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Background: Despite consolidated guidelines, the clinical diagnosis and prognosis of cystic fibrosis (CF) is still challenging mainly because of the extensive phenotypic heterogeneity and the high number of CFTR variants, including their combinations as complex alleles. Results: We report a family with a complicated syndromic phenotype, which led to the suspicion not only of CF, but of a dominantly inherited skeletal dysplasia (SD). Whereas the molecular basis of the SD was not clarified, segregation analysis was central to make a correct molecular diagnosis of CF, as it allowed to identify three CFTR variants encompassing two known maternal mutations and a novel paternal microdeletion. Conclusion: This case well illustrates possible pitfalls in the clinical and molecular diagnosis of CF; presence of complex phenotypes deflecting clinicians from appropriate CF recognition, and/or identification of two mutations assumed to be in trans but with an unconfirmed status, which underline the importance of an in-depth molecular CFTR analysis.

Ethanol is an essential chemical reagent in DNA preparation as its use increases the yield of extraction. All methodologies for DNA isolation involve the use of ethanol in order to prevent DNA dissolution in water and to optimize the binding of DNA to chromatographic membranes. In this note, we show how the presence of ethanol traces in DNA aqueous solution affects ultraviolet Raman spectra, leading to possible misinterpretations. We report a simple method to remove the ethanol Raman features from the spectra, based on heating the DNA sample at 80 ℃, followed by a slow cooling procedure.

Hereditary hearing loss (HHL) is a common disorder characterized by a huge genetic heterogeneity. The definition of a correct molecular diagnosis is essential for proper genetic counseling, recurrence risk estimation, and therapeutic options. From 20 to 40% of patients carry mutations in GJB2 gene, thus, in more than half of cases it is necessary to look for causative variants in the other genes so far identified (~100). In this light, the use of next-generation sequencing technologies has proved to be the best solution for mutational screening, even though it is not always conclusive. Here we describe a combined approach, based on targeted re-sequencing (TRS) of 96 HHL genes followed by high-density SNP arrays, aimed at the identification of the molecular causes of non-syndromic HHL (NSHL). This strategy has been applied to study 103 Italian unrelated cases, negative for mutations in GJB2, and led to the characterization of 31% of them (i.e., 37% of familial and 26.3% of sporadic cases). In particular, TRS revealed TECTA and ACTG1 genes as major players in the Italian population. Furthermore, two de novo missense variants in ACTG1 have been identified and investigated through protein modeling and molecular dynamics simulations, confirming their likely pathogenic effect. Among the selected patients analyzed by SNP arrays (negative to TRS, or with a single variant in a recessive gene) a molecular diagnosis was reached in ~36% of cases, highlighting the importance to look for large insertions/deletions. Moreover, copy number variants analysis led to the identification of the first case of uniparental disomy involving LOXHD1 gene. Overall, taking into account the contribution of GJB2, plus the results from TRS and SNP arrays, it was possible to reach a molecular diagnosis in ~51% of NSHL cases. These data proved the usefulness of a combined approach for the analysis of NSHL and for the definition of the epidemiological picture of HHL in the Italian population.

We describe a real-time polymerase chain reaction (PCR) protocol based on the fluorescent molecule SYBR Green chemistry, for a low- to medium-throughput analysis of Y-chromosome microdeletions, optimized according to the European guidelines and aimed at making the protocol faster, avoiding post-PCR processing, and simplifying the results interpretation. Methods: We screened 156 men from the Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Institute for Maternal and Child Health IRCCS Burlo Garofolo (Trieste, Italy), 150 not presenting Y-chromosome microdeletion, and 6 with microdeletions in different azoospermic factor (AZF) regions. For each sample, the Zinc finger Y-chromosomal protein (ZFY), sex-determining region Y (SRY), sY84, sY86, sY127, sY134, sY254, and sY255 loci were analyzed by performing one reaction for each locus. Results: AZF microdeletions were successfully detected in six individuals, confirming the results obtained with commercial kits. Conclusion: Our real-time PCR protocol proved to be a rapid, safe, and relatively cheap method that was suitable for a low- to medium-throughput diagnosis of Y-chromosome microdeletion, which allows an analysis of approximately 10 samples (with the addition of positive and negative controls) in a 96-well plate format, or approximately 46 samples in a 384-well plate for all markers simultaneously, in less than 2 h without the need of post-PCR manipulation.

