Non-Invasive Prenatal Test Analysis Opens a Pandora's Box: Identification of Very Rare Cases of SRY-Positive Healthy Females, Segregating for Three Generations Thanks to Preferential Inactivation of the XqYp Translocated Chromosome.

The translocation of the testis-determining factor, the SRY gene, from the Y to the X chromosome is a rare event that causes abnormalities in gonadal development. In all cases of males and females carrying this translocation, disorder of sex development is reported. In our study, we described a peculiar pedigree with the first evidence of four healthy females from three generations who are carriers of the newly identified t(X;Y)(q28;p11.2)(SRY+) translocation with no evidence of ambiguous genitalia or other SRY-dependent alterations. Our study was a consequence of a Non-Invasive Prenatal Test (NIPT) showing a sexual chromosomal abnormality (XXY) followed by a chorionic villus analysis suggesting a normal karyotype 46,XX and t(X;Y) translocation detected by FISH. Here, we (i) demonstrated the inheritance of the translocation in the maternal lineage via karyotyping and FISH analysis; (ii) characterised the structural rearrangement via chromosomal microarray; and (iii) demonstrated, via Click-iT® EdU Imaging assay, that there was an absolute preferential inactivation of the der(X) chromosome responsible for the lack of SRY expression. Overall, our study provides valuable genetic and molecular information that may lead personal and medical decisions.

Kinase Inhibitors in Genetic Diseases.

Over the years, several studies have shown that kinase-regulated signaling pathways are involved in the development of rare genetic diseases. The study of the mechanisms underlying the onset of these diseases has opened a possible way for the development of targeted therapies using particular kinase inhibitors. Some of these are currently used to treat other diseases, such as cancer. This review aims to describe the possibilities of using kinase inhibitors in genetic pathologies such as tuberous sclerosis, RASopathies, and ciliopathies, describing the various pathways involved and the possible targets already identified or currently under study.

Germline Testing in a Cohort of Patients at High Risk of Hereditary Cancer Predisposition Syndromes: First Two-Year Results from South Italy.

Germline pathogenic variants (PVs) in oncogenes and tumour suppressor genes are responsible for 5 to 10% of all diagnosed cancers, which are commonly known as hereditary cancer predisposition syndromes (HCPS). A total of 104 individuals at high risk of HCPS were selected by genetic counselling for genetic testing in the past 2 years. Most of them were subjects having a personal and family history of breast cancer (BC) selected according to current established criteria. Genes analysis involved in HCPS was assessed by next-generation sequencing (NGS) using a custom cancer panel with high- and moderate-risk susceptibility genes. Germline PVs were identified in 17 of 104 individuals (16.3%) analysed, while variants of uncertain significance (VUS) were identified in 21/104 (20.2%) cases. Concerning the germline PVs distribution among the 13 BC individuals with positive findings, 8/13 (61.5%) were in the BRCA1/2 genes, whereas 5/13 (38.4%) were in other high- or moderate-risk genes including PALB2, TP53, ATM and CHEK2. NGS genetic testing showed that 6/13 (46.1%) of the PVs observed in BC patients were detected in triple-negative BC. Interestingly, the likelihood of carrying the PVs in the moderate-to-high-risk genes calculated by the cancer risk model BOADICEA was significantly higher in pathogenic variant carriers than in negative subjects. Collectively, this study shows that multigene panel testing can offer an effective diagnostic approach for patients at high risk of hereditary cancers.

The Variant p.Ala84Pro Is Causative of X-Linked Hypophosphatemic Rickets: Possible Relationship with Burosumab Swinging Response in Adults.

