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Background: Studies have suggested that soluble mesothelin-related protein (SMRP) can be used as a serum marker of malignant mesothelioma., Methods: We assessed the analytical performance of the Mesomark (Fujirebio Diagnostic) two-step ELISA on an automated analyser and performed a preliminary clinical evaluation. The precision of the assay and the in vitro effect of interfering substances on SMRP concentrations were investigated. The serum marker was analysed in 109 healthy subjects never exposed to asbestos, 26 healthy subjects exposed to asbestos, 33 patients with asbestosis, 33 with asbestos-related pleural plaques, 10 with non-malignant pleural diseases, 30 with lung cancer and 24 with histological diagnosis of pleural mesothelioma., Results: Using the International Mesothelioma Interest Group classification, there were nine stage IV, two stage III, four stage II and nine stage I patients. SMRP concentrations of 4.75, 11.0 and 14 nmol/mL showed a total imprecision of 3.5%, 3.1% and 3.8%. The detection limit was 0.035 nmol/mL; the mean SMRP concentration of 0.63 nmol/mL was associated with a coefficient of variation of 10%. There was no effect (p>0.05) of interfering substances. Serum samples from patients with established pleural mesothelioma had significantly higher (p<0.05) concentrations of SMRP than control healthy and patient groups., Conclusions: SMRP measured on automated systems could be useful for the diagnosis of mesothelioma in routine clinical practice.

Purpose: To characterize the expression of the visual system homeobox gene (VSX1) in human corneal keratocytes both in vitro and in vivo., Methods: The expression of VSX1 was evaluated through semiquantitative RT-PCR, immunofluorescence and in situ hybridization both in corneas (either freshly obtained or wounded) and in collagenase/hyaluronidase-isolated keratocytes grown in the absence or presence of serum to promote keratocyte-to-myofibroblast differentiation., Results: Quiescent or resting keratocytes normally residing in the corneal stroma or cultured in vitro in the absence of serum did not express VSX1. In wounded corneas or when cultured in the presence of serum to mimic wound-healing responses, keratocytes underwent fibroblastic transformation (with appearance of alpha-SMA and disappearance of CD-34 and keratocan signals) and started expressing VSX1., Conclusions: The results show that VSX1 is expressed in vitro and in vivo during human corneal wound healing, a process in which differentiation of corneal keratocytes into myofibroblasts occurs. These data may help to elucidate the role of VSX1 in cornea physiology suggesting a potential involvement in cornea-related diseases such as keratoconus.

WT1 mutations have been considered a rare cause of nephrotic syndrome but recent reports challenge this assumption. Exclusion of inherited forms is a basic point in any therapeutic strategy to nephrotic syndrome since they do not respond to drugs. We screened for WT1 mutations in 200 patients with nephrotic syndrome: 114 with steroid resistance (SRNS) and 86 with steroid dependence (SDNS) for whom other inherited forms of nephrotic syndrome (NPHS2, CD2AP) had been previously excluded. Three girls out of 32 of the group with steroid resistance under 18 years presented classical WT1 splice mutations (IVS9+5G>A, IVS9+4C>T) of Frasier syndrome. Another one presented a mutation coding for an amino acid change (D396N) at exon 9 that is typical of Denys-Drash syndrome. All presented resistance to drugs and developed end stage renal failure within 15 years. Two girls of the Frasier group presented a 46 XY karyotype with streak gonads while one was XX and had normal gonad morphology. In the two cases with IVS9+5G>A renal pathology was characterized by capillary wall thickening with deposition of IgG and C3 in one that was interpreted as a membrane pathology. Foam cells were diffuse in tubule-interstitial areas. In conclusion, WT1 splice mutations are not rare in females under 18 years with SRNS. This occurs in absence of a clear renal pathology picture and frequently in absence of phenotype change typical of Frasier syndrome. In adults and children with SDNS, screening analysis is of no clinical value. WT1 hot spot mutation analysis should be routinely done in children with SRNS; if the molecular screening anticipates any further therapeutic approach it may modify the long term therapeutic strategy.

Urban air pollution is associated with an increased incidence of allergic respiratory diseases. The aim of this study is to assess the occupational exposure to urban pollution through biological monitoring of PAHs and CO airborne levels in 122 traffic wardens in Bari, Italy and to investigate sensitization to inhaled allergens in a subgroup of workers. After filling in a questionnaire on lifestyle habits and occupational history, a medical examination, spirometry were carried out and blood samples were taken; the measurement of exhaled CO and urinary 1-hydroxypyrene (1-HOP) was performed and data on the air quality of Bari Municipality were obtained. Specific IgE dosage and skin prick tests were done on 18 workers giving altered values of spirometry or anamnestic allergic symptoms. Urinary 1-HOP showed median levels of 0.1 microMol/Mol(creat) (range 0.02-6.68) and was not influenced by smoking habits, work tasks, area of the city and environmental levels of PM10. Exhaled CO, with median value of 1 ppm (range 0-27), was significantly higher in smokers than in non-smokers, while no other variable seemed to play a role in modifying the levels. Specific IgE production versus inhalant allergens was found in 6 cases. Positive skin prick test results were observed in 11 cases. Allergic rhinitis was diagnosed in 6 cases. At least one of the allergometric tests performed was positive in 61 percent of the subjects. In conclusion, our results suggest the importance of introducing allergic status evaluation in this class of workers, exposed to several urban air pollutants.

