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Background: The use of NGS technology has rapidly increased during the last decade, and many new monogenic neurodevelopmental disorders have emerged. Pathogenic variants in the neuronal CAMK2A gene have been recently associated with “intellectual developmental disorder, autosomal dominant 53″ (OMIM#617798), a syndrome characterized by variable clinical manifestations including mild to severe intellectual disability, delayed psychomotor development, delayed or absent speech, delayed walking, seizures, dysmorphic features and behavioral psychiatric manifestations as autism spectrum disorders, aggressive behavior, and hyperactivity. CAMK2A (OMIM*114078) encodes for a subunit of the calcium/calmodulin-dependent serine/threonine kinase II (CaMKII), which is predominately expressed in the brain, where it plays critical roles in synaptic plasticity, learning, and memory as well as in neuronal migration. Methods and Results: We hereby describe a thirty-five-year-old woman affected by severe intellectual disability with epileptic encephalopathy. We performed exome sequencing and found a de novo heterozygous variant in the CAMK2A gene (NM_171825.2: c.874_876delCTT; p.Lys292del), which was fully correlated with her phenotype. This is the first report of an inframe single amino acid deletion in a patient affected by intellectual developmental disorder autosomal dominant 53. The variant is predicted to affect protein structure and function and interaction with other proteins and hits a crucial functional site. Discussion: We discuss our variant in relation to previously reported variants and with the objective of delineating possible genotype–phenotype correlations.

Periodic fever syndromes include autoinflammatory disorders (AID) that involve innate immunity. These disorders are characterized by recurrent fevers and aberrant multi-organ inflammation, without any involvement of T or B cells or the presence of autoantibodies. A complex genetic architecture has been recognized for many AID. However, this complexity has only been partially uncovered for familial Mediterranean fever and other conditions that have a classical monogenic origin and Mendelian transmission. Several gene panels are currently available for molecular diagnosis in patients suspected of having AID. However, even when an extensive number of genes (up to 50–100) are tested in a cohort of clinically selected patients, the diagnostic yield of AID ranges between 15% and 25%, depending on the clinical criteria used for patient selection. In the remaining 75–85% of cases, it is conceivable that the causative gene or genes responsible for a specific condition are still elusive. In these cases, the disease could be explained by variants, either recessive or dominant, that have a major effect on unknown genes, or by the cumulative impact of different variants in more than one gene, each with minor additive effects. In this study, we focused our attention on five familial cases of AID presenting with classical autosomal dominant transmission. To identify the probable monogenic cause, we performed exome sequencing. Through prioritization, filtering, and segregation analysis, we identified a few variants for each family. Subsequent bioinformatics evaluation and pathway analysis helped to narrow down the best candidate genes for each family to FCRL6, PKN1, STAB1, PTDGR, and VCAM1. Future studies on larger cohorts of familial cases will help confirm the pathogenic role of these genes in the pathogenesis of these complex disorders.

Neurodevelopmental Disorders (NDs) are a heterogeneous group of disorders and are considered multifactorial diseases with both genetic and environmental components. Epigenetic dysregulation driven by adverse environmental factors has recently been documented in neurodevelopmental disorders as the possible etiological agent for their onset. However, most studies have focused on the epigenomes of the probands rather than on a possible epigenetic dysregulation arising in their mothers and influencing neurodevelopment during pregnancy. The aim of this research was to analyze the methylation profile of four well-known genes involved in neurodevelopment (BDNF, RELN, MTHFR and HTR1A) in the mothers of forty-five age-matched AS (Asperger Syndrome), ADHD (Attention Deficit Hyperactivity Disorder) and typically developing children. We found a significant increase of methylation at the promoter of the RELN and HTR1A genes in AS mothers compared to ADHD and healthy control mothers. For the MTHFR gene, promoter methylation was significantly higher in AS mothers compared to healthy control mothers only. The observed dysregulation in AS mothers could potentially contribute to the affected condition in their children deserving further investigation.

