Allelic Loss Research Articles

Logging Affects Genetic Diversity Parameters in an Araucaria angustifolia Population: An Endangered Species in Southern Brazil

Araucaria angustifolia is an endangered species with more than 97% of its natural populations extinct. Logging of the species in the few remaining natural populations is highly restricted, though not readily accepted by farmers and logging companies. Consequently, political pressures have emerged for a return to logging of the species. Assessing the sustainability of such logging requires studies of a range of impacts on the remaining populations, including their genetic viability. We investigated the effect of selective logging on genetic diversity, intrapopulation spatial genetic structure (SGS), effective population size (Ne), and pollen and seed dispersal in three A. angustifolia permanent sample blocks established in a remnant of Araucaria Forest in Brazil. In these sample blocks, three logging intensities were applied (LI: 18.4, 31.4, and 32.3% of trees). Microsatellite analysis was performed for all adult and juvenile trees pre- and post-logging saplings. After selective logging, the greatest loss of alleles and the greatest decrease in Ne were observed from the highest LI. Logging increased SGS, while the distance and patterns of pollen and seed dispersal were different for both pre- and post-logging scenarios, with pollen dispersed over greater distances than seed. Pollen dispersal distance post-logging and seed dispersal distance pre- and post-logging decreased with the increased distance between parents. After logging, Ne reduced from 27.7 (LI = 31.4%) to 28.8 (LI = 18.4%) and 39.5% (LI = 32.3%), and some alleles were lost. Despite this, the loss of these alleles may be compensated for in subsequent generations, considering that logging resulted in changes such as an increase in the rate and distance of pollen immigration. Under the conditions evaluated in this study, selective logging of A. angustifolia is not adequate. To achieve truly sustainable forest logging, new rules that combine higher minimum DBH, lower logging intensity, and longer cutting cycles must be adopted. Furthermore, extensive genetic studies must be performed before logging any individual from a natural population.

Paths to adaptation under fluctuating nitrogen starvation: The spectrum of adaptive mutations in Saccharomyces cerevisiae is shaped by retrotransposons and microhomology-mediated recombination.

There are many mechanisms that give rise to genomic change: while point mutations are often emphasized in genomic analyses, evolution acts upon many other types of genetic changes that can result in less subtle perturbations. Changes in chromosome structure, DNA copy number, and novel transposon insertions all create large genomic changes, which can have correspondingly large impacts on phenotypes and fitness. In this study we investigate the spectrum of adaptive mutations that arise in a population under consistently fluctuating nitrogen conditions. We specifically contrast these adaptive alleles and the mutational mechanisms that create them, with mechanisms of adaptation under batch glucose limitation and constant selection in low, non-fluctuating nitrogen conditions to address if and how selection dynamics influence the molecular mechanisms of evolutionary adaptation. We observe that retrotransposon activity accounts for a substantial number of adaptive events, along with microhomology-mediated mechanisms of insertion, deletion, and gene conversion. In addition to loss of function alleles, which are often exploited in genetic screens, we identify putative gain of function alleles and alleles acting through as-of-yet unclear mechanisms. Taken together, our findings emphasize that how selection (fluctuating vs. non-fluctuating) is applied also shapes adaptation, just as the selective pressure (nitrogen vs. glucose) does itself. Fluctuating environments can activate different mutational mechanisms, shaping adaptive events accordingly. Experimental evolution, which allows a wider array of adaptive events to be assessed, is thus a complementary approach to both classical genetic screens and natural variation studies to characterize the genotype-to-phenotype-to-fitness map.

Abstract A004: KRAS allelic imbalance drives an epithelial MAPK-dependent tumor initiation program that is inefficient in provoking metastasis in colorectal cancer in vivo

Abstract Allelic imbalance is reported to be a frequent event in cancers and a common feature associated with oncogenes such as KRAS and BRAF. However the functional and therapeutic consequences of such imbalances are poorly understood. Mutations in the KRAS oncogene are one of the most prevalent events in human cancers and heterozygous KRAS mutations are well-described functionally and are thus viewed as sufficient for tumorigenesis. Recent evidence shows high incidence of KRAS gene dosage changes in human cancers, including loss of the normal wild-type allele of KRAS. However, there is still much debate over the function of wild-type KRAS in tumour initiation, progression and therapy response. Using advanced genetically engineered mouse models of colorectal cancer (VillinCRE Apcfl/fl; LSL-KrasG12D/+ (AK), LSL-KrasG12D/+; Trp53fl/fl; Rosa26N1iCD/+ (KPN)) we investigated the functional impact of wild-type Kras in oncogenic Kras driven tumour initiation (AK), progression and metastasis (KPN) in vivo. Mechanistically, wild-type Kras restrains oncogenic Kras signalling and significantly affects the efficiency of the oncogenic Kras induced transformation and response to therapy. We demonstrate a suppressive role for wild-type Kras during tumorigenesis and highlight the critical impact of wild-type Kras upon therapeutic response and tumour progression in Kras mutant CRC. Wild-type KRAS-deficient colorectal tumours are characterized by increased MAPK signalling and transcriptional activation of MAPK regulators. Importantly, loss of wild-type Kras in oncogenic KRAS-driven aggressive KPN tumours significantly alter tumour progression and liver metastasis, showing increased immune infiltration and WNT signalling. In addition, loss of wild-type Kras modulates response to therapeutic intervention and sensitizes wild-type Kras deficient tumours to MEK1/2 inhibition. This study demonstrates a suppressive role for wild-type Kras during tumour initiation and highlights the critical impact of wild-type Kras upon therapeutic response to MAPK and tumour progression in KRAS mutant CRC. Citation Format: Arafath K. Najumudeen, Sigrid K. Fey, Andrew D. Campbell, Owen J. Sansom. KRAS allelic imbalance drives an epithelial MAPK-dependent tumor initiation program that is inefficient in provoking metastasis in colorectal cancer in vivo [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A004.

