Recommendations for Interpreting and Reporting Silent Carrier and Disease-Modifying Variants in SMA Testing Workflows

Carrier screening; Diagnosis; Spinal muscular atrophyCribado de portadores; Diagnóstico; Atrofia muscular espinalCribratge de portadors; Diagnòstic; Atròfia muscular espinalGenetic testing for SMA diagnosis, newborn screening, and carrier screening has become a significant public health interest worldwide, driven largely by the development of novel and effective molecular therapies for the treatment of spinal muscular atrophy (SMA) and the corresponding updates to testing guidelines. Concurrently, understanding of the underlying genetics of SMA and their correlation with a broad range of phenotypes and risk factors has also advanced, particularly with respect to variants that modulate disease severity or impact residual carrier risks. While testing guidelines are beginning to emphasize the importance of these variants, there are no clear guidelines on how to utilize them in a real-world setting. Given the need for clarity in practice, this review summarizes several clinically relevant variants in the SMN1 and SMN2 genes, including how they inform outcomes for spinal muscular atrophy carrier risk and disease prognosis.This work was partially supported by Grants from Biogen ESP-SMG-17-11256 (to E.F.T. supporting L.B.-P.), Roche and Spanish Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias and co-funded with ERDF funds (Grant No. FIS PI18/000687) (to E.F.T.)

Genetic and epigenetic methylation defects and implication of the ERMN gene in autism spectrum disorders

Autism spectrum disorders (ASD) are highly heritable and genetically complex conditions. Although highly penetrant mutations in multiple genes have been identified, they account for the etiology of <1/3 of cases. There is also strong evidence for environmental contribution to ASD, which can be mediated by still poorly explored epigenetic modifications. We searched for methylation changes on blood DNA of 53 male ASD patients and 757 healthy controls using a methylomic array (450K Illumina), correlated the variants with transcriptional alterations in blood RNAseq data, and performed a case-control association study of the relevant findings in a larger cohort (394 cases and 500 controls). We found 700 differentially methylated CpGs, most of them hypomethylated in the ASD group (83.9%), with cis-acting expression changes at 7.6% of locations. Relevant findings included: (1) hypomethylation caused by rare genetic variants (meSNVs) at six loci (ERMN, USP24, METTL21C, PDE10A, STX16 and DBT) significantly associated with ASD (q-value <0.05); and (2) clustered epimutations associated to transcriptional changes in single-ASD patients (n = 4). All meSNVs and clustered epimutations were inherited from unaffected parents. Resequencing of the top candidate genes also revealed a significant load of deleterious mutations affecting ERMN in ASD compared with controls. Our data indicate that inherited methylation alterations detectable in blood DNA, due to either genetic or epigenetic defects, can affect gene expression and contribute to ASD susceptibility most likely in an additive manner, and implicate ERMN as a novel ASD gene

Polystyrene nanoplastics target lysosomes interfering with lipid metabolism through the PPAR system and affecting macrophage functionalization

Altres ajuts: acords transformatius de la UABNanoplastics (NPs) are currently a main concern for environmental, animal and human health due to their potential to accumulate in different environmental compartments and provoke effects in living organisms. Nevertheless, neither these effects nor the interaction of NPs with the cellular machinery are well characterized, and only scattered information is available. In the present work, we focused on the interaction between NPs and fish cells, both intestinal cells and macrophages, in order to understand which cell organelles are targeted by polystyrene (PS)-NPs and how this could impact cell function. PS-NPs can pass through phospholipid membranes, entering cells via endocytosis, phagocytosis or passive transport. Once internalized, we found that PS-NPs co-localize with lysosomes but not with mitochondria. Moreover, using two types of fluorescent probe (HDCFDA and DHE) we demonstrated that NPs did not trigger the production of reactive oxygen species (ROS), which was corroborated by the fact that neither the oxidative consumption ratio (OCR) nor the extracellular acidification rate (ECAR) in mitochondrial respiration were altered. RNASeq data revealed clear interference by PS-NPs with lipid metabolism, peroxisomes and PPAR signaling. The M1/M2 balance critically determines tissue homeostasis when exposed to exogenous agents such as microorganisms or pollutants. Thus, the expression of different genes (il1β, tnfα, il6, il10, il12, cox2, mmp9, ppar a, b and g) was further assessed to characterize the macrophage phenotype M1 or M2, induced by PS-NPs. Overall, in this study we demonstrate that PS-NPs co-localize within lysosomes, both in macrophages and in intestinal cells of rainbow trout, but do not trigger ROS production nor alter mitochondrial respiration. In macrophages, PS-NPs modulate polarization towards the M2-like phenotype