T helper (h) lymphocytes in pathogenic immune response at mucosal effector site play a key role in IgA nephropathy (IgAN). We evaluated the impact of some Th1/Th2/Th3/T(R)-type, and of monocyte/macrophage cytokines on IgAN susceptibility with a family-based association study including 53 patients, 45 complete trios, 4 incomplete trios and 36 discordant siblings. Cytokine gene polymorphisms with a potential regulatory role on their production were investigated using the family-based association test (FBAT): IFNgamma intron-1 CA repeat at position 1349-1373; IL-13 -1055C/T; TGFbeta +915G/C; IL-10 5'-proximal and distal microsatellites; TNFalpha -308G/A, -238G/A. The FBAT multi-allelic analysis showed an association between IFNgamma polymorphism and susceptibility to IgAN (P=0.03). The bi-allelic analysis evidenced that the 13-CA repeat allele was preferentially transmitted to the affected individuals (P=0.006; Bonferroni P-value=0.04). The direct sequencing of IFNgamma amplicons showed a strict association between the 13-CA repeat allele and the A variant of the +874T/A single nucleotide polymorphism (SNP rs2430561) directly adjacent to the 5' end of the microsatellite. The in vitro production of IFNgamma evaluated in peripheral blood mononuclear cells from 10 genotyped patients demonstrated a correlation between the +874A allele and a lower production of IFNgamma (P=0.028 Mann-Whitney test). This SNP affects IFNgamma production lying within a binding site for the transcription factor NF-kappaB. No significant difference was observed in the 15 years renal survival between IgAN patients carrying different IFNgamma gene polymorphisms. This first family-based association study demonstrates that the +874A allele, strictly associated with IFNgamma 13-CA repeat allele, confers susceptibility to IgAN, without influencing renal survival.

Background: IgA nephropathy (IgAN) or Berger's disease, is the most common glomerulonephritis in the world diagnosed in renal biopsied patients. The involvement of genetic factors in the pathogenesis of the IgAN is evidenced by ethnic and geographic variations in prevalence, familial clustering in isolated populations, familial aggregation and by the identification of a genetic linkage to locus IGAN1 mapped on 6q22-23. This study seems to imply a single major locus, but the hypothesis of multiple interacting loci or genetic heterogeneity cannot be ruled out. The organization of a multi-centre Biobank for the collection of biological samples and clinical data from IgAN patients and relatives is an important starting point for the identification of the disease susceptibility genes., Description: The IgAN Consortium organized a Biobank, recruiting IgAN patients and relatives following a common protocol. A website was constructed to allow scientific information to be shared between partners and to divulge obtained data (URL: http://www.igan.net). The electronic database, the core of the website includes data concerning the subjects enrolled. A search page gives open access to the database and allows groups of patients to be selected according to their clinical characteristics. DNA samples of IgAN patients and relatives belonging to 72 multiplex extended pedigrees were collected. Moreover, 159 trios (sons/daughters affected and healthy parents), 1068 patients with biopsy-proven IgAN and 1040 healthy subjects were included in the IgAN Consortium Biobank. Some valuable and statistically productive genetic studies have been launched within the 5th Framework Programme 1998-2002 of the European project No. QLG1-2000-00464 and preliminary data have been published in "Technology Marketplace" website: http://www.cordis.lu/marketplace., Conclusion: The first world IgAN Biobank with a readily accessible database has been constituted. The knowledge gained from the study of Mendelian diseases has shown that the genetic dissection of a complex trait is more powerful when combined linkage-based, association-based, and sequence-based approaches are performed. This Biobank continuously expanded contains a sample size of adequately matched IgAN patients and healthy subjects, extended multiplex pedigrees, parent-child trios, thus permitting the combined genetic approaches with collaborative studies.

Mutations of CFTR gene are responsible for cystic fibrosis (CF) and other clinical conditions such as congenital absence of the vas deferens (CAVD), chronic pancreatitis (IP), and idiopathic disseminated bronchiectasis (DBE) classified as CFTR-related disorders. The PCR/OLA assay is designed to detect 31 known mutations including the 24 most common CF mutations worldwide, as identified by the CF Consortium. In order to define the CFTR genotype a series of 1812 individuals from central-southern Italy with and without CF manifestations were screened by using the PCR/OLA assay. Here we report the description of five cases of anomalous electropherograms obtained after PCR/OLA analysis, that led to the identification, in the homozygous state, of two point mutations (D110H and S589N) not included in the assay test panel, a large gene deletion (CFTRdel14b&#95;17b), and an exonic polymorphism (c.4002A > G). Haplotype and real time PCR analysis were also performed in the subject carrying the large CFTR deletion. The study demonstrates that the PCR/OLA assay, besides being an efficient and user-friendly method to screen known mutations in the CFTR gene, may also function as a mutation/polymorphism-scanning assay, at least for certain nucleotide changes located in some critical regions of the gene.

