p.F508del in a heterogeneous cystic fibrosis population from Minas Gerais, Brazil.

Cystic fibrosis (CF) is the most common autosomal recessive disease of the Caucasian population. Among the various CF mutations, p.F508del is the most frequent, accounting for two-thirds of the global CF chromosomes, although showing great variability among populations. We have studied 115 unrelated CF patients from a mixed population of Minas Gerais (Brazil). To evaluate part of the DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, blood DNA was obtained and PCR was performed using two pairs of primers that anneal to exons 10 and 24 of the CFTR gene. The PCR product was then submitted to automatic sequencing using the ABI PRISM 310 Genetic Analyzer. The p.F508del mutation was found in 50 (21.7%) of 230 unrelated CF alleles. Fifteen (13.0%) patients were homozygous for this mutation, while 20 (17.4%) were heterozygous; the remaining 80 (69.6%) patients did not carry the p.F508del mutation. Exon 24 sequence had no change in 75 (65.2%) patients, 21 (18.3%) had the sequence variation 4521G/A, 11 (9.6%) had a not yet described sequence variation 4407T/A and 8 (7.0%) patients had both sequence variations (4521G/A and 4407T/A). The polymorphism 4407T/A results in an amino acid modification from aspartic acid to glutamic acid, which will probably have no function effect in CFTR. This low p.F508del prevalence can be due to the variable ethnic origin of this population from Minas Gerais, which may have a high diversity of CF rare mutations.

p.F508del in a heterogeneous cystic fibrosis population from Minas Gerais, Brazil

Cystic fibrosis (CF) is the most common autosomal recessive disease of the Caucasian population. Among the various CF mutations, p.F508del is the most frequent, accounting for two-thirds of the global CF chromosomes, although showing great variability among populations. We have studied 115 unrelated CF patients from a mixed population of Minas Gerais (Brazil). To evaluate part of the DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, blood DNA was obtained and PCR was performed using two pairs of primers that anneal to exons 10 and 24 of the CFTR gene. The PCR product was then submitted to automatic sequencing using the ABI PRISM 310 Genetic Analyzer. The p.F508del mutation was found in 50 (21.7 percent) of 230 unrelated CF alleles. Fifteen (13.0 percent) patients were homozygous for this mutation, while 20 (17.4 percent) were heterozygous; the remaining 80 (69.6 percent) patients did not carry the p.F508del mutation. Exon 24 sequence had no change in 75 (65.2 percent) patients, 21 (18.3 percent) had the sequence variation 4521G/A, 11 (9.6 percent) had a not yet described sequence variation 4407T/A and 8 (7.0 percent) patients had both sequence variations (4521G/A and 4407T/A). The polymorphism 4407T/A results in an amino acid modification from aspartic acid to glutamic acid, which will probably have no function effect in CFTR. This low p.F508del prevalence can be due to the variable ethnic origin of this population from Minas Gerais, which may have a high diversity of CF rare mutations.

Novel vasopressin type 2 (AVPR2) gene mutations in Brazilian nephrogenic diabetes insipidus patients.

Nephrogenic diabetes insipidus (NDI) is an inherited disorder characterized by renal resistance to the antidiuretic effect of arginine vasopressin (AVP), resulting in polyuria, polydipsia, and hypoosmolar urine. In the vast majority of cases, NDI is associated with germ-line mutations in the vasopressin receptor type 2 gene (AVPR2) and in about 8% of the cases with the water channel aquaporin-2 gene (AQP-2) mutations. To date, approximately 277 families with 185 germ-line mutations in the AVPR2 gene have been described worldwide. In the present study, the AVPR2 gene was genotyped in eight unrelated Brazilian kindred with NDI. In five of these NDI families, novel mutations were noted (S54R, I130L, S187R, 219delT, and R230P), whereas three seemingly unrelated probands were found to harbor previously described AVPR2 gene mutations (R106C, R137H, R337X). Additionally a novel polymorphism (V281V) was detected. In conclusion, although NDI is a rare disease, the findings of mutations scattered over the entire coding region of the AVPR2 gene are a valuable model to determine structure function relationship in G-protein-coupled receptor related diseases. Furthermore, our data indicate that in Brazil the spectrum of AVPR2 gene mutations is "family specific".

Investigation of A218C tryptophan hydroxylase polymorphism: association with familial suicide behavior and proband's suicide attempt characteristics.

