Noonan, LEOPARD and cardiofaciocutaneous syndromes (NS, LS and CFCS) are developmental disorders with overlapping features including distinctive facial dysmorphia, reduced growth, cardiac defects, skeletal and ectodermal anomalies, and variable cognitive deficits. Dysregulated RAS-mitogen-activated protein kinase (MAPK) signal traffic has been established to represent the molecular pathogenic cause underlying these conditions. To investigate the phenotypic spectrum and molecular diversity of germline mutations affecting BRAF, which encodes a serine/threonine kinase functioning as a RAS effector frequently mutated in CFCS, subjects with a diagnosis of NS (N= 270), LS (N= 6) and CFCS (N= 33), and no mutation in PTPN11, SOS1, KRAS, RAF1, MEK1 or MEK2, were screened for the entire coding sequence of the gene. Besides the expected high prevalence of mutations observed among CFCS patients (52%), a de novo heterozygous missense change was identified in one subject with LS (17%) and 5 individuals with NS (1.9%). Mutations mapped to multiple protein domains and largely did not overlap with cancer-associated defects. NS-causing mutations had not been documented in CFCS, suggesting that the phenotypes arising from germline BRAF defects might be allele specific. Selected mutant BRAF proteins promoted variable gain of function of the kinase, but appeared less activating compared than the recurrent cancer-associated p.Val600Glu mutant. Our findings provide evidence for a wide phenotypic diversity associated with mutations affecting BRAF, and occurrence of a clinical continuum associated with these molecular lesions.

Noonan syndrome, a developmental disorder characterized by congenital heart defects, reduced growth, facial dysmorphism and variable cognitive deficits, is caused by constitutional dysregulation of the RAS-MAPK signaling pathway. Here we report that germline NRAS mutations conferring enhanced stimulus-dependent MAPK activation account for some cases of this disorder. These findings provide evidence for an obligate dependency on proper NRAS function in human development and growth.

Ectodermal dysplasias form a large disease family with more than 200 members. The combination of hair and tooth abnormalities, alopecia, and cutaneous syndactyly is characteristic of ectodermal dysplasia-syndactyly syndrome (EDSS). We used a homozygosity mapping approach to map the EDSS locus to 1q23 in a consanguineous Algerian family. By candidate gene analysis, we identified a homozygous mutation in the PVRL4 gene that not only evoked an amino acid change but also led to exon skipping. In an Italian family with two siblings affected by EDSS, we further detected a missense and a frameshift mutation. PVRL4 encodes for nectin-4, a cell adhesion molecule mainly implicated in the formation of cadherin-based adherens junctions. We demonstrated high nectin-4 expression in hair follicle structures, as well as in the separating digits of murine embryos, the tissues mainly affected by the EDSS phenotype. In patient keratinocytes, mutated nectin-4 lost its capability to bind nectin-1. Additionally, in discrete structures of the hair follicle, we found alterations of the membrane localization of nectin-afadin and cadherin-catenin complexes, which are essential for adherens junction formation, and we found reorganization of actin cytoskeleton. Together with cleft lip and/or palate ectodermal dysplasia (CLPED1, or Zlotogora-Ogur syndrome) due to an impaired function of nectin-1, EDSS is the second known "nectinopathy" caused by mutations in a nectin adhesion molecule.

Multiplex Ligation-dependent Probe Amplification (MLPA) assay is a recently developed technique able to evidence variations in the copy number of several human genes. Due to this ability, MLPA can be used in the molecular diagnosis of several genetic diseases whose pathogenesis is related to the presence of deletions or duplications of specific genes. Moreover, MLPA assay can also be used in the molecular diagnosis of genetic diseases characterized by the presence of abnormal DNA methylation. Due to the large number of genes that can be analyzed by a single technique, MLPA assay represents the gold standard for molecular analysis of all pathologies derived from the presence of gene copy number variation. In this review, the main applications of the MLPA technique for the molecular diagnosis of human diseases are described.

