Mutational spectrum and clinical signatures in 114 families with hereditary multiple osteochondromas: insights into molecular properties of selected exostosin variants.

Hereditary multiple osteochondromas (HMO) is a rare autosomal dominant skeletal disorder, caused by heterozygous variants in either EXT1 or EXT2, which encode proteins involved in the biogenesis of heparan sulphate. Pathogenesis and genotype-phenotype correlations remain poorly understood. We studied 114 HMO families (158 affected individuals) with causative EXT1 or EXT2 variants identified by Sanger sequencing, or multiplex ligation-dependent probe amplification and qPCR. Eighty-seven disease-causative variants (55 novel and 32 known) were identified including frameshift (42%), nonsense (32%), missense (11%), splicing (10%) variants and genomic rearrangements (5%). Informative clinical features were available for 42 EXT1 and 27 EXT2 subjects. Osteochondromas were more frequent in EXT1 as compared to EXT2 patients. Anatomical distribution of lesions showed significant differences based on causative gene. Microscopy analysis for selected EXT1 and EXT2 variants verified that EXT1 and EXT2 mutants failed to co-localize each other and loss Golgi localization by surrounding the nucleus and/or assuming a diffuse intracellular distribution. In a cell viability study, cells expressing EXT1 and EXT2 mutants proliferated more slowly than cells expressing wild-type proteins. This confirms the physiological relevance of EXT1 and EXT2 Golgi co-localization and the key role of these proteins in the cell cycle. Taken together, our data expand genotype-phenotype correlations, offer further insights in the pathogenesis of HMO and open the path to future therapies.

Customised next-generation sequencing multigene panel to screen a large cohort of individuals with chromatin-related disorder.

BACKGROUND: The regulation of the chromatin state by epigenetic mechanisms plays a central role in gene expression, cell function, and maintenance of cell identity. Hereditary disorders of chromatin regulation are a group of conditions caused by abnormalities of the various components of the epigenetic machinery, namely writers, erasers, readers, and chromatin remodelers. Although neurological dysfunction is almost ubiquitous in these disorders, the constellation of additional features characterizing many of these genes and the emerging clinical overlap among them indicate the existence of a community of syndromes. The introduction of high-throughput next generation sequencing (NGS) methods for testing multiple genes simultaneously is a logical step for the implementation of diagnostics of these disorders. METHODS: We screened a heterogeneous cohort of 263 index patients by an NGS-targeted panel, containing 68 genes associated with more than 40 OMIM entries affecting chromatin function. RESULTS: This strategy allowed us to identify clinically relevant variants in 87 patients (32%), including 30 for which an alternative clinical diagnosis was proposed after sequencing analysis and clinical re-evaluation. CONCLUSION: Our findings indicate that this approach is effective not only in disorders with locus heterogeneity, but also in order to anticipate unexpected misdiagnoses due to clinical overlap among cognate disorders. Finally, this work highlights the utility of a prompt diagnosis in such a clinically and genetically heterogeneous group of disorders that we propose to group under the umbrella term of chromatinopathies.

Dissecting KMT2D missense mutations in Kabuki syndrome patients.

Kabuki syndrome is a rare autosomal dominant condition characterized by facial features, various organs malformations, postnatal growth deficiency and intellectual disability. The discovery of frequent germline mutations in the histone methyltransferase KMT2D and the demethylase KDM6A revealed a causative role for histone modifiers in this disease. However, the role of missense mutations has remained unexplored. Here, we expanded the mutation spectrum of KMT2D and KDM6A in KS by identifying 37 new KMT2D sequence variants. Moreover, we functionally dissected 14 KMT2D missense variants, by investigating their impact on the protein enzymatic activity and the binding to members of the WRAD complex. We demonstrate impaired H3K4 methyltransferase activity in 9 of the 14 mutant alleles and show that this reduced activity is due in part to disruption of protein complex formation. These findings have relevant implications for diagnostic and counseling purposes in this disease.

