Francesco Binni

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.

Sirenomelia and VACTERL Association in the Offspring of a Woman With Diabetes

Sirenomelia and VACTERL association are defects of blastogenesis of unknown cause. Although they appear clinically distinct, some epidemiological and experimental studies suggest a common pathogenetic mechanism. We report on the reproductive history of a 28‐year‐old obese, diabetic mother who had three pregnancies. The first resulted in the birth of a sirenomelic child, the second in a miscarriage, while the third was terminated for fetal malformations, diagnosed post‐mortem as VACTERL association. This observation supports the relationship between sirenomelia and VACTERL, which probably represent the two ends of the same phenotypic spectrum. Their occurrence in the same sibship also indicates a possible common cause. The coexistence with maternal diabetes seems more than a chance occurrence and the constellation of malformations observed in the present family may be explained as the pleiotropic effect of the same teratogenic agent interacting with genetic predisposition to diabetes and/or obesity.

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.

TFR2-related hereditary hemochromatosis as a frequent cause of primary iron overload in patients from Central-Southern Italy

OBJECTIVE Hereditary hemochromatosis (HH) is a common Mendelian disorder of iron metabolism. Eighty percent of northern Europeans descendant HH patients carry the same mutation (p.C282Y) in the HFE gene. Simultaneously, due to a founder effect, its frequency varies considerably between different populations. In Central-Southern Italy the prevalence of p.C282Y mutation is low and in several patients the disease has different causes. Four additional rarer forms have been described. Type 3 HH has been reported in about 50 families and no more than 30 TFR2 pathogenic mutations have been globally identified. The aim of this study is to assess the TFR2 role in non-HFE HH pathogenesis. STUDY DESIGN AND SETTING TFR2 sequence analysis was performed on 45 Italian patients without HFE mutations. RESULTS This study revealed TFR2 biallelic pathogenic mutations in 7/45 (15.6%) individuals. Moreover monoallelic TFR2 deleterious defects (18%) or polymorphisms with unclear meaning (36%) were identified. Besides, we recognized 10 novel variants and 9 described changes. CONCLUSION We believe this to be the largest series of type 3 HH patients described so far. Present findings support the hypothesis of a main role of the TFR2 gene in HH pathogenesis in those regions, such as Central-Southern Italy, where the p.C282Y frequency is low.

