Classical Alleles Research Articles

A cis-eQTL of HLA-DRB1 and a frameshift mutation of MICA contribute to the pattern of association of HLA alleles with cervical cancer.

The association of classic human leukocyte antigen (HLA) alleles with risk of cervical cancer has been extensively studied, and a protective effect has consistently been found for DRB1*1301, DQA1*0103, and/or DQB1*0603 (these three alleles are in perfect linkage disequilibrium [LD] and often occur on the same haplotype in Europeans), while reports have differed widely with respect to the effect of HLA-B*07, DRB1*1501, and/or DQB1*0602 (the last two alleles are also in perfect LD in Europeans). It is not clear whether the reported HLA alleles are responsible for the differences in cervical cancer susceptibility, or if functional variants at other locations within the major histocompatibility complex (MHC) region may explain the effect. In order to assess the relative contribution of both classic HLA alleles and single-nucleotide polymorphisms (SNPs) within the MHC region to cervical cancer susceptibility, we have imputed classic HLA alleles in 1034 cervical cancer patients and 3948 controls in a Swedish population for an integrated analysis. We found that the protective haplotype DRB1*1301-DQA1*0103-DQB1*0603 has a direct effect on cervical cancer and always occurs together with the C allele of a HLA-DRB1 cis-eQTL (rs9272143), which increases the expression of HLA-DRB1. The haplotype rs9272143C-DRB1*1301-DQA1*0103-DQB1*0603 conferred the strongest protection against cervical cancer (odds ratio [OR] = 0.41, 95% confidence interval [CI] = 0.32–0.52, P = 6.2 × 10−13). On the other hand, the associations with HLA-B*0702 and DRB1*1501-DQB1*0602 are attributable to the joint effects of both the HLA-DRB1 cis-eQTL (rs9272143) and a frameshift mutation (G inserion of rs67841474, also known as A5.1) of the MHC class I polypeptide-related sequence A gene (MICA). Variation in LD between the classic HLA loci, rs9272143 and rs67841474 between populations may explain the different associations of HLA-B*07 and DRB1*1501-DQB1*0602 with cervical cancer between studies. The mechanism suggested may also explain similar inconsistent results for other HLA-associated diseases.

Dual-Specificity CAR+ T Cells to Target B-Cell Malignancies and Opportunistic Fungal Infection

s / Biol Blood Marrow Transplant 20 (2014) S128eS150 S132 CTL was characterized as to eps specificity and restricting HLA allele. The pool of CTL donors inherited HLA alleles in frequencies similar to those in caucasian and black populations, with certain alleles overrepresented. In 55% of the CTL lines, the ID T-cell response (TCr) was restricted (restr.) by 3 HLA alleles: A0201(25%), B0702(21%) and B 3501-11(9%); 45% of CTL lines were restr. by other class-I alleles, and 17/123(14%) were restr. by class-II alleles. Certain CMVpp65 15 mers contained overlapping eps presented by both class-I and class-II, which elicitedmore robust TCr. CTLs from all B0702+ donors (26/26) were restr. by B0702. In comparison, CTLs from 30/39(77%) A0201+ donors and 9/19 (47%) HLA B3501-11+ donors were restr. by A0201 and B3501-11 respectively. Donors coinheriting A0201 and B 0702 (9/9) universally demonstrated B0702 restr. ID TCr. In comparison, 11/12 (91.6%) donors coinheriting A0201 and B 4401-04 demonstrated HLA A0201 restr. CTLs. Strikingly, only 1/123 and 0/123 A1101+ and A0301+ donors respectively demonstrated CTLs restr. by these commonly inherited class eI alleles. This analysis thus indicates an immunodominance hierarchy for CMVpp65 eps and their presenting HLA alleles. In a series of 239 MUD, 137 MMUD, and 100 DUCB consecutive HSCT at MSKCC, we could identify an immediately available CMVpp65 specific CTL line matched with the patient at 2-3 alleles and restr. by a shared HLA allele in this GMP-CTL bank in 86%, 89% and 80% cases respectively. This CTL bank thus provides a clinical reagent for the treatment of CMV infections in HSCT recipients. The delineated immunodominance hierarchy for CMVpp65 may also facilitate selection of an appropriately restr. CTL line for treatment with predicted activity in the recipient. We can also successfully generate CMV-CTLs against subdominant CMVpp65 eps presented prevalent HLA alleles using a panel of artificial antigen presenting cells (AAPCs). Expansion of this bank with CTLs generated with AAPCs can broaden its applicability to almost all HSCT recipients.