Pro-Fibrotic Phenotype in a Patient with Segmental Stiff Skin Syndrome via TGF-ß Signaling Overactivation.

Transforming growth factor ß (TGF-ß) superfamily signaling pathways are ubiquitous and essential for several cellular and physiological processes. The overexpression of TGF-ß results in excessive fibrosis in multiple human disorders. Among them, stiff skin syndrome (SSS) is an ultrarare and untreatable condition characterized by the progressive thickening and hardening of the dermis, and acquired joint limitations. SSS is distinct in a widespread form, caused by recurrent germline variants of FBN1 encoding a key molecule of the TGF-ß signaling, and a segmental form with unknown molecular basis. Here, we report a 12-year-old female with segmental SSS, affecting the right upper limb with acquired thickening of the dermis evident at the magnetic resonance imaging, and progressive limitation of the elbow and shoulder. To better explore the molecular and cellular mechanisms that drive segmental SSS, several functional studies on patient's fibroblasts were employed. We hypothesized an impairment of TGF-ß signaling and, consequently, a dysregulation of the associated downstream signaling. Lesional fibroblast studies showed a higher phosphorylation level of extracellular signal-regulated kinase 1/2 (ERK1/2), increased levels of nuclear factor-kB (NFkB), and a nuclear accumulation of phosphorylated Smad2 via Western blot and microscopy analyses. Quantitative PCR expression analysis of genes encoding key extracellular matrix proteins revealed increased levels of COL1A1, COL3A1, AGT, LTBP and ITGB1, while zymography assay reported a reduced metalloproteinase 2 enzymatic activity. In vitro exposure of patient's fibroblasts to losartan led to the partial restoration of normal transforming growth factor ß (TGF-ß) marker protein levels. Taken together, these data demonstrate that in our patient, segmental SSS is characterized by the overactivation of multiple TGF-ß signaling pathways, which likely results in altered extracellular matrix composition and fibroblast homeostasis. Our results for the first time reported that aberrant TGF-ß signaling may drive the pathogenesis of segmental SSS and might open the way to novel therapeutic approaches.

TRIM50 regulates Beclin 1 proautophagic activity.

Autophagy is a catabolic process needed for maintaining cell viability and homeostasis in response to numerous stress conditions. Emerging evidence indicates that the ubiquitin system has a major role in this process. TRIMs, an E3 ligase protein family, contribute to selective autophagy acting as receptors and regulators of the autophagy proteins recognizing endogenous or exogenous targets through intermediary autophagic tags, such as ubiquitin. Here we report that TRIM50 fosters the initiation phase of starvation-induced autophagy and associates with Beclin1, a central component of autophagy initiation complex. We show that TRIM50, via the RING domain, ubiquitinates Beclin 1 in a K63-dependent manner enhancing its binding with ULK1 and autophagy activity. Finally, we found that the Lys-372 residue of TRIM50, critical for its own acetylation, is necessary for its E3 ligase activity that governs Beclin1 ubiquitination. Our study expands the roles of TRIMs in regulating selective autophagy, revealing an acetylation-ubiquitination dependent control for autophagy modulation.

Clock-genes and mitochondrial respiratory activity: Evidence of a reciprocal interplay.

In the past few years mounting evidences have highlighted the tight correlation between circadian rhythms and metabolism. Although at the organismal level the central timekeeper is constituted by the hypothalamic suprachiasmatic nuclei practically all the peripheral tissues are equipped with autonomous oscillators made up by common molecular clockworks represented by circuits of gene expression that are organized in interconnected positive and negative feed-back loops. In this study we exploited a well-established in vitro synchronization model to investigate specifically the linkage between clock gene expression and the mitochondrial oxidative phosphorylation (OxPhos). Here we show that synchronized cells exhibit an autonomous ultradian mitochondrial respiratory activity which is abrogated by silencing the master clock gene ARNTL/BMAL1. Surprisingly, pharmacological inhibition of the mitochondrial OxPhos system resulted in dramatic deregulation of the rhythmic clock-gene expression and a similar result was attained with mtDNA depleted cells (Rho0). Our findings provide a novel level of complexity in the interlocked feedback loop controlling the interplay between cellular bioenergetics and the molecular clockwork. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Clock genes-dependent acetylation of complex I sets rhythmic activity of mitochondrial OxPhos.

