Maddalena Martella

Cx26 deafness: mutation analysis and clinical variability.

Mutations in the gap junction protein connexin 26 (Cx26) gene (GJB2) seem to account for many cases of congenital sensorineural hearing impairment, the reported prevalence being 34-50% in autosomal recessive cases and 10-37% in sporadic cases. The hearing impairment in these patients has been described as severe or profound. We have studied 53 unrelated subjects with congenital non-syndromic sensorineural hearing impairment in order to evaluate the prevalence and type of Cx26mutations and establish better genotype-phenotype correlation. Mutations in the Cx26 gene were found in 53% of the subjects tested, 35.3% of the autosomal recessive and 60% of the sporadic cases in our series. Three new mutations were identified. The hearing deficit varied from mild to profound even in 35delG homozygotes within the same family. No evidence of progression of the impairment was found. Alterations of the Cx26 gene account for a large proportion of cases of congenital non-syndromic sensorineural deafness, so it seems appropriate to extend the molecular analysis even to subjects with mild or moderate prelingual hearing impairment of unknown cause.

Somatic Mosaicism in von Hippel-Lindau Disease

von Hippel‐Lindau (VHL) disease is an autosomal dominant familial cancer syndrome predisposing to the development of retinal and central nervous system haemangioblastomas, pheochromocytomas, renal and pancreatic cancer. In the course of a molecular analysis conducted to detect germline mutations of this gene in von Hippel‐Lindau patients and individuals affected by sporadic tumors, we have identified a case of somatic mosaicism in the asymptomatic mother of a VHL patient who was subsequently diagnosed with pheochromocytoma. This is the first report providing molecular evidence of somatic mosaicism in von Hippel‐Lindau disease. Mosaicism could provide some genetic explanation for the clinical heterogeneity and variable severity of the VHL phenotype, and should be considered, as a possible event when evaluating sporadic cases of VHL or patients with isolated VHL‐related tumors. Hum Mutat 15:114, 2000.

Molecular genetics applied to clinical practice: The Cx26 hearing impairment

Mutations in the Cx26/GJB2 gene account for a large proportion of pre-lingual hearing impairment with a prevalence up to 50% in autosomal recessive cases and a still undefined prevalence in sporadic cases. Ninety-four subjects affected by non-syndromal sensorineural hearing impairment (NSHI) were enrolled in the study. The patients had either a family history of childhood hearing deficit or represented sporadic cases. The risk of an acquired cause of the deficit has been carefully excluded. Audiological characteristics were investigated. Cx26 mutations were found in 50% of subjects. Seventy-three per cent of mutations in this gene were 35delG, with significant geographical variations. In 7% of the putative Cx26 alleles no mutations were detected either in the coding region or in the non-coding exon 1. Cx26 hearing impairment involves all frequencies, is of variable severity, and is very rarely progressive and most frequently symmetrical between the two ears. The high occurrence of this type of pre-lingual hearing impairment argues for modification of the protocols used to investigate the aetiology of childhood hearing impairment. Early screening for Cx26 mutations in all patients with non-syndromal familial and sporadic permanent childhood hearing impairment seems justified.

Molecular Characterization of Large Deletions in the von Hippel-Lindau (VHL) Gene by Quantitative Real-Time PCR

AbstractIntroduction: Mutations of the von Hippel-Lindau (VHL) gene are responsible for VHL disease. This is a familial autosomal-dominant syndrome, predisposing to the development of benign and malignant tumors, including CNS and retinal hemangioblastomas, pheochromocytomas, and clear cell renal carcinomas.At least 30% of the disease-causing mutations in the VHL gene involve large alterations. Identification of these mutations is not possible using PCR-based mutational scanning methods. Quantitative Southern blot analysis has been traditionally employed for the detection of complete or partial deletions and more complex rearrangements of the gene. Methods: An alternative quantitative method was developed using a combination of quantitative Southern blot analysis and real-time PCR. With this approach, we studied 24 large VHL gene alterations to determine the exact nature of the mutations and to possibly characterize the boundaries of the deleted regions. Results: This combined molecular approach showed that all the VHL alterations studied were due to deletions, from which the position in the gene could be more precisely mapped. One of the samples that was completely characterized was found to carry an intragenic 2.2kb deletion with both 5′ and 3′ breakpoints located within Alu-repeat sequences. Conclusion: This is the first report on the molecular analysis of large VHL alterations. The results of our study and the complete characterization of a large deletion lead to the hypothesis that an Alu-mediated mechanism may be responsible for the common occurrence of large alterations in the VHL gene.

