Myoclonic status epilepticus and cerebellar hypoplasia associated with a novel variant in the GRIA3 gene

AMPA-type glutamate receptors (AMPARs) are postsynaptic ionotropic receptors which mediate fast excitatory currents. AMPARs have a heterotetrameric structure, variably composed by the four subunits GluA1-4 which are encoded by genes GRIA1-4. Increasing evidence support the role of pathogenic variants in GRIA1-4 genes as causative for syndromic intellectual disability (ID). We report an Italian pedigree where some male individuals share ID, seizures and facial dysmorphisms. The index subject was referred for severe ID, myoclonic seizures, cerebellar signs and short stature. Whole exome sequencing identified a novel variant in GRIA3, c.2360A > G, p.(Glu787Gly). The GRIA3 gene maps to chromosome Xq25 and the c.2360A > G variant was transmitted by his healthy mother. Subsequent analysis in the family showed a segregation pattern compatible with the causative role of this variant, further supported by preliminary functional insights. We provide a detailed description of the clinical evolution of the index subjects and stress the relevance of myoclonic seizures and cerebellar syndrome as cardinal features of his presentation.

AMH and AMHR2 Involvement in Congenital Disorders of Sex Development

Anti-müllerian hormone (AMH) is 1 of the 2 testicular hormones involved in male development of the genitalia during fetal life. When the testes differentiate, AMH is secreted by Sertoli cells and binds to its specific receptor type II (AMHR2) on the müllerian ducts, inducing their regression. In the female fetus, the lack of AMH allows the müllerian ducts to form the fallopian tubes, the uterus, and the upper part of the vagina. The human <i>AMH</i> gene maps to 19p13.3 and consists of 5 exons and 4 introns spanning 2,764 bp. The <i>AMHR2</i> gene maps to 12q13.13, consists of 11 exons, and is 7,817 bp long. Defects in the AMH pathway are the underlying etiology of a subgroup of disorders of sex development (DSD) in 46,XY patients. The condition is known as the persistent müllerian duct syndrome (PMDS), characterized by the existence of a uterus and fallopian tubes in a boy with normally virilized external genitalia. Approximately 200 cases of patients with PMDS have been reported to date with clinical, biochemical, and molecular genetic characterization. An updated review is provided in this paper. With highly sensitive techniques, AMH and AMHR2 expression has also been detected in other tissues, and massive sequencing technologies have unveiled variants in <i>AMH</i> and <i>AMHR2</i> genes in hitherto unsuspected conditions.

CRISPRCasTyper: An automated tool for the identification, annotation and classification of CRISPR-Cas loci

CRISPR-Cas loci encode for highly diversified prokaryotic adaptive defense systems that have recently become popular for their applications in gene editing and beyond. The increasing demand for bioinformatic tools that systematically detect and classify CRISPR-Cas systems has been largely challenged by their complex dynamic nature and rapidly expanding classification. Here, we developed CRISPRCasTyper, a new automated software tool with improved capabilities for identifying and typing CRISPR arrays and cas loci across prokaryotic sequences, based on the latest classification and nomenclature (39 subtypes/variants) (Makarova et al. 2020; Pinilla-Redondo et al. 2019). As a novel feature, CRISPRCasTyper uses a machine learning approach to subtype CRISPR arrays based on the sequences of the direct repeats. This allows the typing of orphan and distant arrays which, for example, are commonly observed in fragmented metagenomic assemblies. Furthermore, the tool provides a graphical output, where CRISPRs and cas operon arrangements are visualized in the form of colored gene maps, thus aiding annotation of partial and novel systems through synteny. Moreover, CRISPRCasTyper can resolve hybrid CRISPR-Cas systems and detect loci spanning the ends of sequences with a circular topology, such as complete genomes and plasmids. CRISPRCasTyper was benchmarked against a manually curated set of 31 subtypes/variants with a median accuracy of 98.6%. Altogether, we present an up-to-date and freely available software pipeline for significantly improved automated predictions of CRISPR-Cas loci across genomic sequences.ImplementationCRISPRCasTyper is available through conda and PyPi under the MIT license (https://github.com/Russel88/CRISPRCasTyper), and is also available as a web server (http://cctyper.crispr.dk).