Loss of function mutations in the PHEX gene could determine X-linked dominant hypophosphatemia. This is the most common form of genetic rickets. It is characterized by renal phosphate wasting determining an increase in fibroblast growth factor 23 (FGF-23), growth retard, bone deformities and musculoskeletal manifestations. In recent decades, analysis of the PHEX gene has revealed numerous different mutations. However, no clear genotype-phenotype correlations have been reported in patients with hypophosphatemic rickets (XLH). We report two cases of a 28-year-old-male (patient 1) and a 19-year-old male (patient 2) affected by XLH initially treated with phosphate and 1,25-dihydroxyvitamin-D admitted to the Endocrinology unit because of the persistence of muscle weakness, bone pain and fatigue. After phosphate withdrawal, both patients started therapy with burosumab and symptoms ameliorated in three months. However, patient 1's biochemical parameters did not improve as expected so we decided to investigate his genetic asset. We herein describe a possible clinical implication for the missense "de novo" mutation, c.250G>C (p.Ala84Pro) in the PHEX gene, reported in the PHEX database and classified as a variant of uncertain significance (VUS). The clinical implication of this mutation on disease burden and quality of life in adults is still under investigation.

A Familial Form of Epidermolysis Bullosa Simplex Associated with a Pathogenic Variant in KRT5.

Epidermolysis bullosa simplex is a disease that belongs to a group of genodermatoses characterised by the formation of superficial bullous lesions caused by minor mechanical trauma to the skin. The skin fragility observed in the EBS is mainly caused by pathogenic variants in the KRT5 and KRT14 genes that compromise the mechanical stability of epithelial cells. By performing DNA sequencing in a female patient with EBS, we found the pathogenic variant c.967G>A (p.Val323Met) in the KRT5 gene. This variant co-segregated with EBS in the family pedigree and was transmitted in an autosomal dominant inheritance manner. This is the first report showing a familial form of EBS due to this pathogenic variant.

Case Report: Identification of a Novel Pathogenic Germline TP53 Variant in a Family With Li-Fraumeni Syndrome.

Li-Fraumeni syndrome (LFS) is an inherited autosomal dominant disease characterized by a predisposition to many cancers. Germline pathogenic variants in TP53 are primarily responsible for LFS. By performing a targeted sequencing panel in a proband with liver carcinoma having a deceased son affected by osteosarcoma, we found the novel heterozygous frameshift variant c.645del (p.Ser215Argfs*32) in the TP53 gene. This variant co-segregated with typical LFS cancers in the family pedigree, consistent with the pathogenicity of this novel and previously undescribed TP53 variant.

A Novel Splicing Variant of COL2A1 in a Fetus with Achondrogenesis Type II: Interpretation of Pathogenicity of In-Frame Deletions.

Achondrogenesis type II (ACG2) is a lethal skeletal dysplasia caused by dominant pathogenic variants in COL2A1. Most of the variants found in patients with ACG2 affect the glycine residue included in the Gly-X-Y tripeptide repeat that characterizes the type II collagen helix. In this study, we reported a case of a novel splicing variant of COL2A1 in a fetus with ACG2. An NGS analysis of fetal DNA revealed a heterozygous variant c.1267-2_1269del located in intron 20/exon 21. The variant occurred de novo since it was not detected in DNA from the blood samples of parents. We generated an appropriate minigene construct to study the effect of the variant detected. The minigene expression resulted in the synthesis of a COL2A1 messenger RNA lacking exon 21, which generated a predicted in-frame deleted protein. Usually, in-frame deletion variants of COL2A1 cause a phenotype such as Kniest dysplasia, which is milder than ACG2. Therefore, we propose that the size and position of an in-frame deletion in COL2A1 may be relevant in determining the phenotype of skeletal dysplasia.

De novo and bi-allelic variants in AP1G1 cause neurodevelopmental disorder with developmental delay, intellectual disability, and epilepsy.