Identification and In Silico Analysis of Novel von Hippel‐Lindau (VHL) Gene Variants from a Large Population

Mutational inactivation of the VHL gene is the cause of von Hippel‐Lindau (VHL) disease, an autosomal dominant hereditary cancer syndrome predisposing to haemangioblastomas, pheochromocytomas and clear‐cell renal carcinomas. The gene product (pVHL) functions as an adapter in cellular processes including cell growth and apoptosis. VHL mutation analysis was carried out in 426 unrelated subjects with phenotypes ranging from VHL syndrome, to isolated VHL‐related tumours that could represent the first manifestation of the disease. A total of 111 individuals were found to carry alterations, with large deletions representing 40% of the variants. Eighteen of the 95 detected variants were novel, seemingly disease‐causing mutations; their pathogenic role has been evaluated in silico for effects on protein folding and interactions. Putative regions of interaction between pVHL and proteins involved in common pathways have been identified, assessing possible implications for the presence of mutations in these regions. All new variants predicted to truncate or cause complete pVHL loss of structure were associated with phenotypes consistent with VHL type 1. Seven of the new amino acid substitutions are disease‐causing mutations, one is a neutral variant, whereas the results for two remain ambiguous. Our combined molecular and in silico approach for the evaluation of putative disease‐causing mutations contributes to the interpretation of the potential pathogenicity of these novel variants.

Familial nonsyndromic pheochromocytoma.

Abstract:  Judging from recent data, heritable forms account for 30–40% of pheochromocytomas. The molecular basis for the familial pheochromocytoma has been largely elucidated and the role of germline mutation of the VHL, RET, SDHB, and SDHD genes has been established. However, on genotyping a group of 172 sporadic or familial pheochromocytomas, we characterized four unrelated probands with familial pheochromocytomas without any sequence variants of RET (exons 8, 10, 11, 13, 14, 15, and 16) or the entire coding sequence of VHL, SDHB, SDHC, SDHD, and EGLN3 (exon‐intron boundaries included). The proband of family 1 is a man who had a bilateral pheochromocytoma at the age of 32 and a local recurrence at the age of 48 years. His brother died of malignant pheochromocytoma and his nephew died suddenly of an undiagnosed pheochromocytoma. The proband of family 2 is a female who had a 5‐cm benign adrenal pheochromocytoma at the age of 34 years, while her cousin (maternal branch) had a monolateral pheochromocytoma at the age of 42 years. No other tumors had been reported in either family. The proband of family 3 is a female who had a bilateral pheochromocytoma at the age of 66 years. Her sister had a bilateral pheochromocytoma and breast cancer at the age of 54 years. Several other tumors were recorded in this family, including laryngeal cancer, leukemia, and a case of medullary thyroid carcinoma (MTC) in one brother. MTC was naturally ruled out in the proband and her sister. In family 4, the proband was a female who had a bilateral pheochromocytoma at the age of 46 years and a local recurrence a few years later, with liver metastases from the pheochromocytoma. Her brother had a monolateral benign pheochromocytoma. The proband also had a melanoma and bilateral renal cysts. This case revealed a VHL sequence variant IVS2 + 43 A>G, which was also found in one other unrelated sporadic pheochromocytoma. VHL mRNA integrity is currently being evaluated. The proband had no cerebellar or spinal NMR findings or retinal alterations. In family 5, the proband was a female who had a right adrenal pheochromocytoma at the age of 50 years and a breast cancer at 49 years of age. Her mother had had a right adrenal pheochromocytoma at 61 years of age. Although other molecular mechanisms, such as particular variants in untranslated regions or partial gene deletions, cannot be ruled out, we think finding families with nonsyndromic pheochromocytoma without any RET, VHL, SDHB, SDHC, SDHD, or EGLN3 mutation may argue in favor of the presence of other pheochromocytoma susceptibility genes.

