Silvia Majore

NF1 Gene Mutations Represent the Major Molecular Event Underlying Neurofibromatosis-Noonan Syndrome

Neurofibromatosis type 1 (NF1) demonstrates phenotypic overlap with Noonan syndrome (NS) in some patients, which results in the so-called neurofibromatosis-Noonan syndrome (NFNS). From a genetic point of view, NFNS is a poorly understood condition, and controversy remains as to whether it represents a variable manifestation of either NF1 or NS or is a distinct clinical entity. To answer this question, we screened a cohort with clinically well-characterized NFNS for mutations in the entire coding sequence of the NF1 and PTPN11 genes. Heterozygous NF1 defects were identified in 16 of the 17 unrelated subjects included in the study, which provides evidence that mutations in NF1 represent the major molecular event underlying this condition. Lesions included nonsense mutations, out-of-frame deletions, missense changes, small inframe deletions, and one large multiexon deletion. Remarkably, a high prevalence of inframe defects affecting exons 24 and 25, which encode a portion of the GAP-related domain of the protein, was observed. On the other hand, no defect in PTPN11 was observed, and no lesion affecting exons 11-27 of the NF1 gene was identified in 100 PTPN11 mutation-negative subjects with NS, which provides further evidence that NFNS and NS are genetically distinct disorders. These results support the view that NFNS represents a variant of NF1 and is caused by mutations of the NF1 gene, some of which have been demonstrated to cause classic NF1 in other individuals.

Mutation analysis of the NSD1 gene in a group of 59 patients with congenital overgrowth

Sotos syndrome is characterized by pre‐ and post‐natal overgrowth, typical craniofacial features, advanced bone age, and developmental delay. Some degree of phenotypic overlap exists with other overgrowth syndromes, in particular with Weaver syndrome. Sotos syndrome is caused by haploinsufficiency of the NSD1 (nuclear receptor SET domain containing gene 1) gene. Microdeletions involving the gene are the major cause of the syndrome in Japanese patients, whereas intragenic mutations are more frequent in nonJapanese patients. NSD1 aberrations have also been described in some patients diagnosed as Weaver syndrome. Some authors have suggested a certain degree of genotype–phenotype correlation, with a milder degree of overgrowth, a more severe mental retardation, and a higher frequency of congenital anomalies in microdeleted patients. Data on larger series are needed to confirm this suggestion. We report here on microdeletion and mutation analysis of NSD1 in 59 patients with congenital overgrowth. Fourteen novel mutations, two previously described and one microdeletion were identified. All patients with a NSD1 mutation had been clinically classified as “classical Sotos,” although their phenotype analysis demonstrated that some major criteria, such as overgrowth and macrocephaly, could be absent. All patients with confirmed mutations shared the typical Sotos facial gestalt. A high frequency of congenital heart defects was present in patients with intragenic mutations, supporting the relevance of the NSD1 gene in the pathogenesis of this particular defect.

Human RSPO1/R-spondin1 Is Expressed during Early Ovary Development and Augments β-Catenin Signaling

Human testis development starts from around 42 days post conception with a transient wave of SRY expression followed by up-regulation of testis specific genes and a distinct set of morphological, paracrine and endocrine events. Although anatomical changes in the ovary are less marked, a distinct sub-set of ovary specific genes are also expressed during this time. The furin-domain containing peptide R-spondin1 (RSPO1) has recently emerged as an important regulator of ovary development through up-regulation of the WNT/β-catenin pathway to oppose testis formation. Here, we show that RSPO1 is upregulated in the ovary but not in the testis during critical early stages of gonad development in humans (between 6–9 weeks post conception), whereas the expression of the related genes WNT4 and CTNNB1 (encoding β catenin) is not significantly different between these tissues. Furthermore, reduced R-spondin1 function in the ovotestis of an individual (46,XX) with a RSPO1 mutation leads to reduced β-catenin protein and WNT4 mRNA levels, consistent with down regulation of ovarian pathways. Transfection of wild-type RSPO1 cDNA resulted in weak dose-dependent activation of a β-catenin responsive TOPFLASH reporter (1.8 fold maximum), whereas co-transfection of CTNNB1 (encoding β-catenin) with RSPO1 resulted in dose-dependent synergistic augmentation of this reporter (approximately 10 fold). Furthermore, R-spondin1 showed strong nuclear localization in several different cell lines. Taken together, these data show that R-spondin1 is upregulated during critical stages of early human ovary development and may function as a tissue-specific amplifier of β-catenin signaling to oppose testis determination.

