PRRT2 benign familial infantile seizures (BFIS) with atypical evolution to encephalopathy related to status epilepticus during sleep (ESES).

PURPOSE: Heterozygous variants in PRRT2 are mostly associated with benign phenotypes, being the major genetic cause of benign familial infantile seizures (BFIS), as well as in paroxysmal disorders. We report two children from unrelated families with BFIS that evolved to encephalopathy related to status epilepticus during sleep (ESES). METHODS AND RESULTS: Two probands presented with focal motor seizures at 3 months of age, with a limited course. Both children presented, at around 5 years of age, with centro-temporal interictal epileptiform discharges with a source in the frontal operculum, markedly activated by sleep, and associated with stagnation on neuropsychological development. Whole-exome sequencing and co-segregation analysis revealed a frameshift mutation c.649dupC in the proline-rich transmembrane protein 2 (PRRT2) in both probands and all affected family members. CONCLUSION: The mechanism leading to epilepsy and the phenotypic variability of PRRT2 variants remain poorly understood. However, its wide cortical and subcortical expression, in particular in the thalamus, could partially explain both the focal EEG pattern and the evolution to ESES. No variants in the PRRT2 gene have been previously reported in patients with ESES. Due to the rarity of this phenotype, other possible causative cofactors are likely contributing to the more severe course of BFIS in our probands.

Integration of Hi-C with short and long-read genome sequencing reveals the structure of germline rearranged genomes.

Structural variants are a common cause of disease and contribute to a large extent to inter-individual variability, but their detection and interpretation remain a challenge. Here, we investigate 11 individuals with complex genomic rearrangements including germline chromothripsis by combining short- and long-read genome sequencing (GS) with Hi-C. Large-scale genomic rearrangements are identified in Hi-C interaction maps, allowing for an independent assessment of breakpoint calls derived from the GS methods, resulting in >300 genomic junctions. Based on a comprehensive breakpoint detection and Hi-C, we achieve a reconstruction of whole rearranged chromosomes. Integrating information on the three-dimensional organization of chromatin, we observe that breakpoints occur more frequently than expected in lamina-associated domains (LADs) and that a majority reshuffle topologically associating domains (TADs). By applying phased RNA-seq, we observe an enrichment of genes showing allelic imbalanced expression (AIG) within 100 kb around the breakpoints. Interestingly, the AIGs hit by a breakpoint (19/22) display both up- and downregulation, thereby suggesting different mechanisms at play, such as gene disruption and rearrangements of regulatory information. However, the majority of interpretable genes located 200 kb around a breakpoint do not show significant expression changes. Thus, there is an overall robustness in the genome towards large-scale chromosome rearrangements.

Identification and analysis of deletion breakpoints in four Mohr-Tranebjærg syndrome (MTS) patients.

Mohr-Tranebjærg syndrome is an X-linked syndrome characterized by sensorineural hearing impairment in childhood, followed by progressive neurodegeneration leading to a broad phenotypic spectrum. Genetically MTS is caused by pathogenic variants in the TIMM8A gene, including gene deletions and larger contiguous gene deletions. Some of the latter involve the neighboring gene BTK, resulting in agammaglobulinemia. By next-generation mate-pair sequencing we have mapped the chromosomal deletion breakpoints of one MTS case and three XLA-MTS cases and used breakpoint-spanning PCR to fine map the breakpoints by Sanger sequencing. Two of the XLA-MTS cases presented with large deletions (63.5 and 27.2 kb), and the junctional regions were characterized by long stretches of microhomology, indicating that the events have emerged through homologous recombination. Conversely, the MTS case exhibited a small 2 bp region of microhomology, and the regions were not characterized by extensive microhomology. The third XLA-MTS case had a more complex breakpoint, including a 59 bp inverted insertion, thus at least four breakpoints were involved in this event. In conclusion, mate-pair library generation combined with next-generation sequencing is an efficient method for breakpoint identification, also in regions characterized by repetitive elements.

