A candidate transacting modulator of fetal hemoglobin gene expression in the Arab—Indian haplotype of sickle cell anemia

Fetal hemoglobin (HbF) levels are higher in the Arab–Indian (AI) β‐globin gene haplotype of sickle cell anemia compared with African‐origin haplotypes. To study genetic elements that effect HbF expression in the AI haplotype we completed whole genome sequencing in 14 Saudi AI haplotype sickle hemoglobin homozygotes—seven selected for low HbF (8.2% ± 1.3%) and seven selected for high HbF (23.5% ± 2.6%). An intronic single nucleotide polymorphism (SNP) in ANTXR1, an anthrax toxin receptor (chromosome 2p13), was associated with HbF. These results were replicated in two independent Saudi AI haplotype cohorts of 120 and 139 patients, but not in 76 Saudi Benin haplotype, 894 African origin haplotype and 44 AI haplotype patients of Indian origin, suggesting that this association is effective only in the Saudi AI haplotype background. ANTXR1 variants explained 10% of the HbF variability compared with 8% for BCL11A. These two genes had independent, additive effects on HbF and together explained about 15% of HbF variability in Saudi AI sickle cell anemia patients. ANTXR1 was expressed at mRNA and protein levels in erythroid progenitors derived from induced pluripotent stem cells (iPSCs) and CD34⁺ cells. As CD34⁺ cells matured and their HbF decreased ANTXR1 expression increased; as iPSCs differentiated and their HbF increased, ANTXR1 expression decreased. Along with elements in cis to the HbF genes, ANTXR1 contributes to the variation in HbF in Saudi AI haplotype sickle cell anemia and is the first gene in trans to HBB that is associated with HbF only in carriers of the Saudi AI haplotype. Am. J. Hematol. 91:1118–1122, 2016. © 2016 Wiley Periodicals, Inc.     

The susceptibility loci juvenile idiopathic arthritis shares with other autoimmune diseases extend to PTPN2, COG6, and ANGPT1

Objective

To test for associations between non–major histocompatibility complex susceptibility loci previously reported in autoimmune diseases and juvenile idiopathic arthritis (JIA).

Methods

Published autoimmune disease genome‐wide association studies were reviewed, and 519 single‐nucleotide polymorphisms (SNPs) were selected for association testing. The initial cohort included 809 JIA cases and 3,535 controls of non‐Hispanic, European ancestry. Of the SNPs, 257 were successfully genotyped, while 168 were imputed with quality. Based on findings in the initial cohort, replication was sought for 21 SNPs in a second cohort of 1,015 JIA cases and 1,569 controls collected in the US and Germany. For the initial cohort, tests for association were adjusted for potential confounding effects of population structure by including principal components derived from a genome‐wide association study as covariates in logistic regression models. Odds ratios (ORs) and 95% confidence intervals were calculated.

Results

Testing for association of previously reported autoimmune disease genetic associations in the initial cohort suggested associations with JIA in 13 distinct loci. Of these, 7 were validated in the replication cohort. Meta‐analysis results for the replicating loci included PTPN22 (rs6679677 [OR 1.58, P = 1.98 × 10⁻¹²], rs2476601 [OR 1.64, P = 1.90 × 10⁻¹³], and rs2488457 [OR 1.32, P = 6.74 × 10⁻⁸]), PTPN2 (rs1893217 [OR = 1.33, P = 1.60 × 10⁻⁹] and rs7234029 [OR 1.35, P = 1.86 × 10⁻¹⁰]), ADAD1‐IL2‐IL21 (rs17388568 [OR 1.24, P = 1.13 × 10⁻⁶] and rs13143866 [OR 0.83, P = 1.95 × 10⁻⁴]), STAT4 (rs3821236 [OR = 1.27, P = 2.36 × 10⁻⁶] and rs7574865 [OR = 1.31, P = 2.21 × 10⁻⁶]), C12orf30 (rs17696736 [OR = 1.19, P = 2.59 × 10⁻⁵]), COG6 (rs7993214 [OR = 0.76, P = 1.10 × 10⁻⁵]), and ANGPT1 (rs1010824 [OR = 0.79, P = 2.91 × 10⁻⁴]). These polymorphisms have been reported in diseases such as rheumatoid arthritis, type 1 diabetes mellitus, Crohn's disease, and multiple sclerosis.

Conclusion

General susceptibility loci for autoimmunity are shared across diseases, including JIA, suggesting the potential for common therapeutic targets and mechanisms.

