Genetic polymorphisms in IL-2, IL-10 and FOXP3 are associated with autoimmune neutropaenia in early childhood and autoantibody specificity in a Danish cohort.

Autoimmune neutropaenia (AIN) in early childhood is characterized by chronic neutropaenia and positivity for human neutrophil antibodies (HNA), resulting in the excessive destruction of neutrophils. The association between regulatory T cells (Tregs) and AIN has been described, and in this study, we investigated three Treg-associated genes, IL-2, IL-10 and FOXP3. The frequencies of three single nucleotide polymorphisms (SNPs) in IL-2 -330T>G (rs2069762), +114G>T (rs2069763) and IVS3-116 A>G (rs2069772), four SNPs in IL-10 -3575T>A (rs1800890), -1082G>A (rs1800896), -819 C>T (rs1800871) and -592 C>A (rs1800872) and three SNPs in FOXP3 -3499 A>G (rs3761547), -3279 C>A (rs3761548) and -924 A>G (rs2232365) were compared between 166 Danish AIN patients and 358 healthy controls. Disease association was observed for IL-2 IVS3-116 GG (p = 0.0081, OR = 0.35 [0.15-0.80]), IL-10 -3575 TT (p = 0.0078, OR = 1.71 [1.16-2.54]) and IL-10 -1082 AA (p = 0.014, OR = 1.76 [1.14-2.72]) in all patients and FOXP3 -924 (p = 0.0005, A OR = 0.41 [0.25-0.68] and G OR = 2.42 [1.46-4.01]) in male patients. None of the associations were linked to antibody specificity. Disease-associated haplotypes were observed in IL-2 and FOXP3. IL-2 -330T/+114 T/IVS3-116A was associated with anti-FcγRIIIb-positive patients (p = 0.012, OR = 2.07 [1.18-3.62]). FOXP3 -3499A/-3279C/-924A was associated with anti-HNA-1a-positive male patients (p = 0.016, OR = 0.41 [0.20-0.83]), and ACG was associated with female patients, both in the combined group (p = 0.006, OR = NA) and the anti-FcγRIIIb-positive group (p = 0.002, OR = NA). We conclude that our findings reveal a correlation between SNP in Treg-associated genes and AIN, indicating that AIN could be driven by dysfunction of immune homeostatic-evolving Tregs.

High-resolution HLA genotyping identifies risk alleles in both class I and II for primary autoimmune neutropenia in early childhood in a Danish cohort.

HLA studies in patients with autoimmune neutropenia (AIN) have shown very consistent results for the association with HLA class II alleles at low resolution. This study aimed to examine the association of both HLA class I and class II at high resolution to clarify the contribution of risk alleles to the disease. A total of 107 AIN patients were genotyped for six loci of HLA class I (HLA-A, -B and -C) and class II (HLA-DRB1, -DQB1, and -DPB1) genes by a high-resolution (3-field, 6-digit) analysis and compared with HLA typing of 1000 healthy controls. Compared with the controls, the allele frequencies were significantly higher in AIN patients for A*02:17:01G, C*01:02:01G, DRB1*10:01:01G, DRB1*14:01:01G, DRB1*16:01:01G, DQB1*05:02:01G, and DQB1*05:03:01G but lower significant for C*03:04:01G, DRB1*04:01:01G, DRB1*13:02:01G, DQB1*03:02:01G, and DQB1*06:04:01G. Frequently associated two-locus haplotypes were found to be DRB1*10:01:01G-DQB1*05:01:01G and DRB1*16:01:01G-DQB1*05:02:01G, while the S2 (Q- or D-KRAA) shared epitope (SE) was associated with lower risk. A unique association with HLA alleles was observed between patients with specific anti-HNA-1a antibodies and broad-reacting anti-FcγRIIIb. Anti-HNA-1a antibody-positive patients were associated with C*01:02:01G, DRB1*01:01:01G, DRB1*16:01:01G, DQB1*05:01:01G, DQB1*05:02:01G, DQB1*06:04:01G, and DPB1*10:01:01G; the two-locus haplotypes DRB1*01:01:01G-DQB1*05:01:01G and DRB1*16:01:01G-DQB1*05:02:01G; and the S3P (Q- or R-RRAA) SE. Anti-FcγRIIIb antibody-positive patients were associated with the alleles A*02:17:01G, DRB1*10:01:01G, and DQB1*05:02:01G; the haplotypes DRB1*10:01:01G-DQB1*05:01:01G and DRB1*11:01:02G-DQB1*05:02:01G; and the S3D (DRRAA) SE. The different associations regarding FcγRIIIb antibody specificities could indicate disease heterogeneity.

