HLA Typing of Patients with 21-Hydroxylase Deficiency in Iranian Children with Congenital Adrenal Hyperplasia

Congenital adrenal hyperplasia (CAH) is a group of potentially life-threatening disorders, most often caused by deficiency of steroid 21-hydroxylase. Children with ambiguous genitalia, hermaphroditism, or signs and symptoms of CAH admitted to Children's Medical Center were enrolled in the survey, and 101 patients were found. Karyotyping, clinical examination, and paraclinical tests were done. HLA typing was done in patients with proven classical CAH and their parents. HLA antigens were typed in children with CAH-type 21-hydroxylase deficiency. The antigen frequencies were compared with those of the control population. The studies revealed that two HLA antigens, HLA-B18 and HLA-B21, showed a significant increase in frequency. The calculated relative risk value was high, distinguishing the population of patients and their parents. The relative risk among patients was 11.82 for HLA-B18 and 1.75 for HLA-B21 antigens. There was no relationship between HLA-DR antigens and CAH. Studies on the correlation between HLA and CAH indicate an association with HLA-B18 and HLA-B21 antigens, and they can be used as genetic markers of the disorder in the Iranian population, if they are restricted to Iranian patients.     

Restriction fragment analysis of duplication of the fourth component of complement (C4A)

The two genes encoding the fourth component of complement (C4A and C4B) reside between HLA-B and HLA-DR on human chromosome 6. Two kilobases downstream from each C4 gene lies a 21-hydroxylase gene (CA21HA and CA21HB, respectively). Utilizing the method of Southern blotting and a 5'-end 2.4-kb BamHI/KpnI fragment of the C4 cDNA, we have analyzed TaqI-digested DNA from four pedigrees with one or more extended haplotypes containing a C4A duplication, as demonstrated by protein electrophoresis and segregation analysis. Two C4A protein duplications (C4A*2,A*3,C4B*QO and C4A*3,A*5,C4B*QO) segregated with two large TaqI DNA restriction fragments (7.0 and 6.0). In pedigree Fi, one individual homozygous for HLA-A3,B35,C4,DR1,DQ1,BFF,C2C,-C4A2,3,C4BQO had TaqI 7.0- and 6.0-kb restriction fragments with equal hybridization intensities as measured by two-dimensional densitometry (7.0/6.0 kb = 0.83, SD = 0.12, N = 7). A hybridization probe for the 21-hydroxylase gene also demonstrated equal gene dosage (CA21HA/CA21HB = 1.01). DNA from another individual (Ma I-2) with a different C4A gene duplication (C4A*3,A*5,C4B*QO) also had equal densitometry measurements (7.0/6.0 kb = 1.07). We conclude that two extended haplotypes from unrelated pedigrees have two C4 genes and both C4 genes encode separate C4A alleles. These findings are compatible with a gene conversion event of C4B to C4A.     

High frequency of nonclassical steroid 21-hydroxylase deficiency.

Nonclassical steroid 21-hydroxylase deficiency is an autosomal recessive disorder that is defined by clinical and hormonal criteria that distinguishes it from the classical 21-hydroxylase deficiency. No estimates of the gene frequency of nonclassical 21-hydroxylase deficiency, also called attenuated, late-onset, acquired, and cryptic adrenal hyperplasia, have been published thus far. Here, we have used HLA-B genotype data in families containing multiple members affected with nonclassical 21-hydroxylase deficiency together with the results of quantitative hormonal tests to arrive at estimates of gene and disease frequencies for this disorder. We found nonclassical 21-hydroxylase deficiency to be a far more common disorder than classical 21-hydroxylase deficiency, which occurs in 1/8,000 births. The prevalence of the disease in Ashkenazi Jews was 3.7%; in Hispanics, 1.9%; in Yugoslavs, 1.6%; in Italians, 0.3%; and in the diverse Caucasian population, 0.1%. The gene for nonclassical 21-hydroxylase deficiency is in genetic linkage disequilibrium with HLA-B14 in Ashkenazi Jews, Hispanics, and Italians, but not in Yugoslavs or in a diverse, non-Jewish, Caucasian group. The penetrance of nonclassical 21-hydroxylase deficiency gene in the HLA-B14 containing haplotypes was incomplete. Thus, nonclassical 21-hydroxylase deficiency is probably the most frequent autosomal recessive genetic disorder in man and is especially frequent in Ashkenazi Jews, Hispanics, Italians, and Yugoslavs.     