As a consequence of the implementation of NGS technologies, the diagnostic yield of neurodevelopmental disorders has dramatically increased during the past two decades. Among neurodevelopmental genes, transcription-related genes and chromatin remodeling genes are the most represented category of disease-causing genes. Indeed, the term “chromatinopathies” is now widely used to describe epigenetic disorders caused by mutations in these genes. We hereby describe a twenty-seven-year-old female patient diagnosed with moderate intellectual disability comorbid with other neuropsychiatric and behavioral issues carrying a de novo heterozygous stop variant in the KDM5C gene (NM_004187.5: c. 3847G>T, p.Glu1283*), encoding a histone demethylase that specifically acts on the H3K4 lysines. The gene is located on the X chromosome and has been associated with Claes–Jensen-type intellectual disability, an X-linked syndromic disorder. We discuss our case in relation to previously reported affected females harboring pathogenic mutations in the KDM5C gene with the objective of delineating genotype–phenotype correlations and further defining a common recognizable phenotype. We also highlight the importance of reverse phenotyping in relation to whole-exome sequencing results.

The involvement of the Histaminergic System (HS) in neuropsychiatric disease is not well-documented, and few studies have described patients affected by different neuropsychiatric conditions harbouring disruptions in genes involved in the HS. In humans, histamine is synthetised from histidine by the histidine decarboxylase enzyme encoded by the HDC gene (OMIM*142704). This is the sole enzyme in our organism able to synthetise histamine from histidine. Histamine is also contained in many different food types. We hereby describe a twenty-one-year-old female diagnosed with a borderline intellectual disability with autistic traits and other peculiar neuropsychological features carrying a 175-Kb interstitial deletion on chromosome 15q21.2. The deletion was inherited from the mother, who was affected by a severe anxiety disorder. The deleted region contains entirely the HDC and the SLC27A2 genes and partially the ATP8B4 gene. The HDC gene has been previously associated with Tourette Syndrome (TS). Based on the functional role of the HDC, we propose this gene as the best candidate to explain many traits associated with the clinical phenotype of our patient and of her mother.

Asperger syndrome (AS) is a pervasive developmental disorder characterized by general impairment in socialization, stereotypical behavior, defective adaptation to the social context usually without intellectual disability, and some high functioning areas related to memory and mathematics. Clinical criteria are not well defined and the etiology is heterogeneous and mostly unknown. Like in typical autism spectrum disorders (ASD), the genetic background plays a crucial role in AS, and often an almost mendelian segregation can be observed in some families. We performed a whole exome sequencing (WES) in three relatives of a family with vertical transmission of AS-ASD to identify variants in candidate genes segregating with the phenotype. Variant p.(Cys834Ser) in the RADX gene was the only one segregating among all the affected family members. This gene encodes a single-strand DNA binding factor, which mediates the recruitment of genome maintenance proteins to sites of replication stress. Replication stress and genome instability have been reported recently in neural progenitor cells derived from ASD patients, leading to a disruption of long neural genes involved in cell–cell adhesion and migration. We propose RADX as a new gene that when mutated could represent a predisposing factor to AS-ASD.

COVID-19 may be considered a multifactorial disease caused by the interaction between the virus itself, as the environmental contribute, and the genetic background of the host. SARS-CoV-2 infection occurs through the interaction between the spike protein and ACE2, a receptor in the host cells. Clinically, COVID-19 is characterized by a high heterogeneity in symptomatology ranging from asymptomatic to severe symptoms, and even worsening to death. This variability relies on the host genomic profile and other individual comorbidities. We performed exome analysis in one family displaying a variable spectrum of SARS-CoV-2 infection despite a common exposure. After segregation analysis, we found that the c.446CT p.(S149L) in

