The G6055A (G2019S) mutation in LRRK2 is frequent in both early and late onset Parkinson's disease and originates from a common ancestor.

BACKGROUND: Mutations in the gene Leucine-Rich Repeat Kinase 2 (LRRK2) were recently identified as the cause of PARK8 linked autosomal dominant Parkinson's disease. OBJECTIVE: To study recurrent LRRK2 mutations in a large sample of patients from Italy, including early (<50 years) and late onset familial and sporadic Parkinson's disease. RESULTS: Among 629 probands, 13 (2.1%) were heterozygous carriers of the G2019S mutation. The mutation frequency was higher among familial (5.1%, 9/177) than among sporadic probands (0.9%, 4/452) (p<0.002), and highest among probands with one affected parent (8.7%, 6/69) (p<0.001). There was no difference in the frequency of the G2019S mutation in probands with early v late onset disease. Among 600 probands, one heterozygous R1441C but no R1441G or Y1699C mutations were detected. None of the four mutations was found in Italian controls. Haplotype analysis in families from five countries suggested that the G2019S mutation originated from a single ancient founder. The G2019S mutation was associated with the classical Parkinson's disease phenotype and a broad range of onset age (34 to 73 years). CONCLUSIONS: G2019S is the most common genetic determinant of Parkinson's disease identified so far. It is especially frequent among cases with familial Parkinson's disease of both early and late onset, but less common among sporadic cases. These findings have important implications for diagnosis and genetic counselling in Parkinson's disease.

Association between the tumor necrosis factor-alpha -308 G/A gene polymorphism and migraine.

In a group of 299 migraine patients and 306 control subjects, the association of the -308 G/A polymorphism in the tumor necrosis factor-alpha gene (TNFalpha) with the occurrence and clinical characteristics of migraine was tested. Homozygosity for the G allele was associated with an increased risk of migraine (odds ratio [OR] = 2.85, p < 0.001). When the patients were divided into subgroups, the association was confirmed in patients affected by migraine without aura (OR = 3.30, p < 0.001) but not in migraine with aura. These data suggest that the TNFalpha gene or a linked locus significantly modulates the risk for migraine.

Analysis of DNA microarrays by non-destructive fluorescent staining using SYBR green II.

A simple, non-destructive procedure is described to determine the quality of DNA arrays before they are used. It consists of a preliminary staining step of the DNA microarray by using SYBR green II, a fluorophore with specific affinity for ssDNA, followed by a laser scan analysis. The surface quality, integrity and homogeneity of each DNA spot of the array can thus be assessed. After this preliminary control, which may avoid further analytical steps that lead to the waste of precious biological samples, a fully reversible staining procedure is performed that produces an array ready for subsequent use.

Electrospray ionization mass spectrometry of synthetic oligonucleotides using 2-propanol and spermidine

Oligonucleotides have become widely used tools in molecular biology and molecular diagnostics. Their parallel synthesis in large numbers and the increasing interest in microarray technology has raised the requirement for fast and informative analytical tools for their quality control. A direct injection electrospray ionization mass spectrometry (ESI-MS) technique based on the use of aqueous 2-propanol as running eluent, and spermidine (or triethylamine) as DNA modifiers, has been applied to analyze a large set of samples (about 200 synthetic oligonucleotides) ranging from 5 to 15 kDa (17-51mers) with good results in terms of sensitivity, suppression of sodium adduct formation, and speed of analysis. Copyright 2000 John Wiley & Sons, Ltd.

Time-resolved DNA-microarray reading by an intensified CCD for ultimate sensitivity.

We describe a novel technique for DNA-microarray reading based on time-resolved fluorescence measurements. We used an intensified CCD camera with picosecond resolution to acquire a set of time-delayed fluorescence images from a mutation DNA microarray marked with cyanine 3. We measured the fluorescence lifetimes of the marker and the background separately, and we used this information to calculate the amplitude map of the marker, starting from the time-delayed images. This procedure allowed us to identify hybridized spots that are not visible in fluorescence images acquired with continuous-wave detection.

Application of capillary electrophoresis at low pH to oligonucleotides quality control.

A method for oligonucleotides analysis by using capillary electrophoresis at low pH in free solution is described. It may be considered an alternative to classical analytical techniques which use basic buffers and require the presence of sieving media to separate oligonucleotides as a function of their length. On the contrary, at low pH oligo nucleotides can be separated only depending on their base composition. A large set of samples consisting of 72 synthetic oligonucleotides bearing a 5'-alkylamine moiety and designed for HLA genotyping were analysed. The quality of these synthetic oligos was easily assessed, and a single base difference in oligonucleotides of equal sequence was detected. The results suggest the application of this method to the emerging field of mutation detection and single nucleotide polymorfism analysis.

