Permanent congenital hypothyroidism (CH) is a common disease that occurs in 1 of 3,000-4,000 newborns. Except in rare cases due to hypothalamic or pituitary defects, CH is characterized by elevated levels of thyroid-stimulating hormone (TSH) resulting from reduced thyroid function. When thyroid hormone therapy is not initiated within the first two months of life, CH can cause severe neurological, mental and motor damage. In 80-85% of cases, CH is associated with and presumably is a consequence of thyroid dysgenesis (TD). In these cases, the thyroid gland can be absent (agenesis, 35-40%), ectopically located (30-45%) and/or severely reduced in size (hypoplasia, 5%). Familial cases of TD are rare, even though ectopic or absent thyroid has been occasionally observed in siblings. The pathogenesis of TD is still largely unknown. Although a genetic component has been suggested, mutations in the gene encoding the receptor for the thyroid-stimulating hormone (TSHR) have been identified in only two cases of TD with hypoplasia. We report mutations in the coding region of PAX8 in two sporadic patients and one familial case of TD. All three point mutations are located in the paired domain of PAX8 and result in severe reduction of the DNA-binding activity of this transcription factor. These genetic alterations implicate PAX8 in the pathogenesis of TD and in normal thyroid development.

The genes responsible for insulin resistance are poorly defined. Plasma cell differentiation antigen (PC-1) glycoprotein inhibits insulin receptor signaling and is associated with insulin resistance. We describe here a novel polymorphism in exon 4 of the PC-1 gene (K121Q) and demonstrate that it is strongly associated with insulin resistance in 121 healthy nonobese (BMI <30 kg/m2) nondiabetic (by oral glucose tolerance test [OGTT]) Caucasians from Sicily. Compared with 80 KK subjects, Q allele carriers (n = 41, 39 KQ and 2 QQ) showed higher glucose and insulin levels during OGTT (P < 0.001 by two-way analysis of variance) and insulin resistance by euglycemic clamp (M value = 5.25 +/- 1.38 [n = 24] vs. 6.30 +/- 1.39 mg x kg(-1) x min(-1) [n = 49], P = 0.005). Q carriers had higher risk of being hyperinsulinemic and insulin resistant (odds ratio [CI]: 2.99 [1.28-7.0], P < 0.001). Insulin receptor autophosphorylation was reduced (P < 0.01) in cultured skin fibroblasts from KQ versus KK subjects. Skeletal muscle PC-1 content was not different in 11 KQ versus 32 KK subjects (33 +/- 16.1 vs. 17.5 +/- 15 ng/mg protein, P = 0.3). These results suggest a cause-effect relationship between the Q carrying genotype and the insulin resistance phenotype, and raise the possibility that PC-1 genotyping could identify individuals who are at risk of developing insulin resistance, a condition that predisposes to type 2 diabetes and coronary artery disease.

Protein tyrosine phosphatase 1B (PTP1B) inhibits insulin signaling and, when overexpressed, plays a role in insulin resistance (Ahmad et al. 1997). We identified, in the 3' untranslated region of the PTP1B gene, a 1484insG variation that, in two different populations, is associated with several features of insulin resistance: among male individuals, higher values of the insulin resistance HOMA(IR) index (P=.006), serum triglycerides (P=.0002), and total/HDL cholesterol ratio (P=.025) and, among female individuals, higher blood pressure (P=.01). Similar data were also obtained in a family-based association study by use of sib pairs discordant for genotype (Gu et al. 2000). Subjects carrying the 1484insG variant showed also PTP1B mRNA overexpression in skeletal muscle (6,166 plus minus 1,879 copies/40 ng RNA vs. 2,983 plus minus 1,620; P<.01). Finally, PTP1B mRNA stability was significantly higher (P<.01) in human embryo kidney 293 cells transfected with 1484insG PTP1B, as compared with those transfected with wild-type PTP1B. Our data indicate that the 1484insG allele causes PTP1B overexpression and plays a role in insulin resistance. Therefore, individuals carrying the 1484insG variant might particularly benefit from PTP1B inhibitors, a promising new tool for treatment of insulin resistance (Kennedy and Ramachandran 2000).

