Lazaridis, Konstantinos N.

AbstractBackgroundHow variations predicted by pharmacogenomic testing to alter drug metabolism and therapeutic response affect outcomes for patients with disorders of gut‐ brain interaction is unclear.AimsTo assess the prevalence of pharmacogenomics‐predicted drug‐gene interactions and symptom outcomes for patients with disorders of gut‐brain interaction.MethodsPatients who were treated in our clinical practice for functional dyspepsia/bowel disorder underwent pharmacogenomic testing. The change in symptoms from baseline to 6 months was compared for patients with variations in CYP2D6 and CYP2C19, which metabolize neuromodulators, and SLC6A4, which encodes the sodium‐ dependent serotonin transporter.ResultsAt baseline, 79 of 94 participants (84%) had at least one predicted major drug‐ gene interaction, and all 94 (100%) had at least one predicted moderate interaction. For the 44 participants who completed a survey of their symptoms at 6 months, the mean (SD) irritable bowel syndrome‐symptom severity score decreased from 284 (71) at baseline to 231 (95) at 6 months (p < 0.001). Among patients taking selective serotonin reuptake inhibitors, the decrease in symptom severity (p = 0.03) and pain (p = 0.002) scores from baseline to 6 months was greater for patients with a homozygous SLC6A4 long/long genotype (n = 30) (ie, increased serotonin transporter activity) than for patients with homozygous short/short or heterozygous long/short genotypes (n = 64). Symptom outcomes were not affected by CYP2D6 or CYP2C19 variations.ConclusionsThe homozygous SLC6A4 long/long genotype confers better symptom resolution for patients with disorders of gut‐brain interaction who take selective serotonin reuptake inhibitors than do the homozygous short/short or heterozygous long/short genotypes.

Introduction: Telomere biology disorders (TBD) are multisystem diseases often associated with bone marrow failure (BMF) and pulmonary fibrosis. TBD are clinically diagnosed when mean telomere lengths (TL) in blood cells measured by clinical flowFISH assays fall &lt;1 st percentile, in comparison to age-matched reference controls. This information has limitations, e.g. TL in blood cells may not represent TL in other organs (e.g. lung, liver) and moreover, individuals with high clinical suspicion of TBD can show borderline average TL which complicates their diagnosis. The likely reason for this is that cell senescence can be triggered by shortening of a subset, or even a single chromosome arm telomere, a situation that may be missed when measuring average TL. Previous approaches to measure individual chromosome-arm telomere length (CA-TL) are only available for a small number of chromosome arms or can only estimate the number of shortened telomeres without matching them to specific chromosome arms. Taking advantage of recent developments in long-read next generation sequencing (NGS) and the availability of the recently published telomere-to-telomere (T2T) reference human genome, our group developed Telogator, (Stephens Z. et al, 2022) a bioinformatic tool that estimates CA-TL for most chromosome arms using long-read NGS data. Here we use Telogator to describe for the first time the CA-TL pattern in TBD patients in comparison with a control non-TBD cohort. Methods: TBD patients were identified through the Pre-Myeloid and Bone Marrow Failure Specialty Clinic at Mayo Clinic. All patients underwent flowFISH and genetic testing. Age-matched non-TBD patients identified at the Center for Individualized Medicine (Mayo Clinic) were used as reference controls. Informed consent was obtained from all individuals with approval from the Mayo Clinic Institutional Review Board. DNA from peripheral blood mononuclear cells was sequenced using PacBio Sequel II following the manufacturer indications for HiFi sequencing. BAM files were aligned to the T2T reference and CA-TL was calculated using Telogator as described in Stephens Z. et al. 2022. Average TL, CA-TL, and percent of short telomeres (both defined as &lt;5 kb and &lt;3.2 kb) were calculated. Comparisons of average TL and percent of short telomeres were performed using t-test, while Wilcoxon rank-sum test was used to compare CA-TL. Results: Four TBD patients [median age = 58.5 (52-60), 50% female] and 6 non-TBD controls [Median age = 56 (50-63), 33% female] were included in the analysis. All TBD patients presented average TL below the 1 st centile in both lymphocytes and granulocytes, and TBD-related phenotype (100% with pulmonary fibrosis, 50% with BMF, 25% with liver fibrosis). Three had a pathogenic variant in a TBD related genes [2 in TERT (NM_198253.3: c.2030G&gt;A, p.(Gly677Asp) and c.2515_2533del, p.(Thr839Alafs*29)) and 1 in NHP2 (NM_017838.3: c.459_460delAT; p.