Human Lymphocyte Antigen Typing Research Articles

A rapid and sensitive method to detect specific human lymphocyte antigen (HLA) class II alleles associated with celiac disease

Celiac disease (CD) is a complex disorder triggered by gluten affecting genetically predisposed individuals. More than 90% of patients carry human lymphocyte antigen (HLA)-DQ2 (DQA1*05, DQB1*02) and/or HLA-DQ8 (DQA1*03, DQB1*0302). We propose the use of the DQ-CD Typing kit that allows identification of the HLA class II alleles (DQA1*0201,*03,*05, DQB1*02,*0302, DRB1*03,*04,*07) selected to be informative in the CD risk evaluation and of a second kit, namely DQ-CD Zygosis, for DQB1*02 homozygosity determination. The study was performed on a cohort of 100 individuals previously HLA typed with commercial kits. Fresh blood or previously extracted DNA was amplified in a unique PCR program using allele-specific primers and visualized on agarose gel. DNA amplification yielded strong and clear products without non specific signals or ghost bands. All the samples showed the expected alleles in accordance with the previous HLA typing. The DQ-CD Typing and Zygosis kits are fast, simple, economical and accurate tools that can be used to determinate the HLA-DQ2/DQ8 status in laboratory practice addressed for the diagnosis of CD. Molecular HLA testing is considered a valid support in the confirmation/exclusion of CD, especially in high-risk groups, such as CD relatives, or when serological and histological data are ambiguous.

Human Lymphocyte Antigen Typing: Direct DNA Typing-The Only Choice?

Exact human lymphocyte antigen (HLA) allele matching and sequence variation are important for matching organ donors, immune response studies, and disease association investigations. The number of HLAs has reached several hundred within each of the different classes. This level of heterogeneity makes routine DNA typing to the allele level problematic using fixed probe and primer technologies. Routine large-batch screening programs where only intermediate-level typing is required can be performed in automated fashion by several DNA technologies. Screening large numbers of samples with probe-based technologies, even oligo array chips, is cost effective. Allele-specific typing is most easily performed using direct sequencing. New sequencing technologies based on cycle sequencing and high-speed capillary gels have made routine sequencing for clinical typing a reality. The complexity of the class I locus requires a detailed analysis of all the polymorphisms within exons 2 and 3. Sequencing strategies are thus designed to use informative variable regions within the flanking introns and the flanking region as well as the untranslated regions. Simliar strategies are being adapted to the complex class II DRB alleles, which now number about 200 different alleles. Greater understanding of HLA diversity and distribution throughout humans and their relatives facilitates organ matching and the history and origins of human populations. Knowledge of parasites and their role in the selection of alleles will ultimately lead to better prediction and manipulation of the immune system response to these organisms. HLA typing is used to determine relative risk to a variety of autoimmune diseases. Future uses of molecular HLA typing may include the prevention and cessation of these self-destructing diseases.

Neonatal Nutritional Interventions in the Prevention of Type 1 Diabetes

After completing this article, readers should be able to: 1. Outline the current concepts of the pathogenesis of type 1 diabetes (T1D) with regard to dietary triggers and genetic susceptibility. 2. Discuss the evidence, both epidemiologic and that derived from studies of animal models, that supports the association between infant feeding practices and T1D. 3. Describe the roles for intestinal immunity and permeability in supporting the hypothetical model of cow milk protein mediation of T1D autoimmunity. 4. Delineate the goals and rationale for prospective clinical trials evaluating neonatal nutrition and the development of T1D. 5. List the putative dietary modulators of T1D. Type 1 diabetes (T1D), a disease that has unacceptably high morbidity and mortality, is increasing in incidence, prompting the redoubling of efforts toward its prevention. Progress toward prevention and cure relies on elucidation of the disease’s pathogenesis, which, to date, has remained poorly defined. The defining features of T1D, insulin deficiency and hyperglycemia, result from an immune-mediated destruction of insulin-secreting beta cells in the pancreatic islets. The loss of beta cell mass is believed to be gradual for most individuals, accounting for the sometimes prolonged asymptomatic periods of autoimmunity preceding overt diabetes (Fig. 1). Indeed, the chronic autoimmune nature of the disease is well established, as is the genetic predisposition. Specific associations with molecules of the human lymphocyte antigen (HLA) define both susceptibility to and protection from T1D. However, T1D is a polygenic disorder with more than 20 loci associated with susceptibility or resistance to the disease, of which the HLA may account for less than 50% of the genetic predisposition. Genetics clearly comprises a major component of the development of T1D, but the interaction between the environment and the immune system abnormalities is believed to weigh heavily in disease development. Indeed, trends in T1D incidence, both geographic and temporal, suggest a strong …

Human lymphocyte antigen molecular typing: how to identify the 1250+ alleles out there.

