Long read sequencing on its way to the routine diagnostics of genetic diseases.

The clinical application of technological progress in the identification of DNA alterations has always led to improvements of diagnostic yields in genetic medicine. At chromosome side, from cytogenetic techniques evaluating number and gross structural defects to genomic microarrays detecting cryptic copy number variants, and at molecular level, from Sanger method studying the nucleotide sequence of single genes to the high-throughput next-generation sequencing (NGS) technologies, resolution and sensitivity progressively increased expanding considerably the range of detectable DNA anomalies and alongside of Mendelian disorders with known genetic causes. However, particular genomic regions (i.e., repetitive and GC-rich sequences) are inefficiently analyzed by standard genetic tests, still relying on laborious, time-consuming and low-sensitive approaches (i.e., southern-blot for repeat expansion or long-PCR for genes with highly homologous pseudogenes), accounting for at least part of the patients with undiagnosed genetic disorders. Third generation sequencing, generating long reads with improved mappability, is more suitable for the detection of structural alterations and defects in hardly accessible genomic regions. Although recently implemented and not yet clinically available, long read sequencing (LRS) technologies have already shown their potential in genetic medicine research that might greatly impact on diagnostic yield and reporting times, through their translation to clinical settings. The main investigated LRS application concerns the identification of structural variants and repeat expansions, probably because techniques for their detection have not evolved as rapidly as those dedicated to single nucleotide variants (SNV) identification: gold standard analyses are karyotyping and microarrays for balanced and unbalanced chromosome rearrangements, respectively, and southern blot and repeat-primed PCR for the amplification and sizing of expanded alleles, impaired by limited resolution and sensitivity that have not been significantly improved by the advent of NGS. Nevertheless, more recently, with the increased accuracy provided by the latest product releases, LRS has been tested also for SNV detection, especially in genes with highly homologous pseudogenes and for haplotype reconstruction to assess the parental origin of alleles with de novo pathogenic variants. We provide a review of relevant recent scientific papers exploring LRS potential in the diagnosis of genetic diseases and its potential future applications in routine genetic testing.

Ultra-Rare Variants Identify Biological Pathways and Candidate Genes in the Pathobiology of Non-Syndromic Cleft Palate Only.

In recent decades, many efforts have been made to elucidate the genetic causes of non-syndromic cleft palate (nsCPO), a complex congenital disease caused by the interaction of several genetic and environmental factors. Since genome-wide association studies have evidenced a minor contribution of common polymorphisms in nsCPO inheritance, we used whole exome sequencing data to explore the role of ultra-rare variants in this study. In a cohort of 35 nsCPO cases and 38 controls, we performed a gene set enrichment analysis (GSEA) and a hypergeometric test for assessing significant overlap between genes implicated in nsCPO pathobiology and genes enriched in ultra-rare variants in our cohort. GSEA highlighted an enrichment of ultra-rare variants in genes principally belonging to cytoskeletal protein binding pathway (Probability Density Function corrected p-value = 1.57 × 10-4); protein-containing complex binding pathway (p-value = 1.06 × 10-2); cell adhesion molecule binding pathway (p-value = 1.24 × 10-2); ECM-receptor interaction pathway (p-value = 1.69 × 10-2); and in the Integrin signaling pathway (p-value = 1.28 × 10-2). Two genes implicated in nsCPO pathobiology, namely COL2A1 and GLI3, ranked among the genes (n = 34) with nominal enrichment in the ultra-rare variant collapsing analysis (Fisher's exact test p-value < 0.05). These genes were also part of an independent list of genes highly relevant to nsCPO biology (n = 25). Significant overlap between the two sets of genes (hypergeometric test p-value = 5.86 × 10-3) indicated that enriched genes are likely to be implicated in physiological palate development and/or the pathological processes of oral clefting. In conclusion, ultra-rare variants collectively impinge on biological pathways crucial to nsCPO pathobiology and point to candidate genes that may contribute to the individual risk of disease. Sequencing can be an effective approach to identify candidate genes and pathways for nsCPO.

Exploration of Tools for the Interpretation of Human Non-Coding Variants.

The advent of Whole Genome Sequencing (WGS) broadened the genetic variation detection range, revealing the presence of variants even in non-coding regions of the genome, which would have been missed using targeted approaches. One of the most challenging issues in WGS analysis regards the interpretation of annotated variants. This review focuses on tools suitable for the functional annotation of variants falling into non-coding regions. It couples the description of non-coding genomic areas with the results and performance of existing tools for a functional interpretation of the effect of variants in these regions. Tools were tested in a controlled genomic scenario, representing the ground-truth and allowing us to determine software performance.

Defective lipid signalling caused by mutations in PIK3C2B underlies focal epilepsy.