Physiology of living beings show circadian rhythms entrained by a central timekeeper present in the hypothalamic suprachiasmatic nuclei. Nevertheless, virtually all peripheral tissues hold autonomous molecular oscillators constituted essentially by circuits of gene expression that are organized in negative and positive feed-back loops. Accumulating evidence reveals that cell metabolism is rhythmically controlled by cell-intrinsic molecular clocks and the specific pathways involved are being elucidated. Here, we show that in vitro-synchronized cultured cells exhibit BMAL1-dependent oscillation in mitochondrial respiratory activity, which occurs irrespective of the cell type tested, the protocol of synchronization used and the carbon source in the medium. We demonstrate that the rhythmic respiratory activity is associated to oscillation in cellular NAD content and clock-genes-dependent expression of NAMPT and Sirtuins 1/3 and is traceable back to the reversible acetylation of a single subunit of the mitochondrial respiratory chain Complex I. Our findings provide evidence for a new interlocked transcriptional-enzymatic feedback loop controlling the molecular interplay between cellular bioenergetics and the molecular clockwork.

Clinical and Neurobehavioral Features of Three Novel Kabuki Syndrome Patients with Mosaic KMT2D Mutations and a Review of Literature.

Kabuki syndrome (KS) is a rare disorder characterized by multiple congenital anomalies and variable intellectual disability caused by mutations in KMT2D/MLL2 and KDM6A/UTX, two interacting chromatin modifier responsible respectively for 56-75% and 5-8% of the cases. To date, three KS patients with mosaic KMT2D deletions in blood lymphocytes have been described. We report on three additional subjects displaying KMT2D gene mosaics including one in which a single nucleotide change results in a new frameshift mutation (p.L1199HfsX7), and two with already-known nonsense mutations (p.R4484X and p.R5021X). Consistent with previously published cases, mosaic KMT2D mutations may result in mild KS facial dysmorphisms and clinical and neurobehavioral features, suggesting that these characteristics could represent the handles for genetic testing of individuals with slight KS-like traits.

Deregulated expression of cryptochrome genes in human colorectal cancer.

BACKGROUND: Circadian disruption and deranged molecular clockworks are involved in carcinogenesis. The cryptochrome genes (CRY1 and CRY2) encode circadian proteins important for the functioning of biological oscillators. Their expression in human colorectal cancer (CRC) and in colon cancer cell lines has not been evaluated so far. METHODS: We investigated CRY1 and CRY2 expression in fifty CRCs and in the CaCo2, HCT116, HT29, SW480 cell lines. RESULTS: CRY1 (p = 0.01) and CRY2 (p < 0.0001) expression was significantly changed in tumour tissue, as confirmed in a large independent CRC dataset. In addition, lower CRY1 mRNA levels were observed in patients in the age range of 62-74 years (p = 0.018), in female patients (p = 0.003) and in cancers located at the transverse colon (p = 0.008). Lower CRY2 levels were also associated with cancer location at the transverse colon (p = 0.007). CRC patients displaying CRY1 (p = 0.042) and CRY2 (p = 0.043) expression levels over the median were hallmarked by a poorer survival rate. Survey of selected colon cancer cell lines evidenced variable levels of cryptochrome genes expression and time-dependent changes in their mRNA levels. Moreover, they showed reduced apoptosis, increased proliferation and different response to 5-fluorouracil and oxaliplatin upon CRY1 and CRY2 ectopic expression. The relationship with p53 status came out as an additional layer of regulation: higher CRY1 and CRY2 protein levels coincided with a wild type p53 as in HCT116 cells and this condition only marginally affected the apoptotic and cell proliferation characteristics of the cells upon CRY ectopic expression. Conversely, lower CRY and CRY2 levels as in HT29 and SW480 cells coincided with a mutated p53 and a more robust apoptosis and proliferation upon CRY transfection. Besides, an heterogeneous pattern of ARNTL, WEE and c-MYC expression hallmarked the chosen colon cancer cell lines and likely influenced their phenotypic changes. CONCLUSION: Cryptochrome gene expression is altered in CRC, particularly in elderly subjects, female patients and cancers located at the transverse colon, affecting overall survival. Altered CRY1 and CRY2 expression patterns and the interplay with the genetic landscape in colon cancer cells may underlie phenotypic divergence that could influence disease behavior as well as CRC patients survival and response to chemotherapy.