Generation and Characterization of a Third Party GMP Grade Bank of CMV Specific T-Cells for Adoptive Immunotherapy of CMV Infections in Recipients of HSCT from Cord Blood or Seronegative Donors

Adoptive transfer of virus-specific T-cells (CTLs) derived from allogeneic HLA partially matched third party donors can also be effective in a proportion of patients developing EBV lymphomas, or infections due to CMV or adenovirus following transplants from seronegative donors. Such third party donor derived CTLs offer an off the shelf reagent for treatment of viral infections developing after transplant. However, the immunodominant cytotoxic activity exhibited by the CTLs is directed against specific epitopes of the viral protein and restricted by 1-2 HLA alleles. Therefore, it is critical that the T-cells administered from third party donors can recognize viral epitopes presented on shared HLA alleles. We have established a bank of 119 CMV specific T-cell lines (CMV CTLs) generated using autologous APCs loaded with a pool of overlapping peptides spanning the sequence of the dominant immunogenic protein CMVpp65. Each of these 119 CMV CTL lines has been characterized as to the epitope inducing T-cell response as well as the HLA allele restricting the epitope specific T-cell response. Epitopes were identified using an overlapping grid of peptide pools and the HLA restriction by cytotoxic activity against peptide loaded EBVBLCLs matched at a single HLA allele with the T-cell donor. The distribution of the common HLA alleles among the donors for these CTL lines was predominantly within the distribution of HLA allele frequencies represented in the caucasian and black populations, except for HLA A0201 and B0702, which were over represented ( 33% vs 25% and 21% vs 8.7% respectively). In 54% of the CTL lines, the immunodominant T-cell responses were restricted by HLA A0201 (25%), B0702 (21%) and B 3501-11(8%), and in the remaining 50%, the responses were restricted by other HLA class-I alleles, while only 16/119 lines (13%) were restricted by HLA class-II alleles. All 25 donors inheriting HLA B0702 (25/25) demonstrated HLA B0702 restricted CMV CTL responses, while 30/39 (77%) donors inheriting HLA A0201 and 9/19 (47%) donors inheriting HLA B3501-11 demonstrated HLA A0201 and B3501-11 restricted CMV CTL responses. Among all 9 donors co-inheriting HLA A0201 and B 0702, the immunodominant T-cell response was restricted by B0702. Among 12 donors co-inheriting A0201 and B 4401-04, 11/12 (91.6%) demonstrated immunodominant CMV CTL responses restricted by A0201; 1 donor also co-inherited HLA B0702 whose response was restricted by B0702. Therefore, an immunodominance hierarchy for HLA class-I alleles presenting the dominant CMVpp65 epitope was evident through this analysis among these 119 donors and was as follows: B 0702, A0201, B3501-11, A2601, B44, B40, B4201, A0101, B 1801. Strikingly, only 1 of 119 donors demonstrated T-cell responses restricted by A1101; a commonly inherited HLA class –I allele. In a series of 239 consecutive HLA matched related or unrelated transplants (MUD) and 137 HLA mismatched unrelated (MMUD) transplants, and 100 cord blood transplants conducted at our center, in 86%, 89% and 80% of the cases respectively, we could identify a CMV CTL line restricted by a shared HLA allele and matched at 2-3 alleles within this GMP grade CTL bank that would be immediately available for treatment of CMV infection. Appropriately restricted CMV CTLs would only be available in 60-70% of MMUD transplant and none of the cord blood transplants without this approach. This CMV CTL bank therefore represents a readily available clinical reagent for the treatment of resistant CMV infections developing in post transplant patients. The characterization of the CTLs has also enabled the further elucidation of immunodominant CMVpp65 epitopes and hierarchies. Since we have previously shown that CMV CTLs can be generated against subdominant epitopes presented by both common HLA alleles as well as less prevalent HLA alleles using a panel of artificial antigen presenting cells (AAPCs), expansion of this bank using T-cell sensitized against CMVpp65 presented on such AAPCs should broaden the applicability of this bank to all HSCT recipients. Disclosures: No relevant conflicts of interest to declare.

