Computational Analysis of Single Nucleotide Polymorphisms Associated with Altered Drug Responsiveness in Type 2 Diabetes.

Type 2 diabetes (T2D) is one of the most frequent mortality causes in western countries, with rapidly increasing prevalence. Anti-diabetic drugs are the first therapeutic approach, although many patients develop drug resistance. Most drug responsiveness variability can be explained by genetic causes. Inter-individual variability is principally due to single nucleotide polymorphisms, and differential drug responsiveness has been correlated to alteration in genes involved in drug metabolism (CYP2C9) or insulin signaling (IRS1, ABCC8, KCNJ11 and PPARG). However, most genome-wide association studies did not provide clues about the contribution of DNA variations to impaired drug responsiveness. Thus, characterizing T2D drug responsiveness variants is needed to guide clinicians toward tailored therapeutic approaches. Here, we extensively investigated polymorphisms associated with altered drug response in T2D, predicting their effects in silico. Combining different computational approaches, we focused on the expression pattern of genes correlated to drug resistance and inferred evolutionary conservation of polymorphic residues, computationally predicting the biochemical properties of polymorphic proteins. Using RNA-Sequencing followed by targeted validation, we identified and experimentally confirmed that two nucleotide variations in the CAPN10 gene-currently annotated as intronic-fall within two new transcripts in this locus. Additionally, we found that a Single Nucleotide Polymorphism (SNP), currently reported as intergenic, maps to the intron of a new transcript, harboring CAPN10 and GPR35 genes, which undergoes non-sense mediated decay. Finally, we analyzed variants that fall into non-coding regulatory regions of yet underestimated functional significance, predicting that some of them can potentially affect gene expression and/or post-transcriptional regulation of mRNAs affecting the splicing.

Pharmacogenomics of Drug Response in Type 2 Diabetes: Toward the Definition of Tailored Therapies?

Type 2 diabetes is one of the major causes of mortality with rapidly increasing prevalence. Pharmacological treatment is the first recommended approach after failure in lifestyle changes. However, a significant number of patients shows-or develops along time and disease progression-drug resistance. In addition, not all type 2 diabetic patients have the same responsiveness to drug treatment. Despite the presence of nongenetic factors (hepatic, renal, and intestinal), most of such variability is due to genetic causes. Pharmacogenomics studies have described association between single nucleotide variations and drug resistance, even though there are still conflicting results. To date, the most reliable approach to investigate allelic variants is Next-Generation Sequencing that allows the simultaneous analysis, on a genome-wide scale, of nucleotide variants and gene expression. Here, we review the relationship between drug responsiveness and polymorphisms in genes involved in drug metabolism (CYP2C9) and insulin signaling (ABCC8, KCNJ11, and PPARG). We also highlight the advancements in sequencing technologies that to date enable researchers to perform comprehensive pharmacogenomics studies. The identification of allelic variants associated with drug resistance will constitute a solid basis to establish tailored therapeutic approaches in the treatment of type 2 diabetes.

New somatic mutations and WNK1-B4GALNT3 gene fusion in papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is the most frequent thyroid malignant neoplasia. Oncogene activation occurs in more than 70% of the cases. Indeed, about 40% of PTCs harbor mutations in BRAF gene, whereas RET rearrangements (RET/PTC oncogenes) are present in about 20% of cases. Finally, RAS mutations and TRK rearrangements account for about 5% each of these malignancies. We used RNA-Sequencing to identify fusion transcripts and mutations in cancer driver genes in a cohort of 18 PTC patients. Furthermore, we used targeted DNA sequencing to validate identified mutations. We extended the screening to 50 PTC patients and 30 healthy individuals. Using this approach we identified new missense mutations in CBL, NOTCH1, PIK3R4 and SMARCA4 genes. We found somatic mutations in DICER1, MET and VHL genes, previously found mutated in other tumors, but not described in PTC. We identified a new chimeric transcript generated by the fusion of WNK1 and B4GALNT3 genes, correlated with B4GALNT3 overexpression. Our data confirmed PTC genetic heterogeneity, revealing that gene expression correlates more with the mutation pattern than with tumor staging. Overall, this study provides new data about mutational landscape of this neoplasia, suggesting potential pharmacological adjuvant therapies against Notch signaling and chromatin remodeling enzymes.

