A refined physical and transcriptional map of the SPG9 locus on 10q23.3-q24.2

Hereditary spastic paraplegia (HSP) is a genetically heterogeneous disorder characterised by progressive spasticity of the lower limbs. Beside 'pure' forms of HSP, 'complicated' forms are reported, where spasticity occurs associated with additional symptoms. We recently described an Italian family with a complicated dominant form of HSP (SPG9) and we mapped the gene responsible to 10q23.3-q24.2, in a 12cM interval between markers D10S564 and D10S603. The phenotypic manifestations in our family are reminiscent of those already described in a smaller British pedigree. We typed individuals from this British family using markers located in the SPG9 critical interval and haplotype reconstruction showed the disorder co-segregating with SPG9. To characterise the SPG9 region better, we constructed a contig of 22 YACs, assigned it to 18 polymorphic markers and positioned 54 ESTs. Furthermore, we searched for ESTs containing a trinucleotide repeat sequence, since anticipation of symptoms was reported in both families. Finally, analysis of a muscle biopsy specimen from one patient was normal, suggesting that, contrary to SPG7, mitochondrial disturbance could not be a primary feature of SPG9.     

Molecular characterization of a t(2;6) balanced translocation that is associated with a complex phenotype and leads to truncation of the TCBA1 gene

The molecular characterization of balanced chromosomal rearrangements has often been a powerful tool for the positional identification of genes associated with specific diseases. In some instances, these rearrangements may be associated with a variety of different phenotypes, and thus establishing a genotype-phenotype correlation may be a complex process. However, molecular characterization of the rearrangement remains a useful tool for diagnoses or prognoses, or for identifying new genes and establishing a gene-to-function relationship. In this work we describe the characterization of a de novo balanced translocation t(2;6)(q24.3;q22.31) found in a patient with a complex phenotype. The major clinical finding was a severe neurological involvement. Thanks to the molecular characterization of this translocation we found that the rearrangement led to the truncation of the TCBA1 gene on chromosome 6q. We found that the gene is transcribed in different splice variants and is highly specific for the central nervous system. TCBA1 does not show any similarity with other known genes, and no information is available about its function. However, the gene appears to be well conserved among species, and we were able to infer the sequence of a putative mouse homolog of TCBA1. This allowed us to perform a more detailed expression study in mice, thus confirming its specificity for the nervous system. This finding is of particular interest because it suggests that TCBA1 may be correlated with the neurological phenotype of our patient, and possibly mutated in genetic diseases with a neurological phenotype.     

Investigation of some Hypericum species native to Southern of Brazil for antiviral activity

Three plant species, Hypericum connatum, Hypericum caprifoliatum, Hypericum polyanthemum (Guttiferae), growing in Southern of Brazil were chemically investigated and tested for their antiviral activity against feline immunodeficiency virus (FIV). The chemical analysis revealed the presence of polyphenolic compounds such as tannins and flavonoids. Hypericin was not detected in these species. The aqueous extract (AE), the aqueous extract with low tannin concentration (LTCAE) and the methanolic extract (ME) were tested for their cytotoxic properties in concentrations of 50-150 microg/ml. AE was toxic to CRFK for the three species in all concentrations. LTCAE and ME varied between different concentrations being not toxic or allowing 80% of cell growth. LTCAE and ME (10-50 microg/ml) were analyzed for antiviral activity by inhibition of CPE and measuring FIV genome from cell culture supernatant. LTCAE of all species in this work did not cause any inhibition of FIV. Although no difference was seen in CPE, a lower number of viral particles in the supernatant was observed when FIV infected cells were treated with ME of H. connatum. These results suggest that some plants of the genus Hypericum from Southern Brazil contain compounds with potential antiviral activity against lentiviruses.     

Neutrophils from patients with TNFRSF1A mutations display resistance to tumor necrosis factor–induced apoptosis: Pathogenetic and clinical implications

Objective

To explore tumor necrosis factor (TNF)–induced apoptosis in neutrophils from patients with TNF receptor–associated periodic syndrome (TRAPS) and to correlate the results with the different kinds of TNFRSF1A mutations.

Methods

Two hundred sixty‐five patients with clinically suspected inherited autoinflammatory syndrome were screened for mutations of the TNFRSF1A gene. Neutrophils were isolated from heparinized blood by dextran sedimentation and incubated with and without cycloheximide (CHX) and TNFα. Cell apoptosis was assessed by human annexin V binding, and caspase 8 activation was assessed by flow cytometry.

Results

Twenty‐one patients were found to carry a variant of the TNFRSF1A gene: 13 patients had an R92Q substitution, and 8 patients presented other missense substitutions, 1 splicing mutation, and 1 in‐frame interstitial deletion. Neutrophil stimulation with TNF and CHX was associated with induction of apoptosis in 12 normal controls and in 10 subjects with the R92Q mutation. Conversely, neutrophils from 8 TRAPS patients with mutations of cysteine or threonine residues or interstitial deletion did not show any induction of apoptosis after stimulation. The incidence of the R92Q mutation among patients with recurrent autoinflammatory syndromes was similar to that observed in the normal population.

Conclusion

Resistance to TNF‐mediated apoptosis is a feature in TRAPS patients who have mutations of cysteine residues or interstitial deletion, and may play a pathogenic role. The R92Q mutation does not appear to be significantly associated with TRAPS.

     

Fibroblastoid colony-forming cells in myeloproliferative disorders

The properties of human bone marrow fibroblastoid colonies (CFU-F) were studied in normal subjects and in patients with myeloproliferative disorders. Colony incidence was within normal values in all groups of patients analyzed except for myelodisplastic syndromes, with higher mean value. The growth rate of CFU-F is inversely related to the initial colony-forming efficiency both in normal subjects and in patients. Direct correlation between CFU-F and granulocyte-macrophage colony-forming unit (GM-CFU) was detected only in normal subjects, but lacked in patients. Higher number of CFU-F was observed in subjects with increased incidence of bone marrow megakaryocytes or peripheral blood platelets, irrespective of the underlying disorders and the platelet-derived growth-factorenhanced cloning efficiency of bone marrow fibroblasts.