Improving clinical interpretation of five KRIT1 and PDCD10 intronic variants.

Cerebral cavernous malformation (CCM) is a vascular malformation of the central nervous system which may occur sporadically or segregate within families due to heterozygous variants in KRIT1/CCM1, MGC4607/CCM2 or PDCD10/CCM3. Intronic variants are not uncommon in familial CCM, but their clinical interpretation is often hampered by insufficient data supporting in silico predictions. Here, the mRNA analysis for two intronic unpublished variants (KRIT1 c.1147-7 T > G and PDCD10 c.395 + 2 T > G) and three previously published variants in KRIT1 but without data supporting their effects was carried out. This study demonstrated that all variants can induce a frameshift with the lack of residues located in the C-terminal regions and involved in protein-protein complex formation, which is essential for vascular homeostasis. These results support the introduction of mRNA analysis in the diagnostic pathway of familial CCM and expand the knowledge of abnormal splicing patterning in this disorder.

Prenatal genetic diagnosis: Fetal therapy as a possible solution to a positive test.

BACKGROUND: Fetal abnormalities cause 20% of perinatal deaths. Advances in prenatal genetic and other types of screening offer great opportunities for identifying high risk pregnancies. METHODS: Through a literature search, here we summarise what are the prenatal diagnostic technique that are being used and how those techniques may allow for prenatal interventions. RESULTS: Next generation sequencing and non-invasive prenatal testing are fundamental for clinical diagnostics because of their sensitivity and accuracy in identifying point mutations, aneuploidies, and microdeletions, respectively. Timely identification of genetic disorders and other fetal abnormalities enables early intervention, such as in-utero gene therapy, fetal drug therapy and prenatal surgery. CONCLUSION: Prenatal intervention is mainly focused on conditions that may cause death or lifelong disabilities, like spina bifida, congenital diaphragm hernia and sacrococcygeal teratoma; and may be an alternative therapeutic option to termination of pregnancy. However, it is not yet widely available, due to lack of specialized centers.

Propranolol for familial cerebral cavernous malformation (Treat_CCM): study protocol for a randomized controlled pilot trial.

BACKGROUND: Cerebral cavernous malformations (CCMs) are vascular malformations characterized by clusters of enlarged leaky capillaries in the central nervous system. They may result in intracranial haemorrhage, epileptic seizure(s), or focal neurological deficits, and potentially lead to severe disability. Globally, CCMs represent the second most common intracranial vascular malformation in humans, and their familial form (FCCMs) accounts for one-fifth of cases. Neurosurgical excision, and perhaps stereotactic radiosurgery, is the only available therapeutic option. Case reports suggest that propranolol might modify disease progression. METHODS: Treat_CCM is a prospective, randomized, open-label, blinded endpoint (PROBE), parallel-group trial involving six Italian clinical centres with central reading of brain magnetic resonance imaging (MRI) and adverse events. Patients with symptomatic FCCMs are randomized (2:1 ratio) either to propranolol (40-80 mg twice daily) in addition to standard care or to standard care alone (i.e. anti-epileptic drugs or headache treatments). The primary outcome is intracranial haemorrhage or focal neurological deficit attributable to CCMs. The secondary outcomes are MRI changes over time (i.e. de novo CCM lesions, CCM size and signal characteristics, iron deposition, and vascular leakage as assessed by quantitative susceptibility mapping and dynamic contrast enhanced permeability), disability, health-related quality of life, depression severity, and anxiety (SF-36, BDI-II, State-Trait Anxiety Inventory). DISCUSSION: Treat_CCM will evaluate the safety and efficacy of propranolol for CCMs following promising case reports in a randomized controlled trial. The direction of effect on the primary outcome and the consistency of effects on the secondary outcomes (even if none of them yield statistically significant differences) of this external pilot study may lead to a larger sample size in a definitive phase 2 trial. TRIAL REGISTRATION: ClinicalTrails.gov, NCT03589014. Retrospectively registered on 17 July 2018.

Molecular diagnostic workflow, clinical interpretation of sequence variants, and data repository procedures in 140 individuals with familial cerebral cavernous malformations.

