A novel mutation in the TPO gene in goitrous hypothyroid patients with iodide organification defect.

OBJECTIVE: To screen and subsequently sequence the TPO gene for mutations in patients with congenital goitre, hypothyroidism and evidence for an organification defect (positive perchlorate discharge test). PATIENTS: We have studied seven hypothyroid and congenitally goitrous patients from three unrelated families. DESIGN AND MEASUREMENTS: We have measured serum thyroid hormone levels, 131I uptake, serum TSH and serum Tg concentrations. Denaturing gradient gel electrophoresis (DGGE) of PCR amplified genomic DNA was used to screen for mutations in the TPO gene. RESULTS: DGGE identified the presence of two frameshift mutations: a GGCC duplication in exon 8 (homozygous in one family and heterozygous in the other family) and a heterozygous insertion of a single nucleotide (C) at position 2505-2511 in exon 14. In addition, we have detected an alteration in exon 11, not yet described in the literature, derived from a single nucleotide substitution of a C to G at position 2008, altering the well-conserved amino acid domain among the peroxidases superfamily. This mutation in exon 11 was present in two families that showed heterozygous mutation for exon 8 or for exon 14. CONCLUSIONS: These results could support the hypothesis for a putative compound heterozygosity pattern in the affected patients. The altered phenotype (goitre and hypothyroidism since birth) seems justifiable in view of the possible inactivating character of this novel mutation in exon 11.

Thyroid peroxidase in dyshormonogenetic goiters with organification and thyroglobulin defects.

Thyroid peroxidase (TPO) iodide and guaiacol oxidation activities were evaluated in eight dyshormonogenetic goiters. Two of these had a defective thyroglobulin; the TPO iodide oxidation (431 and 316 U/g ptn) and iodination (31 and 8.6 nmol I/mg ptn) activities were within the normal ranges. The goiters from two siblings with positive perchlorate iodide discharge tests also had normal TPO iodide oxidation (602 and 299 U/g ptn) and iodination activities (44 and 11 nmol I/mg ptn). No TPO iodide oxidation activity was found in the goiters from the other four patients with positive perchlorate iodide discharge tests, and TPO iodide oxidation inhibitory activities were detected in both their TPO and thyroglobulin preparations. Three of them had some TPO guaiacol oxidation activity and did not inhibit normal guaiacol oxidation. The TPO preparation immunoblot of these three goiters showed a faintly visible band of normal 100 kDa TPO. However, in the other patient no guaiacol oxidation activity was detected, and only two bands of low-molecular-weight TPO (72 and 43 kDa) were found, again showing that iodine organification defects in dyshormonogenetic goiters can be due to either qualitative or quantitative TPO defects. The TPO inhibition diminished when iodide was increased in the assay, but was not altered by increasing cofactor (H2O2). Our results, so far, suggest that the TPO-inhibitory substance may interact reversibly with a specific iodide site on the enzyme or with the oxidized form of iodide, and/or could bind free iodide, making it unavailable for enzymatic oxidation.

[Specific count of cerebrospinal fluid cells in the counting chamber using supravital staining]. / Contagem específica de células do líquido céfalo raquideano em câmara usando coloração supravital.

The authors adapted to the cerebrospinal fluid (CSF) the staining technic developed by Anguiano and Ancira to perform differential leucocyte counts in the counting chamber. The formula they used is the following: methyl alcohol 1 ml, distilled water 2 ml. To this mixture are added: 6 drops of Leishman stain filtered in Whatman paper number 42; toluidine blue 0.25% aqueous solution (filtered in the same manner), 1 drop; acetate buffer 0.1 M, pH 5.4, 1 drop. The technic varies according to the intensity of pleocytosis. If the CSF is turbid or contains more than 100 cells per c.m. 1 drop is dripped in the bottom of a 10X75 test tube and then 2 drops of the staining fluid are added; the mixture is then shaken; after one or two minutes 1 drop is placed in the Fuchs-Rosenthal counting chamber. If the number of total cells is less than 100 per c.m. different CSF volumes are centrifuged at the rate of 2000 r.p.m., during 6 minutes; the supernatant fluid is poured off and the sedimented cells are suspended in 2 drops of the staining fluid. The morphology of the cells as they appear after they are stained by the supravital staining technic (SVST) is described and illustrated in photomicrographies.(ABSTRACT TRUNCATED AT 250 WORDS)

[Cytologic examination of the cerebrospinal fluid in the diagnosis of primary or metastatic neoplasms of the nervous system]. / Exame citológico do líquido céfalo-raquidiano no diagnóstico de neoplasias primitivas ou metastáticas do sistema nervoso: a propósito de seis casos.

The authors report 6 cases of cerebrospinal fluid (CSF) examinations in which malignant cells were found. In 5 cases the finding was incidental: medulloblastoma (case 1); malignant melanoma (case 2); adenocarcinoma (case 3); meningitis carcinomatosa (case 4) and neuroleukaemia (case 5). In only one case neuroleukaemia was suspected before the study of the CSF (case 6). The unexpected occurrence of malignant cells in the CSF demands the pathologist to be well acquainted with tumor cell cytology, in order to identify them providing so useful information that can decidedly influence subsequent diagnostic and therapeutic procedures. The cell collection technic developed in the authors' laboratory was considered adequate, because both inflammatory and malignant cells were securely identified. Its principle is to obtain thin air-dried smears that dry almost instantaneously on slides, in order the cellular structure be preserved. After centrifuging about 5-8 ml of CSF the tube is inverted so as to pour off the whole of the supernatant fluid. It is kept inverted with the operator's left hand, so that no drop of fluid can run back on to the cells. With his right hand the operator touches the bottom of the inverted tube with a Pasteur micropipette preciously made by distending a microhematocrit tube under the flame of a Bunsen burner and attached with adhesive tape to the handle of a wire loop 2 mm in diameter.(ABSTRACT TRUNCATED AT 250 WORDS)