HLA Typing Studies Research Articles

Next generation sequencing and the classical HLA loci in full heritage Pima Indians of Arizona: Defining the core HLA variation for North American Paleo-Indians

The Pima Indians of the Gila River Indian Community in Arizona have participated in a long-range study of type 2 diabetes mellitus since 1965 and have been the subject of HLA typing and population studies since the early days of serological assays. These data have been in numerous HLA workshops and conferences and have been the source of at least five novel alleles at the classical HLA loci. In recent time nearly the entire study group was subject to next generation sequencing by whole genome or exome technologies, which has allowed us to HLA type over 3000 full heritage persons with recently developed computer algorithms. We present here the results for the classical HLA Loci: HLA-A, B, C, DRA, DRB1, DRB3, DRB4, DRB5, DPA1, DPB1, DQA1, and DQB1 to the third field of resolution for synonymous alleles and type the likely four field resolution alleles from the subset of whole genome sequences. Allele frequencies, and haplotype frequencies at up to five loci, are presented as well as measures of population structure and heterozygosity. We define a core set of HLA variation that approximates the distribution for the Paleo-Indians and impute nine-locus, 4-field haplotypes that are expected to be common in full heritage peoples.

Clinical, molecular, and therapeutic aspects of NDM in ten cases with diabetes in 1st 6 months of life

Neonatal diabetes mellitus (NDM) is defined as hyperglycemia occurring within the first 6 months of life; it may be permanent or transient. Diagnosis of NDM is vital for prognosis, genetic counseling, and treatment. The aim of this study was to study clinical, biochemical, molecular, and therapeutic aspects of diabetes presenting in the first 6 months of life. Ten cases with NDM were studied regarding perinatal and family history, clinical features and biochemical tests on admission and follow-up, HLA typing and molecular studies, and mode of therapy. Molecular studies for the common mutations associated with NDM (KCNJ11, INS, ABCC8 and methylation defects) were done using PCR amplification and gene sequencing. Ten cases developed diabetes in the first 6 months of life (three in the first 8 weeks, seven between 8 and 24 weeks). All presented with ketoacidosis, one had developmental delay with convulsions. Molecular studies revealed methylation defect in two cases, KCNJ11 in three cases, INS mutations in two cases, and no detectable defect in three cases. Insulin was stopped on follow-up in three cases (TND), successfully substituted with sulfonylurea (SU) in three cases, and continued in four cases (PND). Molecular genetic testing is essential in cases with NDM for guiding mode of therapy as SU receptor defects can be successfully treated with oral SU instead of insulin injections. SU was more effective in achieving diabetes control in cases with KCNJ11 mutations.

HLA and MICA polymorphism in Polynesians and New Zealand Maori: Implications for ancestry and health

Data from HLA typing studies have made significant contributions to genetic theories about the Austronesian diaspora and the health of descendant populations. To help further unravel pattern and process elements, we have typed HLA and MICA loci at high resolution in DNA samples from well defined groups of Maori and Polynesian individuals. Our results show a restricted set of HLA class I alleles compared with other well characterised populations. In contrast, the class II HLA-DRB1 locus seems to be diverse in Maori and Polynesians and both groups show high frequencies of HLA-DRB1(∗)04:03, -DRB1(∗)08:03, -DRB1(∗)09:01 and -DRB1(∗)12:01. Our survey also provides the first ever MICA datasets for Polynesians and reveal unusual distributions and associations with the HLA-B locus. Overall, our data provide further support for a hybrid origin for Maori and Polynesians. One novel feature of our study is the finding that the gene sequence of the HLA-B(∗)40:10 allele in Polynesians is a recombinant of HLA-B(∗)55:02 and -B(∗)40:01. HLA-B(∗)40:10 is in close association with HLA-C(∗)04:03, an allele identified as a hybrid of HLA-C(∗)04 and -C(∗)02. In this respect, our data resemble those reports on Amerindian tribes where inter-allele recombination has been a common means of generating diversity. However, we emphasize that Amerindian gene content per se is only a very minor element of the overall Polynesian genepool. The wider significance of HLA and MICA allele frequencies across the Pacific for modern day health is also discussed in terms of the frequency relative to reference populations of disease known to be associated with specific HLA and MICA markers. Thus, Polynesians and Maori are largely unaffected by "European autoimmune diseases" such as ankylosing spondylitis, uveitis and coeliacs disease, yet there are several Maori- and Polynesian-specific autoimmune diseases where the HLA and MICA associations are still to be determined.