Familial cerebral cavernous malformation (FCCM) is an autosomal dominant vascular disorder caused by heterozygous deleterious variants in KRIT1, CCM2 or PDCD10. In a previous study, we presented the clinical and molecular findings in 140 FCCM individuals. In the present work, we report supporting information on (a) applied diagnostic workflow; (b) clinical significance of molecular findings according to the American College of Medical Genetics and Genomics/Association for Molecular Pathology recommendations; (c) standardization of molecular and clinical data according to the Human Phenotype Ontology; (d) preliminary genotype-phenotype correlations on a subgroup of patients by considering sex, age at diagnosis, neurological symptoms, and number and anatomical site(s) of vascular anomalies; (e) datasets submitted to the Leiden Open Variation Database. An overview of the changes of our diagnostic approach before and after the transition to next-generation sequencing is also reported. This work presents the full procedure that we apply for molecular testing, data interpretation and storing in public databases in FCCM.

A Nonsense Mitochondrial DNA Mutation Associates with Dysfunction of HIF1α in a Von Hippel-Lindau Renal Oncocytoma.

The Von Hippel-Lindau (VHL) syndrome has been rarely associated with renal oncocytomas, and tumors usually show HIF1α chronic stabilization. By contrast, oncocytomas mainly associated with respiratory chain (RC) defects due to severe mitochondrial DNA (mtDNA) mutations are incapable of stabilizing HIF1α, since oxygen consumption by the RC is dramatically diminished and prolylhydroxylase activity is increased by α-ketoglutarate accumulation following Krebs cycle slowdown. Here, we investigate the cooccurrence of a pseudohypoxic condition with oncocytic transformation in a case of VHL-associated renal oncocytoma. While HIF1α was abundant in nuclei concordantly with defects in VHL, negative staining of its targets carbonic anhydrase IX (CAIX) and glucose transporter GLUT1, usually overexpressed in VHL-associated neoplasms, suggested HIF1α to be present in its inactive (hydroxylated) form. MtDNA sequencing and immunohistochemistry analyses revealed a MT-CO1 stop-gain mutation and cytochrome c oxidase loss. We suggest that a mitochondrial respiration impairment may lead to hyperhydroxylation of the transcription factor, which we confirmed by specific staining of hydroxylated HIF1α. Such inactive form hence accumulated in the VHL-deficient tumor, where it may contribute to the benign nature of the neoplasm. We propose that the protumorigenic role of HIF1α in VHL cancers may be blunted through drugs inhibiting mitochondrial respiratory complexes, such as metformin.

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.

A single-center study on 140 patients with cerebral cavernous malformations: 28 new pathogenic variants and functional characterization of a PDCD10 large deletion.

Cerebral cavernous malformation (CCM) is a capillary malformation arising in the central nervous system. CCM may occur sporadically or cluster in families with autosomal dominant transmission, incomplete penetrance, and variable expressivity. Three genes are associated with CCM KRIT1, CCM2, and PDCD10. This work is a retrospective single-center molecular study on samples from multiple Italian clinical providers. From a pool of 317 CCM index patients, we found germline variants in either of the three genes in 80 (25.2%) probands, for a total of 55 different variants. In available families, extended molecular analysis found segregation in 60 additional subjects, for a total of 140 mutated individuals. From the 55 variants, 39 occurred in KRIT1 (20 novel), 8 in CCM2 (4 novel), and 8 in PDCD10 (4 novel). Effects of the three novel KRIT1 missense variants were characterized in silico. We also investigated a novel PDCD10 deletion spanning exon 4-10, on patient's fibroblasts, which showed significant reduction of interactions between KRIT1 and CCM2 encoded proteins and impaired autophagy process. This is the largest study in Italian CCM patients and expands the known mutational spectrum of KRIT1, CCM2, and PDCD10. Our approach highlights the relevance of seeking supporting information to pathogenicity of new variants for the improvement of management of CCM.

Novel association of MEN1 gene mutations with parathyroid carcinoma.

