Identification of sixty-two novel and twelve known FBN1 mutations in eighty-one unrelated probands with Marfan syndrome and other fibrillinopathies

Marfan Syndrome (MFS) is an autosomal dominant disorder of the connective tissue due to mutations of Fibrillin-1 gene (FBN1) in more than 90% of cases and Transforming Growth Factor-Beta-Receptor2 gene (TGFB2R) in a minority of cases. Genotyping is relevant for diagnosis and genotype-phenotype correlations. We describe the FBN1 genotypes and related phenotypes of 81 patients who were referred to our attention for MFS or Marfan-like phenotypes. Patients underwent multidisciplinary pertinent evaluation in the adult or paediatric setting, according to their age. The diagnosis relied on Ghent criteria. To optimise DHPLC analysis of the FBN1 gene, all coding regions of the gene were directly sequenced in 19 cases and 10 controls: heterozygous amplicons were used as true positives. DHPLC sensitivity was 100%. Then, DHPLC was used to screen 62 other cases. We identified 74 FBN1 mutations in 81 patients: 64 were novel and 17 known. Of the 81 mutations, 41 were missense (50.6%), 27, either nonsense or frameshift mutations and predicted a premature termination codon (PTC) (33%), 11 affected splice sites (13.6%), and two predicted in-frame deletions (2.5%). Most mutations (67.9%) occurred in cbEGF-like modules. Genotype was clinically relevant for early diagnosis and conclusion of the diagnostic work-up in patients with incomplete or atypical phenotypes.     

Dibasic staining of large epoxy tissue sections and applications to surgical pathology

A variety of normal and pathologic aldehyde-fixed osmium postfixed human tissues were prepared as large sections embedded in Spurr epoxy. They were stained with a sequential basic fuchsin--methylene blue stain which gives "hematoxylin- and eosin-like" staining and additionally functions as several special stains. This technic also allows for electron microscopy directly on the embedded tissue. The histologic and cytologic preservation and overall staining was superior to tissue embedded in glycol methacrylate. The methods and technics presented in this article have important applications in diagnostic surgical pathology and histology in general.     

Reducing the time of sperm-oocyte interaction in human in-vitro fertilization improves the implantation rate

Human oocyte development was evaluated after a reduced timeexposure to spermatozoa in vitro. A total of 119 patients wereassigned to two study groups in a randomized prospective studyin which each patient‘s oocytes were exposed to spermatozoafor either 1 h (group 1 – 58 patients) or the standard16 h incubation period (group 2 – 61 patients). The fertilizationrate obtained in group 1 was higher than in group 2 (285/393,73%, and 272/410, 66% respectively), suggesting that the spermatozoa-oocyteinteraction occurs within 1 h. This was confirmed in a studyin vitro using fluorescently labelled spermatozoa and normaloocyte-cumulus complexes. Spermatozoa enter the cumulus complexwithin 15 min, traverse the cumulus layer within 3 h, and firstappear in the oocyte cortex at 4 h post-insemination. The incidenceof polyspermy was higher in oocytes exposed to spermatozoa for16 h (3%) than for 1 h (1%). There was no difference in thecleavage rate or morphological characteristics of embryos fromboth study groups. However, when evaluating the timing of embryodevelopment, group 1 generated a significantly higher percentageof four to five cell embryos when compared to group 2 (55 versus39%; P < 0.001), documented at 40 h post-insemination. Theimplantation and pregnancy rates for group 1 were 11 and 28%,while the corresponding rates for group 2 were 8 and 15%. Thissuggests that a reduced exposure of oocyte to spermatozoa favoursembryo viability, possibly due to a decrease in potential damagefrom sperm metabolic waste products.     

Common gene polymorphisms in the metabolic folate and methylation pathway and the risk of acute lymphoblastic leukemia and non-Hodgkin's lymphoma in adults.

Folate and methionine metabolism is involved in DNA synthesis and methylation processes. Polymorphisms in the genes of folate metabolism enzymes have been associated with some forms of cancer. In a case-control study, we evaluated whether four common polymorphisms in methylenetetrahydrofolate reductase (MTHFR C677T and A1298C), methionine synthase (MS A2756G), and methionine synthase reductase (MTRR A66G) genes may have a role in altering susceptibility to adult acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL). We analyzed DNA of 120 adult ALL, 200 NHL, and 257 healthy control subjects. Individual carrying the MTHFR 677TT genotype showed a 3.6-fold decreased ALL risk [odds ratio (OR) 0.28, 95% confidence interval (95% CI) 0.12-0.72] than wild-types. Similarly, MS 2756GG individuals showed a 5.0-fold decreased ALL risk (OR 0.20, 95% CI 0.02-1.45) than wild-types. In combined results, subjects with the MTHFR 677CT/TT and MS 2756AG/GG genotypes revealed a 3.6-fold ALL risk reduction (OR 0.28, 95% CI 0.14-0.58) and those with the MTHFR 677TT and MTRR 66AG genotypes revealed a 4.2-fold ALL risk reduction (OR 0.24, 95% CI 0.06-0.81). Finally, those with the MS 2756AG/GG and MTRR 66AG/GG genotypes revealed a 2.2-fold ALL risk reduction (OR 0.45, 95% CI 0.10-0.85). Single analysis for NHL did not show any significant difference for all the polymorphisms investigated, but in the low-grade NHL subgroup, we found a 2.0-fold risk reduction for the MTRR 66GG homozygous genotype (OR 0.50, 95% CI 0.25-0.99), which was higher (OR 0.37, 95% CI 0.14-0.85) when analyzed in combination with MS 2756AA genotype. These data are in accordance with the hypothesis that polymorphisms in the genes for folate and methionine metabolism might play a greater role in the occurrence of ALL than NHL by influencing DNA synthesis and/or DNA methylation.     