Immunochip Analysis Identifies Multiple Susceptibility Loci for Systemic Sclerosis

In this study, 1,833 systemic sclerosis (SSc) cases and 3,466 controls were genotyped with the Immunochip array. Classical alleles, amino acid residues, and SNPs across the human leukocyte antigen (HLA) region were imputed and tested. These analyses resulted in a model composed of six polymorphic amino acid positions and seven SNPs that explained the observed significant associations in the region. In addition, a replication step comprising 4,017 SSc cases and 5,935 controls was carried out for several selected non-HLA variants, reaching a total of 5,850 cases and 9,401 controls of European ancestry. Following this strategy, we identified and validated three SSc risk loci, including DNASE1L3 at 3p14, the SCHIP1-IL12A locus at 3q25, and ATG5 at 6q21, as well as a suggested association of the TREH-DDX6 locus at 11q23. The associations of several previously reported SSc risk loci were validated and further refined, and the observed peak of association in PXK was related to DNASE1L3. Our study has increased the number of known genetic associations with SSc, provided further insight into the pleiotropic effects of shared autoimmune risk factors, and highlighted the power of dense mapping for detecting previously overlooked susceptibility loci.

Allelic Diversity of Polymorphic AMA-1 (Apical Membrane Antigen 1) Vaccine Candidate Antigen of Plasmodium falciparum in Two Population of Imported and Indigenous Cases in South-East of Iran using Nested-PCR and RFLP

The Plasmodium falciparum Apical Membrane Antigen 1 (AMA1) is a leading Malaria Vaccine Candidate Antigen. Antigenic variation is one of the main obstacles in the development of a universal effective malaria vaccine. Antibodies against AMA1 have been shown to block parasite invasion of human erythrocytes. Therefore, detailed studies on the molecular polymorphism PFAMA1 a Geographic area before each experiment or design the vaccine is administered. This study was designed to determine the distribution of AMA1 allele class of Plasmodium falciparum in two population of imported cases and indigenous cases in South-East of Iran .We used the Nested PCR and RFLPs methods with specific primers and restriction enzymes, which improves the three AMA1 allele class(K1-3D7-HB3).Overall the 94 confirmed P. falciparum samples obtained from four different districts, in two population of imported cases(46) and indigenous cases(48) in the south East of Iran. There are three classes of allelic AMA1 were compared in two populations of Indigenous and Imported, 3D7 in Indigenous Population was the higher prevalence than Imported Population. Considering on these results, 3D7 alleles suitable candidates for malaria vaccine design can be. The data reported here will be valuable for the development of AMA1 based malaria vaccine.

KIR-HLA clase i y tuberculosis pulmonar en población amerindia del Chaco, Argentina

IntroductionThe susceptibility to pulmonary tuberculosis (TB) is multifactorial, thus genetic factors such as HLA and immunoglobulins-like killer receptors (KIR) could be predisposed to the development of the disease.Aim To evaluate whether any HLA classi allele and its combination with KIR could be related to the development of TB in the Wichi Amerindian community in north-eastern Argentina. MethodsA cohort study was conducted that included 18 families, 35 individuals affected with TB, 84 cohabiting families, and 63 controls of the same ethnic group.A and B loci of HLA classi were typed by generic PCR followed by reverse hybridization (Dynal), locus C by PCR-SSOP. KIR receptors were studied using sequence specific PCR. ResultsThere was a highly significant association with allele B*35:19/47 in TB vs. household contacts [Pc=0.0051] and vs. controls [Pc=0.0033], and with allele HLA-C*03 in TB vs. household contacts [Pc=0.014] and vs. controls [Pc=0.0033]. KIR receptors had shown increased KIR2DL3/KIR2DL3 frequency in combination with the C1 group of HLA-C (P=.018). HLA-C*03 belongs to C1 group, and this combination could have a strong inhibitory action on the infected cell. ConclusionHLA-B35:19/47-C*03 haplotype could be a susceptibility factor to TB and KIR2DL3-HLA-C1 combination have an inhibitory capacity on NK cells, and might contribute to the course of the infection by Mycobacterium tuberculosis.