The ADAMTS18 gene is responsible for autosomal recessive early onset severe retinal dystrophy.

BACKGROUND: Inherited retinal dystrophies, including Retinitis Pigmentosa and Leber Congenital Amaurosis among others, are a group of genetically heterogeneous disorders that lead to variable degrees of visual deficits. They can be caused by mutations in over 100 genes and there is evidence for the presence of as yet unidentified genes in a significant proportion of patients. We aimed at identifying a novel gene for an autosomal recessive form of early onset severe retinal dystrophy in a patient carrying no previously described mutations in known genes. METHODS: An integrated strategy including homozygosity mapping and whole exome sequencing was used to identify the responsible mutation. Functional tests were performed in the medaka fish (Oryzias latipes) model organism to gain further insight into the pathogenic role of the ADAMTS18 gene in eye and central nervous system (CNS) dysfunction. RESULTS: This study identified, in the analyzed patient, a homozygous missense mutation in the ADAMTS18 gene, which was recently linked to Knobloch syndrome, a rare developmental disorder that affects the eye and the occipital skull. In vivo gene knockdown performed in medaka fish confirmed both that the mutation has a pathogenic role and that the inactivation of this gene has a deleterious effect on photoreceptor cell function. CONCLUSION: This study reveals that mutations in the ADAMTS18 gene can cause a broad phenotypic spectrum of eye disorders and contribute to shed further light on the complexity of retinal diseases.

BBS1 mutations in a wide spectrum of phenotypes ranging from nonsyndromic retinitis pigmentosa to Bardet-Biedl syndrome.

OBJECTIVE: To investigate the involvement of the Bardet-Biedl syndrome (BBS) gene BBS1 p.M390R variant in nonsyndromic autosomal recessive retinitis pigmentosa (RP). METHODS: Homozygosity mapping of a patient with isolated RP was followed by BBS1 sequence analysis. We performed restriction fragment length polymorphism analysis of the p.M390R allele in 2007 patients with isolated RP or autosomal recessive RP and in 1824 ethnically matched controls. Patients with 2 BBS1 variants underwent extensive clinical and ophthalmologic assessment. RESULTS: In an RP proband who did not fulfill the clinical criteria for BBS, we identified a large homozygous region encompassing the BBS1 gene, which carried the p.M390R variant. In addition, this variant was detected homozygously in 10 RP patients and 1 control, compound heterozygously in 3 patients, and heterozygously in 5 patients and 6 controls. The 14 patients with 2 BBS1 variants showed the entire clinical spectrum, from nonsyndromic RP to full-blown BBS. In 8 of 14 patients, visual acuity was significantly reduced. In patients with electroretinographic responses, a rod-cone pattern of photoreceptor degeneration was observed. CONCLUSIONS: Variants in BBS1 are significantly associated with nonsyndromic autosomal recessive RP and relatively mild forms of BBS. As exemplified in this study by the identification of a homozygous p.M390R variant in a control individual and in unaffected parents of BBS patients in other studies, cis - or trans -acting modifiers may influence the disease phenotype. CLINICAL RELEVANCE: It is important to monitor patients with an early diagnosis of mild BBS phenotypes for possible life-threatening conditions.

Molecular diagnosis of Usher syndrome: application of two different next generation sequencing-based procedures.

Usher syndrome (USH) is a clinically and genetically heterogeneous disorder characterized by visual and hearing impairments. Clinically, it is subdivided into three subclasses with nine genes identified so far. In the present study, we investigated whether the currently available Next Generation Sequencing (NGS) technologies are already suitable for molecular diagnostics of USH. We analyzed a total of 12 patients, most of which were negative for previously described mutations in known USH genes upon primer extension-based microarray genotyping. We enriched the NGS template either by whole exome capture or by Long-PCR of the known USH genes. The main NGS sequencing platforms were used: SOLiD for whole exome sequencing, Illumina (Genome Analyzer II) and Roche 454 (GS FLX) for the Long-PCR sequencing. Long-PCR targeting was more efficient with up to 94% of USH gene regions displaying an overall coverage higher than 25×, whereas whole exome sequencing yielded a similar coverage for only 50% of those regions. Overall this integrated analysis led to the identification of 11 novel sequence variations in USH genes (2 homozygous and 9 heterozygous) out of 18 detected. However, at least two cases were not genetically solved. Our result highlights the current limitations in the diagnostic use of NGS for USH patients. The limit for whole exome sequencing is linked to the need of a strong coverage and to the correct interpretation of sequence variations with a non obvious, pathogenic role, whereas the targeted approach suffers from the high genetic heterogeneity of USH that may be also caused by the presence of additional causative genes yet to be identified.