ASD genetic diagnosis has dramatically improved due to NGS technologies, and many new causative genes have been discovered. Consequently, new ASD phenotypes have emerged. An extensive exome sequencing study carried out by the Autism Sequencing Consortium (ASC) was published in February 2020. The study identified 102 genes which are de novo mutated in subjects affected by autism spectrum disorder (ASD) or similar neurodevelopmental disorders (NDDs). The majority of these genes was already known to be implicated in ASD or NDDs, whereas approximately 30 genes were considered “novel” as either they were not previously associated with ASD/NDDs or very little information about them was present in the literature. The aim of this work is to review the current literature since the publication of the ASC paper to see if new data mainly concerning genotype–phenotype correlations of the novel genes have been added to the existing one. We found new important clinical and molecular data for 6 of the 30 novel genes. Though the broad and overlapping neurodevelopmental phenotypes observed in most monogenic forms of NDDs make it difficult for the clinical geneticist to address gene-specific tests, knowledge of these new data can at least help to prioritize and interpret results of pangenomic tests to some extent. Indeed, for some of the new emerging genes analyzed in the present work, specific clinical features emerged that may help the clinical geneticist to make the final diagnosis by associating the genetic test results with the phenotype. The importance of this relatively new approach known as “reverse phenotyping” will be discussed.

Background: Distal renal tubular acidosis (dRTA) is characterized by an impairment of urinary acidification resulting in metabolic acidosis, hypokalemia, and inappropriately elevated urine pH. If not treated, this chronic condition eventually leads to nephrocalcinosis, nephrolithiasis, impaired renal function, and bone demineralization. dRTA is a well-defined entity that can be diagnosed by genetic testing of 5 genes known to be disease-causative. Incomplete dRTA (idRTA) is defined as impaired urinary acidification that does not lead to overt metabolic acidosis and therefore can be diagnosed if patients fail to adequately acidify urine after an ammonium chloride (NH4Cl) challenge or furosemide and fludrocortisone test. It is still uncertain whether idRTA represents a distinct entity or is part of the dRTA spectrum and whether it is caused by mutations in the same genes of overt dRTA. Methods: In this cross-sectional study, we investigated a group of 22 stone formers whose clinical features were suspicious of idRTA. They underwent an NH4Cl challenge and were found to have impaired urinary acidification ability. These patients were then analyzed by genetic testing with sequencing of 5 genes: SLC4A1, ATP6V1B1, ATP6V0A4, FOXI1, and WDR72. Results: Two unrelated individuals were found to have two different variants in SLC4A1 that had never been described before. Conclusions: Our results suggest the involvement of other genes or nongenetic tubular dysfunction in the pathogenesis of idRTA in stone formers. However, genetic testing may represent a cost-effective tool to recognize, treat, and prevent complications in these patients.

Blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES) is due to heterozygous FOXL2 intragenic mutations in about 70% of the patients, whereas total or partial gene deletions account for a minority of cases. Alteration of FOXL2 regulatory elements has been rarely described in patients with BPES. In this study, a prepubertal girl with BPES due to a 197-kb de novo deletion of the regulatory elements upstream of FOXL2 is reported. This girl presented with additional clinical features such as a soft cleft palate and microcephaly; thus, this copy number variant might have other somatic effects. The present deletion encompasses 2 coding genes (MRPS22 and COPB2), whose homozygous mutations have been associated with microcephaly. In our case, the sequences of the non-deleted allele were normal, ruling out a compound genetic defect. Normal levels of new biomarkers of ovarian reserve (anti-müllerian hormone, inhibin B) likely indicate an early diagnosis of type 2 BPES, but an evolutive gonadal damage will be excluded only by long-term follow-up. Additional reports of microdeletions upstream of FOXL2 are needed to better define the underlying genetic mechanism and the related phenotypic spectrum; the ability of the new hormonal markers to predict ovarian function in adolescence and adulthood should be confirmed.