Objective: This study aims at evaluating the occupational exposure to polycyclic aromatic hydrocarbons (PAH) through biological monitoring and at investigating the influence of selected genetic polymorphisms on the levels of internal dose biomarker., Design: A cross-sectional study has been conducted on the basis of biological measurements in order to evaluate the levels of PAH exposure., Setting: PAH exposure assessment has been carried out in a coke-oven of one of the largest European steel plants (ILVA, Taranto), in the context of the program of medical surveillance carried in collaboration with the ILVA Occupational Health Service., Participants: 355 coke-oven workers exposed to PAH were enrolled from the 3 batteries of the coke oven plant. Maintenance workers and industrial cleaners were included., Main Outcome Measures: The measurement of urinary levels of 1-hydroxypyrene (1-OHP) as biomarker of internal dose has been carried out, as well as the evaluation of metabolic polymorphisms of the cytochrome P450 1A1 (CYP1A1), glutathione S-transferases mu (GSTM1) and theta (GSTT1) as biomarkers of susceptibility., Results: The median value of urinary 1-OHP was 1,05 microMol/Molcreat. The difference between the groups was statistically significant, with the highest value observed in the maintenance group (median 1,71, range 0,06-14,69 microMol/Molcreat). It is remarkable that 25% of the workers exceeded the proposed benchmark guideline value of2,3 microMol/Molcreat. No statistical difference was found in relation to smoking habits. Workers with GSTT1 null genotype had higher 1-OHP levels than those with wild type (p = 0,06)., Conclusions: The levels of biomarker of internal dose reflect a critical exposure to PAH in the coke-oven plant. Further observations are needed to better investigate the role of GSTT1 on the 1-OHP levels.

Purpose: Keratoconus is a noninflammatory corneal disorder that is clinically and genetically heterogeneous. Mutations in the VSX1 (visual system homeobox 1) gene have been identified for two distinct, inherited corneal dystrophies: posterior polymorphous corneal dystrophy and keratoconus. To evaluate the possible role of the VSX1 gene in a series of Italian patients, 80 keratoconus-affected subjects were screened for mutations., Methods: The diagnosis of keratoconus was made on the basis of clinical examination and corneal topography. The whole coding region and the exon-intron junctions of the VSX1 gene were analyzed by direct sequencing., Results: Three already-described changes, D144E, G160D, and P247R, and a novel L17P mutation were found in 7 of 80 unrelated patients (8.7%). Two undescribed intronic polymorphisms are also reported., Conclusions: Mutational analysis of the VSX1 gene in a series of Italian patients revealed one novel mutation and confirmed an important role played by this gene in a significant proportion of patients affected by keratoconus, when it is inherited as an autosomal dominant trait with variable expressivity and incomplete penetrance.

The authors report the occupational medicine problems in the Apulia region, which are representative of those in Southern Italy. Late industrialization was associated with an early peak in injuries that was not associated with an increased workforce. Examples of operations adversely affecting worker and population health are presented.

Biological markers can be used at an individual level for medical surveillance or at group level within the context of epidemiological surveillance. However, the use of biological monitoring for medical and epidemiological surveillance is heavily conditioned by the critical issue of biomarker validation. One effective means for combining biological monitoring with medical and epidemiological surveillance is the mandatory annual report on anonymous and collective data; this will be made possible after providing adequate training schemes for the occupational physicians involved and the definition of appropriate guidelines, aimed at improving the quality of occupational health services. It will then be possible to design multicentric surveys to assess the quality of occupational health services using validated indicators.

Implementation of automated external defibrillators (AEDs) in workplace can help to revive victims of sudden cardiac arrest. The American College of Occupational and Environmental Medicine, the American Heart Association have all issued guidelines for medical management of AED programs. The School of Occupational Health-University of Bari--is currently involved in a programme to be developed with the regional 118 emergency team and the Health Department of Region Apulia aiming at establishing a well controlled use of AED in the workplace.

Recent studies of Mendelian disease have begun to clarify the clinical spectrum of the group of disorders that make up familial, focal segmental glomerulosclerosis (FSGS) and nephrotic syndromes. In familial forms of focal segmental glomerulosclerosis (FSGS), both autosomal recessive and dominant inheritance patterns have been reported. At least three genes have been identified which, when defective, cause familial FSGS or nephrosis: the NPHS1 gene, encoding nephrin; the NPHS2 gene, encoding podocin; and the ACTN4 gene, encoding a-actinin-4. Because the majority of FSGS cases occur as sporadic disease, the recently described mutations in the NPHS2 gene "in approximately 25 percent of cases of apparently sporadic, steroid-resistant FSGS in children" have claimed great interest. The applicability of these observations to adults, including the possible importance of the nephrin and alpha-actinin-4 genes in the sporadic disease, remain to be determined. Finally, the mechanisms of podocyte damage and the molecular basis of glomerulosclerosis are reviewed.

Several studies investigating the adverse short-term effects of air pollution indicate that even concentrations of air pollutants close to or lower than air quality standards could negatively affect respiratory and cardiovascular health. Therefore, it seems crucial to develop knowledge about health adverse effects in industrialized urban areas. A time-series study on the association between air pollution levels and daily mortality and hospital admissions for respiratory and cardiovascular diseases is currently carried out in the town of Taranto, "area at high environmental risk" according to WHO because of the presence of several heavy industries and elevated rates for all-causes and all cancer mortality, higher than regional mortality rates, especially for lung cancer.

The development of Occupational Epidemiology in Italy is closely correlated with the political and social awareness of the needs of preventive strategies in the workplace. In the late '60s the Trade Unions supported a model of intervention based on the involvement of the so-called "Homogeneous group of workers" in the validation of the preventive measures taken on the workplace. In spite of the shortcomings of the model, it was extremely effective resulting in enhanced perception of the priority of preventive strategies and in the formation within the National Health Service of the Occupational Health Services. In Italy over the period 1973-2002 there has been an impressive trend of research in field of occupational epidemiology (a search on Medline shows an increasing trend over the years and, in terms of international comparison, higher figures than in Germany, France and Spain). Occupational Epidemiology is now present in the activities of the local Occupational Health Services and in the teaching activities of the Medical Schools throughout the country.

