The landscape of submicroscopic structural variants at the OPN1LW/OPN1MW gene cluster on Xq28 underlying blue cone monochromacy.

Blue cone monochromacy (BCM) is an X-linked retinal disorder characterized by low vision, photoaversion, and poor color discrimination. BCM is due to the lack of long-wavelength-sensitive and middle-wavelength-sensitive cone photoreceptor function and caused by mutations in the OPN1LW/OPN1MW gene cluster on Xq28. Here, we investigated the prevalence and the landscape of submicroscopic structural variants (SVs) at single-base resolution in BCM patients. We found that about one-third (n = 73) of the 213 molecularly confirmed BCM families carry an SV, most commonly deletions restricted to the OPN1LW/OPN1MW gene cluster. The structure and precise breakpoints of the SVs were resolved in all but one of the 73 families. Twenty-two families-all from the United States-showed the same SV, and we confirmed a common ancestry of this mutation. In total, 42 distinct SVs were identified, including 40 previously unreported SVs, thereby quadrupling the number of precisely mapped SVs underlying BCM. Notably, there was no "region of overlap" among these SVs. However, 90% of SVs encompass the upstream locus control region, an essential enhancer element. Its minimal functional extent based on deletion mapping in patients was refined to 358 bp. Breakpoint analyses suggest diverse mechanisms underlying SV formation as well as in one case the gene conversion-based exchange of a 142-bp deletion between opsin genes. Using parsimonious assumptions, we reconstructed the composition and copy number of the OPN1LW/OPN1MW gene cluster prior to the mutation event and found evidence that large gene arrays may be predisposed to the occurrence of SVs at this locus.

Parkin mRNA Expression Levels in Peripheral Blood Mononuclear Cells in Parkin-Related Parkinson's Disease.

INTRODUCTION: Pathogenic variants in parkin (PRKN gene) are the second most prevalent known monogenic cause of Parkinson's disease (PD). How monoallelic or biallelic pathogenic variants in the PRKN gene may affect its transcription in patient-derived biological material has not been systematically studied. METHODS: PRKN mRNA expression levels were measured with real-time polymerase chain reaction (RT-PCR) in peripheral blood mononuclear cells (PBMCs). PBMCs were derived from PRKN-mutated PD patients (PRKN-PD) (n = 12), sporadic PD (sPD) (n = 21) and healthy controls (n = 21). Six of the PRKN-PD patients were heterozygous, four were compound heterozygous, and two were homozygous for PRKN variants. RESULTS: A statistically significant decrease in PRKN expression levels was present, compared to healthy controls and sPD, in heterozygous (P = 0.019 and 0.031 respectively) and biallelic (P < 0.001 for both) PRKN-PD. PRKN expression levels in biallelic PD patients were uniformly very low and were reduced, albeit not significantly, compared to heterozygotes. Based on receiver operating characteristic analysis, low PRKN expression levels were a sensitive and extremely specific indicator for the presence of PRKN pathogenic variants. CONCLUSIONS: Assessment of PRKN mRNA levels in PBMCs may be a useful way to screen for biallelic pathogenic variants in the PRKN gene. Suspicion for certain variants in a heterozygous state may also be raised based on low PRKN mRNA levels. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Exome Sequencing and Optical Genome Mapping in Molecularly Unsolved Cases of Duchenne Muscular Dystrophy: Identification of a Causative X-Chromosomal Inversion Disrupting the DMD Gene.

Duchenne muscular dystrophy (DMD) is a severe progressive muscle disease that mainly affects boys due to X-linked recessive inheritance. In most affected individuals, MLPA or sequencing-based techniques detect deletions, duplications, or point mutations in the dystrophin-encoding DMD gene. However, in a small subset of patients clinically diagnosed with DMD, the molecular cause is not identified with these routine methods. Evaluation of the 60 DMD patients in our center revealed three cases without a known genetic cause. DNA samples of these patients were analyzed using whole-exome sequencing (WES) and, if unconclusive, optical genome mapping (OGM). WES led to a diagnosis in two cases: one patient was found to carry a splice mutation in the DMD gene that had not been identified during previous Sanger sequencing. In the second patient, we detected two variants in the fukutin gene (FKTN) that were presumed to be disease-causing. In the third patient, WES was unremarkable, but OGM identified an inversion disrupting the DMD gene (~1.28 Mb) that was subsequently confirmed with long-read sequencing. These results highlight the importance of reanalyzing unsolved cases using WES and demonstrate that OGM is a useful method for identifying large structural variants in cases with unremarkable exome sequencing.

