Genetic Imbalance in Patients with Cervical Artery Dissection.

BACKGROUND: Genetic and environmental risk factors are assumed to contribute to the susceptibility to cervical artery dissection (CeAD). To explore the role of genetic imbalance in the etiology of CeAD, copy number variants (CNVs) were identified in high-density microarrays samples from the multicenter CADISP (Cervical Artery Dissection and Ischemic Stroke Patients) study and from control subjects from the CADISP study and the German PopGen biobank. Microarray data from 833 CeAD patients and 2040 control subjects (565 subjects with ischemic stroke due to causes different from CeAD and 1475 disease-free individuals) were analyzed. Rare genic CNVs were equally frequent in CeAD-patients (16.4%; n=137) and in control subjects (17.0%; n=346) but differed with respect to their genetic content. Compared to control subjects, CNVs from CeAD patients were enriched for genes associated with muscle organ development and cell differentiation, which suggests a possible association with arterial development. CNVs affecting cardiovascular system development were more common in CeAD patients than in control subjects (p=0.003; odds ratio (OR) =2.5; 95% confidence interval (95% CI) =1.4-4.5) and more common in patients with a familial history of CeAD than in those with sporadic CeAD (p=0.036; OR=11.2; 95% CI=1.2-107). CONCLUSION: The findings suggest that rare genetic imbalance affecting cardiovascular system development may contribute to the risk of CeAD. Validation of these findings in independent study populations is warranted.

Donor-specific HLA antibodies: evaluating the risk for graft loss in renal transplant recipients with isotype switch from complement fixing IgG1/IgG3 to noncomplement fixing IgG2/IgG4 anti-HLA alloantibodies.

Human leukocyte antigen alloantibodies have a multitude of damaging effects on the allograft, both complement (C') activation and Fc-independent ones. To date, the clinical significance of non-C' fixing (NCF) HLA donor-specific antibodies (DSA) is still unclear. In this study, we investigated whether renal transplant recipients with NCF-DSA subclasses (IgG2/IgG4, IgA1/IgA2) are at higher risk of graft loss compared to patients with exclusively C' fixing (IgG1/IgG3). Blood samples from 274 patients were analyzed for HLA IgG and IgA subclasses using a modified single-antigen bead assay. We identified 50 (18.2%) patients with circulating NCF antibodies either DSA (n=17) or against third-party HLA (n=33). NCF-DSAs were preferentially of IgG2/IgG4 isotype (11/17) and were mainly directed against HLA class II (13/17). NCF DSA were present as a mixture with strong C' fixing IgG1/IgG3. Graft survival was similar between patients with exclusively C' fixing antibodies and those with a mixture panel (log rang test P=0.162), and also among patients with different immunoglobulin isotype and subclasses (long-rank test, P=0.732). We conclude that expansion of DSA to NCF subclasses postrenal transplantation does not seem to be associated with worse graft survival as compared to the presence of exclusive C' fixing subclasses.

Correlation of Fc Receptor Polymorphisms with Pneumococcal Antibodies in Vaccinated Kidney Transplant Recipients.

Several polymorphisms within Fc receptors (FCR) have been described, some of which correlate with allograft function. In the current study, we determined three Fcγ receptor and five Fcα receptor dimorphisms in 47 kidney transplant recipients who had been vaccinated against Streptococcus pneumoniae. We analyzed if FCR genotypes correlated with pneumococcal antibodies and their serotype-specific opsonophagocytic function, tested prior to and at months 1 and 12 post-vaccination. In parallel, we assessed antibodies against HLA and MICA and determined kidney function. We observed that IgG2 antibodies against pneumococci at months 1 and 12 after vaccination and IgA antibodies at month 1 differed significantly between the carriers of the three genotypes of FCGR3A rs396991 (V158F, p = 0.02; 0.04 and 0.009, respectively). Moreover, the genotype of FCGR3A correlated with serotype-specific opsonophagocytic function, reaching statistical significance (p < 0.05) at month 1 for 9/13 serotypes and at month 12 for 6/13 serotypes. Heterozygotes for FCGR3A had the lowest antibody response after pneumococcal vaccination. On the contrary, heterozygotes tended to have more antibodies against HLA class I and impaired kidney function. Taken together, our current data indicate that heterozygosity for FCGR3A may be unfavorable in kidney transplant recipients.

