The degree of external genitalia virilization in girls with 21-hydroxylase deficiency appears to be influenced by the CAG repeats in the androgen receptor gene.

BACKGROUND: Women with 21-hydroxylase deficiency present much variability in external genitalia virilization, even among those with similar impairments of 21-hydroxylase (21OH) activity. OBJECTIVE: To evaluate if the number of CAG (nCAG) repeats of the androgen receptor gene influences the degree of external genitalia virilization in women with CYP21A2 mutations, grouped according to impairment of 21OH activity. PATIENTS: The nCAG was determined in 106 congenital adrenal hyperplasia (CAH) patients and in 302 controls. The patients were divided, according to their CYP21A2 genotypes, into Groups A and B, which confer total and severe impairment of 21OH activity, respectively. METHODS: The inactivation pattern of the X-chromosome was studied through genomic DNA digestion with Hpa II. The CAG repeat region was amplified by polymerase chain reaction (PCR) and analysed by GeneScan. RESULTS: The nCAG and the frequency of severe skewed X-inactivation did not differ between normal women and patients. The nCAG median in genotype A was 20.7 (IQR 2.3) for Prader I + II, 22.5 (3.6) for Prader III and 21 (2.9) for Prader IV + V (P < 0.05 for Prader III and Prader IV + V). The nCAG median in genotype B was 21.3 (1.1) for Prader I + II, 20.5 (2.9) for Prader III and 22 (2.8) for Prader IV + V (P > 0.05). A significant difference was found regarding the nCAG median in patients presenting Prader III from genotypes A and B. CONCLUSIONS: We observed great variability in the degree of external genitalia virilization in both CYP21A2 genotypes, and we showed that the CAG repeats of the androgen receptor gene influences this phenotypic variability.

Microconversion between CYP21A2 and CYP21A1P promoter regions causes the nonclassical form of 21-hydroxylase deficiency.

CONTEXT: Most mutations causing 21-hydroxylase deficiency originate from microconversions between CYP21 pseudogenes and active genes. However, around 20% of the alleles in the nonclassical form (NC-21OHD) remain without identified mutations, suggesting the involvement of regulatory regions. The pseudogene promoter is 80% less active than the CYP21A2 due to the presence of -126C>T, -113G>A, -110T>C, and -103A>G mutations. Additionally, mutations in the steroidogenic factor-1 binding sites of the CYP21 distal regulatory region, located at 4676 bases upstream from the cap site of the CYP21A2 gene, decrease its transcription to 35%. OBJECTIVE: The objective of the study was to investigate the CYP21A2 promoter/regulatory regions in NC-21OHD patients with undetermined genotype. SUBJECTS: The study included 17 NC-21OHD patients and 50 controls. METHODS: Promoter/regulatory regions were sequenced from peripheral leukocytes' genomic DNA. The identified substitutions were evaluated through EMSA using -132/-97 wild-type and mutant probes and nuclear extracts from NCI-H295A cells. Transcriptional activity studies were performed with wild-type and mutant constructions transfected in NCI-H295A cells. RESULTS: No mutations were identified in the distal regulatory regions. The -126C>T, -113G>A, -110T>C promoter mutations were found in compound heterozygosity with the V281L mutation in one patient and the -126C>T mutation in compound heterozygosity with the I2 splice in another. The -126T mutation decreases the transcriptional activity to 52%, compatible with the patient's nonclassical phenotype. EMSA demonstrated that the -132/-121 region is important for the DNA interaction with the specificity protein-1 transcription factor. CONCLUSION: Microconversions between CYP21A2 and CYP21A1P promoters could be involved in the nonclassical phenotype. Therefore CYP21A2 promoter analysis should be included in genetic studies of 21OHD.

Molecular CYP21A2 diagnosis in 480 Brazilian patients with congenital adrenal hyperplasia before newborn screening introduction.