OBJECTIVES: Nonclassical major histocompatibility class I HLA-G antigen is a tolerogenic molecule that inhibits lytic activity of natural killer (NK) cells and cytotoxic T lymphocytes. Because of its immunomodulatory and tolerogenic properties, HLA-G molecules may have a role in celiac disease (CD). We analyzed the HLA-G 14 bp deletion/insertion polymorphism, known to have a functional effect on mRNA stability, in a group of 522 CD patients, stratified for the presence of HLA-DQ2 genotype, and 400 healthy individuals to evaluate the possible effect of the polymorphism on the risk to develop the disease. METHODS: HLA-G 14 bp deletion/insertion polymorphism (rs1704) was detected by polymerase chain reaction and double-checked by direct sequencing. RESULTS: The 14 bp inserted (I) allele and the homozygous I/I genotype were significantly more frequent in CD patients than in healthy controls. The presence of I allele was associated with an increased risk of CD (OR 1.35) and the effect of I allele was consistent with a recessive genetic model (P

Objective Purpose of this paper is to analyse OTOF gene in a series of subjects affected by auditory neuropathy. Methods Four children showing mild to profound prelingual deafness, confirmed by the absence of a clear and detectable responses at auditory brainstem responses (ABR), associated with the presence of bilateral OAE, were enrolled in the study. Results and Conclusions Genetic analysis identified five new mutations (a nonsense, a small and a large deletion and two splicing site mutations), and one missense mutation (F1795C) previously described. These results further confirm the role of OTOF gene in auditory neuropathy. In the absence of a context of neurological syndrome, the combination of absent ABR and positive OAE responses should lead to an auditory neuropathy diagnosis and to a mutational screening in OTOF.

Cystic fibrosis (CF) transmembrane regulator protein (CFTR) gene is undoubtedly the main genetic factor involved in the modulation of CF phenotype. However, other factors such as human defensins and the genes encoding for these antimicrobial peptides have been hypothesized as possible modifiers influencing airways infection in CF patients, but their role in the pathogenesis of lung disease is still debated. Since DEFB1 gene encoding for human beta-defensin 1 displays features such as antimicrobial or chemotactic activity playing a role in inflammation, it has been considered as a possible candidate CF modifier gene. We analysed three single nucleotide polymorphisms (SNPs) in the 5'-untranslated region of the DEFB1 gene (namely g-52G>A, g-44C>G and g-20G>A) in a group of 62 CF patients from North Eastern Italy, and in 130 healthy controls, with the aim of verifying the possible association of these functional SNPs with the pulmonary phenotype of CF patients. DEFB1 SNPs have been genotyped by using Taqman allele-specific fluorescent probes and a real-time PCR platform. No significant differences were found for allele, genotype and haplotype frequencies of DEFB1 g-52G>A, g-44C>G and g-20G>A SNPs in CF patients stratified for Pseudomonas aeruginosa infection, as well as in patients with a severe and mild clinical phenotype or in patients stratified for CFTR genotypes. DEFB1 allele, genotype and haplotype frequencies of CF patients globally considered were similar to those of healthy controls. Our findings are discordant with respect to another recent study performed on CF patients coming from Southern Italy, probably due to different ethnicity of the patients.

Problem Mannose-binding lectin (MBL) is an important component of the innate immunity, present at the mucosal level in vagina: a common pathogen’s entry point. Method of study We used a rapid genotyping method based on melting temperature assay to search for three single nucleotide polymorphisms (SNPs) located in the first exon of the MBL2 gene and we also measured MBL serum levels in patients with recurrent bacterial vaginosis (rBV) and recurrent vulvovaginal candidiasis (rVVC). Results Detected frequencies of MBL2 SNPs were comparable to the ones already reported for the Italian population and no significant differences were found between rVVC, rBV and controls. MBL serum levels did not show significant differences between the studied groups. Conclusion No correlation for the screened mutations has been found neither in protecting nor in favoring the infection in rVVC and rBV patients. Our data demonstrate a lack of association between functional polymorphisms in the first exon of MBL2 gene, MBL deficiency,VVC and rBV.