Functional Fc Gamma Receptor Gene Polymorphisms and Long-Term Kidney Allograft Survival.

The functional Fc gamma receptor (FcγR) IIIA polymorphism FCGR3A-V/F158 was earlier suggested to determine the potential of donor-specific HLA antibodies to trigger microcirculation inflammation, a key lesion of antibody-mediated renal allograft rejection. Associations with long-term transplant outcomes, however, have not been evaluated to date. To clarify the impact of FCGR3A-V/F158 polymorphism on kidney transplant survival, we genotyped a cohort of 1,940 recipient/donor pairs. Analyzing 10-year death-censored allograft survival, we found no significant differences in relation to FCGR3A-V/F158. There was also no independent survival effect in a multivariable Cox model. Similarly, functional polymorphisms in two other activating FcγR, FCGR2A-H/R131 (FcγRIIA) and FCGR3B-NA1/NA2 (FcγRIIIB), were not associated with outcome. There were also no significant survival differences among patient subgroups at increased risk of rejection-related injury, such as pre-sensitized recipients (> 0% panel reactivity; n = 438) or recipients treated for rejection within the first year after transplantation (n = 229). Our study results suggest that the earlier shown association of FcγR polymorphism with microcirculation inflammation may not be strong enough to exert a meaningful effect on graft survival.

Pharmacogenetic testing for guiding de novo phenprocoumon therapy in stroke patients.

BACKGROUND: For many conditions causing transient ischemic attack or minor stroke, secondary prevention with early initiation of oral anticoagulation is indicated. The individual response to coumarins is known to vary widely and is not well predicted by clinical variables. Patients' discharge from hospital care is often delayed only because the therapeutic target range has not been reached yet. A feasible tool to guide coumarin dosing and thereby safely shortening time in hospital is required. METHODS: We established a polymerase chain reaction technique for rapid genotyping of the vitamin K epoxide reductase complex (VKORC1), which is the pharmaceutical target of the coumarins. C283 + 837C -> T (rs2359612) genotypes were determined in 49 patients who underwent de novo oral anticoagulation with phenprocoumon for cerebrovascular disease. Other variables potentially affecting phenprocoumon sensitivity were systematically evaluated. RESULTS: Of 49 genotyped patients, 47 were treated in hospital until an international normalized ratio (INR) of 2-3 was reached. The time and the cumulative dose of phenprocoumon necessary to achieve the target INR both were strongly dependent on the individual C283 + 837C -> T genotype (Kruskal-Wallis test p = 0.0002, and p < 0.0001, respectively). Carriers of the TT genotype reached an INR of 2-3 after a mean time of 3.2 days (n = 5), CT carriers after 4.4 days (n = 27), and CC carriers after 6.5 days (n = 15). No other variable, including body weight, was significantly correlated with the treatment response. CONCLUSION: In patients with cerebrovascular disease, genotyping for VKORC1 alone can strongly predict the individual response to de novo phenprocoumon treatment. The size of the pharmacogenetic test's potential effect on a more efficient use of hospital capacities remains to be shown by a controlled interventional study.

Single nucleotide polymorphisms in the VKORC1 gene and the risk of stroke in the Southern German population.

Variation in the gene that encodes the vitamin K epoxide reductase subunit 1 (VKORC1) was recently proposed as a genetic risk factor for stroke in a Chinese population. In this ethnic group, only two common haplotypes were observed, with the C-allele of the polymorphism rs2359612 (VKORC1: c.283+837C>T) associated with stroke and other cardiovascular diseases. Recently, the influence of VKORC1 haplotypes on venous thrombosis and coronary heart disease was analyzed in study populations from France and Northern Germany. We studied the frequencies of the VKORC1 haplotypes in a series of young (<50 years, n = 158) patients with ischemic stroke from Southern Germany. The data were compared with findings from age-matched healthy control subjects from the same population (n = 213). In a replica study we also analysed older stroke patients (>50 years, n = 135) and matched control subjects (n = 113). Neither in the young population, nor in the replica study, we observed significant differences in VKORC1 haplotype distributions between healthy control subjects and patients with ischemic stroke. Our data do not confirm the association between polymorphism in the VKORC1 gene and stroke in the German population.