AXIN2 germline mutations are rare in familial melanoma

In a recent study published in ‘‘Genes Chromosomes and Cancer’’ (Castiglia et al., 2008), one germline Axis inhibition protein 2 (AXIN2) mutation was first described in a sporadic melanoma patient with healthy and noncarrier daughters. This preliminary result made us hypothesize that deregulation of the Wnt/ß-catenin signaling pathway could promote melanoma development and/ or progression and that AXIN2 mutations could be involved in familial melanoma (FAM) susceptibility. AXIN2 is a negative regulator gene of Wnt/ß-catenin signaling, and it is a putative tumor suppressor gene in chromosome band 17q24. The gene product is a scaffold protein that takes part in a multiprotein complex that facilitates ßcatenin phosphorylation and destruction (Kishida et al., 1998; Ikeda et al., 2000). Hence, a role of AXIN2 in human cancer pathogenesis could be suspected. However, available data do not provide a clear picture and the possible role of AXIN2 somatic/germline allelic variants in disease development and/or progression is still largely unknown. Somatic heterozygous mutations in AXIN2 have been described in human cancer including endometrioid adenocarcinoma (Wu et al., 2001), hepatocellular carcinoma (Taniguchi et al., 2002), gastric carcinoma (Kim et al., 2009), and medulloblastoma (Koch et al., 2007). AXIN2 germline truncating mutations were found in patients with syndromic tooth agenesis and colorectal cancer (Lammi et al., 2004). Specific AXIN2 single nucleotide polymorphisms have been reported to correlate with isolated tooth agenesis (Callahan et al., 2009) and to be associated with lung cancer in Japanese and Turkish patients (Kanzaki et al., 2006; Emine et al., 2009). However, no AXIN2 germline mutation was found in non-syndromic colorectal cancer (Peterlongo et al., 2005) and no association was demonstrated with multiple polyposis (Lejeune et al., 2006). However, with the exception of the work by Castiglia et al. (2008), no further study has investigated the role of AXIN2 in human melanoma. In this study, a cohort of 84 Italian FAM patients, 22 of whom with multiple primary melanoma (MPM), were recruited from the Oncological Dermatology Service at the IFO-IRCCS Hospital in Rome (Italy). Former molecular analyses of CDKN2A and CDK4 genes have excluded the presence of pathogenic mutations in all these patients (Majore et al., 2008; Binni et al., 2010). After having obtained informed consent, the entire coding region and splice site junctions of AXIN2 were amplified from the constitutional DNA using 11 primer pairs that anneal on flanking introns, as previously described (Peterlongo et al., 2005). PCR products were purified using Biomek 3000 (Beckman Coulter, Fullerton, CA) according to the manufacturer’s protocol, and sequenced in both directions using the 3130xl Genetic Analyzer (Applied Biosystem). Genomic DNAs from 150 unrelated ethnically matched healthy individuals (corresponding to 300 control chromosomes) were screened for the presence of each identified sequence variation to assess whether it was a real mutation or a polymorphism. Consequences of sequence variations on primary and secondary protein structure were investigated using POLYPHEN (http://genetics. bwh.harvard.edu/pph/), SIFT (blocks.fhcrc.org/ sift/SIFT.html), NetSurfP (Petersen et al., 2009) and Hierarchical Neural Network (http://www. expasy.ch/tools/#second ary) in silico predictors. Putative splice-site mutation effects were analyzed by using the splice site prediction program NNSPLICE ver.0.9 (http://www.fruitfly.org/seq_ tools/splice. html) and ESEFINDER 3.0 (rulai. cshl.edu/tools/ESE/). Molecular analysis disclosed six distinct heterozygous AXIN2 variations in FAM subjects without MPM and with an early age at onset (16–34 years). Two of these, c.1201-84delG and c.1489T >A (p.C497S), were novel heterozygous polymorphic variants, being detected in healthy controls. Moreover, no difference in allele frequencies between the patient group and healthy subjects

A novel heterozygous SOX2 mutation causing anophthalmia/microphthalmia with genital anomalies

Anophthalmia/microphthalmia is a rare developmental craniofacial defect, which recognizes a wide range of causes, including chromosomal abnormalities, single-gene mutations as well as environmental factors. Heterozygous mutations in the SOX2 gene are the most common monogenic form of anophthalmia/microphthalmia, as they are reported in up to 10-15% cases. Here, we describe a sporadic patient showing bilateral anophthalmia/microphthalmia and micropenis caused by a novel mutation (c.59_60insGG) in the SOX2 gene. Morphological and endocrinological evaluations excluded any anomaly of the hypothalamus-pituitary axis. Our finding supports the hypothesis that SOX2 is particularly prone to slipped-strand mispairing, which results in a high frequency of point deletions/insertions.

Type 3 hereditary hemochromatosis in a patient from sub-Saharan Africa: Is there a link between African iron overload and TFR2 dysfunction?