Does rapid sequence divergence preclude RNA structure conservation in vertebrates?

Accelerated evolution of any portion of the genome is of significant interest, potentially signaling positive selection of phenotypic traits and adaptation. Accelerated evolution remains understudied for structured RNAs, despite the fact that an RNA's structure is often key to its function. RNA structures are typically characterized by compensatory (structure-preserving) basepair changes that are unexpected given the underlying sequence variation, i.e., they have evolved through negative selection on structure. We address the question of how fast the primary sequence of an RNA can change through evolution while conserving its structure. Specifically, we consider predicted and known structures in vertebrate genomes. After careful control of false discovery rates, we obtain 13 de novo structures (and three known Rfam structures) that we predict to have rapidly evolving sequences-defined as structures where the primary sequences of human and mouse have diverged at least twice as fast (1.5 times for Rfam) as nearby neutrally evolving sequences. Two of the three known structures function in translation inhibition related to infection and immune response. We conclude that rapid sequence divergence does not preclude RNA structure conservation in vertebrates, although these events are relatively rare.

Rare Pathogenic Variants in Genes Implicated in Glutamatergic Neurotransmission Pathway Segregate with Schizophrenia in Pakistani Families.

Schizophrenia is a disabling neuropsychiatric disorder of adulthood onset with high heritability. Worldwide collaborations have identified an association of ~270 common loci, with small individual effects and hence weak clinical implications. The recent technological feasibility of exome sequencing enables the identification of rare variants of high penetrance that refine previous findings and improve risk assessment and prognosis. We recruited two multiplex Pakistani families, having 11 patients and 19 unaffected individuals in three generations. We performed genome-wide SNP genotyping, next-generation mate pairing and whole-exome sequencing of selected members to unveil genetic components. Candidate variants were screened in unrelated cohorts of 508 cases, 300 controls and fifteen families (with 51 affected and 47 unaffected individuals) of Pakistani origin. The structural impact of substituted residues was assessed through in silico modeling using iTASSER. In one family, we identified a rare novel microduplication (5q14.1_q14.2) encompassing critical genes involved in glutamate signaling, such as CMYA5, HOMER and RasGRF2. The second family segregates two ultra-rare, predicted pathogenic variants in the GRIN2A (NM_001134407.3: c.3505C>T, (p.R1169W) and in the NRG3 NM_001010848.4: c.1951G>A, (p.E651K). These genes encode for parts of AMPA and NMDA receptors of glutamatergic neurotransmission, respectively, and the variants are predicted to compromise protein function by destabilizing their structures. The variants were absent in the aforementioned cohorts. Our findings suggest that rare, highly penetrant variants of genes involved in glutamatergic neurotransmission are contributing to the etiology of schizophrenia in these families. It also highlights that genetic investigations of multiplex, multigenerational families could be a powerful approach to identify rare genetic variants involved in complex disorders.

Acute and persistent symptoms in non-hospitalized PCR-confirmed COVID-19 patients.

Reports of persistent symptoms after hospitalization with COVID-19 have raised concern of a "long COVID" syndrome. This study aimed at determining the prevalence of and risk factors for acute and persistent symptoms in non-hospitalized patients with polymerase chain reaction (PCR) confirmed COVID-19. We conducted a cohort study of non-hospitalized participants identified via the Danish Civil Registration System with a SARS-CoV-2-positive PCR-test and available biobank samples. Participants received a digital questionnaire on demographics and COVID-19-related symptoms. Persistent symptoms: symptoms > 4 weeks (in sensitivity analyses > 12 weeks). We included 445 participants, of whom 34% were asymptomatic. Most common acute symptoms were fatigue, headache, and sneezing, while fatigue and reduced smell and taste were most severe. Persistent symptoms, most commonly fatigue and memory and concentration difficulties, were reported by 36% of 198 symptomatic participants with follow-up > 4 weeks. Risk factors for persistent symptoms included female sex (women 44% vs. men 24%, odds ratio 2.7, 95% CI 1.4-5.1, p = 0.003) and BMI (odds ratio 1.1, 95% CI 1.0-1.2, p = 0.001). In conclusion, among non-hospitalized PCR-confirmed COVID-19 patients one third were asymptomatic while one third of symptomatic participants had persistent symptoms illustrating the heterogeneity of disease presentation. These findings should be considered in health care planning and policy making related to COVID-19.