A 73,128 bp de novo deletion encompassing the OPN1LW/OPN1MW gene cluster in sporadic Blue Cone Monochromacy: a case report.

BACKGROUND: Blue Cone Monochromacy (BCM) is a rare congenital cone dysfunction disorder with X-linked recessive mode of inheritance. BCM is caused by mutations at the OPN1LW/MW cone opsin gene cluster including deletions of the locus control region (LCR) and/or parts of the gene cluster. We aimed at investigating the clinical presentation, genetic cause and inheritance underlying a sporadic case of BCM. CASE PRESENTATION: We report a 24-year-old male presenting with congenital photophobia, nystagmus and colour vision abnormalities. There was no history of retinal dystrophy in the family. Clinical diagnosis of BCM was supported by genetic investigations of the patient and his family members. Molecular genetic analysis of the OPN1LW/OPN1MW gene cluster revealed a novel deletion of about 73 kb in the patient encompassing the LCR. The deletion was absent in the X-chromosomes of both the mother and transmitting grandfather. CONCLUSIONS: The present report provides the clinical findings and the genetic basis underlying a sporadic BCM case which is caused by a de novo deletion within the OPN1LW/MW gene cluster originating from the mother's germline due to Alu-repeat mediated recombination. This is the first report of a de novo deletion resulting in BCM, highlighting the importance to consider BCM and perform genetic testing for this condition in male patients with cone dysfunction also in the absence of a positive family history.

Retinitis pigmentosa: impact of different Pde6a point mutations on the disease phenotype.

Mutations in the PDE6A gene can cause rod photoreceptors degeneration and the blinding disease retinitis pigmentosa (RP). While a number of pathogenic PDE6A mutations have been described, little is known about their impact on compound heterozygous situations and potential interactions of different disease-causing alleles. Here, we used a novel mouse model for the Pde6a R562W mutation in combination with an existing line carrying the V685M mutation to generate compound heterozygous Pde6a V685M/R562W animals, exactly homologous to a case of human RP. We compared the progression of photoreceptor degeneration in these compound heterozygous mice with the homozygous V685M and R562W mutants, and additionally with the D670G line that is known for a relatively mild phenotype. We investigated PDE6A expression, cyclic guanosine mono-phosphate accumulation, calpain and caspase activity, in vivo retinal function and morphology, as well as photoreceptor cell death and survival. This analysis confirms the severity of different Pde6a mutations and indicates that compound heterozygous mutants behave like intermediates of the respective homozygous situations. Specifically, the severity of the four different Pde6a situations may be categorized by the pace of photoreceptor degeneration: V685M (fastest) > V685M/R562W > R562W > D670G (slowest). While calpain activity was strongly increased in all four mutants, caspase activity was not. This points to the execution of non-apoptotic cell death and may lead to the identification of new targets for therapeutic interventions. For individual RP patients, our study may help to predict time-courses for Pde6a-related retinal degeneration and thereby facilitate the definition of a window-of-opportunity for clinical interventions.

The nervous system of Xenacoelomorpha: a genomic perspective.

Xenacoelomorpha is, most probably, a monophyletic group that includes three clades: Acoela, Nemertodermatida and Xenoturbellida. The group still has contentious phylogenetic affinities; though most authors place it as the sister group of the remaining bilaterians, some would include it as a fourth phylum within the Deuterostomia. Over the past few years, our group, along with others, has undertaken a systematic study of the microscopic anatomy of these worms; our main aim is to understand the structure and development of the nervous system. This research plan has been aided by the use of molecular/developmental tools, the most important of which has been the sequencing of the complete genomes and transcriptomes of different members of the three clades. The data obtained has been used to analyse the evolutionary history of gene families and to study their expression patterns during development, in both space and time. A major focus of our research is the origin of 'cephalized' (centralized) nervous systems. How complex brains are assembled from simpler neuronal arrays has been a matter of intense debate for at least 100 years. We are now tackling this issue using Xenacoelomorpha models. These represent an ideal system for this work because the members of the three clades have nervous systems with different degrees of cephalization; from the relatively simple sub-epithelial net of Xenoturbella to the compact brain of acoels. How this process of 'progressive' cephalization is reflected in the genomes or transcriptomes of these three groups of animals is the subject of this paper.