According to WHO, suicide accounts for about 1,000,000 deaths worldwide every year. In view of these dramatic data, several studies have tried to identify possible biological mechanisms and markers of suicide. Genes encoding for proteins involved in the serotonergic transmission are major candidates in association studies of suicidal behavior. The gene that codes for tryptophan hydroxylase (TPH), the rate-limiting enzyme in the biosynthesis of serotonin, is one of these candidates. Two polymorphisms in intron 7 of this gene (A218C and A779C) have been described, but their role in suicidal behavior remains uncertain. TPH A218C polymorphism was analyzed in a sample of 248 psychiatric patients and 63 healthy controls. In addition, at least one close relative member was interviewed to assess family suicidal behavior history. Our research confirmed that a positive history of suicide attempts in a family member is associated with the chance of an individual to attempt suicide. Furthermore, we demonstrated that familial suicide attempts are more lethal and frequently more violent. We were not able to find significant differences of the TPH genotype frequencies between patients and controls. The TPH A218C genotypes were not associated with a history of suicide attempt and the lethality of the most lethal lifetime suicide attempt and suicide attempt method. The authors conclude that the A218C polymorphism of the TPH gene may not be a susceptibility factor for suicidal behavior in this group of psychiatric patients but confirm that a family suicidal behavior history increases the proband's suicide attempt risk.

A homozygous cathepsin C mutation associated with Haim-Munk syndrome.

Haim-Munk syndrome (HMS) is a rare autosomal recessive disorder characterized clinically by abnormal palmoplantar hyperkeratosis and destruction of the periodontium, with hallmarks of onychogryphosis and arachnodactyly. Germline mutations in the lysosomal protease cathepsin C gene (CTSC) have been described in a single patient with HMS and in several individuals with the clinically related disorder Papillon-Lefevre syndrome (PLS). We describe a patient with HMS. We have analysed the cathepsin C gene in the proband and her mother. Sequence analysis of CTSC in the proband revealed a homozygous mutation at codon 196 (587T-->C) within exon 4 that altered the conserved leucine to proline (Leu196Pro), whereas the patient's mother was heterozygous for that mutation. The same mutation has previously been described in an unrelated Brazilian family with PLS. An identical single missense mutation in the cathepsin C gene may underlie both PLS and HMS. These findings confirm that HMS and PLS are allelic variants of cathepsin C gene mutations and suggest that other factors (environmental or genetic) may be important determinants of the clinical phenotype of HMS and PLS.

Familial suicide behaviour: association with probands suicide attempt characteristics and 5-HTTLPR polymorphism.

OBJECTIVE: There is compelling evidence that a serotonergic dysfunction may play a major role in suicide behaviour and it has also been demonstrated that suicide is, at least partially, genetically determined. Thus, the serotonin-related genes are the major candidates. Previously a functional polymorphism in the promoter region of the serotonin transporter gene (5-HTTLPR) was identified and the presence of the short allele (S) was found to be associated with a lower level of expression of the gene and lower levels of 5-HT uptake when compared with the long allele (L). The purpose of this study was to evaluate the association between family suicide behaviour history and probands' suicide attempt (SA) history, SA characteristics and 5-HTTLPR genotype. METHOD: We genotyped 237 probands (major depressed or schizophrenic patients) and used a semistructured interview to determine probands' SA characteristics and first- and second-degree family suicidal behaviour. RESULTS: An association between suicidal family history and proband's SA but not with SA characteristics and probands genotype was found. CONCLUSION: Our results suggest that multiple biological and environmental factors underlie familial transmission of suicidal behaviour.

Thiopurine methyltransferase polymorphisms in a Brazilian population.

Thiopurine methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs. Low-activity phenotypes are correlated with several mutations in the TPMT gene. Polymorphisms of TPMT have been reported for Caucasians, African-Americans and Asians. Since ethnic differences have been demonstrated worldwide, it remains to be elucidated in Brazil. The Brazilian population is the result of five centuries of interethnic crosses between peoples from almost all continents as well as autochthonous Amerindians, all forming the fifth largest and one of the most heterogeneous populations in the world. The frequency of six allelic variants of the TPMT gene, *2 (G238C) (2.2%), *3A (G460A and A719G) (1.5%), *3B (G460A) (0.2%), *3C (A719G) (1.0%), *5 (0%) and *6 (0%) were determined in Brazilian subjects using polymerase chain reaction (PCR)-RFLP and allele-specific PCR-based assays. This study provides the first analysis of TPMT mutant allele frequency in a sample of the Brazilian population.

Analysis of T102C 5HT2A polymorphism in Brazilian psychiatric inpatients: relationship with suicidal behavior.

1. Central serotonergic dysfunction and genetic factors are associated with suicidal behavior in psychiatric patients. The goal of this study was to examine the association between the 5-HT2A gene polymorphism (102T/C) and suicide in a sample of Brazilian psychiatric inpatients. 2. We studied 225 subjects. Genotypic frequencies were obtained after DNA extraction and the region of 5-HT2A/T102C containing the polymorphic site amplified by the polymerase chain reaction and digested with the restriction enzyme HpaII. 3. No differences were found between patients with and without suicide attempt history. Patients with a history of severe suicide attempts also did not exhibit different genotypic frequencies when compared with patients without a suicide attempt history. 4. These results suggest that the 5HT2A gene polymorphism (102T/C) may not be involved in the genetic susceptibility to suicidal behavior.