Predictors of Hypocretin (Orexin) Deficiency in Narcolepsy Without Cataplexy

To compare clinical, electrophysiologic, and biologic data in narcolepsy without cataplexy with low (≤ 110 pg/ml), intermediate (110-200 pg/ml), and normal (> 200 pg/ml) concentrations of cerebrospinal fluid (CSF) hypocretin-1. University-based sleep clinics and laboratories. Narcolepsy without cataplexy (n = 171) and control patients (n = 170), all with available CSF hypocretin-1. Retrospective comparison and receiver operating characteristics curve analysis. Patients were also recontacted to evaluate if they developed cataplexy by survival curve analysis. The optimal cutoff of CSF hypocretin-1 for narcolepsy without cataplexy diagnosis was 200 pg/ml rather than 110 pg/ml (sensitivity 33%, specificity 99%). Forty-one patients (24%), all HLA DQB1*06:02 positive, had low concentrations (≤ 110 pg/ml) of CSF hypocretin-1. Patients with low concentrations of hypocretin-1 only differed subjectively from other groups by a higher Epworth Sleepiness Scale score and more frequent sleep paralysis. Compared with patients with normal hypocretin-1 concentration (n = 117, 68%), those with low hypocretin-1 concentration had higher HLA DQB1*06:02 frequencies, were more frequently non-Caucasians (notably African Americans), with lower age of onset, and longer duration of illness. They also had more frequently short rapid-eye movement (REM) sleep latency (≤ 15 min) during polysomnography (64% versus 23%), and shorter sleep latencies (2.7 ± 0.3 versus 4.4 ± 0.2 min) and more sleep-onset REM periods (3.6 ± 0.1 versus 2.9 ± 0.1 min) during the Multiple Sleep Latency Test (MSLT). Patients with intermediate concentrations of CSF hypocretin-1 (n = 13, 8%) had intermediate HLA DQB1*06:02 and polysomnography results, suggesting heterogeneity. Of the 127 patients we were able to recontact, survival analysis showed that almost half (48%) with low concentration of CSF hypocretin-1 had developed typical cataplexy at 26 yr after onset, whereas only 2% had done so when CSF hypocretin-1 concentration was normal. Almost all patients (87%) still complained of daytime sleepiness independent of hypocretin status. Objective (HLA typing, MSLT, and sleep studies) more than subjective (sleepiness and sleep paralysis) features predicted low concentration of CSF hypocretin-1 in patients with narcolepsy without cataplexy.

Transfusion‐associated graft‐versus‐host disease in immunocompetent patients: case series and review of the literature

Transfusion-associated graft-versus-host disease (TA-GVHD) is a fatal complication of transfusion of blood products that usually affects immunocompromised patients. Articles reporting this condition in immunocompetent recipients are usually from countries that still have problems in irradiation of blood products. This report presents fatal TA-GVHD in four immunocompetent patients referred from rural areas where blood irradiation is still not the routine procedure to our tertiary-care center between July 2004 and July 2005. A similar history and chronological order of events were observed: fresh whole-blood transfusion from relatives, fever, rash, liver dysfunction, diarrhea, and pancytopenia. Skin biopsies demonstrated Grade II to III GVHD involvement. Marrow biopsies showed hypoplasia. In two cases, HLA typing studies were performed. Donors were homozygous for a shared HLA haplotype in the patients. All cases were admitted to the intensive care unit within 3 weeks after transfusions with the diagnosis of sepsis, which rapidly progressed to septic shock and multiorgan failure. Another common observation was Candida albicans growth in blood cultures. Unfortunately, all died despite prompt and appropriate sepsis treatment, along with immunomodulatory therapy. TA-GVHD is probably more prevalent than reported in the literature. It must be considered in the differential diagnosis, if the patient with a recent transfusion history admits with fever, skin rash, abnormal liver function tests, and pancytopenia associated with hypoplastic marrow. In rural areas where gamma irradiation is not possible, the overall policy of transfusion (e.g., restriction of transfusion indications and alternative methods for pathogen inactivation) should be reassessed.

