Publications by authors named "Marcy Richardson"

19 Publications

  • Page 1 of 1

Universal Germline Panel Testing for Individuals With Pheochromocytoma and Paraganglioma Produces High Diagnostic Yield.

J Clin Endocrinol Metab 2022 04;107(5):e1917-e1923

Department of Internal Medicine, Michigan Medicine, University of Michigan, Ann Arbor, MI 48109, USA.

Background: Practice guidelines to identify individuals with hereditary pheochromocytomas and paragangliomas (PPGLs) advocate for sequential gene testing strategy guided by specific clinical features and predate the routine use of multigene panel testing (MGPT).

Objective: To describe results of MGPT for hereditary PPGL in a clinically and ancestrally diverse cohort.

Setting: Commercial laboratory based in the United States.

Methods: Clinical data and test results were retrospectively reviewed in 1727 individuals who had targeted MGPT from August 2013 through December 2019 because of a suspicion of hereditary PPGL.

Results: Overall, 27.5% of individuals had a pathogenic or likely pathogenic variant (PV), 9.0% had a variant of uncertain significance, and 63.1% had a negative result. Most PVs were identified in SDHB (40.4%), followed by SDHD (21.1%), SDHA (10.1%), VHL (7.8%), SDHC (6.7%), RET (3.7%), and MAX (3.6%). PVs in FH, MEN1, NF1, SDHAF2, and TMEM127 collectively accounted for 6.5% of PVs. Clinical predictors of a PV included extra-adrenal location, early age of onset, multiple tumors, and positive family history of PPGL. Individuals with extra-adrenal PGL and a positive family history were the most likely to have a PV (85.9%). Restricting genetic testing to SDHB/C/D misses one-third (32.8%) of individuals with PVs.

Conclusion: Our data demonstrate a high diagnostic yield in individuals with and without established risk factors, a low inconclusive result rate, and a substantial contribution to diagnostic yield from rare genes. These findings support universal testing of all individuals with PPGL and the use of concurrent MGPT as the ideal platform.
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http://dx.doi.org/10.1210/clinem/dgac014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9016434PMC
April 2022

Different Fumarate Hydratase Gene Variants Are Associated With Distinct Cancer Phenotypes.

JCO Precis Oncol 2021 11;5:1568-1578

Division of Population Sciences, Dana-Farber Cancer Institute, Boston, MA.

Purpose: Whether individuals with monoallelic pathogenic variants (PVs) associated with autosomal recessive fumarate hydratase (FH) deficiency are also at risk of autosomal dominant -associated tumors is of paramount clinical importance.

Methods: A retrospective study of individuals with a PV in the gene identified via multigene panel testing from 2012 to 2019 through a single testing laboratory was performed. Cancer histories of individuals with PVs in ( PV) were compared to those with PVs associated only with autosomal recessive FH deficiency (FH-d PV) and to -negative controls. Cancer histories of individuals with truncating versus nontruncating PV were also compared.

Results: Individuals with PV were more likely to have kidney cancer than those with FH-d PV (odds ratio, 9.0; 95% CI, 4.4 to 20.0; < .001) or FH-negative controls (odds ratio, 7.6; 95% CI, 5.2 to 11.2; value < .001). The PV cohort had kidney cancer at a significantly younger age (median age: 35.0 years; interquartile range, 26.0-45.0 years) than the FH-d PV cohort (median age: 44.5 years; interquartile range, 43.5-53.5 years; = .011). Within the PV cohort, there were no differences in the frequency or age at kidney cancer between those with truncating versus nontruncating PV.

Conclusion: Unlike PV, FH-d PV are not associated with kidney cancers at early ages of onset. The FH-d PV cohort had a cancer phenotype that resembled -negative controls. These data may inform genetic counseling and risk assessment of individuals with FH-d PV.
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http://dx.doi.org/10.1200/PO.21.00263DOI Listing
November 2021

Prevalence and spectrum of pathogenic variants among patients with multiple primary cancers evaluated by clinical characteristics.

