Population screening of F508del (DeltaF508), the most frequent mutation in the CFTR gene associated with cystic fibrosis in Argentina.

Cystic fibrosis (CF) is the most common autosomal recessive disease in the Caucasian population. The disease can be caused by one of the more than 900 different mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. However, the deletion of the phe508-codon is the most prevalent mutation observed. Our aim was to perform a screening for this mutation (DeltaF508, or F508del) in the population of Mendoza, Argentina. For the screening, 1,000 blood samples were obtained from CF asymptomatic individuals and combined into 100 pools each containing 10 different blood samples. Pools containing at least one F508del carrier were detected by heteroduplex formation during the PCR amplification of exon 10. The PCR was designed to introduce a recognition site for a restriction enzyme that confirmed the presence of the deletion F508del in the positive pools. The results with this simple method indicate a frequency of carriers in the Mendoza population of 2.1% (1.3%-3.2, 95% confidence limits). The observed frequency of carriers is similar to that reported for European populations. Hum Mutat 18:167, 2001.

High prevalence of vitamin D insufficiency in healthy elderly people living at home in Argentina.

OBJECTIVE: To evaluate the nutritional status of vitamin D in urban populations of healthy elderly people living at home, in different regions of Argentina. DESIGN: Cross-sectional study. SUBJECTS: In total, 386 ambulatory subjects over 65 y of age from seven cities (between latitude 26 degrees S and 55 degrees S) were asked to participate between the end of winter and the beginning of spring. Of these, 369 accepted, 30 were excluded because of medical history or abnormal biochemical determinations. Finally, 339 subjects (226 women and 113 men) (X+/-s.d.) (71.3+/- 5.2 y) were included. RESULTS: Serum 25OHD levels were lowest in the South (latitude range: 41 degrees S-55 degrees S): 14.2+/-5.6 ng/ml (P<0.0001vs North and Mid regions); highest in the North (26 degrees S-27 degrees S): 20.7+/-7.4 ng/ml (P<0.03 vs Mid, P<0.0001vs South); and intermediate in the Mid region (33 degrees S-34 degrees S) 17.9+/-8.2 ng/ml. Serum mid-molecule PTH (mmPTH) and 25OHD were inversely related: (r=-0.24, P<0.001). A cutoff level of 25OHD at which serum mmPTH levels began to increase was established at 27 ng/ml. A high prevalence (87-52%) of subjects with 25OHD levels in the deficiency-insufficiency range (25OHD levels <20 ng/ml) was detected. CONCLUSION: This study shows that vitamin D deficiency/insufficiency in the elderly is a worldwide problem. Correction of this deficit would have a positive impact on bone health of elderly people.

[Diagnosis by directed mutagenesis of a mutation at the hMSH2 gene associated with hereditary nonpolyposis colorectal cancer]. / Diagnóstico por mutagénesis dirigida de una mutación en el gen hMSH2 vinculada a cáncer colorrectal hereditario no poliposo.

Hereditary nonpolyposis colorectal cancer (NHPCC) is the most common form of inherited colon cancer and one of the most frequent autosomal dominant disorders. HNPCC presents an early onset of colorectal cancer (< 50 years), proximal localization of the colonic tumors, and high risk of developing multiple primary colorectal tumors as well as extracolonic tumors. This disease is caused by mutations in at least four DNA mismatch repair genes, (hMSH2, hMLH1, hPMS1 and hPMS2) and estimations indicate that it affects 1:200-1:2,000 people in the Western populations. The identification of the genes responsible for HNPCC has prompted the search for mutations in affected individuals. DNA from an affected member of a family was sent to a Dutch HNPCC Diagnosis Centre. This Centre reported a germinal mutation, which introduces a premature stopcodon and causes the production of a truncated protein. This particular mutation has not been previously registered in the database of mutations related to this disease. After the identification of the mutation in the index patient, we have developed a quick and efficient procedure for detecting mutations in the rest of the family. The methodology is based on the amplification of the exon 13 in the hMSH2 gene using a forward primer that abuts the mutation site and introduces the cutting sequence of the enzyme Haelll++ only in the wild type allele. At present, seventeen members of the family have been diagnosed and nine have been found to be affected. The methodology is simple, specific, sensitive, inexpensive and applicable in low complexity laboratories.

Management of overt and subclinical hypothyroidism. Factors influencing L-thyroxine dosage.