Preliminary Evaluation of the Safety and Efficacy of Standard Intravenous Immunoglobulins in Pregnant Women with Primary Cytomegalovirus Infection

Hyperimmune globulins were reported to prevent and treat fetal cytomegalovirus (CMV) infection during pregnancy. Here, we report that infusions of standard human intravenous immunoglobulin significantly increase CMV IgG titers and avidity indexes in pregnant women, paving the way to their use for passive transfer of maternal CMV humoral immunity to fetuses. Preliminary data on perinatal outcomes of the first 67 newborns are encouraging.     

A Transitional Model for the Evaluation of West Nile Virus Transmission in Italy

In August 2008, after 10 years of apparent silence, West Nile virus (WNV) infection re‐emerged in northern Italy, spreading through the territories of three regions. In the following years, new cases occurred in the same area and additional foci of infection were observed in central and southern Italy, involving also Sicily and Sardinia islands. The Italian Ministry of Health ordered to test by RT‐PCR all blood and organ donors from 15th June to 15th November of each year in the infected areas. The period at risk of WNV transmission was defined on the basis of literature data, but a more scientific estimation of the transmission season, under Italian circumstances, needs to be performed. A transitional model previously developed by other Authors was applied and adapted to Italian circumstances, to describe and quantify the WNV transmission cycle between birds and mosquitoes. Culex spp. was considered the main vector, and mosquito parameters were adapted to this genus. Magpies (Pica pica) were considered the main bird host. The model was partially validated through the results of the entomological surveys carried out in central Italy and in Po Valley. The results of the transitional model permitted to calculate the basic reproduction number (R₀) during 2010 for the whole Italian territory at 1 km of spatial resolution, estimating the risk of WNV transmission during the year and creating detailed risk maps for Italy. The mean values of R₀ for the whole Italy varied between 0.4 and 4.8, with values >1 from the end of May to the middle of September. The coastal and flat zones of Italy showed the highest R₀ values. Although partially validated, the model showed a substantial acceptable capacity of defining the period at major risk of WNV transmission in Italy, helping Public health authorities in the application of appropriate and timely control and preventive measures.     

Changes in Heart Rate Variability in Patients with Spleen-Qi Deficiency Syndrome

Many functional diseases are related to dysautonomia, and heart rate variability has been used to assess dysautonomia. However, heart rate variability has not been studied in Spleen-Qi deficiency syndrome (SQDS). Healthy volunteers (n = 37) and patients with SQDS (n = 67), recruited from the Clinic of the State University of Ecatepec Valley were included in the study. Outcome measures were average heart rate, standard deviation of the normal-to-normal heartbeat intervals, low frequency (LF), high frequency (HF) power, and the LF/HF ratio. Also, intestinal peristalsis, gastrointestinal symptoms (GSs), fatigue, and level of attention were measured. Standard deviation of the normal-to-normal heartbeat intervals (17 ± 2.3%) and HF (14 ± 3.1%) were lower in SQDS patients (17 ± 1.3%) than in healthy volunteers. SQDS patients had higher heart rate, LF power, LF/HF ratio, and fatigue scores (9.6 ± 1.12%, 16 ± 2.1%, 22 ± 3.8%, and 21 ± 4.1%). The fatigue correlated positively with the LF/HF ratio and negatively with HF power. The SQDS group had lower concentration performance (16.2 ± 1.9%) in the d2 test. The intestinal peristalsis showed a reduction (15 ± 1.3%) as compared with control. GS score and peristalsis correlated negatively with HF. Our results suggest that the pathology of SDQS could be associated with a low vagal tone which causes a decrease in peristalsis, increased fatigue, reduced attention, and appearance of GSs.     

Liofilchem? O.A. Listeria agar and direct CAMP test provided sooner Listeria monocytogenes identification from neonatal bacteremia

Listeria monocytogenes infection in pregnant women and newborns is a cause for serious concern, and invasive disease outcome strongly depends on prompt antibiotic therapy. To provide sooner identification from neonatal bacteremia we performed a CAMP test directly on positive blood aliquots and inoculated the Liofilchem^® O.A. Listeria chromogenic agar as well, thus providing a 24-h turn-around time for response.