Characterization of a New Variant in ARHGAP31 Probably Involved in Adams-Oliver Syndrome in a Family with a Variable Phenotypic Spectrum.

Adams-Oliver syndrome is a rare inherited condition characterized by scalp defects and limb abnormalities. It is caused by variants in different genes such as ARHGAP31. Here, we used an interdisciplinary approach to study a family with lower limb anomalies. We identified a novel variant in the ARHGAP31 gene that is predicted to result in a truncated protein with a constitutively activated catalytic site due to the loss of 688 amino acids involved in the C-terminal domain, essential for protein auto-inhibition. Pathogenic variants in ARHGAP31 exon 12, leading to a premature protein termination, are associated with Adams-Oliver syndrome. Bioinformatic analysis was useful to elucidate the impact of the identified genetic variant on protein structure. To better understand the impact of the identified variant, 3D protein models were predicted for the ARHGAP31 wild type, the newly discovered variant, and other pathogenetic alterations already reported. Our study identified a novel variant probably involved in Adams-Oliver syndrome and increased the evidence on the phenotypic variability in patients affected by this syndrome, underlining the importance of translational research, including experimental and bioinformatics analyses. This strategy represents a successful model to investigate molecular mechanisms involved in syndrome occurrence.

OTX Genes in Adult Tissues.

OTX homeobox genes have been extensively studied for their role in development, especially in neuroectoderm formation. Recently, their expression has also been reported in adult physiological and pathological tissues, including retina, mammary and pituitary glands, sinonasal mucosa, in several types of cancer, and in response to inflammatory, ischemic, and hypoxic stimuli. Reactivation of OTX genes in adult tissues supports the notion of the evolutionary amplification of functions of genes by varying their temporal expression, with the selection of homeobox genes from the "toolbox" to drive or contribute to different processes at different stages of life. OTX involvement in pathologies points toward these genes as potential diagnostic and/or prognostic markers as well as possible therapeutic targets.

Occurrence of L1M Elements in Chromosomal Rearrangements Associated to Chronic Myeloid Leukemia (CML): Insights from Patient-Specific Breakpoints Characterization.

Chronic myeloid leukemia (CML) is a rare myeloproliferative disorder caused by the reciprocal translocation t(9;22)(q34;q11) in hematopoietic stem cells (HSCs). This chromosomal translocation results in the formation of an extra-short chromosome 22, called a Philadelphia chromosome (Ph), containing the BCR-ABL1 fusion gene responsible for the expression of a constitutively active tyrosine kinase that causes uncontrolled growth and replication of leukemic cells. Mechanisms behind the formation of this chromosomal rearrangement are not well known, even if, as observed in tumors, repetitive DNA may be involved as core elements in chromosomal rearrangements. We have participated in the explorative investigations of the PhilosoPhi34 study to evaluate residual Ph+ cells in patients with negative FISH analysis on CD34+/lin- cells with gDNA qPCR. Using targeted next-generation deep sequencing strategies, we analyzed the genomic region around the t(9;22) translocations of 82 CML patients and one CML cell line and assessed the relevance of interspersed repeat elements at breakpoints (BP). We found a statistically higher presence of LINE elements, in particular belonging to the subfamily L1M, in BP cluster regions of both chromosome 22 and 9 compared to the whole human genome. These data suggest that L1M elements could be potential drivers of t(9;22) translocation leading to the generation of the BCR-ABL1 chimeric gene and the expression of the active BCR-ABL1-controlled tyrosine kinase chimeric protein responsible for CML.

Soy diet induces intestinal inflammation in adult Zebrafish: Role of OTX and P53 family.