With the aim of establishing optimal dosage schedules, 171 women with either overt (OH, n = 80) or subclinical (SCH, n = 91) hypothyroidism were assessed before and 6 months after starting L-thyroxine (LT4) replacement therapy. Each group was further classified into four subgroups according to post-therapy serum TSH level, as follows: A) complete suppression; B) partial suppression; C) normal range and D) above normal range (insufficient response). In all subgroups, LT4 doses were higher for OH than for SCH, whether expressed as total daily dose (micrograms) or as a function of either actual or ideal body weight (micrograms/kg BW). In OH, LT4 dose was higher for subgroups A or B as compared with either C or D. In SCH, subgroup A received a larger dose than the other subgroups. Post-treatment serum thyroxine levels showed the same pattern for both OH and SCH. Mean LT4 dose was similar in patients with high and normal antithyroid antibodies and in patients with goiter and in those without it. In goitrous patients thyroid volume decreased in subgroup B, particularly in those patients that had elevated antithyroid antibodies, but not in subgroup C. In OH patients a significant negative correlation was found between daily LT4 dose per kg actual BW and actual BW, especially in subgroup C for patients with a body mass index > 27 kg/cm2 (r = -0.90, p < 0.001). In subgroup C of the SCH group, a negative correlation between LT4 dose and age was noticed. Both in OH and in SCH, LT4 dose per kg actual BW required to obtain a serum TSH within the normal range was lower in women with a body mass index (BMI) > 27 kg/m2 than in those with a BMI < or = 27 kg/m2. LT4 doses for subgroup C did not differ from those needed in hypothyroid patients with previous Graves' disease, in either OH or SCH patients.

Changes in the content of rat epididymal fluid induced by prolonged treatment with tamoxifen.

Prolonged treatment with tamoxifen induces changes in the male reproductive tract in rats. In this study changes in the protein content of the rat epididymal fluid as a consequence of prolonged treatment with tamoxifen are reported. Among five lysosomal enzymes measured in the epididymal fluid, alpha-mannosidase (alpha-MAN) significantly diminished, but other enzymes did not. Electrophoretic analysis of fluids showed that proteins of estimated molecular weight 25, 60, 80-85 and 180 kDa decreased in the treated rats. We also detected an increase in the binding of beta-galactosidase (beta-GAL) to caudal spermatozoa in treated rats. These changes may be related in part to the loss of fertilizing capacity of spermatozoa after tamoxifen treatment.

TSH Normal Alta: Su relación con bajo HDL-Colesterol en mujeres con resistencia a la insulina con independencia de otros posibles factores concurrentes / High Normal TSH: Its relationship with low HDL-Cholesterol in women with insulin resistance regardless of other possible concurrent factors

Objetivo: En el hipotiroidismo tanto clínico como subclínico se han descripto alteraciones en el metabolismo lipídico, entre ellas la disminución de colesterol de lipoproteínas de alta densidad (HDL-C). Considerando el rango normal de TSH entre 0.4-3.0 μUI/ml y valores normales altos entre 2.0 y 3.0 μUI/ml, nosotros investigamos la hipótesis de que niveles normales altos de TSH e insulinorresistencia (IR) se encuentran relacionados con HDL-C bajo en mujeres, en ausencia de otros factores concurrentes. Materiales y métodos: Estudiamos en un estudio transversal a 200 mujeres sanas, edad 18-50 años, eutiroideas, normotensas, con anticuerpos antiperoxidasa (ATPO) negativos, no diabéticas, premenopáusicas, IMC 18.0-25.0 Kg/m2, perímetro de cintura ≤ 88 cm; perímetro de cuello ≤ a 35 cm. Se las dividió en 4 grupos, cada uno compuesto por 50 mujeres: Grupo 1 (G1) TSH ≥ 2μU/ml, IR; grupo 2 (G2) TSH ≥ 2μU/ml, no IR; grupo 3 (G3): TSH 0,40 a 1,99 μU/ml, IR; Grupo 4 (G4) TSH 0,40 a 1,99 μU/ml, no IR. Se les midió lípidos, TSH, T4 total y libre (T4L), glucosa e insulina basal y posprandial, índices HOMA y QUICKI y volumen tiroideo (VT). Resultados: Observamos que en el G1 el nivel de HDL-C (46,7± 8,1 mg/dl) fue significativamente menor que en los restantes grupos. (vs G2: 56,8 ± 8,6 mg/dl; vs G3: 51,2 ± 7,6 mg/dl y vs G4: 56,5 ± 9,1 mg/dl. (p<0,01). La frecuencia de pacientes con HDL-C bajo en G1 fue significativamente mayor que en los restantes grupos (vs G2: OR 1,83, IC: 1,23-2,70; vs G3: OR 1,49, IC: 1,04-2,31; vs G4: OR 1,90, IC: 1,29-2,81) . No encontramos diferencias significativas en los niveles de HDL-C entre los restantes grupos. Conclusiones: Observamos en mujeres eutiroideas con TSH normal alta insulinorresistentes, sin otros factores concurrentes, niveles de HDL-C significativamente más bajos que en mujeres no insulinorresistentes y que en mujeres insulinorresistentes con TSH normal baja.

