 Hz). In 43d subgroups, compression was applied for 15 days. In 53d subgroups, compression was followed by 10 days without loading. Disc heights, nucleus/annulus volumetric proportions and nucleus proteoglycan contents were analyzed using one-way ANOVA and post-hoc Tukey comparisons (p < 0.05). Disc heights of 43d and 53d static and dynamic loading rats were lower than shams (p < 0.05). Volumetric proportions remained similar. At 43d, nucleus proteoglycan contents increased in both static and dynamic loading rats. However, at 53d, static loading rats had lower proteoglycan content than dynamic loading rats (p < 0.05). Disc structure is altered following static compression removal, but nucleus proteoglycan content remaining elevated in dynamic group. Dynamic fusionless implants would better preserve disc integrity.

Bone growth resumption following in vivo static and dynamic compression removals on rats.

Mechanical loadings influence bone growth and are used in pediatric treatments of musculoskeletal deformities. This in vivo study aimed at evaluating the effects of static and dynamic compression application and subsequent removal on bone growth, mineralization and neuropathic pain markers in growing rats. Forty-eight immature rats (28 days old) were assigned in two groups (2- and 4 weeks experiment duration) and four subgroups: control, sham, static, and dynamic. Controls had no surgery. A micro-loading device was implanted on the 6th and 8th caudal vertebrae of shams without loading, static loading at 0.2 MPa or dynamic loading at 0.2 MPa ± 30% and 0.1 Hz. In 2-week subgroups, compression was maintained for 15 days prior to euthanasia, while in 4- week subgroups, compression was removed for 10 additional days. Growth rates, histomorphometric parameters and mineralization intensity were quantified and compared. At 2 weeks, growth rates and growth plate heights of loaded groups (static/dynamic)were significantly lower than shams (p b 0.01).However, at 4 weeks, both growth rates and growth plate heights of loaded groups were similar to shams. At 4 weeks, alizarin red intensity was significantly higher in dynamics compared to shams (p b 0.05) and controls (p b 0.01). Both static and dynamic compressions enable growth resumption after loading removal, while preserving growth plate histomorphometric integrity. However, mineralization was enhanced after dynamic loading removal only. Dynamic loading showed promising results for fusionless treatment approaches for musculoskeletal deformities.

Quantitative pivot shift assessment using combined inertial and magnetic sensing.

PURPOSE: The purpose of the study was to demonstrate the feasibility of a new measurement system using micro-electromechanical systems (MEMS)-based sensors for quantifying the pivot shift phenomenon. METHODS: The pivot shift test was performed on 13 consecutive anterior cruciate ligament-deficient subjects by an experienced examiner while femur and tibia kinematics were recorded using two inertial sensors each composed of an accelerometer, gyroscope and magnetometer. The gravitational component of the acquired data was removed using a novel method for estimating sensor orientations. Correlation between the clinical pivot shift grade and acceleration and velocity parameters was measured using Spearman's rank correlation coefficients. RESULTS: The pivot shift phenomenon was best characterized as a drop in femoral acceleration observed at the time of reduction. The correlation between the femoral acceleration drop and the clinical grade was shown to be very strong (r = 0.84, p < 0.0001). CONCLUSIONS: The present study demonstrates the feasibility of quantifying the pivot shift using MEMS-based sensors and removing the gravitational component of acceleration using an estimation of sensor orientation for improved correlation to the clinical grade.

In vivo dynamic loading reduces bone growth without histomorphometric changes of the growth plate.

This in vivo study aimed at investigating the effects of dynamic compression on the growth plate. Rats (28 days old) were divided into three dynamically loaded groups, compared with two groups (control, sham). A device was implanted on the 6th and 8th caudal vertebrae for 15 days. Controls (n = 4) did not undergo surgery. Shams (n = 4) were operated but not loaded. Dynamic groups had sinusoidal compression with a mean value of 0.2 MPa: 1.0 Hz and ± 0.06 MPa (group a, n = 4); 0.1 Hz and ± 0.2 MPa (group b, n = 4); 1.0 Hz and ± 0.14 MPa (group c, n = 3). Growth rates (µm/day) of dynamic groups (a) and (b) were lower than shams (p < 0.01). Growth plate heights, hypertrophic cell heights and proliferative cell counts per column did not change in dynamic (a) and (b) groups compared with shams (p > 0.01). Rats from dynamic group (c) had repeated inflammations damaging tissues; consequently, their analysis was unachievable. Increasing magnitude or frequency leads to growth reduction without histomorphometric changes. However, the combined augmentation of magnitude and frequency alter drastically growth plate integrity. Appropriate loading parameters could be leveraged for developing novel growth modulation implants to treat skeletal deformities.