The authors studied a cohort of 2660 pulp and paper workers in Capitanata, Apulia (Italy). All workers with at least 1 year of employment on January 1 1965 or thereafter until march 30, 2000 where studied. Standardized Mortality Ratios (SMRs) were used to compare the mortality rates of the cohort with those of Apulian population. Ninetyfive percent confidence intervals (CIs) for SMRs were obtained. Cancer risks significantly associated with work where observed: all causes for males (SMR 112.09, 95% CI 104.91-119.77), brain cancer for males (SMR 206.04, 95% CI 103.04-412.01), digestive tract for males (SMR 126.78, CI 100.63-159.73). Association with other malignancies are suggested in this study for Laryngeal and Kidney. These malignancies have been associated with different exposures in the production cycle: biocides (Kidney), Formaldehyde (Kidney, Brain). Unclear is the risk factor for the excess showed in digestive tract cancer.

Recent developments in the genetics and physiology of cystinuria do not support the traditional classification, which is based on the excretion of cystine and dibasic amino acids in obligate heterozygotes. Mutations of only two genes (SLC3A1 and SLC7A9), identified by the International Cystinuria Consortium (ICC), have been found to be responsible for all three types of the disease. The ICC set up a multinational database and collected genetic and clinical data from 224 patients affected by cystinuria, 125 with full genotype definition. Amino acid urinary excretion patterns of 189 heterozygotes with genetic definition and of 83 healthy controls were also included. All SLC3A1 carriers and 14% of SLC7A9 carriers showed a normal amino acid urinary pattern (i.e., type I phenotype). The rest of the SLC7A9 carriers showed phenotype non-I (type III, 80.5%; type II, 5.5%). This makes the traditional classification imprecise. A new classification is needed: type A, due to two mutations of SLC3A1 (rBAT) on chromosome 2 (45.2% in our database); type B, due to two mutations of SLC7A9 on chromosome 19 (53.2% in this series); and a possible third type, AB (1.6%), with one mutation on each of the above-mentioned genes. Clinical data show that cystinuria is more severe in males than in females. The two types of cystinuria (A and B) had a similar outcome in this retrospective study, but the effect of the treatment could not be analyzed. Stone events do not correlate with amino acid urinary excretion. Renal function was clearly impaired in 17% of the patients.

The study describes the mortality of 417 workers employed in a asbestos-cement plant, located in Bari, Puglia, Southern Italy. Follow up started on February 1st 1972. The vital status and cause of death were ascertained at 1995. The mortality experience of the Apulian population was used as comparison. Using 90% confidence limits (CLs), a significant increase in mortality was observed in our cohort from: all causes of death (SMR 118, CL 100-139), pneumoconiosis (SMR 14810, CL 10298-20683), all types of cancer (SMR 139, CL 105-181), lung (SMR 191, CL 126-277), pleural (SMR 1560 CL 431-4081) and peritoneum (SMR 1705, CL 303-5367) malignant neoplasms. In our cohort, the discrepancy between observed and expected mortality for lung and pleural cancer occurred 30 years after the first exposure, after 40 years for all neoplasms and peritoneum cancer. Under the Cox regression model, lung cancer SMR showed a curvilinear trend along time since first exposure, the peak being detected at 35 years. Finally, SMRs from our cohort were compared to a previously described cohort including workers from the same plant compensated for asbestosis by INAIL.

Background: Cystinuria is an inherited disorder of cystine and dibasic amino acids transport that results in urolithiasis because of poor cystine solubility. Three cystinuria phenotypes, differentiated according to urinary amino acid excretion in obligate heterozygotes, were regarded as allelic variants of a monogenic disease. Two mutated amino acid transporter genes, however, have been recently identified as responsible for cystinuria. Mutations in the SLC3A1 gene. encoding for the heavy subunit of the transporter protein rBAT, were associated with type I cystinuria, whereas type II and III cystinuria were associated with mutations in the SLC7A9 gene, encoding for a light subunit of rBAT. Lysine and arginine metabolism have, therefore, been evaluated in cystinuria homozygotes and heterozygotes to better define the cystinuria phenotypes and their correlations with these emerging genotypes., Patients and Methods: Lysine and arginine intestinal absorption and renal excretion were assessed by oral loading and compared to normal controls. Seven cystinuria homozygotes and 7 obligate heterozygotes belonging to the different types received alternately an oral dose of 0.5 mmol/kg body weight lysine or arginine. Plasma concentrations of lysine, arginine, ornithine (derived from rapid arginine conversion) were measured 0, 1, 2, and 3 hours after loading. Their urinary concentrations were measured in morning urine and in urine collected 0-6 hours after loading., Results: Gut lysine absorption was deficient in type II and III, and normal in type I cystinuria homozygotes. Impaired arginine intestinal absorption, as well as massive lysine, arginine, and ornithine hyperexcretion were shared by all homozygotes, irrespective of the type. All heterozygotes shared normal lysine absorption, whereas arginine absorption was slightly impaired in type II and III heterozygotes, which also displayed high lysine, arginine, and ornithine urinary excretion after loading., Conclusions: Two cystinuria phenotypes, type I and non-type I, can be identified in both homozygous and heterozygous cystinuric subjects by oral loading tests with lysine and arginine. In agreement with recent molecular findings, non-type I cystinuria comprises mentioned type II and type III, which constitute allelic variants of a cystine and dibasic amino acid transport disorder distinct from type I cystinuria.