○ The human lymphocyte antigen (HLA) typing community was one of the early groups to adopt molecular testing. This action was borne out of the need to identify the many alleles of the highly polymorphic HLA system. Early paradigms used restriction fragment length polymorphism regimes, but the polymerase chain reaction method of amplification quickly replaced that less-than-discriminating choice. Methods currently in use for HLA typing, with commercial kits available, are sequence-specific oligonucleotide probe (both dot blot and the reverse blot dot), sequence-specific primer amplification, restriction fragment length polymorphism of amplified products, double-stranded sequence conformation polymorphism (with and without reference strand), sequence-based typing, and microarray technologies. More than 1250 alleles are recognized by the World Health Organization and meet their criteria for assignment. These alleles can be identified by molecular methods and represent alleles present at class I and class II loci of the HLA complex. On occasion, ambiguous results still persist, even with the best molecular typing methods. Therefore, it is clear to the HLA typing community that a combination of the above methods may be needed to allow true discrimination of the possible alleles an individual carries in their genetic make-up. It is also clear that a typing laboratory may need to resort to nonmolecular serology to understand the significance and impact of the type generated by the HLA molecular typing laboratory.

Human lymphocyte antigen molecular typing: how to identify the 1250+ alleles out there.

The human lymphocyte antigen (HLA) typing community was one of the early groups to adopt molecular testing. This action was borne out of the need to identify the many alleles of the highly polymorphic HLA system. Early paradigms used restriction fragment length polymorphism regimes, but the polymerase chain reaction method of amplification quickly replaced that less-than-discriminating choice. Methods currently in use for HLA typing, with commercial kits available, are sequence-specific oligonucleotide probe (both dot blot and the reverse blot dot), sequence-specific primer amplification, restriction fragment length polymorphism of amplified products, double-stranded sequence conformation polymorphism (with and without reference strand), sequence-based typing, and microarray technologies. More than 1250 alleles are recognized by the World Health Organization and meet their criteria for assignment. These alleles can be identified by molecular methods and represent alleles present at class I and class II loci of the HLA complex. On occasion, ambiguous results still persist, even with the best molecular typing methods. Therefore, it is clear to the HLA typing community that a combination of the above methods may be needed to allow true discrimination of the possible alleles an individual carries in their genetic makeup. It is also clear that a typing laboratory may need to resort to nonmolecular serology to understand the significance and impact of the type generated by the HLA molecular typing laboratory.

Schizophrenia and the HLA-DRB1 gene in the Japanese population.

Small Japanese studies have suggested that patients with schizophrenia have higher rates of the HLA-DR1 gene than normal subjects. The authors' goal in the present study was to confirm this finding in a larger number of Japanese subjects. They also investigated the rate of DR4 in Japanese patients with schizophrenia because it has been reported that Caucasian patients with schizophrenia have higher rates of DR4. They studied the occurrence of the HLA-DRB1 gene in 233 unrelated Japanese patients with schizophrenia compared with the occurrence of the gene in a group of 493 healthy Japanese volunteers. A larger proportion of the patients with schizophrenia (15.9%) than the comparison subjects (10.5%) were found to have DR1 (DRB1*0101). The proportion of patients (36.9%) and comparison subjects (40.6%) with DR4 did not differ significantly. Consistent with the findings of three other Japanese studies, the findings of the present study suggest that the rate of HLA-DR1 may be higher in Japanese patients with schizophrenia than in normal Japanese subjects. No evidence for an association between schizophrenia and the rate of DR4 was obtained in this study, although the combined data from the present study and other Japanese studies support the finding of lower rates of DR4 among patients with schizophrenia.