Interstitial deletion of the Apc locus in β-catenin-overexpressing cells is a signature of radiation-induced intestinal tumors in C3B6F1 ApcMin/+ mice†.

Recent studies have identified interstitial deletions in the cancer genome as a radiation-related mutational signature, although most of them do not fall on cancer driver genes. Pioneering studies in the field have indicated the presence of loss of heterozygosity (LOH) spanning Apc in a subset of sporadic and radiation-induced intestinal tumors of ApcMin/+ mice, albeit with a substantial subset in which LOH was not detected; whether copy number losses accompany such LOH has also been unclear. Herein, we analyzed intestinal tumors of C3B6F1 ApcMin/+ mice that were either left untreated or irradiated with 2Gy of γ-rays. We observed intratumor mosaicism with respect to the nuclear/cytoplasmic accumulation of immunohistochemically detectable β-catenin, which is a hallmark of Apc+ allele loss. An immunoguided laser microdissection approach enabled the detection of LOH involving the Apc+ allele in β-catenin-overexpressing cells; in contrast, the LOH was not observed in the non-overexpressing cells. With this improvement, LOH involving Apc+ was detected in all 22 tumors analyzed, in contrast to what has been reported previously. The use of a formalin-free fixative facilitated the LOH and microarray-based DNA copy number analyses, enabling the classification of the aberrations as nondisjunction/mitotic recombination type or interstitial deletion type. Of note, the latter was observed only in radiation-induced tumors (nonirradiated, 0 of 8; irradiated, 11 of 14). Thus, an analysis considering intratumor heterogeneity identifies interstitial deletion involving the Apc+ allele as a causative radiation-related event in intestinal tumors of ApcMin/+ mice, providing an accurate approach for attributing individual tumors to radiation exposure.

Case-only exome variation analysis of severe alcohol dependence using a multivariate hierarchical gene clustering approach.

Variation in genes involved in ethanol metabolism has been shown to influence risk for alcohol dependence (AD) including protective loss of function alleles in ethanol metabolizing genes. We therefore hypothesized that people with severe AD would exhibit different patterns of rare functional variation in genes with strong prior evidence for influencing ethanol metabolism and response when compared to genes not meeting these criteria. Leverage a novel case only design and Whole Exome Sequencing (WES) of severe AD cases from the island of Ireland to quantify differences in functional variation between genes associated with ethanol metabolism and/or response and their matched control genes. First, three sets of ethanol related genes were identified including those a) involved in alcohol metabolism in humans b) showing altered expression in mouse brain after alcohol exposure, and altering ethanol behavioral responses in invertebrate models. These genes of interest (GOI) sets were matched to control gene sets using multivariate hierarchical clustering of gene-level summary features from gnomAD. Using WES data from 190 individuals with severe AD, GOI were compared to matched control genes using logistic regression to detect aggregate differences in abundance of loss of function, missense, and synonymous variants, respectively. Three non-independent sets of 10, 117, and 359 genes were queried against control gene sets of 139, 1522, and 3360 matched genes, respectively. Significant differences were not detected in the number of functional variants in the primary set of ethanol-metabolizing genes. In both the mouse expression and invertebrate sets, we observed an increased number of synonymous variants in GOI over matched control genes. Post-hoc simulations showed the estimated effects sizes observed are unlikely to be under-estimated. The proposed method demonstrates a computationally viable and statistically appropriate approach for genetic analysis of case-only data for hypothesized gene sets supported by empirical evidence.

Prognostic biomarkers in and selection of surgical patients with hepatocellular carcinoma.

Prognostic biomarkers in and selection of surgical patients with hepatocellular carcinoma.

Dose-dependent effect of GFI1 expression in the reconstitution and the differentiation capacity of HSCs.