Inflammatory bowel diseases (IBDs) are a group of inflammatory conditions of the colon and small intestine, including Crohn's disease and ulcerative colitis. Since Danio rerio is a promising animal model to study gut function, we developed a soy-dependent model of intestinal inflammation in adult zebrafish. The soya bean meal diet was given for 4 weeks and induced an inflammatory process, as demonstrated by morphological changes together with an increased percentage of neutrophils infiltrating the intestinal wall, which developed between the second and fourth week of treatment. Pro-inflammatory genes such as interleukin-1beta, interleukin-8 and tumour necrosis factor alpha were upregulated in the second week and anti-inflammatory genes such as transforming growth factor beta and interleukin-10. Interestingly, an additional expression peak was found for interleukin-8 at the fourth week. Neuronal genes, OTX1 and OTX2, were significantly upregulated in the first two  weeks, compatible with the development of the changes in the gut wall. As for the genes of the p53 family such as p53, DNp63 and p73, a statistically significant increase was observed after two weeks of treatment compared with controls. Interestingly, DNp63 and p73 were shown an additional peak after four weeks. Our data demonstrate that soya bean meal diet negatively influences intestinal morphology and immunological function in adult zebrafish showing the features of acute inflammation. Data observed at the fourth week of treatment may suggest initiation of chronic inflammation. Adult zebrafish may represent a promising model to better understand the mechanisms of food-dependent intestinal inflammation.

Expression of Otx Genes in Müller Cells Using an In Vitro Experimental Model of Retinal Hypoxia.

INTRODUCTION: Müller glial cells typically activate to react to hypoxic tissue damage in several retinal diseases. We evaluated the in vitro response to a hypoxia-mimicking stimulus on the expression of a set of genes, known to contribute to eye morphogenesis and cell differentiation. MATERIALS AND METHODS: A MIO-M1 Müller cell line was cultured in a hypoxia-mimicking environment by the addition of cobalt chloride to the culture medium, followed by a recovery time in which we mimic restoration from the hypoxic insult. The HIF-1α protein and VEGF-A gene expression were quantified to verify the induction of a hypoxia-like state. RESULTS: Among the genes under study, we did not observe any difference in the expression levels of Otx1 and Otx2 during treatment; conversely, Otx1 was overexpressed during recovery steps. The VEGF-A gene was strongly upregulated at both the CoCl2 and recovery time points. The transactivated isoform (TA) of the TP73 gene showed an overexpression in long-term exposure to the hypoxic stimulus with a further increase after recovery. Discussion. Our molecular analysis is able to describe the activation of a set of genes, never before described, that can drive the response to a hypoxia-like status. The improved comprehension of these cellular events will be useful for designing new therapeutical approaches for retinal pathologies.

A New Targeted CFTR Mutation Panel Based on Next-Generation Sequencing Technology.

Searching for mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) is a key step in the diagnosis of and neonatal and carrier screening for cystic fibrosis (CF), and it has implications for prognosis and personalized therapy. The large number of mutations and genetic and phenotypic variability make this search a complex task. Herein, we developed, validated, and tested a laboratory assay for an extended search for mutations in CFTR using a next-generation sequencing-based method, with a panel of 188 CFTR mutations customized for the Italian population. Overall, 1426 dried blood spots from neonatal screening, 402 genomic DNA samples from various origins, and 1138 genomic DNA samples from patients with CF were analyzed. The assay showed excellent analytical and diagnostic operative characteristics. We identified and experimentally validated 159 (of 188) CFTR mutations. The assay achieved detection rates of 95.0% and 95.6% in two large-scale case series of CF patients from central and northern Italy, respectively. These detection rates are among the highest reported so far with a genetic test for CF based on a mutation panel. This assay appears to be well suited for diagnostics, neonatal and carrier screening, and assisted reproduction, and it represents a considerable advantage in CF genetic counseling.

Whole-gene CFTR sequencing combined with digital RT-PCR improves genetic diagnosis of cystic fibrosis.

Despite extensive screening, 1-5% of cystic fibrosis (CF) patients lack a definite molecular diagnosis. Next-generation sequencing (NGS) is making affordable genetic testing based on the identification of variants in extended genomic regions. In this frame, we analyzed 23 CF patients and one carrier by whole-gene CFTR resequencing: 4 were previously characterized and served as controls; 17 were cases lacking a complete diagnosis after a full conventional CFTR screening; 3 were consecutive subjects referring to our centers, not previously submitted to any screening. We also included in the custom NGS design the coding portions of the SCNN1A, SCNN1B and SCNN1G genes, encoding the subunits of the sodium channel ENaC, which were found to be mutated in CF-like patients. Besides 2 novel SCNN1B missense mutations, we identified 22 previously-known CFTR mutations, including 2 large deletions (whose breakpoints were precisely mapped), and novel deep-intronic variants, whose role on splicing was excluded by ex-vivo analyses. Finally, for 2 patients, compound heterozygotes for a CFTR mutation and the intron-9c.1210-34TG[11-12]T5 allele-known to be associated with decreased CFTR mRNA levels-the molecular diagnosis was implemented by measuring the residual level of wild-type transcript by digital reverse transcription polymerase chain reaction performed on RNA extracted from nasal brushing.