Genome-wide association study signal at the 12q12 locus for Crohn's disease may represent associations with the MUC19 gene.

BACKGROUND: Genome-wide association studies (GWAS) in Crohn's disease (CD) have identified associations with single-nucleotide polymorphism (SNP) rs11175593 at chromosome 12q12. The MUC19 and LRRK2 genes reside close to the GWAS signal, but it is as yet unclear which of the 2 genes represent the CD susceptibility genes. METHODS: We studied associations between nonsynonymous coding variants in the MUC19 (5) and LRRK2 (3) genes in a case-control sample comprising CD cases aged <18 years at diagnosis. The GWAS lead SNP rs11175593 was also investigated. Allelic, genotype, and haplotype associations were examined assuming different models of inheritance. RESULTS: A total of 530 cases and 600 controls were studied. The mean (±SD) age at diagnosis was 12.4 (±3.3). Most cases were male (57.4%). Most patients had ileocolonic disease location (48.8%) and inflammatory behavior at diagnosis (87.0%). Three MUC19 SNPs were nominally significantly associated with CD (rs11564245, Asp→His: P = 0.02; rs4768261, Ser→Phe: P = 0.0008; and rs2933353, Glu→Ala: P = 0.01). Associations with rs4768261 were maintained after corrections for multiple comparisons (permuted, P = 0.007). None of the LRRK2 SNPs were associated with CD. Haplotype analysis supported the single SNP associations noted with the MUC19 gene. CONCLUSIONS: GWAS signal at chromosome 12q12 for CD may represent associations with the MUC19 gene.

A non-synonymous coding variant (L616F) in the TLR5 gene is potentially associated with Crohn's disease and influences responses to bacterial flagellin.

BACKGROUND AND OBJECTIVES: Although numerous studies have implicated TLR5, or its ligands, bacterial flagellins, in the pathogenesis of Crohn's disease (CD), genome-wide association studies (GWAS) have not reported associations with the TLR5 gene. We aimed to examine potential CD-associated TLR5 variants and assess whether they modified inflammatory responses to bacterial flagellins. METHODS AND PRINCIPAL RESULTS: A two-stage study was carried out. In stage 1, we genotyped tagging single-nucleotide polymorphisms (tag-SNPs) in the TLR5 gene in a sample of CD cases (<20 years of age, N = 566) and controls (N = 536). Single SNP and haplotype analysis was carried out. In Stage 2, we assessed the functional significance of potential CD-associated variant(s) vis-à-vis effects on the inflammatory response to bacterial flagellin using HEK293T cells. We observed marginal association between a non-synonymous coding SNP rs5744174 (p = 0.05) and CD. Associations between SNP rs851139 that is in high linkage disequilibrium (LD) with SNP rs5744174 were also suggested (p = 0.07). Haplotype analysis revealed that a 3 marker haplotype was significantly associated with CD (p = 0.01). Functional studies showed that the risk allele (616F) (corresponding to the C allele of SNP rs5744174) conferred significantly greater production of CCL20 in response to a range of flagellin doses than the comparator allele (616L). CONCLUSIONS: Our findings suggest that a non-synonymous coding variation in the TLR5 gene may confer modest susceptibility for CD.

Association between the PTPN2 gene and Crohn's disease: dissection of potential causal variants.

BACKGROUND: Although genome-wide association studies (GWAS) and subsequent meta-analyses have confirmed associations between the PTPN2 (protein tyrosine phosphatase, nonreceptor type 2) gene and Crohn's disease (CD), the potential causal variants remain unidentified. We aimed to dissect potential causal CD-associated PTPN2 variants, assess their functional significance, and relate PTPN2 protein expression with inflammation in CD. METHODS: A 3-stage study was carried out. In stage 1, we genotyped tagging single nucleotide polymorphisms (tag-SNPs) in the PTPN2 gene in a sample of patients with CD (<20 years, n = 556) and controls (n = 602). In stage 2, we resequenced the putative promoter, target exons and introns in the PTPN2 gene, and examined associations with high-frequency variants with CD in the stage 1 cohort. In stage 3 we studied the relationship between PTPN2 protein expression and mucosal inflammation and carried out in silico analyses to study the functional characteristics of the PTPN2 CD-associated SNPs. RESULTS: In stage 1, we observed associations between 5 intronic SNPs and CD including rs1893217 (P = 2 × 10⁻4), the SNP that is in perfect linkage disequilibrium with the lead genome-wide association studies SNP rs2542151. Resequencing revealed 2 known promoter polymorphisms. No associations between these promoter SNPs and CD were evident. In silico analyses revealed that the 5 associated intronic SNPs influenced PTPN2 expression and binding to important transcription factors. PTPN2 protein was overexpressed in inflamed intestinal tissues of patients with CD. CONCLUSIONS: Our findings suggest that noncoding variation in the PTPN2 gene may represent the causal variations influencing susceptibility for CD.