(X154Argext29))]. The average TL calculated by Telogator showed high correlation with flowFISH values (r 2 = 0.9883, p = 0.0059) and was lower in TBD patients than controls (p = 0.0012). Next, when comparing individual CA-TL between groups, only half of CA-TL was statistically different in the TBD group compared to controls (1p, 3q, 4p, 5p, 5q, 6q, 7p, 7q, 8q, 9p, 9q, 10q, 11p, 12q, 12q, 16p, 16q, 18q, 19p, 19q, 20p, 20q, 21q - Figure 1). The percentage of telomeres &lt;5kb and &lt;3 kb was significantly increased in TBD patients compared to controls (p= 0.014 and 0.0333, respectively). Discussion: Using a novel informatic tool, we replicate previous results obtained using classic approaches and were able to expand on these studies by measuring specific CA-TL in TBD patients. We identified statistically significant differences at the CA-TL level in these patients when compared to age-matched controls, while other CA-TL were not statistically different. Future studies and the inclusion of additional TBD patients will help define the clinical relevance of the differential CA-TL shortening and its prevalence among TBD patients. While limited by the small number of samples in the study, we show the feasibility of using this approach to study TL with unprecedented resolution and show that Telogator offers important advantages compared to classic methods to study telomere biology that can be used in other relevant fields like aging and/or cancer. Our results pave the way to advance the mechanistic understanding of TBD.

INTRODUCTION Telomere biology disorders (TBDs) are multisystem diseases caused by accelerated telomere shortening that can present with bone marrow failure and pulmonary fibrosis, among other abnormalities. TBD diagnosis is considered when average blood telomere length (TL) measured by clinical flowFISH is &lt;1 st percentile and/or a pathogenic variant in telomere maintenance genes (most commonly TERT, TERC, RTEL1) is identified. Pathogenic variants in genes coding for the telomerase protein ( TERT) and RNA component ( TERC) decrease telomerase activity. Similarly, variants in RTEL1 result in defective unwinding of the t-loop at telomere ends, increasing the levels of free t-circles in cells. Variants in these genes are often classified as variants of uncertain significance (VUSs by ACMG criteria) with additional efforts needed for variant curation. To address this, we describe results of functional testing performed in TBD patients with VUSs involving TERT, TERC, and RTEL1. METHODS TBD suspected patients were identified through the Pre-Myeloid Cancer and Bone Marrow Failure Clinic (Mayo Clinic) where they underwent clinical flowFISH and genetic testing for TBD related genes. Informed consent was obtained with approval from the Mayo Clinic Institutional Review Board. Viable peripheral blood and bone marrow samples were collected along with age matched healthy volunteers. Telomerase activity was measured in samples with TERT and TERC variants through telomeric repeat amplification protocol (TRAP) assays using the TRAPeze XL Telomerase Detection Kit (MilliporeSigma) following manufacturer instructions. HCT116 cells were used as positive controls while heat inactivated samples served as negative controls. Fluorescence was measured in a SpectraMax plate reader. Percentage of total product generated (TPG) relative to healthy age matched controls was calculated. T-circle detection assays were used on RTEL1 samples based on the protocol by Zhang et al. (2017). Final southern blotting was completed using the TeloTAGGG Telomere Length Assay Kit (Roche), and t-circle bands were visualized in a c600 GEL Imaging System (Azure Biosystems). RESULTS Ten patients (mean age = 53 years (27-71), 50% female) were included in our study. Patients 1 to 5 presented VUS in TERT (n=4) or TERC (n=1) and TL &lt;1 st percentile in lymphocytes and/or granulocytes. Patient 6 carried a known pathogenic variant in RTEL1 and was included as a positive control. Patients 7 to 9 with RTEL1 variants presented TL close to the 10 th or between the 1 st-10 th percentile. No TL results were available for patient 10 but was tested due to the presence of a VUS in RTEL1 (Table 1). Similarly, while patient 7 carried a likely pathogenic variant, it was tested due to presenting lymphocyte TL between the 1 st-10 th percentile. TRAP results indicated decreased telomerase activity compared to age matched controls in all samples with TERT and TERC variants suggesting a pathogenic effect (52.%, 10.7%, 48.7%, 4.4% and 5.2% of TPG compared to age matched controls, respectively) (Table 1). On the other hand, t-circles were only seen in patient 6 (Figure 2). DISCUSSION We used TRAP and t-circle assays to explore the telomerase activity and presence of t-circles in patient samples with TERT/TERC and RTEL1 variants. In the case of TERT/TERC, we observed a reduction of telomerase activity in all samples. Together with blood TL below the 1 st percentile, this suggests that these variants are indeed deleterious (Table 1). However, since patient 4 presented two TERT VUSs, additional testing is needed to conclude how each variant impacts telomerase activity independently. In regard to RTEL1 variants, we observed t-circles in patient 6 as expected. No t-circles could be seen on all remaining samples, including patient 7 who carried a variant classified as likely pathogenic (Figure 1). This in-frame deletion has been described once in a TBD family (Cogan et al. 2015) but was not functionally tested. This result, together with the borderline TL seen in this patient, may suggest that some compensatory mechanism exists on this sample, or that the deleterious effect of the variant is not strong enough to be detected by our assay. Further studies are currently ongoing by our group to validate these results using a cell-based transfection disease model. In summary, we show that functional testing can be a helpful tool to determine pathogenicity of VUSs in TERT/TERC and RTEL1.

