Development of a low-serum medium for the production of monoclonal antibody against congenital adrenal hyperplasia by hybridoma culture.

Statistically designed experiments were used in developing a low-serum medium for the production of a diagnostic monoclonal antibody against congenital adrenal hyperplasia using hybridoma 192. A two-level half-fractional factorial design was used for screening six components (Minimum Essential Medium Eagle amino acids, 2-mercaptoethanol, ethanolamine, ferric citrate, zinc sulfate, and sodium selenite). The experimental design was then augmented to central composite design. The basal Dulbecco's modified Eagle's medium (DMEM; containing 4 mM L-glutamine, 1% antibiotic-antimycotic agent) supplemented with 0.4% by volume fetal bovine serum (FBS), 311.8 mM ferric citrate, 17.3 nM sodium selenite, and 4.5 mM zinc sulfate (LSD) was found to support the growth of the hybridoma. Specific cell growth rate in the LSD (0.033 ± 0.001/h) was slightly lower than in the control medium (i.e., basal DMEM supplemented with 2% FBS; 0.0045 ± 0.003/h). Nevertheless, the specific MAb production rate for LSD was higher (0.057 ± 0.015 pg/cell · h versus 0.004 ± 0.002 pg/cell · h in LSD and control, respectively). The antibody produced in the LSD showed high specificity and no cross-reactivity with the other structural resemblance's steroid hormones, revealing no structural changes owing to the new medium formulation developed. The new medium formulation effectively reduced the medium cost by up to 64.6%.

Complement component 4 copy number variation and CYP21A2 genotype associations in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) is an autosomal recessive disorder of cortisol biosynthesis caused by CYP21A2 mutations. An increase in gene copy number variation (CNV) exists at the CYP21A2 locus. CNV of C4, a neighboring gene that encodes complement component 4, is associated with autoimmune disease susceptibility. In this study, we performed comprehensive genetic analysis of the RP-C4-CYP21-TNX (RCCX) region in 127 unrelated 21-OHD patients (100 classic, 27 nonclassic). C4 copy number was determined by Southern blot. C4 CNV and serum C4 levels were evaluated in relation to CYP21A2 mutations and relevant phenotypes. We found that the most common CYP21A2 mutation associated with the nonclassic form of CAH, V281L, was associated with high C4 copy number (p = 7.13 × 10(-16)). Large CYP21A2 deletion, a common mutation associated with the classic form of CAH, was associated with low C4 copy number (p = 1.61 × 10(-14)). Monomodular RCCX with a short C4 gene, a risk factor for autoimmune disease, was significantly less frequent in CAH patients compared to population estimates (2.8 vs. 10.6 %; p = 1.08 × 10(-4)). In conclusion, CAH patients have increased C4 CNV, with mutation-specific associations that may be protective for autoimmune disease. The study of CYP21A2 in relation to neighboring genes provides insight into the genetics of CNV hotspots, an important determinant of human health.

Complement 4 phenotypes and genotypes in Brazilian patients with classical 21-hydroxylase deficiency.

The aim of this work was to analyse C4 genotypes, C4 protein levels, phenotypes and genotypes in patients with the classical form of 21-hydroxylase deficiency. Fifty-four patients from 46 families (36 female, 18 male; mean age 10.8 years) with different clinical manifestations (31 salt-wasting; 23 simple-virilizing) were studied. Taq I Southern blotting was used to perform molecular analysis of the C4/CYP21 gene cluster and the genotypes were defined according to gene organization within RCCX modules. Serum C4 isotypes were assayed by enzyme-linked immunosorbent assay. The results revealed 12 different haplotypes of the C4/CYP21 gene cluster. Total functional activity of the classical pathway (CH50) was reduced in individuals carrying different genotypes because of low C4 concentrations (43% of all patients) to complete or partial C4 allotype deficiency. Thirteen of 54 patients presented recurrent infections affecting the respiratory and/or the urinary tracts, none of them with severe infections. Low C4A or C4B correlated well with RCCX mono-modular gene organization, but no association between C4 haplotypes and recurrent infections or autoimmunity was observed. Considering this redundant gene cluster, C4 seems to be a well-protected gene segment along the evolutionary process.

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.

Proteomic analysis of peripheral T lymphocytes, suitable circulating biosensors of strictly related diseases.

T lymphocytes and/or their subpopulations from peripheral blood may represent molecular sensors to be used for the evaluation of gene expression modification in physiological and pathological conditions, providing a unique and easily available biological model for integrated studies of gene expression in humans. In this study, a proteomic approach was applied to evaluate the association between changes in T cell protein expression patterns and specific diseased conditions. In particular, two hyperandrogenic syndromes were studied, sharing many clinical and biochemical signs: polycystic ovary syndrome (PCOS) and congenital adrenal hyperplasia (CAH). Comparison of proteomic maps of T lymphocytes derived from patients affected by PCOS or CAH with those derived from healthy subjects showed that 14 proteins are expressed differentially in both PCOS and CAH, 15 exclusively in PCOS and 35 exclusively in CAH. Seventeen of these proteins have been identified by mass spectrometry analysis. Furthermore, proteomic data mining by hierarchical clustering was performed, highlighting T lymphocytes competence as a living biosensor system.

