Expanding the genetic and phenotypic landscape of replication factor C complex-related disorders: RFC4 deficiency is linked to a multisystemic disorder.

The precise regulation of DNA replication is vital for cellular division and genomic integrity. Central to this process is the replication factor C (RFC) complex, encompassing five subunits, which loads proliferating cell nuclear antigen onto DNA to facilitate the recruitment of replication and repair proteins and enhance DNA polymerase processivity. While RFC1's role in cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is known, the contributions of RFC2-5 subunits on human Mendelian disorders is largely unexplored. Our research links bi-allelic variants in RFC4, encoding a core RFC complex subunit, to an undiagnosed disorder characterized by incoordination and muscle weakness, hearing impairment, and decreased body weight. We discovered across nine affected individuals rare, conserved, predicted pathogenic variants in RFC4, all likely to disrupt the C-terminal domain indispensable for RFC complex formation. Analysis of a previously determined cryo-EM structure of RFC bound to proliferating cell nuclear antigen suggested that the variants disrupt interactions within RFC4 and/or destabilize the RFC complex. Cellular studies using RFC4-deficient HeLa cells and primary fibroblasts demonstrated decreased RFC4 protein, compromised stability of the other RFC complex subunits, and perturbed RFC complex formation. Additionally, functional studies of the RFC4 variants affirmed diminished RFC complex formation, and cell cycle studies suggested perturbation of DNA replication and cell cycle progression. Our integrated approach of combining in silico, structural, cellular, and functional analyses establishes compelling evidence that bi-allelic loss-of-function RFC4 variants contribute to the pathogenesis of this multisystemic disorder. These insights broaden our understanding of the RFC complex and its role in human health and disease.

A Comparison of Structural Variant Calling from Short-Read and Nanopore-Based Whole-Genome Sequencing Using Optical Genome Mapping as a Benchmark.

The identification of structural variants (SVs) in genomic data represents an ongoing challenge because of difficulties in reliable SV calling leading to reduced sensitivity and specificity. We prepared high-quality DNA from 9 parent-child trios, who had previously undergone short-read whole-genome sequencing (Illumina platform) as part of the Genomics England 100,000 Genomes Project. We reanalysed the genomes using both Bionano optical genome mapping (OGM; 8 probands and one trio) and Nanopore long-read sequencing (Oxford Nanopore Technologies [ONT] platform; all samples). To establish a "truth" dataset, we asked whether rare proband SV calls (n = 234) made by the Bionano Access (version 1.6.1)/Solve software (version 3.6.1_11162020) could be verified by individual visualisation using the Integrative Genomics Viewer with either or both of the Illumina and ONT raw sequence. Of these, 222 calls were verified, indicating that Bionano OGM calls have high precision (positive predictive value 95%). We then asked what proportion of the 222 true Bionano SVs had been identified by SV callers in the other two datasets. In the Illumina dataset, sensitivity varied according to variant type, being high for deletions (115/134; 86%) but poor for insertions (13/58; 22%). In the ONT dataset, sensitivity was generally poor using the original Sniffles variant caller (48% overall) but improved substantially with use of Sniffles2 (36/40; 90% and 17/23; 74% for deletions and insertions, respectively). In summary, we show that the precision of OGM is very high. In addition, when applying the Sniffles2 caller, the sensitivity of SV calling using ONT long-read sequence data outperforms Illumina sequencing for most SV types.

UK and US risk factors for hearing loss in neonatal intensive care unit infants.

IMPORTANCE: Early detection and intervention of hearing loss may mitigate negative effects on children's development. Children who were admitted to the neonatal intensive care unit (NICU) as babies are particularly susceptible to hearing loss and risk factors are vital for surveillance. DESIGN, SETTING AND PARTICIPANTS: This single-centre retrospective cohort study included data from 142 inborn infants who had been admitted to the NICU in a tertiary regional referral centre. Data were recorded for 71 infants with confirmed permanent congenital hearing loss hearing loss. To determine impact of NICU admission independently of prematurity, babies were individually matched with 71 inborn infants on gestational age, birthweight, and sex. MAIN OUTCOMES AND MEASURES: Neonatal indicators were recorded for all children with permanent congenital hearing loss. Presence of UK and US risk factors for hearing loss were collected on the neonatal population with hearing loss and for the matched controls. RESULTS: A fifth (21%) of babies with hearing loss had one or more UK risk factors whereas most (86%) had at least one US risk factor. False positives would be evident if US factors were used whereas the matched controls had no UK risk factors. Ten babies who at birth had no UK or US risk factors did not have any significant neonatal indicators identified in their records, one was ventilated for one day and two had a genetic anomaly. CONCLUSIONS AND RELEVANCE: Current risk factors for hearing loss we identified for follow-up in this high-risk group are highly specific for congenital hearing loss. UK risk factors were highly specific for hearing loss but not sensitive and conversely, US risk factors are sensitive but not specific so false positives would be recorded. A national study of neonatal indicators could provide the utility to test which combinations of risk factors provide high sensitivity without losing specificity.