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French families (n = 129) with at least one cystic fibrosis (CF) affected child and 44 unrelated subjects from the general population were tested for the presence of the delta F508 mutation by the polymerase chain reaction. The delta F508/CF mutation ratio (CF: uncharacterised CF mutations) was tested in the CF families with and without meconium ileus. The association between delta F508 and CF mutations and restriction fragment length polymorphism haplotypes (XV2c and KM19) has been estimated; these data suggest that the CF chromosomes include a panel of independent and probably different mutations.

The prenatal diagnosis of cystic fibrosis is now routinely performed by using two probes tightly linked to the CF locus (XV2C and KM19). These probes have been shown to exhibit a strong linkage disequilibrium with the CF locus. Our data (103 families) have been pooled with other French data (237 families). They are consistent with the hypothesis of a unique ancestral mutation initially associated with a B (D1E2) restriction fragment length polymorphism (RFLP) haplotype, subsequently reassociated by cross-over with A, C or D haplotypes. Assuming such an hypothesis, the mutation is supposed to be 3000-6000 years old, depending on generation length and the true recombination ratio between the KM19 and CF loci. Up-to-date Spanish, Danish and Greek data are reported together with other previously published population data in order to discuss the geographic origin and age of the mutation in Europe. The action of selection in terms of heterozygote advantage and distorsion of segregation is discussed.

DNAs from unrelated healthy individuals and unrelated individuals affected with 21-hydroxylase deficiency (congenital and late-onset adrenal hyperplasia) were digested with seven restriction enzymes and hybridized with a cDNA probe specific for human 21-hydroxylase genes. Associations were found between restriction fragments and the two forms of the disease: The late onset form is associated with a double dose of a 14 kb fragment generated by EcoRI and with a triple dose of a 3.2 kb fragment generated by Taq I in patients with HLA B14 haplotypes; The classical congenital form is negatively associated with the 14 kb fragment and with a 3.7 kb fragment generated by Taq I in patients with HLA Bw47 haplotypes. A 3.2 kb Taq I fragment is negatively associated with the HLA B8 haplotypes. The other five enzymes tested give no polymorphisms or polymorphisms without correlation with the two forms of the disease.

Steroid 11 beta-hydroxylase (P-450(11) beta) is a mitochondrial cytochrome P-450 enzyme necessary for cortisol biosynthesis. Deficiency of 11 beta-hydroxylase causes a hypertensive form of congenital adrenal hyperplasia. A partial cDNA clone encoding this enzyme has been previously isolated and the corresponding gene, CYP11B1, mapped to human chromosome 8q. This gene has now been isolated along with a second linked homologous gene, CYP11B2. Each gene contains nine exons. The eight introns are identical in location to the introns of the CYP11A gene encoding another mitochondrial P-450 enzyme, cholesterol desmolase, confirming that 11 beta-hydroxylase and cholesterol desmolase are in the same gene family within the P-450 superfamily. The nucleotide sequences of CYP11B1 and B2 are 95% identical in coding regions and about 90% identical in introns. The putative proteins encoded by CYP11B1 and B2 each contain 503 amino acids including a 24-residue signal peptide and have sequences that are 93% identical to each other and 75% identical to the predicted sequence of bovine P-450(11) beta. There are no obviously deleterious mutations in coding sequences of CYP11B2. However, the 5'-flanking regions of CYP11B1 and B2 have diverged considerably, and B2 transcripts were not detected in human adrenal mRNA or among cDNA clones.

High molecular weight DNA extracted from 50 unrelated parisian individuals constitutes a "DNA-thèque" which can be used at the population level. This procedure allows preparation from a variety of tissues such as liver, placenta and leucocytes. The "quality" of the DNA obtained (about 50 Kb, RNA and protein free) and the efficiency of the method (in terms of the yield of DNA obtained) are considered. Blood is the easiest material to obtain and permits the obtaining of a reasonable amount of human and primate DNA, particularly when familial investigation is planned for polymorphism studies. One of the objects of this article is to encourage others to make use of our bank, for studies in population and evolutionary genetics.

We develop here several models concerning strategies for detection of restriction polymorphisms in humans. These different strategies concern the numbers of individuals to test in a DNA collection, the nature of restriction enzyme to utilize, and the length of the probes. For enzyme utilization different strategies are based on (i) the nature of the restriction site; (ii) the mean length of restriction fragments obtained; (iii) the methylated cytosine characteristics in the CG doublets. Each of these approaches are tested in experimental situations.