Extended MHC haplotypes and CYP21/C4 gene organisation in Irish 21-hydroxylase deficiency families

Summary We have analysed fifteen classical 21-hydroxylase deficiency families from throughout Southern Ireland and report the serologically defined HLA-A, HLA-B, HLA-Cw, HLA-DR, C4A and C4B polymorphisms that characterize the inferred disease haplotypes. Additionally, we have used a combination of short and long range restriction mapping procedures in order to characterize the CYP21/C4 gene organization associated with individual serologically defined haplotypes. The results obtained indicate that disease haplotypes are characterized by a high frequency (33%) of CYP21B gene deletion and 8 out of 10 such deletion haplotypes are represented by the extended haplotype HLA-DR1, C4BQo, C4A3, HLA-B40(w60), HLA-Cw3, HLA-A3. Large scale length polymorphism in the CYP21/C4 gene cluster was found to conform strictly to a variable number of tandem repeats model with 4 alleles being detected. Disease haplotypes in which defective CYP21B gene expression is inferred to result from pathological point mutations show extensive diversity of associated HLA markers and include two examples of the extended HLA haplotype HLA-DR3, B8, Cw7, A1 haplotype, which has previously been reported to be negatively associated with 21-hydroxylase deficiency. One unusual disease haplotype has two CYP21 + C4 units, both of which appear to contain CYP21B-like genes.     

TaqI HLA-B and -DRB RFLP analysis can predict disease in siblings of affected children with 21-hydroxylase deficiency

Summary The possibility of using TaqI restriction fragment length polymorphism (RFLP) analysis of the HLA-B locus and the HLA-DR-DQ subregions, flanking the 21-hydroxylase genes, for predicting disease in siblings of children with 21-hydroxylase deficiency was analyzed in 12 nuclear families with at least one affected child and a total of 18 at-risk off-spring. As part of the study allelic TaqI HLA-B RFLP patterns were determined in homozygous cell lines and families. The frequencies of individuals homozygous for TaqI allelic patterns of the different investigated HLA loci, each locus alone and in various combinations, were determined in 100 random controls. In all 12 families it was possible to make correct genetic diagnosis by the use of only one restriction enzyme, TaqI, and two locus-specific HLA cDNA probes, HLA-B and -DRB. In all families four haplotypes were obtained. Thus, affected siblings as well as carriers could be identified. Seven of the eight sibling pairs concordant for 21-hydroxylase deficiency had pairwise identical TaqI HLA-B-DRB-DQA-DQB haplotypes. The last disease-concordant sibling pair had inherited different haplotypes from their mother, who had nonclassical 21-hydroxylase deficiency. None of the ten healthy children shared both haplotypes with their affected sibling(s). Early prenatal suppression of the fetal adrenal cortex with fluorinated corticosteroids can prevent virilization of female fetuses with 21-hydroxylase deficiency. In most cases RFLP analysis of the 21-hydroxylase genes is not informative enough for prenatal diagnosis. Our results from the present retrospective family study indicate that TaqI HLA-B and -DRB RFP analysis will be a valuable tool for first trimester assessment of 21-hydroxylase deficiency. TagI HLA-B and -DRB RFLP analysis can be performed on DNA from chorionic villi biopsies obtained in the 8th week of pregnancy. Supplemented with sex determination, early withdrawal of prophylactic steroid therapy will thus be feasible when the mother carries a male or an unaffected female fetus.     