Discrimination between Celiac and Other Gastrointestinal Disorders in Childhood by Rapid Human Lymphocyte Antigen Typing

Celiac disease (CD) is a genetically complex, multifactorial immune-mediated disease (1). The intestinal damage that characterizes the disorder is induced by dietary gluten ingestion in susceptible individuals (1). A specific HLA DQA10501 / DQB10201 heterodimer, or in a few cases, the HLA DRB104 alleles, is associated with the disease (2). In fact, when presented by these HLA molecules, gluten-derived peptides cause T-cell activation in the intestinal mucosa, which is followed by cytokine production and mucosal intestinal damage (1). The clinical manifestations of CD are very variable, and most of the symptoms are also present in other clinical conditions. Diagnosis of CD includes the presence of circulating gliadin and endomysium antibodies (EMAs) (3) and is traditionally confirmed by intestinal biopsy, according to European Society of Paediatric Gastroenterology and Nutrition (ESPGAN) criteria (4). The genetic diagnostic approach may be very useful in CD patients with IgA deficiency, a condition where the serological tools are less useful; in familial screening; or in cases of latent forms of CD. Regarding the genetic approach, despite numerous reports of HLA class II associations to CD in diverse countries (5)(6)(7)(8)(9), there has been no systematic study to establish the role of HLA typing in the diagnosis of CD, particularly in discriminating CD from other confounding diseases. The aim of this study was to verify, using a PCR-based method that we recently established (10), the prevalence of the HLA heterodimer and of the HLA DRB104 alleles in healthy subjects, in CD-affected children, and in other age-matched subjects affected by confounding disease from South Italy. In a subgroup of CD patients in an active stage of disease, we also compared the diagnostic characteristics of genetic patterns and the presence of the EMAs and gliadin antibodies. We examined two groups of …

Cardiac transplant donor heart allocation based on prospective tissue matching

The present priority scheme for the allocation of donor hearts based on patient acuity and waiting time contributes to the escalating costs of heart transplantation, ignores the potential outcome advantages of prospective tissue matching, and is vulnerable to manipulation. Costs have trebled in recent years, as recipients frequently spend weeks before transplantation as inpatients in intensive care units and become more susceptible to nosocomial complications. The findings from an international cooperative study suggest that patient survival is correlated with the level of histocompatibility (ie, human lymphocyte antigen [HLA]) matching. We observed a similar inverse association between retrospective fortuitous HLA matching and the risk of rejection in 39 patients undergoing heart transplantation over a 29-month period ( p = 0.03 by nonparametric analysis). These observations prompted us to consider the feasibility of donor heart allocation based on the degree of HLA matching and waiting time alone. Current methods permit the accurate determination of HLA type in a matter of hours using donor peripheral blood alone. Human lymphocyte antigen typing, therefore, could be performed locally before organ harvesting, making issues of donor heart preservation irrelevant. We evaluated the extent of HLA matching that might be achieved practically. Forty-seven patients on our waiting list during calendar year 1991 were tested retrospectively for HLA matching with all geographically accessible 1991 heart donors identified by the United Network for Organ Sharing for all donors from hospitals east of the Mississippi River. Patients were matched for compatible blood type and six HLA antigens (two each for A, B, and DR) with 1,068 donors. For patients with blood type O (n = 30), 90% ( 27 / 30 ) had at least one donor with four antigens matched and 37% ( 11 / 30 ) and 20% ( 6 / 30 ) had at least one five- or six-antigen match, respectively. For patients with blood type A (n = 12) 100% ( 12 / 12 ) and 50% ( 6 / 12 ) had four- or five-antigen matches, respectively. Results were similar for blood type B (n = 2) and AB (n = 3) patients. A high degree of prospective HLA matching is achievable in heart transplantation with currently available technology. An allocation system based on matching and waiting time alone would be readily verifiable, would reduce the cost of heart transplantation by directing more hearts to outpatient recipients, and could improve outcome by achieving higher degrees of tissue compatibility. These factors constitute a rationale to consider other than the traditional triage criteria for the allocation of donor hearts.

HLA-type of cerebral vasospasm patients after aneurysmal subarachnoid hemorrhage

We studied human lymphocyte antigen (HLA) types in a group of 45 patients who had aneurysmal subarachnoid hemorrhage (SAH). A significantly increased frequency of HLA antigen A31 and a significantly decreased frequency of HLA antigen B40 were found. In patients with delayed ischemic neurological deficit (DIND) following aneurysmal SAH and HLA typing, HLA-Bw60 antigen showed significant increases; in patients who did not develop HLA-Aw33 and -Cw4 antigens showed significant. Among the patients with Fisher's Group 3 on CT, in particular, these antigens significantly increased when compared with controls from the same geographic area. These results suggest that HLA-Bw60 antigen plays a role as predisposing factor of DIND resulting from vasospasm following aneurysmal SAH, and that HLA-Aw33 and -Cw4 exert protective influence against DIND.

