Balcom, Jessica R.

Abstract Variants in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene, which causes cystic fibrosis, have become increasingly well‐characterized in recent decades. However, clinicians remain challenged by CFTR variants that result in variable expressivity and reduced penetrance, including the variant R117H (c.350G>A) and the intron 9 poly‐thymidine (poly‐T) and poly‐thymidine‐guanosine (TG) tracts, highlighting a knowledge gap that impacts the delivery of accurate information to patients and families. To address this gap, we developed an educational resource that provides descriptive likelihoods of specific phenotypes that may result from genotypes including R117H and the poly‐T and TG tracts. Development of the resource was grounded in an extensive literature review, clinical expertise, and publicly available CFTR variant databases, with feedback incorporated from CF clinicians, genetic counselors, and CF content experts. Genotypes were determined to have very low, low, moderate, high, or very high likelihood of resulting in CF, CFTR‐related disorder (CFTR‐RD), no symptoms, or the designation of CFTR‐related metabolic syndrome/CF‐screen positive, inconclusive diagnosis (CRMS/CFSPID) following CF newborn screening. The resource described in this report is intended to help clinicians navigate the uncertainty surrounding potential diagnostic scenarios to present accurate assessments to patients and families, a critical component of CF genetic testing and counseling.

AbstractOver the past several decades, molecular genetic testing volumes have grown and testing has expanded from single‐gene assays to multigene panels, exome sequencing, and genome sequencing. The number of molecular genetic variants that require manual interpretation has grown simultaneously, resulting in an increased demand for education on molecular variant evaluation (MVE). To meet this growing need, a team of genetic counselors and educational experts undertook a quality improvement (QI) initiative with the objectives of assessing, standardizing, and scaling access to MVE education, without increasing instructor time to deliver the education. Using the Six Sigma define–measure–analyze–improve–control (DMAIC) framework, a flipped learning course with a series of standardized online modules was developed to deliver MVE education in an enduring and accessible format for a diverse group of learners. Outcome measures included the number of online modules developed, the number of individual learners and unique learner groups accessing MVE education, and direct instruction time required to deliver MVE education. Countermeasures to ensure maintenance of educational quality included post‐course learner satisfaction scores and performance on competency assessments. Both the total number of learners and the number of unique learner groups accessing MVE education increased, while instructor time required to deliver content per learner decreased. Learner satisfaction scores remained constant and performance on competency assessments improved. The QI initiative successfully scaled MVE education to a diverse group of learners without decreasing learner outcomes or satisfaction. The flipped learning format provides a scalable and flexible educational model for instructors and learners in a rapidly changing environment that often includes remote work and education.

Lynch syndrome (LS), caused by heterozygous pathogenic variants affecting one of the mismatch repair (MMR) genes (MSH2, MLH1, MSH6, PMS2), confers moderate to high risks for colorectal, endometrial, and other cancers. We describe a four-generation, 13-branched pedigree in which multiple LS branches carry the MSH2 pathogenic variant c.2006G>T (p.Gly669Val), one branch has this and an additional novel MSH6 variant c.3936_4001+8dup (intronic), and other non-LS branches carry variants within other cancer-relevant genes (NBN, MC1R, PTPRJ). Both MSH2 c.2006G>T and MSH6 c.3936_4001+8dup caused aberrant RNA splicing in carriers, including out-of-frame exon-skipping, providing functional evidence of their pathogenicity. MSH2 and MSH6 are co-located on Chr2p21, but the two variants segregated independently (mapped in trans) within the digenic branch, with carriers of either or both variants. Thus, MSH2 c.2006G>T and MSH6 c.3936_4001+8dup independently confer LS with differing cancer risks among family members in the same branch. Carriers of both variants have near 100% risk of transmitting either one to offspring. Nevertheless, a female carrier of both variants did not transmit either to one son, due to a germline recombination within the intervening region. Genetic diagnosis, risk stratification, and counseling for cancer and inheritance were highly individualized in this family. The finding of multiple cancer-associated variants in this pedigree illustrates a need to consider offering multicancer gene panel testing, as opposed to targeted cascade testing, as additional cancer variants may be uncovered in relatives.

AbstractBackgroundMT‐RNR1 variants are a well‐known cause of aminoglycoside‐induced hearing loss (AIHL). Individuals with cystic fibrosis (CF) routinely receive aminoglycosides and are at high risk of AIHL. However, genetic testing before treatment is not routinely performed due to perceived rarity of risk, and cost ineffectiveness with traditional technologies.AimAssess the utility of large‐scale screening for AIHL risk in the CF population, using digital droplet polymerase chain reaction (ddPCR), a novel and scalable low‐cost molecular technique.MethodsUsing a clinically validated ddPCR assay, we performed retrospective testing on 122 and prospective testing on 32 individuals with CF for the two most common pathogenic variants associated with AIHL, MT‐RNR1 m.1555 A > G and m.1494 C > T. Our study screened the largest known cohort of pediatric cases of CF (94/154) for these specific alterations.ResultsWe identified two individuals positive for MT‐RNR1 m.1555 A > G and no positives for m.1494 C > T. Of 32 prospective cases, 17 had aminoglycoside exposure. The positive case in our prospective group recently began inhaled tobramycin and denied hearing issues. The clinician adjusted to care for both the patient and sibling with CF (not included in cohort) who is presumed positive for m.1555 A > G due to the nature of mitochondrial inheritance.ConclusionOur findings demonstrate the utility of pretreatment screening in the cystic fibrosis population for AIHL risk using ddPCR, a scalable and robust testing methodology at a fraction of the cost as compared to other sequencing‐based methods. Therefore, the use of large‐scale screening for AIHL risk in the cystic fibrosis community should be re‐visited.

