Lucia Pedace

Longitudinal hormonal evaluation in a patient with disorder of sexual development, 46,XY karyotype and one NR5A1 mutation

Steroidogenic factor 1 (encoded by the NR5A1 gene) is a critical regulator of reproduction, controlling transcription of key genes involved in sexual dimorphism. To date, NR5A1 variants have been found in individuals with a 46,XY karyotype and gonadal dysgenesis, as well as with a wide spectrum of genital anomalies and, in some patients, with adrenal insufficiency. We describe evolution of gonadal function, from the neonatal period to puberty, in a patient with a 46,XY karyotype, a disorder of sexual development, and a mutation (c.691_699dupCTGCAGCTG) in the NR5A1 gene. The patient, ascertained at birth due to ambiguous genitalia, showed normal values of plasma testosterone in the late neonatal period. Evaluation of the hormonal profile over time indicated severe tubular testicular hypofunction suggestive for a 46,XY disorder of gonadal development. A comprehensive review of published reports of 46,XY and disordered sexual development related to the NR5A1 gene confirmed the clinical and hormonal variability in patients with NR5A1 mutations. Analysis of multiple data allowed us to define the most common features associated with NR5A1 mutations. We further confirmed the indication to perform NR5A1 screening in patients with 46,XY karyotype and disordered sexual development even when Müllerian structures appear to be absent and plasma testosterone levels are within the normal range for age.

Analysis of the miR-34a locus in 62 patients with familial cutaneous melanoma negative for CDKN2A/CDK4 screening

MicroRNAs are small non-coding RNAs, which inhibit expression of specific target genes at the post-transcriptional level and are often misregulated in human cancer. Among them, miR-34a is considered a tumor suppressor with a hypothetical role in melanoma tumorigenesis. In this work, 62 Italian index patients with familial melanoma and negative for CDKN2A/CDK4 screening were investigated for miR-34a germline mutations. Eight novel miR-34a sequence variants were identified at both the heterozygous (c.+259G>A, c.+424G>A, c.+1465C>T, c.+1769C>T, c.+2456T>G, c.+2603C>T, c.+2972T>A, c.+3069T>C) and homozygous (c.+424G>A, c.+1465C>T, c.+1769C>T) states. Molecular screening identified all nucleotide changes in a healthy population of 150 controls and demonstrated that they are common polymorphisms. However, statistically significant differences of allele and genotype frequencies were detected for c.+1465C>T and c.+1769C>T, and borderline values for c.+2456T>G. By stratifying patients by relevant clinical features (presence/absence of multiple primary melanoma, Breslow’s thickness, phototype and number of nevi), no significant findings were noted except for an association between the c.+424G>A (heterozygous individual GA) and multiple primary melanoma and phototype III–IV. Our preliminary study suggests that miR-34a, although having a role in late tumorigenesis, does not contribute to the inherited susceptibility to cutaneous melanoma. A function as phenotypic modulator in familial melanoma cannot be excluded.

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

Molecular characterization of 11 Italian patients with Darier Disease

Darier disease (DD) is an autosomal dominant genodermatosis characterized by multiple warty papules coalescing in seborrheic areas and specific histological skin changes. Heterozygous mutations in ATP2A2, encoding the sarco-endoplasmic reticulum calcium pumping ATPase type 2, are identified as the molecular basis of DD. In this study, molecular features in a large cohort of Italian patients are reported. Molecular data were collected along with the main clinical features. Genomic DNA was used for direct sequencing of ATP2A2. The effect of selected mutations was predicted by in silico analysis or investigated by gene expression studies. 10 different ATP2A2 mutations were identified. Three mutations (c.2300A>G, c.2794G>A, c.569delAins34) have been previously described, while 7, including 2 missense (c.545G>A and c.2116G>A), 2 nonsense (c.1372G>T and c.1675C>T), 1 small deletion (c.142delA), 1 duplication (c.2935_2949dup15) and 1 splice-site mutation (c.2742-1G>A), were novel. Collected data added new variants to the ATP2A2 repertoire and confirmed that ATP2A2 mutations are scattered over the entire gene and, in most cases, private.