Inactivating mutations of the multiple endocrine neoplasia 1 (MEN1) gene cause MEN1 syndrome, characterized by primary hyperparathyroidism (pHPT), and parathyroid and gastro-entero-pancreatic pituitary tumors. At present, only 14 cases of malignant parathyroid tumor have been associated with the syndrome, with 6 cases carrying an inactivating mutation of the MEN1 gene. The present study presents the case of a 48-year-old female who presented with multigland pHPT and multiple pancreatic lesions. The patient underwent surgery several times for the excision of parathyroid hyperplasia, carcinoma and adenoma. The MEN1 gene was screened, revealing three variants (in cis) at the intron/exon 3 boundary (IVS2-3G>C, c.497A>T and c.499G>T) detected on the DNA of the proband, not shared by her relatives. RNA sequencing revealed that the IVS2-3C>G variant caused the skipping of the exon 3. Therefore, the present study reports on a novel rare association of MEN1 syndrome and parathyroid carcinoma. The reported splicing mutation was previously identified in subjects who always developed malignant lesions; thus, a possible genotype-phenotype association may be considered.

Increased prevalence of the GCM2 polymorphism, Y282D, in primary hyperparathyroidism: analysis of three Italian cohorts.

CONTEXT: Glial cells missing-2 (GCM2) is key for parathyroid gland organogenesis. Its persistent expression in the adult parathyroid raises the possibility that overactive forms play a role in the evolution of parathyroid hyperactivity or tumorigenesis. A GCM2 c.844T → G; p.Y282D missense variant has been described within a transactivation inhibitory domain (amino acids 263-352). OBJECTIVE: The aims of the study were to 1) assess the frequency of Y282D in Italian primary hyperparathyroidism (PHPT) and control (C) populations, 2) test for association of 282D with PHPT and its phenotypic features, and 3) compare the transactivation potency of GCM2 282D relative to wild-type Y282. SUBJECTS AND METHODS: Subjects included a large southern Italian cohort (310 PHPT and 433 C) and 2 replication cohorts from northern Italy. Association of 282D with PHPT was tested in all cohorts and with phenotypic features in the larger PHPT cohort. An in vitro GCM promoter-luciferase reporter assay was conducted in HEK293 cells. RESULTS: 282D was significantly increased in the PHPT group, with a minor allele frequency of 0.066 compared with 0.029 in the C group (P = .0008), in the discovery cohort and was more prevalent in the replication cohorts. Combined analysis (510 PHPT and 665 C) yielded a likelihood ratio of 2.27 (95% confidence interval = 1.50-3.42; P < .0001). The 282D variant was not associated with serum calcium, phosphate, creatinine, or PTH levels or with bone mineral density, fractures, or renal stones in the PHPT group. The 282D variant had significantly greater transcriptional activity than the wild-type Y282 (17× basal vs 12× basal; P < 0.05). CONCLUSION: The higher frequency of GCM2 282D in PHPT and enhanced transcriptional activity of this variant supports the notion that it could contribute causally to parathyroid tumorigenesis.

Molecular dissection of the VHL gene in solitary capillary hemangioblastoma of the central nervous system.

Capillary hemangioblastomas (HGBs) of the CNS occur either sporadically or as part of the von Hippel-Lindau (VHL) syndrome. Molecular characterizations of the VHL gene in sporadic HGBs at the somatic level have been limited to date. We investigated the VHL gene in 57 patients most of whom (55 [96%] of 57) had a solitary CNS HGB at the time of surgery. Tissues from 23 HGBs of these patients (2 VHL related and 21 unrelated) were also investigated at genetic and epigenetic levels. Two of the 51 patients with apparently sporadic HGBs and no additional evidence of VHL (∼4%) were found to have a germline VHL gene mutation; both of these patients subsequently developed evidence of VHL syndrome. Somatic VHL gene mutations were found in 11 (52%) of the 21 non-VHL-related cases. A germline mutation was identified in 5 (84%) of 6 VHL-associated HGBs; double gene inactivation was observed in tumor tissue from VHL syndrome patients. Seven different previously unreported VHL gene alterations (6 somatic and 1 germline) were identified; double hits were identified in 7 (12%) of 57 cases. Our findings confirm the usefulness of VHL gene analysis at the germline level in patients who present with apparently solitary HGB. Moreover, the genetic and epigenetic VHL gene investigations performed support a key role for functional alterations of the VHL gene in sporadic neuraxial HGB.