Neural tube defects are a group of birth defects caused by failure of neural tube closure during development. The etiology of NTD, requiring a complex interaction between environmental and genetic factors, is not well understood. We performed whole-exome sequencing (WES) in six trios, with a single affected proband with spina bifida, to identify rare/novel variants as potential causes of the NTD. Our analysis identified four de novo and ten X-linked recessive variants in four of the six probands, all of them in genes previously never implicated in NTD. Among the 14 variants, we ruled out six of them, based on different criteria and pursued the evaluation of eight potential candidates in the following genes: RXRγ, DTX1, COL15A1, ARHGAP36, TKTL1, AMOT, GPR50, and NKRF. The de novo variants where located in the RXRγ, DTX1, and COL15A1 genes while ARHGAP36, TKTL1, AMOT, GPR50, and NKRF carry X-linked recessive variants. This analysis also revealed that four patients presented multiple variants, while we were unable to identify any significant variant in two patients. Our preliminary conclusion support a major role for the de novo variants with respect to the X-linked recessive variants where the X-linked could represent a contribution to the phenotype in an oligogenic model.

In a recent study, we showed that lymphocytes of obese Italian children/adolescents displayed levels of double strand breaks (DSB), assayed as serine 139-phosphorylated histone H2AX (γ-H2AX), about eightfold higher than normal weight controls, and that 30% of this damage-generated micronuclei. These findings suggested that obese children could be at increased risk of obesity-mediated cancer later in life. We therefore aimed to assess the level of γ-H2AX in a genetic animal model of obesity (Zucker rat) to identify a genotoxic/carcinogenic risk in some organs. The DSB marker was studied in 3- to 4-week-old rats and in 9- to 13-week-old rats. Paraffin-embedded sections of heart, thyroid, liver, pancreas, lung, kidney, esophagus, and gut from the fa-/fa- (obese) and the fa+/fa- (lean) control animals were processed for immunohistochemistry detection of γ-H2AX. Pancreas (0.0624 ± 0.0195), lung (0.1197 ± 0.0217), esophagus (0.1230 ± 0.0351), kidney (0.1546 ± 0.0149), and gut (0.1724 ± 0.0352) of 9- to 13-week-old obese rats showed a higher proportion of γ-H2AX-positive nuclei, than their lean counterparts (0.0092 ± 0.0033, 0.0416 ± 0.0185, 0.0368 ± 0.0088, 0.0686 ± 0.0318, and 0.0703 ± 0.0239, respectively). No difference was seen in the 3- to 4-week-old age group with regard to obesity, indicating that the DNA damage increased with older age of the rats. We hypothesize that the organs of the obese animals showing high levels of DSB could represent target tissues for the development of obesity-related cancers. Environ. Mol. Mutagen. 58:477-484, 2017. © 2017 Wiley Periodicals, Inc.

Increasing evidence suggests that early-life events can predispose the newborn to a variety of health issues in later life. In adverse pre- and perinatal conditions, oxidative stress appears to play an important role in the development of future pathological outcomes. From a molecular point of view, oxidative stress can result in genome damage and changes in DNA methylation that can in turn prime pathogenic mechanisms. Interestingly, both alterations have been related to a reciprocal regulation of oxidative stress. The aim of this review is to give a brief overview of the complex relationship linking oxidative stress to DNA damage and methylation and to go through the different sources of exposure that a neonate can encounter in utero or shortly after birth. In this context, the setup of methodologies to monitor the extent of oxidative stress, genomic damage and instability or the presence of altered methylation patterns contributes to the understanding on how the complex events occurring in early life can lead to either a healthy status or a pathological condition.

Blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES) is due to heterozygous FOXL2 intragenic mutations in about 70% of the patients, whereas total or partial gene deletions account for a minority of cases. Alteration of FOXL2 regulatory elements has been rarely described in patients with BPES. In this study, a prepubertal girl with BPES due to a 197-kb de novo deletion of the regulatory elements upstream of FOXL2 is reported. This girl presented with additional clinical features such as a soft cleft palate and microcephaly; thus, this copy number variant might have other somatic effects. The present deletion encompasses 2 coding genes (MRPS22 and COPB2), whose homozygous mutations have been associated with microcephaly. In our case, the sequences of the non-deleted allele were normal, ruling out a compound genetic defect. Normal levels of new biomarkers of ovarian reserve (anti-müllerian hormone, inhibin B) likely indicate an early diagnosis of type 2 BPES, but an evolutive gonadal damage will be excluded only by long-term follow-up. Additional reports of microdeletions upstream of FOXL2 are needed to better define the underlying genetic mechanism and the related phenotypic spectrum; the ability of the new hormonal markers to predict ovarian function in adolescence and adulthood should be confirmed.

