Exonic mutations in cell-cell adhesion may contribute to CADASIL-related CSVD pathology.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a condition caused by mutations in NOTCH3 and results in a phenotype characterised by recurrent strokes, vascular dementia and migraines. Whilst a genetic basis for the disease is known, the molecular mechanisms underpinning the pathology of CADASIL are still yet to be determined. Studies conducted at the Genomics Research Centre (GRC) have also identified that only 15-23% of individuals clinically suspected of CADASIL have mutations in NOTCH3. Based on this, whole exome sequencing was used to identify novel genetic variants for CADASIL-like cerebral small-vessel disease (CSVD). Analysis of functionally important variants in 50 individuals was investigated using overrepresentation tests in Gene ontology software to identify biological processes that are potentially affected in this group of patients. Further investigation of the genes in these processes was completed using the TRAPD software to identify if there is an increased number (burden) of mutations that are associated with CADASIL-like pathology. Results from this study identified that cell-cell adhesion genes were positively overrepresented in the PANTHER GO-slim database. TRAPD burden testing identified n = 15 genes that had a higher number of rare (MAF < 0.001) and predicted functionally relevant (SIFT < 0.05, PolyPhen > 0.8) mutations compared to the gnomAD v2.1.1 exome control dataset. Furthermore, these results identified ARVCF, GPR17, PTPRS, and CELSR1 as novel candidate genes in CADASIL-related pathology. This study identified a novel process that may be playing a role in the vascular damage related to CADASIL-related CSVD and implicated n = 15 genes in playing a role in the disease.

GSTM1 and GSTT1 polymorphisms associated with pain in a chemotherapy-induced peripheral neuropathy cohort.

PURPOSE: Chemotherapy induced peripheral neuropathy (CIPN) is a debilitating condition that is a direct consequence of receiving cancer treatment. The molecular aetiology of CIPN is not well understood, and it is theorised that there may be a genetic component. Genetic polymorphisms in Glutathione-S Transferase (GST) genes, including GSTT1, GSTM1 and GSTP1, encode for enzymes known to metabolise drugs used in chemotherapy, and have been theorised to be associated with CIPN. This study aimed to investigate four markers in these genes for an association in a mixed cancer cohort in relation to CIPN (n = 172). METHODS: CIPN was measured using the neuropathy item from the Patient Reported Outcome Common Terminology Criteria for Adverse Event (PRO-CTCAE) assessment. Genotyping for all samples was performed using PCR for the GSTM1 and GSTT1 null variants and restriction fragment length polymorphisms for the GSTP1 and GSTM1 polymorphisms. RESULTS: No associations were found for the GST gene markers in relation to CIPN within our study, or CIPN severity. Longitudinal stratification of the CIPN phenotypes to examine links for neuropathy, identified nominally significant protective associations with the GSTM* null allele (p-value = 0.038, OR = 0.55) and the presence of pain at month 2 of treatment, as well as a risk factor for pain related month 2 of treatment for individuals with the GSTT1*null allele (p-value = 0.030, OR = 1.64). Higher severity of pain in patients with CIPN persisted at each time-point compared to those without CIPN. CONCLUSION: No significant results for an association between CIPN with polymorphisms in GSTM1, GSTT1 and GSTP1 were identified. However, associations for the GSTM1¬-null and GSTT1-null polymorphisms with pain at month 2 following chemotherapy were identified.

Investigating a Genetic Link Between Alzheimer's Disease and CADASIL-Related Cerebral Small Vessel Disease.

Monogenic forms of Alzheimer's disease (AD) have been identified through mutations in genes such as APP, PSEN1, and PSEN2, whilst other genetic markers such as the APOE ε carrier allele status have been shown to increase the likelihood of having the disease. Mutations in these genes are not limited to AD, as APP mutations can also cause an amyloid form of cerebral small vessel disease (CSVD) known as cerebral amyloid angiopathy, whilst PSEN1 and PSEN2 are involved in NOTCH3 signalling, a process known to be dysregulated in the monogenic CSVD, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). The overlap between AD genes and causes of CSVD led to the hypothesis that mutations in other genes within the PANTHER AD-presenilin pathway may be novel causes of CSVD in a cohort of clinically suspicious CADASIL patients without a pathogenic NOTCH3 mutation. To investigate this, whole exome sequencing was performed on 50 suspected CADASIL patients with no NOTCH3 mutations, and a targeted gene analysis was completed on the PANTHER. ERN1 was identified as a novel candidate CSVD gene following predicted pathogenic gene mutation analysis. Rare variant burden testing failed to identify an association with any gene; however, it did show a nominally significant link with ERN1 and TRPC3. This study provides evidence to support a genetic overlap between CSVD and Alzheimer's disease.

The MinION as a cost-effective technology for diagnostic screening of the SCN1A gene in epilepsy patients.