Fontaine–Farriaux syndrome: A recognizable craniosynostosis syndrome with nail, skeletal, abdominal, and central nervous system anomalies

Craniosynostosis is an etiologically heterogeneous malformation, which may present as an isolated finding or in association with other anomalies. The concurrence of craniosynostosis together with specific central nervous system, abdominal, genital, and limb malformations defines the Fontaine–Farriaux syndrome, described so far in only two patients. We report on a stillborn who mainly presented severe intrauterine growth retardation, bilateral coronal synostosis, generalized nail hypo/aplasia more evident on the posterior side, tapered digits, mild cutaneous syndactyly, abdominal muscle hypoplasia, micropenis and bilateral cryptorchidism. Skeletal radiographs revealed universal platyspondyly and necropsy findings comprised intestinal malrotation, abnormal cortical gyral formation, periventricular heterotopia, and cerebellar hypoplasia. Comparison between the present and the two previously described patients demonstrates that our case shows a combination of features strikingly resembling the original description. Conversely, the second reported patient shows a very atypical phenotype and is, most probably, affected by a distinct clinical entity. The triad of craniosynostosis, anonychia, and abdominal muscle hypo/aplasia emerges as the most consistent core phenotype, although skeletal and brain anomalies are relevant ancillary findings. An in‐depth differential diagnosis with other partially overlapping conditions is carried out.

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.

Identification of a novel duplication in the APC gene using multiple ligation probe amplification in a patient with familial adenomatous polyposis

Germline mutations in the adenomatous polyposis coli (APC) gene cause familial adenomatous polyposis (FAP), an autosomal dominant disease characterized by hundreds to thousands of adenomatous polyps in the colon and rectum, with progression to colorectal cancer. The majority of APC mutations are nucleotide substitutions and frameshift mutations that result in truncated proteins. Recently, large genomic alterations of the APC gene have been reported in FAP. DNA from 15 FAP patients, in whom no APC germline mutations were detected with denaturing high performance liquid chromatography, was analyzed with multiplex ligation-dependent probe amplification (MLPA) to evaluate gross genomic alterations in the APC gene. In one case, MLPA identified a novel duplication of exons 2-6 in one copy of the APC gene. Reverse transcriptase-polymerase chain reaction revealed that the mutant allele contained an in-frame multiexon duplication including 18 nucleotides located in exon 2, upstream of the ATG initiation codon. The presence of a premature stop codon in the duplicated sequence leads to the synthesis of a truncated APC polypeptide. These findings highlight the utility of evaluating infrequent APC mutation events in FAP patients using MLPA.

Jejunal atresia and anterior chamber anomalies: Further delineation of the Strømme syndrome.

Strømme syndrome is a rare multiple congenital malformation syndrome consisting in apple peel intestinal atresia, ocular anomalies, microcephaly and developmental delay. To date, this condition was described in a couple of sibs and 7 additional sporadic patients. We report on a 11-month-old female, who requested surgical correction for jejunal atresia shortly after birth and also presented with megalocornea and persistence of the pupillary membrane. Microcephaly and developmental delay were absent at last examination. An oligonucleotide CGH-array analysis excluded cryptic chromosome rearrangement(s). Comparison of the previously published and present patients added some details on the natural history of Strømme syndrome. Delivery is usually performed preterm possibly due to polyhydramnios. Birth parameters, especially head circumference, are commonly at the lower end of the normal range. Microcephaly is more frequently but not constantly observed in older individuals, thus suggesting a progressive course, and may relate to an underlying neuronal migration defect. Jejunal atresia has an apple peel appearance in most but not all patients and its post-surgical course is usually uneventful. The ocular phenotype comprises a wide range of anterior chamber anomalies with sclerocornea/corneal leukoma being the most common.

A novel germline mutation in CDK4 codon 24 associated to familial melanoma: Letter to the Editor

FIGURE 1 A, Family pedigree: segregation of CDK4 p.Arg24Leu mutation (*). B, CDK4 c.71G>T,p.Arg24Leu mutation. C, Homology model of the wild-type CDK4 (orange) in complex with p16(INK4)a (green). The CDK4 Arg24 residue (spheres) engages intermolecular interactions with the binding surface of the inhibitors. (D) to (G) Arg24 (orange) establishes salt bridges with 2 conserved aspartate residues of (D) p16(INK4)a, (E) p15(INK4)b, (F) p18(INK4)c and (G) p19(INK4)d (green). These interactions are lost in the mutant complexes (white). H, Predicted Gibbs free energy of binding variations (ΔΔG, in kcal/mol) between CDK4 and its partners upon mutations. ΔΔG values indicate decrease affinity Received: 29 June 2017 Revised: 21 August 2017 Accepted: 23 August 2017