NR4A2 as a Novel Target Gene for Developmental and Epileptic Encephalopathy: A Systematic Review of Related Disorders and Therapeutic Strategies.

The NR4A2 gene encodes an orphan transcription factor of the steroid-thyroid hormone-retinoid receptor superfamily. This review focuses on the clinical findings associated with the pathogenic variants so far reported, including three unreported cases. Also, its role in neurodegenerative diseases, such as Parkinson's or Alzheimer's disease, is examined, as well as a brief exploration on recent proposals to develop novel therapies for these neurological diseases based on small molecules that could modulate NR4A2 transcriptional activity. The main characteristic shared by all patients is mild to severe developmental delay/intellectual disability. Moderate to severe disorder of the expressive and receptive language is present in at least 42%, while neuro-psychiatric issues were reported in 53% of patients. Movement disorders, including dystonia, chorea or ataxia, are described in 37% patients, although probably underestimated because of its frequent onset in late adolescence-young adulthood. Finally, epilepsy was surprisingly present in 42% of patients, being drug-resistant in three of them. The age at onset varied widely, from five months to twenty-six years, as did the classification of epilepsy, which ranged from focal epilepsy to infantile spasms or Lennox-Gastaut syndrome. Accordingly, we propose that NR4A2 should be considered as a first-tier target gene for the genetic diagnosis of developmental and epileptic encephalopathy.

N-Type Ca Channel in Epileptic Syndromes and Epilepsy: A Systematic Review of Its Genetic Variants.

N-type voltage-gated calcium channel controls the release of neurotransmitters from neurons. The association of other voltage-gated calcium channels with epilepsy is well-known. The association of N-type voltage-gated calcium channels and pain has also been established. However, the relationship between this type of calcium channel and epilepsy has not been specifically reviewed. Therefore, the present review systematically summarizes existing publications regarding the genetic associations between N-type voltage-dependent calcium channel and epilepsy.

CfDNA Measurement as a Diagnostic Tool for the Detection of Brain Somatic Mutations in Refractory Epilepsy.

Epilepsy is a neurological disorder that affects more than 50 million people. Its etiology is unknown in approximately 60% of cases, although the existence of a genetic factor is estimated in about 75% of these individuals. Hundreds of genes involved in epilepsy are known, and their number is increasing progressively, especially with next-generation sequencing techniques. However, there are still many cases in which the results of these molecular studies do not fully explain the phenotype of the patients. Somatic mutations specific to brain tissue could contribute to the phenotypic spectrum of epilepsy. Undetectable in the genomic DNA of blood cells, these alterations can be identified in cell-free DNA (cfDNA). We aim to review the current literature regarding the detection of somatic variants in cfDNA to diagnose refractory epilepsy, highlighting novel research directions and suggesting further studies.

Noninvasive prenatal testing: How far can we reach detecting fetal copy number variations.

Non-invasive prenatal testing (NIPT) is currently the best screening test for fetal chromosome abnormalities with the highest sensitivity and specificity and can be done from 10 weeks gestation. We report a detection of 44.7 Mb duplication at 11p15.5-p11.2 by NIPT with a fetal fraction (FF) of only 3%. This chromosome abnormality was confirmed after amniocentesis by karyotyping and array comparative genomic hybridization (aCGH) on cultured fetal cells. Further parental investigation showed that the fetal chromosome abnormality was inherited from the mother who was a carrier of a balanced translocation 46,XX,t(11;X)(p11.2;q28). This case highlights the importance of expanded NIPT in the detection of fetal segmental aneuploidy. NIPT together with complementary studies can lead to the detection of parental chromosome rearrangement despite a low FF, which can impact the couple's reproductive plans. We also reviewed other cases with chromosome rearrangement, detected by NIPT, derived from a parental reciprocal translocation.

Candidate Genes for Eyelid Myoclonia with Absences, Review of the Literature.