     

Silent cerebral infarction,income, and grade retention among students with sickle cell anemia

Children with sickle cell anemia have a higher‐than‐expected prevalence of poor educational attainment. We test two key hypotheses about educational attainment among students with sickle cell anemia, as measured by grade retention and use of special education services: (1) lower household per capita income is associated with lower educational attainment; (2) the presence of a silent cerebral infarct is associated with lower educational attainment. We conducted a multicenter, cross‐sectional study of cases from 22 U.S. sites included in the Silent Infarct Transfusion Trial. During screening, parents completed a questionnaire that included sociodemographic information and details of their child's academic status. Of 835 students, 670 were evaluable; 536 had data on all covariates and were used for analysis. The students' mean age was 9.4 years (range: 5–15) with 52.2% male; 17.5% of students were retained one grade level and 18.3% received special education services. A multiple variable logistic regression model identified that lower household per capita income (odds ratio [OR] of quartile 1 = 6.36, OR of quartile 2 = 4.7, OR of quartile 3 = 3.87; P = 0.001 for linear trend), age (OR = 1.3; P < 0.001), and male gender (OR, 2.2; P = 0.001) were associated with grade retention; silent cerebral infarct (P = 0.31) and painful episodes (P = 0.60) were not. Among students with sickle cell anemia, household per capita income is associated with grade retention, whereas the presence of a silent cerebral infarct is not. Future educational interventions will need to address both the medical and socioeconomic issues that affect students with sickle cell anemia. Am. J. Hematol. 89:E188–E192, 2014. © 2014 Wiley Periodicals, Inc.     

Hemorheological risk factors of acute chest syndrome and painful vaso-occlusive crisis in children with sickle cell disease

Background

Little is known about the effects of blood rheology on the occurrence of acute chest syndrome and painful vaso-occlusive crises in children with sickle cell anemia and hemoglobin SC disease.

Design and Methods

To address this issue, steady-state hemorheological profiles (blood viscosity, red blood cell deformability, aggregation properties) and hematologic parameters were assessed in 44 children with sickle cell anemia and 49 children with hemoglobin SC disease (8-16 years old) followed since birth. Clinical charts were retrospectively reviewed to determine prior acute chest syndrome or vaso-occlusive episodes, and rates of these complications were calculated.

Results

Multivariate analysis revealed that: 1) a higher steady-state blood viscosity was associated with a higher rate of vaso-occlusive crises in children with sickle cell anemia, but not in children with hemoglobin SC disease; 2) a higher steady-state red blood cell disaggregation threshold was associated with previous history of acute chest syndrome in children with hemoglobin SC disease and boys with sickle cell anemia.

Conclusions

Our results indicate for the first time that the red blood cell aggregation properties may play a role in the pathophysiology of acute chest syndrome in children with hemoglobin SC disease and boys with sickle cell anemia. In addition, whereas greater blood viscosity is associated with a higher rate of vaso-occlusive crises in children with sickle cell anemia, no association was found in children with hemoglobin SC disease, underscoring differences in the etiology of vaso-occlusive crises between sickle cell anemia and hemoglobin SC disease.Key words: sickle cell anemia, hemoglobin SC disease, red blood aggregation, blood viscosity, red blood cell deformability     

Association of TNFSF4 polymorphisms with systemic lupus erythematosus: a meta-analysis

Abstract

Introduction To more precisely estimate the association between the tumor necrosis factor ligand superfamily member 4 (TNFSF4) gene polymorphisms and systemic lupus erythematosus (SLE) risk, we surveyed studies on the association of the TNFSF4 rs2205960, rs1234315, rs844644, and rs844648 polymorphisms with SLE.

Methods A literature-based search was conducted to identify all relevant studies. A total of eight independent studies were identified and subsequently reviewed in the meta-analysis.

Results The meta-analysis showed an association between the TNFSF4 rs2205960 polymorphism and SLE in all subjects [ odds ratio (OR) 1.327, 95 % confidence interval (CI) 1.227–1.436, P < 0.001]. In a subgroup analysis by ethnicity, a significantly increased risk for SLE was associated with TNFSF4 rs2205960 T allele among patients of European (OR 1.254, 95 % CI 1.185–1.328, P < 0.001) and Asian ethnicity (OR 1.425, 95 % CI 1.352–1.501, P < 0.001). The meta-analysis of the rs1234315 polymorphism revealed no association between SLE and the rs1234315 T allele in all subjects (OR 1.167, 95 % CI 0.874–1.558, P = 0.296), but the results of the subgroup analysis revealed significant association in subjects of Asian ethnicity (OR 1.386, 95 % CI 1.318–1.458, P < 0.001). No association was found between the rs844644 and rs844648 polymorphisms and SLE.

Conclusion The results of our meta-analysis suggest that the TNFSF4 rs2205960 polymorphism may confer susceptibility to SLE in different populations and that the TNFSF4 rs1234315 polymorphism is associated with susceptibility to SLE in Asians.     