BACKGROUND: Most congenital adrenal hyperplasia (CAH) patients carry CYP21A2 mutations derived from conversion events involving the pseudogene, and the remaining carry new mutations. OBJECTIVE: To review causal mutations and genotype-phenotype correlation in 480 Brazilian patients. METHODS: DNA was extracted from 158 salt-wasters (SWs), 116 simple virilizing (SV), and 206 nonclassical (NC) patients. Fourteen point mutations were screened by allele-specific PCR, large rearrangements by Southern blotting/MLPA, and sequencing was performed in those with incomplete genotype. The gene founder effect was analyzed by microsatellite studies. Patients were divided into six genotypes (Null; A: <2%; B: 3-7%; C: >20% of residual enzymatic activity (EA); D: unknown EA; E: incomplete genotype). RESULTS: Targeted methodologies defined genotype in 87.6% of classical and in 80% of NC patients and the addition of sequencing in 100 and 83.5%, respectively. The most frequent mutations were p.V281L (26.6% of alleles), IVS2-13A/C>G (21.1%), and p.I172N (7.5%); seven rare mutations and one novel mutation (p.E351V) were identified. Gene founder effect was observed in all but one (p.W19X) mutation. Null, A, B, and C genotypes correlated with SW (88%), SW (70%), SV (98%), and NC forms (100%), respectively. In group D, the p.E351V mutation correlated with classical form and group E comprised exclusively NC-patients. ACTH-stimulated 17OHP level of 44.3ng/mL was the best cutoff to identify NC-patients carrying severe mutations. CONCLUSIONS: We identified a good genotype-phenotype correlation in CAH, providing useful data regarding prediction of disease's severity; moreover, we suggest that ACTH-stimulated 17OHP levels could predict carrier status for severe mutations. Sequencing is essential to optimize molecular diagnosis in Brazilian CAH patients.

Deletion mapping of chromosome 17 in benign and malignant adrenocortical tumors associated with the Arg337His mutation of the p53 tumor suppressor protein.

The human p53 tumor suppressor gene is located at the short arm of chromosome 17. A germinative mutation (Arg337His) in the tetramerization domain of p53 has been frequently identified in Brazilian children with sporadic adrenocortical tumors. Loss of heterozygosity at this region was demonstrated in the majority of the cases. In the present study, we performed deletion mapping of chromosome 17 in 30 adrenocortical tumors from 29 Brazilian patients (15 children and 14 adults). One boy had bilateral adrenocortical tumor. Sixteen patients had the germinative Arg337His mutation. Loss of heterozygosity analysis using six polymorphic microsatellite markers disclosed loss of the entire chromosome 17 in 18 tumors (10 adenomas and eight carcinomas) from 17 patients. The Arg337His mutation was present in 13 of them. Chromosomal instability involving chromosomes 2, 9, and 11 was also found in 47, 47, and 70% of the 17 patients who exhibited chromosome 17 losses, respectively. The concomitant loss of chromosomes 2, 9, 11, and 17 was evidenced exclusively in malignant tumors. Therefore, chromosomal instability involving three or more chromosomes may contribute to define the malignant adrenocortical lesions. In conclusion, we demonstrated a high frequency of biallelic inactivation of p53 derived from two distinct events, the germinative Arg337His mutation and the acquired loss of the entire chromosome 17. In addition, the isolated loss of the entire chromosome 17 did not correlate with aggressive tumor behavior in these patients with adrenocortical tumors.

[Standardization of DNA extraction with NaCl from oral mucosa cells: application in PROP1 gene study]. / Padronização da técnica de extração de DNA de células de mucosa oral com NaCl: aplicação no estudo do gene PROP1.

DNA extraction of peripheral leukocytes is the most broadly used technique to obtain DNA. However, cell collection from an oral swab, frequently used in forensics, is useful to obtain DNA samples, mainly in newborns, children and patients who live far from the collection sites, where blood sample collection and sending is not feasible. Our objective was to standardize DNA extraction from an oral swab, using the NaCl method, comparing it with a commercial kit. To test DNA quality, we amplified the 3 exons of PROP1 gene of 12 patients with hypopituitarism in DNA obtained from oral cells and peripheral blood cells. Amplification of larger fragments was tested in oral DNA of normal subjects using primers of exon 10 of FSHR gene (1000 bp) and of CYP21A2 gene (1200 bp). Both methods yielded good quality DNA, allowing the amplification of 3 exons of PROP1 gene. The NaCl method showed to be faster and less expensive, resulting in a larger amount of DNA when compared to the commercial kit. We identified the delAG301-302 mutation in 6 patients, 4 in homozygous (33%) and 2 in heterozygous (16%) state and G51A mutation in heterozygous state in a single patient. In conclusion, we standardized the DNA extraction of oral cells with NaCl, which presented lower costs and faster results, when compared with the extraction by a commercial kit indicating that DNA from oral swabs are a reliable source for genetic studies.