NELL1, NCF4, and FAM92B genes are not major susceptibility genes for Crohn's disease in Canadian children and young adults.

BACKGROUND: Genome-wide association studies (GWAS) and replication studies have shown conflicting associations between the NELL1, NCF4, and FAM92B genes and susceptibility for Crohn's disease (CD). We sought to examine whether these genes were associated with CD in Canadian children and young adults. METHODS: A case-control study was carried out at three pediatric gastroenterology clinics across Canada. Patients, ≤20 years at diagnosis, along with controls representative of the general population were selected. Study subjects were genotyped for 22 single nucleotide polymorphisms (SNPs) across the target genes. Allelic and haplotype associations were examined. Odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were estimated. RESULTS: In all, 566 CD cases and 602 controls were investigated. The mean (±SD) age of the patients was 12.3 (±3.3) years. Most patients were male (57.8%), of Caucasian ancestry (98.2%), and had ileocolonic disease location (48.8%). Barring nominal associations with one FAM92B SNP, none of the other 21 SNPs analyzed were associated with CD either at the allelic or haplotype level. Separate analysis for ileal CD (L1 plus L3) also did not reveal significant associations with any of the SNPs. Similarly, a pooled analysis using data from two recent studies did not demonstrate associations between the NCF4 (OR = 1.10, 95% CI = 0.91-1.32, P = 0.32) and FAM92B (OR = 1.05, 95% CI = 0.95-1.17, P = 0.36) GWAS lead SNPs and ileal CD. CONCLUSIONS: GWAS-reported associations in the NELL1, NCF4, and FAM92B genes could not be replicated in Canadian children and young adults. Further investigation in other populations will be required to confirm the presence/absence of associations, if any.

Genes involved in the metabolism of poly-unsaturated fatty-acids (PUFA) and risk for Crohn's disease in children & young adults.

BACKGROUND AND OBJECTIVES: Epidemiological evidence for the role of polyunsaturated fatty-acids (PUFA) in Crohn's disease (CD) is unclear, although the key metabolite leucotriene B4 (LTB(4)) is closely linked to the inflammatory process. We hypothesized that inherited variation in key PUFA metabolic enzymes may modify susceptibility for CD. METHODS AND PRINCIPAL RESULTS: A case-control design was implemented at three pediatric gastroenterology clinics in Canada. Children ≤20 yrs diagnosed with CD and controls were recruited. 19 single nucleotide polymorphisms (SNPs) across the ALOX5 (4) CYP4F3 (5) and CYP4F2 (10) genes, were genotyped. Associations between SNPs/haplotypes and CD were examined. A total of 431 cases and 507 controls were studied. The mean (±SD) age of the cases was 12.4 (±3.3) years. Most cases were male (56.4%), had ileo-colonic disease (L3±L4, 52.7%) and inflammatory behavior (B1±p, 87%) at diagnosis. One genotyped CYP4F3 SNP (rs2683037) not in Hardy-Weinberg Equilibrium was excluded. No associations with the remaining 4 CYP4F3 SNPs with CD were evident. However haplotype analysis revealed associations with a two-marker haplotype (TG) (rs3794987 & rs1290617) (p = 0.02; permuted p = 0.08). CYP4F2 SNPs, rs3093158 (OR (recessive) = 0.56, 95% CI = 0.35-0.89; p = 0.01), rs2074902 (OR (trend) = 1.26, 95% CI = 1.00-1.60; p = 0.05), and rs2108622 (OR (recessive) = 1.6, 95% CI = 1.00-2.57; p = 0.05) were significantly associated whereas rs1272 (OR (recessive) = 0.58, 95% CI = 0.30-1.13; p = 0.10) showed suggestions for associations with CD. A haplotype comprising these 4 SNPs was significantly associated (p = 0.007, permuted p = 0.02) with CD. Associations with SNP rs3780901 in the ALOX5 gene were borderline non-significant (OR (dominant) = 1.29, 95% CI = 0.99-1.67; p = 0.056). A haplotype comprising the 4 ALOX5 SNPs (TCAA, p = 0.036) was associated with CD, but did not withstand corrections for multiple comparisons (permuted p = 0.14). CONCLUSIONS: Inherited variation in enzymes involved in the synthesis/metabolism of LTB(4) may be associated with CD. These findings implicate PUFA metabolism as a important pathway in the CD pathogenesis.