Adaptor protein (AP) complexes mediate selective intracellular vesicular trafficking and polarized localization of somatodendritic proteins in neurons. Disease-causing alleles of various subunits of AP complexes have been implicated in several heritable human disorders, including intellectual disabilities (IDs). Here, we report two bi-allelic (c.737C>A [p.Pro246His] and c.1105A>G [p.Met369Val]) and eight de novo heterozygous variants (c.44G>A [p.Arg15Gln], c.103C>T [p.Arg35Trp], c.104G>A [p.Arg35Gln], c.229delC [p.Gln77Lys∗11], c.399_400del [p.Glu133Aspfs∗37], c.747G>T [p.Gln249His], c.928-2A>C [p.?], and c.2459C>G [p.Pro820Arg]) in AP1G1, encoding gamma-1 subunit of adaptor-related protein complex 1 (AP1γ1), associated with a neurodevelopmental disorder (NDD) characterized by mild to severe ID, epilepsy, and developmental delay in eleven families from different ethnicities. The AP1γ1-mediated adaptor complex is essential for the formation of clathrin-coated intracellular vesicles. In silico analysis and 3D protein modeling simulation predicted alteration of AP1γ1 protein folding for missense variants, which was consistent with the observed altered AP1γ1 levels in heterologous cells. Functional studies of the recessively inherited missense variants revealed no apparent impact on the interaction of AP1γ1 with other subunits of the AP-1 complex but rather showed to affect the endosome recycling pathway. Knocking out ap1g1 in zebrafish leads to severe morphological defect and lethality, which was significantly rescued by injection of wild-type AP1G1 mRNA and not by transcripts encoding the missense variants. Furthermore, microinjection of mRNAs with de novo missense variants in wild-type zebrafish resulted in severe developmental abnormalities and increased lethality. We conclude that de novo and bi-allelic variants in AP1G1 are associated with neurodevelopmental disorder in diverse populations.

Flavonoid supplements increase neurotrophin activity to modulate inflammation in retinal genetic diseases.

Retinal degenerative disorders induce loss of photoreceptors associated with inflammation, and negative remodeling and plasticity of neural retina. Retinal degenerative diseases may have genetic and/or environmental causes. Degeneration of retinal pigment epithelium cells initiates a vicious circle increasing the ongoing inflammation in both retina and choroid. Flavonoids are polyphenolic molecules with antioxidant activity and  dietary intake, specifically of anthocyanins and flavanols, improves oxidative stress and neuro-inflammation. In vitro and ex vivo studies have also revealed biological effects of flavonoids on retinal protection against oxidative stress and inflammation. In this brief review, the protective role of flavonoids against retinal degeneration and inflammation will be discussed along with their therapeutic potential for the treatment of retinal degenerative diseases.

7q35 Microdeletion and 15q13.3 and Xp22.33 Microduplications in a Patient with Severe Myoclonic Epilepsy, Microcephaly, Dysmorphisms, Severe Psychomotor Delay and Intellectual Disability.

Copy number variations (CNVs) play a key role in the pathogenesis of several diseases, including a wide range of neurodevelopmental disorders. Here, we describe the detection of three CNVs simultaneously in a female patient with evidence of severe myoclonic epilepsy, microcephaly, hypertelorism, dimorphisms as well as severe psychomotor delay and intellectual disability. Array-CGH analysis revealed a ∼240 kb microdeletion at the 7q35 inherited from her father, a ∼538 kb microduplication at the 15q13.3 region and a ∼178 kb microduplication at Xp22.33 region, both transmitted from her mother. The microdeletion in 7q35 was included within an intragenic region of the contactin associated protein-like 2 (CNTNAP2) gene, whereas the microduplications at 15q13.3 and Xp22.33 involved the cholinergic receptor nicotinic alpha 7 subunit (CHRNA7) and the cytokine receptor-like factor 2 (CRLF2) genes, respectively. Here, we describe a female patient harbouring three CNVs whose additive contribution could be responsible for her clinical phenotypes.

A novel ABCC6 variant causative of pseudoxanthoma elasticum.