Genetic variations in low-to-medium-affinity Fcγ receptors and autoimmune neutropenia in early childhood in a Danish cohort.

Autoimmune neutropenia (AIN) in early childhood is caused by autoantibodies directed against antigens on the neutrophil membrane and is a frequent cause of neutropenia in children. Association of AIN with Fcγ receptor (FCGR) 3B variants is well described. In this study, we investigate genetic variations in the FCGR locus and copy number variation of FCGR3B. A total of 130 antibody-positive AIN patients, 64 with specific anti-HNA-1a antibodies and 66 with broad-reacting anti-FcγRIIIb antibodies, were genotyped with a multiplex ligation probe assay and compared with healthy controls. Positive findings were confirmed with real-time q-PCR. We determined copy numbers of the FCGR2 and FCGR3 genes and the following SNPs: FCGR2A Q62W (rs201218628), FCGR2A H166R (rs1801274), FCGR2B I232T (rs1050501), FCGR3A V176F (rs396991), haplotypes for FCGR2B/C promoters (rs3219018/rs780467580), FCGR2C STOP/ORF and HNA-1 genotypes in FCGR3B (rs447536, rs448740, rs52820103, rs428888 and rs2290834). Generally, associations were antibody specific, with all associations being representative of the anti-HNA-1a-positive group, while the only association found in the anti-FcγRIIIb group was with the HNA-1 genotype. An increased risk of AIN was observed for patients with one copy of FCGR3B; the HNA genotypes HNA-1a, HNA-1aa or HNA-1aac; the FCGR2A 166H and FCGR2B 232I variations; and no copies of FCGR2B 2B.4. A decreased risk was observed for HNA genotype HNA-1bb; FCGR2A 166R; FCGR2B 232T; and one copy of FCGR2B promoter 2B.4. We conclude that in our Danish cohort, there was a strong association between variation in the FCGR locus and AIN. The findings of different genetic associations between autoantibody groups could indicate the presence of two different disease entities and disease heterogeneity.

ABO, secretor, and Lewis carbohydrate histo-blood groups are associated with autoimmune neutropenia of early childhood in Danish patients.

BACKGROUND: Autoimmune neutropenia of early childhood (AIN) is caused by autoantibodies directed against antigens on the neutrophil membrane. The ABO, secretor, and Lewis histo-blood group systems control the expression of carbohydrate antigens and have previously been linked to autoimmune diseases. We aimed to investigate the association between genotypes and the risk of AIN in Danish patients. STUDY DESIGN AND METHODS: One hundred fifty-four antibody-positive AIN patients were included. Controls (n = 400) were healthy unrelated Danish blood donors. Molecular determination of ABO, secretor (FUT2), and Lewis (FUT3) genotypes were determined using real-time polymerase chain reaction (qPCR) or Sanger sequencing to infer the prevalence of Lewis antigens (Lea and Leb ) and secretor (SeSe or Sese) or nonsecretor (sese) phenotypes. RESULTS: Blood type O was more common in controls (46.8%) than in AIN patients (36.4%) (OR = 0.65; p = 0.028). Secretors of H Leb antigens were less frequent among AIN patients (25.2%) than controls (35.0%) (OR = 0.62; p = 0.037). DISCUSSION: ABO blood group antigens and the secretion of these antigens are associated with a diagnosis of AIN. The mechanism underlying the association between autoimmunity and interaction among ABO, secretor, and Lewis genotypes has not yet been elucidated, but several studies indicate a connection to the gut microbiota.

Association between human leukocyte antigens (HLAs) and human neutrophil antigens (HNAs) and autoimmune neutropenia of infancy in Danish patients.