Epilepsy is one of the most frequent neurological diseases, with focal epilepsy accounting for the largest number of cases. The genetic alterations involved in focal epilepsy are far from being fully elucidated. Here, we show that defective lipid signalling caused by heterozygous ultra-rare variants in PIK3C2B, encoding for the class II phosphatidylinositol 3-kinase PI3K-C2ß, underlie focal epilepsy in humans. We demonstrate that patients' variants act as loss-of-function alleles, leading to impaired synthesis of the rare signalling lipid phosphatidylinositol 3,4-bisphosphate, resulting in mTORC1 hyperactivation. In vivo, mutant Pik3c2b alleles caused dose-dependent neuronal hyperexcitability and increased seizure susceptibility, indicating haploinsufficiency as a key driver of disease. Moreover, acute mTORC1 inhibition in mutant mice prevented experimentally induced seizures, providing a potential therapeutic option for a selective group of patients with focal epilepsy. Our findings reveal an unexpected role for class II PI3K-mediated lipid signalling in regulating mTORC1-dependent neuronal excitability in mice and humans.

unCOVERApp: an interactive graphical application for clinical assessment of sequence coverage at the base-pair level.

MOTIVATION: Next-generation sequencing is increasingly adopted in the clinical practice largely thanks to concurrent advancements in bioinformatic tools for variant detection and annotation. However, the need to assess sequencing quality at the base-pair level still poses challenges for diagnostic accuracy. One of the most popular quality parameters is the percentage of targeted bases characterized by low depth of coverage (DoC). These regions potentially 'hide' clinically relevant variants, but no annotation is usually returned with them. However, visualizing low-DoC data with their potential functional and clinical consequences may be useful to prioritize inspection of specific regions before re-sequencing all coverage gaps or making assertions about completeness of the diagnostic test. To meet this need, we have developed unCOVERApp, an interactive application for graphical inspection and clinical annotation of low-DoC genomic regions containing genes. RESULTS: unCOVERApp interactive plots allow to display gene sequence coverage down to the base-pair level, and functional and clinical annotations of sites below a user-defined DoC threshold can be downloaded in a user-friendly spreadsheet format. Moreover, unCOVERApp provides a simple statistical framework to evaluate if DoC is sufficient for the detection of somatic variants. A maximum credible allele frequency calculator is also available allowing users to set allele frequency cut-offs based on assumptions about the genetic architecture of the disease. In conclusion, unCOVERApp is an original tool designed to identify sites of potential clinical interest that may be 'hidden' in diagnostic sequencing data. AVAILABILITYAND IMPLEMENTATION: unCOVERApp is a free application developed with Shiny packages and available in GitHub (https://github.com/Manuelaio/uncoverappLib). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

SLC12A2 variants cause a neurodevelopmental disorder or cochleovestibular defect.

The SLC12 gene family consists of SLC12A1-SLC12A9, encoding electroneutral cation-coupled chloride co-transporters. SCL12A2 has been shown to play a role in corticogenesis and therefore represents a strong candidate neurodevelopmental disorder gene. Through trio exome sequencing we identified de novo mutations in SLC12A2 in six children with neurodevelopmental disorders. All had developmental delay or intellectual disability ranging from mild to severe. Two had sensorineural deafness. We also identified SLC12A2 variants in three individuals with non-syndromic bilateral sensorineural hearing loss and vestibular areflexia. The SLC12A2 de novo mutation rate was demonstrated to be significantly elevated in the deciphering developmental disorders cohort. All tested variants were shown to reduce co-transporter function in Xenopus laevis oocytes. Analysis of SLC12A2 expression in foetal brain at 16-18 weeks post-conception revealed high expression in radial glial cells, compatible with a role in neurogenesis. Gene co-expression analysis in cells robustly expressing SLC12A2 at 16-18 weeks post-conception identified a transcriptomic programme associated with active neurogenesis. We identify SLC12A2 de novo mutations as the cause of a novel neurodevelopmental disorder and bilateral non-syndromic sensorineural hearing loss and provide further data supporting a role for this gene in human neurodevelopment.

Brain Angiotensinergic Regulation of the Immune System: Implications for Cardiovascular and Neuroendocrine Responses.