Autoimmune disease is not a feature of hepatitis C infection in Ireland.

To determine the prevalence of autoimmune disease, autoantibody positivity, or both in Irish persons with hepatitis C, we surveyed 98 such patients (55 recipients of anti-D, 25 intravenous drug abusers, and 18 blood transfusion recipients). We studied them clinically and tested for anti-nuclear, anti-smooth muscle, and anti-mitochondrial, liver-kidney microsomal, thyroid microsomal, thyroid globulin, and gastric parietal antibodies; and also for rheumatoid factor. In the anti-D antibody group (all female), two patients reported generalized musculoskeletal symptoms but had no demonstrable physical signs. We did not find cryoglobulins in any patient. We detected thyroid microsomal antibodies in only 6 of 55 (10.9%) patients. (In two of these, thyroid globulin antibodies were also positive). These patients were all clinically euthyroid, but two had borderline low-normal thyroid function tests. Titers for anti-nuclear antibodies were weakly positive in 5 of 55 (9.1%) patients, and gastric parietal antibodies were positive in 5 of 55 (9.1%) patients. In particular, we noted no antibodies to liver-kidney microsome. Rheumatoid factor was detected in eight patients. Forty-seven of 55 patients were genotype 1b, and 8 of 55 were genotype 3. In the intravenous drug abusers (8 women, 17 men), we detected no autoantibodies. Seven of the 25 genotypes were tested; three were genotype 3 and four were genotype 1b. In the transfusion group (10 women, 8 men), we detected no autoantibodies apart from weak anti-nuclear antibody Titers (1:10), which we found three patients. Five of 10 genotypes tested were of genotype 3 and the other five were of genotype 1b. These findings suggest that in Irish patients with hepatitis C, neither genotype nor source (and dose) of inoculum contributes to the development of autoimmune disease. How hepatitis C virus is associated with autoimmune disease in other studies remains unknown. The answer may, at least in part, be found in genetic; HLA typing studies should provide useful information.

Immunogenetics in pathogenesis of and protection against toxoplasmosis.

Clinical manifestations and congenital transmission rates vary markedly among individuals with Toxoplasma infection (reviewed in Boyer and McLeod 1996; Remington et al. 1995). An influence of host immune response genes on the outcome of congenital infection is supported by observations of concordance of manifestations in monozygotic versus discordance of manifestations in dizygotic twins (Couvreur et al. 1976; reviewed in Remington et al. 1995), ethnic variations in incidence and severity of manifestations of disease (McAuley et al. 1994) and recent HLA typing studies (Mack et al. 1996b). Data from studies of murine models indicate that variables such as parasite strain (McLeod et al. 1984, 1988; Suzuki et al. 1989; Sibley and Boothroyd 1992; Suzuki and Joh 1994), inoculum size (Johnson et al., unpublished), host age (Johnson et al. unpublished), sex hormones (Roberts et al. 1995), and immune status (Gazzinelli et al. 1993a,b) also clearly modulate and effect outcome of infection. Studies are under way to define precisely how host genetics, as well as certain of the variables mentioned above, determine the small proportion of immunologically competent older children and adults who develop various disease manifestations. These studies are focused on the role of genetics in determining disease manifestations such as those described in systemic (reviewed in Boyer and McLeod 1996), retinal (Couvreur and Thulliez 1996), neurologic disease (Townsend et al. 1975), or lymphadenopathic toxoplasmosis (McCabe et al. 1987; Montoya and Remington 1995). In addition, these studies address whether immunogenetics influence the small proportion of acutely infected women who transmit infection to their fetuses early in gestation (Hohfeld et al. 1994), the 40% of T. gondii seropositive patients with AIDS who develop toxoplasmic encephalitis (Luft and Remington 1992), the severity of manifestations of congenital infection (McAuley et al. 1994; Mack et al. 1996b) or the occurrence or frequency of episodes of reactivation in recurrent retinochoroiditis in individuals with toxoplasmic retinochoroiditis (Mets et al. 1992; Mets et al. 1996).

