Background: No validated renal cell carcinoma (RCC) marker is known for detection of asymptomatic disease in selected populations or for prognostic purposes or treatment monitoring. We identified immunogenic proteins as tumor markers for RCC by combining conventional proteome analysis with serological screening, and we investigated the diagnostic clinical value of such markers in serum. Methods: We studied the immunogenic protein expression profile of CAL 54, a human RCC cell line, by 2-dimensional electrophoresis combined with immunoblotting using sera from healthy donors compared with RCC patients. We developed a homogeneous, fluorescent, dual-monoclonal immunoassay for metalloproteinase 7 (MMP-7) and used it to measure MMP-7 in sera from 30 healthy donors, 30 RCC patients, and 40 control patients. Results: Pro-MMP-7 (29 kDa; pI 7.7) in the CAL 54 cell line secretome was an immunogenic protein reactive with RCC patient sera but not with control sera. The concentrations of pro-MMP-7 were increased (P <0.0001) in sera of RCC patients (median 7.56 μg/L; range 3.12–30.5 μg/L) compared with healthy controls (median 2.13 μg/L; range 0.17–3.5 μg/L). Serum pro-MMP-7 had a sensitivity of 93% (95% CI 78%–99%) at a specificity of 75% (59%–87%) for RCC in the samples tested. Conclusion: Proteomics technology combined with serology led to the identification of serum pro-MMP-7 as a marker of RCC and represents a powerful tool in searching for candidate proteins as biomarkers.

Resistance to thyroid hormone (RTH) is a syndrome of elevated serum thyroxine, inappropriately "normal" serum thyrotropin (TSH) and reduced thyroid hormone responsiveness associated with point mutations in the thyroid hormone receptor-beta (TRbeta) gene. We describe a novel point mutation resulting in a cytosine for adenine substitution at nucleotide 1271 (exon 9) that results in the substitution of threonine for asparagine (T329N). This mutation was identified in a 30-year-old woman who was investigated for recurrent spontaneous abortions and was found to have RTH. Dextrothyroxine (D-T4) therapy was instituted. At 8 mg per day 2 pregnancies followed with the delivery of a healthy boy and an RTH-affected girl another miscarriage occurred on D-T4 treatment at 6 mg per day. The T329N mutation, which was also identified in the daughter, markedly reduces the affinity of TRbeta for triiodothyronine (T3). Formation of T329N mutant TR homodimers and heterodimers with RXRalpha on thyroid hormone response element F2 (TRE F2) was not affected, but the ability of T3 to interrupt T329N mutant TRbeta homodimerization was markedly reduced. The T329N mutant TRbeta was transcriptionally inactive in transient expression assays. In cotransfection assays with wild-type TRbeta1, the mutant TRbeta1 functioned in a dominant negative manner. The results suggest that the T329N mutation in the T3-binding domain of TRbeta is responsible for RTH in the proposita's family.

BackgroundA disintegrin and metalloprotease 12 (ADAM12-S) has previously been reported to be significantly reduced in maternal serum from women with fetal aneuploidy early in the first trimester and to significantly improve the quality of risk assessment for fetal trisomy 21 in prenatal screening. The aim of this study was to determine whether ADAM12-S is a useful serum marker for fetal trisomy 21 using the mixture model.MethodIn this case control study ADAM12-S was measured by KRYPTOR ADAM12-S immunoassay in maternal serum from gestational weeks 8 to 11 in 46 samples of fetal trisomy 21 and in 645 controls. Comparison of sensitivity and specificity of first trimester screening for fetal trisomy 21 with or without ADAM12-S included in the risk assessment using the mixture model.ResultsThe concentration of ADAM12-S increased from week 8 to 11 and was negatively correlated with maternal weight. Log MoM ADAM12-S was positively correlated with log MoM PAPP-A (r = 0.39, P < 0.001), and with log MoM free beta hCG (r = 0.21, P < 0.001). The median ADAM12-S MoM in cases of fetal trisomy 21 in gestational week 8 was 0.66 increasing to approx. 0.9 MoM in week 9 and 10. The use of ADAM12-S along with biochemical markers from the combined test (PAPP-A, free beta hCG) with or without nuchal translucency measurement did not affect the detection rate or false positive rate of fetal aneuploidy as compared to routine screening using PAPP-A and free β-hCG with or without nuchal translucency.ConclusionThe data show moderately decreased levels of ADAM12-S in cases of fetal aneuploidy in gestational weeks 8-11. However, including ADAM12-S in the routine risk does not improve the performance of first trimester screening for fetal trisomy 21.

Resistance to thyroid hormone (RTH) is almost invariably associated with mutations of the thyroid hormone (TH) receptor beta (hTR beta) gene and is inherited as an autosomal dominant disease. Mutations of hTR beta identified in patients affected by RTH cluster generally at two spots of the ligand binding domain. We investigated whether an Italian kindred with RTH had a mutation in the thyroid hormone (TH) receptor beta gene. Blood samples were obtained from the available family members for biochemical and genetic analyses. Thyroid function tests in basal conditions, and in the case of the propositus also following incremental doses of T3, were performed. Exon 4 to 10 of hTR beta gene were amplified using the polymerase chain reaction (PCR) and the mutation was identified by direct sequence analysis. The affinity constant of this mutated receptor for T3 was measured by in vitro transcription-translation and was then compared with that of wild type. We identified a heterozygous G to A transition at nucleotide 1037 of exon 8 at codon 251, resulting in a glycine (G) to glutamic acid (E) substitution (G251E) in the patient affected by RTH and in his affected offspring, but not in the normal family members. This novel mutation represents a de novo mutation since both parents of the index case were unaffected and did not have this genomic mutation. When expressed in vitro, the mutant protein (G251E) showed a marked decrease of the affinity for T3, suggesting an impaired ligand-dependent transactivation activity of this mutant receptor. In vivo studies with incremental doses of L-T3 demonstrated a reduced sensitivity to TH in the index case, in particular at the pituitary level where the thyrotrophs' activity was not completely inhibited even by 200 micrograms/day of L-T3. G251E mutation represents the fourth mutation described up to now in exon 8 of hTR beta among the subjects affected by RTH. A third cluster of mutations of the c-erbA beta gene located proximally with respect to the other two so far described begins to emerge in RTH patients.