Apolipoprotein E genotyping by capillary electrophoretic analysis of restriction fragments.

We present the genotyping of apolipoprotein (apo) E by means of restriction fragment analysis of amplified genomic DNA by high-performance capillary electrophoresis and a replaceable non-gel-sieving matrix. This procedure streamlines the genotyping of apo E in large-scale population studies because of the automation and speed of capillary electrophoresis.

[Surgical staging of pulmonary carcinoma. Probability of error]. / Stadiazione chirurgica del carcinoma polmonare. Probabilità di errore.

234 patients with lung cancer and operated in Thoracic and Cardiovascular Surgery Department of Careggi Hospital in Florence have been evaluated in order to examine surgical staging accuracy in comparison with pathological staging. There is a statistically significative difference between surgical and pathological staging as a datum point. Surgeon is inclined to over-estimate the lymph-nodes involvement and the primitive tumor extension. It is important to bear in mind this bent whenever decisions of surgical strategy have to be taken.

[Features and evolution of postoperative pain in patients undergoing thoracotomy]. / Caratteristiche ed evoluzione del dolore postoperatorio in pazienti sottoposti a toracotomia.

We examined in this perspective study 30 patients (15 males and 15 females) who undergone classic thoracotomy for lung or mediastinal diseases, without postoperative complications. Intensity and different qualitative components of postoperative pain have been evaluated in the 1st, 3rd, 5th, the day after last pleural drainage removal and 2 months after the operation. All patients answered a schedule adherent to Questionario Italiano del Dolore (QUID). Pain became less intense chiefly with drainage removal with the same time trend for both sex (parallel curves). Pain is probably stronger in women. From a qualitative point of view, the sensorial component is the same in both sexes. Affective and evaluative component is greater in women: in other words, women realize more than men the painful stimulus and are more troubled. A computerized analysis of answers to a questionnaire like QUID or, better, its evolution, may be helpful for a more effective pharmacological choice between pure analgesics, sedative analgesics and ataractic drugs.

["N2" carcinoma of the lung. Therapeutic considerations]. / Carcinoma del polmone "N2". Considerazioni terapeutiche.

The follow-up of 240 N2 lung cancer cases operated in Thoracic and Cardiovascular Surgery Department of Florence is examined. The analysis is performed in compliance with global survival, "T", histology, therapeutic choices. Global survival is 81% after 6 months, 60% after 1 year, 37% after 2 years, 26% after 3 years, 23% after 4 years, 23% after 5 years. Significative difference on survival does not exist between principal histologic types (squamous, adenocarcinoma, adenosquamous). Raising the "T" survival decreases, but only for adenocarcinoma. Different therapeutic options (only surgery, surgery+radiotherapy, surgery+chemotherapy, surgery+radio and chemotherapy) do not influence the survival in a way statistically significative. From the literature, any certainty about radiotherapy and chemotherapy associated to surgery for N2 lung cancer treatment does not exist at the moment. Thus radical surgery is essential.

[CT-guided thoracic biopsy. Personal experience with DBNH Haaga 20 G needles]. / Biopsia toracica TC-guidata. Esperienza personale con ago tipo DBNH 20 G.

CT-guided fine-needle biopsy of the chest is nowadays a conventional diagnostic procedure, which can be furtherly improved so as to increase its diagnostic sensitivity and to reduce complications. We studied a series of possible improvements over a 2-year period (1990-1992). In our radiology department 164 CT-guided biopsies were performed in 130 men and 34 women. DBNH Haaga 20-G (15 cm) needles (Cook, Denmark) were used. This kind of needle is a coaxial cutting-edge needle used for simultaneous cytological and histological sampling. Exams were performed with a Philip Tomoscanner CX-S (scanning time: 2.8 seconds). Of 164 tissue samples, 153 (93.4%) were adequate for histological diagnosis. As for suspected neoplastic disease, our results demonstrated 108 true positives, 27 true negatives, 18 false negatives and no false positive. Sensitivity, specificity and diagnostic accuracy were 85.7%, 100% and 88.2%, respectively. In 24 cases there were minor complications--i.e., 5 cases of parenchymal hemorrhage and 19 cases of minor pneumothorax. In one case only there was major pneumothorax which required surgical drainage. On the basis of our experience, we suggest the use of such coaxial catheters as the DBNH Haaga type for the collection of both histological and cytological material because this needle reduces the need for repeated invasive and potentially dangerous maneuvers.