Spectrum of mutations in the renin-angiotensin system genes in autosomal recessive renal tubular dysgenesis

Autosomal recessive renal tubular dysgenesis (RTD) is a severe disorder of renal tubular development characterized by early onset and persistent fetal anuria leading to oligohydramnios and the Potter sequence, associated with skull ossification defects. Early death occurs in most cases from anuria, pulmonary hypoplasia, and refractory arterial hypotension. The disease is linked to mutations in the genes encoding several components of the renin–angiotensin system (RAS): AGT (angiotensinogen), REN (renin), ACE (angiotensin‐converting enzyme), and AGTR1 (angiotensin II receptor type 1). Here, we review the series of 54 distinct mutations identified in 48 unrelated families. Most of them are novel and ACE mutations are the most frequent, observed in two‐thirds of families (64.6%). The severity of the clinical course was similar whatever the mutated gene, which underlines the importance of a functional RAS in the maintenance of blood pressure and renal blood flow during the life of a human fetus. Renal hypoperfusion, whether genetic or secondary to a variety of diseases, precludes the normal development/ differentiation of proximal tubules. The identification of the disease on the basis of precise clinical and histological analyses and the characterization of the genetic defects allow genetic counseling and early prenatal diagnosis. Hum Mutat 33:316–326, 2012.

Screening of mutations in the CFTR gene in 1195 couples entering assisted reproduction technique programs

Genetic testing of the cystic fibrosis transmembrane conductance (CFTR) gene is currently performed in couples undergoing assisted reproduction techniques (ART), because of the high prevalence of healthy carriers in the population and the pathogenic relationship with congenital bilateral absence of vas deferens (CBAVD). However, discordant data have been reported concerning the usefulness of this genetic test in couples with no family history of cystic fibrosis (CF). In this study, we report the results of CFTR molecular screening in 1195 couples entering ART. Genetic testing was initially carried out in a single partner of each couple. CFTR mutations were detected in 55 subjects (4.6%), a percentage that overlaps with the one reported in the general population. However, significantly higher frequencies of were found in CBAVD individuals (37.5%) and in males with nonobstructive azoospermia (6.6%). The 5T allele was found in 78 patients (6.5%). This figure was again significantly different in males with nonobstructive-azoospermia (9.9%) and in those with CBAVD (100%). All together, 139 subjects (11.6%) had either a CFTR mutation or the 5T allele. Subsequent molecular analysis of their partners disclosed a CFTR mutation or 5T allele in nine cases (6.5%). However, none of these couples had CFTR alterations in both members, a CFTR mutation being invariably present in one partner and the 5T allele in the other. In order to improve genetic counselling of these couples, the TG-M470V-5T association was analyzed, and a statistically significant relationship between 12TG-V470 and CBAVD was detected.

6q terminal deletion syndrome associated with a distinctive EEG and clinical pattern: A report of five cases

Summary:  Purpose: Mental retardation, facial dysmorphisms, and neurologic and brain abnormalities are features of 6q terminal deletions. Epilepsy is frequently associated with this chromosome abnormality, but electroclinical findings are not well delineated. We report five unrelated patients with 6q terminal deletions and a peculiar clinical, EEG, and neuroradiologic picture of epilepsy, mental retardation, and colpocephaly.

A novel missense mutation in SLC40A1 results in resistance to hepcidin and confirms the existence of two ferroportin-associated iron overload diseases

Ferroportin‐related iron overload disease differs from haemochromatosis in that it has a dominant mode of inheritance and is usually associated with macrophage iron sequestration. However, it is thought that mutations with opposite effects on protein functions, i.e. loss‐of‐function versus gain‐of‐function mutations, are responsible for variable phenotype presentations. The present study investigated the functional relevance of a novel ferroportin variant: the c.1502 A>G transition, which changes amino acid 501 from tyrosine to cysteine (p.Y501C). This novel variant was identified in a pedigree originating from Central Italy and, although an intra‐familial phenotype heterogeneity was observed, it co‐segregated with an iron overload picture similar to that of the HFE‐related typical haemochromatosis. In cultured cells, the p.Y501C mutant protein reached the plasma membrane and retained a full iron export ability. By contrast, it was resistant to inhibition by hepcidin. These findings confirm that certain ferroportin mutations compromise the activity of hepcidin in iron homeostasis, mimicking hepcidin deficiency as described in all types of hemochromatosis.

Association of hepcidin promoter c.-582 A>G variant and iron overload in thalassemia major

The liver peptide hepcidin is the key regulator of iron homeostasis. This report shows that liver iron content and serum ferritin levels are greater in thalassemia patients with the −582G polymorphic change in the hepcidin promoter as compared with patients with the wild-type (A) sequence. This observation suggests that this change might influence the iron status of irregularly chelated thalassemic patients. See related perspective article on page 1185. Hepcidin is a 25-amino acid peptide, derived from cleavage of an 84 amino acid pro-peptide produced predominantly by hepatocytes. This molecule, encoded by the hepcidin antimicrobial peptide (HAMP) gene shows structural and functional properties consistent with a role in innate immunity. Moreover, as demonstrated in mice and humans, hepcidin is a major regulator of iron metabolism, and acts by binding to ferroportin and controlling its concentration and trafficking. In this study we investigated the influence that mutations in HAMP and/or hemocromatosis (HFE) genes might exert on iron metabolism in a group of poly-transfused thalassemic patients in preparation for bone marrow transplantation. Our results showed that the presence of the c.-582 A>G polymorphism (rs10421768) placed in HAMP promoter (HAMP-P) might play a role in iron metabolism, perhaps varying the transcriptional activation that occurs through E-boxes located within the promoter.