PReS-FINAL-2135: Analysis of the HLA region in a large cohort of juvenile idiopathic arthritis cases identifies independent effects at HLA-DRB1

Juvenile idiopathic arthritis (JIA) is the most common arthritic disease of childhood and is caused by a combination of genes and environment. In the last few years great advances have been made in dissecting the genetic basis of JIA with now 17 confirmed susceptibility loci identified. One of these loci, the MHC region, has been established for many years but the complexity and the broad linkage disequilibrium (LD) across the region has rendered fine-mapping associations challenging. Novel imputation strategies can now be utilized to impute HLA classical alleles and amino acids across the region.

Fine-Mapping the Genetic Association of the Major Histocompatibility Complex in Multiple Sclerosis: HLA and Non-HLA Effects

The major histocompatibility complex (MHC) region is strongly associated with multiple sclerosis (MS) susceptibility. HLA-DRB1*15:01 has the strongest effect, and several other alleles have been reported at different levels of validation. Using SNP data from genome-wide studies, we imputed and tested classical alleles and amino acid polymorphisms in 8 classical human leukocyte antigen (HLA) genes in 5,091 cases and 9,595 controls. We identified 11 statistically independent effects overall: 6 HLA-DRB1 and one DPB1 alleles in class II, one HLA-A and two B alleles in class I, and one signal in a region spanning from MICB to LST1. This genomic segment does not contain any HLA class I or II genes and provides robust evidence for the involvement of a non-HLA risk allele within the MHC. Interestingly, this region contains the TNF gene, the cognate ligand of the well-validated TNFRSF1A MS susceptibility gene. The classical HLA effects can be explained to some extent by polymorphic amino acid positions in the peptide-binding grooves. This study dissects the independent effects in the MHC, a critical region for MS susceptibility that harbors multiple risk alleles.

Aquaporin-4 antibody neuromyelitis optica following anti-NMDA receptor encephalitis

Encephalitis associated with anti-N-Methyl-D-Aspartate Receptor (NMDAR) antibodies is an autoimmune encephalopathy often associated with teratoma [1]. Neuromyelitis optica (NMO) is an inflammatory demyelinating disorder of the central nervous system (CNS) associated with antibodies against aquaporin 4 (AQP4) [2]. Here, we report the case of a patient who developed limbic encephalitis (LE) followed by NMO, whose serum harbored anti-NMDAR and anti-AQP4 IgG antibodies. A 50-year-old woman presented with subacute, shortterm memory loss, confusion, and behavioral changes. Routine blood tests, an infectious disease screening, an electroencephalogram (EEG) and cerebral spinal fluid (CSF) tests were all negative, whereas a brain MRI showed a T2-weighted, hyperintense medial temporal cortex. A pelvic mass was detected by computed tomography (CT) scan. Two months later, she underwent a hystero-adnexectomy with the removal of an ovarian teratoma. A diagnosis of paraneoplastic LE was made. Tests for classical onconeural antibodies (Hu, Ri, Ma2, CV2/CMRP5, amphiphysin, Yo) proved negative. Her memory deficit started to improve 4 months after surgery, when she received repeated courses of low-dose oral steroids. One month later, she had only residual retrograde amnesia (Fig. 1e). Five months later, she developed drowsiness, cervical itching, and impaired gait. Within 2 weeks, she had developed paraplegia and MRI showed multiple T2weighted hyperintense lesions in the pons, hypothalamus, medulla oblongata, and cervical spine (Fig. 1a–c). A CSF analysis proved negative (normal white cell count and proteins, and absence of IgG intrathecal synthesis). Plasmaexchange and high-dose intravenous steroids were initiated, resulting in a slow improvement of strength. Nine months later, she presented with left-eye optic neuritis. High-dose intravenous steroids were partially effective. Three months later, a relapse occurred with weakness of the right limbs. An MRI disclosed new lesions in the pons and dorsal spine (Fig. 1d). Her serum and CSF were tested for AQP4-IgG-Ab by indirect immunofluorescence on a commercial assay (Euroimmun, Lubeck, Germany) [3], proving positive on serum (titer 1:100). The test was extended to NMDAR-IgG-Ab, also proving positive on serum (1:32) and on CSF. The patient was treated with plasma-exchange and oral steroids, with benefit. Three months later, her CSF analysis revealed a normal white cell count and increased protein content (84 mg/dl); matching oligoclonal bands were detected in the CSF and serum. Human leukocyte typing disclosed the presence of the class I allele B8 and class II DR3-DQ2 (DRB1*03-DQB1*02). AQP4-IgG-Aband NMDAR-IgG-Ab-positivity were both confirmed, but a decrease in serum Ab titer was shown (both Abs serum titer 1:10; NMDAR-Ab CSF titer 1:3, 2); VGCC-, AMPAR-, GABAbR-, LGI1and CASPR2-Abs tested negative. Treatment with azathioprine was started. Eight months later, her conditions were stable. This patient presented with LE followed by NMO 1 year later, and tested positive for AQP4and NMDAR-Abs. To M. Zoccarato M. F. Pelizza B. Giometto L. Zuliani (&) Department of Neurology, Ospedale Ca’Foncello, Azienda Unita Locale Socio-Sanitaria 9 Treviso, Piazza Ospedale 1, 31100 Treviso, Italy e-mail: luigizuliani77@gmail.com