Two ABCB4 point mutations of strategic NBD-motifs do not prevent protein targeting to the plasma membrane but promote MDR3 dysfunction.

The ABCB4 gene encodes for MDR3, a protein that translocates phosphatidylcholine from the inner to the outer leaflet of the hepatocanalicular membrane; its deficiency favors the formation of 'toxic bile'. Several forms of hepatobiliary diseases have been associated with ABCB4 mutations, but the detrimental effects of most mutations on the encoded protein needs to be clarified. Among subjects with cholangiopathies who were screened for mutations in ABCB4 by direct sequencing, we identified the new mutation p.(L481R) in three brothers. According to our model of tertiary structure, this mutation affects the Q-loop, whereas the p.(Y403H) mutation, that we already described in two other families, involves the A-loop. This study was aimed at analyzing the functional relevance of these two ABCB4 mutations: MDR3 expression and lipid content in the culture supernatant were evaluated in cell lines stably transfected with the ABCB4 wild-type clone and corresponding mutants. No differences of expression were observed between wild-type and mutant gene products. Instead, both mutations caused a reduction of phosphatidylcholine secretion compared with the wild-type transfected cell lines. On the contrary, cholesterol (Chol) release, after 1 and 3 mM sodium taurocholate stimulation, was higher in the mutant-transfected cell lines than that in the wild-type and was particularly enhanced in cells transfected with the p.Y403H-construct.In summary, our data show that both mutations do not seem to affect protein expression, but are able to reduce the efflux of phosphatidylcholine associated with increase of Chol, thereby promoting the formation of toxic bile.

Fine characterization of the recurrent c.1584+18672A>G deep-intronic mutation in the cystic fibrosis transmembrane conductance regulator gene.

Splicing mutations account for approximately 12% of the 1,890 cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations described in cystic fibrosis (CF). However, their impact on pre-mRNA processing frequently remains unclear. An interesting opportunity to study CFTR transcripts in vivo involves the use of RNA from nasal brushings. Through this approach we previously identified a deep-intronic mutation (c.1584+18672A>G) that activates a 104-base pair (bp) out-of-frame pseudoexon by creating a donor splice site. The screening of 230 patients with CF identified c.1584+18672A>G in three additional individuals, demonstrating that it is a recurrent, and potentially overlooked, mutation among Italian patients. Haplotype analysis suggests that it originated from at least two independent events. To characterize the mutation further, a genomic region, including the activated pseudoexon and surrounding intronic sequences, was cloned into an expression vector and transfected into HeLa cells. RT-PCR analysis identified two alternative splicing products, produced by the activation of two different cryptic acceptor splice sites. One included the 104-bp pseudoexon (78.7% of transcripts), and the other led to the inclusion of a 65-bp pseudoexon (21.3% of mRNAs). The allele-specific measurement of wild-type and aberrant splicings from the nasal-brushing RNA of the three probands with genotype F508del/c.1584+18672A>G demonstrated: (1) a low level of pseudoexon inclusion in the F508del transcript (not containing the splicing mutation); (2) residual wild-type splicing in the c.1584+18672A>G mRNA; (3) the degradation of aberrant transcripts; and (4) the relative strength of the different cryptic splice sites. Interestingly, the residual wild-type splicing detected in transcripts bearing the c.1584+18672A>G mutation correlates well with the milder clinical phenotype of patients.

Cystic fibrosis newborn screening: distribution of blood immunoreactive trypsinogen concentrations in hypertrypsinemic neonates.