Molecular and endocrine characterization of a mutation involving a recombination between the steroid 21-hydroxylase functional gene and pseudogene

The gene encoding steroid 21-hydroxylase activity, P450c21B, is located in the major histocompatibility complex (MHC) class III region, in close proximity to a highly homologous pseudogene, P450c21A. Recombinations between P450c21B and P450c21A have been shown to result in deficiency of 21-hydroxylase activity, the usual cause of congenital adrenal hyperplasia (CAH). A mutant P450c21 gene from a patient with simple virilizing CAH was identified and shown to be consistent with a recombination between P450c21A and P450c21B. Sequence analysis of the mutant gene showed the recombination site to be located between the first exon and the second intron. The mutant gene encodes a leucine instead of the normal proline at codon 31. This mutation resides on a chromosome bearing the HLA-B44 serotype. A comparison of mutation associated with HLA-B44 and that normally found with the HLA-Bw47 serotype suggests that the HLA-B44 mutations are of more ancient origin. The patient's homologous chromosome has a deletion of P450c21B. Endocrinological testing therefore allows for testing of the mutant gene in genetic isolation. Such testing demonstrated that the patient was capable of producing aldosterone and retaining sodium in response to a low-sodium diet, indicating that the mutant gene encodes an enzyme with partial 21-hydroxylase activity.     

Basic and clinical aspects of congenital adrenal hyperplasia

Defective steroid 21-hydroxylation is the most common of the biochemical defects causing hyperplasia of the adrenal cortex. The genetic mode of transmission of all enzyme abnormalities seen in cortisol biosynthesis is autosomal recessive. Steroid 21-hydroxylase deficiency has three currently accepted forms: the simple virilizing and salt-wasting variants of the classical deficiency, and the nonclassical (attenuated) form, which shows a wide clinical range of effects and whose characterization emerged from co-ordinated hormonal testing and family studies. More recent molecular genetic studies have started to identify specific mutations altering 21-hydroxylase activity. Defects in the other enzymes occur more rarely and are less well known, although initial work with abnormal 11 beta-hydroxylase and 3 beta-hydroxylase indicates that allelic gene defects may be correlated with different clinical phenotypes seen for these disorders also. The gene for the enzyme steroid 21-hydroxylase, a cytochrome P-450, is situated within the major histocompatibility complex on the p arm of human chromosome 6, proximal to the HLA-B antigen locus. Linkage disequilibria between certain B and DR alleles and classical and nonclassical 21-hydroxylase deficiency permit the use of HLA genotyping in conjunction with hormonal evaluation for diagnosis of this disorder and for identification of carrier haplotypes in population studies. Test programs have shown the feasibility of neonatal screening for 21-hydroxylase deficiency by blood-spot hormonal assay for elevated 17-hydroxyprogesterone. Prenatal detection of disease currently depends on HLA serotyping of cultured aminocytes jointly with measurement of amniotic 17-hydroxyprogesterone (13-18 week gestation); molecular genetic techniques with more specific nuclear probes will improve the specificity of this test and will in addition permit even earlier definitive fetal genotyping by chorionic villus biopsy (6-10 week gestation).     

Prevalence of polymorphic 21-hydroxylase gene (CA21HB) mutations in salt-losing congenital adrenal hyperplasia

Using genomic restriction analysis of 14 unrelated patients with salt-losing congenital adrenal hyperplasia, we identified three different CA21HB mutation patterns: no detectable restriction fragment abnormalities (16/28 haplotypes), deletion of the active CA21HB gene (9/28), and apparent conversion of the active CA21HB gene to the pseudogene CA21HA (3/28). CA21HB gene deletion was associated with HLA-Bw47 in 6 haplotypes and with absent C4B expression in 7. A variety of HLA and C4 types was associated with the other mutations. Apparent conversion of CA21HB to CA21HA was identified by the disparity between the intensity ratios for the major TaqI and BglII hybridization fragments.     