Frequency of pain crises in sickle cell anemia and its relationship with the sympatho-vagal balance, blood viscosity and inflammation

Background

Recent evidence suggests that autonomic nervous system activity could be involved in the pathophysiology of sickle cell disease, but it is unclear whether differences in autonomic nervous system activity are detectable during steady state in patients with mild and severe disease. The aim of the present study was to compare the autonomic nervous system activity, blood rheology, and inflammation in patients with sickle cell anemia according to the frequency of acute pain crisis.

Design and Methods

Twenty-four healthy volunteers, 20 patients with sickle cell anemia with milder disease, and 15 patients with sickle cell anemia with more severe disease were recruited. Milder disease was defined as having no pain crisis within the previous year. More severe disease was defined as having had within the previous year three or more pain crises which were documented by a physician and required treatment with narcotics. The autonomic nervous system activity was determined by spectral analysis of nocturnal heart rate variability. Blood viscosity determination and measurements of several inflammatory markers (interleukin-6, soluble vascular cell adhesion molecule-1, soluble CD40 ligand and sL-selectin) were made on blood samples collected in steady-state conditions.

Results

Results showed that: 1) patients who had suffered more frequent pain crises had lower parasympathetic activity and greater sympatho-vagal imbalance than both controls and patients with milder disease. However, when adjusted for age, no significant difference was detected between the two sickle cell anemia patient groups; 2) patients who had suffered more frequent pain crises had higher blood viscosity than patients with milder disease, and this was not dependent on age.

Conclusions

Results from the present study indicate that both the autonomic nervous system activity and blood viscosity are impaired in patients with sickle cell anemia exhibiting high frequency of pain crisis in comparison with those who did not experience a crisis within the previous year.     

Nutritional status, hospitalization and mortality among patients with sickle cell anemia in Tanzania

Background

Reduced growth is common in children with sickle cell anemia, but few data exist on associations with long-term clinical course. Our objective was to determine the prevalence of malnutrition at enrolment into a hospital-based cohort and whether poor nutritional status predicted morbidity and mortality within an urban cohort of Tanzanian sickle cell anemia patients.

Design and Methods

Anthropometry was conducted at enrolment into the sickle cell anemia cohort (n=1,618; ages 0.5–48 years) and in controls who attended screening (siblings, walk-ins and referrals) but who were found not to have sickle cell anemia (n=717; ages 0.5–64 years). Prospective surveillance recorded hospitalization at Muhimbili National Hospital and mortality between March 2004 and September 2009.

Results

Sickle cell anemia was associated with stunting (OR=1.92, P<0.001, 36.2%) and wasting (OR=1.66, P=0.002, 18.4%). The greatest growth deficits were observed in adolescents and in boys. Independent of age and sex, lower hemoglobin concentration was associated with increased odds of malnutrition in sickle cell patients. Of the 1,041 sickle cell anemia patients with a body mass index z-score at enrolment, 92% were followed up until September 2009 (n=908) or death (n=50). Body mass index and weight-for-age z-score predicted hospitalization (hazard ratio [HZR]=0.90, P=0.04 and HZR=0.88, P=0.02) but height-for-age z-score did not (HZR=0.93, NS). The mortality rate of 2.5 per 100 person-years was not associated with any of the anthropometric measures.

Conclusions

In this non-birth-cohort of sickle cell anemia with significant associated undernutrition, wasting predicted an increased risk of hospital admission. Targeted nutritional interventions should prioritize treatment and prevention of wasting.     

Genetic variation in CLDN1 and susceptibility to hepatitis C virus infection

Summary. Claudin‐1 is a recently discovered co‐receptor for hepatitis C virus (HCV) that is required for late‐stage binding of the virus. Because variants in the gene that encodes claudin‐1 (CLDN1) could play a role in HCV infection, we conducted a ‘whole gene association study’ among injection drug users (IDUs) to examine whether CLDN1 genetic variants were associated with the risk of HCV infection or with viral clearance. In a cross sectional study, we examined genotype results for 50 single nucleotide polymorphisms (SNPs) across the CLDN1 gene region, comparing genotypes among participants with chronic HCV (n = 658) to those in IDUs who had cleared HCV (n = 199) or remained HCV‐uninfected (n = 68). Analyses were controlled for racial ancestry (African‐American or European‐American) by stratification and logistic regression modeling. We found that participants who remained uninfected more often carried CLDN1 promoter region SNPs ?15312C [odds ratio (OR), 1.72; 95% confidence interval (CI) 1.00–2.94; P = 0.048], ?7153A (OR, 2.13; 95% CI, 1.25–3.62; P = 0.006) and ?5414C (OR, 1.78; 95% CI, 1.06–3.00; P = 0.03). HCV‐uninfected participants less often carried CLDN1 IVS1?2983C (OR, 0.55; 95% CI, 0.31–0.97; P = 0.04), which lies in intron 1. CLDN1 ?15312C, ?7153A and ?5414C formed a haplotype in both the African‐American and European‐American participants and a haplotype analysis supported the association of CLDN1 ?7153A in the HCV‐uninfected participants. The analyses of HCV clearance revealed no associations with any SNP. These results indicate that genetic variants in regulatory regions of CLDN1 may alter susceptibility to HCV infection.     