[Androgen insensitivity syndrome: clinical, hormonal and molecular analysis of 33 cases]. / Síndrome de insensibilidade aos andrógenos: análise clínica, hormonal e molecular de 33 casos.

Androgen insensitivity syndrome (AIS) is a rare X-linked recessive condition in which patients with 46,XY karyotype have a complete (CAIS) or partial (PAIS) impairment of pre- and postnatal virilization due to mutations in the androgen receptor (AR). We present a concise revision of AIS and the AR and report the clinical, hormonal and molecular study of 33 subjects with AIS. The coding region of the AR was analyzed in 33 subjects with clinical and hormonal characteristics that suggested AIS. Eleven patients (9 families) had CAIS and 22 patients (12 families) had PAIS. Mutations in the AR were identified and the molecular diagnosis of AIS established in 100% of families with CAIS and 75% with PAIS. Nine mutations had been previously described (N705S, W741C, M742V, R752X, Y763C, R779W, M807V, R855C e R855H) and 7 mutations were first described in these cohort of patients (S119X, T602P, L768V, R840S, I898F, P904R e IVS3 - 60 G>A).

Familial hyperestrogenism in both sexes: clinical, hormonal, and molecular studies of two siblings.

Familial hyperestrogenism is a rare clinical condition of unknown etiology in which patients present excessive androgen to estrogen conversion. Excessive aromatization is primarily ascribed to abnormalities in the CYP19. Mice that lack steroid 5alpha-reductase type 1 also exhibit hyperestrogenism due to an increased availability of androgen precursors. Here we studied two adult siblings, born to unrelated parents, who presented clinical and hormonal evidence of estrogen excess. The man was treated with topical dihydrotestosterone, which promoted adequate virilization. The woman was treated with anastrazole, a potent aromatase inhibitor, with normalization of menstrual cycles. Genetic linkage to the steroid 5alpha-reductase type 1 gene (SRD5A1) was ruled out in this family. A similar analysis did not rule out linkage to CYP19, although no mutation was identified in the coding region of this gene. Aromatase mRNA was at least 10-fold more abundant in the female patient's skin fibroblasts vs. the control. Southern analysis of genomic DNA did not reveal rearrangements or amplification of the coding region of CYP19. We conclude that the phenotype of familial hyperestrogenism includes prepubertal gynecomastia, hypogonadism, and short stature in men, and precocious thelarche, macromastia, enlarged uterus, and menstrual irregularities in women. Topical dihydrotestosterone is an efficient alternative treatment in men with hyperestrogenism; in addition, second generation aromatase inhibitors are useful in both sexes.

A microdeletion in the ligand binding domain of human steroidogenic factor 1 causes XY sex reversal without adrenal insufficiency.

Steroidogenic factor 1 (SF-1) is an orphan nuclear receptor that plays key roles in endocrine development and function. Knockout mice lacking SF-1 have adrenal and gonadal agenesis, impaired gonadotropin expression, and structural abnormalities of the ventromedial hypothalamic nucleus. Previous studies have identified three human subjects with mutations in SF-1 causing adrenocortical insufficiency with varying degrees of gonadal dysfunction. We now describe a novel 8-bp microdeletion of SF-1, isolated from a 46, XY patient who presented with gonadal agenesis but normal adrenal function, which causes premature termination upstream of sequences encoding the activation function 2 domain. In cell transfection experiments, the mutated protein possessed no intrinsic transcriptional activity but rather inhibited the function of the wild-type protein in most cell types. To our knowledge, this is the first example of an apparent dominant-negative effect of a SF-1 mutation in humans. These findings, which define a SF-1 mutation that apparently differentially affects its transcriptional activity in vivo in the adrenal cortex and the gonads, may be relevant to the cohort of patients who present with 46, XY sex reversal but normal adrenal function.