Abstract The pathogenesis of primary sclerosing cholangitis (PSC) is unclear, although studies implicate IL-17A as an inflammatory mediator in this disease. However, a direct assessment of IL-17 signaling in PSC cholangiocytes is lacking. In this study we aimed to investigate the response of PSC extrahepatic cholangiocyte organoids (ECO) to IL-17A stimulation. Cholangiocytes obtained from PSC and non-PSC patients by endoscopic retrograde cholangiography (ERC) were cultured as ECO. The ECO were treated with vehicle or IL-17A and assessed by transcriptomics, secretome analysis, and genome sequencing (GS). Unsupervised clustering of all integrated scRNA-seq data identified 8 cholangiocyte clusters which did not differ between PSC and non-PSC ECO. However, PSC ECO cells demonstrated a robust response to IL-17 treatment, noted by an increased number of differentially expressed genes (DEG) by transcriptomics, and more abundant chemokine and cytokine expression and secretion. After rigorous filtering, GS identified candidate somatic variants shared among PSC ECO from unrelated individuals. However, no candidate rare variants in genes regulating the IL-17 pathway were identified, but rare variants regulating the MAPK signaling pathway were present in all PSC ECO. In conclusion, PSC and non-PSC patient derived ECO respond differently to IL-17 stimulation implicating this pathway in the pathogenesis of PSC.

Abstract Introduction: Most rare disease patients (75 − 50%) undergoing genomic sequencing remain unsolved, often due to lack of information about variants identified. Data review over time can leverage novel information regarding disease-causing variants and genes, increasing this diagnostic yield. However, time and resource constraints have limited reanalysis of genetic data in clinical laboratories setting. We developed RENEW, (REannotation of NEgative WES/WGS) an automated reannotation procedure that uses relevant new information in on-line genetic databases to enable rapid review of genetic findings. Methods We tested RENEW in an unselected cohort of 1,066 undiagnosed cases from the Mayo Clinic Center for Individualized Medicine using new information in ClinVar, HGMD and OMIM between the date of previous analysis/testing and April of 2022. Results 5,741 variants prioritized by RENEW were rapidly reviewed by variant interpretation specialists. Mean analysis time was approximately 20 seconds per variant (32 hours total time). Reviewed cases were classified as: 879 (93.0%) undiagnosed, 63 (6.6%) putatively diagnosed, and 4 (0.4%) definitively diagnosed. Discussion New strategies are needed to enable efficient review of genomic findings in unsolved cases. We report on a fast and practical approach to address this need and improve overall diagnostic success in patient testing through a recurrent reannotation process.

