Vitek, Carolyn R. Rohrer

Abstract Genetic counseling assistants (GCAs) support genetic counselors (GCs) and genetics clinic workflows, but their potential roles in pretest genetic counseling for rare diseases have not been explored. A pilot within the Mayo Clinic Center for Individualized Medicine's Genetic Testing and Counseling Clinic (GTAC), which offers predefined tests for patients with rare disease, explored the impact of GCA pretest education on appointment time and patient questions. After training, GCAs met GTAC patients prior to the GC to provide scripted information on genetics concepts, the visit purpose, and the test including result types and disclosure plans. Data from the pilot and a control group were collected and analyzed using descriptive statistics and two‐sided t ‐tests. Patient cohort characteristics did not differ between the two models. When a GCA provided pretest education, the GC spent an average of 11.2 minutes less with the patient during their session compared to visits completed solely by the GC ( p < 0.0001). Total appointment time was not impacted by GCA education. Questions asked to GCAs were often not within GCA scope to answer (72.7%), and some patients asked repetitive questions to both the GC and GCA (63.2%). Reduction in GC time per patient could lead to increased accessibility by allowing additional patients to be seen in a day. Similar models may support GCA professional development while allowing GCs to remove repetitive education from their genetic counseling sessions, leading to less burnout and/or increased job satisfaction. Impact on administrative workflows, access, revenue, patient satisfaction and outcomes and GC/GCA satisfaction and benefits can continue to be explored when trialing models incorporating GCAs in pretest education roles.

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 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 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.