A GDF5 frameshift mutation segregating with Grebe type chondrodysplasia and brachydactyly type C+ in a 6 generations family: Clinical report and mini review.

Different mutations in the Growth/Differentiation Factor 5 gene (GDF5) have been associated with varying types of skeletal dysplasia, including Grebe type chondrodysplasia (GTC), Hunter-Thompson syndrome, Du Pan Syndrome and Brachydactyly type C (BDC). Heterozygous pathogenic mutations exert milder effects, whereas homozygous mutations are known to manifest more severe phenotypes. In this study, we report a GDF5 frameshift mutation (c.404delC) segregating over six generations in an extended consanguineous Pakistani family. The family confirmed that both GTC and BDC are part of the GDF5 mutational spectrum, with severe GTC associated with homozygosity, and with a wide phenotypic variability among heterozygous carriers, ranging from unaffected non-penetrant carriers, to classical BDC and to novel unclassified types of brachydactylies.

Nationwide germline whole genome sequencing of 198 consecutive pediatric cancer patients reveals a high incidence of cancer prone syndromes.

PURPOSE: Historically, cancer predisposition syndromes (CPSs) were rarely established for children with cancer. This nationwide, population-based study investigated how frequently children with cancer had or were likely to have a CPS. METHODS: Children (0-17 years) in Denmark with newly diagnosed cancer were invited to participate in whole-genome sequencing of germline DNA. Suspicion of CPS was assessed according to Jongmans'/McGill Interactive Pediatric OncoGenetic Guidelines (MIPOGG) criteria and familial cancer diagnoses were verified using population-based registries. RESULTS: 198 of 235 (84.3%) eligible patients participated, of whom 94/198 (47.5%) carried pathogenic variants (PVs) in a CPS gene or had clinical features indicating CPS. Twenty-nine of 198 (14.6%) patients harbored a CPS, of whom 21/198 (10.6%) harbored a childhood-onset and 9/198 (4.5%) an adult-onset CPS. In addition, 23/198 (11.6%) patients carried a PV associated with biallelic CPS. Seven of the 54 (12.9%) patients carried two or more variants in different CPS genes. Seventy of 198 (35.4%) patients fulfilled the Jongmans' and/or MIPOGG criteria indicating an underlying CPS, including two of the 9 (22.2%) patients with an adult-onset CPS versus 18 of the 21 (85.7%) patients with a childhood-onset CPS (p = 0.0022), eight of the additional 23 (34.8%) patients with a heterozygous PV associated with biallelic CPS, and 42 patients without PVs. Children with a central nervous system (CNS) tumor had family members with CNS tumors more frequently than patients with other cancers (11/44, p = 0.04), but 42 of 44 (95.5%) cases did not have a PV in a CPS gene. CONCLUSION: These results demonstrate the value of systematically screening pediatric cancer patients for CPSs and indicate that a higher proportion of childhood cancers may be linked to predisposing germline variants than previously supposed.

RRP7A links primary microcephaly to dysfunction of ribosome biogenesis, resorption of primary cilia, and neurogenesis.

Primary microcephaly (MCPH) is characterized by reduced brain size and intellectual disability. The exact pathophysiological mechanism underlying MCPH remains to be elucidated, but dysfunction of neuronal progenitors in the developing neocortex plays a major role. We identified a homozygous missense mutation (p.W155C) in Ribosomal RNA Processing 7 Homolog A, RRP7A, segregating with MCPH in a consanguineous family with 10 affected individuals. RRP7A is highly expressed in neural stem cells in developing human forebrain, and targeted mutation of Rrp7a leads to defects in neurogenesis and proliferation in a mouse stem cell model. RRP7A localizes to centrosomes, cilia and nucleoli, and patient-derived fibroblasts display defects in ribosomal RNA processing, primary cilia resorption, and cell cycle progression. Analysis of zebrafish embryos supported that the patient mutation in RRP7A causes reduced brain size, impaired neurogenesis and cell proliferation, and defective ribosomal RNA processing. These findings provide novel insight into human brain development and MCPH.