Three novel mutations in CYP21 gene in Brazilian patients with the classical form of 21-hydroxylase deficiency due to a founder effect.

Three different new mutations were found after CYP21 gene sequencing in three unrelated patients with the classical form of the 21-hydroxylase deficiency. These mutations were also screened in their affected relatives. In one patient and her brother, both affected with the simple virilizing form and in their aunt, with the nonclassical form, an AG>GG transition was found in the acceptor site of intron 2. In another patient with the salt wasting form, we found a 1003 1004 insA, in exon 4, that altered the reading frame and created a stop codon in codon 297. In the third patient and his sister, we found a C>T transition in codon 408. This transition led to the substitution of arginine by cysteine (R408C) in a conserved region where arginine is conserved in at least four different species. These siblings with the R408C mutation, both affected with the salt wasting form, have the IVS2-13A/C>G mutation in the other allele, suggesting that the R408C should lead to complete impairment of enzymatic activity. To rule out the possibility of polymorphism, R408C was screened through allele specific PCR, and it was not found in 100 normal alleles. The screening of these three new mutations by allele-specific PCR or enzymatic restriction in 212 CAH patients disclosed their presence in 2.3% (9/387) of the alleles. All three new mutations were found in compound heterozygous state with previously known mutations. Microsatellite studies, using markers flanking CYP21 gene, revealed that each new mutation presents the same haplotype, suggesting a gene founder effect, similar to what was previously observed with the G424S mutation also described in our population. Although microconversion events are the main cause of mutations in the CYP21 gene, random mutations with a common origin can also be the cause of 21-hydroxylase deficiency.

Clinical, hormonal, behavioral, and genetic characteristics of androgen insensitivity syndrome in a Brazilian cohort: five novel mutations in the androgen receptor gene.

Androgen insensitivity syndrome (AIS) is caused by mutations in the androgen receptor gene and is associated with a variety of phenotypes in 46,XY individuals, ranging from phenotypic women [complete form (CAIS)] to men with minor degrees of undervirilization or infertility [partial form (PAIS)]. We studied 32 subjects with male pseudohermaphroditism from 20 families (9 CAIS, 11 PAIS) with the following criteria for AIS: 46,XY karyotype, normal male basal and human chorionic gonadotropin-stimulated levels of serum testosterone and steroid precursors, gynecomastia at puberty, and, in prepubertal patients, a family history suggestive of X-linked inheritance. The entire coding region of the androgen receptor gene was analyzed, and mutations were found in all families with CAIS and in eight of 11 families with PAIS. Fifteen different mutations were identified, including five (S119X, T602P, L768V, I898F, and P904V) that have not been described previously. Detailed clinical and hormonal features were compared with genotype in 25 subjects with AIS and confirmed by mutational analysis. LH hormone levels and the LH x testosterone product were high in all postpubertal subjects with AIS. All subjects with PAIS maintained at postpubertal age the gender identity and social sex that was assigned to them in infancy, in contrast to other forms of pseudohermaphroditism.

Detection of Y-specific sequences in 122 patients with Turner syndrome: nested PCR is not a reliable method.

The incidence of Y chromosome sequences in patients with Turner syndrome has been evaluated in several studies, and its frequency varied from 0% to 61%, depending on the molecular methodology used. The aim of our study was to screen for Y chromosome sequences in 122 patients with Turner syndrome without cytogenetic evidence of this chromosome. DNA of 100 normal women was also screened and it was used as a negative control. To identify cryptic Y mosaicism, eight regions of Y chromosome were amplified by PCR. In order to increase the sensitivity of Y sequence detection, a nested PCR of the SRY and TSPY genes was also performed. All patients had several stigmata of Turner syndrome and none of them presented with signs of virilization. The most frequent karyotype was 45,X (54.1%), followed by mosaicism involving structural aberration of the X chromosome. There were 12 patients who carried a marker or ring chromosome. First-round PCR identified Y chromosome sequences in only four patients (3%), and all of them had a chromosome mosaicism with at least one cell lineage with a marker chromosome. After nested PCR, 25% of the patients and 14% of the normal women were positive for the presence of Y sequences. Contamination with extraneous genomic DNA was ruled out by microsatellite studies, but we cannot eliminate the possibility of contamination with PCR products, despite careful handling. We conclude that nested PCR overestimated the frequency of Y sequences in patients with Turner syndrome and should be avoided to prevent false positive results, which lead to unnecessary surgical treatment of these patients.