The c-myc protooncogene codes for an evolutionarily conserved nuclear phosphoprotein which is ubiquitously expressed in somatic cells, where it is involved in the control of proliferation and differentiation (1, 2). Alterations in c-myc gene structure and expression caused by retroviral insertion, amplification, and chromosomal translocation are associated with tumorigenesis in different species (1-3). However, the precise function of the c-Myc protein in normal and in neoplastic cells is unknown.Several observations have supported the hypothesis that c-Myc may be a transcription factor. c-Myc can bind DNA in vitro, and a specific sequence (CACGTG) has been identified as its specific binding site (4-6). In addition, c-Myc contains domains which are typical of several types of transcription factors. These include carboxyl-terminal basic, helix-loophelix, and leucine-zipper (b-HLH-LZ) domains which can mediate the formation of oligomeric complexes capable of specific DNA binding (7) and an amino-terminal domain capable of transcriptional transactivation when assayed in experimental chimeric constructs (8,9). Further, a second b-HLH-LZ protein, Max, has been identified which can specifically associate with c-Myc in vitro and in vivo to form heterodimeric complexes capable of specific DNA binding (10-12). However, no direct evidence is available for the function of c-Myc and Max as transcriptional regulators or on their specific role within heterodimeric or homodimeric complexes.In MATERIALS AND METHODSPlasmid Construction and Transient Transfection. The construction of the pHeBo-CMV-Myc2.3 plasmid was previously described (13). To construct the pHeBo-CMV-Max plasmid, we first generated a full-length coding region of human Max cDNA by reverse transcription/PCR amplification of RNA to generate a fragment spanning bp -6 to 465 of the published human Max cDNA sequence (10). The amplified product was cloned into the pGEM-3 plasmid (Promega), completely sequenced to confirm that it matched the published sequence coding for 151 amino acids (10), and then transferred into the pHeBo-CMV expression vector by bluntend ligation into the filled-in Not I site. The p-MMBS-SV1-LUC plasmid was constructed by cloning into the Mlu I site of the pGL2-promoter vector (catalogue no. E1631, Promega) the palindromic self-annealed synthetic double-stranded oligodeoxynucleotide 5'-CGCGGGAAGCAGACCACGTG-GTCTGCTTCC-3', which includes the Myc/Max binding site flanked by the Mlu I site (underlined). Transient transfection of HeLa cells was performed by a calcium phosphate procedure. Luciferase assays were performed according to standard protocols (Promega) using a luminometer.Lymphoblastoid Cell Line (LCL) Transfection and Analysis. LCLs were transfected by electroporation (14). For clonogenicity analysis, transfected LCLs were plated in triplicate agar plates (0.3% in Iscove's modified Dulbecco's medium plus 20% fetal bovine serum) at 5 x 103, 1 x 104, and 2 x 104 cells per ml over a feeder layer (3 x 104 cells) of irradiated (6000 rads; 1 rad...

Insulin resistance plays a major role in dyslipidemia, cardiovascular disease, and type 2 diabetes. TRB3, a mammalian tribbles homolog, whose chromosomal region 20p13-p12 has been linked to human type 2 diabetes, impairs insulin signaling through the inhibition of Akt phosphorylation and is overexpressed in murine models of insulin resistance. We here report that the prevalent TRB3 missense Q84R polymorphism is significantly (P < 0.05) associated with several insulin resistance-related abnormalities in two independent cohorts (n ‫؍‬ 178 and n ‫؍‬ 605) of nondiabetic individuals and with the presence of a cluster of insulin resistancerelated cardiovascular risk factors in 716 type 2 diabetic patients (OR 3.1 [95% CI 1.2-8.2], P ‫؍‬ 0.02). In 100 additional type 2 diabetic patients who suffered from myocardial ischemia, age at myocardial ischemia was progressively and significantly (P ‫؍‬ 0.03) reduced from Q84Q to Q84R to R84R individuals. To test the functional role of TRB3 variants, either Q84 or R84 TRB3 full-length cDNAs were transfected in human HepG2 hepatoma cell lines. As compared with control HepG2 cells, insulin-induced Ser473-Akt phosphorylation was reduced by 22% in Q84-(P < 0.05 vs. control cells) and by 45% in R84-transfected cells (P < 0.05 vs. Q84 transfected and P < 0.01 vs. control cells). These data provide the first evidence that TRB3 gene plays a role in human insulin resistance and related clinical outcomes. Diabetes 54:2807-2811, 2005