Eyelid myoclonia with absences (EMA), also known as Jeavons syndrome (JS) is a childhood onset epileptic syndrome with manifestations involving a clinical triad of absence seizures with eyelid myoclonia (EM), photosensitivity (PS), and seizures or electroencephalogram (EEG) paroxysms induced by eye closure. Although a genetic contribution to this syndrome is likely and some genetic alterations have been defined in several cases, the genes responsible for have not been identified. In this review, patients diagnosed with EMA (or EMA-like phenotype) with a genetic diagnosis are summarized. Based on this, four genes could be associated to this syndrome (SYNGAP1, KIA02022/NEXMIF, RORB, and CHD2). Moreover, although there is not enough evidence yet to consider them as candidate for EMA, three more genes present also different alterations in some patients with clinical diagnosis of the disease (SLC2A1, NAA10, and KCNB1). Therefore, a possible relationship of these genes with the disease is discussed in this review.

Recent Evidence in Epigenomics and Proteomics Biomarkers for Early and Minimally Invasive Diagnosis of Alzheimer's and Parkinson's Diseases.

BACKGROUND: Alzheimer's (AD) and Parkinson's diseases (PD) show deposits of improperly folded modified proteins. Protein expression mechanisms are involved since the early stages. Several studies evaluated epigenomics and proteomics profiles in these patients, with promising results. In general, they focused on early, specific, and minimally invasive biomarkers for the diagnosis and prognosis of AD and PD. OBJECTIVES: This review aimed at summarizing results to find the most reliable evidence in the field. RESULTS: Among epigenomics studies, there is a focus on microRNAs (miRNAs) as candidate diagnostic biomarkers for AD or PD from blood samples like miR-342-3p, miR-107, miR-106a-5p, miR-106b- 5p, miR-195, and miR-19b. In addition, DNA methylation has been tested in a few works, obtaining significant differences in some genes (NCAPH2/LMF2 COASY, SPINT1, BDNFTREM1, TREM2, NPAS2, PDE4D), which could be useful for evaluating the disease progression as well as potential risk factors. Regarding proteomics, most of the studies were untargeted and used plasma or serum samples. In general, they highlighted the importance of coagulation, inflammation pathways, and oxidative stress. Among targeted studies, some proteins (phosphorylated tau, C reactive protein (CRP), interleukins, necrosis factors, transferrin, glial fibrillary acidic protein (GFAP), and neurofilaments) showed different plasma levels in AD and PD patients in comparison with healthy participants. Finally, a few studies have identified specific-AD and PD epigenetic and proteomic biomarkers (ApoE and oxidized DJ-1) in comparison with other similar pathologies. CONCLUSION: In general, there is a common lack of clinical validation of these potential biomarkers because of which its use in clinical practice is still limited.

Noninvasive Prenatal Testing: Comparison of Two Mappers and Influence in the Diagnostic Yield.

OBJECTIVE: The aim of this study was to determine if the use of different mappers for NIPT may vary the results considerably. METHODS: Peripheral blood was collected from 217 pregnant women, 58 pathological (34 pregnancies with trisomy 21, 18 with trisomy 18, and 6 with trisomy 13) and 159 euploid. MPS was performed following a manufacturer's modified protocol of semiconductor sequencing. Obtained reads were mapped with two different software programs: TMAP and HPG-Aligner, comparing the results. RESULTS: Using TMAP, 57 pathological samples were correctly detected (sensitivity 98.28%, specificity 93.08%): 33 samples as trisomy 21 (sensitivity 97.06%, specificity 99.45%), 16 as trisomy 18 (sensibility 88.89%, specificity 93.97%), and 6 as trisomy 13 (sensibility 100%, specificity 100%). 11 false positives, 1 false negative, and 2 samples incorrectly identified were obtained. Using HPG-Aligner, all the 58 pathological samples were correctly identified (sensibility 100%, specificity 96.86%): 34 as trisomy 21 (sensibility 100%, specificity 98.91%), 18 as trisomy 18 (sensibility 100%, specificity 98.99%), and 6 as trisomy 13 (sensibility 100%, specificity 99.53%). 5 false positives were obtained. CONCLUSION: Different mappers use slightly different algorithms, so the use of one mapper or another with the same batch file can provide different results.

