HLA-DR, DQ genotypes of celiac disease patients and healthy subjects from the West of Ireland

Celiac disease (CD) has one of the strongest class II HLA associations of any human illness. We used DNA-RFLP typing to study the class II HLA genotypes of celiac disease patients from the West of Ireland, the geographic area with the highest rate of celiac disease in the world. We confirmed the high frequency of HLA-DR3 in this population, and we were also able to demonstrate the additional risk of developing celiac disease imparted by HLA-DR7. This was done by clearly distinguishing DR7, DQ2 haplotypes from DR7, DQ9 haplotypes, and by "subtraction analysis" of haplotype frequencies. As reported in other populations, most of the patients without DR3 were heterozygous for DR7 and DR11 or 12 (DR5), or had DR4. We used PCR-RFLP and direct sequencing of amplified DNA to examine HLA-DR4 subtypes. The frequency of HLA-DR4 was markedly decreased in patients compared with controls (p=0.000001) and there was a significant alteration of DR4 subtypes of the patients compared with controls (p=0.0227). Moreover, all of the CD patients (5 of 5) with DR4 had a haplotype associated with the DQB1*0302 allele compared with only 11 of 23 control subjects with DR4. Our results in this population with exceptionally high risk of CD strongly support the DQ heterodimer hypothesis and suggest that the recently described sequence difference between the DQB1*02 alleles of DR3 and DR7 may contribute to a synergistic increased risk when these haplotypes are inherited together. In addition, our findings suggest a role for HLA-DQ in DR4-associated CD.     

HLA-B44 subtyping in a Spanish population: further evidence of Caucasian population diversity

HLA-B44 is the most frequent HLA-B allele in Caucasian populations. Several B44 subtypes, B*4402-B*4406, have been identified in individuals with this ethnic origin. Mismatches among B44 subtypes have been described as major targets for allogeneic responses in bone marrow transplantation. We have developed a PCR-SSO method, based on a B12- specific DNA amplification of exon 2 through exon 3 and subsequent non radioactive hybridization with eight probes, which allow us to discriminate all B12 homozygous combinations. We applied this method to determine the frequency of B44 subtypes in a Spanish population, as well as their HLA-A.-C.-DRB1,-DRB3/DRB4/DRB5.-DQA1 and -DQB1 associated haplotypes. A total of 141 healthy unrelated Spanish individuals and 31 B44-bearing haplotypes were investigated. Four B44 alleles were identified, B*4402 (33%), B*4403 (66%), B*4404 (0.7%), and B*4405 (0.7%). Haplotype analysis showed a clear differentiated distribution pattern for the two major B44 subtypes. B*4402 is associated with Cw5 (11/13) and A2 antigens (10/13). In contrast, B*4403 is mainly found together with DRB1*0701 (14/16). An inverted B*4402/B*4403 frequency in comparison with other European and North American Caucasian populations, revealed the existence of an extended haplotype diversity between populations of the same ethnic origin. Apart from anthropological studies, high resolution typing for HLA class I antigens presenting molecular polymorphism will be of great relevance in unrelated bone marrow transplantation.     

Predominant and stable T cell responses to regions of myelin basic protein can be detected in individual patients with multiple sclerosis

T lymphocytes from patients with multiple sclerosis (MS) recognize multiple myelin basic protein (MBP) epitopes. This situation complicates the design of specific immunotherapies. We investigated to which extent the T cell response to MBP is heterogeneous in single subjects in terms of preferentially recognized regions of the molecule, major histocompatibility complex (MHC) restriction, and stability over time. From each of nine patients with MS, a minimum of six MBP-specific T lymphocyte lines (TLL) were assayed for the proliferative response to a panel of overlapping peptides, encompassing the whole MBP. Predominant Tcell recognitions of distinct MBP regions were present in three patients, all HLA-DR2⁺, independently of the clinical features of their disease. Tcell reactivity was preferentially directed to residues 16-38 in one patient. In this case the response was also stable over time, during different phases of the disease. Predominant reactivity to residues 86-99 was detected in the two other DR2⁺ patients. In each of the patients with other HLA-DR haplotypes (DR2^?), as well as in three DR2⁺ non-MS donors, the Tcell response to MBP appeared to be considerably more heterogeneous. The HLA restriction element varied among TLL recognizing the same MBP region, even when raised from the same individual. The genomic HLA typing, performed on the DRB1 and DRB5 genes in the DR2⁺ subjects, showed no obvious correspondence between preferential responses to regions of MBP and HLA-DR2 subtypes. In this context, a simple, new method for the genomic typing of the HLA-DRB1 gene in individuals with the HLA-DR2 serological specificity is also described. We conclude that predominant and stable T cell responses to a single MBP region can be detected in some patients with MS. In these individuals, the MHC restriction of the T cell recognition of predominant regions appears to be variable. Polymorphisms of the HLA-DR2 gene products alone do not account for the selection of the dominant MBP Tcell epitope.     