Identification and functional characterization of three NoLS (nucleolar localisation signals) mutations of the CDC73 gene.

Hyperparathyroidism Jaw-Tumour Syndrome (HPT-JT) is characterized by primary hyperparathyroidism (PHPT), maxillary/mandible ossifying fibromas and by parathyroid carcinoma in 15% of cases. Inactivating mutations of the tumour suppressor CDC73/HRPT2 gene have been found in HPT-JT patients and also as genetic determinants of sporadic parathyroid carcinoma/atypical adenomas and, rarely, typical adenomas, in familial PHPT. Here we report the genetic and molecular analysis of the CDC73/HRPT2 gene in three patients affected by PHPT due to atypical and typical parathyroid adenomas, in one case belonging to familial PHPT. Flag-tagged WT and mutant CDC73/HRPT2 proteins were transiently transfected in HEK293 cells and functional assays were performed in order to investigate the effect of the variants on the whole protein expression, nuclear localization and cell overgrowth induction. We identified four CDC73/HRPT2 gene mutations, three germline (c.679_680delAG, p.Val85_Val86del and p.Glu81_Pro84del), one somatic (p.Arg77Pro). In three cases the mutation was located within the Nucleolar Localisation Signals (NoLS). The three NoLS variants led to instability either of the corresponding mutated protein or mRNA or both. When transfected in HEK293 cells, NoLS mutated proteins mislocalized with a predeliction for cytoplasmic or nucleo-cytoplasmic localization and, finally, they resulted in overgrowth, consistent with a dominant negative interfering effect in the presence of the endogenous protein.

VHL gene alterations in Italian patients with isolated renal cell carcinomas.

Clear cell renal cell carcinoma (ccRCC) is the most common malignant neoplasm of the kidney and belongs to the few human tumors known to develop from mutations of the VHL tumor suppressor gene. VHL germline mutations are associated with hereditary ccRCCs in VHL disease. However, somatic VHL gene defects may also occur in sporadic ccRCCs. In this study, we analyzed the frequency and the spectrum of VHL gene alterations in 35 Italian patients with sporadic renal cell carcinoma (RCC). Tumor-specific intragenic VHL pathogenic mutations were detected in 38% (11/29) of the ccRCC patients and 33% (2/6) of the patients with other types of RCC. One novel 18-bp in-tandem duplication and 4 previously unreported nucleotide changes in the VHL gene were described. Microsatellite analysis showed loss of heterozygosity for at least 1 informative marker in 43% (9/21) of the ccRCCs and 50% (3/6) of the non-ccRCCs; 5 of the 13 tumors (38%) harboring VHL gene alterations also had loss of heterozygosity for at least 1 microsatellite marker. Our results confirm that somatic inactivation of the VHL gene may play a pivotal role in the tumorigenesis of sporadic ccRCCs in Italian patients and suggests that mutation analysis of the VHL gene may be helpful for discriminating sporadic, VHL-gene-related ccRCCs from those related to VHL disease.

20 novel point mutations and one large deletion in EXT1 and EXT2 genes: report of diagnostic screening in a large Italian cohort of patients affected by hereditary multiple exostosis.

BACKGROUND: Hereditary multiple exostosis represents the most frequent bone tumor disease in humans. It consists of cartilage deformities affecting the juxta-ephyseal region of long bones. Usually benign, exostosis could degenerate in malignant chondrosarcoma form in less than 5% of the cases. Being caused by mutations in the predicted tumor suppressor genes, EXT1 (chr 8q23-q24) and EXT2 (chr 11p11-p12) genes, HMEs are usually inherited with an autosomal dominant pattern, although "de novo" cases are not infrequent. AIM: Here we present our genetic diagnostic report on the largest Southern Italy cohort of HME patients consisting of 90 subjects recruited over the last 5years. RESULTS: Molecular screening performed by direct sequencing of both EXT1 and EXT2 genes, by MLPA and Array CGH analyses led to the identification of 66 mutations (56 different occurrences) and one large EXT2 deletion out of 90 patients (74.4%). The total of 21 mutations (20 different occurrences, 33.3%) and the EXT2 gene deletion were novel. In agreement with literature data, EXT1 gene mutations were scattered along all the protein sequence, while EXT2 lesions fell in the first part of the protein. Conservation, damaging prediction and 3-D modeling, in-silico, analyses, performed on three novel missense variants, confirmed that at least in two cases the novel aminoacidic changes could alter the structure stability causing a strong protein misfolding. CONCLUSIONS: Here we present 20 novel EXT1/EXT2 mutations and one large EXT2 deletion identified in the largest Southern Italy cohort of patients affected by hereditary multiple exostosis.

