Drugs acting at dopamine D2-like receptors play a pivotal role in the treatment of both schizophrenia and Parkinson's disease. Recent studies have demonstrated a role for G-protein independent D2 receptor signaling pathways acting through β-arrestin. In this study we describe the establishment of a Bioluminescence Resonance Energy Transfer (BRET) assay for measuring dopamine induced recruitment of human β-arrestin2 to the human dopamine D2 receptor. Dopamine, as well as the dopamine receptor agonists pramipexole and quinpirole, acted as full agonists in the assay as reflected by their ability to elicit marked concentration dependent increases in the BRET signal signifying β-arrestin2 recruitment to the D2 receptor. As expected from their effect on G-protein coupling and cAMP levels mediated through the D2 receptor RNPA, pergolide, apomorphine, ropinirole, bromocriptine, 3PPP, terguride, aripiprazole, SNPA all acted as partial agonists with decreasing efficacy in the BRET assay. In contrast, a wide selection of typical and atypical anti-psychotics was incapable of stimulating β-arrestin2 recruitment to the D2 receptor. Moreover, we observed that haloperidol, sertindole, olanzapine, clozapine and ziprasidone all fully inhibited the dopamine induced β-arrestin2 recruitment to D2 receptor (short variant) in a concentration dependent manner. We conclude that most anti-psychotics are incapable of stimulating β-arrestin2 recruitment to the dopamine D2 receptor, in accordance with their antagonistic properties at the level of G-protein coupling.

BACKGROUND AND OBJECTIVES Hereditary hemochromatosis is a recessive condition characterized by iron accumulation in several organs, followed by organ damage and failure. The disorder is prevalently due to C282Y and H63D mutations in the HFE gene, but additional HFE and TFR2 mutations have been reported. Early iron overload may be assessed by biochemical parameters such as increased transferrin saturation and serum ferritin. DESIGN AND METHODS Taking advantage of the collection of 178 DNA samples selected for increased transferrin saturation (>50% in males and >45% in females) from a previous large scale screening of Italian blood donors, we simultaneously assessed the presence of 14 hemochromatosis-associated molecular defects (11 of HFE and 3 of TFR2) by a reverse hybridization-based strip assay. RESULTS In the series studied the overall C282Y allele frequency was 9% and that of the H63D and S65C was 22.2% and 1.4%, respectively. One rare HFE allele (E168Q), but no TFR2 mutation was detected. When checked at a second examination, transferrin saturation was significantly higher in C282Y homozygotes, H63D/ C282Y compound heterozygotes and H63D homozygotes as compared to wild-type subjects (p<0.05). INTERPRETATION AND CONCLUSIONS Our results confirm previous findings on C282Y and H63D mutations in Italy, show that the C282Y allele frequency is enriched in samples selected for altered iron parameters, and that a few rare genotypes are present in Northern Italy. None of the known TFR2 mutations was identified in this series confirming the preliminary indication of their rare occurrence. Subjects with hemochromatosis-associated genotypes show a persistently higher mean transferrin saturation than do those with wild type genotypes.

Familial Mediterranean fever (FMF) is an autosomal-recessive, inflammatory disorder characterized by short, recurrent attacks of fever, accompanied by pain in the abdomen, chest, or joints and erysipelas-like erythema. Its most severe complication is progressive amyloidosis, leading to end-stage renal failure. FMF predominantly affects Turks, Arabs, Armenians, and Sephardic Jews, with carrier rates reported as high as 1 in 5, but it has been observed in lower frequencies throughout the Mediterranean area (1). It is caused by several mutations within the marenostrin/pyrin-encoding gene MEFV on chromosome 16p13.3, which differently affect the severity of the disease phenotype and the risk of developing renal amyloidosis (2)(3)(4). Although established clinical criteria for FMF exist (5), many patients remain undiagnosed because of rather nonspecific symptoms; therefore, molecular genetic analysis could substantially improve early and correct diagnosis of FMF and allow initiation of lifelong prophylactic treatment of affected individuals with colchicine (6).Aiming at a simple but powerful first-line screening tool for FMF genotyping, we have set up a reverse-hybridization, teststrip-based assay (FMF StripAssay) for the simultaneous detection of 12 MEFV mutations: E148Q in exon 2, P369S in exon 3, F479L in exon 5, and M680I (G/C), M680I (G/A), I692del (2076–2078), M694V, M694I, K695R, V726A, A744S, and R761H in exon 10. For this purpose, we collected DNA samples from individuals who had previously been typed positive for one of these mutations and used them to generate recombinant plasmid clones for the 12 mutant alleles (TOPO TA Cloning Kit; Invitrogen). After confirming the presence of mutations by DNA sequencing, we used these plasmid clones as homozygous reference samples to determine suitable reverse-hybridization probes.We synthesized a series of candidate 15- to 25mer oligonucleotides, selected from the FMF databank sequence (GenBank accession no. AF111163) and encoding all wild-type or mutant alleles, and immobilized …