Association of HLA alleles and haplotypes with CYP21A2 gene p. V282L mutation in the Croatian population.

The CYP21A2 mutations that are in linkage disequilibrium with particular HLA-A, -B, -DRB1 alleles/haplotypes, cause deficiency of the 21-hydroxylase enzyme (21-OHD) and account for the majority of congenital adrenal hyperplasia (CAH) cases. The aim of this study was to investigate those associations with the p.V282L mutation linked to the non-classical (NC) form of CAH among Croatians. The study included parents of patients with the NC form of CAH, positive for the p.V282L mutation (N = 55) and cadaveric donor samples (N = 231). All subjects were HLA-A, -B, and -DRB1 typed and tested for the presence of the p.V282L mutation. Among parents of patients, 92.73% of subjects were positive for the B*14:02 allele and almost half of them carried the HLA-A*33:01-B*14:02-DRB1*01:02 haplotype. Among cadaveric samples 77 out of 96 subjects positive for the B*14:02 allele had the p.V282L mutation. Among them, 37 were positive for the HLA-A*33:01-B*14:02-DRB1*01:02 haplotype, 23 had the HLA-A*33:01-B*14:02-DRB1*03:01 haplotype, 8 had the B*14:02-DRB1*01:02 combination and 5 were carrying the HLA-A*68:02-B*14:02-DRB1*13:03 haplotype. Only 4 of these subjects were positive for the B*14:02 allele. HLA-B*14:02 was the only single allele with association that reached statistically significant P value (RR = 12.00; P = 0.0024). Haplotypes B*14:02-DRB1*01:02 (P < 0.001) and HLA-A*68:02-B*14:02-DRB1*13:03 (P < 0.001) as well as HLA-A*33:01-B*14:02-DRB1*01:02 and HLA-A*33:01-B*14:02-DRB1*03:01 showed high relative risks (RR = 45.00, RR = 41.63 and RR = 36.96, respectively). Our data support the previously documented association of the HLA-A*33:01-B*14:02-DRB1*01:02 haplotype with the p.V282L mutation, but also point out a high frequency of the p.V282L mutation among Croatians with HLA-A*33:01-B*14:02-DRB1*03:01 and HLA-A*68:02-B*14:02-DRB1*13:03 haplotypes.

Molecular heterogeneity of the fourth component of complement (C4) and its genes in vitiligo.

In view of evidence suggesting vitiligo is an autoimmune disease, we investigated whether vitiligo is associated with inherited deficiencies of the fourth (C4) and second (C2) component of complement and with certain human leukocyte antigens (HLA). Analysis of functional activities of C4 and C2 in sera of patients with vitiligo (n = 42) showed that 17% of them had a heterozygous C4 deficiency and 5% had a heterozygous C2 deficiency. In the normal control group (n = 30), 3% had a heterozygous C4 deficiency and none had a C2 deficiency. C4 typing by Western blot analysis showed the frequency of the C4A*Q0 allele in the vitiligo patient group to be close to normal. However, the frequency of one C4B*Q0 allele was three times higher, and that of two C4B*Q0 alleles five times higher in the vitiligo patient group than the reported frequencies in normal control groups. Southern blot analysis of Taq1 digests of DNA using C4 and 21-hydroxylase probes showed that two patients with two C4B*Q0 alleles had a deletion of a 21-OHA-C4B segment. In the other patients, having one or two C4B*Q0 alleles, these null alleles probably occurred due to a loss of C4 gene expression. HLA analysis did not show any allelic association of C4A*Q0 or C4B*Q0 with any HLA antigen in vitiligo, but confirmed the previous findings of a negative association with HLA-DR3 and a positive association with HLA-DR4. These results suggest that abnormalities of the C4B gene and the above-mentioned associations with HLA antigens may be some of the risk factors in vitiligo.

Hormonal studies in obligate heterozygotes and siblings of patients with 11 beta-hydroxylase deficiency congenital adrenal hyperplasia.

Hormonal response to ACTH stimulation and HLA genotyping were determined in families of patients with 11 beta-hydroxylase deficiency congenital adrenal hyperplasia. Neither hormonal measurements nor HLA genotyping were useful for the detection of heterozygosity in the families.