Cancer 2022 03 7;128(6):1275-1283. Epub 2021 Dec 7.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.

Background: Multiple primary cancers (MPCs) are a hallmark of cancer predisposition syndromes. Here the frequency of germline pathogenic variants (PVs) among patients with MPCs is reported.

Methods: Patients with MPCs who underwent multigene panel testing from March 2012 to December 2016 were studied. Eligible patients had an analysis of 21 genes: ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, PTEN, RAD51C, RAD51D, STK11, and TP53. The frequencies of PVs by sex, number of cancers, and age at diagnosis were compared with 2-sided χ tests or Fisher exact tests when the number was <10.

Results: Among the 9714 patients analyzed, most were female (91.1%) and White (71.0%); the median age at testing was 63 years, and the median ages at first and second cancer diagnoses were 49 and 58 years, respectively. Overall, 1320 (13.6%) had PVs. The prevalence of PVs increased with the number of primary cancers (PCs): 13.1% with 2 PCs, 15.9% with 3 PCs, and 18.0% with ≥4 PCs (P = .00056). Differences in the prevalence of PVs by age at diagnosis were significant: 14.7% with 2 PCs at an age < 50 years, 15.8% with 1 PC at an age < 50 years, and 12.0% with all PCs at an age ≥ 50 years (P = 2.07E-05). PVs by the age at second cancer diagnosis were also significant: 14.7% at an age < 50 years, 13.9% at an age of 50 to 69 years, and 11.4% at an age ≥ 70 years (P for trend = .005).

Conclusions: Among patients with MPCs, there is a high frequency of germline PVs, with a higher frequency found among patients with a higher number of PCs. These findings suggest that genetic testing should be considered even among patients who are older at the diagnosis of an additional primary malignancy.
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http://dx.doi.org/10.1002/cncr.34056DOI Listing
March 2022

Closing the gap: Systematic integration of multiplexed functional data resolves variants of uncertain significance in BRCA1, TP53, and PTEN.

Am J Hum Genet 2021 12 17;108(12):2248-2258. Epub 2021 Nov 17.

Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA; Brotman Baty Institute for Precision Medicine, Seattle, WA 98195, USA. Electronic address:

Clinical interpretation of missense variants is challenging because the majority identified by genetic testing are rare and their functional effects are unknown. Consequently, most variants are of uncertain significance and cannot be used for clinical diagnosis or management. Although not much can be done to ameliorate variant rarity, multiplexed assays of variant effect (MAVEs), where thousands of single-nucleotide variant effects are simultaneously measured experimentally, provide functional evidence that can help resolve variants of unknown significance (VUSs). However, a rigorous assessment of the clinical value of multiplexed functional data for variant interpretation is lacking. Thus, we systematically combined previously published BRCA1, TP53, and PTEN multiplexed functional data with phenotype and family history data for 324 VUSs identified by a single diagnostic testing laboratory. We curated 49,281 variant functional scores from MAVEs for these three genes and integrated four different TP53 multiplexed functional datasets into a single functional prediction for each variant by using machine learning. We then determined the strength of evidence provided by each multiplexed functional dataset and reevaluated 324 VUSs. Multiplexed functional data were effective in driving variant reclassification when combined with clinical data, eliminating 49% of VUSs for BRCA1, 69% for TP53, and 15% for PTEN. Thus, multiplexed functional data, which are being generated for numerous genes, are poised to have a major impact on clinical variant interpretation.
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http://dx.doi.org/10.1016/j.ajhg.2021.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8715144PMC
December 2021

Classification of the canonical splice alteration c.934-2A > G is likely benign based on RNA and clinical data.

Cold Spring Harb Mol Case Stud 2022 01 10;8(1). Epub 2022 Jan 10.

Ambry Genetics, One Enterprise, Aliso Viejo, California 92656, USA.

