Search

Introduction: The field of forensic DNA analysis has undergone rapid advancements in recent decades. The integration of massively parallel sequencing (MPS) has notably expanded the forensic toolkit, moving beyond identity matching to predicting phenotypic traits and biogeographical ancestry. This shift is of particular significance in cases where conventional DNA profiling fails to identify a single suspect. Supplementing forensic analyses with estimated biological age may be valuable but involves a complex and time-consuming DNA methylation analysis. This study explores and validates the performance of a comprehensive forensic third-generation sequencing assay utilizing Oxford Nanopore Technologies (ONT) in an adaptive and direct sequencing approach. We incorporated the most widely used forensic markers, i.e., STRs, SNPs, InDels, mitochondrial DNA (mtDNA), and two methylation-based clock classifiers, thereby combining forensic genetic and epigenetic analysis in one single workflow., Methods and Results: In our investigation, DNA from six anonymous individuals was sequenced using the ONT standard adaptive direct sequencing approach, reaching a mean percentage of on-target reads ranging from 6.6 % to 7.7 % per sample. ONT data was compared to standard MPS data and Illumina EPIC DNA methylation profiles. Basecalling employed recommended ONT software packages. TREAT was used for ONT-based analysis of autosomal and Y-chromosome STRs, achieving 90-92 % correct calls depending on allelic read depth thresholds. InDel analyses for two lower-quality samples proved challenging due to inadequate read depth, while the remaining four samples significantly contributed to the observed percentage markers (60.9 %) and correct calls (97.8 %). SNP analysis achieved a 98 % call rate, with only two mismatches and two missed alleles. ONT-generated DNA methylation data demonstrated Pearson's correlation coefficients with EPIC data ranging from 0.67 to 0.97 for Horvath's clock. Additional age-associated markers exhibited Pearson's correlation coefficients with chronological age between 0.14 (ELOVL2) and 0.96 (FHL2) at read depths of <30 and <20, respectively. Despite excluding mtDNA from our targeted sequencing approach, adaptive proof-reading fragments covered the complete mtDNA with an average read depth of 21-72, showing 100 % concordance with reference data., Discussion: Our exploratory study using ONT adaptive sequencing for conventional forensic and age associated DNA methylation markers showed high sequencing accuracy for a significant number of markers, showcasing ONT as a promising (epi)genetic forensic method. Future studies must address three critical aspects: determining clear quantity and quality measures and detection thresholds for accuracy, optimizing input DNA quantity for forensic casework expectations, and addressing ethical considerations associated with phenotype and ancestry analysis to prevent ethnic biases., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The zoonotic pathogen Streptococcus suis can cause septicemia and meningitis in humans. We report five complete genomes of Streptococcus suis serotype 2 and serotype 9, covering the complete phylogeny of serotype 9 Dutch porcine isolates and zoonotic isolates. The isolates include the model strain S10 and the Dutch emerging zoonotic lineage., (Copyright © 2020 van der Putten et al.)

Perrault syndrome (PS) is a rare recessive disorder characterized by ovarian dysgenesis and sensorineural deafness. It is clinically and genetically heterogeneous, and previously mutations have been described in different genes, mostly related to mitochondrial proteostasis. We diagnosed three unrelated females with PS and set out to identify the underlying genetic cause using exome sequencing. We excluded mutations in the known PS genes, but identified a single homozygous mutation in the ERAL1 gene (c.707A > T; p.Asn236Ile). Since ERAL1 protein binds to the mitochondrial 12S rRNA and is involved in the assembly of the small mitochondrial ribosomal subunit, the identified variant represented a likely candidate. In silico analysis of a 3D model for ERAL1 suggested that the mutated residue hinders protein-substrate interactions, potentially affecting its function. On a molecular basis, PS skin fibroblasts had reduced ERAL1 protein levels. Complexome profiling of the cells showed an overall decrease in the levels of assembled small ribosomal subunit, indicating that the ERAL1 variant affects mitochondrial ribosome assembly. Moreover, levels of the 12S rRNA were reduced in the patients, and were rescued by lentiviral expression of wild type ERAL1. At the physiological level, mitochondrial respiration was markedly decreased in PS fibroblasts, confirming disturbed mitochondrial function. Finally, knockdown of the C. elegans ERAL1 homologue E02H1.2 almost completely blocked egg production in worms, mimicking the compromised fertility in PS-affected women. Our cross-species data in patient cells and worms support the hypothesis that mutations in ERAL1 can cause PS and are associated with changes in mitochondrial metabolism., (© The Author 2017. Published by Oxford University Press.)