Histocompatibility antigens in vitiligo: Hamburg study on 102 patients from northern Germany.

One-hundred and two patients from the area of Hamburg, Germany, with vitiligo (photo-skin types II and III, Fitzpatrick classification, 1979), were examined for a possible association of the human lymphocyte antigen (HLA) complex with this disease. Antisera panels for the detection of 75 HLA antigens were utilized for this HLA typing (WHO Bulletin, 1988). The results were compared with 400 unrelated age- and sex-matched controls with photo-skin types II and III from the same geographical area. Nine of 76 vitiligo patients showed a significantly increased expression of the rare antigen HLA DRW12 (pc = 0.000708), and 65/102 patients showed a marginally significant increased frequency of HLA A2 (pc = 0.04850) compared with controls. For HLA BW60 (40), a significant increase in frequency was shown only in the adult vitiligo group (pc = 0.0126). A 'preventive' antigen for the manifestation of vitiligo has not been identified in this study. A comparative analysis of all published data on the possible association of HLA with vitiligo does not support a definitive linkage to the MHC so far.

Identification, Characterization, and Quantitation of Soluble HLA Antigens in the Circulation and Peritoneal Dialysate of Renal Patients

Human lymphocyte antigen (HLA) class I and class II antigens and beta 2 microglobulin (B2M) were identified in peritoneal dialysate (PD) and serum from patients with end-stage renal disease (ESRD) using monoclonal antibodies in an enzyme-linked immunoassay. The HLA class I and class II antigens each exhibited approximate molecular weights of 50,000 to 60,000 daltons by chromatography on Sepharose CL 6B. Class I antigens in serum and PD fluid were associated with B2M. Free B2M (Mr 11,500) also was detected in both sera and PD fluids. Unlike class I antigens, class II antigens were not found to have attached B2M. Class I and class II antigens eluted from 2-diethylaminoethanol ion exchange gradient columns at 0.07 mol/L (molar) phosphate buffer pH 7.2 and migrated with alpha 2-beta 1 mobility in agarose electrophoresis. Class I antigens were purified from ESRD patients' PD fluid by solid-phase immunoaffinity chromatography. Enzyme-linked immunoassay demonstrated that this purified protein was composed of a class I heavy chain and B2M. Class I allospecificity was confirmed by neutralization on known HLA typing antisera in a microcytotoxicity assay. Soluble HLA class I antigen preparations specifically inhibited blast transformation of responder lymphocytes in mixed lymphocyte culture reactions. Inhibition was dose dependent and ranged from 0% to 95%. The presence of soluble HLA antigens in body fluids may play an important part in the immunologic tolerance to self. This study demonstrates a ready source of large quantities of soluble HLA for detailed analysis.

HLA typing in actinic prurigo

Thirty-two actinic prurigo patients of Cree ancestry underwent human lymphocyte antigen (HLA) typing and were compared with 32 control subjects of Cree ancestry. We found a significantly increased frequency of HLA-A24 and Cw4 antigens and a significant decrease in the frequency of the A3 antigen in actinic prurigo patients. These HLA associations may be helpful in determining whether actinic prurigo is a distinct disease or a variant of polymorphous light eruption.

HLA histocompatibility affects cardiac transplant rejection and may provide one basis for organ allocation