GFI1 is a transcriptional repressor and plays a pivotal role in regulating the differentiation of hematopoietic stem cells (HSCs) towards myeloid and lymphoid cells. Serial transplantation of Gfi1 deficient HSCs repopulated whole hematopoietic system but in a competitive setting involving wild-type HSCs, they lose this ability. The underlying mechanisms to this end are poorly understood. To better understand this, we used different mouse strains that express either loss of both Gfi1 alleles (Gfi1-KO), with reduced expression of GFI1 (GFI1-KD) or wild-type Gfi1/GFI1 (Gfi1-/GFI1-WT; corresponding to the mouse and human alleles). We observed that loss of Gfi1 or reduced expression of GFI1 led to a two to four fold lower number of HSCs (defined as Lin-Sca1+c-Kit+CD150+CD48-) compared to GFI1-WT mice. To study the functional influence of different levels of GFI1 expression on HSCs function, HSCs from Gfi1-WT (expressing CD45.1 + surface antigens) and HSCs from GFI1-KD or -KO (expressing CD45.2 + surface antigens) mice were sorted and co-transplanted into lethally irradiated host mice. Every 4weeks, CD45.1+ and CD45.2 + on different lineage mature cells were analyzed by flow cytometry. At least 16weeks later, mice were sacrificed, and the percentage of HSCs and progenitors including GMPs, CMPs and MEPs in the total bone marrow cells was calculated as well as their CD45.1 and CD45.2 expression. In the case of co-transplantation of GFI1-KD with Gfi1-WT HSCs, the majority of HSCs (81% ± 6%) as well as the majority of mature cells (88% ± 10%) originated from CD45.2 + GFI1-KD HSCs. In the case of co-transplantation of Gfi1-KO HSCs with Gfi1-WT HSCs, the majority of HSCs originated from CD45.2+ and therefore from Gfi1-KO (61% ± 20%); however, only a small fraction of progenitors and mature cells originated from Gfi1-KO HSCs (<1%). We therefore in summary propose that GFI1 has a dose-dependent role in the self-renewal and differentiation of HSCs.

Abstract 3318: Frequent loss of CACNA1C, a calcium voltage-gated channel subunit is associated with lung adenocarcinoma progression and poor prognosis

Abstract Lung adenocarcinoma (LUAD) is a common non-small cell lung cancer disease most commonly found among non-smokers. This cancer is usually diagnosed at an advanced stage and can recur frequently and quickly with low chances of survival. Moreover, LUAD patients live with the fear of developing second primary tumors in their lifetime. A high-resolution SNP array (Affymetrix 250 NspI) analysis of primary LUAD tumors and their follow-up mucosal biopsies identified frequent copy neutral loss of heterozygosity (LOH, loss of 59 SNPs) of CACNA1C, a calcium voltage-gated channel subunit, located to human chromosome 12p13.33. Transcriptomic profiling utilizing The Cancer Genome Atlas (TCGA) revealed frequent loss of mRNA expression of CACNA1C in LUAD tumors (n=515) compared to the normal counterparts (n=59). Loss of CACNA1C mRNA expression was significantly associated with stage, grade, lymph node metastasis, P53 gene mutation, and worst survival of the LUAD patients. Epigenetic alteration such as DNA methylation could be a potential mechanism of the inactivation of a tumor suppressor gene (TSG) other than LOH, frequently observed in human cancers. We also recorded significantly higher promoter methylation of CACNA1C in the above LUAD tumors compared to the normal counterparts. Higher promoter methylation of CACNA1C was significantly associated with stage, grade, lymph node metastasis, P53 gene mutation, and poor survival of the LUAD patients. Thus, both allelic loss and methylation appear to be potential mechanisms behind the loss of function of CACNA1C in LUAD patients. Further validation of CACNA1C expression at the protein level confirmed its high membranous expression in normal lung tissues. Analysis of an independent cohort of LUAD subjects revealed significant loss of CACNA1C in primary tumors (n=30) and lymph node metastases (n=10) of LUAD patients compared to their normal counterparts. Loss of CACNA1C protein expression was associated with lymph node metastasis and stage-wise progression of the LUAD patients. Further validation of CACNA1C expression in larger cohorts and functional characterization would be beneficial for potential biomarkers and therapeutic development in the longer run. On the other hand, epigenetically silenced TSGs can be reactivated by CRISPR-CAS9-based technology or demethylating agents, implicating the therapeutic promise of targeting TSGs. Citation Format: Saanvi Dasgupta, Srijan Acharya, Mohammad A. Khan, Paramahansa Pramanik, Stephen M. Marbut, Furhan Yunus, Jose N. Galeas, Seema Singh, Ajay P. Singh, Santanu Dasgupta. Frequent loss of CACNA1C, a calcium voltage-gated channel subunit is associated with lung adenocarcinoma progression and poor prognosis [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 3318.

Abstract 5214: Expanding the spectrum of germline-driven cancers by leveraging population-scale targeted tumor and normal sequencing

