A new gene for autosomal dominant facial palsy/migraine identified in a family by whole exome sequencing.

BACKGROUND: Facial palsy manifests as unilateral or bilateral weakness and inability to move some of the facial muscles. The aetiology may be different including idiopathic, trauma, infections or brain tumours or it can be associated with chronic neurological diseases. For instance, in recurrent migraine, an increased risk of idiopathic facial palsy (often unilateral) has been observed. Migraine is a neurovascular disorder characterized by mild to severe intensity of headaches, often associated with neuro-ophthalmological symptoms. METHODS: A family is reported where five members were affected by facial palsy associated with other clinical features including migraine, diplopia, facial swelling, eye conjunctivitis following a vertical transmission. Whole exome sequencing was performed in three members (two affected and one healthy) in order to identify potential variants causative of their phenotype. RESULTS: A missense variant c.304G>A was found leading to the p.(Ala102Thr) substitution in the TRPM8 gene, previously related to migraine by genome wide association studies. This variant was classified as deleterious by several predictor tools, and the mutant residue was predicted to alter the protein structure in terms of flexibility and interactions with the surrounding residues. CONCLUSION: These findings suggest that TRPM8 could be a new causative gene further linking migraine and recurrent facial palsy.

Results of a Gene Panel Approach in a Cohort of Patients with Incomplete Distal Renal Tubular Acidosis and Nephrolithiasis.

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.

Identification of new candidate genes for spina bifida through exome sequencing.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

A 6.5 mb deletion at 3q24q25.2 narrows Wisconsin syndrome critical region to a 750 kb interval: A potential role for MBNLI.

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.

Exploring the Role of the MUTYH Gene in Breast, Ovarian and Endometrial Cancer.

BACKGROUND: MUTYH germline monoallelic variants have been detected in a number of patients affected by breast/ovarian cancer or endometrial cancer, suggesting a potential susceptibility role, though their significance remains elusive since the disease mechanism is normally recessive. Hence, the aim of this research was to explore the hypothesis that a second hit could have arisen in the other allele in the tumor tissue. METHODS: we used Sanger sequencing and immunohistochemistry to search for a second MUTYH variant in the tumoral DNA and to assess protein expression, respectively. RESULTS: we detected one variant of unknown significance, one variant with conflicting interpretation of pathogenicity and three benign/likely benign variants; the MUTYH protein was not detected in the tumor tissue of half of the patients, and in others, its expression was reduced. CONCLUSIONS: our results fail to demonstrate that germinal monoallelic MUTYH variants increase cancer risk through a LOH (loss of heterozygosity) mechanism in the somatic tissue; however, the absence or partial loss of the MUTYH protein in many tumors suggests its dysregulation regardless of MUTYH genetic status.

Kinetics of nuclear phosphorylation (γ-H2AX) in human lymphocytes treated in vitro with UVB, bleomycin and mitomycin C.

After double-strand break induction, formation of γ-H2AX foci due to phosphorylation at Ser-139 of histone H2AX represents an early event of the DNA damage response (DDR). γ-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 γ-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 γ-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 γ-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.

RADX Gene Variant May Predispose to Familial Asperger Syndrome.

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.

Deletion of a Single Lysine Residue at Position 292 of CAMK2A Disrupts Protein Function, Causing Severe Epileptic Encephalopathy and Intellectual Disability.

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.

Melkersson-Rosenthal Syndrome and Migraine: A New Phenotype Associated with SCN1A Variants?

Peripheral facial palsy rarely occurs as part of Melkersson-Rosenthal syndrome (MRS), which is characterized by the classical triad of tongue cheilitis, recurrent episodes of orofacial swelling, and palsy. MRS is a disorder with variable expressivity and clinical as well as genetic heterogeneity; however, the causative gene remains to be identified. Migraine is a common neurological disorder, presenting with or without aura, which may be associated with neurological symptoms. The classical example of monogenic migraine is familial hemiplegic migraine (FHM), which has phenotypic variability in carriers of variants in the same gene or even carriers of the same variant. We present a family in which two sisters displayed recurrent migraines, one of which presented recurrent facial palsy and had clinical diagnosis of MRS. We performed WES and Sanger sequencing for segregation analysis in the available family members. We identified a c.3521C>G missense heterozygous variant in SCN1A carried only by the affected sister. Variants in the SCN1A gene can cause a spectrum of early-onset epileptic encephalopathies, in addition to FHM; therefore, our finding reasonably explains the proband phenotype, in which the main symptom was recurrent facial palsy. This report also adds knowledge to the clinical spectrum of SCN1A alterations and suggests a potential overlap between MRS and FHM.

Identification by Exome Sequencing of Predisposing Variants in Familial Cases of Autoinflammatory Recurrent Fevers.

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.

Maternal Epigenetic Dysregulation as a Possible Risk Factor for Neurodevelopmental 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.

A pathogenic variant in the FLCN gene presenting with pure dementia: is autophagy at the intersection between neurodegeneration and cancer?