We report on a patient with a 6.5 Mb interstitial de novo deletion in 3q24q25.2, characterized by array CGH. The patient is a 4-year and 2-month-old girl, who presented to us with mild developmental delay, absence of language, facial dysmorphism, hirsutism, strabismus, and Dandy-Walker Malformation. The main clinical signs typical of WS (Wisconsin syndrome) are evident in the patient. The molecular mapping of WS in 3q23q25 allowed geneticists to define the syndrome more accurately. Comparing the present patient's phenotype with that of cases with a molecular characterization so far reported, it was possible to narrow the critical region for WS to an interval of 750 Kb, where two genes (MBNL1 and TMEM14E) are harbored. The potential role of MBNL1 in causing the WS phenotype is discussed. © 2016 Wiley Periodicals, Inc.

Bystander effect is a known radiobiological effect, widely described using ionizing radiations and which, more recently, has also been related to chemical mutagens. In this study, we aimed to assess whether or not a bystander response can be induced in cultured human peripheral lymphocytes by vincristine, a chemotherapeutic mutagen acting as spindle poison, and by mitomycin-C, an alkylating agent already known to induce this response in human lymphoblastoid cells. Designing a modified ad hoc protocol for the cytokinesis blocked micronucleus (MN) assay, we detected the presence of a dose-dependent bystander response in untreated cultures receiving the conditioned medium (CM) from mitomycin-C (MMC) or vincristine (VCR) treated cultures. In the case of MMC, MN frequencies, expressed as micronucleated binucleates, were: 13.5±1.41 at 6μM, 22±2.12 at 12μM or 28.25±5.13 at 15μM vs. a control value of 4.75±1.59. MN levels for VCR, expressed as micronucleated mononucleates were: 2.75±0.88 at 0.0μM, 27.25±2.30 at 0.4μM, 46.25±1.94 at 0.8μM, 98.25±7.25 at 1.6μM. To verify that no mutagen residual was transferred to recipient cultures together with the CM, we evaluated MN levels in cultures receiving the medium immediately after three washings following the chemical treatment (unconditioned medium). We further confirmed these results using a cell-mixing approach where untreated lymphocytes were co-cultured with donor cells treated with an effect-inducing dose of MMC or VCR. A distinct production pattern of both reactive oxygen species and soluble mediator proteins by treated cells may account for the differences observed in the manifestation of the bystander effect induced by VCR. In fact, we observed an increased level of ROS, IL-32 and TGF-β in the CM from VCR treated cultures, not present in MMC treated cultures.

In 2013, 42 million children under the age of 5 years were overweight or obese. In the context of obesity, we recently showed that (1) peripheral lymphocytes of obese children/adolescents had an 8-fold increase in double strand breaks (DSBs), expressed as g-H2AX foci, than normal weight adolescents, and (2) 30% of the damage was retained into chromosome mutations. Thus, we investigated DSBs repair efficiency in a group of obese adolescents assessing the kinetic of H2AX phosphorylation in mitomycin C (MMC)-treated lymphocytes harvested 2 h- or 4 h-post mutagen treatment. According to our previous studies, these harvesting times represent the peak of DSBs induction and the time in which an appreciable DSBs reduction was observed. In addition, we evaluated the expression of the high mobility group box-1 protein (HMGB1), a chromatin remodelling protein involved in DSBs repair and obesity. Compared to normal weight adolescents, obese subjects 1) showed higher levels of g-H2AX foci at either 2 h- (0.239±0.041 vs. 0.473±0.048, P=0.0016) or 4 h- (0.150±0.026 vs. 0.255±0.030, P=0.0198) post mutagen treatment, and 2) have repaired a greater amount of the initial lesions (0.088±0.033 vs. 0.218±0.045, P=0.0408). Concordantly, 1) HMGB1 levels of obese individuals increased and decreased at 2h- or 4 h-post mutagen treatment, respectively, and 2) the opposite occurred for the normal weight adolescents where the protein was down-expressed at 2h and over-expressed at 4h. In conclusion, lymphocytes of obese and normal weight adolescents showed a distinct temporal kinetic of repairing MMC-induced DSBs, together with a different expression of HMGB1. The finding that obesity may modulate the repair of DNA damage induced in lymphocytes by genotoxic agents should be confirmed by further experiments.