PRENATAL DIAGNOSIS OF CONGENITAL ADRENAL HYPERPLASIA DUE TO 21-HYDROXYLASE DEFICIENCY

Prenatal diagnosis of the classic forms of the congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency has progressed at the same time as the advances made in the field of both endocrinology and molecular genetics. In 1975, prenatal diagnosis of CAH was first made in the second trimester on the basis of raised levels of 17-hydroxyprogesterone (17-OHP) in the amniotic fluid (AF). Later an indirect prenatal diagnosis based on the genetic tracking of CAH was greatly facilitated by its linkage to HLA. By 1979, to obviate the need for corrective genital surgery the virilized genitalia in CAH female fetuses, a prenatal treatment was proposed by giving dexametfiasone to the mother in early pregnancy, and later was shown to be successful. Rapidly, there was a demand for an early prenatal diagnosis of the affection. This was rendered possible by the advances in molecular biology associated with the practice of chorionic villus biopsy. Prenatal diagnosis was then done in the first trimester of the pregnancy, by either HLA typing using HLA-DNA probes or direct studies of the genetic abnormalities of the C4-CYP21 locus. In practice, four methods have been, and can still be used : measurement of AF levels of 17-OHP, HLA typing, molecular studies of the C4-CYP21 genes, all in association with fetal sex determination (measurement of AF testosterone levels, karyotype and more recently detection of Y-chromosome specific sequences). Steroid analysis remains a simple and safe method in the absence of prenatal treatment and the only informative method when there is no previous family study. Nowadays, an early and accurate prenatal diagnosis can be achieved by molecular genetic studies on DNA extracted from chorion villus samples, but it requires the previous study of the nuclear family. The choice between HLA typing and direct C4-CYP21 loci studies depend on the possibilities and expertise of a given laboratory. HLA typing is still more often used with the improvement of using molecular studies instead of serological methods because almost all families are then informative. Nevertheless, HLA typing is unreliable in some circumstances: in case of a recombination event (1 % in our studies of over 250 families) or de novo mutation(s) and when the index case is deceased or not available. Studies of the C4-CYP21 gene locus by Southern blotting and the CYP21B gene mutations by PCR method have not these disadvantages. In our hands, Southern blotting study of the duplicated C4-CYP21 region has shown some complex genetic rearrangements and is fully informative method for prenatal diagnosis in about 60% of CAH families. The 9 most common point mutations of the CYP21B gene causing CAH (and also present in the CYP21A pseudogene) can be detected after specific PCR amplification of the functional CYP21B gene. In these conditions, almost all families are informative in our experience. Moreover, using this direct genetic analysis associated with the possibility to detect the heterozygotes in a non related-CAH population, a prenatal diagnosis could be done in a family without a previous CAH affected child. A prenatal diagnosis in the first trimester is required in order to stop unnecessary prenatal treatments (if unaffected female fetuses and all males fetuses), or when the parents wish to interrupt the pregnancy in case of a CAH affected fetus. In our experience using molecular genetics of the CYP21-C4 loci and sex determination by PCR method (SRY, Y-chromosome repeated sequences) or karyotype, prenatal diagnosis has been successfully done in 23 pregnancies at risk 1:4. In one case, there was no previous family study because the index c;>se was dead; in an other family, the only informations were the state of heterozygoty of the parents: one being a relative of a characterized CAH family, the other being detected by a systematic ACTH test and confirmed by mutation studies.