The IRT screening test for the use in diagnosing newborns with CF has a high sensitivity but is not very specific resulting in a large number of screened positive infants found to have a normal sweat test. The aim of this study was to analyze the differences in b-IRT levels among different groups of newborns positive to NBS.Population data included all b-IRT positive (>99th centile) neonates born in Lombardia from 2000 to 2007. The hypertrypsinemic newborns were divided into four groups, according to CF status (noncarrier, carrier, CFTR-RD, CF).Among a total of 717,172 newborns screened within the study period, 7,354 newborns were found positive to NBS and were included in the study. An overall statistically significant difference in b-IRT levels was found among the four groups (p < 0.001), while b-IRT values did not differ between noncarriers and carriers. b-IRT levels had a low predictive accuracy in correctly identifying the four different groups (c-index: 0.60), but the accuracy was high in discriminating between classic CF and carrier or noncarrier status in neonates positive to NBS. The IRT level on the initial blood specimen obtained at birth differs based on the CF genotype, although a wide range of individual variation may occur.

A wide methodological approach to identify a large duplication in CFTR gene in a CF patient uncharacterised by sequencing analysis.

BACKGROUND: PCR-based diagnostic procedures are not able to characterise 6% of CF alleles. Recently, the application of array-CGH and of CFTR mRNA analysis has allowed the identification of new copy number mutations and splicing defects, that account for 2% and 13% of CF alleles, respectively, in the Italian population. METHODS: Here, we report the characterisation of a large duplication in CFTR gene through different methods: MLPA assay, RT-PCR and high-resolution array-CGH. RESULTS: We identified a large duplication, involving exons 6b-16, in a patient heterozygous for F508del mutation. This duplication produces an abnormal transcript with an out of frame addition of 2244 nucleotides and leads to the insertion of 8 amino-acid residues in the protein, followed by a stop codon. CONCLUSIONS: We propose a wide methodological approach based on MLPA assay, RT-PCR and high-resolution array-CGH to routinely analyse CF patients uncharacterised for one or both CFTR alleles.

Clinical features and genotype-phenotype correlations in children with progressive familial intrahepatic cholestasis type 3 related to ABCB4 mutations.

OBJECTIVES: The aim of the study was to estimate the frequency of ABCB4 mutations among children with chronic intrahepatic cholestasis with elevated gamma-glutamyl-transpeptidase (γ-GT) activity and to characterize the genotypes with respect to severity of symptoms, response to ursodeoxycholic acid therapy, and outcome. PATIENTS AND METHODS: Molecular analysis of ABCB4 in 133 Italian children was performed, and ABCB4 mutations were classified as disease-causing mutations or benign substitutions according to the prediction algorithm PolyPhen. RESULTS: : Twenty-eight patients were identified carrying 31 mutations (20 disease causing). Twenty patients carried 2 mutated alleles and 8 only 1. At presentation (1-204 months), 20 children were symptomatic with jaundice and/or pruritus, whereas in 8 biochemical cholestasis was a fortuitous finding. Cirrhosis developed in 15 and 6 progressed to terminal liver failure. Disease-causing mutations on both alleles were found to be associated with reduced liver expression of ABCB4 protein, lack of response to ursodeoxycholic acid therapy, and progression to cirrhosis and end-stage liver disease, whereas mild genotypes, including single heterozygous mutations, were generally associated with less severe disease and, often, absence of symptoms. CONCLUSIONS: ABCB4 mutations are responsible for a chronic liver disease in more than one-third of patients with chronic intrahepatic cholestasis and elevated γ-GT activity. In patients with severe ABCB4 genotype, the disease is often progressive with risk of developing cirrhosis and liver failure during the first 2 decades of life. Patients with mild genotypes, including single heterozygous mutations, have variable expressions of liver disease that may be influenced by comorbidity factors and modulated by still unknown genetic modifiers.

A novel donor splice site characterized by CFTR mRNA analysis induces a new pseudo-exon in CF patients.

BACKGROUND: The CFTR gene is tightly regulated and differentially expressed in many mucosal epithelial cell types. There is evidence of an increasing number of genomic variations in the intronic regions influencing mRNA splicing, and also the level of normal CFTR transcript. METHODS: In the present study, we investigate the molecular defect by RT-PCR analyzing the mRNA of 25 cystic fibrosis (CF) patients in whom only one or no CF allele had been identified after DNA analysis (of all the exons of the CFTR gene). RESULTS: mRNA analysis led to the detection of a cryptic exon in two patients: the new exon is a 104 bp insertion between exons 10 and 11 and is caused by a new point mutation c.1584+18672 bp A>G (http://www.hgvs.org/mutnomen/) discovered in intron 10; moreover, they showed the absence of exon 9 skipping. CONCLUSIONS: Our results confirm the utility of RNA analysis in discovering new mutations and in investigating their effect on normal splicing processes.