Paroxysmal Cranial Dyskinesia and Nail-Patella Syndrome Caused by a Novel Variant in the LMX1B Gene.

BACKGROUND: In a Danish family, multiple individuals in five generations present with early-onset paroxysmal cranial dyskinesia, musculoskeletal abnormalities, and kidney dysfunction. OBJECTIVE: To demonstrate linkage and to identify the underlying genetic cause of disease. METHODS: Genome-wide single-nucleotide polymorphisms analysis, Sequence-Tagged-Site marker analyses, exome sequencing, and Sanger sequencing were performed. RESULTS: Linkage analyses identified a candidate locus on chromosome 9. Exome sequencing revealed a novel variant in LMX1B present in all affected individuals, logarithm of the odds (LOD) score of z = 6.54, predicted to be damaging. Nail-patella syndrome (NPS) is caused by pathogenic variants in LMX1B encoding a transcription factor essential to cytoskeletal and kidney growth and dopaminergic and serotonergic network development. NPS is characterized by abnormal musculoskeletal features and kidney dysfunction. Movement disorders have not previously been associated with NPS. CONCLUSIONS: Paroxysmal dyskinesia is a heretofore unrecognized feature of the NPS spectrum. The pathogenic mechanism might relate to aberrant dopaminergic circuits. © 2020 International Parkinson and Movement Disorder Society.

Chromothripsis and DNA Repair Disorders.

Chromothripsis is a mutational mechanism leading to complex and relatively clustered chromosomal rearrangements, resulting in diverse phenotypic outcomes depending on the involved genomic landscapes. It may occur both in the germ and the somatic cells, resulting in congenital and developmental disorders and cancer, respectively. Asymptomatic individuals may be carriers of chromotriptic rearrangements and experience recurrent reproductive failures when two or more chromosomes are involved. Several mechanisms are postulated to underlie chromothripsis. The most attractive hypothesis involves chromosome pulverization in micronuclei, followed by the incorrect reassembly of fragments through DNA repair to explain the clustered nature of the observed complex rearrangements. Moreover, exogenous or endogenous DNA damage induction and dicentric bridge formation may be involved. Chromosome instability is commonly observed in the cells of patients with DNA repair disorders, such as ataxia telangiectasia, Nijmegen breakage syndrome, and Bloom syndrome. In addition, germline variations of TP53 have been associated with chromothripsis in sonic hedgehog medulloblastoma and acute myeloid leukemia. In the present review, we focus on the underlying mechanisms of chromothripsis and the involvement of defective DNA repair genes, resulting in chromosome instability and chromothripsis-like rearrangements.

Multigenic truncation of the semaphorin-plexin pathway by a germline chromothriptic rearrangement associated with Moebius syndrome.

Moebius syndrome (MBS) is a congenital disorder caused by paralysis of the facial and abducens nerves. Although a number of candidate genes have been suspected, so far only mutations in PLXND1 and REV3L are confirmed to cause MBS. Here, we fine mapped the breakpoints of a complex chromosomal rearrangement (CCR) 46,XY,t(7;8;11;13) in a patient with MBS, which revealed 41 clustered breakpoints with typical hallmarks of chromothripsis. Among 12 truncated protein-coding genes, SEMA3A is known to bind to the MBS-associated PLXND1. Intriguingly, the CCR also truncated PIK3CG, which in silico interacts with REVL3 encoded by the other known MBS-gene REV3L, and with the SEMA3A/PLXND1 complex via FLT1. Additional studies of other complex rearrangements may reveal whether the multiple breakpoints in germline chromothripsis may predispose to complex multigenic disorders.

