Viral load, organ distribution, histopathological lesions, and cytokine mRNA expression in goats infected with a molecular clone of the caprine arthritis encephalitis virus

Caprine arthritis encephalitis virus (CAEV) is a lentivirus of goats that causes persistent infection characterized by the appearance of inflammatory lesions in various organs. To define the sites of persistence, 5 goats were infected with a molecular clone of CAEV, and the viral load was monitored by real-time-PCR and RT-PCR in different sites 8 years after infection. The lymph nodes proved to be an important virus reservoir, with moderate virus replication relative to what is reported for lentiviruses of primates. Mammary gland and milk cells were preferred sites of viral replication. The viral load varied significantly between animals, which points to an important role of the genetic background. We found a clear association between occurrence of histopathological lesions and viral load in specific sites. The mRNA expression analysis of several cytokines did not reveal differences between animals that could explain the considerable individual variations in viral load observed.     

Structure-activity relationship of 1,4-dihydropyridines as potentiators of the cystic fibrosis transmembrane conductance regulator chloride channel

Mutations occurring in the CFTR gene, encoding for the cystic fibrosis transmembrane conductance regulator chloride channel, cause cystic fibrosis (CF). Mutations belonging to class II, such as DeltaPhe508, give rise to a protein with both a defective maturation and altered channel gating. Mutations belonging to class III, such as G551D and G1349D, cause only a gating defect. We have previously identified antihypertensive 1,4-dihydropyridines (DHPs), a class of drugs that block voltage-dependent Ca(2+) channels, as effective potentiators of CFTR gating, able to correct the defective activity of CFTR mutants (Mol Pharmacol 68:1736-1746, 2005). However, optimization of potency for CFTR versus Ca(2+) channels is required to design selective compounds for CFTR pharmacotherapy. In the present study, we have established DHP structure-activity relationship for both CFTR potentiation and Ca(2+) channel inhibition using cell-based assays for both types of channels. A panel of 333 felodipine analogs was studied to understand the effect of various substitutions and modifications in the DHP scaffold. Our results show that alkyl substitutions at the para position of the 4-phenyl ring lead to compounds with very low activity on Ca(2+) channels and strong effect as potentiators on the DeltaPhe508, G551D, and G1349D CFTR mutants.     

In vitro drug treatments reduce the deleterious effects of aggregates containing polyAla expanded PHOX2B proteins

Heterozygous in frame duplications of the PHOX2B gene, leading to polyalanine (polyAla) expansions ranging from + 5 to + 13 residues of a 20-alanine stretch, have been identified in the vast majority of patients affected with Congenital Central Hypoventilation Syndrome (CCHS), a rare neurocristopathy characterized by absence of adequate autonomic control of respiration with decreased sensitivity to hypoxia and hypercapnia. Ventilatory supports such as tracheostomy, nasal mask or diaphragm pacing represent the only options available for affected. We have already shown that the severity of the CCHS phenotype correlates with the length of polyAla expansions, ultimately leading to formation of toxic intracytoplasmic aggregates and impaired PHOX2B mediated transactivation of target gene promoters, such as DBH. At present, there is no specific treatment to reduce cell aggregates and to ameliorate patients' respiration. In this work, we have undertaken in vitro analyses aimed at assessing the effects of molecules on the cellular response to polyAla PHOX2B aggregates. In particular, we tested 17-AAG, ibuprofen, 4-PBA, curcumin, trehalose, congo red and chrysamine G for their ability to i) recover the nuclear localisation of polyAla expanded PHOX2B, ii) rescue of PHOX2B mediated transactivation of the DBH promoter, and iii) clearance of PHOX2B (+ 13 Ala) aggregates. Our data have suggested that 17-AAG and curcumin are effective in vitro in both rescuing the nuclear localization and transactivation activity of PHOX2B carrying the largest expansion of polyAla and promoting the clearance of aggregates of these mutant proteins inducing molecular mechanisms such as ubiquitin-proteasome (UPS), autophagy and heat shock protein (HSP) systems.     