Clonal composition of human adamantinomatous craniopharyngiomas and somatic mutation analyses of the patched (PTCH), Gsalpha and Gi2alpha genes.

Craniopharyngioma is the most common childhood tumor and thought to arise from embryonic remnants of Rathke's pouch. The paucity of published data on the molecular basis of these tumors prompted us to examine 22 adamantinomatous craniopharyngiomas looking for genetic abnormalities. Using the X-linked polymorphic androgen receptor gene as a tool for X-chromosome inactivating analysis, we found that a subset of craniopharyngiomas are monoclonal and therefore are probably due to acquired somatic genetic defects. Thus, we investigated these tumours for mutations within three candidate genes, Gsalpha, Gi2alpha and patched (PTCH). Using single stranded conformational polymorphism (SSCP), denaturing gradient gel electrophoresis and direct sequencing, the presence of somatic mutations in these genes could not be demonstrated in any tumor. Our data indicate that a subset of craniopharyngiomas are monoclonal and the mutations in the PTCH, Gsalpha, and Gi2alpha contribute little if any to craniopharyngioma development.

PTCH gene mutations in odontogenic keratocysts.

An odontogenic keratocyst (OKC) is a benign cystic lesion of the jaws that occurs sporadically or in association with nevoid basal cell carcinoma syndrome (NBCCS). Recently, the gene for NBCCS was cloned and shown to be the human homologue of the Drosophila segment polarity gene Patched (PTCH), a tumor suppressor gene. The PTCH gene encodes a transmembrane protein that acts in opposition to the Hedgehog signaling protein, controlling cell fates, patterning, and growth in numerous tissues, including tooth. We investigated three cases of sporadic odontogenic keratocysts and three other cases associated with NBCCS, looking for mutations of the PTCH gene. Non-radioactive single-strand conformational polymorphism and direct sequencing of PCR products revealed a deletion of 5 base pairs (bp) in exon 3 (518delAAGCG) in one sporadic cyst as well as mutations in two cysts associated with NBCCS, a nonsense (C2760A) and a missense (G3499A) alteration. This report is the first to describe a somatic mutation of PTCH in sporadic odontogenic keratocysts as well as two novel mutations in cysts associated with NBCCS, indicating a similar pathogenesis in a subset of sporadic keratocysts.

Microsatellite instability in sporadic parathyroid adenoma.

Parathyroid adenomas are usually benign uniglandular tumors, and inactivation of several tumor suppressor genes, notably the MEN 1 gene, or activation of oncogenes have been implicated in the tumorigenesis. Genomic instability, indicative of the involvement of DNA mismatch repair genes, has not been previously described in parathyroid adenomas. A single large parathyroid adenoma was resected from an 8.5-yr-old Brazilian patient with no personal or family history of other endocrinopathies. Analysis of paired tumor-nontumor DNA using 23 microsatellite markers, located on chromosomes 1, 10, and 11 was carried out. Microsatellite instability was detected in nine markers (D1S191, D1S212, D1S413, D1S2848, RET, D11S901, D11S903, INSR, and INT2), whereas no allelic loss was detected with any of the analyzed markers. Immunohistochemical analysis of retinoblastoma protein expression revealed low levels of expression, but no histopathological signs of malignancy. We conclude that in this single, apparently sporadic parathyroid adenoma, DNA mismatch repair genes might be involved in parathyroid tumorigenesis.

Molecular analyses of the vasopressin type 2 receptor and aquaporin-2 genes in Brazilian kindreds with nephrogenic diabetes insipidus.

Nephrogenic diabetes insipidus (NDI) is associated with germline mutations in two genes: vasopressin receptor type 2 (V2(R)) in X-linked NDI, and the water channel aquaporin-2, in autosomal-recessive disease. Genetic heterogeneity is further emphasized by reports of phenotypically abnormal individuals with normal structural genes. We analyzed both genes in five Brazilian families and the aquaporin-2 gene in two Swedish families with clinical and laboratory diagnosis of NDI, by a combination of denaturing gradient gel electrophoresis (DGGE) and direct DNA sequencing. A novel polymorphism in the aquaporin-2 gene (S167S), but no disease-associated mutations in any tested individual from all seven families, was detected. In two Brazilian families, frameshift mutations were detected in the V2(R) gene: one leading to a premature stop after codon 36 and the other to a longer peptide (462 aa instead of the 373 aa wild-type protein). In two other Brazilian families, probable disease-associated missense mutations were detected: an alanine to proline at codon 163 (A163P) and an asparagine to aspartic acid at codon 85 (D85N). In one Brazilian family, both genes were structurally normal and the aquaporin-2 gene was also normal in the two Swedish kindreds. This report further extends the mutational spectrum of NDI and suggests that there are other mutational or epigenetic events inactivating the two known genes or even novel genes that underlie NDI.

