Guidelines for NGS procedures applied to prenatal diagnosis by the Spanish Society of Gynecology and Obstetrics and the Spanish Association of Prenatal Diagnosis.

OBJECTIVE: This document addresses the clinical application of next-generation sequencing (NGS) technologies for prenatal genetic diagnosis and aims to establish clinical practice recommendations in Spain to ensure uniformity in implementing these technologies into prenatal care. METHODS: A joint committee of expert obstetricians and geneticists was created to review the existing literature on fetal NGS for genetic diagnosis and to make recommendations for Spanish healthcare professionals. RESULTS: This guideline summarises technical aspects of NGS technologies, clinical indications in prenatal setting, considerations regarding findings to be reported, genetic counselling considerations as well as data storage and protection policies. CONCLUSIONS: This document provides updated recommendations for the use of NGS diagnostic tests in prenatal diagnosis. These recommendations should be periodically reviewed as our knowledge of the clinical utility of NGS technologies, applied during pregnancy, may advance.

Creation of an Isogenic Human iPSC-Based RGC Model of Dominant Optic Atrophy Harboring the Pathogenic Variant c.1861C>T (p.Gln621Ter) in the OPA1 Gene.

Autosomal dominant optic atrophy (ADOA) is a rare progressive disease mainly caused by mutations in OPA1, a nuclear gene encoding for a mitochondrial protein that plays an essential role in mitochondrial dynamics, cell survival, oxidative phosphorylation, and mtDNA maintenance. ADOA is characterized by the degeneration of retinal ganglion cells (RGCs). This causes visual loss, which can lead to legal blindness in many cases. Nowadays, there is no effective treatment for ADOA. In this article, we have established an isogenic human RGC model for ADOA using iPSC technology and the genome editing tool CRISPR/Cas9 from a previously generated iPSC line of an ADOA plus patient harboring the pathogenic variant NM_015560.3: c.1861C>T (p.Gln621Ter) in heterozygosis in OPA1. To this end, a protocol based on supplementing the iPSC culture media with several small molecules and defined factors trying to mimic embryonic development has been employed. Subsequently, the created model was validated, confirming the presence of a defect of intergenomic communication, impaired mitochondrial respiration, and an increase in apoptosis and ROS generation. Finally, we propose the analysis of OPA1 expression by qPCR as an easy read-out method to carry out future drug screening studies using the created RGC model. In summary, this model provides a useful platform for further investigation of the underlying pathophysiological mechanisms of ADOA plus and for testing compounds with potential pharmacological action.

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.

Heterozygous and Homozygous Variants in SORL1 Gene in Alzheimer's Disease Patients: Clinical, Neuroimaging and Neuropathological Findings.

In the last few years, the SORL1 gene has been strongly implicated in the development of Alzheimer's disease (AD). We performed whole-exome sequencing on 37 patients with early-onset dementia or family history suggestive of autosomal dominant dementia. Data analysis was based on a custom panel that included 46 genes related to AD and dementia. SORL1 variants were present in a high proportion of patients with candidate variants (15%, 3/20). We expand the clinical manifestations associated with the SORL1 gene by reporting detailed clinical and neuroimaging findings of six unrelated patients with AD and SORL1 mutations. We also present for the first time a patient with the homozygous truncating variant c.364C>T (p.R122*) in SORL1, who also had severe cerebral amyloid angiopathy. Furthermore, we report neuropathological findings and immunochemistry assays from one patient with the splicing variant c.4519+5G>A in the SORL1 gene, in which AD was confirmed by neuropathological examination. Our results highlight the heterogeneity of clinical presentation and familial dementia background of SORL1-associated AD and suggest that SORL1 might be contributing to AD development as a risk factor gene rather than as a major autosomal dominant gene.

First female with Allan-Herndon-Dudley syndrome and partial deletion of X-inactivation center.