TRIM8 downregulation in glioma affects cell proliferation and it is associated with patients survival.

BACKGROUND: Human gliomas are a heterogeneous group of primary malignant brain tumors whose molecular pathogenesis is not yet solved. In this regard, a major research effort has been directed at identifying novel specific glioma-associated genes. Here, we investigated the effect of TRIM8 gene in glioma. METHODS: TRIM8 transcriptional level was profiled in our own glioma cases collection by qPCR and confirmed in the independent TCGA glioma cohort. The association between TRIM8 expression and Overall Survival and Progression-free Survival in TCGA cohort was determined by using uni-multivariable Cox regression analysis. The effect of TRIM8 on patient glioma cell proliferation was evaluated by performing MTT and clonogenic assays. The mechanisms causing the reduction of TRIM8 expression were explored by using qPCR and in vitro assays. RESULTS: We showed that TRIM8 expression correlates with unfavorable clinical outcome in glioma patients. We found that a restored TRIM8 expression induced a significant reduction of clonogenic potential in U87MG and patient's glioblastoma cells. Finally we provide experimental evidences showing that miR-17 directly targets the 3' UTR of TRIM8 and post-transcriptionally represses the expression of TRIM8. CONCLUSIONS: Our study provides evidences that TRIM8 may participate in the carcinogenesis and progression of glioma and that the transcriptional repression of TRIM8 might have potential value for predicting poor prognosis in glioma patients.

TBC1D7 mutations are associated with intellectual disability, macrocrania, patellar dislocation, and celiac disease.

TBC1D7 forms a complex with TSC1 and TSC2 that inhibits mTORC1 signaling and limits cell growth. Mutations in TBC1D7 were reported in a family with intellectual disability (ID) and macrocrania. Using exome sequencing, we identified two sisters homozygote for the novel c.17_20delAGAG, p.R7TfsX21 TBC1D7 truncating mutation. In addition to the already described macrocephaly and mild ID, they share osteoarticular defects, patella dislocation, behavioral abnormalities, psychosis, learning difficulties, celiac disease, prognathism, myopia, and astigmatism. Consistent with a loss-of-function of TBC1D7, the patient's cell lines show an increase in the phosphorylation of 4EBP1, a direct downstream target of mTORC1 and a delay in the initiation of the autophagy process. This second family allows enlarging the phenotypic spectrum associated with TBC1D7 mutations and defining a TBC1D7 syndrome. Our work reinforces the involvement of TBC1D7 in the regulation of mTORC1 pathways and suggests an altered control of autophagy as possible cause of this disease.

Molecular analysis, pathogenic mechanisms, and readthrough therapy on a large cohort of Kabuki syndrome patients.