Background: Myoclonus-dystonia (M-D) is a hyperkinetic movement disorder with predominant myoclonic symptoms combined with dystonia of the upper part of the body. A proportion of M-D cases are caused by mutations in the epsilon-sarcoglycan gene. In remaining M-D patients, no genetic factor has been established, indicating genetic heterogeneity., Methods: Patients were included in a prospective clinical database and recruited from referral centers and general neurology clinics in The Netherlands. To investigate new genetic causal factors in M-D syndrome, we performed homozygosity mapping combined with exome sequencing in a three-generation M-D family and genetically screened 24 additional patients with M-D., Results: We found co-segregation of the rare missense variant Thr1904Met in the RELN gene. By additional screening of an M-D cohort, we identified co-segregation of RELN variants in two families (Thr1904Met, Ile1217Met) and identified two sporadic RELN mutation carriers (Pro1703Arg, Leu411Ile). Taken together, five of 25 SGCE-negative M-D patients carried RELN rare missense variants., Conclusion: We propose that RELN mutations contribute to the genetic heterogeneity of M-D. Reelin is a large secreted glycoprotein that plays essential roles in the cytoarchitecture of laminated brain structures and modulation of synaptic transmission and plasticity., (© 2015 International Parkinson and Movement Disorder Society.)

The Hennekam lymphangiectasia-lymphedema syndrome is a genetically heterogeneous disorder. It can be caused by mutations in CCBE1 which are found in approximately 25 % of cases. We used homozygosity mapping and whole-exome sequencing in the original HS family with multiple affected individuals in whom no CCBE1 mutation had been detected, and identified a homozygous mutation in the FAT4 gene. Subsequent targeted mutation analysis of FAT4 in a cohort of 24 CCBE1 mutation-negative Hennekam syndrome patients identified homozygous or compound heterozygous mutations in four additional families. Mutations in FAT4 have been previously associated with Van Maldergem syndrome. Detailed clinical comparison between van Maldergem syndrome and Hennekam syndrome patients shows that there is a substantial overlap in phenotype, especially in facial appearance. We conclude that Hennekam syndrome can be caused by mutations in FAT4 and be allelic to Van Maldergem syndrome.

Background: Genetic evaluation of cardiomyopathies poses a challenge. Multiple genes are involved but no clear genotype-phenotype correlations have been found so far. In the past, genetic evaluation for hypertrophic (HCM) and dilated (DCM) cardiomyopathies was performed by sequential screening of a very limited number of genes. Recent developments in sequencing have increased the throughput, enabling simultaneous screening of multiple genes for multiple patients in a single sequencing run., Objective: Development and implementation of a next generation sequencing (NGS) based genetic test as replacement for Sanger sequencing., Methods and Results: In order to increase the number of genes that can be screened in a shorter time period, we enriched all exons of 23 of the most relevant HCM and DCM related genes using on-array multiplexed sequence capture followed by massively parallel pyrosequencing on the GS-FLX Titanium. After optimisation of array based sequence capture it was feasible to reliably detect a large panel of known and unknown variants in HCM and DCM patients, whereby the unknown variants could be confirmed by Sanger sequencing., Conclusions: The rate of detection of (pathogenic) variants in both HCM and DCM patients was increased due to a larger number of genes studied. Array based target enrichment followed by NGS showed the same accuracy as Sanger sequencing. Therefore, NGS is ready for implementation in a diagnostic setting.