Enhancer–gene maps in the human and zebrafish genomes using evolutionary linkage conservation

The spatiotemporal expression of genes is controlled by enhancer sequences that bind transcription factors. Identifying the target genes of enhancers remains difficult because enhancers regulate gene expression over long genomic distances. To address this, we used an evolutionary approach to build two genome-wide maps of predicted enhancer–gene associations in the human and zebrafish genomes. Evolutionary conserved sequences were linked to their predicted target genes using PEGASUS, a bioinformatics method that relies on evolutionary conservation of synteny. The analysis of these maps revealed that the number of predicted enhancers linked to a gene correlate with its expression breadth. Comparison of both maps identified hundreds of putative vertebrate ancestral regulatory relationships from which we could determine that predicted enhancer–gene distances scale with genome size despite strong positional conservation. The two maps represent a resource for further studies, including the prioritization of sequence variants in whole genome sequence of patients affected by genetic diseases.

Enhancer-gene maps in the human and zebrafish genomes using evolutionary linkage conservation

The spatiotemporal expression of genes is controlled by enhancer sequences that bind transcription factors. Identifying the target genes of enhancers remains difficult because enhancers regulate gene expression over long genomic distances. To address this, we used an evolutionary approach to build two genome-wide maps of enhancer-gene associations in the human and zebrafish genomes. Enhancers were identified using sequence conservation, and linked to their predicted target genes using PEGASUS, a bioinformatics method that relies on evolutionary conservation of synteny. The analysis of these maps revealed that the number of enhancers linked to a gene correlate with its expression breadth. Comparison of both maps identified hundreds of vertebrate ancestral regulatory relationships from which we could determine that enhancer-gene distances scale with genome size despite strong positional conservation. The two maps represent a resource for further studies, including the prioritisation of sequence variants in whole genome sequence of patients affected by genetic diseases.

Organization of the multigene families of African Swine Fever Virus

African swine fever virus is a complex DNA virus that infects swine and is spread by ticks. Mortality rates in domestic pigs are very high and the virus is a significant threat to pork farming. The genomes of 16 viruses have been sequenced completely, but these represent only a few of the 23 genotypes. The viral genome is unusual in that it contains 5 multigene families, each of which contain 3-19 duplicated copies (paralogs). There is significant sequence divergence between the paralogs in a single virus and between the orthologs in the different viral genomes. This, together with the fact that in most of the multigene families there are numerous gene indels that create truncations and fusions, makes annotation of these regions very difficult; it has led to inconsistent annotation of the 16 viral genomes. In this project, we have created multiple sequence alignments for each of the multigene families and have produced gene maps to help researchers more easily understand the organization of the multigene families among the different viruses. These gene maps will help researchers ascertain which members of the multigene families are present in each of the viruses. This is critical because some of the multigene families are known to be associated with virus virulence.

Reduced DOCK4 expression leads to erythroid dysplasia in myelodysplastic syndromes

Anemia is the predominant clinical manifestation of myelodysplastic syndromes (MDS). Loss or deletion of chromosome 7 is commonly seen in MDS and leads to a poor prognosis. However, the identity of functionally relevant, dysplasia-causing, genes on 7q remains unclear. Dedicator of cytokinesis 4 (DOCK4) is a GTPase exchange factor, and its gene maps to the commonly deleted 7q region. We demonstrate that DOCK4 is underexpressed in MDS bone marrow samples and that the reduced expression is associated with decreased overall survival in patients. We show that depletion of DOCK4 levels leads to erythroid cells with dysplastic morphology both in vivo and in vitro. We established a novel single-cell assay to quantify disrupted F-actin filament network in erythroblasts and demonstrate that reduced expression of DOCK4 leads to disruption of the actin filaments, resulting in erythroid dysplasia that phenocopies the red blood cell (RBC) defects seen in samples from MDS patients. Reexpression of DOCK4 in −7q MDS patient erythroblasts resulted in significant erythropoietic improvements. Mechanisms underlying F-actin disruption revealed that DOCK4 knockdown reduces ras-related C3 botulinum toxin substrate 1 (RAC1) GTPase activation, leading to increased phosphorylation of the actin-stabilizing protein ADDUCIN in MDS samples. These data identify DOCK4 as a putative 7q gene whose reduced expression can lead to erythroid dysplasia.