Effect of donor cell type on complement-dependent cytotoxicity crossmatch outcome for patients immunized against HLA class-II antigens.

OBJECTIVES: In the Eurotransplant zone, the crossmatch serum exchange program was established to reduce unnecessary organ shipment, using the complement-dependent cytotoxicity crossmatch as the reference to make the decision. Crossmatching at the donor center dictates whether the transplant should be shipped to the recipient center where a decisive crossmatch would then be done. However, in recent years, the target cell used for the crossmatching has changed from spleen cells to peripheral blood lymphocytes. In this study, we assess the impact of this change on the outcome of complement-dependent cytotoxicity crossmatches for patients immunized against HLA-class II. MATERIALS AND METHODS: The influence of the donor cell type was analyzed by crossmatching unseparated peripheral blood lymphocytes, separated T and B lymphocytes, as well as spleen cells from 12 organ donors with sera from 40 immunized kidney retransplant candidates. Negative sera and sera harboring only anti-HLA class-II antibodies were used as additional controls. We did more than 1200 complement-dependent cytotoxicity crossmatches. RESULTS: Crossmatches with sera containing anti-HLA class-I plus class-II alloantibodies (n=113 per cell type) were positive in 42% of peripheral blood lymphocytes, 72% of spleen cells, and 81% of B cells. Crossmatches with sera containing exclusively anti- HLA class-II antibodies (n=89 per cell type) were positive in 1% of peripheral blood lymphocytes, 30% of spleen cells, and in 31% of B cells. Overall, spleen or separated B cells identified approximately 30% more positive donor-recipient pairs. CONCLUSIONS: The data show that the change from spleen cells to peripheral blood lymphocytes as donor target cells for complement-dependent cytotoxicity crossmatching increased risk of false negative results for patients harboring anti-HLA class-II antibodies.

Immunoglobulin isotype-specific characterization of anti-human leukocyte antigen antibodies eluted from explanted renal allografts.

To evaluate the immunoglobulin isotypes of anti-human leukocyte antigen (HLA) antibodies harbored in rejected renal allografts, we isolated proteins by acid elution accumulated in 94 rejected and explanted kidneys and characterized their antibody specificities by complement-dependent cytotoxicity, enzyme-linked immunosorbent assay, and flow cytometry (Luminex) techniques. In addition, we differentially analyzed non-complement-binding immunolglobulin (Ig) G2/4 and IgA1/2 antibodies in the eluates using two modified solid phase assays. We found non-complement-binding IgG2 and IgG4 antibodies in 16/58 (28%) of the IgGall-positive eluates, 15 eluates with anti-HLA class I and 4 with anti-HLA class II specificities, respectively. Anti-HLA class I IgG2/4 antibodies directed against the donor were found in 7 eluates (54% of the IgG2/4-pos. eluates), whereas 2 eluates (50%) had class II IgG2/4 antibodies directed against the donor. IgA1/2 antibodies could be detected in 9 eluates (16%); 5 of them had anti-HLA class I and 5 anti-HLA class II antibodies. We could clearly exhibit that explanted kidney allografts harbor anti-HLA antibodies. Moreover, our study demonstrates that non-complement-binding anti-HLA antibodies accumulate in rejected renal allografts.

Genetic analysis of familial connective tissue alterations associated with cervical artery dissections suggests locus heterogeneity.