Molecular epidemiology of Usher syndrome in Italy.

PURPOSE: Usher syndrome is an autosomal recessive disorder characterized by hearing and vision loss. Usher syndrome is divided into three clinical subclasses (type 1, type 2, and type 3), which differ in terms of the severity and progression of hearing loss and the presence or absence of vestibular symptoms. Usher syndrome is defined by significant genetic heterogeneity, with at least 12 distinct loci described and 9 genes identified. This study aims to provide a molecular epidemiology report of Usher syndrome in Italy. METHODS: Molecular data have been obtained on 75 unrelated Italian patients using the most up-to date technology available for the screening of Usher syndrome gene mutations, i.e., the genotyping microarray developed by Asper Biotech (Tartu, Estonia), which simultaneously investigates 612 different marker positions using the well established arrayed primer extension methodology (APEX). RESULTS: Using this method, we found that 12% of cases (9 out of 75) harbored homozygous or compound heterozygous mutations in the gene positions analyzed, whereas 20% (15 out of 75) of the patients were characterized by the presence of only one mutated allele based on the positions analyzed. One patient was found to be compound heterozygous for mutations in two different genes and this represents an example of possible digenic inheritance in Usher syndrome. A total of 66.6% of cases (50 out of 75) were found to be completely negative for the presence of Usher syndrome gene mutations in the detected positions. Mutations detected by the array were confirmed by direct sequencing. CONCLUSIONS: These findings highlight the efficacy of the APEX-based genotyping approach in the molecular assessment of Usher patients, suggesting the presence of alleles not yet identified and/or the involvement of additional putative genes that may account for the pathogenesis of Usher syndrome.

Evaluation of Italian patients with leber congenital amaurosis due to AIPL1 mutations highlights the potential applicability of gene therapy.

PURPOSE: To evaluate the suitability of gene delivery-based approaches as potential treatment of Leber congenital amaurosis 4 (LCA4) due to AIPL1 mutations. METHODS: Genomic DNA from patients was analyzed using a microarray chip and direct sequencing. A detailed clinical evaluation including fundus autofluorescence (FAF) and optical coherence tomography (OCT) was performed in patients with AIPL1 mutations. Aipl1 null mice and porcine eyes were subretinally injected with adeno-associated viral (AAV) vectors harboring the human AIPL1 coding sequence. RESULTS: We identified 10 LCA4 patients with mutations in AIPL1. The p.W278X sequence variation was the one most frequently found. Clinical assessment revealed common features including diffuse retinal dystrophies and maculopathy. However, optical coherence tomography showed partially retained photoreceptors in extramacular regions at all ages. The fundus autofluorescence was elicitable at the posterior pole and absent in the fovea. AAV-mediated gene transfer in Aipl1 -/- mice was associated with restoration of AIPL1 and ßPDE expression in photoreceptors and protection from degeneration. Administration of a clinically relevant dose of AAV2/8-AIPL1 to the preclinical large porcine retina resulted in high level of AIPL1 photoreceptor expression in the absence of toxicity. CONCLUSIONS: Using advanced imaging diagnostics we showed that maculopathy is a main feature of LCA4. We identified retinal areas at the posterior pole with surviving photoreceptors present even in adult LCA4 patients, which could be the target of gene therapy. The possible use of gene therapy for LCA4 is additionally supported by the protection from photoreceptor degeneration observed in Aipl 1-/- mice and by the high levels of photoreceptor transduction in the absence of toxicity observed after AAV2/8 delivery to the large porcine retina.

Gene therapy for Leber's congenital amaurosis is safe and effective through 1.5 years after vector administration.