Strong association between HLA-Cw*0706 and HLA-B*44032 in the Bubi population from Equatorial Guinea

Unrelated Bubi, native to the island of Bioko (Equatorial Guinea), were previously typed by low-resolution polymerase chain reaction using sequence-specific primers (PCR-SSP) and serology for HLA-A, -B and -C. HLA-B*44 was found frequently and associated with Cw*07. We have studied the HLA subtypes of 20 B*44pos/Cw*07pos Bubi individuals. HLA-B and -C were typed by sequencing exons 2 and 3. To distinguish the alleles Cw*1701/02/03, Cw*07011/012/06 and Cw*1801/02 additional sequencing of exon 1 or 5 was performed. All 20 B*44pos/Cw*07pos individuals of the Bubi population were typed Cw*0706 positive. Nineteen of them carried the B*44032 allele and one B*4407. In addition, 19 B*44neg/ Cw*07pos Bubi individuals were typed for HLA-C and none of them proved Cw*0706 positive. To determine whether the association between Cw*0706 and B*44032 was limited to the Bubi, 19 individuals from Dutch Caucasian families were typed in which B44 and Cw7 segregated on one haplotype. None of these individuals showed the presence of B*44032 or Cw*0706. The haplotypes found in the Dutch Caucasians were B*4402-Cw*0704, B*44031-Cw*07011 and B*44031-Cw*0702. The present observation indicates a strong association between B*44032 and Cw*0706 in the Bubi population.     

EFI region 8 Balkan external proficiency testing for HLA typing: Results of 15 years

Istanbul Medical Faculty, Department of Medical Biology, as the first EFI‐accredited HLA laboratory in Turkey since 1999 has been organizing both national and international quality control tests for HLA typing under the banner of the “Balkan external proficiency testing (BEPT)” encompassing countries from EFI regions 8. The first round of BEPT in 2004 was organized for low‐resolution HLA‐A,‐B typing by molecular methods and 12 centres participated in the exercise. In 2005, low‐resolution HLA‐DR typing was added, and in 2007, low‐resolution HLA‐C and DQ typing were added to the exercise and 28 centres participated. In 2015, high‐resolution (four digits or higher typing) HLA‐A,‐B,‐C,‐DR and –DQ typing added to the exercise and 40 centres participated. In the last 2 years, 2017 and 2018, the number of participating centres increased to 48 and 52, respectively. When the distribution of low‐resolution typing methods applied in the exercises were investigated, it was found that 82% of the centres in the first round of BEPT in 2004 used PCR‐SSP, whereas in the last round in 2018, 26% of the centres preferred SSP, while the rest used SSO (50%) or SSP and SSO (24%) methods. Methods for high‐resolution typing were SBT (41%), NGS (18%), SSO (18%), SSP (6%), SBT + NGS (6%) and SBT + SSP (11%). In 15th trial for BEPT, nomenclature mistake rate was 12%, and erroneous result rate was 6% for HLA samples. The most common mistakes in the exercises were nomenclature mistakes. The EFI Standards Version 7.0 is stated "HLA type can be reported using a hyphen if homozygosity is not proven by family studies." In order to provide standard results among HLA laboratories and since accurate HLA typing leads to significant increase of graft and patient survival, quality control exercises should be performed in all HLA‐based tests periodically.     