Prospective human lymphocyte antigen (HLA) typing is not performed for heart transplantation, and the relation between HLA matching and cardiac graft rejection is unclear. Recipient and donor HLA matching were analyzed retrospectively in 51 patients undergoing orthotopic cardiac transplantation. Immunosuppression was based on cyclosporine and prednisone. During the mean follow-up of 34 months (range, 16 to 63 months), the 46 operative survivors had an average of 3.95 rejection episodes (range, zero to 11 episodes). Twenty-one patients had steroid-resistant rejection requiring treatment with polyclonal or monoclonal antithymocyte globulin. Human lymphocyte antigen typing was available for 44 patients, and antigens were grouped in broad specificities. Patients with two or more HLA-A or HLA-B matches had a reduced number of rejection episodes ( 3 / 10 versus 19 / 34 ) and a lower incidence of steroid-resistant rejection ( 1 / 10 versus 18 / 34 ; p = 0.01). Inclusion of HLA-DR matches did not alter the findings. There was a strong correlation between the increased frequency of rejection and the incidence of steroid-resistant rejection ( p < 0.0001). Four of six late deaths occurred in patients with steroid-resistant rejection; four were due to acute rejection and two to graft atherosclerosis. Although not currently done, prospective HLA matching is feasible with present typing methods. Our results suggest a rationale for prospective histocompatibility testing in cardiac transplantation with allocation of donor hearts to patients with two or more HLA matches.

HLA Compatibility and Cardiac Transplant Recipient Survival

Although the major histocompatibility complex has been linked with the control and expression of immune response in mammalian species, its importance for heart transplantation has not been demonstrated. The relationship of patient survival to human lymphocyte antigen (HLA) (A and B loci) compatibility was studied in 164 consecutive cyclosporine-treated patients who underwent orthotopic heart transplantation between 1980 and 1986 at Stanford University Medical Center. All patients receiving a transplant within this time frame were included except those for whom HLA typing was unavailable. A mismatched antigen was defined as an antigen present in the donor but not in the recipient. The actuarial four-year survival (Cutler-Ederer) for the 19 patients with 0 or 1 mismatch was 88 ± 8%; for the 39 patients with 2 mismatches, 70 ± 12%; for the 73 patients with 3 mismatches, 59 ± 7%; and for the 33 patients with 4 mismatches, 54 ± 14%. Both actuarial and linear rate analyses revealed no significant correlation between HLA mismatching and rejection rate, likelihood of death from rejection, or length of time to first episode of rejection. Patients with 3 or 4 mismatches had significantly ( p < 0.05) more infections than those with fewer mismatches. By actuarial analysis, a trend toward a higher number of deaths from rejection and infection was observed in the groups with 3 and 4 mismatches, but it did not achieve statistical significance. The data demonstrate that well-matched HLA grafts are associated with better long-term survival and fewer infections in cardiac transplant patients.

Gene conversion in salt-losing congenital adrenal hyperplasia with absent complement C4B protein.

Two of four siblings expressed the salt-losing form of congenital adrenal hyperplasia due to 21-hydroxylase deficiency (CAH) and had identical human lymphocyte antigen (HLA) and complement C4 (fourth component of complement) types (HLA-A3,C4,B35,C4A3,C4BQO,DR1/A2,C-,B18,C4A3, C4BQO,DR6). The father and one unaffected sibling were heterozygous carriers of CAH, as determined by a 30-min iv ACTH stimulation test and HLA typing. In addition, the iv ACTH stimulation test revealed that the mother and the other unaffected sibling also carried an allele for an attenuated form of CAH. Restriction endonuclease digests of genomic DNA obtained from members of this family and from normal unrelated subjects were hybridized with cDNA probes encoding human 21-hydroxylase and C4. With the 21-hydroxylase probe, Southern blots prepared from control DNA samples revealed two major restriction fragments in each of four restriction endonuclease digests; TaqI produced major bands at 3.7 and 3.2 kilobases (kb), KpnI at 4.0 and 2.9 kb, EcoRI at 18 and 13 kb, and BglII at 15 and 12.5 kb. Southern blots prepared from DNA of the two patients lacked the 3.7-kb TaqI and 2.9-kb KpnI fragments, but had increased hybridization intensity (relative to control DNA samples) in the 3.2-kb TaqI and 4.0-kb KpnI fragments. By contrast, blots with EcoRI or BglII had two large hybridization fragments not different from control DNA samples. These data indicate the presence of two different 21-hydroxylase genes. Additional mapping studies revealed that the two genes had the restriction pattern of the inactive 21-hydroxylase gene. When genomic DNA that had been isolated from all members of this family and from normal subjects was hybridized with the human C4 cDNA probe, the restriction fragment hybridization patterns for all four endonuclease digests were similar in the two groups. Hence, our results suggest that the 21-hydroxylase deficiency of our patients is due to conversion of the active 21-hydroxylase gene to the inactive gene. This gene conversion was associated with absence of functional C4B protein, without any detectable alterations in the restriction fragment pattern of the C4 genes.