Abstract Large case-control and familial studies have established clear cancer-specific risk profiles for several key cancer predisposition genes (CPGs). For example, germline pathogenic variants (PVs) in BRCA1/2 (gBRCA) are associated with increased risk for developing breast, ovarian, pancreatic, and prostate cancers. However, the extent to which gBRCA mutations are involved in mediating the tumorigenesis of other cancer types remains challenging to characterize. We hypothesized that integrating orthogonal features such as selection for biallelic inactivation of the PVs and depletion of canonical somatic drivers among the carriers can enrich the signal for identifying novel gene and cancer type associations. We then extend this framework to identify novel CPGs as well as to understand how tumors arise in patients with PVs in oncogenes. To study this, we leveraged the prospective MSK-IMPACT matched tumor-normal sequencing cohort of 49,291 patients across 77 major cancer types. We study 90 well-known CPGs as well as &amp;gt;300 cancer genes not previously associated with cancer predisposition. Overall, 8% (N=3,964) of patients harbored a PV in high or moderate penetrance CPGs. We identified 90 gene and cancer type associations with enrichment for biallelic inactivation (q&amp;lt;0.05), including 19 novel findings among clinically actionable genes. For example, we find enrichment for biallelic inactivation of BRCA1/2 in unexpected lineages such as hepatobiliary, endometrioid, and ampullary cancers. These tumors were also significantly depleted for somatic gain-of-function driver alterations. Hepatobiliary cancers with gBRCA1 mutations were also enriched for somatic loss of NF1. Among carriers of PVs in oncogenes, we observe two possible mechanisms of first somatic hit towards malignant transformation. We find enrichment for copy number gain or copy neutral loss of heterozygosity of the germline PV in thyroid cancers with a PV in RET. We also find that lung cancers with a germline PV in EGFR frequently developed additional somatic point mutations located in cis with the PV. Investigating genes with no prior association with germline predisposition to cancer, we find evidence for KEAP1 and CIC as likely novel CPGs. Lung (n=8) and thyroid (n=4) cancers with deleterious germline variants in KEAP1 were characterized by loss of the wild-type allele, co-occurring somatic STK11 mutations, and depletion of canonical drivers such as EGFR. We also found biallelic loss of CIC in two patients with Neuroblastoma, each carrying a different germline loss-of-function mutation in CIC. Both tumors were also negative for MYCN and ALK defects. Collectively, our findings expand our understanding of cancer predisposition in cancer, shed new insights into how tumors arise in germline carriers, and provide a framework for identifying new CPGs using population scale tumor-normal paired clinical sequencing data. Citation Format: Miika Mehine, Rebecca Caeser, Yelena Kemel, Daniel Muldoon, Sebastià Franch-Expósito, A. Rose Brannon, Aijazuddin Syed, Ozge Ceyhan-Birsoy, Maksym Misyura, Panieh Terraf, David B. Solit, Marc Ladanyi, Kenneth Offit, Zsofia K. Stadler, Diana L. Mandelker, Yonina R. Murciano-Goroff, Charles M. Rudin, Michael F. Berger, Chaitanya Bandlamudi. Expanding the spectrum of germline-driven cancers by leveraging population-scale targeted tumor and normal sequencing. [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 5214.

Abstract 15: Investigating the spectrum of brain tumors associated with Adgrb3 and Tp53 loss in a mouse model of Li-Fraumeni syndrome

Abstract Adhesion G protein-coupled receptor 3 (ADGRB3), also known as brain specific angiogenesis inhibitor (BAI3) is a member of the ADGRB1-3 subfamily of adhesion transmembrane proteins, which are highly expressed in the brain specifically in cerebellum and hippocampal neurons. ADGRB3 has been shown to play diverse roles under physiological and pathological conditions which include dendritic morphogenesis, synaptic plasticity, synaptogenesis, and myogenesis. Loss of ADGRB3 expression and point mutations in the gene have been observed in sporadic tumors, including brain tumors, but the significance of this observation has not been investigated. Moreover, no mouse models to understand the role of ADGRB3 in brain tumor susceptibility and pathobiology have been developed thus far. Li-Fraumeni syndrome (LFS) is a rare inherited autosomal dominant disorder caused by a germline mutation in one TP53 allele, predisposing patients to the development of a variety of tumors from a pediatric age, including gliomas and medulloblastoma. The secondary molecular alterations that predispose LFS patients to brain tumors are currently unknown. Interestingly, a LFS patient was reported to carry a translocation in ADGRB3 and HGMLL genes. To investigate the molecular mechanisms underlying brain tumor formation in LFS patients and the role of p53 and ADGRB3 in the process, we have generated a LFS mouse model. The mice harbor a germline p53 deleted allele and a second floxed allele. Loss of the second allele is induced in the brain by crossing with mice harboring a Nestin promoter driven Cre recombinase, since Nestin is expressed along the craniospinal axis and in glial precursor cells. We observed that the majority of nestin-Cre p53m/f mice all died between 8 and 10 months of age while nestin-Cre p53m/+ mice did not. About 20% of the mice developed hind leg paralysis and harbored large gliomas, which likely caused their demise. The remainder mice lacked brain tumors, but had other malignancies (sarcomas, etc.) as observed in patients. Remarkably, the addition of Adgrb3 deletion to the genotype led to a dramatic increase (from 20% to 60%??) in the number of brain tumors, including medulloblastoma formation, which are also reported in LFS patients. The Adgrb3-/- p53+/- Nestin-Cre mouse model constitutes a useful tool to understand the tumorigenic landscape caused by the loss of Adgrb3. We are now performing genomic analyses on the excised tumors and derived neurosphere cultures to further study the transformation process and the molecular changes induced by Adgrb3 loss. Citation Format: Alex B. Torrelli-Diljohn, Svetlana Komarova, Vipul Sheth, Benjamin Lin, Paran Goel, Ryan Miller, Robert Welner, Erwin G. Van Meir. Investigating the spectrum of brain tumors associated with Adgrb3 and Tp53 loss in a mouse model of Li-Fraumeni syndrome [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 15.

