Variable clinical presentation and outcome in pediatric patients with resistance to thyroid hormone (RTH).

Resistance to thyroid hormone (RTH) is characterized by elevated levels of thyroid hormones, normal or slightly increased TSH levels respondent to TRH, resistance to thyroid hormone administration, and variable clinical expression. To describe the diverse clinical and biochemical findings of six children from five unrelated families with molecular diagnosis of RTH (0.5-12.7 years) and their follow-up (3-20 years). All RTH patients and 4 affected parents' harbored mutations in exons 9 or 10 of the thyroid receptor ß gene: p.M313T (de novo), pN331D, p.L341P, p.L346F, and p.P453L. At consultation 5/6 had goiter, 4/6 tachycardia, and 3/5 learning disabilities. Median hormone levels were: T(4) 257.4 nmol/l (NR: 77.2-180.2); FreeT(4) 39.9 pmol/(NR:10.3-28.3); T(3) 4.28 nmol/l (NR:1.23-3.39) TSH 2.8 mUI/l (NR: 0.5-5) always responsive to TRH. TSH levels remained detectable after supraphysiologic T(3) administration while SHBG levels showed a paradoxical decrease in 4/6. Thyroid antibodies, initially present in two subjects, became positive in other two during follow-up. All patients grew normally and presented variable symptoms that were treated according to need. Two patients developed psychiatric disorders. Only one of the four affected parents exhibited clinical signs of RTH (tachycardia and depression). Parent's thyroid profile showed similar TSH and T(3) levels but lower T(4) and FT(4) than their children. RTH has a distinctive biochemical profile with highly variable clinical manifestations and outcomes. Its recognition and molecular characterization avoid misleading diagnosis. Treatment has to be instituted according to each subject's own clinical requirements.

Detection of the nucleophosmin gene mutations in acute myelogenous leukemia through RT-PCR and polyacrylamide gel electrophoresis.

OBJECTIVES: Mutations in the C-terminal region of the nucleophosmin (NPM1) gene occur in approximately 60% of acute myeloid leukemia (AML) cases with normal karyotype and represent the most common genetic lesion presently known in this disease. Because of their frequency and favorable impact on prognostic outcome, screening for this aberration is currently recommended in routine diagnostic characterization of AML. Several techniques enabling to detect NPM1 mutation have been reported, but all require sophisticated equipment, which represent an obstacle particularly in countries with limited resources. METHODS: We designed an RT-PCR strategy to amplify NPM1 exon 12 followed by electrophoresis and fragment visualization on polyacrylamide gels to discriminate a 4-5 bp size difference resulting from mutations in this gene. A hemi-nested method was designed to increase sensitivity for the study of minimal residual disease (MRD). RESULTS: The assay enabled specific detection of NPM1 mutations in 12/36 patients. A 10(-2) sensitivity level was obtained using one amplification round, while the hemi-nested PCR approach yielded a 10(-5) sensitivity level, therefore proving useful to assess MRD in patients carrying the mutation. The results were independently validated in 24 AML cases by sequencing analysis. CONCLUSIONS: This simple and low-cost assay may integrate diagnostic work-up of AML and could be used for assessment of response to therapy in patients with NPM1 mutations.

No evidence of association of CTLA-4 -318 C/T, 159 C/T, 3' STR and SUMO4 163 AG polymorphism with autoimmune diabetes.

Autoimmune diabetes is an organ specific and multifactorial disorder including insulin dependent diabetes mellitus (Type 1 Diabetes) and latent autoimmune diabetes in adults (LADA), which progresses to insulin dependency because of the beta cells destruction. Several polymorphisms in different genes have been associated with diabetes. The CTLA4 gene is considered a down regulator of T cell function, and the SUMO4 gene encodes a small ubiquitin-like modifier implicated in the intensity and duration of the immune response. We selected 62 LADA patients, 123 patients with Type 1 diabetes patients and 136 unrelated volunteers to study CTLA4 -318 C/T, 159 C/T, 3' STR and SUMO4 163 A/G polymorphisms by PCR. There was a statistical difference significant in the frequency of the allele 209pb for the 3'STR between LADA and Type 1 diabetes patients but not with respect the normal group, the frequencies were found to be 6.9%, 1.0% and 1.9%, respectively. However, no association with either of the polymorphisms has been found in the studied population. The knowledge of the several susceptibility loci in autoimmune diabetes will enhanced the prediction of individuals at high risk of developing the disease in order to establish the best treatment and the prevention of autoimmune diabetes.

Cytotoxic T lymphocyte antigen 4 heterozygous codon 49 A/G dimorphism is associated to latent autoimmune diabetes in adults (LADA).