BACKGROUND AND PURPOSE: Cervical artery dissections (CAD) can be associated with connective tissue aberrations in skin biopsies. The analysis of healthy relatives of patients suggested that the connective tissue phenotype is familial with an autosomal dominant inheritance. METHODS: We performed genetic linkage studies in 3 families of patients with CAD. Connective tissue phenotypes for the patients and all family members were assessed by electron microscopic study of skin biopsies. A genome-wide linkage analysis of 1 family (1 patient with 8 healthy relatives) indicated 2 candidate loci. Three genes were subsequently studied by sequence analysis. Part of the genome was also studied by linkage analysis in 2 further families. RESULTS: The genome-wide scan in a single family suggested linkage between the hypothetical mutation causing the connective tissue phenotype and informative genetic markers on chromosome 15q24 (logarithm of the odds score: Z= +2.1). A second possible candidate locus (Z=+1.9) was found on chromosome 10q26. Sequence analysis of 3 candidate genes in the suggestive locus (chondroitin sulfate proteoglycan4 [CSPG4], lysyl oxidase-like1 [LOXL1] and fibroblast growth factor receptor2 [FGFR2]) did not lead to the identification of a mutation responsible for connective tissue alterations. In 2 additional smaller families the loci on chromosome 15q24 and 10q26 were excluded by linkage analysis. CONCLUSIONS: Linkage analysis of a large family with CAD-associated connective tissue alterations suggested the presence of a candidate locus on chromosome 15q2 or on chromosome 10q26. Sequence analysis did not lead to the identification of a mutated candidate gene in 1 of these loci. The study of 2 additional pedigrees indicated locus heterogeneity for the connective tissue phenotype of CAD patients.

Association of a coding polymorphism in Fc gamma receptor 2A and graft survival in re-transplant candidates.

The family of Fc gamma receptors (FcγRs) is involved in mediating immunological effector functions. FcγRs are differentially expressed on immune cells and can act either activating or inhibitory, with FcγR2A belonging to the first group. The polymorphism H131R (rs1801274) in FCGR2A has been associated with acute rejection and can shift the overall balance between activating and inhibitory FcγRs. Anti-HLA allo-antibodies in transplant recipients have been identified as risk factor for organ survival after transplantation. In this study we genotyped FCGR2A H131R in 200 patients who had undergone kidney transplantation and experienced loss of graft function. FCGR2A polymorphism was related to graft survival and anti-HLA antibodies. Graft survival was calculated as the time interval between transplantation and return to chronic dialysis after transplantation. The gene frequency of FCGR2A R/R131 was found significantly more often in patients with earlier (⩽60months) compared to patients with later (>60months) graft failure. Overall patients homozygous for R/R131 had a significantly shorter graft survival, compared to H/H131 or H/R131 which is even more pronounced, when anti-HLA antibodies were present. These data suggest, that FCGR2A polymorphisms constitute a risk factor for graft loss following kidney transplantation and that this effect is related to anti-HLA antibodies.

The Karlsburg type 1 diabetes risk study of a normal schoolchild population: association of beta-cell autoantibodies and human leukocyte antigen-DQB1 alleles in antibody-positive individuals.

The intent of this study was to analyze the prevalence of diabetes-associated autoantibodies (AAbs) at or above the 99(th) percentile as well as their association with human leukocyte antigen (HLA)-DQB1 alleles in a normal population of 6,337 schoolchildren. AAbs against glutamic acid decarboxylase (GADA), tyrosine phosphatase IA-2 (IA-2A), and/or insulin (IAA) were detected by (125)I-antigen binding and islet cell antibodies (ICA) immunohistochemically in 181 (2.86%) schoolchildren. HLA-DQB1 alleles were analyzed in 178/181 children and subsequently compared with 119 controls. 2.37% (150/6,337) possessed only one AAb, whereas 0.49% (31/6,337) had multiple AAbs but at increased levels (P < 0.001). Subjects with GADA, IA-2A, or IAA revealed an increased frequency of the diabetes-associated HLA-DQB1 alleles *0302 and/or *02 (P = 0.001-0.006) as well as a decreased frequency in the protective allele *0602 (P < 0.001-0.022). DQB1*0602 was completely absent within children with multiple AAbs or with GADA, IA2-A, or IAA at or above the 99.9(th) percentile. In comparison to children with single AAbs, the frequency of associated/protective alleles of children with multiple AAbs was enhanced/diminished (P = 0.004-0.009). The study shows that also in the general population the multiple AAbs or high level single AAbs predict rather certainly a HLA-DQB1-mediated diabetes susceptibility as shown for first degree relatives of type 1 diabetic patients.

Detection and specification of noncomplement binding anti-HLA alloantibodies.