-associated polyposis (MAP) is an autosomal recessive disorder characterized by the development of multiple adenomatous colonic polyps and an increased lifetime risk of colorectal cancer. Germline biallelic pathogenic variants in are responsible for MAP. The c.934-2A > G (NM_001128425.1) variant, which is also known as c.850-2A > G for NM_001048174.2, has been identified in our laboratory in more than 800 patients, including homozygous and compound heterozygote carriers. The variant was initially classified as a variant of uncertain significance (VUS) because of lack of a MAP phenotype in biallelic carriers. In two unrelated female patients who were heterozygous carriers of this variant, further testing by RNA sequencing identified an aberrant transcript with a deletion of 9 nt at the start of exon 11 ( r.934_942del9). This event is predicted to lead to an in-frame loss of three amino acids in a noncritical domain of the protein. This was the only splice defect identified in these patients that was not present in the controls, and the aberrant transcript is derived exclusively from the variant allele, strongly supporting the cause of this splice defect as being the intronic variant, c.934-2A > G. The splicing analysis demonstrating a small in-frame skipping of three amino acids in a noncritical domain, along with the absence of a MAP phenotype in our internal cohort of biallelic carriers, provides evidence that the variant is likely benign and not of clinical significance.
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http://dx.doi.org/10.1101/mcs.a006152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8744492PMC
January 2022

Strong functional data for pathogenicity or neutrality classify BRCA2 DNA-binding-domain variants of uncertain significance.

Am J Hum Genet 2021 03 19;108(3):458-468. Epub 2021 Feb 19.

Mayo Clinic, Rochester, MN 55905, USA. Electronic address:

Determination of the clinical relevance of rare germline variants of uncertain significance (VUSs) in the BRCA2 cancer predisposition gene remains a challenge as a result of limited availability of data for use in classification models. However, laboratory-based functional data derived from validated functional assays of known sensitivity and specificity may influence the interpretation of VUSs. We evaluated 252 missense VUSs from the BRCA2 DNA-binding domain by using a homology-directed DNA repair (HDR) assay and identified 90 as non-functional and 162 as functional. The functional assay results were integrated with other available data sources into an ACMG/AMP rules-based classification framework used by a hereditary cancer testing laboratory. Of the 186 missense variants observed by the testing laboratory, 154 were classified as VUSs without functional data. However, after applying protein functional data, 86% (132/154) of the VUSs were reclassified as either likely pathogenic/pathogenic (39/132) or likely benign/benign (93/132), which impacted testing results for 1,900 individuals. These results indicate that validated functional assay data can have a substantial impact on VUS classification and associated clinical management for many individuals with inherited alterations in BRCA2.
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http://dx.doi.org/10.1016/j.ajhg.2021.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8008494PMC
March 2021

RNA-Seq Analysis Is a Useful Tool in Variant Classification.

JCO Precis Oncol 2020 Nov;4:1226-1227

Rachid Karam, MD, PhD; Holly LaDuca, MS; Marcy E. Richardson, PhD; and Tina Pesaran, MS, Ambry Genetics, Aliso Viejo, CA and Elizabeth Chao, MD, Ambry Genetics, Aliso Viejo, CA, and University of California, Irvine, CA.

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http://dx.doi.org/10.1200/PO.20.00310DOI Listing
November 2020

Integration of functional assay data results provides strong evidence for classification of hundreds of BRCA1 variants of uncertain significance.

Genet Med 2021 02 22;23(2):306-315. Epub 2020 Oct 22.

Cancer Epidemiology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA.

Purpose: BRCA1 pathogenic variant heterozygotes are at a substantially increased risk for breast and ovarian cancer. The widespread uptake of testing has led to a significant increase in the detection of missense variants in BRCA1, the vast majority of which are variants of uncertain clinical significance (VUS), posing a challenge to genetic counseling. Here, we harness a wealth of functional data for thousands of variants to aid in variant classification.