Generation and Characterization Of a Third Party GMP Grade Bank Of CMV Specific T-Cells For Adoptive Immunotherapy Of CMV Infections In Recipients Of HSCT From Cord Blood Or Seronegative Donors

Adoptive transfer of virus-specific T-cells (CTLs) derived from allogeneic HLA partially matched third party donors can also be effective in a proportion of patients developing EBV lymphomas, or infections due to CMV or adenovirus following transplants from seronegative donors. Such third party donor derived CTLs offer an off the shelf reagent for treatment of viral infections developing after transplant. However, the immunodominant cytotoxic activity exhibited by the CTLs is directed against specific epitopes of the viral protein and restricted by 1-2 HLA alleles. Therefore, it is critical that the T-cells administered from third party donors can recognize viral epitopes presented on shared HLA alleles. We have established a bank of 119 CMV specific T-cell lines (CMV CTLs) generated using autologous APCs loaded with a pool of overlapping peptides spanning the sequence of the dominant immunogenic protein CMVpp65. Each of these 119 CMV CTL lines has been characterized as to the epitope inducing T-cell response as well as the HLA allele restricting the epitope specific T-cell response. Epitopes were identified using an overlapping grid of peptide pools and the HLA restriction by cytotoxic activity against peptide loaded EBVBLCLs matched at a single HLA allele with the T-cell donor.The distribution of the common HLA alleles among the donors for these CTL lines was predominantly within the distribution of HLA allele frequencies represented in the caucasian and black populations, except for HLA A0201 and B0702, which were over represented ( 33% vs 25% and 21% vs 8.7% respectively). In 54% of the CTL lines, the immunodominant T-cell responses were restricted by HLA A0201 (25%), B0702 (21%) and B 3501-11(8%), and in the remaining 50%, the responses were restricted by other HLA class-I alleles, while only 16/119 lines (13%) were restricted by HLA class-II alleles.All 25 donors inheriting HLA B0702 (25/25) demonstrated HLA B0702 restricted CMV CTL responses, while 30/39 (77%) donors inheriting HLA A0201 and 9/19 (47%) donors inheriting HLA B3501-11 demonstrated HLA A0201 and B3501-11 restricted CMV CTL responses. Among all 9 donors co-inheriting HLA A0201 and B 0702, the immunodominant T-cell response was restricted by B0702. Among 12 donors co-inheriting A0201 and B 4401-04, 11/12 (91.6%) demonstrated immunodominant CMV CTL responses restricted by A0201; 1 donor also co-inherited HLA B0702 whose response was restricted by B0702. Therefore, an immunodominance hierarchy for HLA class-I alleles presenting the dominant CMVpp65 epitope was evident through this analysis among these 119 donors and was as follows: B 0702, A0201, B3501-11, A2601, B44, B40, B4201, A0101, B 1801. Strikingly, only 1 of 119 donors demonstrated T-cell responses restricted by A1101; a commonly inherited HLA class –I allele.In a series of 239 consecutive HLA matched related or unrelated transplants (MUD) and 137 HLA mismatched unrelated (MMUD) transplants, and 100 cord blood transplants conducted at our center, in 86%, 89% and 80% of the cases respectively, we could identify a CMV CTL line restricted by a shared HLA allele and matched at 2-3 alleles within this GMP grade CTL bank that would be immediately available for treatment of CMV infection. Appropriately restricted CMV CTLs would only be available in 60-70% of MMUD transplant and none of the cord blood transplants without this approach. This CMV CTL bank therefore represents a readily available clinical reagent for the treatment of resistant CMV infections developing in post transplant patients. The characterization of the CTLs has also enabled the further elucidation of immunodominant CMVpp65 epitopes and hierarchies. Since we have previously shown that CMV CTLs can be generated against subdominant epitopes presented by both common HLA alleles as well as less prevalent HLA alleles using a panel of artificial antigen presenting cells (AAPCs), expansion of this bank using T-cell sensitized against CMVpp65 presented on such AAPCs should broaden the applicability of this bank to all HSCT recipients. Disclosures:No relevant conflicts of interest to declare.