Mutational analyses of candidate genes in human squamous cell carcinomas.

OBJECTIVES AND STUDY DESIGN: Squamous cell carcinomas are common malignancies and a major cause of mortality. The molecular mechanisms involved in tumorigenesis remain largely unknown, but sequence alterations have been identified in coding regions of several genes. Primary squamous cell carcinomas of various tissues (skin, head and neck, esophagus, lung, penis, uterus, and vagina) from 52 patients were analyzed for the presence of mutations within several candidate genes presumably involved in tumorigenesis: Gsalpha, Gi2alpha, GTPase activating protein (GAP), and patched (PTCH) genes. METHODS: Mutational analysis scheme included polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), single stranded conformational polymorphism (SSCP), and selected sequence analysis. RESULTS AND CONCLUSION: No tumor had any evidence of mutations in any of these analyzed genes. Mutations within these genes do not occur frequently in an unselected population of patients with squamous cell carcinomas.

Odontogenic myxomas are not associated with activating mutations of the Gs alpha gene.

BACKGROUND: Myxoma is a rare bone tumor of the mandible and maxillary sinus whose etiology and underlying molecular mechanisms remain unknown. Mutations that inhibit the GTPase activity of the a subunit of the stimulating G protein (Gsa) have been demonstrated in the myocardium of patients with McCune-Albright syndrome. The histopathological similarities shared by cardiac and jaw myxomas coupled with the paucity of reported candidate genes involved in jaw tumor pathogenesis, prompted us to investigate for the presence of gsp mutations in 23 sporadic jaw myxomas. MATERIALS AND METHODS: We used the polymerase chain reaction (PCR) to amplify the appropriate genomic fragments, followed by denaturing gradient gel electrophoresis (DGGE) analysis. RESULTS: No gsp mutations could be demonstrated in any of tumors analyzed, while the technique has a proven capability to detect these specific mutations. CONCLUSIONS: We conclude that mutations of the Gs alpha gene rarely, if ever, are associated with sporadic jaw myxomas tumorigenesis.

Sporadic cardiac myxomas and tumors from patients with Carney complex are not associated with activating mutations of the Gs alpha gene.

Cardiac myxomas are rare tumors that may be encountered sporadically or in the context of the Carney complex. The molecular basis for the development of cardiac myxomas and Carney complex tumors is unclear. Pathological myocardial function and myocardial hypertrophy have been associated with alterations in the heterotrimeric GTP-binding proteins. The postulated proto-oncogenic character of the gene encoding the alpha sub-unit of the stimulatory GTP-binding protein Gs alpha (gsp) in pituitary and thyroid tumors, the finding of identical somatic gsp mutations in the myocardium of patients with McCune-Albright syndrome, and the associated endocrine anomalies of the Carney complex prompted us to investigate the occurrence of activating missense mutations in the Gs alpha gene in 10 sporadically occurring atrial myxomas and in 8 tumors from 7 patients with Carney complex. No gsp mutations could be demonstrated by using the polymerase chain reaction and denaturing gradient gel electrophoresis complemented by direct DNA sequencing. Thus, activating Gs alpha mutations neither are associated with the development of atrial myxomas, nor can be demonstrated in other tumors from patients with Carney complex. The significance of these mutations in the myocardium of asymptomatic patients with McCune-Albright syndrome remains to be determined.

Nephrogenic diabetes insipidus: an X chromosome-linked dominant inheritance pattern with a vasopressin type 2 receptor gene that is structurally normal.

Nephrogenic diabetes insipidus is a rare hereditary disorder, most commonly transmitted in an X chromosome-linked recessive manner and characterized by the lack of renal response to the action of antidiuretic hormone [Arg8]vasopressin. The vasopressin type 2 receptor (V2R) has been suggested to be the gene that causes the disease, and its role in disease pathogenesis is supported by mutations within this gene in affected individuals. Using the PCR, denaturing gradient gel electrophoresis, and direct DNA sequencing, we examined the V2R gene in four unrelated kindreds. In addition, linkage analysis with chromosome Xq28 markers was done in one large Brazilian kindred with an apparent unusual X chromosome-linked dominant inheritance pattern. In one family, a mutation in codon 280, causing a Tyr-->Cys substitution in the sixth transmembrane domain of the receptor, was found. In the other three additional families with nephrogenic diabetes insipidus, the V2R-coding region was normal in sequence. In one large Brazilian kindred displaying an unusual X chromosome-linked dominant mode of inheritance, the disease-related gene was localized to the same region of the X chromosome as the V2R, but no mutations were found, thus raising the possibility that this disease is caused by a gene other than V2R.