CDKN2A/CDK4 molecular study on 155 Italian subjects with familial and/or primary multiple melanoma.

Dear Sir, During recent decades the incidence of cutaneous malignant melanoma has increased dramatically worldwide. At the same time, the survival rate has shown a remarkable improvement, mainly due to a higher proportion of cases presenting with early-stage lesions. Between 5% and 10% of melanoma occurs in a familial setting and to date only two high-penetrance loci responsible for melanoma susceptibility have been identified, CDKN2A and CDK4. CDKN2A is the major locus for melanoma predisposition, although alterations have been detected in only 20–40% of melanoma-prone families (Goldstein et al., 2006; Newton-Bishop et al., 1999). CDKN2A encodes two different proteins, p16INK4A and p14ARF, both tumor suppressors. The p16INK4A protein inhibits the cyclin D1-cyclin-dependent kinase 4 (CDK4) complex, which controls cell cycle progression by phosphorylating the retinoblastoma protein; while p14ARF is involved in the cell-cycle regulation by binding to MDM2, thereby inhibiting p53 degradation. Mutations of CDK4 seem to be a rare cause of melanoma susceptibility (Goldstein et al., 2002) and to date only seven families: two American, two French, one Norwegian, one Australian and one English have been described (Molven et al., 2005; Soufir et al., 1998, 2007; Zuo et al.,1996). All mutations found so far affect codon 24 with arginine substituted by histidine or cysteine. This aminoacid exchange is critical because it abrogates p16INKA-CDK4 binding, essential for CDK4 inactivation. The aim of our work was to assess the prevalence of CDKN2A and CDK4 mutations in a sample of Italian melanoma-prone families and cases with multiple primary melanoma (MPM). We identified 15 ⁄ 155 (9.6%) patients with CDKN2A mutations: 4 ⁄ 76 (5.2%) affected by familial melanoma, 8 ⁄ 18 (44.4%) affected by familial and MPM, and 3 ⁄ 61 (4.9%) with sporadic MPM (Table 1). The most common mutation was G101W, a documented founder mutation in Italy (Mantelli et al., 2002). CDK4 exon 2 was analyzed in 140 subjects affected by melanoma (73 from pedigrees with familial melanoma, 54 with MPM, 13 with both familial and MPM), negative for CDKN2A mutations, using the following PCR primers: CDK4-2F 5¢-TGATTGTAGGGTCTCCCTTGA and CDK4-2R 5¢-CAACCCTCCACCACCTTCT (GenBank: U37022). Haplotype analysis was performed, as reported by Molven et al. (2005), by analysing three microsatellite markers upstream (D12S1700, D12S305; CDK4M4) and three markers downstream (CDK4M1, D12S1691, D12S1632) the CDK4 region, as well as three intragenic SNPs (rs2270777, rs2069502, rs2069506) and one SNP in the promoter region (rs2072052). Details of primers for SNPs and microsatellite analysis are available on request. We found one subject with a CDK4 R24H substitution. This 70-year-old male, with skin phototype IV, presented with an in situ cutaneous melanoma of the shoulder at the age of 59 years. After eleven years follow-up he is free from relapse. His 68-year-old sister was affected by melanoma of the foot (SSM of the instep) at the age of 53 years. His son developed two primary melanomas, firstly, an in situ melanoma of the leg (at age 35 years) and, subsequently, (at age of 38 years) a superficial spreading melanoma (SSM) of the thorax (0.5 mm Breslow thickness, Clark level III). The proband’s daughter, skin phototype III, presented with a SSM of the right leg (0.6 mm Breslow thickness, Clark level IV), at the age of 38 years. The healthy grand-daughter carried the CDK4 R24H mutation while the grandson was wildtype. The proband’s father was reported to have died at the age of 42 years as a consequence of liver cancer (histological result was not available). The mother was negative for CDK4 mutation, thus supporting paternal segregation of the R24H variant. The average age of onset of melanoma in this family was 46 years with a difference between the first and the second generation of >15 years (59 for the first generation and 38 for the second). Our results confirm the role of the CDK4 gene in melanoma susceptibility despite the low frequency

Novel and recurrent p14ARF mutations in Italian familial melanoma

Binni F, Antigoni I, De Simone P, Majore S, Silipo V, Crisi A, Amantea A, Pacchiarini D, Castori M, De Bernardo C, Catricalà C, Grammatico P. Novel and recurrent p14ARF mutations in Italian familial melanoma.