Emergence of Vibrio cholerae O1 classical biotype in 2012 in Iran

Cholera outbreaks annually occur in many parts of Iran. The aim of this study was to investigate the biotype and genotype diversity of V. cholerae isolates from recent outbreak (2012) in Iran and to characterize the ctxB allelic sequence of isolates. The ctxB sequence of all isolates was analyzed and compared with the reference ctxB sequences for El Tor and classical biotypes in GenBank database. The PFGE genotype specification of isolates was determined and genetic relatedness among isolates and also with those previously reported from Iran was assessed. Ten out of eleven isolates were identified as El Tor biotype and one single isolate belonged to classical biotype. All isolates except three possessed tcpA, ctxA, ctxB and wbeT genes. All the ctxB(+) isolates in this study (classical and El Tor biotypes) possessed the ctxB sequence of classical biotype allele providing evidences of El Tor variants. Eight out of 11 isolates (73%) showed identical pulsotypes (P1). Each of the remaining three isolates showed distinct pulsotypes (P2-P4) with more than three band differences. Pulsotype P2 was corresponded to an isolate (9%) with classical biotype. The result demonstrated diversity in virulence gene content of strains with identical PFGE patterns and also provided evidences of the import of a V. cholerae strain with classical biotype from out of our country as no classical biotype strain has been previously (1998-2011) reported from Iran. Emergence of V. cholerae with Classical biotype after at least one decade in Iran is alarming due to fear of expansion of V. cholerae strains with high virulence potential and signifies the need to monitor and analyze all new cases in countries with cholera outbreaks or even sporadic cases as this could conceivably affect the neighbouring countries and may expose the world to the seventh pandemic with Classical biotype strains.

HLA-F and MHC Class I Open Conformers Are Ligands for NK Cell Ig-like Receptors

Killer Ig-like receptors (KIRs) are innate immune receptors expressed by NK and T cells classically associated with the detection of missing self through loss of their respective MHC ligand. Some KIR specificities for allelic classical class I MHC (MHC-I) have been described, whereas other KIR receptor-ligand relationships, including those associated with nonclassical MHC-I, have yet to be clearly defined. We report in this article that KIR3DL2 and KIR2DS4 and the nonclassical Ag HLA-F, expressed as a free form devoid of peptide, physically and functionally interact. These interactions extend to include classical MHC-I open conformers as ligands, defining new relationships between KIR receptors and MHC-I. The data collectively suggest a broader, previously unrecognized interaction between MHC-I open conformers--including prototypical HLA-F--and KIR receptors, acting in an immunoregulatory capacity centered on the inflammatory response.

Development and validation of a multiplex-PCR assay for X-linked intellectual disability