Hybridization of albumin clones cDNA with human DNAs digested by several restriction endonucleases reveals two HaeIII polymorphisms. The first polymorphism, H1, is of low frequency (f1 = 0.05); the second, which is validated by family analysis, occurs frequently (f2 = 0.21) and is an intronic polymorphism, probably of substitution--base type.

Amniotic fluid levels of 21-deoxycortisol (21-DOF) and 17-hydroxyprogesterone (17-OHP) were measured in 49 pregnancies, including 31 pregnancies at risk for CAH. The results were compared with those obtained by HLA typing and linkage analysis to a HLA DNA probe. The mean amniotic fluid levels in the control pregnancies were 0.28 nmol/L for 21-DOF and 4.1 nmol/L for 17-OHP. The levels were similar in early and midpregnancy for 21-DOF (0.29 vs. 0.27 nmol/L) and 17-OHP (3.4 vs. 4.2 nmol/L). The amniotic fluid 21-DOF level was 1.75 nmol/L in affected pregnancies, significantly higher than in the control pregnancies (mean, 0.28 nmol/L). The mean amniotic fluid 17-OHP level in the affected pregnancies (30.5 nmol/L) also was significantly higher than that in the control pregnancies (4.10 nmol/L). Simultaneous measurement of 21-DOF and 17-OHP levels in amniotic fluid from 10-18 weeks of gestation can be used for early diagnosis of congenital adrenal hyperplasia.

21-deoxycortisol is a steroid produced mainly by the adrenal gland. Its normal plasma baseline concentrations (0.03 to 0.30 n/ml) and its concentrations after tetracosactide injection (0.15 to 0.76 ng/ml) do not significantly vary with age, sex and phases of the menstrual cycle. 21-deoxycortisol was assayed in plasma by a specific radioimmunological method and its values were compared with those of 17-OH progesterone in heterozygous subjects with the classical and non-classical forms of 21-hydroxylase deficiency, and in the amniotic fluid of foetuses with this deficiency. Baseline concentrations of 21-deoxycortisol in the classical forms of 21-hydroxylase deficiency (n = 12; 55.36 to 186.6 ng/ml) and post-tetracosactide concentrations in non-classical late onset forms (n = 31; 4.04 to 47 ng/ml) were much higher than in normal subjects, thus making this steroid as sensitive as, or even more sensitive than 17-OH progesterone in diagnosing 21-hydroxylase deficiency. Post-tetracosactide assays of 21-deoxycortisol in 84 heterozygous subjects with 21-hydroxylase deficiency (0.70 to 5.40 ng/ml) enabled these subjects to be detected with a more than 90 percent sensitivity, which cannot be obtained with 17-OH progesterone assays. 21-deoxycortisol concentrations in the amniotic fluid of foetuses with 21-hydroxylase deficiency (n = 11; 0.391 to 0.930 ng/ml) were constantly superior to those observed in normal foetuses (n = 38; 0.034 to 0.221 ng/ml), so that the deficiency can be diagnosed with the steroid as easily as with 17-OH progesterone.

The close genetic linkage between the gene for congenital adrenal hyperplasia due to 21-hydroxylase (21-OH) deficiency and HLA genes allowed us to use the polymorphism of this system as a marker of the disease. HLA genotyping can be performed by using restriction enzyme fragments hybridized with specific probes instead of serologic methods. In seven pregnancies at risk for 21-OH deficiency, a first trimester prenatal diagnosis has been performed by determining the fetal genotype by linkage analysis of DNA from chorionic villi using HLA class I and class II probes. In four of these pregnancies, determination of 17-OH progesterone in first trimester amniotic fluid afforded a complementary approach to the diagnosis.

Incidence of congenital adrenal hyperplasia due to 21 hydroxylase deficiency was studied in France. Five hundred and twenty six patients born during the period 1963-1979 were found. Assuming complete ascertainment in females, the incidence of the disease is 0.43 X 10(-4) or 1: 23,044. The frequency of the carriers is 0.013 (1/76). The birth places of patients show an unequal geographic distribution. The mean inbreeding coefficient is 270 X 10(-5), a figure higher than the mean coefficient of France. The frequency of marriages between first cousins is slightly raised, 0.3%.