C4A gene deletion and HLA associations in black Americans with systemic lupus erythematosus

In North America and European Caucasoids with systemic lupus erythematosus (SLE) there is an increased frequency of aC4A, CYP21A gene deletion, largely associated with theHLA-B8,DR3,C4A ^* QO extended haplotype. There have been no consistent HLA associations reported for SLE in blacks, although an increased frequency of serologically determinedC4A null alleles has been reported in two studies. We studied 79 black American SLE patients and 68 black controls by restriction fragment length polymorphism analysis to dermine if aC4A gene deletion was a genetic risk factor for SLE. Moreover, the nature of the deletion and any HLA phenotypic associations were sought. Nineteen of 79 (24%) patients compared to 5 of 68 (7.4%) controls had a phenotypicC4A,CYP21A gene deletion (P=.005; RR=4). A homozygous deletion in four patients gave a genotypic frequency of 23/158 (14.5%) SLE patients vs 5/136 (3.7%) controls (P=.001; RR=4.5). The deletion was associated with HLA-DR2 (P=.03) and HLA-DR3 (P=.03). Moreover, all subjects with the deletion had HLA-DR2 or DR3 (P=7.7×10⁻⁶). HLA-B44 was also associated with the deletion (P=.02), and eight of the nine HLA-B44 positives also carried HLA-DR2. HLA-B8 approached significance (P=.08) and was always accompanied by HLA-DR3. Finally, this black population demonstrated a uniqueC4B gene size polymorphism with 80% C4B “short” as compared to the 40% C4B “short” frequency reported in whites. We conclude that a largeC4A,CYP21A gene deletion, particularly associated with theHLA-B44,-DR2, and-DR3 alleles, is the strongest genetic risk factor thus far identified for SLE susceptibility in black Americans. Furthermore, the unique preponderance of theC4B “short” gene form may be a factor in the actual formation of the deletion.     

HLA linkage and B14, DR1, BfS haplotype association with the genes for late onset and cryptic 21-hydroxylase deficiency.

Classical congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21-OH-def) has been established to be an HLA-linked, recessive monogenetic disease. However, two nonclassical forms of 21-OH-def have also been described: "cryptic" 21-OH-def, which has been shown to be HLA-linked, and "late onset" 21-OH-def, for which the status of linkage to HLA has been less certain. We now describe studies of eight additional unrelated probands with symptomatic, "late onset" 21-OH-def, and conclude that this form is also HLA-linked. Both "late onset" and "cryptic" 21-OH-def are highly associated with the same HLA antigens and markers (HLA-B14, HLA-DR1, and Bf type S) in individuals from different ethnic and geographical backgrounds. Since both "late onset" and "cryptic" 21-OH-def appear to occur in individuals with one classical 21-OH-def (21-OHCAH) allele who in addition have another 21-OH-def allele, as well as in individuals who appear to be homozygous for variant 21-PH-def alleles, and since both late onset and cryptic 21-OH-def appear to occur in the same families, our data suggest that these syndromes may represent different clinical expressions of similar or identical nonclassical 21-OH-def alleles.     

A segregation and linkage study of classical and nonclassical 21-hydroxylase deficiency.

The segregation of classical and nonclassical 21-hydroxylase deficiency (21-OHD) and its linkage to HLA-B was investigated in 220 families. First, the surprisingly high frequency of the nonclassical 21-OHD gene estimated elsewhere was confirmed using a different methodology which avoided particular assumptions concerning the classification of an individual''s genotype. In the present study the gene frequency was found to be .103 +/- .020 in an ethnically pooled sample and was as high as .223 +/- .062 among Ashkenazi Jews. Second, the segregation analysis of families ascertained through a nonclassical 21-OHD proband and those ascertained through a classical 21-OHD proband showed essentially identical results. A partial recessive model with no recombination between 21-OHD and HLA-B fitted the data better than did a complete recessive model with approximately 0.5% recombination between 21-OHD and HLA-B. The support for the partial over the complete recessive model depended on the assumed ascertainment probability, an unknown parameter in these data. Four families provided most of the evidence against the complete recessive model. All these included an unaffected sib who shared both HLA-B specificities in common with the affected proband. Possible explanations for the condition in these families include recombination, gene conversion, mutation in one of the parental gametes, or technical errors.     