Molecular analysis of two uncharacterized sequence variants of the VHL gene

AbstractMutations in the VHL gene cause von Hippel-Lindau disease, a cancer predisposing syndrome characterized by a variety of benign and malignant neoplasms. We report the molecular characterization of two sequence variants of the VHL gene: a synonymous substitution c.462 A>C in exon 2 and a duplication of 11 bp in the promoter region (c.−65_−55dup11). The first variant is a pathogenic mutation because, although it does not change the sense of the affected codon, it causes skipping of exon 2 in the affected allele by altering the splicing consensus site at the 3′ end of exon 2. The 11 bp duplication represents a nonpathogenic variant. In fact, although it affects a critical region of the VHL promoter, it was found in healthy controls, and we show that carrier individuals express both VHL alleles at equimolar levels. Our data underline the importance of careful evaluation of the potential pathogenicity of sequence variants that may not belong to the obvious disease-causing mutation categories, or that affect relevant regulatory regions. mRNA analysis will be required to ultimately resolve this issue.

Connexin 26 preverbal hearing impairment: mutation prevalence and heterozygosity in a selected population.

The objective of this investigation was to determine the prevalence of Cx26 mutations in familial and sporadic cases of non-syndromic preverbal hearing impairment (HI). Molecular analysis of the Connexin 26 (Cx26/GJB2) gene was performed in 271 non-consanguineous individuals from the north of Italy, enrolled in the study because of the presence of non-syndromic preverbal sensorineural HI. One hundred and fortysix subjects (group 1) were referred from different ENT, paediatric and clinical genetic services, while 125 individuals (group 2) underwent Cx26 analysis based on precise anamnestic and clinical criteria for non-syndromic HI and low risk of acquired deficit. All of the cases were also classified as familial or sporadic due to the presence or absence of other documented childhood HI in the family. Of the total 271 individuals, 36.9% were positive for Cx26 mutations: 37 belonged to group 1 and 63 to group 2, which delineates a statistically significant difference between the two groups. The difference is mainly attributable to sporadically occurring cases. No significant differences between group 1 and group 2 were found regarding the prevalence of the common 35delG variant and the number of unidentified putative Cx26 alleles, although these latter were shown to be higher in sporadically occurring cases of the unselected group 1. The difference observed in Cx26 prevalence can be explained by the clinical selection of group 2, which ensures minimum risk of including cases of acquired HI. In particular, in cases of sporadically occurring HI, the use of a defined protocol increases the chances of a positive molecular result, improving genetic counselling and the possibility of establishing better genotype-phenotype correlation. Our data raise questions about the possible interpretation of Cx26 heterozygosity in a selected population of hearing-impaired individuals. Sumario El objetivo de esta investigatión fue determinar la prevalencia de las mutaciones de la Cx26 en casos familiares y esporádicos de impedimentos auditivos (HI) no sindrómicos preverbales. El análisis molecular del gen de la Conexina 26 (Cx26/GJB2) se llevó a cabo en 271 sujetos no consanguíneos del norte de Italia incluídos en el estudio por presentar un HI sensorineural no sindrómico. 146 sujetos (grupo 1) fueron referidos por servicios de ORL, de Pediatría o de Genética Clínica, mientras que 125 del grupo 2, siguieron el análisis de Cx26 con base en criterios anamnésicos y clínicos precisos para HI no sindrómicos y con bajo riesgo de deficit adquirido. Todos los casos fueron clasificados como familiares o esporádicos por la presencia o ausencia de otros HI infantiles documentados en la familia. Del total de 271 individuos, 36.9% fueron positivos para mutaciones de Cx26: 37 pertenecían al grupo 1 y 63 al grupo 2, lo que delínea una diferencia estadisticamente significativa entre los dos grupos. La diferencia es sobre todo atribuible a casos que ocurrieron esporadicamente. No hubo diferencias significativas entre ambos grupos en cuanto a la prevalencia de la variante 35delG y el numero de alelos supuestamente no identificados, a pesar de que estos últimos mostraron ser más en los casos que ocurrieron esporadicamente del grupo 1 no selecto. La diferencia observada en la prevalencia de la Cx26 puede explicarse por la selectión clínica del grupo 2, que asegura un minimo de riesgo de inclusion de casos de HI adquirido. En particular, en los casos de HI de ocurrencia esporadica, el uso de un protocolo definido aumenta las posibilidades de un resultado molecular positivo, con lo que mejora el consejo genetico y la posibilidad de establecer una mejor correlation genotipo-fenotipo. Nuestros datos plantean interrogantes sobre la posible interpretatión de la heterocigosidad de la Cx26 en una población seleccionada de individuos con impedimentos auditivos.