OBJECTIVE: The Renin-Angiotensin-Aldosterone System (RAAS) plays a major role in the regulation of cardiovascular functions, water and electrolytic balance, and hormonal responses. We perform a review of the literature, aiming at providing the current concepts regarding the angiotensin interaction with the immune system in the brain and the related implications for cardiovascular and neuroendocrine responses. METHODS: Appropriate keywords and MeSH terms were identified and searched in Pubmed. Finally, references of original articles and reviews were examined. RESULTS: Angiotensin II (ANG II), beside stimulating aldosterone, vasopressin and CRH-ACTH release, sodium and water retention, thirst, and sympathetic nerve activity, exerts its effects on the immune system via the Angiotensin Type 1 Receptor (AT 1R) that is located in the brain, pituitary, adrenal gland, and kidney. Several actions are triggered by the binding of circulating ANG II to AT 1R into the circumventricular organs that lack the Blood-Brain-Barrier (BBB). Furthermore, the BBB becomes permeable during chronic hypertension thereby ANG II may also access brain nuclei controlling cardiovascular functions. Subfornical organ, organum vasculosum lamina terminalis, area postrema, paraventricular nucleus, septal nuclei, amygdala, nucleus of the solitary tract and retroventral lateral medulla oblongata are the brain structures that mediate the actions of ANG II since they are provided with a high concentration of AT 1R. ANG II induces also T-lymphocyte activation and vascular infiltration of leukocytes and, moreover, oxidative stress stimulating inflammatory responses via inhibition of endothelial progenitor cells and stimulation of inflammatory and microglial cells facilitating the development of hypertension. CONCLUSION: Besides the well-known mechanisms by which RAAS activation can lead to the development of hypertension, the interactions between ANG II and the immune system at the brain level can play a significant role.

Neuroendocrine Mechanisms Involved in Male Sexual and Emotional Behavior.

OBJECTIVE: The aim of this narrative review was to analyze the role played by brain areas, neurohormones and neurotransmitters in the regulation of emotional and sexual behavior in the male. METHODS: We analyzed the currently available literature dealing with brain structures, neurotransmitters and neurohormones involved in the regulation of emotional and sexual behavior in the male. RESULTS: A common brain pathway is involved in these two aspects. The Hippocampus seems to control the signals coming from the external environment, while the amygdala and the hypothalamus control the response to social stimuli. Stimulation of amygdala in the animal models increases sexual performance, while it triggers violent emotional responses. Stimulation of the hypothalamus causes reactions of violent anger and increases sexual activity. Catecholaminergic stimulation of the amygdala and hypothalamus increases emotional and sexual behavior, while serotonin plays an inhibitory role. Cholinergic inhibition leads to a suppression of copulatory activity, while the animal becomes hyperemotive. Opioids, such as ß-endorphin and met-enkephalin, reduce copulatory activity and induce impotence. Gonadal steroid hormones, such as estrogen in female and testosterone in male, which play a major role in the control of sexual behavior and gender difference have been highlighted in this review. Vasopressin, oxytocin and their receptors are expressed in high density in the "social behavior neural network" and play a role as signal system controlling social behavior. Finally, the neuropeptide kisspeptin and its receptors, located in the limbic structures, mediate olfactory control of the gonadotropic axis. CONCLUSION: Further studies are needed to evaluate possible implications in the treatment of psychosexual and reproductive disorders.

Vigilance States: Central Neural Pathways, Neurotransmitters and Neurohormones.

BACKGROUND AND OBJECTIVE: The sleep-wake cycle is characterized by a circadian rhythm involving neurotransmitters and neurohormones that are released from brainstem nuclei and hypothalamus. The aim of this review is to analyze the role played by central neural pathways, neurotransmitters and neurohormones in the regulation of vigilance states. METHOD: We analyzed the literature identifying relevant articles dealing with central neural pathways, neurotransmitters and neurohormones involved in the control of wakefulness and sleep. RESULTS: The reticular activating system is the key center in the control of the states of wakefulness and sleep via alertness and hypnogenic centers. Neurotransmitters and neurohormones interplay during the dark-light cycle in order to maintain a normal plasmatic concentration of ions, proteins and peripheral hormones, and behavioral state control. CONCLUSION: An updated description of pathways, neurotransmitters and neurohormones involved in the regulation of vigilance states has been depicted.

Vasopressin in Heart Failure.

BACKGROUND AND OBJECTIVE: The nonapeptide hypothalamic hormone vasopressin (VP), exerts important effects on cardiovascular system via its receptors V1, V2 and V3. Patients with congestive heart failure (CHF) present elevated plasma VP levels. Aim of this paper is to review the role of vasopressin in CHF. METHODS: We analyzed the best of published literature dealing with the role of VP in patients affected by CHF, identifying keywords and MeSH terms in Pubmed and then searching them. The last search was performed on August 2017. RESULTS: Scientific articles dealing with the relationship between VP and CHF show that circulating high VP levels found in CHF despite an exaggerated increase in circulatory blood volume can contribute to CHF exacerbation. In particular, the stimulation of V1R induces vascular constriction responsible for increased systemic vascular resistance and afterload, and, in addition, coronary vasoconstriction with consequent reduced coronary circulation and cardiac contractility, whereas the stimulation of V2R induces free water reabsorption and this is responsible of preload increase and congestion of pulmonary vascular bed with edema and hyponatremia, markers of advanced CHF. CONCLUSION: VP can play an important role among the derangements of the endocrine system in CHF even being a possible target in the treatment of this condition. Vaptans, antagonists of VP receptors, in fact, are able to increase urine output and plasma sodium levels without the increased risk of arrhythmic death induced by diuretics, even though, further studies are needed to establish a possible role of these drugs in the treatment of CHF.