PFAPA is an autoinflammatory syndrome characterized by periodic fever, aphthous stomatitis, sterile pharingitis, and adenitis, with an onset usually before the age of five. While the condition is most commonly sporadic, a few cases are familial and are usually compatible with an autosomal dominant (AD) transmission pattern, with reduced penetrance in some pedigrees. We performed exome analysis in a family where PFAPA was present in three relatives in two generations showing apparent AD segregation, identifying several rare and/or novel heterozygous variants in genes involved in the autoinflammatory pathway. Following segregation analysis of candidate variants, only one, c. 2770T>C p.(S924P) in the ALPK1 gene, was found to be consistently present in affected family members. ALPK1 is broadly expressed in different tissues and its protein is the intracellular kinase activated by the bacterial ADP-heptose bisphosphate that phosphorylates and activates TRAF-Interacting protein with Forkhead-Associated domain (TIFA) and triggers the immediate response to Gram-negative bacterial invasion. Sequencing analysis of 13 additional sporadic cases and 10 familial PFAPA cases identified two additional heterozygous missense variants c.1024G>C p.(D342H) and c.710C>T p.(T237M) in two sporadic patients, suggesting that rare variants in ALPK1 may represent a predisposing factor for recurrent periodic fever in a pediatric population.

Terminal deletions of long arm of chromosome 13 are rare and poorly characterized by cytogenetic studies, making for difficult genotype-phenotype correlations. We report two siblings presenting generalized epilepsy, intellectual disability, and genitourinary tract defects. Array CGH detected a 1.3 Mb deletion at 13q34; it contains two protein-coding genes, SOX1 and ARHGEF7, whose haploinsufficiency can contribute to the epileptic phenotype.

Obesity has been implicated in the development of many cancers. This can lead to genome damage, especially in the form of double-strand break, the presence of which is now easily detected through nuclear phosphorylation of histone H2AX (γ-H2AX) focus assay. Recently, the endothelin (ET) axis has also been shown to have a role in the growth and progression of several tumors, including lung cancer. The aim of this study was to evaluate the ET-1 system transcriptional alterations and γ-H2AX in lung tissue of Zucker rats subdivided into obese (O, n=22) and controls (CO, n=18) rats: under either fasting conditions (CO(fc)-O(fc)) or acute hyperglycemia (CO(AH)-O(AH)). Significantly higher prepro-ET-1 (p=0.05) and ET-converting enzyme (ECE)-2 mRNA expression was observed in O with respect to CO. A significant positive association was observed between prepro-ET-1 and ET-A in the whole rat population (p=0.009) or in the obese group alone (p=0.007). The levels of γ-H2AX in O and in O(AH) rats were significantly higher (p=0.019) than in the corresponding CO and CO(AH) rats (p=0.038). The study shows an inappropriate secretion of ET-1 in O animals with a parallel DNA damage in their lungs, providing novel mechanisms by which ET receptor antagonist may exert organ protection.