BACKGROUND: Autoimmune neutropenia of infancy (AIN) is a frequent cause of neutropenia in children. The disease is caused by antibodies against epitopes on the immunoglobulin G (IgG) Fc receptor type 3b (FcγIIIb). We investigated the possible association of human neutrophil antigens (HNA), human leukocyte antigen (HLA)-DR, and HLA-DQ alleles with AIN and the association of these genotypes with the presence of autoantibodies. METHODS: Eighty AIN cases with a median age of 13.5 months were included. Controls were healthy unrelated Danish blood donors. Anti-HNA-1a autoantibodies were detected using a flow cytometric granulocyte immunofluorescence test (Flow-GIFT) with phenotyped donor cells for detection of antibody specificity. Molecular determination of HNA genotypes was determined using real-time polymerase chain reaction (q-PCR). High-resolution HLA-DRB1 and HLA-DQB1 were determined by next-generation sequencing. RESULTS: Antibodies against HNA-1a were detected in 51% (n = 41) of AIN patients, and anti-HNA-1b was detected in 3% (n = 2) of cases. In 46% of cases, the antibodies were anti-FcγIIIb-reactive. FCGR3B*01+,*02-,*03- was more common (odds ratio, 6.70; P < .0001), and FCGR3B*01-,*02+,*03- was less common (odds ratio, 0.30; P < .0001) among AIN cases. HNA-1a antibodies were significantly more frequent among AIN cases with the FCGR3B*01+,*02-,*03- genotype (odds ratio, 3.86; P < .007). The HLA-DRB1*14 - HLA-DQB1*05:03 haplotype was significantly more common (odds ratio, 7.44; P < .0001) in AIN patients. CONCLUSION: The HLA haplotype HLA-DRB1*14 - DQB1*05:03 is associated with Danish AIN cases. Among Danish AIN patients, anti-HNA-1a is the most common autoantibody, and the antibody is more common in cases with the FCGR3B*01+,*02-,*03- genotype.

[Treatment of monogenic disorders with viral transduced haematopoietic stem cells].

Infusion of ex vivo transduced haematopoietic stem cells (HSC) has emerged as a promising new treatment of certain monogenetic disorders. Since early clinical studies on patients with severe combined immune deficiency were halted due to de novo leukaemia, the technology has matured. Thus, treatment of transfusion-dependent thalassaemia and adenosine deaminase deficient severe combined immunodeficiency by using lentiviral vectors for gene correction of autologous HSC can induce expression of the deficient protein and thus potentially cure the patients. The review summarises recent advances allowing for clinical implementation of the treatment in Denmark.

Is microchimerism a sign of imminent disease recurrence after allogeneic hematopoietic stem cell transplantation? A systematic review of the literature.

Chimerism analysis following hematopoietic stem cell transplantation (HSCT) for leukemia is routinely applied in parallel with quantification of minimal residual disease (MRD) to identify imminent relapse. In the past decades, new methods with a lower limit of detection compared to standard methods have been developed, so-called microchimerism analysis. Microchimerism analysis is fast, simple, applicable across pre-HSCT disease-type and can be applied on peripheral blood allowing frequent testing during follow-up. Monitoring of microchimerism in blood could replace repeated bone marrow analysis for MRD and allow earlier detection of imminent relapse or graft failure. Clinical studies in single center cohorts have shown conflicting but promising results. There is currently no consensus on the interpretation of microchimerism analysis and heterogeneity of studies remains a major obstacle for inter-study comparisons and meta-analysis in this field. We have conducted a systematic review of studies investigating associations between microchimerism and relapse of leukemia post-HSCT. We summarize current evidence and provide suggestions for future research.

Highly sensitive chimerism detection in blood is associated with increased risk of relapse after allogeneic hematopoietic cell transplantation in childhood leukemia.

Analysis of chimerism in blood post-HCT using STR-PCR is routinely applied in parallel with quantification of MRD to predict relapse of leukemia. RQ-PCR chimerism is 10- to 100-fold more sensitive, but clinical studies in children are sparse. We analyzed IMC in blood samples following transplantation for acute lymphoblastic or myeloid leukemia in 56 children. IMC was defined as a minimum increase of (a) 0.1% or (b) 0.05% recipient DNA between two samples. The risk of relapse was higher in children with IMC of both 0.1% and 0.05% compared to children without IMC (HR 12.8 [95% CI: 3.9-41.4; P < .0001] and 7.6 [95% CI: 2.2-26.9; P < .01], respectively). The first IMC was detected at a median of 208 days prior to relapse. The 5-year cumulative incidence of relapse for children with a single IMC was 45.5% (CI 12.3-74.4) and 41.0% (14.2-66.6) for IMC above 0.1% and 0.05%, respectively. However, in 47 and 38 children never attaining IMC > 0.1% and >0.05%, 10 and 8 children relapsed, respectively. In a landmark analysis, no association was found between IMC prior to 90 days post-HCT and subsequent relapse by either classification of IMC and AUC for RQ-PCR chimerism was 54.2% (95 CI 27.7- 84.8). Although limited by a retrospective design, these results indicate that monitoring of RQ-PCR chimerism in peripheral blood may have a role in early detection of relapse in acute childhood leukemia.