Allan-Herndon-Dudley is an X-linked recessive syndrome caused by pathogenic variants in the SLC16A2 gene. Clinical manifestations are a consequence of impaired thyroid metabolism and aberrant transport of thyroid hormones to the brain. Carrier females are generally asymptomatic and may show subtle symptoms of the disease. We describe a female with a complete Allan-Herndon-Dudley phenotype, carrying a de novo 543-kb deletion of the X chromosome. The deletion encompasses exon 1 of the SLC16A2 gene and JPX and FTX genes; it is known that the latter two genes participate in the X-inactivation process upregulating XIST gene expression. Subsequent studies in the patient demonstrated the preferential expression of the X chromosome with the JPX and FTX deletion.

First patient with mosaic NOTCH3 gene pathogenic variant. Unrevealed mosaicisms and importance of their detection.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited small vessel disease caused predominantly by pathogenic variants in NOTCH3 gene. Neither germline nor somatic mosaicism has been previously published in NOTCH3 gene. CADASIL is inherited in an autosomal dominant manner; only rare cases have been associated with de novo pathogenic variants. Mosaicism is more common than previously thought because mosaic variants often stay unrevealed. An apparently de novo variant might actually be a consequence of a parental mosaicism undetectable with Sanger sequencing, especially in the case of low grade mosaicism. Parental testing by sensitive tools like deep targeted next-generation sequencing (NGS) analysis could detect cases of unrevealed medium or low level mosaicism in patients tested by Sanger sequencing. Here, we report the first patient with mosaic NOTCH3 gene pathogenic variant to our knowledge; the allelic fraction in the leucocyte DNA was low (13%); the pathogenic variant was inhered by his two daughters. The patient was diagnosed by deep targeted NGS analysis after studying his two affected daughters. This report highlights the importance of parental testing by sensitive tools like deep targeted NGS analysis. Detection of mosaicism is of great importance for diagnosis and adequate family genetic counseling.

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.

Genetic diagnosis of childhood sensorineural hearing loss.

INTRODUCTION: Congenital/early-onset sensorineural hearing loss (SNHL) is one of the most common hereditary disorders in our environment. There is increasing awareness of the importance of an etiologic diagnosis, and genetic testing with next-generation sequencing (NGS) has the highest diagnostic yield. Our study shows the genetic results obtained in a cohort of patients with bilateral congenital/early-onset SNHL. MATERIALS AND METHODS: We included 105 children with bilateral SNHL that received genetic testing between 2019 and 2022. Genetic tests were performed with whole exome sequencing, analyzing genes related to hearing loss (virtual panel with 244 genes). RESULTS: 48% (50/105) of patients were genetically diagnosed. We identified pathogenic and likely pathogenic variants in 26 different genes, and the most frequently mutated genes were GJB2, USH2A and STRC. 52% (26/50) of variants identified produced non-syndromic hearing loss, 40% (20/50) produced syndromic hearing loss, and the resting 8% (4/50) could produce both non-syndromic and syndromic hearing loss. CONCLUSIONS: Genetic testing plays a vital role in the etiologic diagnosis of bilateral SNHL. Our cohort shows that genetic testing with NGS has a high diagnostic yield and can provide useful information for the clinical workup of patients.

Diagnostic yield of genetic testing in adults with sensorineural hearing loss.

INTRODUCTION: The contribution of genetic causes to sensorineural hearing loss (SNHL) in adults is less clear than in children, and genetic diagnosis is still not standardized in adults. In this study we present the genetic results obtained in a cohort of adult patients with SNHL. MATERIALS AND METHODS: We included 63 adults with SNHL that received genetic testing between 2019 and 2022. Whole exome sequencing was performed and variants in genes related to hearing loss (virtual panel with 244 genes) were prioritised and analysed. RESULTS: 24% (15/63) of patients were genetically diagnosed: 87% (13/15) of patients had non-syndromic hearing loss and 13% (2/15) had syndromic hearing loss. We identified pathogenic and likely pathogenic variants in 11 different genes. CONCLUSIONS: Our results show that a significant proportion of adults with SNHL have a genetic origin, and that implementation of genetic testing improves diagnostic accuracy and allows personalized management of these patients.