In humans, the most common genomic disorder is the hemizygous deletion of the chromosome 22q11.2 region, that results in the “22q11.2 deletion syndrome” (22q11.2DS). A peculiarity of 22q11.2DS is its great phenotypic variability that makes this pathology a classic example of a syndrome with variable expressivity and incomplete penetrance. The reasons for this variability have not been elucidated yet, and the molecular substrates underlying the different clinical features of 22q11.2DS are still debated. A cohort of 21 patients has been analyzed by array CGH in order to detect some of the genetic differences that may influence this variability. Two aspects have been investigated: (1) the precise localization of the deletion breakpoints within the low copy repeats (LCRs), (2) the additional Copy Number Variations (CNVs) elsewhere in the genome, by analyzing their gene content. Both protein-coding genes and miRNAs were considered, in order to discover possible epistatic interactions between genes of the 22q11.2 region and the rest of the genome. Eighteen out of twenty-one patients had a deletion of ~3 Mb mediated by LCR22-A and D, whereas 3/21 had a smaller deletion. The breakpoints within the LCR22-A and D do not have a major role in the phenotypic variability since they are rather clustered and the small differences concern genes that are not directly related to clinical signs of 22q11.2DS. A detailed analysis of the gene content of 22q11.2 deleted region indicates that this syndrome could be a bioenergetic disorder or consequence of an altered post-transcriptional gene regulation, due to the presence of DGCR8, a major player of the microRNA (miRNA) biogenesis. Only four genes with mitochondrial function are harbored in the additional CNVs, whereas 11 miRNA, all related to biological pathways present in the 22q11.2DS, have been detected in 19/21 patients. CNVs and miRNAs are new entities that have changed the order of complexity at the level of gene expression and regulation, thus CNV-miRNAs (miRNA harbored in the CNVs) are potential functional variants that should be considered high priority candidate variants in genotype-phenotype association studies. Deletion of DGCR8, the main actor in miRNA biogenesis, amplifies this variability. To our knowledge, this is the first report that focus on the miRNA-CNVs in 22q11.2DS, with the aim of trying to better understand their role in the variable expressivity and incomplete penetrance.

'22q11 deletion syndrome' (22q11DS) is a rare genetic syndrome, in which most patients share the same deletion, but their clinical features may vary a great deal. The genetic mechanisms underlying the variable expressivity and reduced penetrance of 22q11DS still have to be fully elucidated. Epilepsy has been reported in about 15.2% of the patients, however, few studies have focused on this topic and in most cases a detailed epileptic profile is missing. Since only a minority of patients experience epileptic seizures, 22q11deletion can be considered a predisposing factor, which is not sufficient ‘per se’ to cause epilepsy; to date, no candidate gene for epilepsy has been identified in the deleted region. We report on a 6-year-old girl with 22q11DS presenting a form of epilepsy that can be classified as ‘Panayiotopoulos syndrome’. Array CGH revealed an additional microduplication of 172 kb in 2q37, harboring three genes. One of these, DGKD (diacylglycerol kinase delta), is interrupted by the distal breakpoint of the duplication. DGKD encodes a cytoplasmic enzyme that phosphorylates diacylglycerol to produce phosphatidic acid. This is an important second messenger in a pathway of lipid signaling that has been implicated in epilepsy and other neurological diseases. Disruption of DGKD by a t(X;2) has been previously reported in a patient with epilepsy. The 2q37 microduplication was inherited from her mother, who never experienced epileptic seizures, thus this imbalance is not 'per se' sufficient to cause epilepsy. It can be hypothesized that the epileptic phenotype is provoked by the simultaneous presence of 22q11.2 deletion and 2q37 duplication. It has been shown that rare additional copy number variations outside the 22q11.2 region may modulate the risk of congenital heart defects. It is possible that also for the epileptic phenotype, the additional CNVs may represent an important modifying factor underlying the variable expressivity and incomplete penetrance in the 22q11DS.

We present on a new case of myelodysplastic syndrome characterized by array Comparative Genomic Hybridization. This technique confirmed the monosomy 7, detected by conventional cytogenetics, and revealed also a deletion on the short arm of chromosome 12. This deletion extends for about 14.8 Mb and breaks ETV6 gene. 12p deletion extents in hematological malignancies may vary, but the minimally deleted region almost invariably contains ETV6, that is considered the main candidate tumor suppressor genes within the region for tumor progression. It has been shown that levels of ETV6 were significantly decreased in cases with 12p13 deletions, whereas expression of other genes in the deleted region, like BCL2L14, LRP6, DUSP16 and GPRC5D, did not show any variation, independently of their copy number status. This observation strengthens the fact that ETV6 may play a potential role in the tumorigenesis process. The role of ETV6 in our patient myelodysplastic syndrome is showed by his clinical history and his poor prognosis.