A synonymous mutation in the CFTR gene causes aberrant splicing in an italian patient affected by a mild form of cystic fibrosis.

Mutations within exons are responsible for aberrant splicing of pre-mRNA in several human disease genes and in some viral systems. Nonsense, missense, and even synonymous mutations can induce aberrant skipping of the mutant exon, producing nonfunctional proteins. In this paper, we describe the effect on the splicing efficiency of the synonymous variant 2811 G>T [Gly893Gly] detected in a patient of Italian descent affected by a mild form of cystic fibrosis, until now mentioned as sequence variation with unknown functional consequences. The study, performed through DNA as well as RNA analyses, shows that this mutation creates a new 5' splice site within exon 15, resulting in a transcript lacking 76 amino acid residues. Although this aberrant splicing causes a shorter exon 15, the downstream exonic sequence from exon 16 to the end of the open reading frame is in frame. This study indicates that apparently neutral polymorphism, which may be erroneously classified as nonpathogenic, may indeed led to aberrant splicing thereby resulting in defective protein.

Borderline sweat test: Utility and limits of genetic analysis for the diagnosis of cystic fibrosis.

OBJECTIVE: The sweat test remains the gold standard for the diagnosis of Cystic Fibrosis (CF) even despite the availability of molecular analysis of Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR). We investigated the relationship between CFTR mutation analysis and sweat chloride concentration in a cohort of subjects with borderline sweat test values, in order to identify misdiagnosis of CF. DESIGN AND METHODS: In the period between March 2006 and February 2008 we performed 773 sweat tests in individuals referred for suspect CF. Ninety-one subjects had chloride values in the border-line range. Clinicians required CFTR gene complete scanning on 66 of them. RESULTS: The mean value of sweat chloride in the DNA negative subjects was lower than in those with at least one CFTR mutation. Our data indicate that 39 mEq/l is the best sensitivity trade off for the sweat test with respect to genotype. CONCLUSIONS: To optimise diagnostic accuracy of reference intervals, it may be useful to modify from 30 to 39 mEq/l the threshold for sweat chloride electrolytes.

Occult hepatitis B virus in liver tissue of individuals without hepatic disease.

BACKGROUND/AIMS: While many data are available concerning occult hepatitis B virus (HBV) infection in patients with hepatic disorders, there is little information about this cryptic infection in individuals without liver disease. The aim of this study was to investigate the prevalence of occult HBV in the general population by examining liver specimens from a large series of HBV-surface-antigen negative individuals with no clinical and biochemical evidence of liver disease. METHODS: The presence of HBV DNA was evaluated by testing, through polymerase chain reaction techniques, DNA extracts from 98 liver-disease-free individuals who underwent liver resection or needle biopsy during abdominal surgery. Sixteen of them were anti-HBV-core antigen (anti-HBc) positive and 82 were HBV serum-marker negative. All patients were negative for antibody to hepatitis C virus. RESULTS: Occult HBV infection was revealed in 16 of the 98 cases (16.3%). In particular, 10/16 anti-HBc positive (62.5%) versus 6/82 (7.3%) HBV-seronegative individuals were occult carriers (p<0.0001). CONCLUSIONS: This study revealed that about 1/6 of the Italian general population might be carriers of occult HBV infection, and this condition is significantly associated with the anti-HBc positive status.

Molecular characterization and structural implications of 25 new ABCB4 mutations in progressive familial intrahepatic cholestasis type 3 (PFIC3).