The Role of Neurohypophyseal Hormones Vasopressin and Oxytocin in Neuropsychiatric Disorders.

BACKGROUND AND OBJECTIVE: Although the neurohypophyseal hormones vasopressin (VP) and oxytocin (OT) are mostly known for their role respectively in antidiuresis, and in labour, lactation and maternal behavior, both might exert widespread influences either on emotion and cognition in healthy subjects, showing some gender-related differences. They interact with each other facilitating shifts between positive socially- oriented and defensive states. In fact, VP amplifies the reactivity to stressors showing also beneficial effects on attention, verbal learning as well as memory, whereas OT reduces the amplitude of the stress response, improves emotion processing, and can play a negative effect on memory and verbal learning in healthy individuals. Several data indicate the possible involvement of these neuropeptides in the pathophysiology of psychiatric conditions involving social interactions, such as autism, as well as in schizophrenia and depression. The aim of this paper is to review the literature relating to the role played by neurohypophyseal hormones in neuropsychiatric disorders. METHODS: We analyzed the best of published literature dealing with the relationships between neurohypophyseal hormones and neuropsychiatric conditions like autism (AD), major depressive disorder (MDD), bipolar disorder (BD) and schozophrenia, identifying keywords and MeSH terms in Pubmed and then searching them. The last search was performed on December 2017. RESULTS: Several studies indicate a role played by OT and VP in AD, schizophrenia, MDD and BD. Even if conflicting data have been reported, several mechanisms may be involved in these behavioral diseases, such as differences in aminoacid sequence and peptide biological activity, neurotransmission and genetic disorders involving OT and VP receptors. CONCLUSION: The involvment of VP and OT in neurpopsychiatric disorders can support a possible beneficial therapy with OT or with VP antagonists. The target may be obtained using effective drug delivery methods as well as the association with other drugs.

Synaptic Inputs of Neural Afferent Pathways to Vasopressin- and Oxytocin-Secreting Neurons of Supraoptic and Paraventricular Hypothalamic Nuclei.

BACKGROUND: Magnocellular neurosecretory neurons of the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei synthesize vasopressin and oxytocin in response to signals generated by osmoreceptors and baroreceptors and, respectively, by receptors of the nipples and cervix. METHODS: We analyzed the literature identifying relevant articles dealing with synaptic inputs of neural afferent pathways to Vasopressin-and Oxytocin-secreting neurons of SON and PVN. RESULTS: This article focuses on the multisynaptic pathways involved in the regulation of Vasopressin and Oxytocin secretion. CONCLUSION: An updated topographic description of the afferent pathways involved in the regulation of VPergic and OTergic neurons and their synaptic inputs inducing the stimulus-secretion-coupling has been depicted.

Role of central and peripheral chemoreceptors in vasopressin secretion control.

In this review, we analyzed the role played by central and peripheral chemoreceptors (CHRs) in vasopressin (AVP) secretion control. Central neural pathways subserving osmotic and non-osmotic control of AVP secretion are strictly correlated to brain areas participating in chemoreception mechanisms. Among the different brain areas involved in central chemoreception, the most important site has been localized in the retrotrapezoid nucleus of the rostral ventrolateral medulla. These central CHRs are able to detect very small pH/CO2 fluctuations, participating in brain blood flow regulation, acid-base balance and blood pressure control. Decreases in arterial pH and increases in arterial pCO2 stimulate AVP release by the Supraoptic and Paraventricular Nuclei. Carotid CHRs transduce low arterial O2 tension into increased action potential activity, leading to bradycardia and coronary vasodilatation via vagal stimulation, and systemic vasoconstriction via catecholaminergic stimulation. Stimulation of carotid CHRs by hypoxia increases neurohypophyseal blood flow and AVP release, an effect inhibited by CHRs denervation. Two renal CHRs have been identified: Type R1 CHRs do not have a resting discharge but are activated by renal ischemia and hypotension; Type R2 CHRs have a resting discharge and respond to backflow of urine into the renal pelvis. Signals arising from renal CHRs modulate the activity of hypothalamic AVPergic neurons: activation of R1 and R2 CHRs, following increased intrapelvic pressure with solutions of mannitol, NaCl and KCl, produces a significant increase of AVP secretion and the same effect has been obtained by the intrarenal infusion of bradykinin, which excites afferent renal nerves, as well as by the electrical stimulation of these nerves.