Methods: We have collected, curated, and harmonized functional data for 2701 missense variants representing 24.5% of possible missense variants in BRCA1. Results were harmonized across studies by converting data into binary categorical variables (functional impact versus no functional impact). Using a panel of reference variants we identified a subset of assays with high sensitivity and specificity (≥80%) and apply the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) variant interpretation guidelines to assign evidence criteria for classification.

Results: Integration of data from validated assays provided ACMG/AMP evidence criteria in favor of pathogenicity for 297 variants or against pathogenicity for 2058 representing 96.2% of current VUS functionally assessed. We also explore discordant results and identify limitations in the approach.

Conclusion: High quality functional data are available for BRCA1 missense variants and provide evidence for classification of 2355 VUS according to their pathogenicity.
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http://dx.doi.org/10.1038/s41436-020-00991-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862071PMC
February 2021

Prevalence of pathogenic variants in DNA damage response and repair genes in patients undergoing cancer risk assessment and reporting a personal history of early-onset renal cancer.

Sci Rep 2020 08 11;10(1):13518. Epub 2020 Aug 11.

Cancer Prevention and Control Program, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA, 19111-2497, USA.

Pathogenic variants (PVs) in multiple genes are known to increase the risk of early-onset renal cancer (eoRC). However, many eoRC patients lack PVs in RC-specific genes; thus, their genetic risk remains undefined. Here, we determine if PVs in DNA damage response and repair (DDRR) genes are enriched in eoRC patients undergoing cancer risk assessment. Retrospective review of de-identified results from 844 eoRC patients, undergoing testing with a multi-gene panel, for a variety of indications, by Ambry Genetics. PVs in cancer-risk genes were identified in 12.8% of patients-with 3.7% in RC-specific, and 8.55% in DDRR genes. DDRR gene PVs were most commonly identified in CHEK2, BRCA1, BRCA2, and ATM. Among the 2.1% of patients with a BRCA1 or BRCA2 PV, < 50% reported a personal history of hereditary breast or ovarian-associated cancer. No association between age of RC diagnosis and prevalence of PVs in RC-specific or DDRR genes was observed. Additionally, 57.9% patients reported at least one additional cancer; breast cancer being the most common (40.1% of females, 2.5% of males). Multi-gene testing including DDRR genes may provide a more comprehensive risk assessment in eoRC patients. Further validation is needed to characterize the association with eoRC.
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http://dx.doi.org/10.1038/s41598-020-70449-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7419503PMC
August 2020

Curated multiple sequence alignment for the Adenomatous Polyposis Coli (APC) gene and accuracy of in silico pathogenicity predictions.

PLoS One 2020 4;15(8):e0233673. Epub 2020 Aug 4.

Department of Medicine, University of Vermont, Larner College of Medicine, Burlington, Vermont, United States of America.

Computational algorithms are often used to assess pathogenicity of Variants of Uncertain Significance (VUS) that are found in disease-associated genes. Most computational methods include analysis of protein multiple sequence alignments (PMSA), assessing interspecies variation. Careful validation of PMSA-based methods has been done for relatively few genes, partially because creation of curated PMSAs is labor-intensive. We assessed how PMSA-based computational tools predict the effects of the missense changes in the APC gene, in which pathogenic variants cause Familial Adenomatous Polyposis. Most Pathogenic or Likely Pathogenic APC variants are protein-truncating changes. However, public databases now contain thousands of variants reported as missense. We created a curated APC PMSA that contained >3 substitutions/site, which is large enough for statistically robust in silico analysis. The creation of the PMSA was not easily automated, requiring significant querying and computational analysis of protein and genome sequences. Of 1924 missense APC variants in the NCBI ClinVar database, 1800 (93.5%) are reported as VUS. All but two missense variants listed as P/LP occur at canonical splice or Exonic Splice Enhancer sites. Pathogenicity predictions by five computational tools (Align-GVGD, SIFT, PolyPhen2, MAPP, REVEL) differed widely in their predictions of Pathogenic/Likely Pathogenic (range 17.5-75.0%) and Benign/Likely Benign (range 25.0-82.5%) for APC missense variants in ClinVar. When applied to 21 missense variants reported in ClinVar and securely classified as Benign, the five methods ranged in accuracy from 76.2-100%. Computational PMSA-based methods can be an excellent classifier for variants of some hereditary cancer genes. However, there may be characteristics of the APC gene and protein that confound the results of in silico algorithms. A systematic study of these features could greatly improve the automation of alignment-based techniques and the use of predictive algorithms in hereditary cancer genes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0233673PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402488PMC
October 2020

Rare Missense Alleles Confer Risk for Ovarian and Breast Cancer.