Kabuki syndrome (KS) is a multiple congenital anomalies syndrome characterized by characteristic facial features and varying degrees of mental retardation, caused by mutations in KMT2D/MLL2 and KDM6A/UTX genes. In this study, we performed a mutational screening on 303 Kabuki patients by direct sequencing, MLPA, and quantitative PCR identifying 133 KMT2D, 62 never described before, and four KDM6A mutations, three of them are novel. We found that a number of KMT2D truncating mutations result in mRNA degradation through the nonsense-mediated mRNA decay, contributing to protein haploinsufficiency. Furthermore, we demonstrated that the reduction of KMT2D protein level in patients' lymphoblastoid and skin fibroblast cell lines carrying KMT2D-truncating mutations affects the expression levels of known KMT2D target genes. Finally, we hypothesized that the KS patients may benefit from a readthrough therapy to restore physiological levels of KMT2D and KDM6A proteins. To assess this, we performed a proof-of-principle study on 14 KMT2D and two KDM6A nonsense mutations using specific compounds that mediate translational readthrough and thereby stimulate the re-expression of full-length functional proteins. Our experimental data showed that both KMT2D and KDM6A nonsense mutations displayed high levels of readthrough in response to gentamicin treatment, paving the way to further studies aimed at eventually treating some Kabuki patients with readthrough inducers.

Interplay between SOX9, ß-catenin and PPARγ activation in colorectal cancer.

Colorectal carcinogenesis relies on loss of homeostasic mechanisms regulating cell proliferation, differentiation and survival. These cell processes have been reported to be influenced independently by transcription factors activated downstream of the Wnt pathway, such as SOX9 and ß-catenin, and by the nuclear receptor PPARγ. The purpose of this study was to explore the expression levels and functional link between SOX9, ß-catenin and PPARγ in the pathogenesis of colorectal cancer (CRC). We evaluated SOX9, ß-catenin and PPARγ expression levels on human CRC specimens by qPCR and immunoblot detection. We tested the hypothesis that PPARγ activation might affect SOX9 and ß-catenin expression using four colon cancer cell lines (CaCo2, SW480, HCT116, and HT29 cells). In CRC tissues SOX9 resulted up-regulated at both mRNA and protein levels when compared to matched normal mucosa, ß-catenin resulted up-regulated at protein levels, while PPARG mRNA and PPARγ protein levels were down-regulated. A significant relationship was observed between high PPARG and SOX9 expression levels in the tumor tissue and female gender (p=0.005 and p=0.04, respectively), and between high SOX9 expression in the tumor tissue and age (p=0.04) and microsatellite instability (MSI), in particular with MSI-H (p=0.0002). Moreover, treatment with the synthetic PPARγ ligand rosiglitazone induced different changes of SOX9 and ß-catenin expression and subcellular localization in the colon cancer cell lines examined. In conclusion, SOX9, ß-catenin and PPARγ expression levels are deregulated in the CRC tissue, and in colon cancer cell lines ligand-dependent PPARγ activation unevenly influences SOX9 and ß-catenin expression and subcellular localization, suggesting a variable mechanistic role in colon carcinogenesis.

DPP6 gene disruption in a family with Gilles de la Tourette syndrome.

Gilles de la Tourette syndrome (TS) is a neurodevelopmental disorder characterized by multiple motor and vocal tics, frequently associated with psychiatric co-morbidities. Despite the significant level of heritability, the genetic architecture of TS still remains elusive. Herein, we investigated an Italian family where an 8-year-old boy, his father, and paternal uncle have a diagnosis of TS. Array-CGH and high resolution SNP-array analyses revealed a heterozygous microdeletion of ∼135 kb at the 7q36.2 locus in the proband and his father. Fluorescent in situ hybridization and quantitative PCR (qPCR) analyses confirmed the presence of the alteration also in the paternal uncle. The deletion selectively involves the first exon of the DPP6 gene, leading to a down-regulation of its expression, as demonstrated by the reduced messenger RNA (mRNA) levels assessed by RT-qPCR. The DPP6 gene encodes for a type II membrane glycoprotein expressed predominantly in the central nervous system. To date, a de novo DPP6 exonic duplication, of uncertain significance, was reported in one patient with TS. Moreover, the DPP6 gene has been implicated in the pathogenesis of autism spectrum disorder (ASD) and, notably, in haloperidol-induced dyskinesia. This first familial case provides evidence for association between DPP6 haploinsufficiency and TS, further suggesting a plausible molecular link between TS and ASD, and might shed some light on the efficacy and tolerability profiles of antidopaminergic agents used for tic management, thus prompting further studies on a larger cohort of patients.