The prognostic value of YKL-40 concentrations in nonmyeloablative conditioning allogeneic hematopoietic cell transplantation.

Increased plasma concentrations of YKL-40, also called chitinase-3-like-1 protein (CHI3L1), have been correlated with disease severity in a variety of malignant and inflammatory diseases. The objective of the current study was to assess pretransplant recipient and donor CHI3L1 polymorphisms and plasma YKL-40 concentrations as prognostic biomarkers in a cohort of 149 patients treated with hematopoietic cell transplantation (HCT) after nonmyeloablative conditioning for hematologic malignancies. Recipients with pretransplant YKL-40 concentrations above the age-adjusted 95th percentile (high) had higher relapse-related mortality (33% versus 18%, P = .04; hazard ratio (HR) = 4.41, P = .01), lower progression-free survival (38% versus 64%, P < .01; HR = 2.84, P = .01), and overall survival (42% versus 69%, P = .01; HR = 3.09, P = .01). Recipients transplanted with donors with high YKL-40 concentrations had an increased probability and risk of grade 2-4 acute graft-versus-host disease (aGVHD) (93% versus 62%, P < .01; HR = 2.25, P = .02). CHI3L1 polymorphisms were associated with plasma YKL-40 concentrations, but not with clinical outcomes. In conclusion, our study suggests that plasma YKL-40 could function as a biomarker for relapse risk and treatment-related toxicity, and possibly as a tool complementing clinical risk scores such as the HCT comorbidity index.

Allogeneic haematopoietic cell transplantation with nonmyeloablative conditioning in Denmark.

INTRODUCTION: Haematopoietic cell transplantation with nonmyeloablative conditioning (NMC-HCT) is used in the treatment of haematological malignancies. MATERIAL AND METHODS: Use of NMC-HCT in Denmark from 2000-07 was examined. RESULTS: Unrelated donor searches resulted in a suitable donor in 75% of cases of which 36% were transplanted. Among 244 patients referred for NMC-HCT, 72% were transplanted. There was a significant difference in the number of NMC-HCTs between national regions. Increasing waiting time resulted in 22% of the referred patients being taken off the waiting list without NMC-HCT. CONCLUSION: Some patients may have had a chance of cure if they had been transplanted without delay.

[Health status and degree of traumatisation among newly arrived asylum seeker--secondary publication]. / Nyankomne asylansøgeres helbredsstatus og traumatiseringsgrad--sekundaerpublikation.

An unknown number of asylum seekers arriving in Denmark have been exposed to torture. Amnesty International's Danish Medical Group examined 142 asylum seekers, of whom 45% had been exposed to torture. Physical and psychological symptoms were 2-3 times as frequent among torture survivors as among non-tortured asylum seekers. Among the torture survivors, 63% fulfilled the criteria of post-traumatic stress disorder, 58% had objective psychological findings, and 42% had torture-related scars. Identification of torture survivors is important in order to initiate the necessary medical treatment.

Degree of predicted minor histocompatibility antigen mismatch correlates with poorer clinical outcomes in nonmyeloablative allogeneic hematopoietic cell transplantation.