Encefalopatía epiléptica del lactante: lo prioritario es un buen estudio genético / Infantile epileptic encephalopathy: a good genetic study should be a priority

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Chimeric Genes in Deletions and Duplications Associated with Intellectual Disability.

We report on three nonrelated patients with intellectual disability and CNVs that give rise to three new chimeric genes. All the genes forming these fusion transcripts may have an important role in central nervous system development and/or in gene expression regulation, and therefore not only their deletion or duplication but also the resulting chimeric gene may contribute to the phenotype of the patients. Deletions and duplications are usually pathogenic when affecting dose-sensitive genes. Alternatively, a chimeric gene may also be pathogenic by different gain-of-function mechanisms that are not restricted to dose-sensitive genes: the emergence of a new polypeptide that combines functional domains from two different genes, the deregulated expression of any coding sequence by the promoter region of a neighboring gene, and/or a putative dominant-negative effect due to the preservation of functional domains of partially truncated proteins. Fusion oncogenes are well known, but in other pathologies, the search for chimeric genes is disregarded. According to our findings, we hypothesize that the frequency of fusion transcripts may be much higher than suspected, and it should be taken into account in the array-CGH analyses of patients with intellectual disability.

High diagnostic yield of syndromic intellectual disability by targeted next-generation sequencing.

BACKGROUND: Intellectual disability is a very complex condition where more than 600 genes have been reported. Due to this extraordinary heterogeneity, a large proportion of patients remain without a specific diagnosis and genetic counselling. The need for new methodological strategies in order to detect a greater number of mutations in multiple genes is therefore crucial. METHODS: In this work, we screened a large panel of 1256 genes (646 pathogenic, 610 candidate) by next-generation sequencing to determine the molecular aetiology of syndromic intellectual disability. A total of 92 patients, negative for previous genetic analyses, were studied together with their parents. Clinically relevant variants were validated by conventional sequencing. RESULTS: A definitive diagnosis was achieved in 29 families by testing the 646 known pathogenic genes. Mutations were found in 25 different genes, where only the genes KMT2D, KMT2A and MED13L were found mutated in more than one patient. A preponderance of de novo mutations was noted even among the X linked conditions. Additionally, seven de novo probably pathogenic mutations were found in the candidate genes AGO1, JARID2, SIN3B, FBXO11, MAP3K7, HDAC2 and SMARCC2. Altogether, this means a diagnostic yield of 39% of the cases (95% CI 30% to 49%). CONCLUSIONS: The developed panel proved to be efficient and suitable for the genetic diagnosis of syndromic intellectual disability in a clinical setting. Next-generation sequencing has the potential for high-throughput identification of genetic variations, although the challenges of an adequate clinical interpretation of these variants and the knowledge on further unknown genes causing intellectual disability remain to be solved.

Intragenic CNVs for epigenetic regulatory genes in intellectual disability: Survey identifies pathogenic and benign single exon changes.

The disruption of genes involved in epigenetic regulation is well known to cause Intellectual Disability (ID). We reported a custom microarray study that interrogated among others, the epigenetic regulatory gene-class, at single exon resolution. Here we elaborate on identified intragenic CNVs involving epigenetic regulatory genes; specifically discussing those in three genes previously unreported in ID etiology-ARID2, KDM3A, and ARID4B. The changes in ARID2 and KDM3A are likely pathogenic while the ARID4B variant is uncertain. Previously, we found a CNV involving only exon 6 of the JARID2 gene occurred apparently de novo in seven patients. JARID2 is known to cause ID and other neurodevelopmental conditions. However, exon 6 of this gene encodes one of a series of repeated motifs. We therefore, investigated the impact of this variant in two cohorts and present a genotype-phenotype assessment. We find the JARID2 exon 6 CNV is benign, with a high population frequency (>14%), but nevertheless could have a contributory effect. We also present results from an interrogation of the exomes of 2,044 patients with neurocognitive phenotypes for the incidence of potentially damaging mutation in the epigenetic regulatory gene-class. This paper provides a survey of the fine-scale CNV landscape for epigenetic regulatory genes in the context of ID, describing likely pathogenic as well as benign single exon imbalances. © 2016 Wiley Periodicals, Inc.