Pseudoxanthoma elasticum is an autosomal recessive heritable disorder caused by mutations in ABCC6. We describe two siblings showing typical skin lesions and a clinical diagnosis of pseudoxanthoma elasticum. Genetic analysis of ABCC6 revealed a novel homozygous c.4041G > A variant located in the last position of exon 28 that compromises the splicing donor site, resulting in a shorter messenger RNA. The deletion impairs the nucleotide-binding fold region, which is crucial for ABCC6 function.

Biallelic variants in the ciliary gene TMEM67 cause RHYNS syndrome.

A rare syndrome was first described in 1997 in a 17-year-old male patient presenting with Retinitis pigmentosa, HYpopituitarism, Nephronophthisis and Skeletal dysplasia (RHYNS). In the single reported familial case, two brothers were affected, arguing for X-linked or recessive mode of inheritance. Up to now, the underlying genetic basis of RHYNS syndrome remains unknown. Here we applied whole-exome sequencing in the originally described family with RHYNS to identify compound heterozygous variants in the ciliary gene TMEM67. Sanger sequencing confirmed a paternally inherited nonsense c.622A > T, p.(Arg208*) and a maternally inherited missense variant c.1289A > G, p.(Asp430Gly), which perturbs the correct splicing of exon 13. Overall, TMEM67 showed one of the widest clinical continuum observed in ciliopathies ranging from early lethality to adults with liver fibrosis. Our findings extend the spectrum of phenotypes/syndromes resulting from biallelic TMEM67 variants to now eight distinguishable clinical conditions including RHYNS syndrome.

Internet Use and Access, Behavior, Cyberbullying, and Grooming: Results of an Investigative Whole City Survey of Adolescents.

BACKGROUND: According to the Digital Agenda for Europe, the way children use the Internet and mobile technologies has changed dramatically in the past years. OBJECTIVE: The aims of this study were to: (1) breakdown the modalities of access and use of the Internet by teenagers to assess risks and risky behaviors; and (2) provide scientific data to evaluate and counsel safe use of the Internet and new technologies by teenagers. METHODS: The study was conducted under the program "Strategies for a Better Internet for Children" started in May 2012 by the European Commission. It represents the main result of the project launched by Telecom Italia, "Anche io ho qualcosa da dire" (I too have something to say), thanks to which many contributions were collected and used to develop a survey. The questionnaire was structured in 45 questions, covering three macro areas of interest. It was approved by the Department Board at University of Magna Graecia's School of Medicine. After authorization from the regional high school authority, it was administered to all 1534 students (aged 13-19 years) in the city of Catanzaro, Italy. RESULTS: The data was broken down into three main groups: (1) describing education and access to the Internet; (2) methods of use and social networking; and (3) perception and evaluation of risk and risky behaviors. Among noteworthy results in the first group, we can mention that the average age of first contact with information technologies was around 9 years. Moreover, 78.87% (1210/1534) of the interviewed students reported having access to a smartphone or a tablet. Among the results of the second group, we found that the most used social networks were Facebook (85.78%, 1316/1534), YouTube (61.14%, 938/1534), and Google+ (51.56%, 791/1534). About 71.31% (1094/1534) of the interviewed teenagers use their name and surname on social networks, and 40.09% (615/1534) of them knew all their Facebook contacts personally. Among the results of the third group, we found that 7.69% (118/1534) of the interviewed teenagers have uploaded pictures or movies of which they felt ashamed; 27.05% (415/1534) have received invitations from people they met on the Internet to meet in real life; and 8.67% (133/1534) have accepted such invitations. CONCLUSIONS: The results offer a breakdown of the teenagers' use of the Internet, focusing on how teenagers learn to use and access it while taking into account factors such as parental coaching, schooling, or self-education. It describes how they approach and interact with social networks and how they perceive risks and risky behaviors on the Internet. Information technology must be seen as an instrument and not as a hindrance. For this to happen, parental guidance, schooling, and medical counseling are needed for a sound development of the child in this critical stage.

New SLC12A3 disease causative mutation of Gitelman's syndrome.

