Suemi Marui

Apparent mineralocorticoid excess in a Brazilian kindred: hypertension in the heterozygote state

Background Apparent mineralocorticoid excess (AME) is a cause of low-renin, low-aldosterone hypertension in which cortisol acts as a mineralocorticoid. The condition reflects an inability to inactivate cortisol to cortisone due to defective activity of the type 2 isozyme of 11b-hydroxysteroid dehydrogenase (11β-HSD2). Homozygous mutations in 11b-HSD2 gene in patients with AME have been described. A 7-year-old Brazilian girl had previously been found to have AME. Her father recently presented with mineralocorticoid hypertension at age 38 years. Objective To describe the clinical details, to perform steroid analyses and to assess the molecular basis for the hypertension in this kindred. Methods The 11βHSD2 gene was amplified from genomic DNA by the polymerase chain reaction and sequenced by direct chain-termination sequencing on an automatic DNA sequencer. The sequencing results were validated by restriction-site polymorphism. The mutant 11β-HSD2 protein was expressed in Chinese hamster ovary polyoma cells and enzymatic activity was assessed by metabolizing cortisol in vitro. Results Sequence analysis of genomic DNA revealed a novel C1061T point mutation in exon V of the human 11β-HSD2 gene, resulting in an amino acid substitution of alanine by valine at codon 328 of the enzyme protein (A328V). Expression studies confirmed that the mutant protein was devoid of 11β-HSD2 activity. A HhaI restriction-site polymorphism confirmed that the proband was homozygous for the mutation whereas both parents were heterozygotes. The father of the proband had hypertension, a normal serum potassium level, suppressed plasma renin activity and plasma aldosterone level and a moderately elevated urinary cortisol: cortisone metabolite ratio. Conclusions AME in this kindred is caused by a novel mutation in the 11β-HSD2 gene. Detection of hypokalaemia, at least in this kindred, is an insensitive screening test for mineralocorticoid-based hypertension. In contrast to results from previously investigated kindreds, we have demonstrated that this kindred has an abnormal phenotype in the heterozygote state. Further studies are now required in order to evaluate the role of 11β-HSD2 activity in the pathophysiology of ‘essential’ hypertension.

GH Values after Clonidine Stimulation Measured by Immunofluorometric Assay in Normal Prepubertal Children and GH-Deficient Patients

Objective: To establish the cut-off values of GH measured by immunofluorometric assay, a more sensitive and specific assay, in normal prepubertal children and compare their values with those of proven GH-deficient patients. Methods: 30 normal children (20 males) and 26 patients with known causes of GH deficiency were submitted to the clonidine test and their GH values were compared. A powdered clonidine tablet (0.1 mg/m2) was given orally and blood samples for GH measurements were drawn at times –30, 0, 60, 90 and 120 min. Results: GH peak values presented a wide variation ranging from 1.7 to 25 µg/l (mean ± SD = 12.87 ± 5.8 µg/l) in the normal group. The cut-off values for the 5th and 10th percentiles of the distribution curve were 3.3 and 5.5 µg/l, respectively. In the GH deficiency group, maximum GH levels after clonidine stimulation ranged from <0.1 to 2.1 µg/l (0.56 ± 0.58 µg/l). Conclusions: The cut-off values obtained with the immunofluorometric method are lower than the ones obtained by radioimmunoassay. We suggest a cut-off value of 3.3 µg/l (5th percentile) that ensures 100% of sensitivity along with 93% of specificity to exclude the diagnosis of GH deficiency when using this immunofluorometric method.

Hormonal, pituitary magnetic resonance, LHX4 and HESX1 evaluation in patients with hypopituitarism and ectopic posterior pituitary lobe

Objective  LHX4 and HESX1 are important in early stages of pituitary development and their mutations can be associated with an ectopic posterior lobe (EPL) in the pituitary of patients with hypopituitarism. The EPL can be located at the median eminence or at the path of the pituitary stalk. The aim of this study was to analyse LHX4 and HESX1 and characterize the hormonal deficiency profiles, establishing relationships with magnetic resonance imaging (MRI) findings in these patients.

Correlations of CTLA‐4 gene polymorphisms and hepatitis C chronic infection

Cytotoxic T lymphocyte‐associated factor 4 (CTLA‐4) functions as a negative regulator of T cell‐mediated immune response. Molecular changes associated to CTLA‐4 gene polymorphisms could reduce its ability to suppress and control lymphocyte proliferation.

Pre-operative role of BRAF in the guidance of the surgical approach and prognosis of differentiated thyroid carcinoma

OBJECTIVE The p.V600E BRAF and RAS mutations are found in 30-80% of differentiated thyroid carcinoma (DTC). BRAF mutation has been associated with poor prognosis. This study investigated the role of molecular studies in preoperative diagnosis of DTC and the association of p.V600E mutation with prognostic factors. DESIGN Prospective study. METHODS A total of 202 patients with cytological diagnosis of Bethesda III-VI underwent preoperative molecular studies and subsequent thyroidectomy. p.V600E and RAS mutations were studied in the cytology smears, using real-time PCR genotyping technique. The BRAF mutation (BRAF(+) or BRAF(-)) was correlated with histological and clinical findings. RESULTS Molecular study of 172 nodules with Bethesda III-V cytology improved negative predictive value and accuracy of Bethesda III and IV diagnosis. BRAF mutation was present in 65% of 94 DTC and p.Q61R NRAS in one. Except for age, BRAF(+) and BRAF(-) did not differ in sex, tumor size, histological subtype, multifocality, vascular invasion, extrathyroidal extension, or prognostic staging. Among papillary carcinomas, lymph node (LN) metastasis was diagnosed in 23% BRAF(+) and 37% BRAF(-). Distant metastasis occurred in four BRAF(-). Recurrent or persistent disease was more frequent in BRAF(-) (26.7 vs 3.3% BRAF(+), P=0.002) along follow-up of 29.8±10 months. BRAF(+) patients without LN metastasis by pre-operative evaluation submitted to thyroidectomy with central neck dissection (CND) had more frequent LN metastasis (45 vs 5% no CND, P=0.002), but no difference in clinical outcome was observed. CONCLUSIONS Pre-operative identification of BRAF mutation improved cytological diagnosis of DTC, but it was not associated with poor prognostic factors. Prophylactic CND did not guarantee better outcome in BRAF(+) patients.