A Novel Pathogenic Variant in the MN1 Gene in a Patient Presenting with Rhombencephalosynapsis and Craniofacial Anomalies, Expanding MN1 C-terminal Truncation Syndrome.

Meningioma-1 is a transcription activator that regulates mammalian palate development and is required for appropriate osteoblast proliferation, motility, differentiation, and function. Microdeletions involving the MN1 gene have been linked to syndromes including craniofacial anomalies, such as Toriello-Carey syndrome. Recently, truncating variants in the C-terminal portion of the MN1 transcriptional factor have been linked to a characteristic and distinct phenotype presenting with craniofacial anomalies and partial rhombencephalosynapsis, a rare brain malformation characterized by midline fusion of the cerebellar hemispheres with partial or complete loss of the cerebellar vermis. It has been called MN1 C-terminal truncation (MCTT) syndrome or CEBALID (Craniofacial defects, dysmorphic Ears, Brain Abnormalities, Language delay, and Intellectual Disability) and suggested to be caused by dominantly acting truncated protein MN1 instead of haploinsufficiency. As a proto-oncogene, MN1 is also involved in familial meningioma. In this study, we present a novel case of MCTT syndrome in a female patient presenting with craniofacial anomalies and rhombencephalosynapsis, harboring a de novo pathogenic variant in the MN1 gene: c.3686_3698del, p.(Met1229Argfs*87).

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.

Fragile X Syndrome Caused by Maternal Somatic Mosaicism of FMR1 Gene: Case Report and Literature Review.

Fragile X syndrome (FXS) is caused by an abnormal expansion of the number of trinucleotide CGG repeats located in the 5' UTR in the first exon of the FMR1 gene. Size and methylation mosaicisms are commonly observed in FXS patients. Both types of mosaicisms might be associated with less severe phenotypes depending on the number of cells expressing FMRP. Although this dynamic mutation is the main underlying cause of FXS, other mechanisms, including point mutations or deletions, can lead to FXS. Several reports have demonstrated that de novo deletions including the entire or a portion of the FMR1 gene end up with the absence of FMRP and, thus, can lead to the typical clinical features of FXS. However, very little is known about the clinical manifestations associated with FMR1 gene deletions in mosaicism. Here, we report an FXS case caused by an entire hemizygous deletion of the FMR1 gene caused by maternal mosaicism. This manuscript reports this case and a literature review of the clinical manifestations presented by carriers of FMR1 gene deletions in mosaicism.

Towards a Change in the Diagnostic Algorithm of Autism Spectrum Disorders: Evidence Supporting Whole Exome Sequencing as a First-Tier Test.

Autism spectrum disorder (ASD) is a prevalent and extremely heterogeneous neurodevelopmental disorder (NDD) with a strong genetic component. In recent years, the clinical relevance of de novo mutations to the aetiology of ASD has been demonstrated. Current guidelines recommend chromosomal microarray (CMA) and a FMR1 testing as first-tier tests, but there is increasing evidence that support the use of NGS for the diagnosis of NDDs. Specifically in ASD, it has not been extensively evaluated and, thus, we performed and compared the clinical utility of CMA, FMR1 testing, and/or whole exome sequencing (WES) in a cohort of 343 ASD patients. We achieved a global diagnostic rate of 12.8% (44/343), the majority of them being characterised by WES (33/44; 75%) compared to CMA (9/44; 20.4%) or FMR1 testing (2/44; 4.5%). Taking into account the age at which genetic testing was carried out, we identified a causal genetic alteration in 22.5% (37/164) of patients over 5 years old, but only in 3.9% (7/179) of patients under this age. Our data evidence the higher diagnostic power of WES compared to CMA in the study of ASD and support the implementation of WES as a first-tier test for the genetic diagnosis of this disorder, when there is no suspicion of fragile X syndrome.

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.