De novo mutations in genes of mediator complex causing syndromic intellectual disability: mediatorpathy or transcriptomopathy?

BACKGROUND: Mutations in the X-linked gene MED12 cause at least three different, but closely related, entities of syndromic intellectual disability. Recently, a new syndrome caused by MED13L deleterious variants has been described, which shows similar clinical manifestations including intellectual disability, hypotonia, and other congenital anomalies. METHODS: Genotyping of 1,256 genes related with neurodevelopment was performed by next-generation sequencing in three unrelated patients and their healthy parents. Clinically relevant findings were confirmed by conventional sequencing. RESULTS: Each patient showed one de novo variant not previously reported in the literature or databases. Two different missense variants were found in the MED12 or MED13L genes and one nonsense mutation was found in the MED13L gene. CONCLUSION: The phenotypic consequences of these mutations are closely related and/or have been previously reported in one or other gene. Additionally, MED12 and MED13L code for two closely related partners of the mediator kinase module. Consequently, we propose the concept of a common MED12/MED13L clinical spectrum, encompassing Opitz-Kaveggia syndrome, Lujan-Fryns syndrome, Ohdo syndrome, MED13L haploinsufficiency syndrome, and others.

Identification of Intellectual Disability Genes in Female Patients with a Skewed X-Inactivation Pattern.

Intellectual disability (ID) is a heterogeneous disorder with an unknown molecular etiology in many cases. Previously, X-linked ID (XLID) studies focused on males because of the hemizygous state of their X chromosome. Carrier females are generally unaffected because of the presence of a second normal allele, or inactivation of the mutant X chromosome in most of their cells (skewing). However, in female ID patients, we hypothesized that the presence of skewing of X-inactivation would be an indicator for an X chromosomal ID cause. We analyzed the X-inactivation patterns of 288 females with ID, and found that 22 (7.6%) had extreme skewing (>90%), which is significantly higher than observed in the general population (3.6%; P = 0.029). Whole-exome sequencing of 19 females with extreme skewing revealed causal variants in six females in the XLID genes DDX3X, NHS, WDR45, MECP2, and SMC1A. Interestingly, variants in genes escaping X-inactivation presumably cause both XLID and skewing of X-inactivation in three of these patients. Moreover, variants likely accounting for skewing only were detected in MED12, HDAC8, and TAF9B. All tested candidate causative variants were de novo events. Hence, extreme skewing is a good indicator for the presence of X-linked variants in female patients.

Novel mutations of NFIX gene causing Marshall-Smith syndrome or Sotos-like syndrome: one gene, two phenotypes.

BACKGROUND: Only 15 point mutations in NFIX gene have been reported so far, nine of them cause the Marshall-Smith syndrome (MSS) and the remaining mutations lead to an overgrowth disorder with a less severe phenotype, defined as Sotos-like. METHODS: The clinical findings in three patients with MSS and two patients with a Sotos-like phenotype are presented. Analysis of the NFIX gene was performed both by conventional or next-generation sequencing. RESULTS: Five de novo mutations in NFIX gene were identified, four of them not previously reported. Two frameshift mutations and a donor-splice one caused MSS, while two missense mutations in the DNA binding/dimerisation domain entailed an overgrowth syndrome with some clinical features resembling Sotos syndrome, accompanied by a marfanoid habitus, very low BMI, long narrow face, or arachnodactyly. CONCLUSION: Marshall-Smith mutations are scattered through exons 6-10 of NFIX gene, while most point mutations causing an overgrowth syndrome are clustered in exon 2. Clinical features of this overgrowth syndrome may well be considered an intermediate phenotype between Sotos and Marfan syndromes.