A novel FAM83G variant from palmoplantar keratoderma patient disrupts WNT signalling via loss of FAM83G-CK1α interaction

Palmoplantar keratoderma (PPK) is a multi-faceted skin disorder characterized by the thickening of the epidermis and abrasions on the palms and soles of the feet. Among the genetic causes, biallelic pathogenic variants in the FAM83G gene have been associated with PPK in dogs and humans. Here, a novel homozygous variant (c.794G&gt;C, p.Arg265Pro) in the FAM83G gene, identified by whole exome sequencing in a 60-year-old female patient with PPK, is reported. The patient exhibited alterations in the skin of both hands and feet, dystrophic nails, thin, curly and sparse hair, long upper eyelid eyelashes, and poor dental enamel. FAM83G activates WNT signalling through association with ser/thr protein kinase CK1α. When expressed in FAM83G−/− DLD1 colorectal cancer cells, the FAM83GR265P variant displayed poor stability, a loss of interaction with CK1α and attenuated WNT signalling response. These defects persisted in skin fibroblast cells derived from the patient. Our findings imply that the loss of FAM83G-CK1α interaction and subsequent attenuation of WNT signalling underlie the pathogenesis of PPK caused by the FAM83GR265P variant

An Atypical Presentation of Upper Motor Neuron Predominant Juvenile Amyotrophic Lateral Sclerosis Associated with TARDBP Gene: A Case Report and Review of the Literature

Juvenile amyotrophic lateral sclerosis; Predominant disorderEsclerosi lateral amiotròfica juvenil; Trastorn predominantEsclerosis lateral amiotrófica juvenil; Trastorno predominanteAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that can rarely affect young individuals. Juvenile ALS (JALS) is defined for individuals with an onset of the disease before the age of 25. The contribution of genetics to ALS pathology is a field of growing interest. One of the differences between adult-onset ALS and JALS is their genetic background, with a higher contribution of genetic causes in JALS. We report a patient with JALS and a pathogenic variant in the TARDBP gene (c.1035C > G; p.Asn345Lys), previously reported only in adult-onset ALS, and with an atypical phenotype of marked upper motor neuron predominance. In addition, the proband presented an additional variant in the NEK1 gene, c.2961C > G (p.Phe987Leu), which is classified as a variant of unknown significance. Segregation studies showed a paternal origin of the TARDBP variant, while the variant in NEK1 was inherited from the mother. We hypothesize that the NEK1 variant acts as a disease modifier and suggests the possibility of a functional interaction between both genes in our case. This hypothesis could explain the peculiarities of the phenotype, penetrance, and the age of onset. This report highlights the heterogeneity of the phenotypic presentation of ALS associated with diverse pathogenic genetic variants

Integrated analysis of whole-exome sequencing and transcriptome profiling in males with autism spectrum disorders

BACKGROUND: Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders with high heritability. Recent findings support a highly heterogeneous and complex genetic etiology including rare de novo and inherited mutations or chromosomal rearrangements as well as double or multiple hits. METHODS: We performed whole-exome sequencing (WES) and blood cell transcriptome by RNAseq in a subset of male patients with idiopathic ASD (n = 36) in order to identify causative genes, transcriptomic alterations, and susceptibility variants. RESULTS: We detected likely monogenic causes in seven cases: five de novo (SCN2A, MED13L, KCNV1, CUL3, and PTEN) and two inherited X-linked variants (MAOA and CDKL5). Transcriptomic analyses allowed the identification of intronic causative mutations missed by the usual filtering of WES and revealed functional consequences of some rare mutations. These included aberrant transcripts (PTEN, POLR3C), deregulated expression in 1.7% of mutated genes (that is, SEMA6B, MECP2, ANK3, CREBBP), allele-specific expression (FUS, MTOR, TAF1C), and non-sense-mediated decay (RIT1, ALG9). The analysis of rare inherited variants showed enrichment in relevant pathways such as the PI3K-Akt signaling and the axon guidance. CONCLUSIONS: Integrative analysis of WES and blood RNAseq data has proven to be an efficient strategy to identify likely monogenic forms of ASD (19% in our cohort), as well as additional rare inherited mutations that can contribute to ASD risk in a multifactorial manner. Blood transcriptomic data, besides validating 88% of expressed variants, allowed the identification of missed intronic mutations and revealed functional correlations of genetic variants, including changes in splicing, expression levels, and allelic expression. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13229-015-0017-0) contains supplementary material, which is available to authorized users

Deep Molecular Characterization of Milder Spinal Muscular Atrophy Patients Carrying the c.859G>C Variant in SMN2