新等位基因人类白细胞抗原B*4609的发现和确认

目的 鉴定一个人类白细胞抗原(human leukocyte antigen,HLA)新等位基因HLA-B*4609.方法 使用序列特异性寡核苷酸PCR技术进行HLA基因分型,发现反应格局异常的可疑新等位基因,应用分子克隆和DNA双向测序技术测定新等位基因的核苷酸序列,并与已知等位基因进行序列比对分析.结果 检出1个样本HLA-B位点反应格局异常,DNA测序分型结果一个为B*151101,另一个的核苷酸序列与已知的HLA等位基因均不同,该基因序列与同源性最高的HLA-B*460101基因序列相比,在第3外显子区域中527位碱基发生A→T突变,导致176位编码氨基酸由谷氨酸(GAG)变成缬氨酸(GTG).结论 样本中含有HLA-B新等位基因序列.该序列申报后,被世界卫生组织HLA因子命名委员会正式命名为HLA-B*4609.     

Development of a PCR-SSOP approach capable of defining the natural killer cell inhibitory receptor (KIR) gene sequence repertoires

A molecular typing method based on polymerase chain reaction (PCR) amplification of three different target domains (immunoglobulin domains 1 and 3, and the transmembrane-cytoplasmic domain), followed by hybridisation with 26 digoxigenin-labelled sequence-specific oligonucleotide probes (SSOP) has been established for the polymorphic killer inhibitory receptor (KIR) genes. In addition to identifying the 12 KIR subfamilies, our PCR-SSOP typing approach could also distinguish the putative alleles, NKB1 and NKAT3, that comprise the KIR3DL1 subfamily. Ninety unrelated blood donors and 13 families (52 individuals), including both parents, were subjected to our KIR PCR-SSOP typing approach. All 12 KIR subfamilies, including a 2DS5 variant sequence, were present in the 90 individuals and displayed varied phenotype frequencies: 2DL1 (0.96), 2DL2 (0.31), 2DL3 (0.95), 2DS1 (0.56) 2DS2 (0.51), 2DS3 (0.27), 2DS4 (0.96), 2DS5v (0.35), 3DS1 (0.47), 3DL1 (0.96), 3DL2 (1.0) and 2DL4 (1.0). A total of 23 different KIR phenotypes were defined in this study, and 10 of these were only found on one occasion in one individual, indicating considerable diversity in the KIR phenotype profiles within the Irish population. Most individuals (93%) possessed the complement of inhibitory KIR specificities for the three well-defined HLA-B and -C ligands. An unusual probe pattern for 3DS1 was observed in 3 individuals indicating a variant 3DS1 gene sequence with changes at nucleotide positions 1185-1186, within the cytoplasmic domain. Sequencing analysis revealed a new single nucleotide polymorphism in exon 3 of 3DL1 NKB1(195, G-A) and a 22-bp deletion polymorphism in exon 5 of 2DS4 (nucleotides 777-798 deleted). A number of strong KIR associations were observed, namely 2DL1 with 2DL3, 2DS4 with 3DL1, 2DL2 with 2DS1/2DS2/2DS3, 2DS1 with 2DS3/2DS5v/3DS1, 2DS2 with 2DS3 and 2DS5v with 3DS1. Analysis of the KIR segregation observed in the 13 families confirmed these strong associations and permitted the definition of a number of partial KIR haplotypes, e.g. 2DL2-2DS1-2DS2-2DS3-3DL1. The segregation analysis concluded that at least 3 distinct gene loci encode 2DL1-4 and at least 4 gene loci encode the non-inhibitory KIR2DS1-2DS5. In the case of 3DL1-2 and 3DS1, our data suggests 3 gene loci, one for each subfamily.     

Identification of a proteomic biomarker associated with invasive ST1, serotype VI Group B Streptococcus by MALDI-TOF MS

Background

Group B Streptococcus (GBS) is an important invasive pathogen in neonates, pregnant women and the elderly. Serotype VI GBS, which has been rarely reported globally, has emerged as a significant pathogen in Asia. However, traditional serologic latex agglutination (LA) methods may fail to type isolates that lack of or low expression of CPS.