The safety and efficacy of gene therapy for inherited retinal diseases is being tested in humans affected with Leber's congenital amaurosis (LCA), an autosomal recessive blinding disease. Three independent studies have provided evidence that the subretinal administration of adeno-associated viral (AAV) vectors encoding RPE65 in patients affected with LCA2 due to mutations in the RPE65 gene, is safe and, in some cases, results in efficacy. We evaluated the long-term safety and efficacy (global effects on retinal/visual function) resulting from subretinal administration of AAV2-hRPE65v2. Both the safety and the efficacy noted at early timepoints persist through at least 1.5 years after injection in the three LCA2 patients enrolled in the low dose cohort of our trial. A transient rise in neutralizing antibodies to AAV capsid was observed but there was no humoral response to RPE65 protein. The persistence of functional amelioration suggests that AAV-mediated gene transfer to the human retina does not elicit immunological responses which cause significant loss of transduced cells. The persistence of physiologic effect supports the possibility that gene therapy may influence LCA2 disease progression. The safety of the intervention and the stability of the improvement in visual and retinal function in these subjects support the use of AAV-mediated gene augmentation therapy for treatment of inherited retinal diseases.

A homozygous missense mutation in the IRBP gene (RBP3) associated with autosomal recessive retinitis pigmentosa.

PURPOSE: Interphotoreceptor retinoid-binding protein (IRBP) has been considered essential for normal rod and cone function, as it mediates the transport of retinoids between the photoreceptors and the retinal pigment epithelium. This study was performed to determine whether mutations in the IRBP gene (RBP3) are associated with photoreceptor degeneration. METHODS: A consanguineous family was ascertained in which four children had autosomal recessive retinitis pigmentosa (RP). Homozygosity mapping performed with SNP microarrays revealed only one homozygous region shared by all four affected siblings. Sequencing of RBP3, contained in this region, was performed in this family and others with recessive RP. Screening was also performed on patients with various other forms of retinal degeneration or malfunction. RESULTS: Sequence analysis of RBP3 revealed a homozygous missense mutation (p.Asp1080Asn) in the four affected siblings. The mutation affects a residue that is completely conserved in all four homologous modules of the IRBP protein of vertebrate species and in C-terminal-processing proteases, photosynthesis enzymes found in bacteria, algae, and plants. Based on the previously reported crystal structure of Xenopus IRBP, the authors predict that the Asp1080-mediated conserved salt bridge that appears to participate in scaffolding of the retinol-binding domain is abolished by the mutation. No RBP3 mutations were detected in 395 unrelated patients with recessive or isolate RP or in 680 patients with other forms of hereditary retinal degeneration. CONCLUSIONS: Mutations in RBP3 are an infrequent cause of autosomal recessive RP. The mutation Asp1080Asn may alter the conformation of the IRBP protein by disrupting a conserved salt bridge.

Identification and expression analysis of novel Jakmip1 transcripts.

Janus kinase and microtubule interacting protein 1, (Jakmip1) conserved in vertebrates and predominantly expressed in neural tissues, was identified for its ability to bind Tyk2, a member of the Janus kinase (Jak) family of non-receptor tyrosine kinases. Recently Jakmip1 was also identified as an interacting partner of GABA(B)R1 and as a regulatory protein of GABA(B)R2 mRNA. We have confirmed that this gene is highly expressed in brain and retina tissues and it is also present at lower levels in other tissues. We have identified four new transcripts of 2975 bp, 1743 bp, 2189 bp and 2420 bp respectively, named Jakmip1B, Jakmip1C, Jakmip1D and Jakmip1E. The involvement of the Janus kinase pathway in the development of mouse retina and in the control of survival and proliferation of human retinal ganglion cells, together with the restricted Jakmip1 gene expression pattern, may suggest this gene is a putative candidate for neuro-degenerative and retinal diseases. For this reason, a mutation analysis of the Jakmip1 gene in a panel of 50 unrelated patients with retinitis pigmentosa has been performed, revealing no pathogenic mutations.

Clinical and molecular genetics of Leber's congenital amaurosis: a multicenter study of Italian patients.