Abstract Two inherited autosomal dominant cancer predisposition conditions - BRCA related hereditary breast and ovarian cancer (HBOC) and Lynch syndrome (LS) - are termed Centers for Disease Control and Prevention Tier 1 (CDCT1) genetic conditions, for which early identification and intervention have a positive impact on public health. Selection of individuals for genetic testing for these conditions is based on personal and family history. The goal of this study was to evaluate whether screening in a tertiary medical center using exome sequencing could efficiently identify carriers of HBOC and LS and to determine the frequency of incremental carriers identified outside of traditional clinical practice guidelines. Participants from three geographically diverse Mayo Clinic practices in the USA (Rochester MN, Phoenix AZ, Jacksonville FL) consented to clinical “Exome+” sequencing (Helix, San Mateo, CA; Mayo GeneGuide, Rochester, MN) in the TAPESTRY study (NCT05212428), which links sequencing data with electronic health records and the return of CDCT1 genetic findings. For this study we focused on two inherited cancer predisposition conditions: BRCA-related HBOC (BRCA1 and BRCA2) and LS (MLH1, MSH2, MSH6, PMS2 and EPCAM). Detailed chart review to collect demographic information, personal and family history, and assessment of clinical practice guidelines for genetic evaluation (National Cancer Control Network 2021). To date, 44,306 patients have enrolled and sequenced in TAPESTRY. Annotation and interpretation of all variants in 7 genes for HBOC and LS resulted in identification of 550 carriers (prevalence 1.24%) which included 387 with HBOC (27.2% BRCA1, 42.8% BRCA2) and 163 with LS (12.3% MSH6, 8.8% PMS2, 4.5% MLH1, 3.8% MSH2 and 0.2% EPCAM). Demographics of the cohort included: 62.7% female, mean age 55.2 years, non-white race 9.6% and 3.8% Hispanic/Latino ethnicity. A personal history of cancer was present in 46.4% of test positive patients, including 22.5% of HBOC patients with breast or ovarian cancer and 20.9% of LS patients with colorectal or uterine cancer. More than half of the patients (52.1%) were newly diagnosed with HBOC and LS based on the results of this study. Overall, 39.2% of HBOC/LS carriers identified through Exome+ sequencing did not satisfy NCCN criteria for genetic evaluation, this was higher for LS (56.2%) compared to HBOC (32%). Amongst those newly diagnosed with HBOC/LS (n=286), NCCN criteria were not satisfied in 60% of cases (78% for LS and 51% for HBOC). Of the pathogenic germline variant carriers who met NCCN guidelines for testing, 34.2% were not aware of their diagnosis prior to participation in this study. Our results emphasize the need for wide genomic screening for CDCT1 cancer predisposition syndromes. Such screening could identify at-risk carriers of HBOC and LS, who would not otherwise be identified through clinical practice guidelines. Citation Format: Emily Gay, Niloy Jewel Samadder, Michelle L. Bublitz, Melanie M. Peterson, Tammy A. Wilson, Lorelei A. Bandel, Sebastian M. Armasu, Robert A. Vierkant, Matthew J. Ferber, Eric W. Klee, Nicholas B. Larson, Teresa M. Kruisselbrink, Timothy B. Curry, Jan B. Egan, Jennifer L. Kemppainen, Jessa S. Bidwell, Jennifer L. Anderson, Tammy M. McAllister, T'Nita S. Walker, Katie L. Kunze, Vanda Lindpere, Michael A. Golafshar, Margaret Klint, Richard J. Presutti, William V. Bobo, Aleksander Sekulic, Jolene M. Summer Bolster, Cheryl L. Willman, Konstantinos N. Lazaridis. Genetic screening in a tertiary medical center identifies carriers of cancer predisposition diseases that would be missed by clinical guidelines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5768.

Abstract Hereditary factors play a role in the development of several cancers. Identification of germline predisposition can have implications for treatment and cancer prevention. While previous studies have found that universal multigene panel testing in patients with cancer yields increased detection of actionable heritable variants compared to targeted testing, patients belonging to racial and ethnic minority groups have been underrepresented in these studies. We undertook a prospective multi-site study of germline alternations among solid tumor cancer patients from under-represented minority (URM) populations receiving care at Mayo Clinic Comprehensive Cancer Centers between January 2020 and November 2022. Patients with a new or active cancer diagnosis (all stages) not selected for family history of cancer were tested with a &gt;80-gene next generation sequencing panel. Of 532 patients (mean [SD] age, 57.2 [12.7] years; 320 (60.2%) female), 247 (46.4%) were Hispanic/Latino, 130 (24.4%) Black/African American, 80 (15.0%) Asian, 