Klee, Eric W.

Problem Case-based learning is an established means of educating students in law, business, and medicine; however, this methodology is not often applied to educating translational biomedical researchers. The application of case-based learning to translational biomedical research education allows scholars to actively engage with real-world material and apply their newfound knowledge as it is acquired. Approach Through the Mayo Clinic Center for Clinical and Translational Science (CCaTS), three courses were delivered in 2009–2017 which emphasized case-based learning in clinical and translational science, entrepreneurship, and individualized medicine. Quantitative measures collected in student course reviews upon course completion were analyzed. Additionally, products arising from each course were identified, including publications and startups pitched. Outcomes Analyses demonstrate that case-based learning techniques are well suited to graduate biomedical research education. Furthermore, case studies can be employed throughout the entire clinical and translational spectrum, from basic and preclinical work through to clinical and population-based learning. Next Steps Within CCaTS, next steps include creating case-based courses in regulatory and team science to continue to allow scholars to learn and apply these critical skills to real-world material. The goal is to continue to provide immersive training opportunities in areas of clinical and translational science that cannot be readily learned in a traditional lecture-based class setting.

AbstractBackgroundBeta‐galactosidase‐1 (GLB1) is a lysosomal hydrolase that is responsible for breaking down specific glycoconjugates, particularly GM1 (monosialotetrahexosylganglioside). Pathogenic variants in GLB1 cause two different lysosomal storage disorders: GM1 gangliosidosis and mucopolysaccharidosis type IVB. In GM1 gangliosidosis, decreased β‐galactosidase‐1 enzymatic activity leads to the accumulation of GM1 gangliosides, predominantly within the CNS. We present a 22‐month‐old proband with GM1 gangliosidosis type II (late‐infantile form) in whom a novel homozygous in‐frame deletion (c.1468_1470delAAC, p.Asn490del) in GLB1 was detected.MethodsWe used an experimental protein structure of β‐galactosidase‐1 to generate a model of the p.Asn490del mutant and performed molecular dynamic simulations to determine whether this mutation leads to altered ligand positioning compared to the wild‐type protein. In addition, residual mutant enzyme activity in patient leukocytes was evaluated using a fluorometric assay.ResultsMolecular dynamics simulations showed the deletion to alter the catalytic site leading to misalignment of the catalytic residues and loss of collective motion within the model. We predict this misalignment will lead to impaired catalysis of β‐galactosidase‐1 substrates. Enzyme assays confirmed diminished GLB1 enzymatic activity (~3% of normal activity) in the proband.ConclusionsWe have described a novel, pathogenic in‐frame deletion of GLB1 in a patient with GM1 gangliosidosis type II.

Objectives: 1) Describe the genetic alterations discovered in a series of sporadic vestibular schwannomas (VS). 2) Identify if more clinically aggressive variants possess different genetic alterations compared to more indolent-behaving VS. Methods: Fresh frozen tumor and matched peripheral blood leukocytes from 23 individuals with sporadic VS were analyzed using whole-exome sequencing, tumor whole transcriptome expression profiling (mRNA-Seq), and tumor mate-pair analysis. Source cases included tumors with fast preoperative growth, giant tumors in young patients, tumors with macrocystic change, recurrent tumors following radiation or microsurgery, and indolent small tumors with minimal or no growth before surgery. Somatic and germ-line alterations of the NF2 gene and beyond the NF2 locus were identified and analyzed using complementing analyses. Results: Biallelic somatic events involving the NF2 gene were discovered in every analyzed tumor specimen with no concurrent NF2 variants identified in matching peripheral blood specimens. Thirteen tumors showed loss of one chromosome 22 (ch22), 4 tumors showed copy-neutral 22q loss of heterozygosity, and 31 unique small variants in the NF2 gene were discovered. Of the latter, 10 were essential splice site, 11 frame shift, 7 stop gain, 2 missense, and 1 in-frame mutation. No other common or recurring NF2 mutations were identified. However, several other notable large chromosomal aberrations were discovered including 2 tumors with loss of a chromosome 21, 3 with loss of an X or Y chromosome, 1 with copy-neutral loss of heterozygosity in chromosome 15, and 1 with loss of 18p and 16q. All of these other major chromosomal abnormalities only occurred in tumors demonstrating a more aggressive phenotype. Conclusions: To date, few studies have used whole-exome sequencing, mate-pair analysis, and RNA-seq to profile genome-wide alterations in sporadic VS. Using high-throughput deep sequencing, “two-hit” alterations in the NF2 gene were identified in every tumor and were not present in peripheral blood supporting that all events were somatic. Type of NF2 gene alteration and accessory mutations outside the NF2 locus may predict phenotypic expression and clinical course.