Abstract 6095: A DNA damaging agent affects BRCA2 exon 3-skipping

Abstract Introduction: BRCA2 is a tumor suppressor that participates in DNA repair. Inherited pathogenic mutations in BRCA2 are associated with a 38%-84% risk for breast cancer and a 16.5%-27% risk for ovarian cancer. BRCA2 exon 3, which encodes a binding site for the interacting proteins EMSY and PALB2, is frequently skipped during alternative splicing events, often due to spliceogenic mutations, but also for as yet uncharacterized environmental conditions. A genomic deletion that results in complete allelic loss of exon 3 has been shown to be pathogenic, yet sequence variants that result in increased frequency of exon 3-skipping have been shown to be non-pathogenic. Thus, the allele-specific ability of BRCA2 to contribute to DNA repair is affected by the level of exon 3-skipping. In this study we explore the effects of the topoisomerase inhibitor doxorubicin on the frequency of BRCA2 exon 3-skipping. Isoform-specific end-point RT-PCR shows that doxorubicin increases the frequency of BRCA2 exon 3-skipping in the breast cancer cell line MCF7 at concentrations below those required for promoting apoptosis. Methods: MCF7 cells were treated with doxorubicin according to standard protocols. IC50 levels of doxorubicin were determined using a crystal violet assay. Flow cytometry was performed using standard protocols. RNA was prepared using Qiagen reagents and amplified with Invitrogen One-Step RT-PCR reagents. Isoform-specific RT-PCR primers were designed to amplify the full-length region surrounding BRCA2 exon 3 independently from the region left by an exon-skipping event (∆3). RT-PCR products were visualized using agarose gel electrophoresis. Results and Conclusion: Results suggest that doxorubicin increases the level of BRCA2 exon 3-skipping in MCF7 at concentrations below those required for promoting apoptosis. This suggests doxorubicin may compromise the DNA repair functions of BRCA2 and other alternatively spliced mRNAs, which may contribute to the therapeutic benefits and/or toxicity of the drug. Citation Format: Jordan R. Werner, Hiba Siddiqui, Ethan Castillo, Mohammed Ansari, James D. Fackenthal. A DNA damaging agent affects BRCA2 exon 3-skipping [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 6095.

Abstract 2601: Lrig1 is a haplo-insufficient tumor suppressor and Lrig1-expressing cells can function as cells-of-origin of tumor development

Abstract LRIG1, leucine-rich repeats and immunoglobulin-like domains protein 1, is deleted or downregulated in many cancer types and has been reported to function as a tumor suppressor. LRIG1 is preferentially expressed in adult stem/progenitor cells and regulates their biological properties and quiescence by repressing the ERBB signaling. We recently reported LRIG1 to be overexpressed in human prostate cancer and to function as a feedback tumor suppressor. Human LRIG1 transgenically expressed in the mouse prostate inhibited the development and growth of both Hi-Myc and TRAMP tumors. The relationship between Lrig1-expressing adult stem/progenitor cells and cells-of-origin for cancer remains poorly understood. To address this, we deleted the Pten tumor suppressor gene in Lrig1-expressing cells by crossing Lrig1-CreERT2 mouse with Ptenfl/fl animals followed by Cre activation via tamoxifen administration. This knockout of Pten in Lrig1-expressing cells allowed us to assess the tumor suppressive function of Lrig1 as Lrig1-CreERT2 is a knock-in model in which one allele of Lrig1 is inactivated. We also generated a Lrig1-lineage tracing mouse model to label endogenous Lrig1-expressing cells by crossing Lrig1-CreERT2 mouse with CAG-tdTomato animals. Cre recombinase in these animals was activated by tamoxifen and epithelial tissues were harvested and analyzed for tomato signal by fluorescent microscopy. Lrig1-lineage tracing model showed that Lrig1-expressing cells in mouse prostate and oral epithelia are of CK5+ and CK8- phenotype suggesting their basal lineage. Targeted deletion of Pten in the Lrig1-expressing cellular compartments resulted in hyperplasic lesions in oral mucosa as early as 4 weeks and prevalent papilloma by 8 weeks. Pten deletion in the mouse prostate led to hyperplasia and high-grade prostate intraepithelial neoplasia (HGPIN) in a time-dependent manner. Consistent with these hyperplastic and tumor phenotypes, we observed elevated Ki-67 and pAkt staining indicating increased cell proliferation. Since Lrig1-CreERT2 is a knock-in model in which one allele of Lrig1 is inactivated, our data suggest that even one allelic loss of Lrig1 promotes more aggressive tumor phenotypes compared to those reported earlier with Pten deletion in CK5+ basal cells (CK5-CreERT2:Ptenfl/fl) in which similar lesions in oral mucosa and prostate tissues developed only after 20-24 weeks of Pten deletion. These results suggested that LRIG1 is a haplo-insufficient tumor suppressor as mono-allelic deletion of Lrig1 is sufficient to promote the tumorigenic process in mouse epithelial tissues induced by Pten loss. Our study also suggests that the Lrig-1 expressing cells can function as the cells-of-origin of cancer. We propose that Lrig1 may be used as a prognostic marker in cancers of epithelial origin and higher levels may portend a better prognosis. Citation Format: Moyi Wang, Qiuhui Li, Amanda Tracz, Jason Kirk, Anmbreen Jamroze, Rahul Kumar, Dean G. Tang. Lrig1 is a haplo-insufficient tumor suppressor and Lrig1-expressing cells can function as cells-of-origin of tumor development [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 2601.