U1RNP Antibody-Positive Neonatal Lupus

Neonatal lupus erythematosus (NLE) is a distinct subset of lupus characterized by cutaneous findings (50%), cardiac conduction defects (50%), and autoantibodies to Ro (SS-A) antigen. HLA typing studies of Ro (SS-A) antibody-positive mothers of infants with NLE have shown an association with the HLA-DR3 phenotype. We report the clinical and serologic features of two infant-mother pairs who are U1RNP antibody positive and Ro (SS-A) antibody negative. HLA typing is reported on these infants, their mothers, and two additional infant-mother pairs with U1RNP antibody-positive lupus whose clinical features have been reported previously. Cutaneous findings included malar erythema, annular and polycyclic plaques, and scales that resolved with residual telangiectasia and hyperpigmentation 6 months after birth. Systemic abnormalities, including complete heart block, were absent. HLA typing revealed HLA-DR3 in two of four mothers, HLA-DR4 and HLA-DRw53 in two of four mothers, and either HLA-DQ1 or HLA-DQ3 in four of four mothers. No distinct HLA associations were seen in the three infants examined. The spectrum of cutaneous disease in U1RNP antibody-positive infants is similar to Ro (SS-A) antibody-positive infants with NLE. Complete heart block was not a feature of U1RNP antibody-positive NLE. HLA typing studies show a more diverse immunogenetic pattern in U1RNP antibody-positive mothers of infants with NLE compared with Ro (SS-A) antibody-positive mothers.

Detection of hereditary haemochromatosis in an HLA-identical pedigree showing discordance between HLA class I genes and the disease locus

Hereditary haemochromatosis is a recessive disease in which primary hepatocellular carcinoma, complicating cirrhosis, is responsible for about one-third of deaths in affected homozygotes. We describe a unique HLA haplo-identical pedigree showing parent-to-offspring transmission of hereditary haemochromatosis in whom HLA typing studies, including class I and class II allogenotype analysis, were of no benefit in identifying affected homozygotes. However, affected siblings in the pre-cirrhotic stage of haemochromatosis, with apparent discordance between the haemochromatosis allele and class I loci on chromosome 6, were detected by undertaking a family study, using analysis of serum parameters of iron status in combination with magnetic resonance imaging (MRI). This pedigree emphasises the critical importance of genetic and non-invasive methods for the identification of asymptomatic homozygotes before cirrhosis develops.

Isolation of a human T cell line specific for a streptococcal cell surface antigen

A streptococcal cell surface antigen of Mr 185,000 (SAI/II) expressed by Streptococcus mutans has previously been well characterised. A T cell line specific for native SAI/II has been isolated from peripheral blood mononuclear cells (PBMC) of a naturally sensitised normal individual. This line has been maintained in culture for several months and was shown to be highly specific, not only for different preparations of native antigen but also for recombinant SAI/II protein. It did not respond to a homologous antigen SpaA (Mr 210,000), extracted from Strep. sobrinus. The phenotype of the line was CD3+ CD4+ CD8- TcR alpha beta +. HLA typing and inhibition studies showed that the response was restricted by both DR and DP encoded class II.

Successful HLA Nonidentical Bone Marrow Transplantation in Three Patients With the Leukocyte Adhesion Deficiency

AbstractThree consecutive patients with the severe phenotype of leukocyte adhesion deficiency characterized by a defective expression of LFA-1, Mac-1 (CR3), and p150.95 on leukocytes have received HLA partially incompatible bone marrow transplantation (BMT). The degree of HLA incompatibility between related donors and recipients was 2 HLA antigens in one and one full haplotype in the two others. Graft-v-host disease (GVHD) prophylaxis consisted in T- cell depletion of the bone marrow inoculum and a 60-day course of cyclosporin A. A first attempt led to autologous recovery in one patient. The second transplant in this patient and the first transplant in the two others led to stable partial engraftment of lymphocytes and phagocytic cells, as shown by expression of adhesion molecules (LFA-1, Mac-1) on leukocytes and by HLA typing and restriction fragment-length polymorphism studies using minisatellite probes. Although the level of mixed chimerism was lower in one patient (7% to 30% donor cells) and >50% in the two others, recovery of lymphocyte and phagocytic cell functions was sufficient enough to allow the patient to lead a normal life, infection free in the three cases. These patients, now 57,32, and 19 months post-transplant, are in good condition without any therapy. These results lead us to propose that the LFA-1 molecule plays a role in HLA- incompatible graft rejection, probably by mediating adhesion of cytotoxic T and non-T lymphocytes to their targets.