RETINAL ABNORMALITIES ASSOCIATED WITH A MUTATION OF THE NUCLEOTIDE 683 IN VON HIPPEL-LINDAU DISEASE

Abstract Background: von Hippel-Lindau disease (VHL) is a hereditary cancer syndrome in which affected individuals are at risk of developing tumors in multiple organs, including eyes, cerebellum, spinal cord, kidneys, inner ear, adrenal glands and pancreas. Methods: We performed a fundus examination and fluorescein and indocyanine green (ICG) angiography in both eyes of a young woman affected by VHL with bilateral pheochromocytoma, retinal angioma, retinal microaneurysms and unusual alterations of the deep retinal layers. A molecular analysis of the VHL gene was carried out. Results: Ophthalmoscopy disclosed in her right eye a small retinal hemangioma, some microaneurysms in both eyes at the posterior pole and multiple, small, whitish, dome-shaped lesions scattered in the retinal pigment epithelium (RPE) of the posterior retina. Fluorescein angiograms revealed in the early phase multiple hyperfluorescent spots that showed progressive discoloration in the late phase of angiography. Some of these spots were ophthalmoscopically undetectable. The late phase of ICG angiography showed some small hyperfluorescent points located at the level of the RPE, and some of them corresponded to the hyperfluorescent spots seen on fluorescein angiography. The molecular analysis revealed the presence of a „missense” mutation of the VHL gene at nucleotide 683. Conclusions: Alterations in the RPE have never been observed in the VHL syndrome. We describe an unusual case of VHL with a capillary hemangioma associated to diffuse alterations with the RPE of the posterior retina. The possibility exists that these lesions form part of the eye modifications in VHL.

Association between a combination of single nucleotide polymorphisms and large vessel cerebral vasculopathy in African children with sickle cell disease.

Sickle Cell Disease (SCD) exhibits a high phenotypic heterogeneity: the frequency and severity of clinical complications can be extremely diverse leading to variable risks of disability or death [1]. One of the major complications of SCD is represented by acute cerebrovascular events: 11% of children presents overt stroke in childhood if unscreened and untreated and 30–40% have silent cerebral infarcts [2]. Currently, Transcranial Doppler (TCD) is the gold standard to identify patients at high risk of stroke offering them the opportunity for primary prevention through a chronic transfusion program [2]. Despite the high sensitivity, TCD has low specificity with the risk of over treating some patients. Hence the need for new prognostic tools that can be used together with TCD to enable the identification of individuals at the highest risk whowould really benefit from blood transfusion therapy. Moreover, although screening with Magnetic resonance Imaging (MRI) and Angiography (MRA) is recommended for early diagnosis of silent infarcts and recognition of large vessel stenosis, MRI/MRA are not useful to identify patients at risk of developing silent infarcts or large vessel stenosis. A tool that is able to stratify patients at risk for silent infarcts is not yet available. The phenotypic heterogeneity of SCD has been attributed to various factors, and several candidate genetic polymorphisms have been proposed to affect the risk of stroke and of cerebral vasculopathy, but the studies conducted so far have yielded conflicting results [3,4]. Flanagan et al. [3], conducted a study on a large cohort of SCA pediatric patients (n.677). Patients were mainly African Americans and the authors reported the association of GOLGB1 Y1212C (rs3732410) and ENPP1 K173Q (rs1044498) mutations with a decreased risk of clinically overt stroke, whereas PON1 Q192R (rs662) was associated with an increased risk of stroke. In the same paper GOLGB1was linked to a lower frequency of silent infarcts and to reduced TCD velocities. Belisario et al. [4], on the contrary, in their Brazilian cohort (n.395) reported only the association of ENPP1 Q173 with an increased risk of stroke and the development of high-risk TCD velocities. These apparently opposite results in populations of different ethnic background raise the need for further studies assessing the role of Single Nucleotide Polymorphisms (SNPs) in influencing not only the risk of stroke itself but of cerebral vasculopathy overall in SCD. In fact, recent evidence suggests that cerebral vasculopathy and stroke risk in different populations could vary according to the polymorphism in several genes [5–8]. We report on the association between genetic variants GOLGB1 Y1212C, ENPP1 K173Q and PON1 Q192R and cerebrovascular complications in our cohort of children with SCD, attending the Sickle