Founder effect for the highly prevalent R337H mutation of tumor suppressor p53 in Brazilian patients with adrenocortical tumors.

The incidence of adrenocortical tumors in children from the Southern region of Brazil is higher than in other parts of the world. This fact has been related to the identification of an inherited missense mutation of the p53 (R337H) at high frequency (78-97%) in Brazilian children with adrenocortical tumors. Given the high frequency of this germline mutation in the Brazilian population, it is very likely that the R337H mutation has arisen from a common origin. In this study, we analyzed two highly polymorphic intragenic markers (VNTRp53 and p53CA) in 22 patients (16 children and 6 adults) with adrenocortical tumors carrying the germline R337H mutation and 60 normal individuals using GeneScan Fragment Analysis software. We found six and sixteen different alleles for the VNTRp53 and p53CA polymorphic markers, respectively. Two distinct alleles, both with 122 bp, were found in 56.8% (VNTRp53) and 54.5% (p53CA) of the 44 alleles from patients with adrenocortical tumors associated with the R337H mutation. Differently, these same VNTRp53 and p53CA alleles were found in 18.3% and 14.2% of 120 alleles from normal individuals, respectively (p<0.01, Chi-square test). An identical haplotype for p53 locus was also identified in 95% of the apparently unrelated Brazilian patients with adrenocortical tumors carrying the R337H mutation. In conclusion, we demonstrated a strong evidence of co-segregation between two intragenic polymorphic p53 markers and the germline R337H mutation, indicating that this mutation has originated from a single common ancestral in the great majority of the Brazilian patients with adrenocortical tumors.

[Multicentric study of Brazilian patients with 21-hydroxylase deficiency: a genotype-phenotype correlation]. / Estudo multicêntrico de pacientes brasileiros com deficiência da 21-hidroxilase: correlação do genótipo com o fenótipo.

We analyzed the clinical and molecular data of 205 patients with the three different clinical forms of 21-hydroxylase deficiency, in whom the clinical and molecular diagnosis were already defined. The most frequent mutations were I2 splice in the salt wasting form, I172N in the simple virilizing and V281L in the nonclassical form, presenting similar frequencies as those observed in other populations. We found a lower frequency of 21-hydroxylase gene deletion, similar to that previously identified in Argentinean and Mexican populations. Five new mutations were described in our population: G424S, H28+C, Ins 1003 1004 A, R408C and IVS2-2A>G. The genotype was classified in three groups according to the impairment of enzymatic activity observed in vitro, Group A: 0-2%, Group B: 3-7% and Group C: >20%. Group A mutations correlated with the salt wasting form, the Group B with simple virilizing form and Group C with the non classical form. The severity of genotype showed a positive correlation with higher 17OH-progesterone and testosterone levels. The I2 splice mutation in homo or hemizygosis confers classical form phenotype with both salt wasting and simple virilizing forms, precluding the prediction of the clinical form through genotype in pre and neonatal diagnosis. The good genotype-phenotype correlation in patients with 21-hydroxylase deficiency shows the usefulness of genotype to predict the clinical form for genetic counseling, prenatal diagnosis and to confirm neonatal screening diagnosis, except in cases with I2 splice mutation.

Insulin-like growth factor 1 gene (CA)n repeats and a variable number of tandem repeats of the insulin gene in Brazilian children born small for gestational age.