A GGC-repeat expansion in ZFHX3 encoding polyglycine causes spinocerebellar ataxia type 4 and impairs autophagy.

Despite linkage to chromosome 16q in 1996, the mutation causing spinocerebellar ataxia type 4 (SCA4), a late-onset sensory and cerebellar ataxia, remained unknown. Here, using long-read single-strand whole-genome sequencing (LR-GS), we identified a heterozygous GGC-repeat expansion in a large Utah pedigree encoding polyglycine (polyG) in zinc finger homeobox protein 3 (ZFHX3), also known as AT-binding transcription factor 1 (ATBF1). We queried 6,495 genome sequencing datasets and identified the repeat expansion in seven additional pedigrees. Ultrarare DNA variants near the repeat expansion indicate a common distant founder event in Sweden. Intranuclear ZFHX3-p62-ubiquitin aggregates were abundant in SCA4 basis pontis neurons. In fibroblasts and induced pluripotent stem cells, the GGC expansion led to increased ZFHX3 protein levels and abnormal autophagy, which were normalized with small interfering RNA-mediated ZFHX3 knockdown in both cell types. Improving autophagy points to a therapeutic avenue for this novel polyG disease. The coding GGC-repeat expansion in an extremely G+C-rich region was not detectable by short-read whole-exome sequencing, which demonstrates the power of LR-GS for variant discovery.

Posterior regeneration in Isodiametra pulchra (Acoela, Acoelomorpha).

INTRODUCTION: Regeneration is a widespread phenomenon in the animal kingdom, but the capacity to restore damaged or missing tissue varies greatly between different phyla and even within the same phylum. However, the distantly related Acoelomorpha and Platyhelminthes share a strikingly similar stem-cell system and regenerative capacity. Therefore, comparing the underlying mechanisms in these two phyla paves the way for an increased understanding of the evolution of this developmental process.To date, Isodiametra pulchra is the most promising candidate as a model for the Acoelomorpha, as it reproduces steadily under laboratory conditions and is amenable to various techniques, including the silencing of gene expression by RNAi. In order to provide an essential framework for future studies, we report the succession of regeneration events via the use of cytochemical, histological and microscopy techniques, and specify the total number of cells in adult individuals. RESULTS: Isodiametra pulchra is not capable of regenerating a new head, but completely restores all posterior structures within 10 days. Following amputation, the wound closes via the contraction of local muscle fibres and an extension of the dorsal epidermis. Subsequently, stem cells and differentiating cells invade the wound area and form a loosely delimited blastema. After two days, the posterior end is re-patterned with the male (and occasionally the female) genital primordium being apparent. Successively, these primordia differentiate into complete copulatory organs. The size of the body and also of the male and female copulatory organs, as well as the distance between the copulatory organs, progressively increase and by nine days copulation is possible. Adult individuals with an average length of 670 µm consist of approximately 8100 cells. CONCLUSION: Isodiametra pulchra regenerates through a combination of morphallactic and epimorphic processes. Existing structures are "re-modelled" and provide a framework onto which newly differentiating cells are added. Growth proceeds through the intercalary addition of structures, mirroring the embryonic and post-embryonic development of various organ systems. The suitability of Isodiametra pulchra for laboratory techniques, the fact that its transcriptome and genome data will soon be available, as well as its small size and low number of cells, make it a prime candidate subject for research into the cellular mechanisms that underlie regeneration in acoelomorphs.

Accurate long-read sequencing identified GBA1 as major risk factor in the Luxembourgish Parkinson's study.