The aim of the study was to investigate the distribution of human leukocyte antigen (HLA) -specific immunoglobulin (Ig) isotypes/subclasses in alloimmunized patients awaiting a kidney retransplant. Sera from 102 patients were analyzed for the presence of anti-HLA-A, anti-HLA-B alloantibodies by complement-dependent cytotoxicity test with the addition of dithiothreitol (CDC+DTT). Furthermore, anti-HLA class I and class II alloantibodies were determined using a commercial solid-phase (enzyme-linked immunosorbent assay [ELISA]) system. The respective isotypes/subclasses were defined by replacing the IgG1-4 secondary antibody with IgG1-, IgG2-, IgG3-, IgG4-, IgA1-, IgA2-, and IgM-specific antibodies. The HLA specificities of the noncomplement-binding IgG2 and IgG4 antibodies were determined and compared with the mismatches from the failed transplants. Thirty-eight of 102 (37%) sera were positive in the class I CDC+DTT, in contrast to 41 of 102 (40%) detected by class I ELISA and 47 of 102 (46%) by class II ELISA. Seventeen of 102 (17%) positive reaction were observed for the IgM-isotype, whereas none were detected for the IgA-isotype. Twenty-five of 102 (25 %) sera contained noncomplement-binding IgG2 and/or IgG4 antibodies; in the majority of the cases, 22 of 25 (88%) were directed against the organ donor antigen. These data show that donor-specific, noncomplement-binding IgG2 and IgG4 alloantibodies exist with high prevalence in HLA-immunized retransplant candidates. Therefore, a thorough antibody screening workup, including CDC with or without DTT and ELISA screening should be performed for patients before they reenter the waiting list. Defining the Ig isotypes and subclasses can be helpful to explain inconsistent results.

Copy number variation in patients with cervical artery dissection.

Cervical artery dissection (CeAD) occurs in healthy young individuals and often entails ischemic stroke. Skin biopsies from most CeAD-patients show minor connective tissue alterations. We search for rare genetic deletions and duplication that may predispose to CeAD. Forty-nine non-traumatic CeAD-patients with electron microscopic (EM) alterations of their dermal connective tissue (EM+ patients) and 21 patients with normal connective tissue in skin biopsies (EM- patients) were analyzed. Affymetrix 6.0 microarrays (Affymetrix) from all patients were screened for copy number variants (CNVs). CNVs absent from 403 control subjects and from 2402 published disease-free individuals were considered as CeAD-associated. The genetic content of undentified CNVs was analyzed by means of the Gene Ontology (GO) Term Mapper to detect associations with biological processes. In 49 EM+ patients we identified 13 CeAD-associated CNVs harboring 83 protein-coding genes. In 21 EM- patients we found five CeAD-associated CNVs containing only nine genes (comparison of CNV gene density between the groups: Mann-Whitney P=0.039). Patients' CNVs were enriched for genes involved in extracellular matrix organization (COL5A2, COL3A1, SNTA1, P=0.035), collagen fibril organization COL5A2, COL3A1, (P=0.0001) and possibly for genes involved in transforming growth factor beta (TGF)-beta receptor signaling pathway (COL3A1, DUPS22, P=0.068). We conclude that rare genetic variants may contribute to the pathogenesis of CeAD, in particular in patients with a microscopic connective tissue phenotype.

Pretransplant IgG subclasses of donor-specific human leukocyte antigen antibodies and development of antibody-mediated rejection.