Brief Report: Single‐nucleotide polymorphisms in VKORC1 are risk factors for systemic lupus erythematosus in Asians

Objective

The increased risk of thrombosis in systemic lupus erythematosus (SLE) may be partially explained by interrelated genetic pathways for thrombosis and SLE. The present study was undertaken to investigate whether 33 established and novel single‐nucleotide polymorphisms (SNPs) in 20 genes involved in hemostasis pathways that have been associated with deep venous thrombosis (DVT) in the general population are risk factors for SLE among Asian subjects.

Methods

Patients in the discovery cohort were enrolled in 1 of 2 North American SLE cohorts. Patients in the replication cohort were enrolled in 1 of 4 Asian or 2 North American cohorts. We first genotyped 263 Asian patients with SLE and 357 healthy Asian control subjects for 33 SNPs in the discovery phase, and then genotyped 5 SNPs in up to an additional 1,496 patients and 993 controls in the replication phase. Patients were compared to controls for bivariate association with minor alleles. Principal components analysis was used to control for intra‐Asian ancestry in the replication cohort.

Results

Two genetic variants in the gene VKORC1 were highly significant in both the discovery and replication cohorts: rs9934438 (in the discovery cohort, odds ratio [OR] 2.45, P = 2 × 10⁻⁹; in the replication cohort, OR 1.54, P = 4 × 10⁻⁶) and rs9923231 (in the discovery cohort, OR 2.40, P = 6 × 10⁻⁹; in the replication cohort, OR 1.53, P = 5 × 10⁻⁶). These associations were significant in the replication cohort after adjustment for intra‐Asian ancestry: for rs9934438, OR 1.34, P = 0.0029; for rs9923231, OR 1.34, P = 0.0032.

Conclusion

Genetic variants in VKORC1, which are involved in vitamin K reduction and associated with DVT, correlate with SLE development in Asian subjects. These results suggest that there may be intersecting genetic pathways for the development of SLE and thrombosis.

     

A hybrid approach reveals the allosteric regulation of GTP cyclohydrolase I

Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) catalyzes the conversion of GTP to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). Besides other roles, BH4 functions as cofactor in neurotransmitter biosynthesis. The BH4 biosynthetic pathway and GCH1 have been identified as promising targets to treat pain disorders in patients. The function of mammalian GCH1s is regulated by a metabolic sensing mechanism involving a regulator protein, GCH1 feedback regulatory protein (GFRP). GFRP binds to GCH1 to form inhibited or activated complexes dependent on availability of cofactor ligands, BH4 and phenylalanine, respectively. We determined high-resolution structures of human GCH1−GFRP complexes by cryoelectron microscopy (cryo-EM). Cryo-EM revealed structural flexibility of specific and relevant surface lining loops, which previously was not detected by X-ray crystallography due to crystal packing effects. Further, we studied allosteric regulation of isolated GCH1 by X-ray crystallography. Using the combined structural information, we are able to obtain a comprehensive picture of the mechanism of allosteric regulation. Local rearrangements in the allosteric pocket upon BH4 binding result in drastic changes in the quaternary structure of the enzyme, leading to a more compact, tense form of the inhibited protein, and translocate to the active site, leading to an open, more flexible structure of its surroundings. Inhibition of the enzymatic activity is not a result of hindrance of substrate binding, but rather a consequence of accelerated substrate binding kinetics as shown by saturation transfer difference NMR (STD-NMR) and site-directed mutagenesis. We propose a dissociation rate controlled mechanism of allosteric, noncompetitive inhibition.

Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) (EC:3.5.4.16) catalyzes the conversion of GTP to dihydroneopterin triphosphate (H2NTP). This reaction is the first and rate-limiting step involved in the de novo synthesis of tetrahydrobiopterin (BH4) (1). BH4 plays key roles in phenylalanine catabolism and the biosynthesis of serotonin and catecholamine-type neurotransmitters like dopamine or norepinephrine by functioning as an essential cofactor for hydroxylases of the aromatic amino acids phenylalanine, tyrosine, and tryptophan (2, 3). Further, BH4 is cofactor for the family of nitric oxide synthases (4), which produce the cellular signaling molecule nitric oxide that helps to modulate vascular tone and insulin secretion and affects inflammation as well as the regulation of immune responses (5).Several lines of evidence, including human genetic data that show that a GCH1-deficient haplotype is pain resistant, suggest that selective inhibition of GCH1 is an attractive target to treat neuropathic and inflammatory pain disorders (6–8). Abnormalities in the control mechanisms of GCH1 or the activities in other enzymes of its biosynthetic pathway leads to BH4 deficiency, which is linked to a variety of vascular diseases such as diabetes, atherosclerosis, and hypertension (9–14) and to neurological disorders, including Parkinson’s disease (15, 16). These examples impressively show the serious consequences of nonphysiological levels of BH4. Nature therefore evolved a highly regulated mechanism of BH4 homeostasis.In a seminal paper (17) by Harada et al., the molecular basis of BH4 homeostasis was uncovered and shown to involve GCH1 and a regulatory protein, now known as GTP-cyclohydrolase-I-feedback-regulatory protein (GFRP), which simultaneously functions as a positive and negative regulator of GCH1 (17). The effects of GFRP on GCH1 occur via formation of heteromeric protein complexes between GCH1 and GFRP, which are dependent on the intracellular concentrations of the effector molecules phenylalanine or BH4. Elevated phenylalanine levels lead to stimulation of GCH1 activity, whereas BH4, the end product of the biosynthesis pathway, inhibits GCH1 in a feedback inhibition type mode (18). Mammalian GCH1 shows cooperative enzymatic activity. Complex formation with GFRP-Phe leads to increased activity at lower substrate concentrations and eliminates substrate cooperativity. Conversely, GCH1 alone is allosterically inhibited by BH4. In the presence of GFRP, the inhibitory effect of BH4 is boosted and occurs at lower, physiologically relevant BH4 concentrations. The GCH1−GFRP system can therefore be regarded as a metabolic sensor that establishes BH4 and aromatic amino acid homeostasis.The human GCH1 sequence comprises 250 amino acids and forms a 270-kDa, D5-symmetric homodecameric functional enzyme complex in solution (19, 20). GFRP occurs as a pentamer of 50 kDa (5 × 10 kDa). GCH1−GFRP complexes consist of one GCH1 decamer flanked by two pentameric GFRP complexes. Association is along the particle fivefold axes, and the complexes are ∼370 kDa in size (21).Structural information on GCH1 was first obtained on the Escherichia coli enzyme (19, 22, 23); later structures of the human GCH1 (24) and rat GCH1−GFRP complexes (18, 25) were determined. The structures revealed the subunit fold and quaternary structure of the functional complex and established GCH1 as a Zn(II)-dependent hydrolase. The X-ray structures of stimulatory and inhibitory rat GCH1−GFRP complexes show that phenylalanine binds to a surface pocket on GFRP close to the protein−protein interaction interface with GCH1, whereas BH4 binds to an allosteric pocket on GCH1 close to the GFRP interface (18, 25, 26). Structural differences between stimulatory and inhibitory complexes were found to be minor. The medium resolution (2.8 Å) of the studies and the circumstance that, for this particular case, folding and unfolding events play a major role, and are impacted by crystal packing artifacts, did not allow for detailed insights into the structural basis of allosteric control mechanisms.We therefore decided to investigate this unresolved issue. First, we determined the structures of human GCH1−GFRP complexes by cryoelectron microscopy (cryo-EM) at high resolution. Further, we conducted elaborate structural, enzyme kinetics, biophysics, and mutagenesis studies on human GCH1 and used the results in conjunction with the cryo-EM data to identify the key structural features responsible for the allosteric inhibition of GCH1.     

Genomic ancestry and somatic alterations correlate with age at diagnosis in Hispanic children with B‐cell acute lymphoblastic leukemia

Hispanic children have a higher incidence of acute lymphoblastic leukemia (ALL) than non‐Hispanic whites but tend to be diagnosed at older ages. In genome‐wide association studies, Native American ancestry and polymorphisms in six genes have been associated with ALL risk. In multivariable regression models, we investigated whether genomic ancestry, inherited risk SNPs, or acquired somatic alterations were associated with differences in age at diagnosis in Hispanic children with B‐cell ALL. Genome‐wide array data were used to estimate each participant's percent membership in the three Hispanic ancestral populations: Native American, African, and European. Each 20% increase in European ancestry was associated with a six month younger age at diagnosis (95% CI = 0.36–11.6 months, P = 0.037). Correspondingly, each 20% increase in Native American ancestry was associated with a six‐month older age at diagnosis (P = 0.037). Both the TEL‐AML1 translocation and high‐hyperdiploidy were associated with younger age at diagnosis (24.4 months, P = 2.0 x 10^?4 and 12.4 months, P = 0.011, respectively), while CDKN2A and IKZF1 deletions were associated with older age at diagnosis (19.7 months, P = 7.0 x 10^?4 and 18.1 months, P = 0.012, respectively). No associations with age at diagnosis were observed for RAS mutation, PAX5 deletion or for known heritable risk alleles in IKZF1, CDKN2A, PIP4K2A, GATA3, ARID5B, or CEBPE. Because younger age at diagnosis is associated with improved treatment outcomes for children with ALL, the effect of European ancestry on ALL survival may be mediated by its effect on age at diagnosis, or by proxy, its association with more treatable molecular subtypes of ALL. Am. J. Hematol. 89:721–725, 2014. © 2014 Wiley Periodicals, Inc.     