A novel in-frame mutation in CLN3 leads to Juvenile neuronal ceroid lipofuscinosis in a large Pakistani family.

Aim: Neuronal ceroid lipofuscinosis (NCLs) are the most common neurodegenerative disorders, with global incidence of 1 in 100,000 live births. NCLs affect central nervous system, primarily cerebellar and cerebral cortices. Juvenile neuronal ceroid lipofuscinosis (JNCL), also known as Batten disease, is the most common form of NCLs. JNCL is primarily caused by pathogenic mutations in CLN3 gene, which encodes a transporter transmembrane protein of uncertain function. The 1.02 kb deletion is the most common mutation in CLN3 that results in frame shift and a premature termination leading to nonfunctional protein. Here, we invetigated a large consanguineous family consisting of four affected individuals with clincal symptoms suggestive of Juvenile neuronal ceroid lipofuscinosis. Materials and methods: We conducted clinial and radilogical investigation of the family and performed NGS based Gene Panel sequencing comprising of five hundred and forty five candidate genes to characterize it at genetic level. Results: We identified a novel homozygous c.181_183delGAC mutation in the CLN3 gene seggregating witht the disorder in the family. The mutation induces in-frame deletion, deleting one amino acid (p.Asp61del) in CLN3 protein. The deleted amino acid aspartic acid plays an important role as general acid in enzymes active centers as well as in maintaining the ionic character of proteins. Conclusion: Our finding adds to genetic variability of Juvenile neuronal ceroid lipofuscinosis associated with CLN3 gene and a predicted CLN3 protein interacting domain site.

Haploinsufficiency of ARHGAP42 is associated with hypertension.

Family studies have established that the heritability of blood pressure is significant and genome-wide association studies (GWAS) have identified numerous susceptibility loci, including one within the non-coding part of Rho GTPase-activating protein 42 gene (ARHGAP42) on chromosome 11q22.1. Arhgap42-deficient mice have significantly elevated blood pressure, but the phenotypic effects of human variants in the coding part of the gene are unknown. In a Danish cohort of carriers with apparently balanced chromosomal rearrangements, we identified a family where a reciprocal translocation t(11;18)(q22.1;q12.2) segregated with hypertension and obesity. Clinical re-examination revealed that four carriers (age 50-77 years) have had hypertension for several years along with an increased body mass index (34-43 kg/m2). A younger carrier (age 23 years) had normal blood pressure and body mass index. Mapping of the chromosomal breakpoints with mate-pair and Sanger sequencing revealed truncation of ARHGAP42. A decreased expression level of ARHGAP42 mRNA in the blood was found in the translocation carriers relative to controls and allele-specific expression analysis showed monoallelic expression in the translocation carriers, confirming that the truncated allele of ARHGAP42 was not expressed. These findings support that haploinsufficiency of ARHGAP42 leads to an age-dependent hypertension. The other breakpoint truncated a regulatory domain of the CUGBP Elav-like family member 4 (CELF4) gene on chromosome 18q12.2 that harbours several GWAS signals for obesity. We thereby provide additional support for an obesity locus in the CELF4 domain.

A splice-site variant in the lncRNA gene RP1-140A9.1 cosegregates in the large Volkmann cataract family.