BackgroundX-linked intellectual disability is a common cause of inherited cognitive deficit affecting mostly males. There are several genetic causes implicated in this condition, which has hampered the establishment of an accurate diagnosis. We developed a multiplex-PCR assay for the mutational hotspot regions of the FMR1, AFF2 and ARX genes.MethodsThe multiplex-PCR was validated in a cohort of 100 males selected to include known alleles for the FMR1 repetitive region: five full mutations (250–650 CGGs), ten premutations (70–165 CGGs) and eighty-five in the normal range (19–42 CGGs). Sequencing or Southern blotting was used to confirm the results, depending on the allele class. In this cohort, with the exception of one sample showing an AFF2 intermediate-sized allele, all other samples were normal (8–34 CCGs). No ARX variant was found besides the c.429_452dup. The validated assay was applied to 5000 samples (64.4% males and 35.6% females).ResultsThe normal-allelic range of both FMR1 and AFF2 genes as well as the nature of ARX variants identified was similar in both genders. The rate of homozygosity observed in female samples, 27.5% for FMR1 and 17.8% for AFF2 alleles, is comparable to that published by others. Two FMR1 premutations were identified, in a male (58 CGGs) and a female case [(CGG)47/(CGG)61], as well as several FMR1 or AFF2 intermediate-sized alleles. One AFF2 premutation (68 CCGs) and two putative full expansions were picked up in male subjects, which seems relevant considering the rarity of reported AFF2 mutations found in the absence of a family history.ConclusionsWe developed a robust multiplex-PCR that can be used to screen the mutational hotspot regions of FMR1, AFF2 and ARX genes. Moreover, this strategy led to the identification of variants in all three genes, representing not only an improvement in allele-sizing but also in achieving a differential diagnosis. Although the distinction between females who are truly homozygous and those with a second pre- or full mutation sized allele, as well as a definitive diagnosis, requires a specific downstream technique, the use of this multiplex-PCR for initial screening is a cost-effective approach which widens the scope of detection.

HLA-A*68

Human leukocyte allele (HLA) class I polymorphism has the greatest impact of human genetic variation on viral load set point. A substantial part of this effect is due to the action of HLA-B and HLA-C alleles. With few exceptions the role of HLA-A molecules in immune control of HIV is unclear. We here study HLA-A*68:02, one of the most highly prevalent HLA-A alleles in C-clade infected sub-Saharan African populations, and one that plays a prominent role in the HIV-specific CD8 T-cell responses made against the virus. We define eight epitopes restricted by this allele and propose the peptide binding motif for HLA-A*68:02. Although one of these epitopes almost exactly overlaps an HLA-B*57-restricted epitope in Gag linked with immune control of HIV, this HLA-A*68:02-restricted Gag-TA10 response imposed only weak selection pressure on the virus and was not associated with significantly lower viral setpoint. The only HLA-A*68:02-restricted responses imposing strong selection pressure on HIV were in the flanking regions of Pol-EA8 and Pol-EA11 and within the Vpr-EV10 epitope (P = 8 × 10). However, targeting of this latter epitope was associated with significantly higher viral loads (P = 0.003), suggesting lack of efficacy. This study is consistent with previous data showing that HLA-A-restricted Gag-specific responses can impose selection pressure on HIV. In the case of HLA-A*68:02 the Gag response is subdominant, and apparently has little impact in natural infection. However, these data suggest the potential for high frequency vaccine-induced Gag responses restricted by this allele to have significant antiviral efficacy in vaccine recipients.

Correlation between relatives given complete genotypes: From identity by descent to identity by function

In classical quantitative genetics, the correlation between the phenotypes of individuals with unknown genotypes and a known pedigree relationship is expressed in terms of probabilities of IBD states. In existing approaches to the inverse problem where genotypes are observed but pedigree relationships are not, dependence between phenotypes is either modeled as Bayesian uncertainty or mapped to an IBD model via inferred relatedness parameters. Neither approach yields a relationship between genotypic similarity and phenotypic similarity with a probabilistic interpretation corresponding to a generative model. We introduce a generative model for diploid allele effect based on the classic infinite allele mutation process. This approach motivates the concept of IBF (Identity by Function). The phenotypic covariance between two individuals given their diploid genotypes is expressed in terms of functional identity states. The IBF parameters define a genetic architecture for a trait without reference to specific alleles or population. Given full genome sequences, we treat a gene-scale functional region, rather than a SNP, as a QTL, modeling patterns of dominance for multiple alleles. Applications demonstrated by simulation include phenotype and effect prediction and association, and estimation of heritability and classical variance components. A simulation case study of the Missing Heritability problem illustrates a decomposition of heritability under the IBF framework into Explained and Unexplained components.