Mutations in the GJB2 gene, which encodes the gap junction protein Connexin 26 (Cx26), are the major cause of genetic non-syndromic hearing loss. The role of several GJB2 mutations in causing hereditary deafness remains controversial. By combining genetic, clinical and biochemical studies, we have analysed the possible pathogenetic role of the V153I mutation. Molecular epidemiological studies revealed a V153I allele frequency in the normal hearing population of 0.49. The same allele has been detected at the homozygous state in a normal hearing subject. Finally, functional data obtained in transiently transfected HeLa cells showed that V153I was correctly synthesized and targeted to the plasma membrane and the formed intercellular channels function was not disturbed, as the intercellular diffusion of Lucifer Yellow remained unchanged. These findings provide new insights against a possible pathogenetic role of the V153I mutation of the GJB2 gene, and indicate it as a possible neutral variant. Furthermore, t...

Usher syndrome is an autosomal recessive disorder characterized by retinitis pigmentosa, sensorineural hearing loss and, in some cases, vestibular dysfunction. The disorder is clinically and genetically heterogeneous and, to date, mutations in 11 genes have been described. This finding makes difficult to get a precise molecular diagnosis and offer patients accurate genetic counselling. To overcome this problem and to increase our knowledge of the molecular basis of Usher syndrome, we designed a targeted resequencing custom panel. In a first validation step a series of 16 Italian patients with known molecular diagnosis were analysed and 31 out of 32 alleles were detected (97% of accuracy). After this step, 31 patients without a molecular diagnosis were enrolled in the study. Three out of them with an uncertain Usher diagnosis were excluded. One causative allele was detected in 24 out 28 patients (86%) while the presence of both causative alleles characterized 19 patients out 28 (68%). Sixteen novel and 27 known alleles were found in the following genes: USH2A (50%), MYO7A (7%), CDH23 (11%), PCDH15 (7%) and USH1G (2%). Overall, on the 44 patients the protocol was able to characterize 74 alleles out of 88 (84%). These results suggest that our panel is an effective approach for the genetic diagnosis of Usher syndrome leading to: 1) an accurate molecular diagnosis, 2) better genetic counselling, 3) more precise molecular epidemiology data fundamental for future interventional plans.

Cystic fibrosis (CF) is a common recessive genetic disease caused by mutations in the gene encoding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. More than 1800 different mutations have been described to date. Here, we report 3 novel mutations in CFTR in 3 Italian CF patients. To detect and identify 36 frequent mutations in Caucasians, we used the INNO-LiPA CFTR19 and INNO-LiPA CFTR17+Tn Update kits (Innogenetics; Ghent, Belgium). Our first analysis did not reveal both of the responsible mutations; thus, direct sequencing of the CFTR gene coding region was performed. The 3 patients were compound heterozygous. In one allele, the F508del (c.1521_1523delCTT, p.PHE508del) mutation in exon 11 was observed in each case. For the second allele, in patient No.1, direct sequencing revealed an 11-base pair deletion (GAGGCGATACT) in exon 14 (c.2236_2246del; pGlu746Alafs*29). In patient No. 2, direct sequencing revealed a nonsense mutation at nucleotide 3892 (c.3892G>T) in exon 24. In patient No. 3, direct sequencing revealed a deletion of cytosine in exon 27 (c.4296delC; p.Asn1432Lysfs*16). These 3 novel mutations indicate the production of a truncated protein, which consequently results in a non-functional polypeptide.