Background Cahan's criteria have been utilized since 1948 to establish causality between prior radiation treatment and the development of secondary malignancy. One major criterion specifies that histological and radiographic evidence collected before and after radiation treatment must confirm separate tumor types; however, pretreatment biopsy is rarely obtained prior to radiosurgery for vestibular schwannoma and many other skull base and cranial lesions. Therefore, in these cases Cahan's criteria cannot be validly applied. Objective This article proposes an update to Cahan's criteria using modern molecular genetic analysis for cases lacking baseline histopathology. Methods Mate-pair sequencing and whole exome sequencing of a cerebellopontine angle undifferentiated high-grade pleomorphic sarcoma (UHGPS) that developed after stereotactic radiosurgery of a presumed benign vestibular schwannoma. Results Mate-pair sequencing and whole exome sequencing of the sarcoma revealed complex chromosomal aberrations. Notably, the tumor contained a deletion in the NF2 gene at 22q12 and an in-frame deletion on exon 5 of the remaining copy of NF2. Biallelic events impacting NF2 are atypical for UHGPS but are characteristic for vestibular schwannoma. These findings help support the conclusion that the UHGPS arose from a benign vestibular schwannoma all along. Conclusions Next-generation sequencing can be successfully applied to a radiation-induced sarcoma when both the original and malignant tumors harbor separate signature genetic markers. As our understanding of the genetic profile of various tumors expand, we believe that next-generation sequencing and other genomic tools will play an increasingly important role in establishing causality between radiation and the development of secondary malignancy.

Abstract Background: Established laboratory methods, including next-generation sequencing (NGS), can accurately and inexpensively detect certain classes of clinically important mutations in readily accessible protein coding regions of a patient's DNA. These events include single nucleotide variants (SNVs), small insertions/deletions (indels), and sometimes medium sized copy-number deletions or duplications (del/dups). However, other mutation types with clinical relevance are known, and indeed data on over 80,000 patients show that pathogenic, medically important variants of other, more technically challenging types are prevalent in genes related to hereditary breast and ovarian cancer (see associated abstract by Lincoln et al.). We sought to develop and evaluate a reference standard by which laboratories may be able to assess and improve their performance on representative examples of complex, technically “hard” mutations in medically important genes associated with breast, ovarian, and other cancers. Methods: We selected a diverse set of 23 challenging alterations, uncovered by the aforementioned clinical study, all of which are considered pathogenic and potentially actionable under ACMG and NCCN guidelines when uncovered in the germline DNA of a patient. These mutations are in the BRCA1, BRCA2, MLH1, MSH2, MSH6, and PMS2 genes. We generated a single synthetic DNA specimen with all 23 mutations introduced into a known genomic background. Following presentation of this general strategy at a 2016 meeting, 7 laboratories volunteered to collaboratively evaluate this approach. This DNA was created, validated, blinded and provided to these laboratories who sequenced it using a total of 9 different NGS based workflows, including 5 validated clinical tests with customized methods and two sequencing technology vendor (Illumina, Ion Torrent) default pipelines. Multiple target capture biochemical methods were used, as was whole genome sequencing. Results: Twelve of the 23 variants were detected by all 9 laboratory workflows, but just 2 workflows detected all 23. Many, but not all, of these test limitations were previously known. Importantly, evidence of each variant was present in the raw data, suggesting that this strategy is compatible with the diverse biochemical methods used in NGS laboratories today. Raw data for the synthetic variants mimicked that of the endogenous ones (including presenting similar technical artifacts which make these variants “hard”) demonstrating that controls such as these may be useful in the development of methods with improved sensitivity. The vendor-supplied bioinformatics pipelines fared the worst, reinforcing the importance of carefully selecting bioinformatics algorithms and parameters in any laboratory developed test. Discussion: Medically important but technically challenging mutations are prevalent in genes involved in hereditary breast, ovarian, and other cancers. Although patient specimens are also critical, synthetic controls may help efficiently assess the analytic range of any clinical test, highlighting certain strengths and limitations, and can help laboratories develop new methods to improve sensitivity for these challenging variants. Citation Format: Lincoln S, Zook J, Truty R, Chowdhury S, Fellowes A, Mahamdallie S, Ferber M, Cleveland M, Huang C, Tomson F, Klee E, DeSilva W, Seal S, Aradhya S, Nussbaum R, Garlick R, Kingsmore S, Rahman N, Salit M, Shirts B. An interlaboratory study of complex mutation detection in genes associated with hereditary breast and ovarian cancer highlights both successes and current challenges [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-06-08.

ACTB encodes the β-actin, and pathogenic variations in this gene have typically been associated with Baraitser-Winter cerebrofrontofacial syndrome, a congenital malformation syndrome characterized by short stature, craniofacial anomalies, and cerebral anomalies. Here, we describe the third case with the p.Arg183Trp variant in ACTB causing juvenile-onset dystonia. Our patient has severe, intractable dystonia, developmental delay, and sensorineural hearing loss, besides hyperintensities in the caudate nuclei and putamen on the brain MRI, which is a distinct but overlapping phenotype with the previously reported case of identical twins with the same alteration in ACTB.

Abstract Background Pathogenic variants in ryanodine receptor 1 ( RYR 1, MIM # 180901) are the cause of congenital myopathy with fiber‐type disproportion, malignant hyperthermia susceptibility type 1, central core disease of muscle, multiminicore disease and other congenital myopathies. Methods We present a patient with global developmental delay, hypotonia, myopathy, joint hypermobility, and multiple other systemic complaints that were noted early in life. Later she was found to have multiple bone deformities involving her spine, with severe scoliosis that was corrected surgically. She was also diagnosed with ophthalmoplegia, chronic hypercapnic respiratory failure, and hypertension. At 22 years of age she presented to the genetics clinic with a diagnosis of mitochondrial myopathy and underwent whole exome sequencing ( WES ). Results Whole exome sequencing revealed two novel compound heterozygous variants in RYR 1 (c.7060_7062del, p.Val2354del and c.4485_4500del, p.Tyr1495X). Conclusion Review of her clinical, pathologic, and genetic findings pointed to a diagnosis of a congenital myopathy with fiber‐type disproportion.