Heterozygous variants in the glucocerebrosidase GBA1 gene are an increasingly recognized risk factor for Parkinson's disease (PD). Due to the GBAP1 pseudogene, which shares 96% sequence homology with the GBA1 coding region, accurate variant calling by array-based or short-read sequencing methods remains a major challenge in understanding the genetic landscape of GBA1-associated PD. We analyzed 660 patients with PD, 100 patients with Parkinsonism and 808 healthy controls from the Luxembourg Parkinson's study, sequenced using amplicon-based long-read DNA sequencing technology. We found that 12.1% (77/637) of PD patients carried GBA1 variants, with 10.5% (67/637) of them carrying known pathogenic variants (including severe, mild, risk variants). In comparison, 5% (34/675) of the healthy controls carried GBA1 variants, and among them, 4.3% (29/675) were identified as pathogenic variant carriers. We found four GBA1 variants in patients with atypical parkinsonism. Pathogenic GBA1 variants were 2.6-fold more frequently observed in PD patients compared to controls (OR = 2.6; CI = [1.6,4.1]). Three novel variants of unknown significance (VUS) were identified. Using a structure-based approach, we defined a potential risk prediction method for VUS. This study describes the full landscape of GBA1-related parkinsonism in Luxembourg, showing a high prevalence of GBA1 variants as the major genetic risk for PD. Although the long-read DNA sequencing technique used in our study may be limited in its effectiveness to detect potential structural variants, our approach provides an important advancement for highly accurate GBA1 variant calling, which is essential for providing access to emerging causative therapies for GBA1 carriers.

Impact of COVID-19 in nutritional and functional status of survivors admitted in intensive care units during the first outbreak. Preliminary results of the NUTRICOVID study.

BACKGROUND & AIMS: COVID-19 patients present a high hospitalization rate with a high mortality risk for those requiring intensive care. When these patients have other comorbid conditions and older age, the risk for severe disease and poor outcomes after ICU admission are increased. The present work aims to describe the preliminary results of the ongoing NUTRICOVID study about the nutritional and functional status and the quality of life of adult COVID-19 survivors after ICU discharge, emphasizing the in-hospital and discharge situation of this population. METHODS: A multicenter, ambispective, observational cohort study was conducted in 16 public hospitals of the Community of Madrid with COVID-19 survivors who were admitted to the ICU during the first outbreak. Preliminary results of this study include data retrospectively collected. Malnutrition and sarcopenia were screened at discharge using MUST and SARC-F; the use of healthcare resources was measured as the length of hospital stay and requirement of respiratory support and tracheostomy during hospitalization; other study variables were the need for medical nutrition therapy (MNT); and patients' functional status (Barthel index) and health-related quality of life (EQ-5D-5L). RESULTS: A total of 176 patients were included in this preliminary analysis. Most patients were male and older than 60 years, who suffered an average (SD) weight loss of 16.6% (8.3%) during the hospital stay, with a median length of stay of 53 (27-89.5) days and a median ICU stay of 24.5 (11-43.5) days. At discharge, 83.5% and 86.9% of the patients were at risk of malnutrition and sarcopenia, respectively, but only 38% were prescribed MNT. In addition, more than 70% of patients had significant impairment of their mobility and to conduct their usual activities at hospital discharge. CONCLUSIONS: This preliminary analysis evidences the high nutritional and functional impairment of COVID-19 survivors at hospital discharge and highlights the need for guidelines and systematic protocols, together with appropriate rehabilitation programs, to optimize the nutritional management of these patients after discharge.

Splicing mutations in inherited retinal diseases.

Mutations which induce aberrant transcript splicing represent a distinct class of disease-causing genetic variants in retinal disease genes. Such mutations may either weaken or erase regular splice sites or create novel splice sites which alter exon recognition. While mutations affecting the canonical GU-AG dinucleotides at the splice donor and splice acceptor site are highly predictive to cause a splicing defect, other variants in the vicinity of the canonical splice sites or those affecting additional cis-acting regulatory sequences within exons or introns are much more difficult to assess or even to recognize and require additional experimental validation. Splicing mutations are unique in that the actual outcome for the transcript (e.g. exon skipping, pseudoexon inclusion, intron retention) and the encoded protein can be quite different depending on the individual mutation. In this article, we present an overview on the current knowledge about and impact of splicing mutations in inherited retinal diseases. We introduce the most common sub-classes of splicing mutations including examples from our own work and others and discuss current strategies for the identification and validation of splicing mutations, as well as therapeutic approaches, open questions, and future perspectives in this field of research.

Efficient hybrid de novo assembly of human genomes with WENGAN.