Introduction: Folliculin, encoded by FLCN gene, plays a role in the mTORC1 autophagy cascade and its alterations are responsible for the Birt-Hogg-Dubé (BHD) syndrome, characterized by follicle hamartomas, kidney tumors and pneumothorax. Patient and results: We report a 74-years-old woman diagnosed with dementia and carrying a FLCN alteration in absence of any sign of BHD. She also carried an alteration of MAT1A gene, which is also implicated in the regulation of mTORC1. Discussion: The MAT1A variant could have prevented the development of a FLCN-related oncological phenotype. Conversely, our patient presented with dementia that, to date, has yet to be documented in BHD. Folliculin belongs to the DENN family proteins, which includes C9orf72 whose alteration has been associated to neurodegeneration. The folliculin perturbation could affect the C9orf72 activity and our patient could represent the first human model of a relationship between FLCN and C9orf72 across the path of autophagy.

Genetic Dysruption of the Histaminergic Pathways: A Novel Deletion at the 15q21.2 locus Associated with Variable Expressivity of Neuropsychiatric Disorders.

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.

Genetic Characterization in Familial Rotator Cuff Tear: An Exome Sequencing Study.

Background: multiple gene variants seem to contribute to rotator cuff (RC) tear susceptibility. The aim of the study is to perform an exome sequencing analysis within a family to identify rare gene variants predisposing to the development of RC tear. Material and methods: the exome sequencing was conducted in a family consisting of four individuals, two healthy and the remaining ones with bilateral RC tears. Variants in common among the two affected subjects were selected, and those in common with the healthy subject and those with a frequency >1% were removed. The potential pathogenicity of the variants was investigated using the predictions of several in silico tools from VarSome. Results: the exome sequencing yielded approximately 600,000 variants per patient, subsequently filtered according to frequency <1% and absence of association with other diseases. Removing variants common with the healthy subject, 348 rare variants among 248 genes were identified. Based on the risk of damaging, three candidate genes for RC tear were found: COL23A1, EMILIN3, and HDAC10. Conclusion: this is the first whole-exome sequencing analysis within a family to explore genetic predisposition in RC tear. The results reveal the presence of common damaging variants among affected individuals in the COL23A1, EMILIN3, and HDAC10 genes.

Whole exome sequencing identifies a rare variant in MAS1 gene in a subject with lethal COVID-19.

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.446C>T p.(S149L) in MAS1 gene was exclusively present in the individual with severe COVID-19, who died because of pneumonia and multiple thrombotic events. MAS1 encodes a receptor for Ang1-7 in the renin-angiotensin system (RAS) with an anti-inflammatory, anti-fibrotic and anti-angiogenic effect. We hypothesize that downregulation of RAS, due to this rare variant, might impair the protective effect and concur to the clinical severity of the disease. Our results support the protective role of the ACE2/Ang-(1-7)/Mas1 axis and the potential danger of its dysregulation leading to severe COVID-19 disease; if further confirmed, these findings will be useful for management of critically ill patients.

Genetic variants determine intrafamilial variability of SARS-CoV-2 clinical outcomes in 19 Italian families.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection results in a wide range of outcomes characterized by a high heterogeneity in both symptomatology and susceptibility to the disease. In such a perspective, COVID-19 may be considered as a multifactorial disease featured by the interaction between the environment, which is the virus itself, and the genetic profile of the host. Our analysis aimed at investigating the transmission dynamics of SARS-CoV-2 within families whose members responded in different ways to the infection, although the exposure was common to the entire group and occurred before the availability of any vaccine. The goal was to understand how the genetic background of each subject can influence the viral infection outcome and hence the above-mentioned clinical variability. We performed a segregation analysis in 19 Italian families with a designed custom panel of 42 genes involved in immunity and virus entry and which have also been shown to be related to SARS-CoV-2 host response. We carried out both a familial segregation analysis and a global statistical analysis. In the former we identified eighteen risk variants co-segregating with a COVID-positive status and six variants with a possible protective effect. In addition, sixteen variants showed a trend of association to a severe phenotype. Together with common SNPs, we detected private rare variants that may also provide insight into the observed clinical COVID-19 heterogeneity. The global statistical analysis confirmed statistically significant positive associations between SARS-CoV-2 individual response and some specific gene variants identified in familial analysis. In conclusion our data confirm that the clinical expression of COVID-19 is markedly influenced by the host genetic profile both with a mendelian transmission pattern and a polygenic architecture.

Expanding the Spectrum of KDM5C Neurodevelopmental Disorder: A Novel De Novo Stop Variant in a Young Woman and Emerging Genotype-Phenotype Correlations.

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.

Genotype-Phenotype Correlations in Relation to Newly Emerging Monogenic Forms of Autism Spectrum Disorder and Associated Neurodevelopmental Disorders: The Importance of Phenotype Reevaluation after Pangenomic Results.

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.

Role of oxidative stress, genome damage and DNA methylation as determinants of pathological conditions in the newborn: an overview from conception to early neonatal stage.

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.