Clinical and molecular description of the first Italian cohort of 33 subjects with hypophosphatasia.

Introduction: Hypophosphatasia (HPP) is a rare genetic disease caused by inactivating variants of the ALPL gene. Few data are available on the clinical presentation in Italy and/or on Italian HPP surveys. Methods: There were 30 suspected HPP patients recruited from different Italian tertiary cares. Biological samples and related clinical, biochemical, and anamnestic data were collected and the ALPL gene sequenced. Search for large genomic deletions at the ALPL locus (1p36) was done. Phylogenetic conservation and modeling were applied to infer the effect of the variants on the protein structure. Results: There were 21 ALPL variants and one large genomic deletion found in 20 out of 30 patients. Unexpectedly, NGS-driven differential diagnosis allowed uncovering three hidden additional HPP cases, for a total of 33 HPP subjects. Eight out of 24 coding variants were novel and classified as "pathogenic", "likely pathogenic", and "variants of uncertain significance". Bioinformatic analysis confirmed that all the variants strongly destabilize the homodimer structure. There were 10 cases with low ALP and high VitB6 that resulted negative to genetic testing, whereas two positive cases have an unexpected normal ALP value. No association was evident with other biochemical/clinical parameters. Discussion: We present the survey of HPP Italian patients with the highest ALPL mutation rate so far reported and confirm the complexity of a prompt recognition of the syndrome, mostly for HPP in adults. Low ALP and high VitB6 values are mandatory for the genetic screening, this latter remaining the gold standard not only to confirm the clinical diagnosis but also to make differential diagnosis, to identify carriers, to avoid likely dangerous therapy in unrecognized cases.

Absence of deletion and duplication of MLL2 and KDM6A genes in a large cohort of patients with Kabuki syndrome.

Kabuki syndrome is a rare, multiple congenital anomaly/mental retardation syndrome caused by MLL2 point mutations and KDM6A microdeletions. We screened a large cohort of MLL2 mutation-negative patients for MLL2 and KDM6A exon(s) microdeletion and microduplication. Our assays failed to detect such rearrangements in MLL2 as well as in KDM6A gene. These results show that these genomic events are extremely rare in the Kabuki syndrome, substantiating its genetic heterogeneity and the search for additional causative gene(s).

Circadian Genes Expression Patterns in Disorders Due to Enzyme Deficiencies in the Heme Biosynthetic Pathway.

Heme is a member of the porphyrins family of cyclic tetrapyrroles and influences various cell processes and signalling pathways. Enzyme deficiencies in the heme biosynthetic pathway provoke rare human inherited metabolic diseases called porphyrias. Protein levels and activity of enzymes involved in the heme biosynthetic pathway and especially 5'-Aminolevulinate Synthase 1 are featured by 24-h rhythmic oscillations driven by the biological clock. Heme biosynthesis and circadian pathways intermingle with mutual modulatory roles. Notably, heme is a ligand of important cogs of the molecular clockwork, which upon heme binding recruit co-repressors and inhibit the transcription of numerous genes enriching metabolic pathways and encoding functional proteins bringing on crucial cell processes. Herein, we assessed mRNA levels of circadian genes in patients suffering from porphyrias and found several modifications of core clock genes and clock-controlled genes expression, associated with metabolic and electrolytic changes. Overall, our results show an altered expression of circadian genes accompanying heme biosynthesis disorders and confirm the need to deepen the knowledge of the mechanisms through which the alteration of the circadian clock circuitry could take part in determining signs and symptoms of porphyria patients and then again could represent a target for innovative therapeutic strategies.