Generating accurate genome assemblies of large, repeat-rich human genomes has proved difficult using only long, error-prone reads, and most human genomes assembled from long reads add accurate short reads to polish the consensus sequence. Here we report an algorithm for hybrid assembly, WENGAN, that provides very high quality at low computational cost. We demonstrate de novo assembly of four human genomes using a combination of sequencing data generated on ONT PromethION, PacBio Sequel, Illumina and MGI technology. WENGAN implements efficient algorithms to improve assembly contiguity as well as consensus quality. The resulting genome assemblies have high contiguity (contig NG50: 17.24-80.64 Mb), few assembly errors (contig NGA50: 11.8-59.59 Mb), good consensus quality (QV: 27.84-42.88) and high gene completeness (BUSCO complete: 94.6-95.2%), while consuming low computational resources (CPU hours: 187-1,200). In particular, the WENGAN assembly of the haploid CHM13 sample achieved a contig NG50 of 80.64 Mb (NGA50: 59.59 Mb), which surpasses the contiguity of the current human reference genome (GRCh38 contig NG50: 57.88 Mb).

Immunostaining and In Situ Hybridization of the Developing Acoel Nervous System.

The study of acoel morphologies has been recently stimulated by the knowledge that this group of animals represents an early offshoot of the Bilateria. Understanding how organ systems and tissues develop and the molecular underpinnings of the processes involved has become an area of new research. The microscopic anatomy of these organisms is best understood through the systematic use of immunochemistry and in situ hybridization procedures. These methods allow us to map, in precise detail, the expression patterns of genes and proteins, in space and time. With the additional use of genomic resources, they provide us with insights on how a group of "early" bilaterians have diversified over time. As these animals are new to the world of molecular studies, the protocols have involved a lot of new and specific adaptations to their specific anatomical-histological characteristics. Here we explain some of these protocols in detail, with the aim that should prove useful in our much-needed understanding of the origins of bilaterian animals. An anatomical sketch is provided at the beginning as a necessary guide for those not familiar with the Acoela.

Xenacoelomorpha: a case of independent nervous system centralization?

Centralized nervous systems (NSs) and complex brains are among the most important innovations in the history of life on our planet. In this context, two related questions have been formulated: How did complex NSs arise in evolution, and how many times did this occur? As a step towards finding an answer, we describe the NS of several representatives of the Xenacoelomorpha, a clade whose members show different degrees of NS complexity. This enigmatic clade is composed of three major taxa: acoels, nemertodermatids and xenoturbellids. Interestingly, while the xenoturbellids seem to have a rather 'simple' NS (a nerve net), members of the most derived group of acoel worms clearly have ganglionic brains. This interesting diversity of NS architectures (with different degrees of compaction) provides a unique system with which to address outstanding questions regarding the evolution of brains and centralized NSs. The recent sequencing of xenacoelomorph genomes gives us a privileged vantage point from which to analyse neural evolution, especially through the study of key gene families involved in neurogenesis and NS function, such as G protein-coupled receptors, helix-loop-helix transcription factors and Wnts. We finish our manuscript proposing an adaptive scenario for the origin of centralized NSs (brains).

De novo intrachromosomal gene conversion from OPN1MW to OPN1LW in the male germline results in Blue Cone Monochromacy.

X-linked cone dysfunction disorders such as Blue Cone Monochromacy and X-linked Cone Dystrophy are characterized by complete loss (of) or reduced L- and M- cone function due to defects in the OPN1LW/OPN1MW gene cluster. Here we investigated 24 affected males from 16 families with either a structurally intact gene cluster or at least one intact single (hybrid) gene but harbouring rare combinations of common SNPs in exon 3 in single or multiple OPN1LW and OPN1MW gene copies. We assessed twelve different OPN1LW/MW exon 3 haplotypes by semi-quantitative minigene splicing assay. Nine haplotypes resulted in aberrant splicing of ≥20% of transcripts including the known pathogenic haplotypes (i.e. 'LIAVA', 'LVAVA') with absent or minute amounts of correctly spliced transcripts, respectively. De novo formation of the 'LIAVA' haplotype derived from an ancestral less deleterious 'LIAVS' haplotype was observed in one family with strikingly different phenotypes among affected family members. We could establish intrachromosomal gene conversion in the male germline as underlying mechanism. Gene conversion in the OPN1LW/OPN1MW genes has been postulated, however, we are first to demonstrate a de novo gene conversion within the lineage of a pedigree.