CYP3A5 Polymorphism in Renal Transplantation: A Key to Personalized Immunosuppression

BackgroundTacrolimus is essential for the maintenance of immunosuppression after a kidney transplant. CYP3A5 is the gene that metabolizes tacrolimus, and polymorphism in this gene affects the metabolizing status. AimTo assess the genetic polymorphism status of patients undergoing kidney transplantation and determine its impact on graft function and complications in the post-transplant period. MethodsWe retrospectively included the patients who had undergone a kidney transplant and had positive genetic polymorphism of the CYP3A5 gene. Based on loss of alleles, patients were categorized as non-expresser (loss of both alleles), intermediate expresser (loss of one allele), and expresser (no loss of allele) denoted by CYP3A5*3/*3, CYP3A5*1/*3, and CYP3A5*1/*1, respectively. Data were analyzed with descriptive statistics. ResultsOf 25 patients, 60%, 32%, and 8% were non-expressers, intermediate-expressers, and expressers, respectively. The mean tacrolimus trough concentration to dose ratio after 6 months of the transplant was higher in non-expressers than intermediate-expressers and expressers (213 vs 85 and 46 ng/mL/mg/kg/d, respectively). The graft function was normal in all 3 groups except for graft rejection 1 patient in the expresser group. Compared with expressers, urinary tract infections (42.9% and 62.5%) and new-onset diabetes after transplantation (28.6% and 12.5%) were more frequent in non-expresser and intermediate expressers, respectively. The proportion of patients developing new-onset diabetes after transplantation was lower with the pre-transplant diagnosis of CYP3A5 polymorphism (16.7% vs 23.1%). ConclusionGenotype-based dosing of tacrolimus helps achieve the desired therapeutic concentrations that can help to optimize graft outcomes and reduce the tacrolimus-related adverse effects. Pre-transplant evaluation of CYP3A5 can be more helpful in planning treatment strategies for optimized outcomes after kidney transplantation.

Genetic analyses of DNA repair pathway associated genes implicate new candidate cancer predisposing genes in ancestrally defined ovarian cancer cases.

Not all familial ovarian cancer (OC) cases are explained by pathogenic germline variants in known risk genes. A candidate gene approach involving DNA repair pathway genes was applied to identify rare recurring pathogenic variants in familial OC cases not associated with known OC risk genes from a population exhibiting genetic drift. Whole exome sequencing (WES) data of 15 OC cases from 13 families tested negative for pathogenic variants in known OC risk genes were investigated for candidate variants in 468 DNA repair pathway genes. Filtering and prioritization criteria were applied to WES data to select top candidates for further analyses. Candidates were genotyped in ancestry defined study groups of 214 familial and 998 sporadic OC or breast cancer (BC) cases and 1025 population-matched controls and screened for additional carriers in 605 population-matched OC cases. The candidate genes were also analyzed in WES data from 937 familial or sporadic OC cases of diverse ancestries. Top candidate variants in ERCC5, EXO1, FANCC, NEIL1 and NTHL1 were identified in 5/13 (39%) OC families. Collectively, candidate variants were identified in 7/435 (1.6%) sporadic OC cases and 1/566 (0.2%) sporadic BC cases versus 1/1025 (0.1%) controls. Additional carriers were identified in 6/605 (0.9%) OC cases. Tumour DNA from ERCC5, NEIL1 and NTHL1 variant carriers exhibited loss of the wild-type allele. Carriers of various candidate variants in these genes were identified in 31/937 (3.3%) OC cases of diverse ancestries versus 0-0.004% in cancer-free controls. The strategy of applying a candidate gene approach in a population exhibiting genetic drift identified new candidate OC predisposition variants in DNA repair pathway genes.

SDHA Germline Mutations in SDH-Deficient GISTs: A Current Update.

Loss of function of the succinate dehydrogenase complex characterizes 20-40% of all KIT/PDGFRA-negative GIST. Approximately half of SDH-deficient GIST patients lack SDHx mutations and are caused by a hypermethylation of the SDHC promoter, which causes the repression of SDHC transcription and depletion of SDHC protein levels through a mechanism described as epimutation. The remaining 50% of SDH-deficient GISTs have mutations in one of the SDH subunits and SDHA mutations are the most common (30%), with consequent loss of SDHA and SDHB protein expression immunohistochemically. SDHB, SDHC, and SDHD mutations in GIST occur in only 20-30% of cases and most of these SDH mutations are germline. More recently, germline mutations in SDHA have also been described in several patients with loss of function of the SDH complex. SDHA-mutant patients usually carry two mutational events at the SDHA locus, either the loss of the wild type allele or a second somatic event in compound heterozygosis. This review provides an overview of all data in the literature regarding SDHA-mutated GIST, especially focusing on the prevalence of germline mutations in SDH-deficient GIST populations who harbor SDHA somatic mutations, and offers a view towards understanding the importance of genetic counselling for SDHA-variant carriers and relatives.