OBJECTIVE: To investigate the influence of (CA)n repeats in the insulin-like growth factor 1 gene and a variable number of tandem repeats of the insulin gene on birth size in children who are small or adequate-sized for gestational age and to correlate these polymorphisms with serum insulin-like growth factor 1 levels and insulin sensitivity in children who are small for gestational age, with and without catch-up growth. PATIENTS AND METHODS: We evaluated 439 infants: 297 that were adequate-sized for gestational age and 142 that were small for gestational age (66 with and 76 without catch-up). The number of (CA)n repeat in the insulin-like growth factor 1 gene and a variable number of tandem repeats in the insulin gene were analyzed using GENESCAN software and polymerase chain reaction followed by enzymatic digestion, respectively. Clinical and laboratory data were obtained from all patients. RESULTS: The height, body mass index, paternal height, target height and insulin-like growth factor 1 serum levels were higher in children who were small for gestational age with catch-up. There was no difference in the allelic and genotypic distributions of both polymorphisms between the adequate-sized and small infants or among small infants with and without catch-up. Similarly, the polymorphisms were not associated with clinical or laboratory variables. CONCLUSION: Polymorphisms of the (CA)n repeats of the insulin-like growth factor 1 gene and a variable number of tandem repeats of the insulin gene, separately or in combination, did not influence pre- or postnatal growth, insulin-like growth factor 1 serum levels or insulin resistance.

Could the leukocyte x chromosome inactivation pattern be extrapolated to hair bulbs?

BACKGROUND: The androgen receptor gene is located on the X chromosome with a polymorphic tract of CAG repeats that is inversely correlated to the receptor's transactivation activity. A short CAG tract is associated with hyperandrogenic disorders. In women, one of the X chromosomes is inactivated and the X chromosome inactivation (XCI) pattern varies among tissues. Previous studies of hyperandrogenic disorders only evaluated XCI in leukocytes. OBJECTIVE: To evaluate whether the XCI pattern in leukocytes could be extrapolated to those in hair bulbs. MATERIAL: A total of 58 healthy women were used for this study. DNA was extracted from leukocytes (n = 58 women) and pubic (n = 53 women) and scalp hair (n = 21 women). METHODS: Hpa II digested and undigested DNA samples underwent fluorescence PCR GeneScan analysis. RESULTS: A significant and positive correlation of XCI was found between leukocytes and hair bulbs. However, individual comparisons showed that 13 and 19% of the women presented a different leukocyte XCI pattern in pubic hair and scalp hair, respectively. CONCLUSION: The XCI pattern was similar in leukocytes and hair bulbs of normal women indicating that leukocyte DNA is useful for XCI analysis. However, the XCI pattern could vary among tissues from the same subject, indicating that care should be taken when extrapolating individual leukocyte XCI patterns to other tissue.

Insulin-like growth factor 1 gene (CA)n repeats and a variable number of tandem repeats of the insulin gene in Brazilian children born small for gestational age

OBJECTIVE: To investigate the influence of (CA)n repeats in the insulin-like growth factor 1 gene and a variable number of tandem repeats of the insulin gene on birth size in children who are small or adequate-sized for gestational age and to correlate these polymorphisms with serum insulin-like growth factor 1 levels and insulin sensitivity in children who are small for gestational age, with and without catch-up growth. PATIENTS AND METHODS: We evaluated 439 infants: 297 that were adequate-sized for gestational age and 142 that were small for gestational age (66 with and 76 without catch-up). The number of (CA)n repeat in the insulin-like growth factor 1 gene and a variable number of tandem repeats in the insulin gene were analyzed using GENESCAN software and polymerase chain reaction followed by enzymatic digestion, respectively. Clinical and laboratory data were obtained from all patients. RESULTS: The height, body mass index, paternal height, target height and insulin-like growth factor 1 serum levels were higher in children who were small for gestational age with catch-up. There was no difference in the allelic and genotypic distributions of both polymorphisms between the adequate-sized and small infants or among small infants with and without catch-up. Similarly, the polymorphisms were not associated with clinical or laboratory variables. CONCLUSION: Polymorphisms of the (CA)n repeats of the insulin-like growth factor 1 gene and a variable number of tandem repeats of the insulin gene, separately or in combination, did not influence pre- or postnatal growth, insulin-like growth factor 1 serum levels or insulin resistance. .