Autoimmune diabetes is an organ specific and multifactorial disorder with a classical onset as insulin dependent diabetes mellitus (IDDM) and with another form of onset as latent autoimmune diabetes in adults (LADA), which has a slower onset and a later progress to insulin dependency as a result of the beta cells destruction. The cytotoxic T lymphocyte-antigen 4 (CTLA4) has been identified as a susceptible marker of the disease; it is considered a down regulator of T cell function, playing a key role in autoimmunity. We analyzed CTLA4 codon 49 A/G polymorphism in 123 IDDM patients, 63 LADA patients and 168 healthy non-diabetic control individuals. The frequency of the heterozygous A/G genotype in LADA patients was significantly increased compared to IDDM patients (55.6 vs. 39.8%, p = 0.0415). There was no statistical significant difference in the distribution of the A/G dimorphism between autoimmune diabetes patients (LADA or IDDM) and non-diabetic control individuals. HLA DQ region is responsible for the genetic susceptibility to autoimmune diabetes in IDDM patients in about 50% and it has a lower effect in genetic susceptibility in LADA patients. Several other genetic loci are needed to develop autoimmune diabetes in adult patients. Therefore, LADA may be the result of a combined minor risk loci effect in a major risk haplotype.

Variable number of tandem repeats of the insulin gene determines susceptibility to latent autoimmune diabetes in adults.

BACKGROUND: The different clinical presentations of latent autoimmune diabetes in adults (LADA) and type 1 diabetes mellitus may be the result of susceptibility genes in determining the mode of onset. We analyzed the 5' polymorphisms of the insulin mini-satellite region (INS), a variable number of tandem repeats (VNTR) [repeat units; RU]. We evaluated the association of the different INS-VNTR alleles in patient susceptibility to LADA autoimmune diabetes. To our knowledge, this constitutes the first study of this kind performed in a Caucasian population. METHODS: From an group of 160 Argentinean patients previously characterized as having LADA, we selected 44 patients who presented with humoral autoimmunity for genotyping and compared them to 88 patients with type 1 diabetes and 138 healthy individuals. The INS-VNTR allele classes were determined by Southern blotting (class I: 21-44RU; class III: 138-159RU). Subjects with class I alleles were further studied using PCR amplification to determine the exact length of the alleles (short 1S: 22-37RU; medium 1M: 38-41RU; large 1L: 42-43RU). Allelic and genotype frequencies were estimated by chi(2) tests for independence with 2 x 2 contingency tables and the relative risks (RR) were determined using GraphPad InStat software. RESULTS: We observed differential associations among the class I alleles when comparing patients with LADA (80.6%) and type 1 diabetes (81.3%) with the controls (70%; p < 0.005). This increase was largely due to the high frequency of the 1S/S genotype (63.6% LADA vs 37% controls, with a p-value of 0.0019 [p1]; 53.4% type 1 diabetes vs 37% controls, with a p-value of 0.0149 [p2]). Remarkably, all LADA patients genotyped as class I homozygous had the shorter (S) class I allele (100%). Differences in the overall 1S distribution were observed: in LADA the 94.4% of the alleles were equal to or smaller than 35RU, while in patients with type 1 diabetes it was 78.3% and in controls 74.1%. Moreover, the relative risks associated with the 1S/S genotype for patients with LADA showed a substantial increase with respect to those with type 1 diabetes (52%) when we compare them to the controls (1S/S LADA/control, 2.282 [RR1] vs type 1 diabetes/control, 1.497 [RR2]). CONCLUSION: The presence of the 1S allele could be considered a risk factor in LADA patients, as previously reported for type 1 diabetes. The class I INS-VNTR allele in LADA increases genetic susceptibility to disease development.

A novel mutation in exon 5 of the glucokinase gene in an Argentinian family with maturity onset diabetes of the young.

Maturity onset diabetes of the young (MODY) is caused by mutations in at least six different genes, including the glucokinase gene (MODY 2) and genes encoding the tissue-specific transcription factors (MODY 1 and MODY 3-6). To determine the presence of mutations in MODY 2 in four members of a family who have the clinical characteristics of MODY, we performed polymerase chain reaction and single strand conformation polymorphism screening, followed by DNA sequencing. We found a novel mutation which consisted of the deletion of a cytosine in the position 2 of the exon 5 codon 168. This mutation produced a frame shift which determines a stop codon at position 203 in exon 6. The identification of a mutation in glucokinase gene and transcription factor genes in patients with early-onset diabetes confirms the diagnosis of MODY and has important implications for clinical management.