Interleukin (IL)-10 is a major anti-inflammatory cytokine that has been associated with obesity and type 2 diabetes. The three polymorphisms ؊1082G/A, ؊819C/T, and ؊592C/A in the IL10 promoter were reported to influence IL10 transcription. We investigated whether these polymorphisms were associated with type 2 diabetes and related traits in a cohort of Italian Caucasians comprising 551 type 2 diabetic and 1,131 control subjects. The ؊819C/T and ؊592C/A polymorphisms were in perfect linkage disequilibrium (r 2 ‫؍‬ 1.0). The ؊1082G/A polymorphism was not associated with type 2 diabetes or related traits. Although the ؊592C/A polymorphism was not associated with type 2 diabetes, nondiabetic homozygous carriers of the A allele showed increased BMI and insulin resistance and lower plasma IL-10 levels compared with the other genotypes. In the nondiabetic group, the ATA haplotype was associated with an increased risk for obesity

Aquaporin 7 (AQP7), the gateway protein controlling glycerol release, has recently emerged as a modulator of adipocyte metabolism. AQP7 knockout mice develop obesity and hyperglycemia. The contribution of AQP7 to these abnormalities in humans is unknown. We examined whether common single nucleotide polymorphisms (SNPs) in the AQP7 gene modulate the risk of obesity and related abnormalities. Among several SNPs we identified, A-953G in the AQP7 promoter was associated with type 2 diabetes in 977 (530 female/447 male) Caucasians: odds ratio for XG (i.e., AG؉GG) versus AA individuals was 1.36 (95% CI 1.01-1.84), P ‫؍‬ 0.04. This finding was entirely due to the association among females (1.8 [1.2-2.6], P ‫؍‬ 0.004), which was no longer significant when adjusted for BMI. In fact, BMI was higher in XG than in AA females (30.8 ؎ 6.6 vs. 28.9 ؎ 5.2, P ‫؍‬ 0.002). This association was confirmed in independent case-control study (n ‫؍‬ 299 female subjects) for morbid obesity (1.66 [1.01-2.74], P ‫؍‬ 0.04). Luciferase and mobility shift assays showed that, compared with ؊953A, the ؊953G promoter had reduced transcriptional activity (P ‫؍‬ 0.001) and impaired ability to bind CCAAT/ enhancer binding protein (C/EBP)␤ transcription factor (P ‫؍‬ 0.01). Finally, AQP7 expression in adipose tissue decreased from AA to AG to GG individuals (P ‫؍‬ 0.036). These data strongly suggest that AQP7 downregulation is pathogenic for obesity and/or type 2 diabetes. Diabetes

Newborns with high TSH at birth and with normal free T(4) and normal or slightly elevated TSH at the confirmatory examination are considered false positive for congenital hypothyroidism. We evaluated thyroid function, thyroid antibodies, thyroid volume and morphology, thyroperoxidase and TSH receptor genes, and auxological data in 56 false positive children at 16-44 months of age. In these children thyroid function at confirmatory examination was fully normal in 33 (TSH, 0.8-4.9 mU/liter; group I) and nearly normal (borderline elevated TSH, 5.0-11.7 mU/liter) in the other 23 (group II). Compared with 65 control children with normal TSH at birth, false positive children had significantly higher basal serum TSH (mean +/- SD, 4.38 +/- 2.2 vs. 1.4 +/- 0.8 mU/liter; P < 0.01). Subclinical hypothyroidism, indicated by increased basal TSH and/or increased TSH response to TRH, was present in 36% children in group I and 70% in group II. Free T(4) was within the normal range in all children. Compared with the control group, false positive children had significantly higher free T(3) values (4.9 +/- 0.8 vs. 3.7 +/- 1.0 pmol/liter; P < 0.01) and a higher prevalence of antithyroid antibodies (25% vs. 1.5%; P < 0.001). Frequent thyroid morphology abnormalities and frequent thyroperoxidase and TSH receptor gene sequence variations were also observed. In conclusion, newborns classified false positive at congenital hypothyroidism screening have a very high risk of subclinical hypothyroidism in infancy and early childhood.