Genetic linkage and HLA association in congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

Twenty-eight families of patients with congenital adrenal hyperplasia due to 21-hydroxylase (21-OH) deficiency were studied to evaluate the specific HLA linkage relationship and HLA antigen association to the 21-OH deficiency gene. Genotype assignment, based on hormonal studies (ACTH stimulation) and HLA genotyping, correlated very well (p less than 0.01) in 23 unaffected sibs of children with 21-OH deficiency further supporting the genetic linkage of the 21-OH deficiency gene to the HLA complex. One family was informative for the placement of the 21-OH deficiency gene outside the HLA complex on the HLA-DR locus side. In this family HLA-A, B, C, DR, MT, MB, and glyoxylase typing and mixed lymphocyte culture was performed. An association of 21-OH deficiency and the HLA-A3 antigen was noted in the 28 families. This association is not secondary to the association of the 21-OH deficiency gene with HLA-BW47.

Diagnosis of classical steroid 21-hydroxylase deficiency using an HLA-B locus-specific DNA-probe.

The HLA-B and steroid 21-hydroxylase loci are known to be closely linked. Restriction fragment length polymorphisms seen after digestion of genomic DNA with MspI and TaqI with the HLA-B locus-specific DNA-probe, pHLA-1.1, were examined in 7 nuclear families with classical steroid 21-hydroxylase deficiency. In each family 2 polymorphic hybridizing bands (corresponding to the 2 HLA-B genes) were seen. In all families, TaqI-generated polymorphisms allowed for identification of children previously shown on clinical and biochemical criteria to be affected by 21-hydroxylase deficiency from their unaffected sibs. The results were in complete agreement with the clinical diagnoses. Among the unaffected children, carriers could be distinguished from non-carriers in all cases by TaqI polymorphisms. MspI-generated polymorphisms allowed for full identification of genotypes in 5 families. In one family, MspI-generated polymorphisms could be used to identify affected from unaffected children, but could not distinguish between carriers and non-carriers. In another family, no identification of genotypes was possible by MspI-generated polymorphisms alone. The HLA-B locus-specific DNA-probe, pHLA-1.1, can be used for diagnosis and genotyping of individuals from families with 21-hydroxylase deficiency. This technique can be used as an alternative to HLA-serotyping, or in situations where HLA-serotyping is technically difficult, for example in chorionic villus samples.

Vaccine tolerance in steroid substituted patients with congenital adrenal hyperplasia.

The tolerance and side effects of vaccinations were determined in patients with congenital adrenal hyperplasia (CAH) who receive physiological corticosteroid substitution. In a retrospective approach, questionnaires about the frequencies of vaccinations and observed side effects were sent to CAH patients, and medical records were reviewed. We received 82 questionnaires from 63 patients with CAH and salt-losing and 19 patients without salt-losing. Patients age ranged from 2-40 years. No statistical differences were found for vaccination frequencies between patients with or without salt-losing. CAH patients had received complete vaccinations against diphtheria, tetanus and poliomyelitis in 79%, 85% and 78%, respectively, whereas pertussis vaccination was complete in only 23%. Live vaccination against measles, mumps and rubella was performed in 63%, 50% and 38%. Side effects of vaccination were indicated in 5 out of 82 questionnaires who all belonged to CAH patients with salt-losing. Transient side effects were an anaphylactic reaction, probably to tetanus immunoglobulin, in 1 case, and fever and convulsions after diphtheria, pertussis and tetanus (DPT) vaccine in 2 cases. In 2 further patients putative complications were noted. Encephalitis with permanent disabilities was observed after the third DPT vaccination, but a causative relation could not be established. In another boy, encephalopathy noticed after measles vaccination was induced by previous toxicosis. Although encephalopathy was described in 2 patients after vaccinations, no vaccination damage could be proven in our retrospective study. As expected, an increased vaccination risk in CAH patients was not demonstrated.

Polymorphism of the complement components in human pathology.

The complement system is an important part of non clonal or innate immunity that collaborates with acquired immunity to kill pathogens and to facilitate the clearance of immune complexes. The complement is made up of 20 distinct plasma proteins and 9 different membrane proteins. Three components, factor B, C2 and C4 (with 2 isotypes), are coded by polymorphic HLA-linked genes and are sometimes referred to as class III antigens, inherited as compact units called complotypes. The C4 genes are the most polymorphic, including a common null allele (Q0) at both the C4A and C4B loci. Other polymorphic complement factors (not linked to HLA) are C3 (2 common alleles), C6 and C7 (closely linked, with 3 and 2 alleles, respectively). A certain degree of polymorphism has also been described for complement receptors and membrane control proteins. No differences in functional activity are usually detected among different alleles. Immune-mediated diseases are associated with C4Q0, in particular: systemic lupus erythematosus and discoid-systemic lupus erythematosus, insulin-dependent diabetes mellitus, liver cirrhosis, celiac disease and IgA/IgG4 deficiency. Even if optimal HLA markers do become available, genetic counselling is usually not the ultimate goal for dealing with most of the HLA-associated common diseases, although their study could help to better delineate disease pathogenesis.