Abstract Polymorphous low-grade neuroepithelial tumor of the young (PLNTY) is a recently described epileptogenic tumor characterized by oligodendroglioma-like components, aberrant CD34 expression, and frequent mitogen-activated protein kinase (MAPK) pathway activation. We molecularly profiled 13 cases with diagnostic histopathological features of PLNTY (10 female; median age, 16 years; range, 5–52). Patients frequently presented with seizures (9 of 12 with available history) and temporal lobe tumors (9 of 13). MAPK pathway activating alterations were identified in all 13 cases. Fusions were present in the 7 youngest patients: FGFR2-CTNNA3 (n = 2), FGFR2-KIAA1598 (FGFR2-SHTN1) (n = 1), FGFR2-INA (n = 1), FGFR2-MPRIP (n = 1), QKI-NTRK2 (n = 1), and KIAA1549-BRAF (n = 1). BRAF V600E mutation was present in 6 patients (17 years or older). Two fusion-positive cases additionally harbored TP53/RB1 abnormalities suggesting biallelic inactivation. Copy number changes predominantly involving whole chromosomes were observed in all 10 evaluated cases, with losses of chromosome 10q occurring with FGFR2-KIAA1598 (SHTN1)/CTNNA3 fusions. The KIAA1549-BRAF and QKI-NTRK2 fusions were associated respectively with a 7q34 deletion and 9q21 duplication. This study shows that despite its name, PLNTY also occurs in older adults, who frequently show BRAF V600E mutation. It also expands the spectrum of the MAPK pathway activating alterations associated with PLNTY and demonstrates recurrent chromosomal copy number changes consistent with chromosomal instability.

Current guidelines suggest screening all patients with idiopathic pulmonary arterial hypertension for genetic aberrations, particularly mutations in Bone Morphogenic Protein Receptor Type II (BMPR2), the gene most commonly implicated in the pathogenesis of PAH. Herein, we present a novel technique used to identify a pathogenic germline BMPR2 alteration in a 36‐year‐old female and family members with hereditary pulmonary arterial hypertension who each screened negative by standard cytogenetics and molecular genetics testing.

Abstract BACKGROUND Molecular parameters have been incorporated into the 2016 WHO Classification of CNS Tumors and subsequent cIMPACT-NOW updates to facilitate clinical management of glioma patients. However, there have been few reports of the overall clinical utility of comprehensive genetic testing in adult glioma patients. We report the results of sequencing and chromosomal microarray analysis of adult gliomas seen at the Mayo Clinic and through the Mayo Clinic Laboratories (MCL) reference practice. METHODS A consecutive series of 379 Mayo Clinic adult glioma patients were consented to receive targeted next generation sequencing and chromosomal microarray analysis, regardless of standard clinical ordering practices. Known diagnostic, prognostic, and predictive alterations were annotated for each case. These results were compared to the larger MCL reference practice of 2400 adult glioma samples that received one or both tests clinically. RESULTS Of the consecutive Mayo Clinic cases, 67% had alterations that fell into at least one of the diagnostic, prognostic, and/or predictive categories. Molecular testing generated diagnostic, prognostic, and predictive information in 44%, 34%, and 36% of cases, respectively. Diagnostically, molecular testing mainly aided in arriving at a final integrated diagnosis, and only a small number of cases (n= 5; 1%) had a change in diagnosis. The consecutive cases represent the typical distribution of adult glioma types (47% IDH-wildtype glioblastoma/astrocytoma, 22% IDH-mutant astrocytoma/glioblastoma, 13% IDH-mutant 1p/19q-codeleted oligodendroglioma, and 18% other tumor types). The MCL practice is enriched in tumors with atypical molecular patterns (37% IDH-wildtype glioblastoma/astrocytoma, 17% IDH-mutant astrocytoma/glioblastoma, 18% IDH-mutant 1p/19q-codeleted oligodendroglioma, and 29% other tumor types). CONCLUSIONS Molecular testing provides useful diagnostic, prognostic, and predictive information for the clinical management of adult glioma patients. While the current guidelines for ordering molecular testing are effective for diagnostic purposes, additional prognostic and predictive information can be gleaned from comprehensive molecular testing of adult gliomas.

AbstractMAPK pathway activation has been recurrently observed in desmoplastic infantile ganglioglioma/astrocytoma (DIG/DIA) with reported disproportionally low mutation allele frequencies relative to the apparent high tumor content, suggesting that MAPK pathway alterations may be subclonal. We sought to expand the number of molecularly profiled cases and investigate if tumor cell composition could account for the observed low mutation allele frequencies. Molecular (targeted neuro-oncology next-generation sequencing/RNA sequencing and OncoScan microarray) and immunohistochemical (CD68-PGM1/CD163/CD14/CD11c/lysozyme/CD3/CD20/CD34/PD-L1) studies were performed in 7 DIG. Activating MAPK pathway alterations were identified in 4 (57%) cases: 3 had a BRAF mutation (V600E/V600D/V600_W604delinsDQTDG, at 8%–27% variant allele frequency) and 1 showed a TPM3-NTRK1 fusion. Copy number changes were infrequent and nonrecurrent. All tumors had at least 30% of cells morphologically and immunophenotypically consistent with microglial/macrophage lineage. Two subtotally resected tumors regrew; 1 was re-excised and received adjuvant treatment (chemotherapy/targeted therapy), with clinical response to targeted therapy only. Even with residual tumor, all patients are alive (median follow-up, 83 months; 19–139). This study further supports DIG as another MAPK pathway-driven neuroepithelial tumor, thus expanding potential treatment options for tumors not amenable to surgical cure, and suggests that DIG is a microglia/macrophage-rich neuroepithelial tumor with frequent low driver mutation allele frequencies.