Hereditary hemochromatosis (HHC) is a common genetic trait that increases the risk of developing systemic iron overload. The disorder is caused bymutations in genes coding various proteins involved in the regulation of iron access into the blood. In HHC, excessive iron storage may result in progressivemultiorgandamage,which can be treatedwith phlebotomy. HHC iswell defined inNorthern Europe. In this population,most affected individuals share the HFE recessive p.C282Y mutation, which is the most common genetic cause for classic (type 1) HHC [1]. It is presumed that p.C282Y appeared for the first time in the Celtic or Viking populationmore than 2000 years ago and followed their migrations. Accordingly, p.C282Y is less frequent in Southern Europeans, Native Americans, American of African descent, Pacific Islanders, and Asians, and nearly absent in Southern Africans [2]. Mutations in 4 additional genes (namely HJV, HAMP, TFR2 and SLC40A1) account for different, rarer forms of HHC (type 2A, type 2B, type 3, type 4), and can be found also in patients outside Northern Europe. Nevertheless, taken together, the 5 HHC-related genes do not explain the entire spectrum of iron overload in humans, especially in Southern Europe and in non-Caucasians [1,3]. In fact, iron overload is a complex phenotype resulting from the contribution of a wide range of factors, genetic and environmental, that can operate synergistically in the same individual. Accordingly, in sub-Saharan Africa, where iron overload has the highest prevalence, it is thought to be exacerbated by chronic excessive intake of dietary iron (African iron overload; AIO). AIO is a condition characterized by hepatic and probably, systemic iron overload, and is particularly common in rural areas. It is a frequent cause of cirrhosis and hepatocellular carcinoma and confers susceptibility to tuberculosis and other infections. Whether a genetic factor contributes or not to AIO is still a matter of debate [4,5]. We report a sub-Saharan African man with severe HHC carrying two novel TFR2mutations. To the best of our knowledge, this is the first case of molecularly characterized HHC in sub-Saharan Africans. The patient was a 49-year-old African originating from Burundi and referring to us for biochemical evidence of severe iron overload. He denied alcohol consumption and had normal dietary habits. At 46 years of age, the patient was hospitalized for malaria infection. Due to persistence of chronic fatigue and hypersomnia, he underwent further examinations revealing liver enlargement and steatosis at ultrasound, hyperferritinemia (3.516 μg/L), highly increased transferrin saturation (>100%) and microcytemia without other hematologic disorders. Liver cytonecrosis indexes were slightly increased (Table 1). Viral hepatitis was excluded by serum testing and a diagnosis of HHC was made on clinical grounds. In order to confirm the diagnosis, liver biopsy was proposed and, than, refused by the patient. Liver iron accumulation was measured by the superconducting quantum interference device (SQUID) method, underlining severe hepatic overload (estimated hepatic iron concentration: 16.8 mg/g dry weight; normal values≤3 mg/g). Measurement of liver

Monozygotic Twin Discordance for Phacomatosis Cesioflammea Further Supports the Post-Zygotic Mutation Hypothesis

Phacomatosis pigmentovascularis (PPV) is a group of sporadic skin disorders combining widespread cutaneous capillary malformations and pigmented nevi. Etiopathogenesis of the various forms of PPV is unknown, although a non‐allelic twin spotting has been proposed as the most likely underlying mechanism. We report on the second instance of monozygotic twin discordance for PPV. Identical twins were observed shortly after birth, one affected by PPV and the other healthy, except for a standard Mongolian spot. Membrane examination was compatible with a monochorionic diamniotic pregnancy, and microsatellite analysis demonstrated monozygosity. This report confirms that PPV likely originates from a post‐zygotic mutation rising shortly after conception and affecting different cell lineages. Speculations about mechanisms linked to phenotypic discrepancies among identical twins were also put forward.

Early Ultrasound Suspect of Thanatophoric Dysplasia Followed by First Trimester Molecular Diagnosis

Early Ultrasound Suspect of Thanatophoric Dysplasia Followed by First Trimester Molecular Diagnosis Antonella Giancotti,* Marco Castori,* Antonella Spagnuolo, Francesco Binni, Valentina D’Ambrosio, Gaia Pasquali, Antonio Pizzuti, and Paola Grammatico Department of Obstetrics and Gynecology, Sapienza University, Policlinico Umberto I Hospital, Rome, Italy Medical Genetics, Department of Molecular Medicine, Sapienza University, San Camillo-Forlanini Hospital, Rome, Italy Medical Genetics, Department of Experimental Medicine, Sapienza University, Policlinico Umberto I Hospital, Rome, Italy