CDC73 mutations and parafibromin immunohistochemistry in parathyroid tumors: clinical correlations in a single-centre patient cohort.

OBJECTIVE: To determine if molecular and immunohistochemical (IHC) features of the HRPT2/CDC73 gene and its product, parafibromin, predict the natural history of parathyroid malignancy, particularly atypical adenoma, as seen in a single-centre patient cohort. METHODS: Matched tumor and non-tumor tissues were obtained from 46 patients with parathyroid carcinoma (CA) (n = 15), atypical adenoma (AA) (n = 14) and typical adenoma (TA) (n = 17), as defined by standardized histopathological criteria. Exons and exon-intron boundaries of the CDC73 gene were sequenced to identify germline or somatic mutations. IHC staining for parafibromin was performed and scored as positive if nuclear staining was at least partially IHC-positive. RESULTS: Mutations of CDC73 were observed in 9/15 (60 %) CA, 2/14 (14 %) AA, and 1/17 (6 %) TA tumors. A recurrent two basepair mutation in exon 7 -- c.679_680delAG -- accounted for half of all identified mutations. Absence of parafibromin nuclear staining was noted in 8/12 (67 %) CA, 2/13 (15 %) AA, and 3/17 (18 %) TA tumors. Median follow up times were 88 months for CA, 76 months for AA, and 104 months for TA patients. One patient, a member of a previously reported multiplex family with a germline CDC73 mutation was found to have a second adenoma after removal of an atypical adenoma. CONCLUSIONS: Molecular screening and IHC are both useful tools in the differential diagnosis of parathyroid tumors, but both have limited sensitivity and specificity. CDC73 mutations and negative immunostaining were common in atypical adenomas, but no local recurrence was observed in any case with successful surgical removal after follow-up periods of 27 to 210 months.

Coexistence of multiple endocrine neoplasia type 1 and type 2 in a large Italian family.

To describe the coexistence of mutations of both the multiple endocrine neoplasia type 1 (MEN1) and type 2 (MEN2) genes in a large Italian family and evaluate if it could be associated with more aggressive clinical manifestations of the two syndromes. Blood samples were obtained for genetic and biochemical analyses. The RET gene exons (8, 10, 11, 13, 14, 15, 16, 18) and the MEN1 coding regions, including the exon-intron boundaries, were amplified by PCR and directly sequenced. We identified two germline mutations in the proband: the first one, K666M, located at the exon 11 of RET proto-oncogene and the second one, IVS4+1G>T, located in the MEN1 gene. The functional characterization of IVS4+1G>T variation, located in the splicing donor site of exon 4 of MEN1 gene, caused the in-frame junction of exon 3 to exon 5, thus obtaining a shorter protein. The same proband's germline mutations were found in 16 relatives out of 21 screened subjects: 8 carried IVS4+1G>T, 4 RET K666M, and 4 both the mutations. This is the second report in literature of coexistence in the same family of germline mutations of both RET proto-oncogene and MEN1 gene. The simultaneous presence of the two mutations was not apparently associated with more aggressive diseases, since at last follow-up all patients appeared to be disease-free or well compensated by medical therapy; finally, no one exhibited metastatic diseases.

Small deletion at the 7q21.2 locus in a CCM family detected by real-time quantitative PCR.