Background. IgA nephropathy (IgAN) occurs sporadically in unrelated individuals. Several different polymorphic genes have been investigated in recent years in order to demonstrate their possible association with IgAN. Three recent, different studies with conflicting conclusions have discussed the role of the mannose binding lectin (MBL), a serum lectin involved in natural immunity, in the IgAN pathogenesis by examination of MBL deposits in biopsies. In the present study we investigated several polymorphisms of the MBL gene located in the promoter region and in the first exon. Methods. MBL polymorphism detection was performed in 22 Italian patients with familial IgA nephropathy and in 138 Italian patients with the sporadic form of the disease. The polymorphisms in the MBL2 promoter region and in the exon 1 were investigated, respectively, by direct sequencing and by amplification refractory mutation system-polymerase chain reaction on genomic DNA collected from peripheral blood. Seventy-four unrelated healthy subjects matched for ethnic origin were used as controls. Results. Allelic and genotypic frequencies of the polymorphisms at position -550, -328, -221 and at codon 54 did not show any differences between patients and controls. Similar frequency distributions of these polymorphisms were also found in the subgroups of IgAN patients subdivided according to the clinical manifestations and the progression of the disease. Conclusions. This study indicates that the analysed polymorphisms of the MBL gene do not appear to be primarily involved in the susceptibility and severity of IgAN.

(CD) is a T-cell-mediated chronic inflammatory disease characterized by autoimmune, immunological and environmental components, where genetic factors in addition to the main known risk factors (gliadin and human leukocyte antigen (HLA)-DQ haplotypes) are supposed to be involved. CD14 is a multifunctional receptor involved in the bacterial lipopolysaccharides-dependent signal transduction. The CD14 gene maps on the long arm of chromosome 5 (5q22-q32), a 'hotbed' region for CD; promoter polymorphisms are known to influence its expression. In this study we analyzed three CD14 promoter polymorphisms (c.-1359G>T, c.-1145A>G and c.-159C>T, ) in 938 CD Italian patients and 533 healthy controls, with known HLA-DQ haplotypes, with the aim of evaluating their possible association with the disease. The c.-1145A>G G and c.-159C>T T alleles (as well as the combination of the two alleles in the GT haplotype), were identified as susceptibility factors for CD development, being significantly more frequent in CD patients than in healthy controls. This association was also confirmed when the analysis was restricted to only those subjects characterized by HLA-DQ risk haplotypes. Our results indicate the involvement of CD14 gene polymorphisms in the susceptibility to CD.Genes and Immunity advance online publication, 31 May 2012; doi:10.1038/gene.2012.23.

The influence of thrombophilia on fertility and on IVF outcome is very controversial. The objectives of this study were: (i) to compare the prevalence of Factor V Leiden (FVL) and prothrombin gene G20210A mutation (PGM) in women undergoing IVF to women with spontaneous pregnancy; (ii) to compare the IVF outcomes and the risk of complications in FVL and PGM carrier to non-carrier women.From March 2005 to December 2009, a total of 510 women requiring IVF were recruited in a prospective cohort study. A separate population of 490 nulliparous women who conceived naturally was also evaluated as fertile controls. All women were tested for the presence of FVL and PGM.The prevalence of thrombophilic mutations was the same among women requiring IVF (6.9%) and women with spontaneous pregnancy (6.9%). A total of 480 patients underwent 1105 IVF cycles. There were 30 women carriers (86 IVF cycles) and 450 non-carriers for thrombophilic mutations (1019 IVF cycles). No significant differences in the mean number of oocytes retrieved and the number of good quality embryos transferred were found between the mutation carrier and non-mutation carrier women; likewise the reproductive outcome and the IVF complications were not statistically different between the two groups. The cumulative live birth rate after six IVF cycles was similar in the mutation carrier and non-mutation carrier women. For the mutation carrier women, the optimistic estimate of cumulative live birth rate after six IVF cycles was 60.8% and the conservative estimate was 50.0%. Corresponding rates for the non-mutation carrier women were 56.8 and 36.2%, respectively.The results of this study suggest that FVL and PGM presence in asymptomatic women and in the absence of other risk factors do not influence IVF outcome, or represent risk factors for ovarian hyperstimulation syndrome (OHSS), or favour thrombosis after IVF. Screening for FVL and PGM does not appear to be justified to identify the patients at the risk for IVF failure, and/or for OHSS, and/or for thrombotic complications.