Substitutions in the CYP21A2 promoter explain the simple-virilizing form of 21-hydroxylase deficiency in patients harbouring a P30L mutation.

The classical and nonclassical phenotypes of 21-hydroxylase deficiency represent a continuous spectrum of the impairment of 21-hydroxylase activity due to mutations between the CYP21A2 gene. These mutations occur mainly by microconversion in the homologous nonfunctional CYP21A1P gene. The P30L mutation is associated with the nonclassical form, and it reduces the activity to 30-40% of the normal enzyme. We have described three female patients exhibiting a simple virilizing phenotype and bearing the P30L mutation in compound heterozygosis with a severe mutation. To identify additional mutations causing this phenotype, the promoter region was sequenced and four mutations were identified: -126C --> T, -113G --> A, -110T --> C and -103 A --> G. These substitutions are normally present in the promoter region of the pseudogene and in vitro studies demonstrated that they reduced the transcriptional activity fivefold. They might have been converted to the CYP21A2 promoter together with the P30L mutation in these patients. Therefore, these substitutions in synergism with the P30L mutation might decrease the enzyme activity resulting in a more severe phenotype, and a DNA sequence of -167 bases of the CYP21A2 gene should be performed in patients with 21-hydroxylase deficiency in whom the phenotype is more severe than predicted by the genotype.

Use of nonradioactive labeling to detect large gene rearrangements in 21-hydroxylase deficiency.

PURPOSE: To establish the Southern blotting technique using hybridization with a nonradioactive probe to detect large rearrangements of CYP21A2 in a Brazilian cohort with congenital adrenal hyperplasia due to 21-hydroxylase deficiency (CAH-21OH). METHOD: We studied 42 patients, 2 of them related, comprising 80 non-related alleles. DNA samples were obtained from peripheral blood, digested by restriction enzyme Taq I, submitted to Southern blotting and hybridized with biotin-labeled probes. RESULTS: This method was shown to be reliable with results similar to the radioactive-labeling method. We found CYP21A2 deletion (2.5%), large gene conversion (8.8%), CYP21AP deletion (3.8%), and CYP21A1P duplication (6.3%). These frequencies were similar to those found in our previous study in which a large number of cases were studied. Good hybridization patterns were achieved with a smaller amount of DNA (5 mug), and fragment signs were observed after 5 minutes to 1 hour of exposure. CONCLUSIONS: We established a non-radioactive (biotin) Southern blot/hybridization methodology for CYP21A2 large rearrangements with good results. Despite being more arduous, this technique is faster, requires a smaller amount of DNA, and most importantly, avoids problems with the use of radioactivity.

Brazilian population data on the polymerase chain reaction-based loci LDLR, GYPA, HBGG, D7S8, and Gc.

Gene and genotype frequencies in relation to the low density lipoprotein receptor (LDLR), glycophorin A (GYPA), hemoglobin G gammaglobin (HBGG), D7S8, and group specific component (Gc) loci were determined in a sample of 344 unrelated individuals (250 whites and 94 mulattoes) living in the city of São Paulo, Brazil. DNA was extracted from 5 mL of peripheral blood obtained from each of the 344 volunteers by the salting-out procedure. Polymerase chain reaction and reverse dot-blot analysis were performed with the Amplitype PM PCR Amplification and Typing Kit (Polymarker Multiplex; Applied Biosystems, Foster City, CA) under conditions recommended by the manufacturer. Estimated allele frequencies in the white sample were in the usual range of that of other United States and European population groups. In any case, genotype distributions for these loci did not deviate significantly from Hardy-Weinberg equilibrium proportions. Only 1 marginally significant (0.01 < P < 0.05) association, between loci HBGG and Gc, was detected in our mulatto sample out of a total of 20 possible pairwise comparisons of the 5 loci for both data sets. Allele frequencies were significantly different (P < 0.001) at the HBGG and Gc loci when the white and mulatto samples were compared. Biologic relationship exclusion probabilities (test powers) were calculated for the data. A Brazilian database has thus been established for the loci LDLR, GYPA, HBGG, D7S8, and Gc, 5 polymerase chain reaction-based loci systems that have been shown to be a useful tool for biologic relationship identification and exclusion.