Next-generation sequencing; Phenotype–genotype correlations; Spinal muscular atrophySeqüenciació de nova generació; Correlacions fenotip-genotip; Atròfia muscular espinalSecuenciación de nueva generación; Correlaciones fenotipo-genotipo; Atrofia muscular espinalSpinal muscular atrophy (SMA) is a severe neuromuscular disorder caused by biallelic loss or pathogenic variants in the SMN1 gene. Copy number and modifier intragenic variants in SMN2, an almost identical paralog gene of SMN1, are known to influence the amount of complete SMN proteins. Therefore, SMN2 is considered the main phenotypic modifier of SMA, although genotype–phenotype correlation is not absolute. We present eleven unrelated SMA patients with milder phenotypes carrying the c.859G>C-positive modifier variant in SMN2. All were studied by a specific NGS method to allow a deep characterization of the entire SMN region. Analysis of two homozygous cases for the variant allowed us to identify a specific haplotype, Smn2-859C.1, in association with c.859G>C. Two other cases with the c.859G>C variant in their two SMN2 copies showed a second haplotype, Smn2-859C.2, in cis with Smn2-859C.1, assembling a more complex allele. We also identified a previously unreported variant in intron 2a exclusively linked to the Smn2-859C.1 haplotype (c.154-1141G>A), further suggesting that this region has been ancestrally conserved. The deep molecular characterization of SMN2 in our cohort highlights the importance of testing c.859G>C, as well as accurately assessing the SMN2 region in SMA patients to gain insight into the complex genotype–phenotype correlations and improve prognostic outcomes.This research was funded by grants from Biogen (ESP-SMG-17-11256), Roche, GaliciAME and Spanish Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias and co-funded with ERDF funds (grant no. FIS PI18/000687). A grant from Horizon 2020 IMI2 Screen4Care is acknowledged by E.B., and L.T., E.F.T., R.J., J.S., L.C.-C., F.M., E.B., and L.T. are members of the ERN NMD Network for Rare Diseases. E.F.T. is a member of the ERN ITHACA Network for Rare Diseases

Deep Molecular Characterization of Milder Spinal Muscular Atrophy Patients Carrying the c.859G&gt;C Variant in SMN2

Spinal muscular atrophy (SMA) is a severe neuromuscular disorder caused by biallelic loss or pathogenic variants in the SMN1 gene. Copy number and modifier intragenic variants in SMN2, an almost identical paralog gene of SMN1, are known to influence the amount of complete SMN proteins. Therefore, SMN2 is considered the main phenotypic modifier of SMA, although genotype-phenotype correlation is not absolute. We present eleven unrelated SMA patients with milder phenotypes carrying the c.859G>C-positive modifier variant in SMN2. All were studied by a specific NGS method to allow a deep characterization of the entire SMN region. Analysis of two homozygous cases for the variant allowed us to identify a specific haplotype, Smn2-859C.1, in association with c.859G>C. Two other cases with the c.859G>C variant in their two SMN2 copies showed a second haplotype, Smn2-859C.2, in cis with Smn2-859C.1, assembling a more complex allele. We also identified a previously unreported variant in intron 2a exclusively linked to the Smn2-859C.1 haplotype (c.154-1141G>A), further suggesting that this region has been ancestrally conserved. The deep molecular characterization of SMN2 in our cohort highlights the importance of testing c.859G>C, as well as accurately assessing the SMN2 region in SMA patients to gain insight into the complex genotype-phenotype correlations and improve prognostic outcomes

Detecting PKD1 variants in polycystic kidney disease patients by single-molecule long-read sequencing

A genetic diagnosis of autosomal-dominant polycystic kidney disease (ADPKD) is challenging due to allelic heterogeneity, high GC content, and homology of the PKD1 gene with six pseudogenes. Short-read next-generation sequencing approaches, such as whole-genome sequencing and whole-exome sequencing, often fail at reliably characterizing complex regions such as PKD1. However, long-read single-molecule sequencing has been shown to be an alternative strategy that could overcome PKD1 complexities and discriminate between homologous regions of PKD1 and its pseudogenes. In this study, we present the increased power of resolution for complex regions using long-read sequencing to characterize a cohort of 19 patients with ADPKD. Our approach provided high sensitivity in identifying PKD1 pathogenic variants, diagnosing 94.7% of the patients. We show that reliable screening of ADPKD patients in a single test without interference of PKD1 homologous sequences, commonly introduced by residual amplification of PKD1 pseudogenes, by direct long-read sequencing is now possible. This strategy can be implemented in diagnostics and is highly suitable to sequence and resolve complex genomic regions that are of clinical relevance