Lack of association of Toll-like receptor 9 polymorphisms with susceptibility to systemic lupus erythematosus in an Asian population: a meta-analysis

Abstract

The association of Toll-like receptor 9 (TLR9) gene polymorphisms with systemic lupus erythematosus (SLE) risk remains controversial and ambiguous. To more precisely estimate the relationship between TLR9 gene polymorphisms and the susceptibility to SLE, a meta-analysis was performed. A total of seven independent studies were involved in this analysis. Meta-analysis was performed for three TLR9 gene polymorphisms (rs187084, rs352139, and rs352140). We have compared allele or genotype frequencies of the polymorphisms in SLE patients and controls. When available studies were pooled into the meta-analysis, there was no evidence showing a significant association between rs187084 and SLE risk in an Asian population (for C vs. T: OR = 0.81, P = 0.117; for CC vs. TT: OR = 0.71, P = 0.158; for CT vs. TT: OR = 0.86, P = 0.085; for CC + CT vs. TT: OR = 0.78, P = 0.093; for CC vs. CT + TT: OR = 0.81, P = 0.285). Similar results were found between rs352139 and SLE. No significant association was detected in any genetic model in the Asian population either (for G vs. A: OR = 1.11, P = 0.095; for GG vs. AA: OR = 1.32, P = 0.238; for GA vs. AA: OR = 1.17, P = 0.084; for GG + GA vs. AA: OR = 1.17, P = 0.073; for GG vs. GA + AA: OR = 1.17, P = 0.404). We found no association between TLR9 gene rs352140 polymorphism and SLE in the Asian population (for A vs. G: OR = 1.02, P = 0.728). In conclusion, there is still not enough evidence to indicate an association between TLR9 gene rs187084, rs352139, and rs352140 polymorphisms and the development of SLE in the Asian population.     

Leg Amputation for an Extensive,Severe and Intractable Sickle Cell Anemia Ulcer in a Brazilian Patient

A 35-year-old African Brazilian patient had sickle cell anemia complicated with recurrent vasoocclusive (VOC) crises and refractory painful leg ulcers for 16 years. The ulcers started over both medial malleoli and expanded gradually. The ulcer on the left leg spread from the foot to the knee circumferentially and was refractory to all forms of therapy within the frame work of multi-disciplinary care. The patient agreed to a below the knee amputation of the left leg. He felt much better after the amputation but developed severe neuropathic phantom pain that was well controlled medically. He could differentiate the sickle cell anemia and ulcer pain from the neuropathic pain. About 6 months after the amputation he had dengue fever with fatal outcome. This is the first report of treatment of refractory sickle cell anemia leg ulcer with amputation and probably the first report of a Brazilian patient with sickle cell anemia and dengue fever.     

Association of IL-1R2 genetic polymorphisms with the susceptibility of ankylosing spondylitis in Northern Chinese Han population

Objectives. Previous Genome-wide association studies (GWAS) have demonstrated Interleukin-1 receptor 2 (IL-1R2) was strongly associated with susceptibility to ankylosing spondylitis (AS). The aim of this study was to replicate the association of IL-1R2 single-nucleotide polymorphisms (SNPs) with AS in the northern Han Chinese.

Methods. A total of 490 AS patients and 580 matched healthy controls were enrolled in our study. Six tagSNPs in IL-1R2: rs4851526, rs4851527, rs2302589, rs2072476, rs2072472, and rs2310173 were selected and genotyped by Taqman SNP genotyping method. The differences of allele and genotype frequencies were analyzed by use of PLINK 1.07.

Results. Logistic regression analysis showed that one tagSNP rs2302589 in IL-1R2 was significantly associated with AS susceptibility (OR 0.77, 95% CI = 0.64–0.92, P = 0.005). However, no significant association was observed on the other tagSNPs for AS risk. The haplotype analysis further showed that the haplotype “GCGCGG” of IL-1R2 was also associated with the increased risk of AS (OR 1.362, P = 0.0207).

Conclusions. This is the first detection that the genetic variation rs2302589 in IL-1R2 gene was associated with AS in Northern Han Chinese. This result confirmed that IL-1R2 may be genetic biomarker for susceptibility to AS.     