MHC susceptibility genes to IgA deficiency are located in different regions on different HLA haplotypes

Familial predisposition to IgA deficiency (IgAD) suggests that genetic factors influence susceptibility. Most studies support a polygenic inheritance with a susceptibility locus (designated IGAD1) in the MHC, but its exact location is still controversial. This study aimed to map the predisposing IGAD1 locus (or loci) within the MHC by investigating the pattern of association of the disease with several markers in the region. DNA-based techniques were used to type individual alleles of four polymorphic HLA genes (HLA-DR, -DQA1, -DQB1, and HLA-B), six microsatellites (all located between HLA-DR and HLA-B), and three single nucleotide polymorphisms on the TNF gene. The frequencies of these alleles were compared among ethnically matched populations comprising 182 patients and 343 controls. Additionally, we investigated parents and siblings of 100 of these patients. All four parental haplotypes were established in each family (n = 400), and transmission disequilibrium tests were performed. Surprisingly, our results did not support the hypothesis of a unique susceptibility gene being shared by all MHC susceptibility haplotypes. On HLA-DR1 and -DR7-positive haplotypes IGAD1 mapped to the class II region, whereas on haplotypes carrying HLA-DR3 the susceptibility locus mapped to the telomeric end of the class III region, as reported previously. Our results show how, in complex diseases, individuals may be affected for different genetic reasons and a single linkage signal to a region of a chromosome may actually be the result of disease-predisposing alleles in different linked genes in different pedigrees.     

CYP21/C4 gene organisation in Italian 21-hydroxylase deficiency families

Summary A total of 33 Italian 21-hydroxylase (21-OH) deficiency families were investigated using a combination of short and long range restriction mapping of the CYP21/C4 gene cluster. The analyses revealed that large-scale length polymorphism in this gene cluster strictly conformed to a compound variable number of tandem repeats (VNTR) plus insertion system with between one and four CYP21 + C4 units and seven BssHII restriction fragment length polymorphisms (RFLPs) (75kb, 80kb, 105kb, 110kb, 135kb, 140kb and 180kb). A total of 9/66 disease haplotypes, but only 1/61 nondisease haplotypes, showed evidence of gene addition by exhibiting three or more CYP21 + C4 repeat units. Of these, two were identified in one 21-OH deficiency patient who has a total of eight CYP21 + C4 units, being homozygous for the HLA haplotype DR2 DQ2 B5 A28. This haplotype carries four CYP21 + C4 units, three of which contain CYP21A-like genes and one of which contains a CYP21B-like gene that presumably carries a pathological point mutation. Of the other gene addition haplotypes associated with 21-OH deficiency, four show three CYP21 + C4 units flanked by HLA-DR1 and HLA-B14 markers. Although such haplotypes have commonly been associated with non-classical 21-OH deficiency, three examples in the present study are unexpectedly found in two salt-wasting patients, who are respectively homozygous or heterozygous for this haplotype. Only 7/66 disease haplotypes showed evidence of a CYP21B gene deletion.     