GH-releasing hormone receptor gene: a novel splice-disrupting mutation and study of founder effects.

Background: Mutations in GH-releasing hormone receptor gene (GHRHR) are emerging as the most common cause of autosomal recessive isolated GH deficiency (IGHD). Objective: To search for GHRHR mutations in patients with familial or sporadic IGHD and to investigate founder effects in recurring mutations. Methods: The coding region of GHRHR was entirely amplified and sequenced from DNA of 18 patients with IGHD (16 unrelated) with topic posterior pituitary lobe on MRI. Haplotypes containing promoter SNPs and microsatellites flanking GHRHR were analyzed in patients with c.57+1G>A (IVS1+1G>A) mutation of our previously published kindred and also a Brazilian patient and 2 previously reported Japanese sisters with c.1146G>A (p.E382E) mutation. Results: A novel homozygous intronic GHRHR c.752-1G>A (IVS7-1G>A) mutation, predicting loss of the constitutive splice acceptor site, was identified in two siblings with IGHD. A compound heterozygous c.[57+1G>A];[1146G>A] and a heterozygous c.527C>T (p.A176V) were found in two sporadic cases. Haplotype analysis provided evidence for a founder effect for the c.57+1G>A mutation and independent recurrence for the c.1146G>A mutation. Conclusion: We report a novel splice-disrupting mutation in GHRHR in 2 siblings and provide evidence that all c.57+1G>A (IVS1+1G>A) mutant chromosomes have the same haplotype ancestor, indicating the occurrence of a founder effect in Brazilian patients with IGHD.

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.

Novel mutation in MCT8 gene in a Brazilian boy with thyroid hormone resistance and severe neurologic abnormalities

MCT8 is a cellular transporter of thyroid hormones important in their action and metabolization. We report a male patient with the novel inactivating mutation 630insG in the coding region in exon 1 of MCT8. He was characterized clinically by severe neurologic impairment (initially with global hypotonia, later evolving with generalized hypertonia), normal growth during infancy, reduced weight gain, and absence of typical signs and symptoms of hypothyroidism, while the laboratory evaluation disclosed elevated T3, low total and free T4, and mildly elevated TSH serum levels. Treatment with levothyroxine improved thyroid hormone profile but was not able to alter the clinical picture of the patient. These data reinforce the concept that the role of MCT8 is tissue-dependent: while neurons are highly dependent on MCT8, bone tissue, adipose tissue, muscle, and liver are less dependent on MCT8 and, therefore, may suffer the consequences of the exposition to high serum T3 levels.

High Prevalence of Pituitary Magnetic Resonance Abnormalities and Gene Mutations in a Cohort of Brazilian Children with Growth Hormone Deficiency and Response to Treatment

Data were retrospectively collected from 69 Brazilian patients (45 boys) with growth hormone deficiency (GHD) who received exogenous growth hormone (GH) for a median duration of 4 years (range 1-13 years). Forty-two patients had multiple pituitary hormone deficiencies and 27 had isolated GHD. Peak GH was < 7 ng/ml (IRMA) or < 3.2 ng/ml (IFMA) after two stimulation tests. Therapy was started at median age of 10.0 years (range 2.2-21.6 years), bone age of 5.8 years (0.5-13.5 years) and height standard deviation score -4.4 (range -9.3 to -1.6). MRI revealed pituitary abnormalities in 87% of patients. Homozygous mutations in PROP-1, GHRH-R, GH-1 or HESX-1 genes were found in 12 patients. Mean height velocities were 3.3 pretreatment and 10.3, 7.8, 7.4 and 6.4 cm/yr, respectively, during 1-4 years of treatment with GH. In conclusion, the high prevalence (96%) of genetic and/or pituitary abnormalities probably reflects the stringent diagnostic criteria used, and GH replacement resulted in significant catch-up growth.

Factitious Thyrotoxicosis Induced by Mesotherapy: A Case Report

CONTEXT Mesotherapy consists of cutaneous injections of a mixture of compounds and has recently been used for cosmetic purposes to reduce local fat and cellulite. To date, several reports have described only local adverse events related to this therapy. We describe the first report of a female patient who developed thyrotoxicosis due to cosmetic mesotherapy with triiodothyroacetic acid in its formulation. Apart from mechanical rupture of the epidermal barrier, a disturbance of type III deiodinase activity or skin fibroblast paracrine function and vascular alterations related to simultaneously injected vasoactive compounds were observed. These findings could be related to thyroid hormone metabolite absorption and systemic consequences in the reported case. CONCLUSION We describe factitious thyrotoxicosis induced by mesotherapy, to raise awareness of a systemic adverse effect resulting from this widespread cosmetic practice.