We analyzed HLA-G 3777G > C, HLA-G 14 bp deletion/insertion and HLA-G*0105N polymorphisms in HIV-positive white adult participants, infected through horizontal heterosexual transmission, and unexposed uninfected individuals, all from north eastern Italy. We report a new association between the HLA-G*0105N allele and HIV infection in adult white female participants, being HLA-G*0105N null allele correlated with an augmented risk (odds ratio = 4.35, 95% confidence interval = 1.38-18.07, P = 0.005) for HIV infection.

To measure epidermal thickness by using skin ultrasonography (US) in a series of healthy control subjects and obligate carriers for the worldwide most frequent form of congenital hearing loss owing to the mutated alleles of the connexin 26 gene (GJB2).The patent for the protocol, coupled with a new sonographic probe specifically designed to analyze epidermal thickness and a dedicated algorithm to classify individuals in groups, is pending. Institutional ethics committee approval and patient consent were obtained. After a preliminary study in 23 subjects aimed to define the best body site and instrument and protocol for US, a total of 303 individuals (237 healthy subjects, 51 carriers, and 15 homozygotes) were tested at midline forehead by using a linear large-band probe with a frequency ranging from 6 to 15 MHz to determine epidermal thickness. Variance and linear regression analyses were performed. Regression coefficients were then used to obtain measurements of thickness corrected for age and sex.GJB2 obligate carriers had a significant increase in epidermal thickness compared with control subjects. GJB2 status explains about 50.0% of this variability, whereas an additional 25.0% is explained by sex and age. Results led to the development of a possible screening protocol with a 98.0% sensitivity and 92.8% specificity in subjects aged 2080 years, with a likelihood ratio of a positive test of 14:1. Even better results (100% sensitivity and 98.9% specificity) were obtained in an analysis of people of only reproductive age.Epidermal thickening in the white population owing to GJB2 carrier status can be detected by using US. This measurement could provide a simple, noninvasive, rapid, and sensitive test for carrier screening.

In the post-human-genome area, the challenge is to derive details of heritable variation in relation to how human variation reflects adaptation to the different environments. Heterozygote advantage represents a superior genetic adaptation presumably explaining the presence of the allele at frequencies above those expected from a simple replacement of a homozygous lethal allele by mutation alone. A successful adaptation requires natural selection acting on that part of the body that makes a difference in survival. There is ongoing discussion as to what extent inherited diseases may be phenotypically balanced by a selective heterozygous advantage. We know for sure that sickle cell anemia and some other red blood cell disorders are selected for by malaria,1 whereas a selective carrier advantage for cystic fibrosis has been recently shown.2 For other common inherited diseases counterbalancing mechanisms have been only hypothesized. Congenital hearing loss accounts for about 1 in 1000 infants and approximately 80% of the cases are inherited as an autosomal recessive trait. Connexin 26 (GJB2) gene, a component of a gap junction, is a major gene for these forms, and a large proportion of recessive cases are due to mutations in this gene.3 Different common population-specific mutations have been so far described.4, 5 Among them, mutation 35delG explains up to 70% of the alleles detected in Caucasians, with carrier frequencies ranging from 1/30 in the Mediterranean area to 1/70 in northern Europe.5 The high carrier frequency of the GJB2 mutations in many ethnic groups strongly suggests that there may be a heterozygous advantage. Why GJB2 carriers are so frequent? Quite recently, it has been reported that carriers of R134W allele, present in some African countries such as Ghana,6, 7 show a thicker epidermis than wild-type ones.8 Moreover, in vitro studies have further supported this finding showing that cells expressing the R134W allele: (a) form a significantly thicker epidermis in an organotypic co-culture skin model, (b) show an increased migration, (c) are significantly less susceptible to cellular invasion by the enteric pathogen Shigella flexneri than wild-type cells.9 Another study has shown that the transfection of some deafness-associated mutant constructs resulted in a statistically significant reduction in cell death when compared with wild-type ones.10 Thus, researchers hypothesized that an increased cell survival may explain a thicker epidermis due to an extended terminal differentiation program leading to an improved barrier against infection. Moreover, some specific GJB2 mutated alleles lead to skin diseases in which abnormal thickening of the skin is present.11 If epidermal thickening is the phenomenon underlying heterozygote advantage, should we be able to find an increased thickness of the epidermis in GJB2 carriers? To answer this question and possibly to explain the increased 35delG carrier frequency, we have developed an accurate sonographic protocol to measure epidermal thickness able to discriminate between normal controls and GJB2 carriers.12 Table 1 report the data on epidermal thickness in a series of 240 Italian normal controls (134 females and 106 males) aged from 20 to 86 years. They have been then compared with those obtained in a series of fifty-four 35delG obligate carriers (29 females and 25 males). Results of the analysis are shown in Figure 1, in which two regression lines are calculated for each of the two groups (normal controls and carriers). A clear tendency to thickening along with age is present in both sexes (P