Cerebral cavernous malformations (CCMs) represent a common autosomal dominant disorder that predisposes patients to haemorrhagic strokes and focal neurological signs. About 56% of the hereditary forms of CCMs have been so far associated with mutations in the KRIT1 (Krev Interaction Trapped 1) gene, located at 7q21.2 (CCM1 locus). We described the complete loss of 7q21.2 locus encompassing the KRIT1 gene and 4 flanking genes in a CCM family by using a dense set of 12 microsatellite markers. The complete loss of the maternal copy of KRIT1 gene region was confirmed by Real-Time Quantitative Polymerase Chain Reaction (RT-QPCR) and the same approach was used for expression analysis. Additional RT-QPCR analysis showed the extension of the deletion, for a total of 700 kb, to the adjacent downstream and upstream-located genes, MTERF, AKAP9, CYP51A1, as well as a partial loss of the ANKIB1 gene. Here we report the molecular characterization of an interstitial small genomic deletion of the 7q21.2 region in a CCMs affected family, encompassing the KRIT1 gene. Our findings confirm the loss of function mechanism for the already known CCM1 locus, without any evident involvement of the other deleted genes. Moreover, our investigations highlight the usefulness of the RT-QPCR to the molecular characterization of the breakpoints genomic deletions and to the identification of internal deleted genes involved in the human genetic diseases.

Sporadic and MEN1-related primary hyperparathyroidism: differences in clinical expression and severity.

Primary hyperparathyroidism (PHPT) is a common endocrine disease that is associated with multiple endocrine neoplasia type 1 (MEN1) in approximately 2% of PHPT cases. Lack of a family history and other specific expressions may lead to underestimated MEN1 prevalence in PHPT. The aim of this study was to identify clinical or biochemical features predictive of MEN1 and to compare the severity of the disease in MEN1-related versus sporadic PHPT (sPHPT). We performed a 36-mo cross-sectional observational study in three tertiary referral centers on an outpatient basis on 469 consecutive patients with sporadic PHPT and 64 with MEN1-related PHPT. Serum calcium, phosphate, PTH, 25(OH)D(3), and creatinine clearance were measured, and ultrasound examination of the urinary tract/urography was performed in all patients. In 432 patients, BMD was measured at the lumbar spine (LS) and femoral neck (FN). MEN1 patients showed lower BMD Z-scores at the LS (-1.33 +/- 1.23 versus -0.74 +/- 1.4, p = 0.008) and FN (-1.13 +/- 0.96 versus -0.6 +/- 1.07, p = 0.002) and lower phosphate (2.38 +/- 0.52 versus 2.56 +/- 0.45 mg/dl, p = 0.003) and PTH (113.8 +/- 69.5 versus 173.7 +/- 135 pg/ml, p = 0.001) levels than sPHPT patients. Considering probands only, the presence of MEN1 was more frequently associated with PTH values in the normal range (OR, 3.01; 95% CI, 1.07-8.50; p = 0.037) and younger age (OR, 1.61; 95% CI, 1.28-2.02; p = 0.0001). A combination of PTH values in the normal range plus age <50 yr was strongly associated with MEN1 presence (OR, 13.51; 95% CI, 3.62-50.00; p = 0.0001). In conclusion, MEN1-related PHPT patients show more severe bone but similar kidney involvement despite a milder biochemical presentation compared with their sPHPT counterparts. Normal PTH levels and young age are associated with MEN1 presence.

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.

An 11-bp duplication in the promoter region of the VHL gene in a patient with cerebellar hemangioblastoma and renal oncocytoma.

Central nervous system hemangioblastomas are benign vascular tumours that may present sporadically or as manifestation of the von Hippel-Lindau (VHL) disease. VHL Syndrome is a rare autosomal dominant disorder characterized, besides hemangioblastomas, by susceptibility to multifocal and bilateral renal cell carcinoma and cysts, retinal angiomas, pheochromocytoma, epididymis cystoadenoma, pancreatic cysts and/or islet cell tumours. Germline mutations of VHL tumour suppressor gene cause the VHL disease, while somatic mutations have been associated with sporadic hemangioblastomas and clear-cell renal carcinomas. We identified an 11-bp duplication in the promoter region of the VHL gene in a VHL-affected individual. Functional analysis revealed that this variant affects the binding or the binding affinity of one or more transcription factors that regulate the transcription of VHL in vivo, reducing the endogenous levels of VHL mRNA. Moreover, consistent with the "two hits" model, microsatellite analysis of hemangioblastoma tissue from this patient revealed Allelic Imbalance for the chromosomal region near the VHL gene. We propose that these molecular events, through a loss of pVHL function, lead to the onset of the VHL-related tumours in that individual.