Imputing Amino Acid Polymorphisms in Human Leukocyte Antigens

DNA sequence variation within human leukocyte antigen (HLA) genes mediate susceptibility to a wide range of human diseases. The complex genetic structure of the major histocompatibility complex (MHC) makes it difficult, however, to collect genotyping data in large cohorts. Long-range linkage disequilibrium between HLA loci and SNP markers across the major histocompatibility complex (MHC) region offers an alternative approach through imputation to interrogate HLA variation in existing GWAS data sets. Here we describe a computational strategy, SNP2HLA, to impute classical alleles and amino acid polymorphisms at class I (HLA-A, -B, -C) and class II (-DPA1, -DPB1, -DQA1, -DQB1, and -DRB1) loci. To characterize performance of SNP2HLA, we constructed two European ancestry reference panels, one based on data collected in HapMap-CEPH pedigrees (90 individuals) and another based on data collected by the Type 1 Diabetes Genetics Consortium (T1DGC, 5,225 individuals). We imputed HLA alleles in an independent data set from the British 1958 Birth Cohort (N = 918) with gold standard four-digit HLA types and SNPs genotyped using the Affymetrix GeneChip 500 K and Illumina Immunochip microarrays. We demonstrate that the sample size of the reference panel, rather than SNP density of the genotyping platform, is critical to achieve high imputation accuracy. Using the larger T1DGC reference panel, the average accuracy at four-digit resolution is 94.7% using the low-density Affymetrix GeneChip 500 K, and 96.7% using the high-density Illumina Immunochip. For amino acid polymorphisms within HLA genes, we achieve 98.6% and 99.3% accuracy using the Affymetrix GeneChip 500 K and Illumina Immunochip, respectively. Finally, we demonstrate how imputation and association testing at amino acid resolution can facilitate fine-mapping of primary MHC association signals, giving a specific example from type 1 diabetes.

THU0015 Association of acid phosphatase locus 1*C ALLELE with the risk of cardiovascular events in rheumatoid arthritis patients

BackgroundAcid phosphatase locus 1 (ACP1) encodes a low molecular weight phosphotyrosine phosphatase implicated in different biological functions in the cell.ObjectivesThe aim of the present study was to determine the contribution...

AB0011 Impact of HLA-DRB1 shared epitope alleles according the new classification on the susceptibility and structural severity in rheumatoid arthritis

Backgroundrheumatoid arthritis (RA) is a complex polygenic disease. HLA-DRB1 alleles encoding the shared epitope (SE) are associated with RA susceptibility and severity. Recently HLA-DRB1 SE alleles has been reclassified into...

Identification of two novel waxy alleles and development of their molecular markers in sorghum

High amylopectin grains of waxy sorghum have a high economic value in the food and bioenergy industries because of their increased starch digestibility and higher ethanol conversion rate compared with wild-type sorghum grains. Mutation in the granule-bound starch synthase (GBSS) gene contributes to the waxy phenotype. Two classes of waxy alleles, wx(a) and wx(b), have been characterized previously. In the present work, we identified two novel types of waxy mutations in the sorghum GBSS gene, designated as wx(c) and wx(d). The wx(c) allele has a G deletion at the 5' splicing site of the ninth intron, causing a shift of the 5' cleavage site; in turn, a reading frame shift occurred and resulted in an early translation termination. The wx(d) allele contained a mutation at the 3' splicing site of the 10th intron, which led to a splicing site shift and resulted in the deletion of five amino acids (GTGKK) in the predicted translation product. Furthermore, cleaved amplified polymorphic sequence (CAPS) markers were developed to detect the wx(c) and wx(d) alleles. With these markers, classification of waxy alleles was performed in nearly 100 sorghum accessions from our breeding program. Most waxy sorghum cultivars in China were either wx(a) or wx(c), implying that these two mutations are preferentially maintained during domestic selection in glutinous sorghum production.

HLA-F and MHC-I open conformers are ligands for natural killer cell receptor KIR3DL2. (P5004)

Abstract Killer Immunoglobulin-like receptors (KIR) are innate immune receptors expressed by NK and T cells classically associated with the detection of missing-self through loss of their respective MHC ligand. Some KIR specificities for allelic classical class I MHC (MHC-I) have been described, while other KIR receptor-ligand relationships, including those associated with non-classical MHC-I, have yet to be clearly defined. We report here that KIR3DL2 and the non-classical antigen HLA-F, expressed as a free form devoid of peptide, physically and functionally interact. We further extend those results to include classical MHC-I open conformers as ligands, defining new allelic relationships with KIR3DL2 and MHC-I. The data collectively suggest a broader, previously unrecognized interaction between MHC-I open conformers - with HLA-F as a prototypical example - and KIR receptors, acting in an immunoregulatory capacity centered on the inflammatory response.