In fully HLA-matched allogeneic hematopoietic cell transplantation (HCT), the main mechanism of the beneficial graft-versus-tumor (GVT) effect and of detrimental graft-versus-host disease (GVHD) is believed to be caused by donor cytotoxic T cells directed against disparate recipient minor histocompatibility antigens (miHAs). The most common origin of disparate miHAs is nonsynonymous single nucleotide polymorphism (nsSNP) differences between donors and patients. To date, only some 30 miHAs have been identified and registered, but considering the many different HLA types in the human population, as well as all the possible nsSNP differences between any 2 individuals, it is likely that many miHAs have yet to be discovered. The objective of the current study was to predict novel HLA-A- and HLA-B-restricted miHAs in a cohort of patients treated with nonmyeloablative conditioning allogeneic HCT (matched related donor, n = 70; matched unrelated donor, n = 56) for a hematologic malignancy. Initially, the cohort was genotyped for 53 nsSNPs in 11 known miHA source proteins. Twenty-three nsSNPs within 6 miHA source proteins showed variation in the graft-versus-host (GVH) direction. No correlation between the number of disparate nsSNPs and clinical outcome was seen. Next, miHAs in the GVH direction were predicted for each patient-donor pair. Using the NetMHCpan predictor, we identified peptides encompassing an nsSNP variant uniquely expressed by the patient and with predicted binding to any of the HLA-A or -B molecules expressed by the patient and donor. Patients with more than the median of 3 predicted miHAs had a significantly lower 5-year overall survival (42% vs 70%, P = .0060; adjusted hazard ratio [HR], 2.6, P = .0047) and significantly higher treatment-related mortality (39% vs 10%, P = .0094; adjusted HR, 4.6, P = .0038). No association between the number of predicted miHAs and any other clinical outcome parameters was observed. Collectively, our data suggest that the clinical outcome of HCT is affected not by disparate nsSNPs per se, but rather by the HLA-restricted presentation and recognition of peptides encompassing these. Our data also suggest that 6 of the 11 proteins included in the current study could contain more miHAs yet to be identified, and that the presence of multiple miHAs confers a higher risk of mortality after nonmyeloablative conditioning HCT. Furthermore, our data suggest a possible role for in silico based miHA predictions in donor selection as well as in selecting candidate miHAs for further evaluation in in vitro and in vivo experiments.

Asylum seekers in Denmark--a study of health status and grade of traumatization of newly arrived asylum seekers.

BACKGROUND: An unknown number of asylum seekers arriving in Denmark have been exposed to torture or have experienced other traumatising events in their country of origin. The health of traumatised asylum seekers, both physically and mentally, is affected upon arrival to Denmark, and time in asylum centres leads to further deterioration in health. METHODS: One hundred forty-two (N=142) newly arrived asylum seekers were examined at Center Sandholm by Amnesty International Danish Medical Group from the 1st of September until the 31st of December 2007. FINDINGS: The asylum seekers came from 33 different countries, primarily representing Afghanistan, Iraq, Iran, Syria, and Chechnya. Of the asylum seekers, 45 percent had been exposed to torture--approximately one-third within the year of arrival to Denmark. Unsystematic blows, personal threats or threats to family, degrading treatment, isolation, and witnessing torture of others were the main torture methods reported. The majority of the asylum seekers had witnessed armed conflict, persecution, and imprisonment. The study showed that physical symptoms were approximately twice as frequent and psychological symptoms were approximately two to three times as frequent among torture survivors as among non-tortured asylum seekers. However, even the health of non-tortured asylum seekers was affected. Among the torture survivors, 63 percent fulfilled the criteria for post-traumatic stress disorder, and 30-40 percent of the torture survivors were depressed, in anguish, anxious, and tearful in comparison to 5-10 percent of the non-tortured asylum seekers. Further, 42 percent of torture survivors had torture-related scars. INTERPRETATION: Torture survivors amid newly arrived asylum seekers are an extremely vulnerable group, hence examination and inquiry about the torture history is extremely important in order to identify this population to initiate the necessary medical treatment and social assistance. Amnesty International Danish Medical group is currently planning a follow-up study of the present population which will focus on changes in health status during their time in Denmark.

The social situation of motherless children in rural and urban areas of Guinea-Bissau.

With the increasing prevalence of HIV infection and the high maternal mortality, orphans are a rapidly growing problem in Africa. However, few studies describe the social conditions of these children. Our study focuses on motherless children in urban and rural areas of Guinea-Bissau. A rural and an urban cohort of children (128 and 192, respectively) that had been followed by demographic surveillance since 1990 were identified and the relatives of these children interviewed. A control cohort of 808 individuals was also identified. Although orphan children remained disadvantaged, there were few differences between surviving motherless and control children in nutritional status, use of health care services, school attendance, quality of housing, and clothing. Motherless children moved more frequently and were more likely to live in small families, often with an older grandmother. The traditional extended family system appears to be capable of handling motherless children in a non-discriminatory fashion. However, the AIDS epidemic will continue to stress the extended family system and social services to the limit.