66 (12.4%) American Indian/Alaskan Native, 2 (0.4%) Native Hawaiian/Pacific Islander, and 7 (1.3%) other. PGV were found in 43 patients (8.1%), including 29 moderate- or high- penetrance cancer susceptibility genes. Variants of uncertain significance (VUS) were found in 272 patients (51.1%). Of those with a PGV, 29 (67.4%) had incremental clinically actionable findings that would not have been detected by established testing criteria, of which 16 were moderate- or high-penetrance variants. Younger age of diagnosis, gender, race, ethnicity, and stage of disease were not predictive of PGV. In this study of under-represented minority cancer patients, universal genetic testing found that 1 in 12 patients harbor a PGV, more than two-thirds of whom would remain undetected using current guidelines. The paucity of genomic data in URM populations also results in a large proportion of VUS which may lead to under-estimation of disease associated variants. Table 1. Clinical and Demographic Characteristics of Included Patients Positive Negative VUS Total N (%) 43 217 272 532 (8.1%) (40.8%) (51.1%) Age - mean (SD) 53.7 57.0 57.8 57.2 (12.7) (12.6) (12.8) (12.7) Gender - Female 26 132 162 320 (60.5%) (60.8%) (59.6%) (60.2%) Race/Ethnicity Hispanic/Latino 24 110 113 247 (55.8%) (50.7%) (41.5%) (46.4%) Black/African 5 56 69 130 American (11.6%) (25.8%) (25.4%) (24.4%) Asian 10 (23.3%) 23 (10.6%) 47 (17.3%) 80 (15.0%) American Indian/Alaskan Native 4 (9.3%) 26 (12.0%) 36 (13.2%) 66 (12.4%) Native Hawaiian/Pacific Islander 0 (0.0%) 0 (0.0%) 2 (0.7%) 2 (0.4%) Other 0 (0.0%) 2 (0.9%) 5 (1.8%) 7 (1.3%) Tumor Types Breast 19 (44.2%) 80 (36.9%) 110 (40.4%) 209 (39.3%) CNS 1 (2.3%) 2 (0.9%) 1 (0.4%) 4 (0.8%) GI 7 (16.3%) 46 (21.2%) 58 (21.3%) 111 (20.9%) GU 8 (18.6%) 57 (26.3%) 62 (22.8%) 127 (23.9%) GYN 0 (0.0%) 8 (3.7%) 10 (3.7%) 18 (3.4%) Head/Neck 5 (11.6%) 8 (3.7%) 6 (2.2%) 19 (3.6%) Lung 0 (0.0%) 6 (2.8%) 11 (4.0%) 17 (3.2%) Melanoma 0 (0.0%) 2 (0.9%) 1 (0.4%) 3 (0.6%) Sarcoma 2 (4.7%) 4 (1.8%) 4 (1.5%) 10 (1.9%) Other 1 (2.3%) 4 (1.8%) 9 (3.3%) 14 (2.6%) Citation Format: Kevork A. Khadarian, Sailaja Pisipati, Jeremy C. Jones, Gerardo Colon-Otero, Donald W. Northfelt, Lindsey Gary, Idara U. Ekpoh, Giovanna G. Moreno, Edward D. Esplin, Robert Nussbaum, Brandie Heald, Margaret A. Klint, Kathleen A. Barrus, Tanios S. Bekaii-Saab, Aleksandar Sekulic, Michael A. Golafshar, Katie L. Kunze, Cheryl L. Willman, Konstantinos N. Lazaridis, Niloy Jewel Samadder. Universal genetic testing for hereditary cancer syndromes in an under-represented minority population [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1921.

Abstract Background In the United States, rare disease (RD) is defined as a condition that affects fewer than 200,000 individuals. Collectively, RD affects an estimated 30 million Americans. A significant portion of RD has an underlying genetic cause; however, this may go undiagnosed. To better serve these patients, the Mayo Clinic Program for Rare and Undiagnosed Diseases (PRaUD) was created under the auspices of the Center for Individualized Medicine (CIM) aiming to integrate genomics into subspecialty practice including targeted genetic testing, research, and education. Methods Patients were identified by subspecialty healthcare providers from 11 clinical divisions/departments. Targeted multi-gene panels or custom exome/genome-based panels were utilized. To support the goals of PRaUD, a new clinical service model, the Genetic Testing and Counseling (GTAC) unit, was established to improve access and increase efficiency for genetic test facilitation. The GTAC unit includes genetic counselors, genetic counseling assistants, genetic nurses, and a medical geneticist. Patients receive abbreviated point-of-care genetic counseling and testing through a partnership with subspecialty providers. Results Implementation of PRaUD began in 2018 and GTAC unit launched in 2020 to support program expansion. Currently, 29 RD clinical indications are included in 11 specialty divisions/departments with over 142 referring providers. To date, 1,152 patients have been evaluated with an overall solve rate of 14.1% and as high as 66.7% depending on the phenotype. Noteworthy, 42.7% of the solved or likely solved patients underwent changes in medical management and outcome based on genetic test results. Conclusion Implementation of PRaUD and GTAC have enabled subspecialty practices advance expertise in RD where genetic counselors have not historically been embedded in practice. Democratizing access to genetic testing and counseling can broaden the reach of patients with RD and increase the diagnostic yield of such indications leading to better medical management as well as expanding research opportunities.