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.

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.

Usefulness of MLPA in the detection of SHOX deletions.

SHOX haploinsufficiency causes a wide spectrum of short stature phenotypes, such as Leri-Weill dyschondrosteosis (LWD) and disproportionate short stature (DSS). SHOX deletions are responsible for approximately two thirds of isolated haploinsufficiency; therefore, it is important to determine the most appropriate methodology for detection of gene deletion. In this study, three methodologies for the detection of SHOX deletions were compared: the fluorescence in situ hybridization (FISH), microsatellite analysis and multiplex ligation-dependent probe amplification (MLPA).

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.

Tall stature and poor breast development after estrogen replacement in a hypergonadotrophic hypogonadic patient with a 45,X/46,X,der(X) karyotype with SHOX gene overdosage.

SHOX is exclusively expressed in the developing distal limb bones of human embryos and in the first and second pharyngeal arches. It works as a promoter for linear growth and as a repressor of growth plate fusion. It was reported, recently, that SHOX overdosage and gonadal estrogen deficiency have led to tall stature due to continued growth.

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.

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.

Y-STRs in forensic medicine: DNA analysis in semen samples of azoospermic individuals.

The incidence of rape has increased, especially in metropolitan areas, such as the city of São Paulo. In Brazil, studies about it have shown that the majority of this type of crime is committed by the relatives and persons close to the victim. This has made the crime more difficult to be denounced, as only 10% of the cases are reported to competent police authorities.

[Complete form of androgen insensitivity syndrome in Brazilian patients due to P766A mutation in the androgen receptor].

Androgen insensitivity syndrome (AIS) is a rare X-linked disorder, caused by mutations in the androgen receptor gene (AR), associated with a variety of phenotypes in 46,XY individuals. We studied two 23 year-old twin-sisters with female social sex referred due to primary amenorrhea, who exhibited bilateral palpable gonads in the inguinal region and a 46,XY karyotype. The uterus was absent in pelvic sonograms.

[Androgen insensitivity syndrome: clinical, hormonal and molecular analysis of 33 cases].

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.

[Standardization of DNA extraction with NaCl from oral mucosa cells: application in PROP1 gene study].

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.

Frequency of the allelic variant (Trp8Arg/Ile15Thr) of the luteinizing hormone gene in a Brazilian cohort of healthy subjects and in patients with hypogonadotropic hypogonadism.

Purpose: To evaluate the frequency of allelic variant Trp8Arg/Ile15Thr in the luteinizing hormone beta-subunit gene in a Brazilian population of healthy subjects and in patients with hypogonadotropic hypogonadism.

Subjects And Methods: Two hundred and two adults (115 women) with normal sexual function and 48 patients (24 women) with hypogonadotropic hypogonadism underwent a molecular study of the the luteinizing hormone beta-subunit gene using a polymerase chain reaction technique followed by enzymatic digestion with the restriction enzymes Nco I (for detection of the Trp8Arg point mutation) and Fok I (for detection of the Ile15Thr point mutation). Basal luteinizing hormone and FSH, testosterone, or estradiol levels were measured in 37 healthy subjects (21 women) and in 27 hypogonadotropic hypogonadism patients (13 women) by immunofluorometric methods (hLH-Spec and hFSH-Spec, AutoDELFIA, Wallac Oy, Turku, Finland).

[Multicentric study of Brazilian patients with 21-hydroxylase deficiency: a genotype-phenotype correlation].

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Population genetics of nine short tandem repeat loci: allele frequency distribution in a Brazilian population sample.

Gene and genotype frequencies in relation to the D3S1358, vWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317, and D7S820 loci were determined in a sample of 290 unrelated individuals (204 Caucasians and 86 mulattoes) living in the city of São Paulo, Brazil. The sex test Amelogenin was also performed in all subjects from our sample, revealing the expected sex in all instances. Allele frequency data obtained from the analysis of these samples were in the usual range of other population groups with similar racial background.