Progressive familial intrahepatic cholestasis type 3 (PFIC3) is an autosomal-recessive disorder due to mutations in the ATP-binding cassette, subfamily B, member 4 gene (ABCB4). ABCB4 is the liver-specific membrane transporter of phosphatidylcholine, a major and exclusive component of mammalian bile. The disease is characterized by early onset of cholestasis with high serum gamma-glutamyltranspeptidase activity, which progresses into cirrhosis and liver failure before adulthood. Presently, about 20 distinct ABCB4 mutations associated to PFIC3 have been described. We report the molecular characterization of 68 PFIC3 index cases enrolled in a multicenter study, which represents the largest cohort of PFIC3 patients screened for ABCB4 mutations to date. We observed 31 mutated ABCB4 alleles in 18 index cases with 29 distinct mutations, 25 of which are novel. Despite the lack of structural information on the ABCB4 protein, the elucidation of the three-dimensional structure of bacterial homolog allows the three-dimensional model of ABCB4 to be built by homology modeling and the position of the mutated amino-acids in the protein tertiary structure to be located. In a significant fraction of the cases reported in this study, the mutation should result in substantial impairment of ABCB4 floppase activity. The results of this study provide evidence of the broad allelic heterogeneity of the disease, with causative mutations spread along 14 of the 27 coding exons, but with higher prevalence on exon 17 that, as recently shown for the closely related paralogous ABCB1 gene, could contain an evolutionary marker for mammalian ABCB4 genes in the seventh transmembrane segment.

Molecular and functional analysis of occult hepatitis B virus isolates from patients with hepatocellular carcinoma.

UNLABELLED: Occult HBV infection is characterized by the persistence of HBV DNA in the liver of individuals negative for HBV surface antigen (HBsAg). Occult HBV may exist in the hepatocytes as a free genome, although the factors responsible for the very low viral replication and gene expression usually observed in this peculiar kind of infection are mostly unknown. Aims of this study were to investigate whether the viral genomic variability might account for the HBsAg negativity and the inhibition of the viral replication in occult HBV carriers, and to verify in vitro the replication capability of occult HBV strains. We studied liver viral isolates from 17 HBV patients, 13 with occult infection and 4 HBsAg-positive. Full-length HBV genomes from each case were amplified and directly sequenced. Additionally, full-length HBV DNA from eight occult-HBV and two HBsAg-positive cases were cloned and sequenced. Finally, three entire, linear HBV genomes from occult cases were transiently transfected in HuH7 cells. Direct sequencing showed the absence of mutations capable of interfering with viral replication and gene expression in the major viral population of each case. Cloning experiments showed highly divergent HBV strains both in HBsAg-positive and HBsAg-negative individual cases (range of divergence 1.4%-7.1%). All of the 3 transfected full-length HBV isolates showed normal patterns of replication in vitro. CONCLUSION: Multiple viral variants accumulate in the liver of occult HBV-infected patients. Occult HBV strains are replication-competent in vitro, suggesting that host, rather than viral factors are responsible for cryptic HBV infection.

Virological profiles in hepatitis B virus inactive carriers: monthly evaluation in 1-year follow-up study.

UNLABELLED: STUDY SUBJECT: We longitudinally evaluated the virological behaviour and the hepatitis B virus (HBV) genomic variability in inactive HBV surface antigen (HBsAg) chronic carriers. PATIENTS AND METHODS: Fourteen HBsAg-positive healthy workers (13 inactive carriers and 1 with active HBV infection) were followed up for 12 months by monthly evaluation of aminotransferase, HBV DNA, and IgM anti HBV core antigen (IgM anti-HBc) values. Moreover, HBV serum isolates from each case were amplified, cloned and sequenced to evaluate the presence of the potentially clinical relevant core-promoter and precore mutations. The same technical procedures were used to examine the S gene of isolates from 3 randomly selected inactive carriers and the patients with active HBV infections. RESULTS: Aminotransferase values were constantly normal in all cases. Viremia levels appear to fluctuate widely over time in each individual case, although the HBV DNA remained below 2 x 10(4) copies/ml in all samples. Only four serum samples from two inactive carriers had IgM anti-HBc values higher than the specific cut-off limit of the assay. Either wild type or core-promoter/precore HBV variants or a mixture of them were detected in the inactive carriers. S gene nucleotide homology among the clones from the three inactive carriers and the subject with active infection was 98.9%, 98.3%, 98.1% and 98.2%, respectively. CONCLUSIONS: The degree of suppression of HBV replication in inactive carriers is variable over time, and the entity and quality of HBV variability is comparable between active and inactive carriers.