Parent education and biologic factors influence on cognition in sickle cell anemia

Children with sickle cell anemia have a high prevalence of silent cerebral infarcts (SCIs) that are associated with decreased full‐scale intelligence quotient (FSIQ). While the educational attainment of parents is a known strong predictor of the cognitive development of children in general, the role of parental education in sickle cell anemia along with other factors that adversely affect cognitive function (anemia, cerebral infarcts) is not known. We tested the hypothesis that both the presence of SCI and parental education would impact FSIQ in children with sickle cell anemia. A multicenter, cross‐sectional study was conducted in 19 US sites of the Silent Infarct Transfusion Trial among children with sickle cell anemia, age 5–15 years. All were screened for SCIs. Participants with and without SCI were administered the Wechsler Abbreviated Scale of Intelligence. A total of 150 participants (107 with and 43 without SCIs) were included in the analysis. In a multivariable linear regression model for FSIQ, the absence of college education for the head of household was associated with a decrease of 6.2 points (P = 0.005); presence of SCI with a 5.2 point decrease (P = 0.017); each $1000 of family income per capita with a 0.33 point increase (P = 0.023); each increase of 1 year in age with a 0.96 point decrease (P = 0.023); and each 1% (absolute) decrease in hemoglobin oxygen saturation with 0.75 point decrease (P = 0.030). In conclusion, FSIQ in children with sickle cell anemia is best accounted for by a multivariate model that includes both biologic and socioenvironmental factors. Am. J. Hematol. 89:162–167, 2014. © 2013 Wiley Periodicals, Inc.     

Unusual β-Globin Haplotype Distribution in Newborns from Bengo,Angola

Abstract

Mutations on the HBB gene are a common cause of hemoglobinopathies, including sickle cell anemia, a severe genetic condition that constitutes a major public health concern. The aim of this study was to determine the prevalence of sickle cell anemia and β-globin haplotype distribution in newborns from the Bengo region. The first two exons of β-globin gene were sequenced, and the variability at the single nucleotide polymorphism (SNP) defining the Hb S (HBB: c.20A>T) haplotypes, was analyzed by a SNaPshot^® Multiplex system. About 3.3% of the children were homozygous for Hb S, and 82.2% had as background the Bantu/Central African Republic (BAN/CAR) haplotype, 11.2% the Benin (BEN) and 6.6% the Senegal (SEN). The estimate of Hb S reached the very high value of 0.1476?±?0.0133, with the aggravating factor of 82.2% of the sickle alleles being anchored in the BAN/CAR haplotype, associated with the more severe sickle cell anemia phenotypes. Also, the high prevalence of the SEN haplotype was not expected, having therapeutic consequences since is associated with more severe outcomes. In addition, two β-thalassemia (β-thal) variants were also detected, IVS I-110 (G>A) (HBB: c.93-21G>A) and codon 39 (C>T) (HBB: c.118C>T), together totaling a frequency of 1.3%. Some of the newborns with these mutations were compound heterozygotes for Hb S, likely carrying genotypes consistent with sickle cell disease. As a whole, infants molecularly diagnosed with sickle cell disease accounted for 4.5% of newborns from Bengo, Angola, a figure that per se, highlights the urgent need of implementing policies warranting surveillance of these children, in parallel with community education in the region.     

Study of functional variants of the BANK1 gene in rheumatoid arthritis

Objective

To investigate 1 functional (rs17266594) and 2 potentially functional (rs10516487 and rs3733197) BANK1 variants, which were previously identified as systemic lupus erythematosus (SLE) susceptibility markers, to test whether they are associated with rheumatoid arthritis (RA).

Methods

Four different cohorts were included in the study: 1,080 RA patients and 1,368 healthy controls from Spain, 278 RA patients and 568 healthy controls from Sweden, 288 RA patients and 287 healthy controls from Argentina, and 288 RA patients and 288 healthy controls from Mexico. Samples were genotyped for BANK1 single‐nucleotide polymorphisms (SNPs) using a TaqMan 5′‐allele discrimination assay. Statistical analysis comparing allele and genotype distributions was performed with the chi‐square test.

Results

We did not find a significant association between RA and the rs10516487 and rs17266594 BANK1 polymorphisms. However, there was an increase in the major alleles among RA patients. Similarly, for rs3733197, there was an increase in the major allele among patients in every cohort. Nevertheless, this skewing reached statistical significance in the Spanish (P = 0.01, odds ratio [OR] 1.17 [95% confidence interval (95% CI) 1.03–1.32]) and Argentinean (P = 0.04, OR 1.31 [95% CI 1.00–1.72]) populations. We found a significant association of rs10516487 (P = 0.005, OR 1.15 [95% CI 1.04–1.28]) and rs3733197 (P = 0.0009, OR 1.17 [95% CI 1.07–1.29]) with RA in the pooled analysis. In a 3‐SNP haplotype analysis, we found that the major TGG haplotype was significantly associated with RA (P = 0.005, OR 1.14 [95% CI 1.04–1.25]). In addition, we found a common CAA haplotype that was protective against RA (P = 0.0004, OR 0.82 [95% CI 0.74–0.92]).