Successful HLA nonidentical bone marrow transplantation in three patients with the leukocyte adhesion deficiency

Three consecutive patients with the severe phenotype of leukocyte adhesion deficiency characterized by a defective expression of LFA-1, Mac-1 (CR3), and p150.95 on leukocytes have received HLA partially incompatible bone marrow transplantation (BMT). The degree of HLA incompatibility between related donors and recipients was 2 HLA antigens in one and one full haplotype in the two others. Graft-v-host disease (GVHD) prophylaxis consisted in T-cell depletion of the bone marrow inoculum and a 60-day course of cyclosporin A. A first attempt led to autologous recovery in one patient. The second transplant in this patient and the first transplant in the two others led to stable partial engraftment of lymphocytes and phagocytic cells, as shown by expression of adhesion molecules (LFA-1, Mac-1) on leukocytes and by HLA typing and restriction fragment-length polymorphism studies using minisatellite probes. Although the level of mixed chimerism was lower in one patient (7% to 30% donor cells) and greater than 50% in the two others, recovery of lymphocyte and phagocytic cell functions was sufficient enough to allow the patient to lead a normal life, infection free in the three cases. These patients, now 57, 32, and 19 months post- transplant, are in good condition without any therapy. These results lead us to propose that the LFA-1 molecule plays a role in HLA- incompatible graft rejection, probably by mediating adhesion of cytotoxic T and non-T lymphocytes to their targets.

Meiotic recombination in the beta globin gene cluster causing an error in prenatal diagnosis of beta thalassaemia.

In the course of a prenatal diagnosis for beta thalassaemia by linkage analysis of restriction fragment length polymorphisms, a homozygous beta thalassaemia fetus was misdiagnosed as beta thalassaemia trait. Extensive studies of the polymorphic sites within the beta globin gene cluster in all the members of the family resulted in the conclusion that the paternal chromosome 11 of the newborn was different from that expected. Paternity was confirmed by HLA typing and blood group studies. The analysis of another polymorphic locus on chromosome 11 within the family was in agreement with the possibility of a crossing over between the two paternal chromosomes in a region 5' to the beta gene, previously indicated to contain a 'hot spot' area for recombination. This report underlines the risk of performing prenatal diagnosis using restriction polymorphisms 5' to the beta gene.

Gamma interferon induces detectable serological and functional expression of DR and DP but not DQ antigens on cultured amniotic fluid cells.

Cultured amniotic fluid cells, which are used for HLA typing studies for the prenatal diagnosis of HLA linked diseases and for prenatal determination of paternity, usually contain mixtures of fibroblastic and epithelioid cells. Cells of both types lack constitutive expression of HLA class II antigens, but these can be induced by pretreatment with gamma interferon. Both serological and functional studies indicate that detectable levels of DR and DP but not DQ antigens can be thereby induced. DR and DP cannot, however, be induced on chorionic villus cells.

抗ＨＬＡモノクローナル抗体による輸血後ＧＶＨＤの解析

Immunohistochemical and HLA typing studies of a premature baby (female, 24 gestation weeks, 651g at birth) with post-transfusion graft-versus-host disease were presented. After clinical manifestation of the disease, HLA typing of the patient blood showed positivity for HLA-A24 which was theoretically absent from HLA typing of her parents. It was suggested that HLA-A24 positive lymphocytes must have been derived from one of the blood donors. In order to confirm these results, immunohistochemical study was performed on biopsied skin by using anti-HLA-A24 monoclonal antibody, HU-49. Lymphocytes infiltrating in the skin, mainly at the epidermo-dermal juncton showed strong positivity for HU-49 reactive antigen. In addition, the majority of those lymphocytes was Leu-2a positive. The collective evidence strongly indicates that the HLA-A24 and Leu-2a positive T cells derived from blood transfusion caused GVHD in this case. Lastly immunohistochemical analysis of the skin biopsy material using monoclonal anti-HLA antibodies appears to be useful for the early diagnosis of post-transfusion GVHD.

Allospecific DR gene expression on the human thyroid cell.