In Pursuit of New Imprinting Syndromes by Epimutation Screening in Idiopathic Neurodevelopmental Disorder Patients.

Alterations of epigenetic mechanisms, and more specifically imprinting modifications, could be responsible of neurodevelopmental disorders such as intellectual disability (ID) or autism together with other associated clinical features in many cases. Currently only eight imprinting syndromes are defined in spite of the fact that more than 200 genes are known or predicted to be imprinted. Recent publications point out that some epimutations which cause imprinting disorders may affect simultaneously different imprinted loci, suggesting that DNA-methylation may have been altered more globally. Therefore, we hypothesised that the detection of altered methylation patterns in known imprinting loci will indirectly allow identifying new syndromes due to epimutations among patients with unexplained ID. In a screening for imprinting alterations in 412 patients with syndromic ID/autism we found five patients with altered methylation in the four genes studied: MEG3, H19, KCNQ1OT1, and SNRPN. Remarkably, the cases with partial loss of methylation in KCNQ1OT1 and SNRPN present clinical features different to those associated with the corresponding imprinting syndromes, suggesting a multilocus methylation defect in accordance with our initial hypothesis. Consequently, our results are a proof of concept that the identification of epimutations in known loci in patients with clinical features different from those associated with known syndromes will eventually lead to the definition of new imprinting disorders.

A novel missense mutation in the NSDHL gene identified in a Lithuanian family by targeted next-generation sequencing causes CK syndrome.

The NSDHL gene encodes 3ß-hydroxysteroid dehydrogenase involved in one of the later steps of the cholesterol biosynthetic pathway. Mutations in this gene can cause CHILD syndrome (OMIM 308050) and CK syndrome (OMIM 300831). CHILD syndrome is an X-linked dominant, male lethal disorder caused by mutations in the NSDHL gene that result in the loss of the function of the NSDHL protein. CK syndrome is an allelic X-linked recessive disorder. So far, 13 patients with CK syndrome from two families have been reported on. We present a new five-generation family with affected males manifesting clinical features of CK syndrome. Next generation sequencing was targeted to a custom panel of 542 genes with known or putative implication on intellectual disability. Missense mutation p.Gly152Asp was identified in the NSDHL gene in the DNA sample of the affected male. Mutation carrier status was confirmed for all the obligate carriers in the family. The clinical features of the affected males in the family manifested as weak fetal movements, severe intellectual disability, seizures, spasticity, atrophy of optic discs, microcephaly, plagiocephaly, skeletal abnormalities, and minor facial anomalies, including a high nasal bridge, strabismus, and micrognathia. A highly significant preferential transmission of the mutation was observed in this and previous families segregating CK syndrome. Our report expands the clinical spectrum of this syndrome to include weak fetal movements, spasticity, and plagiocephaly, and transmission ratio distortion. The various findings in these patients increase our understanding of the diversity of the clinical presentation of cholesterol biosynthesis disorders.

Pure duplication of 19p13.3 in three members of a family with intellectual disability and literature review. Definition of a new microduplication syndrome.

This paper describes the presence of an interstitial pure duplication of 19p13.3 (4.95 Mb) in a patient with intellectual disability studied by array-CGH which was initially considered as a de novo alteration. The discovery of the same chromosomal alteration in a first-degree cousin of this patient led us to investigate the presence of insertional translocations, which were consequently found in three family generations. The same duplication was found in three intellectually disabled patients and among the translocation carrier family members a very high incidence of miscarriages are reported. A review of other published cases has allowed us to find three other patients with a similar pure duplication, all of them sharing some common clinical findings such as intrauterine growth retardation, microcephaly, motor and speech delay, moderate to severe intellectual disability, and dysmorphic features. These findings allow us to suggest the presence of a new microduplication syndrome in chromosomal region 19p13.3.