[Genetic of the 21 hydroxylase deficiency]. / Génétique du déficit en 21-hydroxylase.

A dose genetic linkage exist between the HLA complex (especially HLA-B), and the 21 hydroxylase deficiency form of adrenal hyperplasia. By their polymorphisms HLA antigens can be used as "markers" to follow the segregation of 21-OH deficiency in families, to diagnose the heterozygous offspring and eventually to offer a prenatal diagnosis to couples at risk. In late onset forms of 21-OH deficiency the same genetic linkage has been demonstrated with a high frequency of HLA-B14 antigen.

[Analysis of the association of HLA-DQ1 alleles with mutation of the 21-hydroxylase gene in patients with congenital adrenal hyperplasia]. / Analiz assotsiatsii allelei HLA-DQA1 s mutatsiiami gena 21-gidroksilazy u bol'nykh s vrozhdenno i giperplaziei kory nadpochechnikov.

In 96 patients with congenital adrenal hyperplasia (CAH) and 50 healthy donors from northwestern Russia the distribution of the HLA-DQA1 alleles and the mutation spectrum and frequency at the CYP21B gene were examined. In the patients with nonclassical (NC) CAH, the distribution of the HLA-DQA1 polymorphic alleles was similar to that in the population sample. In the patients with the salt-wasting form of the disease a statistically significant decrease of the *0401 or *501 major allele frequency was observed. The prevalence of certain HLA-DQA1 genotypes, namely, HLA5, HLA3, and HLA4, was observed in the patients with the NC, salt-wasting (SW), and simple virilizing CAH, respectively. Each clinical group was characterized by a specific spectrum of clinically valuable mutations. An association between the CYP21B mutations most frequently found in case of SW and SV CAH (delB, I2splice, and I172N) and certain HLA-DQA1 alleles was demonstrated. The necessity of more precise clinical diagnostics of the NC CAH cases along with detailed examination of this group for determination of the major mutations typical of the NC CAH cases from northwestern Russia is discussed.

[Classical form of congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Genetic considerations in light of personal studies]. / Klasyczna postac wrodzonego przerostu kory nadnerczy w niedoborze 21-hydroksylazy. Rozwazania genetyczne w swietle badan wlasnych.

Long-term immunogenetic studies on HLA and GLOI systems in the families of probands with classic 21-hydroxylase related adrenal hyperplasia served as a basis for a complex analysis. The analyzed factors included the phenotype and haplotype incidence, HLA-ABC homozygosity, and -in informational families-the strength of genetic linkage for recombinant fractions (lod score). The results suggest the usefulness of routine determinations of class I HLA antigens in families of patients with 21-hydroxylase deficiency.

[Class I HLA antigens in children from families with congenital adrenal hyperplasia]. / Antygeny HLA klasy I w rodzinach dzieci z wrodzonym przerostem nadnerczy.

Congenital adrenal hyperplasia (CAH) is a syndrome of adrenal steroid metabolism errors with an autosomal inheritance model. The most common metabolic defect is 21-hydroxylase deficiency. It has been demonstrated that 21-hydroxylase genes are in close association with HLA antigens. I HLA antigens were typed in a group of 32 families of children with CAH-type 21-hydroxylase deficiency with salt loss. The antigen frequencies were determined and compared to those of the control population. The studies revealed that two HLA antigens determined by the B Locus, i.e. HLA-B47 and HLA-B61, showed a highly significant frequency (chi 2 corresponding to 404,5259 and 23,7808, respectively). The calculated relative risk and etiologic fraction values were extremely high, distinguishing the population of patients and their parents. The RR value among patients was 427.1 for HLA-B47 and 7.8 for HLA-B61 antigen. Studies on the correlation between HLA and CAH indicate an association with HLA-B47 and HLA-B61 antigens.

[HLA haplotypes in families of children with congenital adrenal hyperplasia]. / Haplotypy HLA w rodzinach dzieci z wrodzonym przerostem nadnerczy.

The haplotypes of class I HLA system were determined in a group of 32 children with congenital adrenal hyperplasia and classic form of 21-hydroxylase deficiency. The haplotype frequencies were analyzed and compared to those of the control population. Haplotypes containing the HLA-B-47 antigen were significantly more frequent among the families of patients than among the control population. Among the patients, haplotypes HLA A3-B47-Cw6 and HLA A3-B47-Cwx constituted 50% of all the observed haplotypes with the HLA-B47 antigen. It is stressed that the frequency of homozygotic systems for antigens determined by locus HLA-A (33.34%) and for antigens with locus HLA-B (22.23) were much higher in the affected children.