Mutations in the unconventional myosin VI gene, Myo6, are associated with deafness and vestibular dysfunction in the Snell's waltzer (sv) mouse. The corresponding human gene, MYO6, is located on chromosome 6q13. We describe the mapping of a new deafness locus, DFNA22, on chromosome 6q13 in a family affected by a nonsyndromic dominant form of deafness (NSAD), and the subsequent identification of a missense mutation in the MYO6 gene in all members of the family with hearing loss.

Cystinuria (OMIM 220100) is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids that results in nephrolithiasis of cystine. Mutations in SLC3A1, which encodes rBAT, cause Type I cystinuria, and mutations in SLC7A9, which encodes a putative subunit of rBAT (b(o,+)AT), cause non-Type I cystinuria. Here we describe the genomic structure of SLC7A9 (13 exons) and 28 new mutations in this gene that, together with the seven previously reported, explain 79% of the alleles in 61 non-Type I cystinuria patients. These data demonstrate that SLC7A9 is the main non-Type I cystinuria gene. Mutations G105R, V170M, A182T and R333W are the most frequent SLC7A9 missense mutations found. Among heterozygotes carrying these mutations, A182T heterozygotes showed the lowest urinary excretion values of cystine and dibasic amino acids. Functional analysis of mutation A182T after co-expression with rBAT in HeLa cells revealed significant residual transport activity. In contrast, mutations G105R, V170M and R333W are associated to a complete or almost complete loss of transport activity, leading to a more severe urinary phenotype in heterozygotes. SLC7A9 mutations located in the putative transmembrane domains of b(o,+)AT and affecting conserved amino acid residues with a small side chain generate a severe phenotype, while mutations in non-conserved residues give rise to a mild phenotype. These data provide the first genotype-phenotype correlation in non-Type I cystinuria, and show that a mild urinary phenotype in heterozygotes may associate with mutations with significant residual transport activity.

Cystinuria is an autosomal recessive aminoaciduria in which two clinical types have been described (type I and non-type I). Cystinuria type I is caused by mutations in SLC3A1, a gene located in 2p16 coding for an amino acid transporter named rBAT. Using multiplex semi-quantitative fluorescent PCR, we amplified the ten exons of SLC3A1 together with exon 5 of DSCR1 (located on chromosome 21) as a double-dose control gene. We detected two large novel deletions in a Belgian family, one comprising exons 2-10 and another one at exon 10. The method described here can be used to detect a range of deletions from single-base differences in size to entire missing exons, making it useful for scanning genes with a small to medium number of exons., (Copyright 2000 Wiley-Liss, Inc.)

An increasing body of evidence supports the concept of genetic heterogeneity within inflammatory bowel disease (IBD). In this study, a polymorphism of the motilin gene, which determines an amino acid substitution in the motilin protein, has been investigated in IBD patients. Fifty patients with ulcerative colitis (UC), and 52 with Crohn's disease (CD) were investigated for anti-neutrophil cytoplasmatic antibodies (ANCA) and the polymorphism in the second exon of the motilin gene. Sixty unrelated blood donors served as controls. ANCA were found in 30% of UC and 13% of CD. In controls the DNA polymorphism identified two alleles (1 and 2) at a frequency of 42% and 58%, respectively. Patients with either UC or CD showed a slight increase in the frequency of allele 2 (69% and 60%, respectively; P > 0.05 vs controls). This allele was predominant in ANCA-positive CD patients (86%; P < 0.04) while in UC it did not differ. All ANCA-positive CD patients had the disease confined to the colon. A polymorphism of second exon of the motilin gene, leading to a protein variant, is significantly more frequent in the subset of ANCA-positive CD patients. This subgroup of patients appears to share peculiar genetic and clinical features.

Iron availability regulates ferritin synthesis posttranscriptionally by the interaction between iron-regulatory proteins (IRPs) and an iron responsive element (IRE), a stem-loop sequence located on the 5' untranslated region of ferritin mRNA. IRPs recognize IREs as a sequence/structure motif, blocking ferritin translation. Recently, we and others independently described families with a combination of hyperferritinemia (serum L-ferritin > or = 1,000 microg/L, without iron overload) and congenital bilateral cataract, transmitted as an autosomal-dominant trait. The molecular basis were two distinct point mutations in the highly conserved CAGUG(X) hexaloop of L-ferritin IRE on chromosome 19. A new three-generation family with a similar phenotype and a unique genotype is here reported. DNA amplification by polymerase chain reaction and sequence analysis showed a 29-base pair deletion in the L-ferritin IRE, involving the whole 5' sequence essential to the base pairing of the IRE stem. This deletion is predicted to cause the disruption of IRE stem-loop secondary structure and the nearly complete abolition of the negative control of ferritin synthesis by IRE/IRP binding. Hereditary Hyperferritinemia-Cataract Syndrome (HHCS) appears as a new genetic disorder with a unique phenotype associated with at least four different mutations in the L-ferritin IRE. Hematologists should take into account HHCS in the differential diagnosis of unexplained hyperferritinemia.