Conclusion

These results suggest that BANK1 SNPs and haplotypes may contribute to RA susceptibility with a low risk.

     

Association study of IL10 and IL23R–IL12RB2 in Iranian patients with Behçet's disease

Objective

Independent replication of the findings from genome‐wide association studies (GWAS) remains the gold standard for results validation. Our aim was to test the association of Behçet's disease (BD) with the interleukin‐10 gene (IL10) and the IL‐23 receptor–IL‐12 receptor β2 (IL23R–IL12RB2) locus, each of which has been previously identified as a risk factor for BD in 2 different GWAS.

Methods

Six haplotype‐tagging single‐nucleotide polymorphisms (SNPs) in IL10 and 42 in IL23R–IL12RB2 were genotyped in 973 Iranian patients with BD and 637 non‐BD controls. Population stratification was assessed using a panel of 86 ancestry‐informative markers.

Results

Subtle evidence of population stratification was found in our data set. In IL10, rs1518111 was nominally associated with BD before and after adjustment for population stratification (odds ratio [OR] for T allele 1.20, 95% confidence interval [95% CI] 1.02–1.40, unadjusted P [P_unadj] = 2.53 × 10⁻²; adjusted P [P_adj] = 1.43 × 10⁻²), and rs1554286 demonstrated a trend toward association (P_unadj = 6.14 × 10⁻²; P_adj = 3.21 × 10⁻²). Six SNPs in IL23R–IL12RB2 were found to be associated with BD after Bonferroni correction for multiple testing, the most significant of which were rs17375018 (OR for G allele 1.51, 95% CI 1.27–1.78, P_unadj = 1.93 × 10⁻⁶), rs7517847 (OR for T allele 1.48, 95% CI 1.26–1.74, P_unadj = 1.23 × 10⁻⁶), and rs924080 (OR for T allele 1.29, 95% CI 1.20–1.39, P = 1.78 × 10⁻⁵). SNPs rs10489629, rs1343151, and rs1495965 were also significantly associated with BD in all tests performed. Results of meta‐analyses of our data combined with data from other populations further confirmed the role of rs1518111, rs17375018, rs7517847, and rs924080 in the risk of BD, but no epistatic interactions between IL10 and IL23R–IL12RB2 were detected. Results of imputation analysis highlighted the importance of IL23R regulatory regions in the susceptibility to BD.

Conclusion

These findings independently confirm, extend, and refine the association of BD with IL10 and IL23R–IL12RB2. These associations warrant further validation and investigation in patients with BD, as they may have implications for the development of novel therapies (e.g., immunosuppressive therapy targeted at IL‐23p19).

     

The HLA–DRB1 shared epitope is associated with susceptibility to rheumatoid arthritis in African Americans through European genetic admixture

Objective

To determine whether shared epitope (SE)–containing HLA–DRB1 alleles are associated with rheumatoid arthritis (RA) in African Americans and whether their presence is associated with higher degrees of global (genome‐wide) genetic admixture from the European population.

Methods

In this multicenter cohort study, African Americans with early RA and matched control subjects were analyzed. In addition to measurement of serum anti–cyclic citrullinated peptide (anti‐CCP) antibodies and HLA–DRB1 genotyping, a panel of >1,200 ancestry‐informative markers was analyzed in patients with RA and control subjects, to estimate the proportion of European ancestry.

Results

The frequency of SE‐containing HLA–DRB1 alleles was 25.2% in African American patients with RA versus 13.6% in control subjects (P = 0.00005). Of 321 patients with RA, 42.1% had at least 1 SE‐containing allele, compared with 25.3% of 166 control subjects (P = 0.0004). The mean estimated percent European ancestry was associated with SE‐containing HLA–DRB1 alleles in African Americans, regardless of disease status (RA or control). As reported in RA patients of European ancestry, there was a significant association of the SE with the presence of the anti‐CCP antibody: 86 (48.9%) of 176 patients with anti‐CCP antibody–positive RA had at least 1 SE allele, compared with 36 (32.7%) of 110 patients with anti‐CCP antibody–negative RA (P = 0.01, by chi‐square test).

Conclusion

HLA–DRB1 alleles containing the SE are strongly associated with susceptibility to RA in African Americans. The absolute contribution is less than that reported in RA among populations of European ancestry, in which ∼50–70% of patients have at least 1 SE allele. As in Europeans with RA, the SE association was strongest in the subset of African American patients with anti‐CCP antibodies. The finding of a higher degree of European ancestry among African Americans with SE alleles suggests that a genetic risk factor for RA was introduced into the African American population through admixture, thus making these individuals more susceptible to subsequent environmental or unknown factors that trigger the disease.