Studies on resources of genetic diversity in conservative flocks of geese using microsatellite DNA polymorphic markers

The studies conducted aimed at evaluating the genetic diversity within and between varieties of conservative flocks of geese, using the polymorphism of 14 microsatellite sequences. The experimental material included conservative flocks of geese the following indigenous breeds and varieties kept in Poland: Kielecka (Ki), Kartuska (Ka), Lubelska (Lu), Suvalska (Su), Rypinska (Ry), Sub-Carpathian (SC), Hunched Beak (HB) and Pomeranian (Po). Among the 14 microsatellite sequences a total of 97 microsatellite alleles were identified. The number of alleles at one locus ranged from 3 to 19. In the overall pool of 97 alleles, 26 (26.8%) were specific for individual breeds and varieties of geese. The values of the expected heterozygosity (He) for individual geese ranged from 0.38 (Sub-Carpathian) to 0.51 (HB). Similarly, the mean values for the observed heterozygosity (Ho) ranged from 0.45 (Po) to 0.55 (Ki and Su). The polymorphic information content reached the highest value of 0.80 at loci CKW21 (Ki) and TTUCG5 (Po and Su). The greatest genetic distance was observed between the HB and Ry (0.44) and between the HB and Po (0.39) varieties, while the smallest–between the Lu and Po as well as Lu and Ki (0.028) varieties. The phylogenetic tree, elaborated on the basis of the genetic distances, clearly confirms the specificity of the HB goose as compared to the remaining breeds and varieties.     

Spline methods for the comparison of physical and genetic maps.

The first genetic maps were constructed by linkage analysis. Physical mapping techniques, such as radiation hybrids and complete sequencing, produce a different picture. For the purposes of population genetics, clinical genetics, and genetic epidemiology, it is important to harmonize and amalgamate existing genetic and physical maps. Among other things, comparisons of the two kinds of maps promotes better understanding of the wide variation in local recombination rates per unit physical length of DNA. The current paper presents methods for estimating recombination intensity as a function of physical distance along a chromosome. Genetic map distance is the integral of intensity. We derive fast reliable estimation algorithms based on a Poisson process model, penalized likelihoods, and cubic spline interpolation. Our methods provide a rigorous and statistically sound foundation for comparing physical and genetic maps. To illustrate the possibilities, we apply the methods to published recombination data on CEPH families and the complete sequences of chromosomes 21 and 22. Our results are in good agreement with previous studies and the biological data.     

Gene conversion in steroid 21-hydroxylase genes.

The steroid 21-hydroxylase gene, CYP21B, encodes cytochrome P450c21, which mediates 21-hydroxylation. The gene is located about 30 kb downstream from pseudogene CYP21A. The CYP21A gene is homologous to the CYP21B gene but contains some mutations, including a C----T change which leads a termination codon, TAG, in the eighth exon. We found the same change in a mutant CYP21B gene isolated from a patient with 21-hydroxylase deficiency. Furthermore, a reciprocal change--i.e., a T----C change in the eighth exon of the CYP21A gene--was observed in the Japanese population and was associated with the two HLA haplotypes, HLA-B44-DRw13 and HLA-Bw46-DRw8. These changes may be considered the result of gene conversion-like events.     

Molecular genetics of 21-hydroxylase deficiency in congenital adrenal hyperplasia

21-hydroxylase gene analysis was performed on the genomic DNA from patients with congenital adrenal hyperplasia (CAH), their siblings, their parents as well as from a healthy individual serving as control. After digestion by the Taq I and Bgl II restriction enzymes, DNA was hybridized with specific nucleotidic probes: pC21a for the 21-hydroxylase genes, pAT-A for the C4 component Complement genes, closely linked to the 21-hydroxylase genes on the 6 chromosome. Likewise the pFB3B probe was used for the B factor gene located 80 kilobases upstream the 21-hydroxylase gene. From this molecular analysis on 11 families, we report here 4 investigations showing the most frequent genetic abnormalities we have encountered: gene deletions, gene conversions and point mutations. These data show that the molecular approach is a powerful tool for studying this endocrine disease at the clinical, genetic and fundamental point of view.     