We evaluated the possible association of human leukocyte antigen-G (HLA-G) 3777G>C and 14-bp deletion/insertion (D/I) polymorphisms haplotypes and combined genotypes with perinatal HIV transmission in Brazilian children. The 3777G>C polymorphism alone has no effect on HIV vertical transmission but, when linked with the D allele, exerts a positive role in the protection. Indeed, we identified the DC HLA-G haplotype as significantly associated with a protective effect towards HIV vertical transmission.

Objectives: The aim of our study was to verify the possible association between an HLA-G 14-bp deletion/insertion polymorphism and perinatal HIV transmission in Brazilian children. Design: We analyzed the 14-bp deletion/insertion polymorphisms in seronegative (i.e., exposed uninfected, N = 71) and seropositive (exposed infected, N = 175) Brazilian children born from HIV-positive mothers and in healthy controls (n = 175). Methods: HLA-G 14-bp deletion/insertion polymorphism (rs16375) was detected by PCR amplification of the target sequence followed by agarose gel electrophoresis. All the samples were also analyzed by direct sequencing in order to validate the genotyping results. Results: HIV-exposed uninfected children showed significant differences in their allele and genotype frequencies of the HLA-G 14-bp polymorphism when compared to both seropositive children and healthy controls. The 14-bp-deleted (D) allele was more frequent in exposed uninfected children (79%) than in healthy controls (60%) and HIV-positive children (58%); the higher percentage of the D allele found in the exposed uninfected children with respect to HIV-positive individuals was significantly associated with a reduced risk of vertical transmission. This effect was ascribable to the presence of the D/D homozygous genotype. Conclusion: Our findings support the possible role for the HLA-G 14-bp deletion/insertion polymorphism in the HIV vertical transmission in Brazilian children. The presence of the D allele and D/D genotype is associated with a protective effect toward HIV perinatal infection.

Mannose-binding lectin (MBL) is an important component of the innate immunity, present at the mucosal level in vagina: a common pathogen's entry point.We used a rapid genotyping method based on melting temperature assay to search for three single nucleotide polymorphisms (SNPs) located in the first exon of the MBL2 gene and we also measured MBL serum levels in patients with recurrent bacterial vaginosis (rBV) and recurrent vulvovaginal candidiasis (rVVC).Detected frequencies of MBL2 SNPs were comparable to the ones already reported for the Italian population and no significant differences were found between rVVC, rBV and controls. MBL serum levels did not show significant differences between the studied groups.No correlation for the screened mutations has been found neither in protecting nor in favoring the infection in rVVC and rBV patients. Our data demonstrate a lack of association between functional polymorphisms in the first exon of MBL2 gene, MBL deficiency, VVC and rBV.

The X-linked form of Charcot-Marie-Tooth disease (CMTX) is caused by mutations in connexin32 (Cx32), a gap junction protein expressed by Schwann cells where it forms reflexive channels that allow the passage of ions and signaling molecules across the myelin sheath. Although most mutations result in loss of function, several studies have reported that some retain the ability to form homotypic intercellular channels. To gain insight into the molecular defect of three functional CMTX variants, S26L, Delta111-116 and R220stop, we have used several fluorescent tracers of different size and ionic charge to compare their permeation properties to those of wild-type Cx32. Although all mutations allowed the passage of the dye with the smallest molecular mass, they exhibited a clear reduction in the permeability of either one or all of the probes with respect to wild-type channels, as assessed by the percentage of injections showing dye coupling. These data reveal that a lower size cutoff distinguishes these functional CMTX variants from wild-type channels and suggest that this defect may be of pathophysiological relevance.