The detection of carrier status in female relatives of Duchenne/Becker muscular dystrophy patients is not always possible and this poses a problem in genetic counseling. We have developed a simple method that can be used in families in which affected males are characterized by the presence of a deletion within the dystrophin gene. PCR fragments, corresponding to the deleted regions are used as fluorescent probes for hybridization of peripheral lymphocytes nuclei of female relatives. The results obtained clearly demonstrate the feasibility of this method for detecting female DMD/BMD carriers.

Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) is an autosomal dominant condition consisting of congenital dysplasia of the eyelids with a reduced horizontal diameter of the palpebral fissures, droopy eyelids and epicanthus inversus. Two clinical entities have been described: type I and type II. The former is distinguished by female infertility, whereas the latter presents without other symptoms. Both type I and type II were recently mapped on the long arm of chromosome 3 (3q22-q23), suggesting a common gene may be affected. The centromeric and the telomeric limits of this region are well defined between loci D3S1316 and D3S1615, which reside approximately 5 cM apart. Here, we present the construction of a YAC contig spanning the entire BPES locus using 17 polymorphic markers, 2 STS and 28 ESTs. This region of approximately 5 Mb was covered by 31 YACs, and was supported by detailed FISH analysis. In addition, we have precisely mapped the propionyl-CoA carboxylase beta polypeptide (PCCB), the gene mutated in propionic acidemia, within this contig. Apart from providing a framework for the identification of the BPES gene, this contig will also be useful for the future identification of defects and genes mapped to this region, and for developing template resources for genomic sequencing.

Cystinuria is an autosomal recessive aminoaciduria in which three urinary phenotypes (I, II, and III) have been described. An amino acid transporter gene, SLC3A1 (formerly rBAT), was found to be responsible for this disorder. Mutational and linkage analysis demonstrated the presence of genetic heterogeneity in which the SLC3A1 gene is responsible for type I cystinuria but not for type II or type III. In this study, we report the identification of the cystinuria type III locus on the long arm of chromosome 19 (19q13.1), obtained after a genomewide search. Pairwise linkage analysis in a series of type III or type II families previously excluded from linkage to the cystinuria type I locus (SLC3A1 gene) revealed a significant maximum LOD score (zeta max) of 13.11 at a maximum recombination fraction (theta max) of .00, with marker D19S225. Multipoint linkage analysis performed with the use of additional markers from the region placed the cystinuria type III locus between D19S414 and D19S220. Preliminary data on type II families also seem to place the disease locus for this rare type of cystinuria at 19q13.1 (significant zeta max = 3.11 at theta max of .00, with marker D19S225).

A new genetic disorder (Hyperferritinemia and Cataract Syndrome) characterized by a combination of high serum ferritin level and congenital bilateral nuclear cataract has been recently described. This disease is transmitted as autosomal dominant trait and is due to mutations in the ferritin L gene (FTL). FTL gene has been localized to 19q13.3-qter by somatic cell hybrids. In this work we present the precise mapping of FTL gene on chromosome 19q13.3 using in situ fluorescence hybridization.

Cystinuria is an autosomal recessive disorder of amino acid transport, manifesting as three phenotypes (I, II, and III). An amino acid transport gene, rBAT, is responsible for cystinuria. Mutation and linkage analyses have demonstrated the disease to be heterogeneous, with rBAT being the defective gene in type I cystinuria. The genomic structure of the human rBAT gene (HGMW-approved symbol SLC 3A1) has been established via two strategies: (i) construction of two different genomic libraries by subcloning the Mega-YAC921B6 (CEPH), containing rBAT, in Lambda ZAP and screening using rBAT cDNA and different PCR products; and (ii) generation and sequencing of genomic fragments by long PCR using rBAT cDNA-derived primers. The rBAT gene spans approximately 45 kb and consists of 10 exons. The introns range from 500 to 13,000 bp. All splice sites conform to the GT/AG rule. The promoter region has been further analyzed, and a predicted TATA box 98 bp upstream of the first coding ATG was identified. In addition an Alu repeat has been detected 72 bp upstream of the predicted TATA box.

A cystinuria disease gene (rBAT) has recently been identified, but evidence strongly suggests that only Type-I cystinuria is due to mutations in this gene. Sixteen point mutations and a large deletion causing the disease have so far been described in the rBAT gene sequence. To identify new mutated alleles, genomic DNA was analyzed, after the determination of the entire genomic structure of the rBAT gene, by RNA-single strand conformation polymorphism analysis, an accurate and sensitive method able to detect nucleotide changes. Four new point mutations, a large deletion, and a common intragenic polymorphism were detected. These new mutations increase to 22 the number of mutated alleles so far characterized in rBAT. In addition, the frequency of 21 mutations was assessed in a sample of accurately defined Type-I cystinuria chromosomes. They account for about 58% of all Type-I chromosomes, mutation M467T being the most common (0.26).