A polymorphic variant inside the osteopontin gene shows association with disease course in oligoarticular juvenile idiopathic arthritis

BACKGROUND: Oligoarticular onset juvenile idiopathic arthritis (JIA) has a variable disease course. In some patients the disease remains confined to a few joints (persistent oligoarticular) while in others it extends to affect more joints (oligoarticular extended). Osteopontin is thought to play a role in the pathogenesis. OBJECTIVE: To investigate whether a polymorphic variant in the human osteopontin gene, which is in linkage disequilibrium with recently characterised promoter variants, is associated with the disease course in oligoarticular JIA. METHODS: Genotyping of the two base pair insertion/deletion variant at +245 in the first intron was undertaken by polymerase chain reaction (PCR) amplification of DNA fragments, using a fluorescently labelled primer, followed by allele detection after rapid separation of PCR products on an automated DNA sequencer. RESULTS: Allele 2 of the polymorphic variant in the osteopontin first intron was significantly associated with the persistent oligoarticular form rather than the extended form of JIA. This was verified at the level of genotype and allele frequencies. CONCLUSIONS: The results suggest that osteopontin gene polymorphism is associated with the disease course in oligoarticular JIA and might therefore represent a useful genetic marker to characterise patients with oligoarticular JIA who are at risk of a worse outcome.     

Familial vesicoureteral reflux: testing replication of linkage in seven new multigenerational kindreds

Vesicoureteral reflux (VUR) (OMIM %193000), a common cause of childhood renal failure, is strongly influenced by hereditary factors. Familial VUR most closely conforms to autosomal-dominant inheritance, but because of variable penetrance and expressivity, large multigenerational pedigrees tractable to linkage analysis have been difficult to ascertain. A single genome-wide study of familial VUR has demonstrated linkage to chromosome 1p13, with 78% locus heterogeneity. Previous studies in humans have also suggested loci on chromosomes 6p21, 10q26, and 19q13, whereas mutations in ROBO2 were recently reported in some patients with VUR. Replication of these studies was attempted in seven previously undescribed families from Italy and the United States. Simulation studies, assuming 50% locus heterogeneity, showed that these kindreds had 85% power to replicate linkage and 53% power to achieve genome-wide significance at candidate intervals. Thirty-five markers on chromosomes 1p13, 3p12, 6p21, 10q26, and 19q13 were genotyped and analysis of linkage under a variety of models was performed. Parametric analysis excluded linkage to all candidate loci under genetic homogeneity; moreover, the data did not support statistically significant linkage under models of locus heterogeneity. Similarly, nonparametric, allele-sharing analysis did not reveal any evidence of linkage at any of the loci tested. Thus, despite sufficient power, linkage of familial VUR to previously reported candidate intervals could not be replicated. These data demonstrate substantial genetic heterogeneity of VUR and suggest that mapping strategies relying on a large number of kindreds or single "loaded" pedigrees will be most effective to achieve replication or detection of linkage.     

crv4, a mouse model for human ataxia associated with kyphoscoliosis caused by an mRNA splicing mutation of the metabotropic glutamate receptor 1 (Grm1)

We describe a novel spontaneous autosomal recessive mutation, cervelet-4 (crv4), which arose in a BALB/c strain. Mice homozygous for the mutation exhibit principally a reduced body size, a congenital neurological phenotype characterized by ataxic gait and intention tremor, with no gross anomalies observed in brain or cerebellum, and skeletal anomalies. Using linkage analysis, we mapped the crv4 locus to the proximal region of chromosome 10, at the location of the Grm1 gene. Genetic complementation crosses between crv4 and Grm1 KO mice confirmed that crv4 is a new allele of Grm1. Molecular analysis of the Grm1 gene in mutant mice revealed the insertion of a 190-bp LTR fragment in intron 4. Our results also indicated that the presence of the LTR fragment caused the disruption of the Grm1 normal splicing process and complete absence of the wild-type protein. crv4 is an interesting model to extend the study of Grm1 function and the pathological effects of Grm1 deficiency in vivo.     

Parental origin and somatic mosaicism of PHOX2B mutations in Congenital Central Hypoventilation Syndrome

Heterozygous polyalanine repeat expansions of PHOX2B have been associated with Congenital Central Hypoventilation Syndrome, a rare neurocristopathy characterized by absence of adequate control of respiration during sleep. Here we report a PHOX2B mutational screening in 63 CCHS patients, 58 of whom presenting with poly-A expansions or frameshift, missense and nonsense mutations. To assess a somatic or germline occurrence of poly-A length variations, the relative amounts of mutant and wild type alleles have been quantified in 20 selected CCHS patients presenting with an expansion, and in their parents. Somatic mosaicism was shown in four parents, while no mosaic was found among CCHS patients. Moreover, while co-segregation analysis of the PHOX2B poly-A expansions with selected marker alleles in the same 20 CCHS trios has not demonstrated any parent-of-origin effect of the mutations, it has provided further clues to clarify the molecular mechanism underlying the expansion occurrence. Finally, the segregation of PHOX2B poly-A anomalous tracts within family members has allowed us to exclude tendency of polymorphic variations towards expansion. This strengthens the notion that expanded polyalanine tracts, identified as frequent disease-causing mutations also in other human diseases, are mitotically and meiotically stable.