Detection of Microsatellite Instability in Colonoscopic Biopsies and Postal Urine Samples from Lynch Syndrome Cancer Patients Using a Multiplex PCR Assay.

Identification of mismatch repair (MMR)-deficient colorectal cancers (CRCs) is recommended for Lynch syndrome (LS) screening, and supports targeting of immune checkpoint inhibitors. Microsatellite instability (MSI) analysis is commonly used to test for MMR deficiency. Testing biopsies prior to tumour resection can inform surgical and therapeutic decisions, but can be limited by DNA quantity. MSI analysis of voided urine could also provide much needed surveillance for genitourinary tract cancers in LS. Here, we reconfigure an existing molecular inversion probe-based MSI and BRAF c.1799T > A assay to a multiplex PCR (mPCR) format, and demonstrate that it can sample >140 unique molecules per marker from <1 ng of DNA and classify CRCs with 96−100% sensitivity and specificity. We also show that it can detect increased MSI within individual and composite CRC biopsies from LS patients, and within preoperative urine cell free DNA (cfDNA) from two LS patients, one with an upper tract urothelial cancer, the other an undiagnosed endometrial cancer. Approximately 60−70% of the urine cfDNAs were tumour-derived. Our results suggest that mPCR sequence-based analysis of MSI and mutation hotspots in CRC biopsies could facilitate presurgery decision making, and could enable postal-based screening for urinary tract and endometrial tumours in LS patients.

Improving the identification of patients with a genetic diagnosis of familial hypercholesterolaemia in primary care: A strategy to achieve the NHS long term plan.

BACKGROUND AND AIMS: We aimed to validate a nurse-led process using electronic health records to identify those at risk of familial hypercholesterolaemia (FH) for genetic diagnosis in primary care. METHODS: Those at risk of FH were identified using searches developed and refined locally and implemented in primary care by a trained nurse; they were invited for further assessment and genetic testing if indicated. Family members at risk of FH were identified and invited for cascade testing. RESULTS: In total 94,444 patient records were screened (expected prevalence of FH (1 in 250); 377). Of 176 records which already had a diagnostic for FH, 15 had been genetically confirmed and one was undergoing DNA testing. A further 572 (0.61%) were identified as high risk of FH. After desktop screening, 113 (15%) were invited for further assessment. Of these, 73 individuals attended the primary care clinic (64%) of whom 61 (54%) underwent proband genetic testing. Pathogenic variants were detected in 22 cases (36%) and variants of unknown significance in a further 4 cases; a total of 26 probands (43%) were therefore referred for family cascade testing. CONCLUSIONS: An optimised FH identification pathway, based on the NICE CG71 recommendations for systematic searching of primary care electronic health records, can be deployed successfully in primary care settings.

Sequencing-based microsatellite instability testing using as few as six markers for high-throughput clinical diagnostics.

Microsatellite instability (MSI) testing of colorectal cancers (CRCs) is used to screen for Lynch syndrome (LS), a hereditary cancer-predisposition, and can be used to predict response to immunotherapy. Here, we present a single-molecule molecular inversion probe and sequencing-based MSI assay and demonstrate its clinical validity according to existing guidelines. We amplified 24 microsatellites in multiplex and trained a classifier using 98 CRCs, which accommodates marker specific sensitivities to MSI. Sample classification achieved 100% concordance with the MSI Analysis System v1.2 (Promega) in three independent cohorts, totaling 220 CRCs. Backward-forward stepwise selection was used to identify a 6-marker subset of equal accuracy to the 24-marker panel. Assessment of assay detection limits showed that the 24-marker panel is marginally more robust to sample variables than the 6-marker subset, detecting as little as 3% high levels of MSI DNA in sample mixtures, and requiring a minimum of 10 template molecules to be sequenced per marker for >95% accuracy. BRAF c.1799 mutation analysis was also included to streamline LS testing, with all c.1799T>A variants being correctly identified. The assay, therefore, provides a cheap, robust, automatable, and scalable MSI test with internal quality controls, suitable for clinical cancer diagnostics.

Juvenile idiopathic arthritis, mitral valve prolapse and a familial variant involving the integrin-binding fragment of FBN1.

Mutations in Fibrillin 1 (FBN1) are associated with Marfan syndrome and in some instances with the MASS phenotype (myopia, mitral valve prolapse, borderline non-progressive aortic root dilatation, skeletal features, and striae). Potential confusion over diagnosis and management in patients with borderline features has been addressed through the revised Ghent nosology, which emphasizes the importance of aortic root dilatation and ectopia lentis as features of Marfan syndrome. The overlapping and more common mitral valve prolapse syndrome is precluded by ectopia lentis or aortic dilatation. Among these clinically related conditions, there is no compelling evidence that genotype predicts phenotype, with the exception of neonatal Marfan syndrome, mutations in which cluster within FBN1 exons 24-32. Recent reports also link two very different phenotypes to changes in FBN1. Heterozygous mutations in transforming growth factor ß-binding protein-like domain 5 (TB5) can cause acromicric or geleophysic dysplasias-and mutations in the TB4 domain, which contains an integrin binding RGD loop, have been found in congenital scleroderma/stiff skin syndrome. We report on a variant in an evolutionarily conserved residue that stabilizes the integrin binding fragment of FBN1, associated with juvenile idiopathic arthritis, mitral valve prolapse or apparently normal phenotype in different family members.

Utility of genetic testing in suspected familial cranial diabetes insipidus.

AIM: Differentiating familial cranial diabetes insipidus (CDI) from primary polydipsia can be difficult. We report the diagnostic utility of genetic testing as a means of confirming or excluding this diagnosis. PATIENT AND METHODS: The index case presented at 3 months with polydipsia. He was diagnosed with familial CDI based on a positive family history combined with what was considered to be suspicious symptomatology and biochemistry. He was treated with desmopressin (DDAVP) but re-presented at 5 months of age with hyponatraemia and the DDAVP was stopped. Gene sequencing of the vasopressin gene in father and his offspring was undertaken to establish the underlying molecular defect. RESULTS: Both father and daughter were found to have the pathogenic mutation c.242T>C (p.Leu81Pro) in exon 2 of the AVP gene consistent with a diagnosis of familial diabetes insipidus. The index case did not have the pathogenic mutation and the family could be reassured that he would not require intervention with DDAVP. CONCLUSIONS: Gene sequencing of AVP gene can have a valuable role in predicting whether or not a child is at risk of developing CDI in future. This can help to prevent family uncertainty and unnecessary treatment with its associated risks. LEARNING POINTS: Differentiating patients with familial cranial diabetes insipidus from those with primary polydipsia is not always straightforward.Molecular genetic analysis of the vasopressin gene is a valuable way of confirming or refuting a diagnosis of familial CDI in difficult cases and is a valuable way of identifying individuals who will develop CDI in later childhood. This information can be of great value to families.