Gitelman's syndrome (GS) is a salt-losing tubulopathy with an autosomal recessive inheritance caused by mutations of SLC12A3, which encodes for the thiazide-sensitive NaCl cotransporter. In this study we report a new mutation of SLC12A3 found in two brothers affected by GS. Hypokalemia, hypocalciuria and hyper-reninemia were present in both patients while hypomagnesemia was detected only in one. Both patients are compound heterozygotes carrying one well known GS associated mutation (c.2581 C > T) and a new one (c.283delC) in SLC12A3 gene. The new mutation results in a possible frame-shift with a premature stop-codon (pGln95ArgfsX19). The parents of the patients, heterozygous carriers of the mutations found in SLC12A3, have no disease associated phenotype. Therefore, the new mutation is causative of GS.

Novel Mutation in the CHRDL1 Gene Detected in Patients With Megalocornea.

PURPOSE: The aim of this study was to determine the mutation associated with X-linked megalocornea (MGC1) found in 2 patients from the same area in southern Italy. METHODS: Diagnosis of megalocornea was confirmed by detailed ophthalmic examination in 2 probands from independent families and in another 3 affected family members. Genomic DNA of the probands was used to amplify and sequence all the coding regions of CHRDL1. RESULTS: Megalocornea diagnosis was associated with a novel mutation found in the probands and affected kindreds (5 subjects). The mutation is an 11-base pair deletion that leads to a stop codon in the second coding exon of the CHRDL1 gene. Research on the CHRDL1 mutation was also performed on other family members (11 subjects) not affected by MGC1, and the mutation was not detected in unaffected male family members. CONCLUSIONS: The detection of mutations in the CHRDL1 gene is useful for differential diagnosis with different forms of megalocornea.

The sodium-phosphate co-transporter SLC34A2, and pulmonary alveolar microlithiasis: Presentation of an inbred family and a novel truncating mutation in exon 3.

Pulmonary alveolar microlithiasis is a disorder in which many tiny fragments (microliths) of calcium phosphate gradually accumulate in alveoli. Loss of function mutations in the gene SLC34A2 coding for the sodium phosphate co-transporter (NaPi-IIb) are responsible for genetic forms of alveolar microlithiasis. We now report a consanguineous Italian family from Calabria with two affected members segregating alveolar microlithiasis in a recessive fashion. We describe, for the first time, a novel loss of function mutation in the gene coding for NaPi-IIb. A careful description of the clinical phenotype is provided together with technical details for direct sequencing of the gene.

A case of premature ovarian failure in a 33-year-old woman.

Objective. To assess aetiology of a POF in a 33-year-old woman and, if possible, plan a cure. Design. Case report. Setting. medical genetics diagnostic unit in a university hospital. Patient. A 33-year-old woman with premature ovarian failure (POF). Intervention(s). Genetic counseling, karyotyping, FISH study. Result(s). Turner-like diagnosis. Conclusion(s). Most cases of POF remain idiopathic. Turner syndrome can occur in very different phenotypes; cytogenetic and molecular profiling can provide a definitive diagnosis in cases with nonclassical phenotype.

Identification of a new mutation in the gene coding for hairless protein responsible for alopecia universalis: The importance of direct gene sequencing.

Mutations in the gene HR coding for the hairless protein are associated with atrichia with papular lesions (APL), an autosomal recessive form of alopecia universalis that is characterized by generalized scalp and body atrichia with papular lesions. We here describe a South Italian family of ancient Albanian heritage. The full phenotype with complete atrichia was expressed in 2 siblings, whereas the parents and one sister were unaffected. Direct sequencing of the gene coding for the hairless protein allowed the identification of a new mutation in exon 17. Consistent with the recessive inheritance of the disease, both the siblings were homozygous for the mutation, whereas the parents and the unaffected sister where heterozygous. A relevant discrepancy with a haplotype linkage study is reported, stressing the importance of gene sequencing in genetic diagnosis and counseling because linkage studies can be biased by recombination events.