The cDNAs of mammalian amino acid transporters already identified could be grouped into four families. One of these protein families is composed of the protein rBAT and the heavy chain of the cell surface antigen 4F2 (4F2hc). The cRNAs of rBAT and 4F2hc induce amino acid transport activity via systems b(0,+) -like and y(+)L -like inXenopus oocytes respectively. Surprisingly, neither rBAT nor 4F2hc is very hydrophobic, and they seem to be unable to form a pore in the plasma membrane. This prompted the hypothesis that rBAT and 4F2hc are subunits or modulators of the corresponding amino acid transporters. The association of rBAT with a light subunit of ~40kDa has been suggested, and such an association has been demonstrated for 4F2hc.The b(0,+)-like system expressed in oocytes by rBAT cRNA transports L-cystine, L-dibasic and L-neutral amino acids with high-affinity. This transport system shows exchange of amino acids through the plasma membrane ofXenopus oocytes, suggesting a tertiary active transport mechanism. The rBAT gene is mainly expressed in the outer stripe of the outer medulla of the kidney and in the mucosa of the small intestine. The protein localizes to the microvilli of the proximal straight tubules (S3 segment) of the nephron and the mucosa of the small intestine. All this suggested the participation of rBAT in a high-affinity reabsorption system of cystine and dibasic amino acids in kidney and intestine, and indicated rBAT (named SLC3A1 in Gene Data Bank) as a good candidate gene for cystinuria. This is an inherited aminoaciduria due to defective renal and intestinal reabsorption of cystine and dibasic amino acids. The poor solubility of cystine causes the formation of renal cystine calculi. Mutational analysis of the rBAT gene of patients with cystinuria is revealing a growing number (~20) of cystinuria-specific mutations, including missense, nonsense, deletions and insertions. Mutations M467T (substitution of methionine 467 residue for threonine) and R270X (stop codon at arginine residue 270) represent approximately half of the cystinuric chromosomes where mutations have been found. Mutation M467T reduces transport activity of rBAT in oocytes. All this demonstrates that mutations in the rBAT gene cause cystinuria.Three types of cystinuria (types, I, II and III) have been described on the basis of the genetic, biochemical and clinical manifestations of the disease. Type I cystinuria has a complete recessive inheritance; type I heterozygotes are totally silent. In contrast, type II and III heterozygotes show, respectively, high or moderate hyperaminoaciduria of cystine and dibasic amino acids. Type III homozygotes show moderate, if any, alteration of intestinal absorption of cystine and dibasic amino acids; type II homozygotes clearly show defective intestinal absorption of these amino acids. To date, all the rBAT cystinuria-specific mutations we have found are associated with type I cystinuria (~70% of the chromosomes studied) but not to types II or III. This strongly suggests genetic heterogeneity for cystinuria. Genetic linkage analysis with markers of the genomic region of rBAT in chromosome 2 (G band 2p16.3) and intragenic markers of rBAT have demonstrated genetic heterogeneity for cystinuria; the rBAT gene is linked to type I cystinuria, but not to type III. Biochemical, genetic and clinical studies are needed to identify the additional cystinuria genes; a low-affinity cystine reabsortion system and the putative light subunit of rBAT are additional candidate genes for cystinuria.

Recently, we described a new genetic disorder (the "hereditary hyperferritinemia-cataract syndrome") clinically characterized by the combination of elevated serum ferritin and congenital bilateral nuclear cataract, both cotransmitted as an autosomal dominant trait. In affected subjects, hyperferritinemia (ranging from 950 to 2,259 micrograms/L) is typically not related to iron overload. Differently from subjects with hereditary hemochromatosis, they have normal to low levels of serum iron and percent of transferrin saturation and absence of iron overload in parenchymal organs. When unnecessary phlebotomies are performed, they rapidly develop iron-deficient anemia, with persistently elevated levels of serum ferritin. By RNA-single-strand conformation polymorphism screening of the L-subunit ferritin gene on chromosome 19, we were able to identify in affected subjects a mutation in the 5' untranslated region. This mutation involves the five nucleotides sequence [CAGUG] of the iron-responsive element (IRE), which is critical for the posttranscriptional regulation of ferritin synthesis by means of IRE-binding protein (IRE-BP). Thus, it is very likely to provide the molecular basis for the iron-insensitive upregulation of ferritin synthesis in affected subjects.

Several gene transfer techniques that employ 'naked DNA' molecules have recently been developed and numerous gene therapy protocols that make use of 'naked-DNA' have been proposed. We studied the possibility of enhancing the stability of 'naked DNA vectors' and thus also gene transfer and expression efficiencies, by constructing phosphorothioate (PS-) double strand DNA molecules and functional transcription units. We first synthesized short PS-double strand DNA molecules by the annealing of two complementary, 35 nt long, oligonucleotides. The accessibility of DNA modifying enzymes to this molecule was significantly decreased: T4-ligase and kinase activity were respectively reduced up to 1/2 and to 1/6, as compared to the normal phosphodiester molecule. Nucleolytic stability was increased either to purified enzymes (DNase I and Bal31) or to incubations in fresh serum, cell culture medium or in muscle protein extract. Phosphorothioate end-capped complete eukaryotic transcription units (obtained by Taq polymerase amplification with PS-primers) were not significantly protected from nucleolytic attack. On the contrary, synthetic transcription units, 'mini genes', obtained by Taq amplification with 1, 2 or 3 PS-dNTP substitutions, were resistant to DNase I and Bal31 nucleolytic activity. Transcription efficiency, driven by the T7 promoter, was 96.5, 95 and 33.5% (respectively with 1, 2 or 3 substitutions), as compared to the normal phosphodiester molecules.

Cystinuria is an autosomal recessive amino-aciduria where three urinary phenotypes have been described (I, II, and III). An amino acid transporter gene, SLC3A1 (formerly rBAT), was found to be responsible for this disorder. To assess whether mutations in SLC3A1 are involved in different cystinuria phenotypes, linkage with this gene and its nearest marker (D2S119) was analyzed in 22 families with type I and/or type III cystinuria. Linkage with heterogeneity was proved (alpha = 0.45; P < 0.008). Type I/I families showed homogeneous linkage to SLC3A1 (Zmax > 3.0 at theta = 0.00; alpha = 1), whereas types I/III and III/III were not linked. Our data suggest that type I cystinuria is due to mutations in the SLC3A1 gene, whereas another locus is responsible for type III. This result establishes genetic heterogeneity for cystinuria, classically considered as a multiallelic monogenic disease.