Studies of HLA, factor B (Bf), complement C2 and C4 haplotypes in type 1 diabetic and control families from northern Sweden

The HLA-A,-B,-C,-DR antigens and the complement factors C2, C4 and Bf were determined in 30 insulin-dependent diabetes mellitus (IDDM) patients and 30 healthy controls from northern Sweden. Family studies allowed the deduction of extended haplotypes in the HLA and complement systems. Phenotype studies revealed significant associations between IDDM and HLA-DR4 (p less than 0.001), HLA-DR3 (p less than 0.05), HLA-DR3/4 (p less than 0.025), C4-B3 (p less than 0.001) and Bf-S (p less than 0.025). Haplotype studies showed that the extended haplotype [HLA-B15, C2-1, C4-A3B3, Bf-S, HLA-DR4] had a particularly strong association to IDDM. This haplotype was found in 10 out of 30 IDDM probands but in none of 30 control children and accounts for practically all the C4-B3 allotypes among the 30 IDDM probands. The C4-B3 gene therefore seems to be a valuable marker for IDDM. No haplotype containing HLA-DR3 was increased in frequency among the IDDM probands. The extended haplotype [HLA-B7, C2-1, C4-A3B1, Bf-S, HLA-DR2] present among the controls was absent in the IDDM probands. The frequency of the extended haplotype [HLA-B15, C2-1, C4-A3B3, Bf-S, HLA-DR4] was increased also among the parents to the IDDM probands compared to those of the control parents, whereas the frequency of [HLA-B7, C2-1, C4-A3B1, Bf-S, HLA-DR2] was decreased. The extended haplotype [HLA-B8, C2-1, C4-B1, Bf-S, HLA-DR3] was more common among the males (p less than 0.05) compared to the females in the total material. The family analysis showed that 3 out of 5 affected sibs shared both haplotypes with their IDDM proband. This was the case for only 3 out of 35 unaffected sibs.     

Decreased fecundability in Hutterite couples sharing HLA-DR.

To study the effects of parental HLA sharing on pregnancy outcome, we initiated population-based studies in the Hutterites. We previously reported longer intervals from marriage to each birth among couples sharing HLA, particularly HLA-DR. In the present report, we present the results of a prospective, 5-year study of fecundability and fetal loss rates in this population. Between April 1986 and April 1991, 154 pregnancies were observed in 104 couples. The median number of months of unprotected intercourse to a positive pregnancy test was significantly longer among couples sharing HLA-DR who stopped nursing prior to the first menses as compared with couples not sharing HLA-DR who stopped nursing prior to the first menses (5.1 vs. 2.0 mo, respectively; P = .016). Fetal loss rates were increased among couples sharing HLA-B as compared with couples not sharing HLA-B (.23 vs. .12, respectively; P = .041, adjusted for age, gravidity, and kinship). These data suggest that our earlier observations of increased birth interval lengths among Hutterite couples sharing HLA were predominantly due to longer intervals until a clinical pregnancy among couples sharing HLA-DR and, to a lesser degree, were due to increased fetal loss rates among couples sharing HLA-B.     

Assignment of the gene coding for both the beta-subunit of prolyl 4-hydroxylase and the enzyme disulfide isomerase to human chromosome region 17p11----qter

The chromosomal location of the human gene coding for both the beta-subunit of prolyl 4-hydroxylase (P4HB) and the enzyme disulfide isomerase (PDI) was determined using mouse x human somatic cell hybrids and three different methods for identifying either the human P4HB/PDI protein or the respective gene: (1) immunoblotting with species-specific monoclonal antibodies; (2) radioimmunoassay with species-specific polyclonal antibodies; and (3) Southern blotting after cleavage of the DNA with EcoRI, HindIII, or BamHI, followed by hybridization with a mixture of two cDNA probes for human P4HB. All three methods gave identical data, demonstrating complete cosegregation of the human protein or its gene in all 17 cell hybrids tested with human chromosome 17. A cell hybrid lacking an intact chromosome 17 localized the gene to 17p11----qter.