Human thyroid epithelial cells, stimulated with recombinant gamma interferon (IF) (100 U/ml), were HLA-DR typed by means of antibody-dependent, complement-mediated cytotoxicity. The thyroid cell lysis reaction patterns were found to be similar to those obtained with autologous peripheral B cells in four of five separate experiments. Since the cytotoxic reactions with thyroid cells were sometimes incomplete with many antisera, a two-color fluorescence technique was developed to measure the specificity of the antibody-dependent, complement-mediated lysis. Using constitutively HLA-D antigen-positive Raji B lymphoblastoid cells as controls, we showed that non-polymorphic HLA-D and HLA-DR determinants could be detected on the thyroid cell surfaces by this cell lysis approach. In addition, gamma IF stimulated thyroid cells from a DR3 positive individual were lysed to a significantly greater than unstimulated autologous thyroid cells with the use of a specific monoclonal antibody to HLA-DR3, thus confirming the original HLA typing studies. These data demonstrate, for the first time, that the expression of HLA-DR antigen on the surface of human thyroid cells is allospecific and may, therefore, play an important role in the immunopathology of autoimmune thyroid disease.

Molecular genetics and the characterization of steroid 21-hydroxylase deficiency.

Classical 21-hydroxylase deficiency congenital adrenal hyperplasia is a monogenic autosomal recessive disorder that has been conclusively shown by family HLA-typing studies to be in close genetic linkage with the human major histocompatibility complex. More recently recognized is the nonclassical disorder, an attenuated form of 21-hydroxylase deficiency characterized variably by late onset or absence of symptoms. Certain of the mild 21-hydroxylase deficiency allotypes involved in the nonclassical disorder have also been shown to be genetically linked with HLA, exhibiting distinct (B and B,DR) antigen associations. The nonclassical disorder is now also known to result from different genotypes: two mild 21-hydroxylase defects in conjunction, or a mild defect occurring with a sever (classical) defect. Restriction mapping and hybridization analysis have located two highly homologous base sequences, one structural gene coding for 21-hydroxylase and one pseudogene, in the Class III region of the MHC in tandem with the A and B genes for C4, the fourth component of complement. Current work documenting and characterizing gene abnormalities, as well as elucidating the molecular genetic basis of the mutations that have arisen, is aimed at developing better cDNA probes for prenatal diagnosis by amniocentesis and chorionic villus biopsy. In addition, because of the close association of the C4 and 21-hydroxylase genes, coordination of data on C4 variants and null alleles with altered 21-hydroxylase activity is improving understanding of the genetic mechanisms generating disease alleles of this enzyme crucial for normal endocrine function.

The use of phytohemaglutinin (PHA) and T cell SRBC rosette selection for induction of class II antigens on T cells for HLA typing studies involving CML patients : Cynthia Callaway, Denola Brown, Kim Fehir, and Marilyn S. Pollack; The Methodist Hospital, Houston, TX

The use of phytohemaglutinin (PHA) and T cell SRBC rosette selection for induction of class II antigens on T cells for HLA typing studies involving CML patients : Cynthia Callaway, Denola Brown, Kim Fehir, and Marilyn S. Pollack; The Methodist Hospital, Houston, TX

Recurrent spontaneous abortion in human pregnancy: results of immunogenetical, cellular, and humoral studies.

We have confirmed that many women who suffer from recurrent spontaneous abortions have an increased incidence of histocompatibility (HLA-A, HLA-B, HLA-C, and HLA-DR) antigen-sharing with their husbands. We also found abortion-prone women who did not share HLA with their mates; however, they failed to respond or responded suboptimally to their husbands when compared to third-party controls in the mixed lymphocyte culture (MLC) reactions. Some aborting patients manifested a husband-specific MLC blocking factor in their plasma which was not present in their serum. In contrast, nonpregnant childbearing wives did not share as many of their husbands' HLA antigens as frequently, had normal or heightened MLC responses to their husbands, and showed no evidence of humoral blocking factors. We propose that HLA typing, MLC reactions, and blocking factor studies will serve as useful laboratory indices in future clinical studies of immunogenetical disorders associated with idiopathic habitual abortion.