PURPOSE: To identify the molecular basis of Leber's congenital amaurosis (LCA) in a cohort of Italian patients and to perform genotype-phenotype analysis. METHODS: DNA samples from 95 patients with LCA were analyzed by using a microarray chip containing disease-associated sequence variants in eight LCA genes. In addition, all patients in whom no mutations were identified by microarray were subjected to sequence analysis of the CEP290 gene. Patients with mutations identified underwent a detailed ophthalmic evaluation. RESULTS: Disease-causing mutations were identified in 28% of patients, and twelve novel variants were identified. Mutations occurred more frequently in the RPE65 (8.4%), CRB1 (7.4%), and GUCY2D (5.2%) genes. Mutations in CEP290 were found in only 4.2% of the patients analyzed. Clinical assessment of patients carrying RPE65 or CRB1 mutations revealed the presence of retained visual capabilities in the first decade of life. RPE65 mutations were almost always associated with normal macular thickness, as assessed by optical coherence tomography (OCT), whereas CRB1 mutations were associated with reduced retinal thickness and a coarsely laminated retina. Fundus autofluorescence was mostly observed in patients with RPE65 and GUCY2D mutations and was not elicitable in patients carrying CRB1. CONCLUSIONS: RPE65 gene mutations represented a significant cause of LCA in the Italian population, whereas GUCY2D and CEP290 mutations had a lower frequency than that found in other reports. This finding suggests that the genetic epidemiology of LCA in Italy is different from that reported in the United States and in northern European countries. Autofluorescence in patients with RPE65 mutations was more frequently associated with preserved retinal thickness, which suggests that these mutations are not associated with progression of retinal degeneration. Therefore, normal retinal thickness (identified with OCT) and fundus autofluorescence may be the means with which to identify patients with LCA who carry RPE65 mutations, which are expected to be a potential gene therapy target in the near future.

Identification and characterization of C1orf36, a transcript highly expressed in photoreceptor cells, and mutation analysis in retinitis pigmentosa.

By means of computational methods, we identified an uncharacterized human transcript, Chromosome 1 open reading frame 36 (C1orf36), that is expressed in the retina and that maps to 1q32.3. The cDNA contains an open reading frame of 585bp that encodes a 195-aminoacid protein with a predicted mass of 22.7kDa. An alternatively spliced transcript in a retinoblastoma cell line, encoding for a truncated peptide, was also identified. PCR experiments performed using human cDNA from several sources indicate that C1orf36 has a preferential expression in the retina. Accordingly, in situ hybridization experiments, performed using as probe a murine C1orf36 cDNA fragment, detected a hybridization signal on mouse retinal adult sections. The C1orf36 protein shares homology with putative proteins in Mus musculus and Fugu rubripes, suggesting evolutionary conservation of its function. Additional sequence analysis of the C1orf36 gene product predicts its subcellular mitochondrial localization and the presence of both evolutionary conserved phosphorylation sites and regions adopting a coiled-coil conformation. We also defined the genomic structure of the gene. This enabled us to perform a mutational analysis of the C1orf36 coding region of about 300 patients affected by retinitis pigmentosa. No pathological mutations were detected in this analysis.

Identification and characterisation of the retinitis pigmentosa 1-like1 gene (RP1L1): a novel candidate for retinal degenerations.

Retinitis pigmentosa (RP) is the most common form of inherited retinopathy, with an approximate incidence of 1 in 3700 individuals worldwide. Mutations in the retinitis pigmentosa 1 (RP1) gene are responsible for about 5-10% cases of autosomal dominant RP. The RP1 gene is specifically expressed in the photoreceptor layers of the postnatal retina and encodes a predicted protein characterised by the presence of two doublecortin (DC) domains, known to be implicated in microtubule binding. We identified and characterised, both in human and in mouse, a novel mammalian gene, termed Retinitis Pigmentosa1-like1 (RP1L1), because of its significant sequence similarity to the RP1 gene product. The sequence homology between RP1 and RP1L1 was found to be mostly restricted to the DC domains and to the N-terminal region, including the first 350 amino acids. The RP1L1 gene was also found to be conserved in distant vertebrates, since we identified a homologue in Fugu rubripes (pufferfish). Similar to RP1, RP1L1 expression is restricted to the postnatal retina, as determined by semiquantitative reverse transcriptase-PCR and Northern analysis. The retina-specific expression and the sequence similarity to RP1 render RP1L1 a potential candidate for inherited retinal disorders.