A cystinuria disease gene (rBAT) has been recently identified, and some mutations causing the disease have been described. The frequency of these mutations has been investigated in a large sample of 51 Italian and Spanish cystinuric patients. In addition, to identify new mutated alleles, genomic DNA has been analyzed by an accurate and sensitive method able to detect nucleotide changes. Because of the lack of information available on the genomic structure of rBAT gene, the study was carried out using the sequence data so far obtained by us. More than 70% of the entire coding sequence and 8 intron-exon boundaries have been analyzed. Four new mutations and seven intragenic polymorphisms have been detected. All mutations so far identified in rBAT belong only to cystinuria type I alleles, accounting for approximately 44% of all type I cystinuric chromosomes. Mutation M467T is the most common mutated allele in the Italian and Spanish populations. After analysis of 70% of the rBAT coding region, we have detected normal sequences in cystinuria type II and type III chromosomes. The presence of rBAT mutated alleles only in type I chromosomes of homozygous (type I/I) and heterozygous (type I/III) patients provides evidence for genetic heterogeneity where rBAT would be responsible only for type I cystinuria and suggests a complementation mechanism to explain the intermediate type I/type III phenotype.

RNA single-strand conformation polymorphism (rSSCP) is a recently developed method for detecting genetic defects. This technique requires DNA amplification with a polymerase chain reaction making use of one T7 promoter-containing primer. Amplification products are subsequently transcribed in vitro and the labelled transcripts are analysed for single-strand conformation changes. rSSCP has been applied to mutation screening of the phenylalanine hydroxylase gene and rBAT cDNA, from PKU and cystinuric patients, respectively. Experimental evidence shows that 83% and 86% of screened PKU and cystinuric mutations, respectively, give rise to detectable rSSCP signals. Thus, results obtained show that RNA single-strand conformation polymorphism analysis is generally applicable and is a suitable technique for detecting genetic disease causing mutations, both in basic research and in clinical practice.

A complete coding-region analysis on 225 cystic fibrosis (CF) chromosomes from a cohort that includes all the affected subjects born in two North-Eastern Italian regions over eight years was performed. In a previous study, we identified mutations on 166/225 (73.8%) CF chromosomes after screening for 62 mutations. To characterise the remaining 59 CF chromosomes, we carried out automated direct DNA sequencing (exons 9 and 13), RNA single-strand conformation polymorphism (exons 1-8 and 10-12) and denaturing gradient gel electrophoresis (exons 14a-24) of the 27 exons and flanking regions of the CF transmembrane conductance regulator gene. We identified 22 mutations, four of which are novel, viz. 711 + 5G-->A, R709X, 3132delTG and 2790-2A-->G, and we characterised 90.2% (203/225) of the CF chromosomes. Taking advantage of the homogeneity of the sample, an evaluation of the most important clinical parameters, assessed at the age of 12 years, is presented. We confirm some previously reported genotype-phenotype correlations and we report a new nonsense mutation (R709X) associated with a pancreatic sufficient phenotype.

We have been screening a cohort of 225 chromosomes from cystic fibrosis patients for mutations in the cystic fibrosis transmembrane regulator gene using a combination of DGGE,RNA-SSCP and DNA sequencing. A novel splice site mutation was detected by multiplex DGGE in a homozygous patient. Restriction-site generating PCR (RG-PCR) analysis demonstrated that both parents carried the same mutation. The molecular haplotype was the same. All the known ancestors came from the same (Veneto) region, and no consanguinity was documented up to the sixth generation.

The product of the human motilin gene (MLN) has an important role in regulating gastrointestinal motility. The precise chromosomal localisation and expression of this gene are still unresolved. Here, we report a detailed study assigning MLN to 6p21.3; MLN is tightly linked to the HLA-DQalpha locus. Moreover, MLN expression has been evaluated in a large series of tissues. Positive signals have been obtained for brain, bronchi and a gastrointestinal malignancy. Direct sequencing exon by exon of the codifying region, intron/exon boundaries and promoter has allowed the identification of three DNA polymorphisms, one of which corresponds to a common protein variant. The chromosomal localisation of MLN, and its expression in broncoepithelial cells suggests that this gene is involved in immotile-cilia syndrome (ICS) disease. Sequence and segregation analysis of the MLN gene carried out in two families, in which the disease locus was previously assigned to 6p21.3, exclude MLN as a candidate gene for the HLA-associated form of ICS.

A strategy is described that allows a rapid and accurate identification and screening of cystic fibrosis gene mutations. It consists of setting up and developing RNA single strand conformation polymorphism (rSSCP) protocols, a technique based on the large repertoire of secondary structure of single-stranded RNA. By incorporating the T7 phage promoter sequence into PCR primers, it is possible to carry out rSSCP and compare it to standard single-strand conformation polymorphism (SSCP). Several parallel tests indicate that rSSCP detects a higher fraction of single base changes, and is less time consuming than SSCP since it requires only one fairly short electrophoretic run. Using this technique we were able to identify two new splicing mutations in introns 5 (711 + 5G-->A) and 10 (1717-8G-->A) of the CFTR gene.