Mutations of the GJB2 gene, encoding connexin 26, are the most common cause of hereditary congenital hearing loss in many countries and account for up to 50% of cases of autosomal-recessive non-syndromic deafness. By contrast, only a few GJB2 mutations have been reported to cause an autosomal-dominant form of non-syndromic deafness. Here, we report a family from Southern Italy affected by non-syndromic autosomal dominant post-lingual hearing loss, due to a novel missense mutation in the GJB2 gene, a threonine to asparagine amino acid substitution at codon 55 (T55N). Functional studies indicated that the mutation T55N produces a protein that, although expressed to levels similar to those of the wt counterpart, is deeply impaired in its intracellular trafficking and fails to reach the plasma membrane. The mutation T55N is located at the apex of the first extracellular loop of the protein, a region suggested to play a role in protein targeting and a site for other two mutations, G59A and D66H, causing dominant forms of deafness.

We performed MBL2 genotyping in 47 CF patients—cared of at the regional CF Centre of Trieste—trying to establish a correlation within allelic variants of MBL2 and modification of patients' clinical outcome. FEV1 values were significantly lowered and a significantly earlier age at onset of Pseudomonas aeruginosa colonisation was found in CF patients with at least one MBL2 variant.

In the present study, we report the genomic reconstruction of the human homeobox-containing gene HHEX by the use of the data available in public databases. This analysis allowed characterization of the gene organization showing that it is very similar to the mouse gene. Moreover the gene was mapped using FISH to 10q24.

We have studied the expression of ornithine decarboxylase (ODC) mRNA by in situ hybridization and in situ RT-PCR in human breast cancer MCF-7 cell line. In situ RT-PCR demonstrated the overexpression of ODC mRNA, localized over the cytoplasm, while a very low signal was detected by in situ hybridization. Our findings indicate that in situ RT-PCR could represent a useful tool to study different levels of ODC expression in normal and tumor tissues. Since ODC expression is regulated by c-Myc oncoprotein, this model could be useful to monitor in vivo the effects of new anti-neoplastic molecules, specific inhibitors of c-Myc.

A new highly repeated DNA fragment isolated from Macaca fascicularis (MFASAT) is described. Our findings obtained by sequencing, Southern blot analysis, and fluorescent in situ hybridization (FISH) on metaphasic chromosomes strongly suggest that MFASAT can be considered as a member of the alphoid DNA family characteristic of Old World monkeys. The chromosomal localization of MFASAT, obtained by FISH, showed that this alphoid DNA is present in the peri-centromeric area of all the chromosomes. MFASAT showed a high degree of conservation when compared, by sequence alignment, to other Macaca species and Papio papio as expected for species with considerable genome conservation. A low degree of homology has been found comparing M. fascicularis alphoid DNA with a more distantly related Cercopithecidae species such as Cercopithecus aethiops. Resume : Une nouvelle sequence d'ADN repetitif isole chez le Macaca fascicularis (MFASAT) est decrite. Les resultats du sequencage, de l'analyse par hybridations Southern et in situ en fluorescence (FISH) sur des chromosomes en metaphase suggerent fortement que MFASAT peut etre considere comme un membre de la famille d'ADN alphoide caracteristique des singes du monde antique. La localisation chromosomique du MFASAT, obtenue par FISH, a montre que cet ADN alphoide etait present dans la zone pericentromerique de tous les chromosomes. MFASAT a montre un haut niveau de conservation lorsque compare, par alignement de sequences, a d'autres especes du genre Macaca et le Papio papio tel qu'attendu pour des especes montrant une forte conservation genomique. Un faible degre d'homologie a ete trouve en comparant l'ADN alphoide du M. fascicularis avec celui d'especes moins apparentees parmi les Cercopithecidae telles que le Cercopithecus aethiops. Mots cles : ADN alphoide, Macaca fascicularis, FISH, alignement de sequences. (Traduit par la Redaction) 1070