RAB5C is a TBC1D21 interactor involved in sperm morphology formation

&lt;p&gt;背景與目的: 不孕是重要的公眾健康議題，造成不孕的因素其中一半與男性相關。其中造成男性不孕的一項重要病因是畸型精子症。在先前的研究中，我們以cDNA微晶片比較造精功能正常與異常的睪丸組織中各別基因的表現量。其中發現一個與不孕相關Rab GTPase激活蛋白，名為TBC1D21蛋白。TBC1D21特異的表現於減數分裂後期精細胞中。而於Tbc1d21基因剔除小鼠模式中發現嚴重精子尾部的中節區域損壞。進一步我們以蛋白質質體學方法鑑定出與TBC1D21結合蛋白。於本研究中我們聚焦於其中一個TBC1D21結合蛋白RAB5C。研究方法與結果: RAB5C蛋白表現於小鼠睪丸組織切片中的精子細胞的頂體與尾部。於成熟精子中，RAB5C蛋白濃縮於頭部頂體與散落於尾部中節區域。其中有趣的是精子經過頂體反應後，於頂體的RAB5C蛋白量明顯下降消失。最後我們利用共同免疫染色法確認RAB5C與TBC1D21共同散落於成熟精子尾部中節區域。結論:基於以上結果，我們發現TBC1D21 交互作用蛋白RAB5C參與於精子頭部頂體與尾部中節的形成。&lt;/p&gt; &lt;p&gt;&amp;nbsp;&lt;/p&gt;&lt;p&gt;Background and purpose: Infertility is a critical public health issue, and half of its causes are related to male factors. Teratozoospermia is one of major cause of male infertility. In our previous studies, we identified the TBC1 domain family member 21 (TBC1D21) gene, a Rab GTPase-activating protein (GAP), as a sterility-related gene in cDNA microarray compared to normal and spermatogenic testicular tissues. TBC1D21 is specifically expressed in post-meiotic male germ cells. Furthermore, the loss of Tbc1d21 allele in mice results in severe defects in the midpiece of the sperm&amp;rsquo;s tail. Additionally, TBC1D21 interactors were selected using proteomic assays. Herein, we focused on RAB5C, which is a TBC1D21 interactor. Methods and Results: RAB5C was localized in the acrosomal region and elongating tail in spermatids of murine testicular sections. In a mature sperm cell, RAB5C signals were concentrated in the acrosome and were scattered around the midpiece. Interestingly, RAB5C pro-tein levels decreased in the acrosome after triggering acrosomal reaction. Finally, we confirmed that the interspersed RAB5C signals were co-localized with TBC1D21 within the midpieces of the mature sperm cells. Conclusion: Based on these results, we suggest RAB5C, a TBC1D21 interactor, was involved in acrosomal and midpiece formation.&lt;/p&gt; &lt;p&gt;&amp;nbsp;&lt;/p&gt;

Abstract P5-02-54: Application of low-pass whole genome sequencing for the detection of Homologous Recombination Deficiency in breast cancer

Abstract Background: Homologous recombination deficiency (HRD), broadly defined as a loss of the cellular mechanism underlying homologous recombination, is often observed in breast cancer. HRD causes distinctive perturbations to tumor genomic architecture that allow for its molecular identification, while also rendering HRD+ cancers vulnerable to specific chemotherapeutic interventions. This makes the molecular identification of HRD a promising avenue in precision medicine of breast cancer. Specific features of HRD include the presence of large-scale transitions (LST), telomeric allelic imbalance (TAI) and Loss of Heterozygosity (LOH). Each are readily detectable via targeted next generation sequencing (tNGS) or via array-based genotyping. However, genome-wide approaches for HRD detection using cost-effective methods, such as low-pass sequencing (LP-WGS), remain relatively under-explored. Here, we investigated whether HRD signals can be successfully re-capitulated using LP-WGS technology and benchmarked our results against the current field standard (both tNGS and array genotyping). Methods: LP-WGS and tNGS was performed on 96 samples across a range of tumor types (including N=17 breast cancer samples). LP-WGS libraries were prepared using Nextera (Illumina) using 0.4ng DNA input, and sequenced to 0.5-1x coverage. tNGS libraries were prepared using TSO500 (Illumina) using 40-80ng input, and sequenced to &amp;gt;150x unique read coverage. Regions of CNV were estimated using CNVKit v0.9.6, and regions of LOH were estimated using a novel ancestry-aware method. Small variant detection was performed using the TSO500 v2.2.0.12 analysis pipeline. SNP array analysis of 12 tumor samples using Oncoscan (ThermoFisher) was also performed. CNV and LOH estimates derived from LP-WGS, TSO500 and SNP array data were calculated using Jaccard similarity, treating the SNP array data as the “ground truth”. Results: We benchmarked HRD signals derived from LP-WGS compared to the array-based calls and observed near perfect sensitivity for CNV gains across samples (Jaccard index=1.0), as well as for CNV losses between LP-WGS and SNP array (Jaccard index=1.0). We additionally noted that LPS-WGS calls captured both CNV loss and gains that were not detectable via the SNP array. For TAI, LP-WGS re-capitulated 7/10 unique signals also identified via array. We also observed high concordance between regions of the genome called LOH between both platforms (median Jaccard index=0.70, IQR=0.254), but noted an attenuation of sensitivity in samples where estimated tumor heterogeneity was high. We also evaluated LP-WGS CNV calls against the TSO500 assay and noted high sensitivity (96%; 94%) and specificity (89%; 91%) for both CNV gains and losses, respectively. Conclusions: Workflows incorporating LP-WGS can support the detection of HRD genome-wide, paving the way for a more affordable assay that may help to inform clinical decision making in the future treatment of breast cancer. Citation Format: Gillian Belbin, Jie An, Chase Mazur, Joseph Pickrell, Jeremy Li, Daniel Metzger, Shuang Gao, Erik Van Roey, Robert Seager, Sarabjot Pabla, Durga Prasad Dash, Jeffrey M. Conroy. Application of low-pass whole genome sequencing for the detection of Homologous Recombination Deficiency in breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-54.