BACKGROUND: The subclass of IgG antibodies contributes to their capability to activate complement. It is currently unknown whether the pretransplant IgG subclass composition allows distinguishing harmful from presumably irrelevant donor-specific human leukocyte antigen (HLA) antibodies (HLA-DSA) detected by single-antigen flow beads (SAFB). METHODS: Seventy-four patients transplanted in the presence of HLA-DSA were investigated. HLA-DSA characteristics were not different between patients experiencing antibody-mediated rejection (AMR) (n=40) and patients who did not (n=34) experience AMR. Sera were reanalyzed using SAFB with IgG subclass-specific reporter antibodies. RESULTS: The 74 patients had in total 141 HLA-DSA. IgG1 was the predominant subclass (78%), followed by IgG2 (49%), IgG3 (36%), and IgG4 (20%). When grouped according to the complement-activating capability, only 4 of 74 patients (5%) had exclusively weak/no complement-activating HLA-DSA (i.e., IgG2 and IgG4), 21 of 74 patients (28%) had isolated strong complement-activating HLA-DSA (i.e., IgG1 and IgG3), and 46 of 74 patients (62%) had a mixture of both. There was no difference between the strong complement-activating and the mixture group regarding incidence of AMR (57% vs. 54%; P=0.81), phenotypes of AMR (P=0.70), and death-censored allograft survival at 5 years (78% vs. 78%; P=0.74). Interestingly, patients with exclusively weak/no complement-activating HLA-DSA (n=4) had a numerically lower incidence of AMR (25%) and no allograft loss has occurred yet. CONCLUSION: In 90% of patients, pretransplant HLA-DSA are composed of isolated strong or a mixture of strong and weak/no complement-activating IgG subclasses. Because outcomes in these two groups were similar, pretransplant IgG subclass analysis is likely not providing substantial value beyond the standard IgG SAFB assay for pretransplant risk stratification.

Influence of major HIV-1 protease inhibitor resistance mutations on CTL recognition.

BACKGROUND: HIV-1 protease is subjected to dual selection pressure exerted by protease inhibitors (PIs) and cytotoxic T lymphocytes (CTL). Recently, we identified KMIGGIGGF (KF9) as a HLA-B*1501-restricted CTL epitope, including several major PI resistance mutations (M46I/L, I47A/V, G48V, I50V). To assess potential interactions between KF9-specific CTL and emergence of these important resistance mutations, we studied CTL recognition of the mutations and analyzed protease sequences in an HLA-I­typed patient cohort. METHODS: CTL recognition of KF9 and resistance mutations in KF9 were studied in 38 HLA-B*1501-positive HIV-1­infected patients using variant KF9 peptides in interferon-g enzyme-linked immunospot assays. Protease sequences were analyzed in 302 HLA-I­typed HIV-1­infected patients. RESULTS: G48V abolished KF9 recognition by CTL in all patients. Furthermore, M46I, I47A, and I50V could impair or abolish CTL recognition in many patients. In contrast, M46L and I47V showed good CTL recognition in nearly all patients. HIV-1 protease sequence analysis showed no statistical correlation between the occurrence of resistance mutations in KF9 and HLA-B*1501. Viral load in patients failing therapy with KF9 mutations was significantly lower in HLA-B*1501-positive patients in comparison with HLA-B*1501-negative patients. CONCLUSIONS: PI mutations, G48V, M46I, and I47A, can abrogate CTL recognition, indicating potential interactions between development of drug resistance and CTL response. However, we could not find evidence that development of these PI mutations is influenced by KF9-specific CTL.

Characterization of anti-HLA antibodies eluted from explanted renal allografts.

To evaluate the immunoglobulin isotypes of anti-HLA antibodies harbored in rejected renal allografts, we isolated proteins by acid elution accumulated in 94 rejected and explanted kidneys and characterized their antibody specificities by CDC, ELISA, and flow cytometry (Luminex) techniques. In addition, we differentially analyzed non-complement binding IgG2/4 and IgA1/2 antibodies in the eluates using two modified solid phase assays. The comparative analysis of anti-HLA class I and II antibodies with donor specificity (ELISA vs. Luminex) revealed 30% vs. 33% class I IgGall positive eluates, whereas 26% vs. 15% of the eluates had donor-specific anti-HLA class II IgGaII antibodies. Regarding the non-complement binding antibodies we could show IgG2 and IgG4 antibodies in 16/58 (28%) of the IgGaII positive eluates: 15 eluates with anti-HLA class I and 4 with anti-HLA class II specificities, respectively. Anti-HLA class I IgG2/4 antibodies directed against the donor were found in 7 eluates (54% of the IgG2/4 pos. eluates), whereas 2 eluates (50%) had class II IgG2/4 antibodies directed against the donor. IgA1/2 antibodies could be detected in 9 eluates (16%), 5 of them had anti-HLA class I and 5 anti-HLA class II antibodies. We could clearly show that explanted kidney allografts harbor anti-HLA antibodies, when sensitive antibody detection techniques are used. Moreover, our study demonstrates that non-complement binding anti-HLA antibodies also accumulate in renal allografts.