The MinION is a portable DNA sequencer that allows real time sequencing at low capital cost investment. We assessed accuracy and cost-effectivess of the MinION for genetic diagnostic testing of known SCN1A mutations that cause Dravet Syndrome (DS). DNA samples (n = 7) from DS patients previously shown to carry SCN1A mutations via Ion Torrent and Sanger sequencing were sequenced using the MinION. SCN1A amplicons for 8 exons were sequenced using the MinION with 1D chemistry on an R9.4 flow cell. All known missense mutations were detected in all samples showing 100 % concordance with results from other methods. However, the MinION failed to detect the insertions/deletions (INDELs) present in these patients. Nevertheless, these results indicate that MinION is a cost-effective platform for use as an initial screening step in the detection of nucleotide substitution mutations in in SCN1A, especially in under-resourced laboratories or hospitals. Further improvements are required to reliably detect INDELS in this gene.

Tiered analysis of whole-exome sequencing for epilepsy diagnosis.

It is thought that despite highly variable phenotypic expression, 70-80% of all epileptic cases are caused by one or more genetic mutations. Next generation sequencing technologies, such as whole exome sequencing (WES), can be used in a diagnostic or research setting to identify genetic mutations which may have significant prognostic implications for patients and their families. In this study, 398 genes associated with epilepsy or recurrent seizures were stratified into tiers based on genotype-phenotype concordance, tissue gene expression, frequency of affected individuals with mutations and evidence from functional and family studies. WES was completed on 14 DNA samples (2 with known mutations in SCN1A and 12 with no known mutations) from individuals diagnosed with epilepsy using an Ion AmpliSeq approach. WES confirmed positive SCN1A mutations in two samples. In n = 5/12 samples (S-3 to -14) we identified potentially causative mutations across five different genes. S-5 was identified to have a novel missense mutation in CCM2; S-6 a novel frameshift mutation identified in ADGRV1; S-10 had a previously reported pathogenic mutation in PCDH19, whilst a novel missense mutation in PCDH19 was shown in S-12; and S-13 identified to have separate missense mutations in KCNA2 and NPRL3. The application of WES followed by a targeted variant prioritization approach allowed for the discovery of potentially causative mutations in our cohort of previously undiagnosed epilepsy patients.

Variant Call Format-Diagnostic Annotation and Reporting Tool: A Customizable Analysis Pipeline for Identification of Clinically Relevant Genetic Variants in Next-Generation Sequencing Data.

In this article, we introduce the variant call format-diagnostic annotation and reporting tool (VCF-DART), a customized analysis pipeline tool for the rapid annotation of variants from exome or genome sequencing and the generation of reports for genetic diagnostics. VCF-DART uses custom gene lists to categorize variants into specific analysis tiers and to subcategorize them on the basis of standard parameters to facilitate the rapid interrogation of potentially pathogenic variants by human operators. The tool uses publicly available databases to identify a range of data to assist with variant classification and curation processes and includes robust logging of parameters and database versions to allow comparison of analyses performed at different times. VCF-DART-an online analysis pipeline for next-generation sequencing data-is a platform agnostic tool that leverages the use of publicly available databases to improve a laboratory's calling ability and reduce analysis times long-term. It also runs highly efficiently and scales from desk and laptop machines to servers. Overall, VCF-DART provides a simple, customizable, and entirely open-source method to identify genetic variants that may be of clinical importance in a variety of genetically important conditions.

Three cases of an unusual pattern of invasion in malignant Brenner tumors.

Ovarian malignant Brenner tumors are rare neoplasms that are typically admixed with benign and borderline Brenner tumor elements. We report 3 cases of an unusual variant of malignant Brenner tumor where the infiltrative malignant component arose directly from a benign Brenner tumor rather than from borderline elements and did not exhibit a desmoplastic stromal response. Borderline elements were present in 1 case, but the invasive component did not arise from these. Our cases highlight that an absence of a borderline element should not dissuade the pathologist from diagnosing a malignant Brenner tumor.

Epstein-Barr virus-positive mucocutaneous ulcer of the oral cavity: the importance of having a detailed clinical history to reach a correct diagnosis.

Epstein-Barr virus-positive mucocutaneous ulcer (EBVMCU) is a recently recognized clinicopathological entity. It presents as an ulcerative lesion with lymphoma-like histologic features and is clinically associated with various types of immunosuppression. EBVMCU lesions respond well to conservative measures aimed at correcting the underlying immunosuppression. The case described clearly demonstrates the importance of appropriately completing biopsy submission forms with a detailed clinical history, thus aiding the histopathologist in reaching the correct diagnosis, particularly in pathologic conditions that have similar or overlapping histopathological features. Failure to do so may lead to incorrect diagnosis and management.