It has been described abnormalities in lipid metabolism in clinical and subclinical hypothyroidism, including the reduction of high-density lipoprotein cholesterol (HDL-C). Considering the normal range for TSH between 0.4-3.0 uUI / ml and high-normal values between 2.0 and 3.0 uUI / ml, we investigated the hypothesis that in euthyroid women, high-normal TSH levels and insulin resistance (IR) are associated with low HDL-C. We observed in euthyroid women with high-normal TSH and insulin resistance, without other factors, a significantly lower level of HDL-C than in non-insulin or insulin resistance women with low-normal TSH.

Detección de una mutación no estándar en el proto-oncogen ret por mutagénesis dirigida / Detection of a non-standard mutation in the ret protoncogene by site directed mutagenesis

MEN2A is an autosomic dominant disease, characterized by medullary thyroid cancer, pheochromocytoma and parathyroid hyperplasia. Mutations in the ret proto-oncogene are associated with this disease, with almost 100% of penetrance. The gene, situated on chromosome 10q11.2, codes for a transmembrane protein with a tyrosinkinase-like receptor function. Mutations that affect its extracellular domain, stimulate spontaneous homodimerization and elevate the basal tyrosinkinase activity. The codon 634 of the gene is considered a hot-spot site, since it is mutated in 85% of the MEN2A families. Our group developed in 2002 an indirect and costless strategy to detect alterations in this site. We present a family suspected of having MEN2A. We applied our PCR based indirect strategy on the DNA of the index patient and found that there was no mutation in that site. Posterior sequencing of exon 10 and 11 confirmed that the mutation affecting this family was in codon 611. Thus, we developed a new costless family-specific strategy based on mutagenic PCR and enzymatic cuts to diagnose all the family members. A seven-year old boy with this mutation was preventively thyroidectomized. In this way, combining the indirect methodology for codon 634 previously developed by our group, and a posterior family-specific mutation detection strategy, we were able to diagnose and intervene presymptomatically the family members, avoiding sending all the samples to foreign centers.

El síndrome de MEN2A es una enfermedad autosómica dominante que se caracteriza por el desarrollode cáncer medular de tiroides, feocromocitoma e hiperplasia de paratiroides. Mutaciones en elret proto-oncogén se asocian con MEN2A, con una penetrancia cercana al 100%. El gen se encuentra en elcromosoma 10q11.2 y codifica para una proteína transmembrana con función de receptor del tipo tirosina quinasa.Mutaciones que afectan el dominio extracelular de la proteína estimulan la dimerización espontánea del receptory un aumento de la actividad de tirosina quinasa basal. El codón 634 codifica para una cisteína, y es consideradoun sitio hot-spot por encontrarse mutado en el 85% de las familias con MEN2A. Para este sitio, nuestro grupo desarrolló en 2002 una metodología de detección indirecta y económica. Ante una familia sospechada de MEN2A, se aplicó esta estrategia, que reveló un codón 634 sano. Por posterior secuenciación se confirmó que el paciente índice portaba una mutación en el codón 611. Se desarrolló una nueva estrategia familiaespecífica por PCR mutagénica, que permitió diagnosticar en nuestro país a todos los integrantes de la familiacon costos accesibles. Un niño en el cual se halló la mutación, fue tiroidectomizado preventivamente, y a lafecha goza de buena salud. De esta manera, combinando la estrategia de detección de mutaciones en el sitiohot-spot y un posterior diseño de otra metodología familia-específica se pudo diagnosticar e intervenir preventivamente a la familia, sin enviar todas las muestras al extranjero.