Maternal uniparental disomy of chromosome 14 (upd(14)mat) or Temple syndrome is an imprinting disorder associated with a relatively mild phenotype. The absence of specific congenital malformations makes this condition underdiagnosed in clinical practice. A boy with a de novo robertsonian translocation 45,XY,rob(13;14)(q10;q10) is reported; a CGH/SNP array showed a loss of heterozygosity in 14q11.2q13.1. The final diagnosis of upd(14)mat was made by microsatellite analysis, which showed a combination of heterodisomy and isodisomy for different regions of chromosome 14. Obesity after initial failure to thrive developed, while compulsive eating habits were not present, which was helpful for the clinical differential diagnosis of Prader-Willi syndrome. In addition, the boy presented with many phenotypic features associated with upd(14)mat along with hypoesthesia to pain, previously unreported in this disorder, and bilateral cryptorchidism, also rarely described. These features, as well as other clinical manifestations (i.e., truncal obesity, altered pubertal timing), may suggest a hypothalamic-pituitary involvement. A detailed cytogenetic and molecular characterization of the genomic rearrangement is presented. Early genetic diagnosis permits a specific follow-up of children with upd(14)mat in order to optimize the long-term outcome.

After double-strand break induction, formation of ?-H2AX foci due to phosphorylation at Ser-139 of histone H2AX represents an early event of the DNA damage response (DDR). ?-H2AX foci are then rapidly dephosphorylated as signal for the subsequent recruitment of effector proteins. The induction and disappearance of the foci can be, therefore, used to monitor the functioning of the DDR machinery in a cell population exposed to genotoxic stress. Here, we investigated the time-course of ?-H2AX in unstimulated or cultured peripheral lymphocytes in vitro treated with UVB, bleomycin and mitomycin C (MMC). Once the mutagen exposure was performed, cells were harvested at different interval times from 0.5 to 5h. The results show that (i) in 20-h stimulated peripheral lymphocytes, UVB irradiation caused extensive and dose-dependent increases in nuclear phosphorylation, and disappearance of ?-H2AX foci progressed, proportionally to the UV fluence, with increasing the harvesting time; (ii) UVB-exposed G0 cells cultured for 20-h post-irradiation displayed low amounts of DNA phosphorylation, depicting a time-course in which the maximum effect was reached at 0.5h and dephosphorylation started after 1h; (iii) treatment of unstimulated lymphocytes with bleomycin sulphate induced an increase in nuclear phosphorylation of several folds higher than that of untreated cells, depicting kinetics comparable to those observed for UVB-exposed G1 cells; (iv) in stimulated cells, MMC caused a severe and dose-dependent high degree of H2AX phosphorylation together with a very slower kinetic of dephosphorylation with respect to the other experimental treatments. This study confirms the feasibility of the ?-H2AX focus assay as a genotoxic end-point and supports the view that the proposed type of analysis should be introduced in biomonitoring studies of human populations. This could also represent a feasible and useful tool in the screening and diagnosis of precancerous states or very early stages of other diseases.

Among natural chemicals used as cancer chemo-preventive and/or chemotherapeutic agents, saponins represent one of the most promising and interesting family of compounds. In this work, we aimed to elucidate the biological effects on human cells of six saponin fractions (SFs) obtained from in vitro cultures of Astragalus verrucosus Moris, a poorly characterized species. Interestingly, SF (3) showed a strongly inhibitory effect on the proliferation of human colon adenocarcinoma cell line (HCT116) via activation of a p53-dependent apoptotic pathway. In addition, SF (3) and the other SFs did not display genotoxic activity in human peripheral lymphocytes.