Genome-wide analysis of NBS-LRR genes revealed contribution of disease resistance from Saccharum spontaneum to modern sugarcane cultivar.

During plant evolution, nucleotide-binding sites (NBS) and leucine-rich repeat (LRR) genes have made significant contributions to plant disease resistance. With many high-quality plant genomes sequenced, identification and comprehensive analyses of NBS-LRR genes at whole genome level are of great importance to understand and utilize them. In this study, we identified the NBS-LRR genes of 23 representative species at whole genome level, and researches on NBS-LRR genes of four monocotyledonous grass species, Saccharum spontaneum, Saccharum officinarum, Sorghum bicolor and Miscanthus sinensis, were focused. We found that whole genome duplication, gene expansion, and allele loss could be factors affecting the number of NBS-LRR genes in the species, and whole genome duplication is likely to be the main cause of the number of NBS-LRR genes in sugarcane. Meanwhile, we also found a progressive trend of positive selection on NBS-LRR genes. These studies further elucidated the evolutionary pattern of NBS-LRR genes in plants. Transcriptome data from multiple sugarcane diseases revealed that more differentially expressed NBS-LRR genes were derived from S. spontaneum than from S. officinarum in modern sugarcane cultivars, and the proportion was significantly higher than the expected. This finding reveals that S. spontaneum has a greater contribution to disease resistance for modern sugarcane cultivars. In addition, we observed allelespecific expression of seven NBS-LRR genes under leaf scald, and 125 NBS-LRR genes responding to multiple diseases were identified. Finally, we built a plant NBS-LRR gene database to facilitate subsequent analysis and use of NBSLRR genes obtained here. In conclusion, this study complemented and completed the research of plant NBS-LRR genes, and discussed how NBS-LRR genes responding to sugarcane diseases, which provided a guide and genetic resources for further research and utilization of NBS-LRR genes.

Assessing genetic diversity patterns at neutral and adaptive loci to inform population reinforcement of an endangered migratory vulture

One of the primary goals of conservation translocation programs should be the maintenance of both population demographic stability and genetic diversity. Here, we provide genetic management recommendations to inform a population reinforcement of the declining Egyptian Vulture population in the Balkans. Specifically, we examined whether the number of released individuals is sufficient to prevent genetic diversity loss due to random genetic drift and what the origin of the individuals should be that comprise the captive breeding pool. To this aim, we estimated and assessed genetic diversity levels and genetic structure of Egyptian Vulture populations across much of the species’ range using both neutral and non-neutral candidate loci involved in migration. We then evaluated the effects of the currently proposed population management scheme and candidate source populations on retaining allelic diversity. Our results show low differentiation values among populations and absence of genetic structure which point to past high gene flow. Furthermore, there was no predicted significant impact of different source populations on the genetic diversity of the recipient Balkan population. We also found that the declining Egyptian Vulture population in the Balkans still retains high levels of genetic diversity and therefore genetic diversity restoration is not currently needed. However, without any management, diversity is likely to decrease fast because of increased genetic drift as the population size continues to decline. Population reinforcement with nine birds per year for 20 years would provide sufficient demographic support for the population to retain > 85% of rare allelic diversity. Birds originating from the Balkans would ensure ecological and behavioral similarity and thus would be the best option for reinforcement. Nevertheless, our results demonstrate that to prevent further population contraction and loss of adaptive alleles, releasing individuals of different origin would also be appropriate.

Pervasiveness of HLA allele-specific expression loss across tumor types

BackgroundEfficient presentation of mutant peptide fragments by the human leukocyte antigen class I (HLA-I) genes is necessary for immune-mediated killing of cancer cells. According to recent reports, patient HLA-I genotypes can impact the efficacy of cancer immunotherapy, and the somatic loss of HLA-I heterozygosity has been established as a factor in immune evasion. While global deregulated expression of HLA-I has also been reported in different tumor types, the role of HLA-I allele-specific expression loss — that is, the preferential RNA expression loss of specific HLA-I alleles — has not been fully characterized in cancer.MethodsHere, we use RNA and whole-exome sequencing data to quantify HLA-I allele-specific expression (ASE) in cancer using our novel method arcasHLA-quant.ResultsWe show that HLA-I ASE loss in at least one of the three HLA-I genes is a pervasive phenomenon across TCGA tumor types. In pancreatic adenocarcinoma, tumor-specific HLA-I ASE loss is associated with decreased overall survival specifically in the basal-like subtype, a finding that we validated in an independent cohort through laser-capture microdissection. Additionally, we show that HLA-I ASE loss is associated with poor immunotherapy outcomes in metastatic melanoma through retrospective analyses.ConclusionsTogether, our results highlight the prevalence of HLA-I ASE loss and provide initial evidence of its clinical significance in cancer prognosis and immunotherapy treatment.