Biology subjects

Santibanez-Koref, M.

Publications and source records attributed to Santibanez-Koref, M..

3 recordsLinked to original sources

bioRxiv2018

A molecular inversion probe and sequencing-based microsatellite instability assay for high throughput cancer diagnostics and Lynch syndrome screening

BackgroundClinical guidelines recommend microsatellite instability (MSI) and BRAF V600E testing of all colorectal cancers (CRCs) to screen for Lynch syndrome (LS), a hereditary predisposition to cancer. MSI is also associated with response to immunotherapy. However, uptake of MSI testing is poor and current assays are not suitable for high throughput diagnostics.\n\nWe aimed to develop a cheap and scalable sequencing assay for MSI classification, which is robust to variables in clinical samples and simultaneously tests for BRAF V600E to streamline the LS screening pipeline.\n\nMethods24 short (7-12bp) microsatellites and the BRAF V600E locus were amplified in multiplex using single molecule molecular inversion probes (smMIPs) and sequenced using the Illumina MiSeq platform. Reads were aligned to reference genome hg19. An MSI classifier was trained from 98 CRCs and validated in 99 independent CRCs collected in pathology laboratories in Edinburgh, Spain and Newcastle.\n\nResultsThe smMIP-based MSI assay has 100% accuracy for MSI status relative to MSI Analysis System (Promega). MSI classification is reproducible (100% concordance) and is robust to sample variables, detecting less than 5% MSI-high content in template DNA and giving reliable classification from sequencing only 75 DNA molecules per marker. BRAF V600E was detected with mutant allele frequencies down to 1.7%.\n\nConclusionsOur short microsatellite, smMIP-based, MSI assay provides a cheap and fully automatable assessment of MSI status and BRAF mutation. It is readily scalable to high throughput cancer diagnostics, and is suitable both as a companion diagnostic for immunotherapy and for streamlined LS screening.

cancer biology↗

bioRxiv2018

A novel panel of short mononucleotide repeats linked to informative polymorphisms enabling effective high volume low cost discrimination between mismatch repair deficient and proficient tumours

Somatic mutations in mononucleotide repeats are commonly used to assess the mismatch repair status of tumours. Current tests focus on repeats with a length above 15bp, which tend to be somatically more unstable than shorter ones. These longer repeats also have a substantially higher PCR error rate and tests that use capillary electrophoresis for fragment size analysis often require expert interpretation. In this communication, we present a panel of 17 short repeats (length 7-12bp) for sequence-based microsatellite instability (MSI) testing. Using a simple scoring procedure that incorporates the allelic distribution of the mutant repeats, and analysis of two cohort of tumours totalling 209 samples, we show that this panel is able to discriminate between MMR proficient and deficient tumours, even when constitutional DNA is not available. In the training cohort, the method achieved 100% concordance with fragment analysis, while in the testing cohort, 4 discordant samples were observed (corresponding to 97% concordance). Of these, 2 showed discrepancies between fragment analysis and immunohistochemistry and one was reclassified after re-testing using fragment analysis. These results indicate that our approach offers the option of a reliable, scalable routine test for MSI.

genomics↗

bioRxiv2018

Deleterious genetic variants in NOTCH1 are a major contributor to the incidence of non-syndromic Tetralogy of Fallot

AimsFamilial recurrence studies provide strong evidence for a genetic component to the predisposition to sporadic, non-syndromic Tetralogy of Fallot (TOF), the most common cyanotic congenital heart disease (CHD) phenotype. Rare genetic variants have been identified as important contributors to the risk of CHD, but relatively small numbers of TOF cases have been studied to date. Here, we use whole exome sequencing to assess the prevalence of rare, potentially deleterious variants in candidate genes previously associated with both syndromic and non-syndromic TOF, in the largest cohort of non-syndromic TOF patients reported to date.\n\nMethods & Results829 non-syndromic TOF patients underwent whole exome sequencing. A systematic review of the literature was conducted which revealed 77 genes in which mutations had been reported in patients with TOF. The presence of rare, deleterious variants in the 77 candidate genes was determined, defined by a minor allele frequency of [≤] 0.001 and scaled combined annotation-dependent depletion (CADD) score of [≥] 20. We found a clustering of heterozygous rare, deleterious variants in NOTCH1 (P=1.89E-15), DOCK6 (P=2.93E-07), MYOM2 (P= 7.35E-05), TTC37 (P=0.016), MESP1 (P=0.024) and TBX1 (P=0.039), after correcting for multiple testing. NOTCH1 was most frequently found to harbour deleterious variants. Changes were observed in 49 patients (6%; 95% confidence interval [CI]: 4.5% - 7.8%) and included six truncating/frameshift variants and forty missense variants. Sanger sequencing of the unaffected parents of thirteen cases identified five de novo variants. Variants were not confined to a single functional domain of the NOTCH1 protein but significant clustering of variants was evident in the EGF-like repeats (P=0.018). Three NOTCH1 missense variants (p.G200R, p.C607Y and de novo p.N1875S) were subjected to functional evaluation and showed a reduction in Jagged1 ligand-induced NOTCH signalling. p.C607Y, which exhibited the most significant reduction in signalling, also perturbed S1 cleavage of the NOTCH1 receptor in the Golgi.\n\nConclusionThe NOTCH1 locus is a frequent site of genetic variants predisposing to non-syndromic TOF with 6% of patients exhibiting rare, deleterious variants. Our data supports the polygenic origin of TOF and suggests larger studies may identify additional loci.

genetics↗