Expanding the phenotype associated to KMT2A variants: overlapping clinical signs between Wiedemann-Steiner and Rubinstein-Taybi syndromes.

Lysine-specific methyltransferase 2A (KMT2A) is responsible for methylation of histone H3 (K4H3me) and contributes to chromatin remodeling, acting as "writer" of the epigenetic machinery. Mutations in KMT2A were first reported in Wiedemann-Steiner syndrome (WDSTS). More recently, KMT2A variants have been described in probands with a specific clinical diagnosis comprised in the so-called chromatinopathies. Such conditions, including WDSTS, are a group of overlapping disorders caused by mutations in genes coding for the epigenetic machinery. Among them, Rubinstein-Taybi syndrome (RSTS) is mainly caused by heterozygous pathogenic variants in CREBBP or EP300. In this work, we used next generation sequencing (either by custom-made panel or by whole exome) to identify alternative causative genes in individuals with a RSTS-like phenotype negative to CREBBP and EP300 mutational screening. In six patients we identified different novel unreported variants in KMT2A gene. The identified variants are de novo in at least four out of six tested individuals and all of them display some typical RSTS phenotypic features but also WDSTS specific signs. This study reinforces the concept that germline variants affecting the epigenetic machinery lead to a shared molecular effect (alteration of the chromatin state) determining superimposable clinical conditions.

Large rearrangements detected by MLPA, point mutations, and survey of the frequency of mutations within the SLC3A1 and SLC7A9 genes in a cohort of 172 cystinuric Italian patients.

Cystinuria is a rare inherited disorder characterized by defective renal reabsorption of cystine and the dibasic amino acids. SLC3A1 and SLC7A9 have been identified as responsible genes. The large majority of the more than 200 mutations so far identified in the two genes are point mutations, while only few alleles carrying gross genomic alterations have been reported. We screened 39 cystinuric patients for large rearrangements, by two home-made multiplex ligation-dependent probe amplification (MLPA) assays. MLPA analysis led to the identification of 6 different alleles in SLC3A1 and 2 in SLC7A9 accounting for a total of 25 copy number changes, 11 in SLC3A1 and 14 in SLC7A9. Three large rearrangements in SLC3A1, deletion of exons 2-4 (E2_E4del), deletion of exons 5-6 (E5_E6del) and duplication of exons 8-9 (E8_E9dup) are novel. A complete SLC7A9 gene deletion was found in three patients. In addition, we report the identification of three novel point mutations in SLC7A9 (p.G105E, p.R250K, c.1416_1417insAC), the frequency and the occurrence of cystinuria mutations in a cohort of 172 Italian patients. In conclusion, we developed a reliable and robust MLPA analytic method for SLC3A1 and SLC7A9 genes that represents an optimal complement to DNA sequence analysis in patients with cystinuria, enabling the screening for deletions and duplications.

VHL frameshift mutation as target of nonsense-mediated mRNA decay in Drosophila melanogaster and human HEK293 cell line.

There are many well-studied examples of human phenotypes resulting from nonsense or frameshift mutations that are modulated by Nonsense-Mediated mRNA Decay (NMD), a process that typically degrades transcripts containing premature termination codons (PTCs) in order to prevent translation of unnecessary or aberrant transcripts. Different types of germline mutations in the VHL gene cause the von Hippel-Lindau disease, a dominantly inherited familial cancer syndrome with a marked phenotypic variability and age-dependent penetrance. By generating the Drosophila UAS:Upf1(D45B) line we showed the possible involvement of NMD mechanism in the modulation of the c.172delG frameshift mutation located in the exon 1 of Vhl gene. Further, by Quantitative Real-time PCR (QPCR) we demonstrated that the corresponding c.163delG human mutation is targeted by NMD in human HEK 293 cells. The UAS:Upf1(D45B) line represents a useful system to identify novel substrates of NMD pathway in Drosophila melanogaster. Finally, we suggest the possible role of NMD on the regulation of VHL mutations.