Purpose: To identify the mutation for Volkmann cataract (CTRCT8) at 1p36.33. Methods: The genes in the candidate region 1p36.33 were Sanger and parallel deep sequenced, and informative single nucleotide polymorphisms (SNPs) were identified for linkage analysis. Expression analysis with reverse transcription polymerase chain reaction (RT-PCR) of the candidate gene was performed using RNA from different human tissues. Quantitative transcription polymerase chain reaction (qRT-PCR) analysis of the GNB1 gene was performed in affected and healthy individuals. Bioinformatic analysis of the linkage regions including the candidate gene was performed. Results: Linkage analysis of the 1p36.33 CCV locus applying new marker systems obtained with Sanger and deep sequencing reduced the candidate locus from 2.1 Mb to 0.389 Mb flanked by the markers STS-22AC and rs549772338 and resulted in an logarithm of the odds (LOD) score of Z = 21.67. The identified mutation, rs763295804, affects the donor splice site in the long non-coding RNA gene RP1-140A9.1 (ENSG00000231050). The gene including splice-site junctions is conserved in primates but not in other mammalian genomes, and two alternative transcripts were shown with RT-PCR. One of these transcripts represented a lens cell-specific transcript. Meta-analysis of the Cross-Linking-Immuno-Precipitation sequencing (CLIP-Seq) data suggested the RNA binding protein (RBP) eIF4AIII is an active counterpart for RP1-140A9.1, and several miRNA and transcription factors binding sites were predicted in the proximity of the mutation. ENCODE DNase I hypersensitivity and histone methylation and acetylation data suggest the genomic region may have regulatory functions. Conclusions: The mutation in RP1-140A9.1 suggests the long non-coding RNA as the candidate cataract gene associated with the autosomal dominant inherited congenital cataract from CCV. The mutation has the potential to destroy exon/intron splicing of both transcripts of RP1-140A9.1. Sanger and massive deep resequencing of the linkage region failed to identify alternative candidates suggesting the mutation in RP1-140A9.1 is causative for the CCV phenotype.

Small supernumerary marker chromosomes: A legacy of trisomy rescue?

We studied by a whole genomic approach and trios genotyping, 12 de novo, nonrecurrent small supernumerary marker chromosomes (sSMC), detected as mosaics during pre- or postnatal diagnosis and associated with increased maternal age. Four sSMCs contained pericentromeric portions only, whereas eight had additional non-contiguous portions of the same chromosome, assembled together in a disordered fashion by repair-based mechanisms in a chromothriptic event. Maternal hetero/isodisomy was detected with a paternal origin of the sSMC in some cases, whereas in others two maternal alleles in the sSMC region and biparental haplotypes of the homologs were detected. In other cases, the homologs were biparental while the sSMC had the same haplotype of the maternally inherited chromosome. These findings strongly suggest that most sSMCs are the result of a multiple-step mechanism, initiated by maternal meiotic nondisjunction followed by postzygotic anaphase lagging of the supernumerary chromosome and its subsequent chromothripsis.

Replicative and non-replicative mechanisms in the formation of clustered CNVs are indicated by whole genome characterization.

Clustered copy number variants (CNVs) as detected by chromosomal microarray analysis (CMA) are often reported as germline chromothripsis. However, such cases might need further investigations by massive parallel whole genome sequencing (WGS) in order to accurately define the underlying complex rearrangement, predict the occurrence mechanisms and identify additional complexities. Here, we utilized WGS to delineate the rearrangement structure of 21 clustered CNV carriers first investigated by CMA and identified a total of 83 breakpoint junctions (BPJs). The rearrangements were further sub-classified depending on the patterns observed: I) Cases with only deletions (n = 8) often had additional structural rearrangements, such as insertions and inversions typical to chromothripsis; II) cases with only duplications (n = 7) or III) combinations of deletions and duplications (n = 6) demonstrated mostly interspersed duplications and BPJs enriched with microhomology. In two cases the rearrangement mutational signatures indicated both a breakage-fusion-bridge cycle process and haltered formation of a ring chromosome. Finally, we observed two cases with Alu- and LINE-mediated rearrangements as well as two unrelated individuals with seemingly identical clustered CNVs on 2p25.3, possibly a rare European founder rearrangement. In conclusion, through detailed characterization of the derivative chromosomes we show that multiple mechanisms are likely involved in the formation of clustered CNVs and add further evidence for chromoanagenesis mechanisms in both "simple" and highly complex chromosomal rearrangements. Finally, WGS characterization adds positional information, important for a correct clinical interpretation and deciphering mechanisms involved in the formation of these rearrangements.