Gestational Diabetes Mellitus (GDM) is the most common metabolic condition during pregnancy and may result in short- and long-term complications for both mother and offspring. The complexity of phenotypic outcomes seems influenced by genetic susceptibility, nutrient-gene interactions and lifestyle interacting with clinical factors. There is strong evidence that not only the adverse genetic background but also the epigenetic modifications in response to nutritional and environmental factors could influence the maternal hyperglycemia in pregnancy and the foetal metabolic programming. In this view, the correlation between epigenetic modifications and their transgenerational effects represents a very interesting field of study. The present review gives insight into the role of gene variants and their interactions with nutrients in GDM. In addition, we provide an overview of the epigenetic changes and their role in the maternal-foetal transmission of chronic diseases. Overall, the knowledge of epigenetic modifications induced by an adverse intrauterine and perinatal environment could shed light on the potential pathophysiological mechanisms of long-term disease development in the offspring and provide useful tools for their prevention.

Stem cells isolated from amniotic fluid are known to be able to differentiate into different cells types, thus being considered as a powerful tool for cellular therapy of different human diseases. In the last 4 years, amniotic fluid-derived stem (AFS) cells have been shown to express embryonic and adult stem cell markers. These cells can be considered an intermediate stage between embryonic stem cells and adult stem cells. AFS cells can give rise to adipogenic, osteogenic, myogenic, endothelial, neurogenic, and hepatic lineages, inclusive of all embryonic germ layers. AFS cells have a high renewal capacity and can be expanded for over 250 doublings without any detectable loss of chromosomal telomere length. Taken together, all these data provide evidence that amniotic fluid represents a new and very promising source of stem cells for research, as well as clinical applications. Certainly stem cells from amniotic fluid will be useful both for a customized cell supply for newly born children and for banking cells to be used for therapeutic cell transplantation in immunogically matched recipients. Further investigations are also warranted to fully explore the amniotic cells' potential for adult human disorders.

The correlation between epigenetics and human reproduction represents a very interesting field of study, mainly due to the possible transgenerational effects related to epigenetic modifications of male and female gametes. In the present review, we focused our attention to the role played by epigenetics on male reproduction, evidencing at least four different levels at which sperm epigenetic modifications could affect reproduction: (1) spermatogenesis failure; (2) embryo development; (3) outcome of assisted reproduction technique (ART) protocols, mainly as concerning genomic imprinting; and (4) long-term effects during the offspring lifetime. The environmental agents responsible for epigenetic modifications are also examined, suggesting that the control of paternal lifestyle prior to conception could represent in the next future a novel hot topic in the management of human reproduction.

Maternal impairments in folate metabolism and elevated homocysteinemia are known risk factors for having a child with Down syndrome (DS) at a young age. The 80G>A polymorphism of the reduced folate carrier gene (RFC-1) has been recently demonstrated to affect plasma folate and homocysteine levels, alone or in combination with the 677C>T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene. We performed the present study on 80 Italian mothers of DS individuals, aged less than 35 at conception, and 111 Italian control mothers, to study the role of the RFC-1 80G>A, MTHFR 677C>T, and MTHFR 1298A>C genotypes to the risk of a DS offspring at a young maternal age. When polymorphisms were considered alone, both allele and genotype frequencies did not significantly differ between DS mothers and control mothers. However, the combined MTHFR677TT/RFC-1 80GG genotype was borderline associated with an increased risk (OR 6 (CI 95%: 1.0-35.9), P = 0.05), and to be MTHF1298AA/RFC-1 80(GA or AA) was inversely associated with the risk (OR 0.36 (CI 95%: 0.14-0.96), P = 0.04). Present results seem to indicate that none of the RFC-1 80G>A, MTHFR 677C>T, and MTHFR 1298A>C polymorphisms is an independent risk factor for a DS offspring at a young maternal age; however, a role for the combined MTHFR/RFC-1 genotypes in the risk of DS pregnancies among young Italian women cannot be excluded.