Cancer Res 2020 02 10;80(4):857-867. Epub 2019 Dec 10.

James Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio.

Germline loss-of-function mutations in BRCA1 interacting protein C-terminal helicase 1 (BRIP1) are associated with ovarian carcinoma and may also contribute to breast cancer risk, particularly among patients who develop disease at an early age. Normal BRIP1 activity is required for DNA interstrand cross-link (ICL) repair and is thus central to the maintenance of genome stability. Although pathogenic mutations have been identified in , genetic testing more often reveals missense variants, for which the impact on molecular function and subsequent roles in cancer risk are uncertain. Next-generation sequencing of germline DNA in 2,160 early-onset breast cancer and 1,199 patients with ovarian cancer revealed nearly 2% of patients carry a very rare missense variant (minor allele frequency < 0.0001) in . This is 3-fold higher than the frequency of all rare BRIP1 missense alleles reported in more than 60,000 individuals of the general population ( < 0.0001, test). Using CRISPR-Cas9 gene editing technology and rescue assays, we functionally characterized 20 of these missense variants, focusing on the altered protein's ability to repair ICL damage. A total of 75% of the characterized variants rendered the protein hypomorph or null. In a clinical cohort of >117,000 patients with breast and ovarian cancer who underwent panel testing, the combined OR associated with BRIP1 hypomorph or null missense carriers compared with the general population was 2.30 (95% confidence interval, 1.60-3.30; < 0.0001). These findings suggest that novel missense variants within the helicase domain of BRIP1 may confer risk for both breast and ovarian cancer and highlight the importance of functional testing for additional variants. SIGNIFICANCE: Functional characterization of rare variants of uncertain significance in revealed that 75% demonstrate loss-of-function activity, suggesting rare missense alleles in confer risk for both breast and ovarian cancer.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-1991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8722358PMC
February 2020

Assessment of Diagnostic Outcomes of RNA Genetic Testing for Hereditary Cancer.

JAMA Netw Open 2019 10 2;2(10):e1913900. Epub 2019 Oct 2.

Ambry Genetics, Aliso Viejo, California.

Importance: Performing DNA genetic testing (DGT) for hereditary cancer genes is now a well-accepted clinical practice; however, the interpretation of DNA variation remains a challenge for laboratories and clinicians. Adding RNA genetic testing (RGT) enhances DGT by clarifying the clinical actionability of hereditary cancer gene variants, thus improving clinicians' ability to accurately apply strategies for cancer risk reduction and treatment.

Objective: To evaluate whether RGT is associated with improvement in the diagnostic outcome of DGT and in the delivery of personalized cancer risk management for patients with hereditary cancer predisposition.

Design, Setting, And Participants: Diagnostic study in which patients and/or families with inconclusive variants detected by DGT in genes associated with hereditary breast and ovarian cancer, Lynch syndrome, and hereditary diffuse gastric cancer sent blood samples for RGT from March 2016 to April 2018. Clinicians who ordered genetic testing and received a reclassification report for these variants were surveyed to assess whether RGT-related variant reclassifications changed clinical management of these patients. To quantify the potential number of tested individuals who could benefit from RGT, a cohort of 307 812 patients who underwent DGT for hereditary cancer were separately queried to identify variants predicted to affect splicing. Data analysis was conducted from March 2016 and September 2018.

Main Outcomes And Measures: Variant reclassification outcomes following RGT, clinical management changes associated with RGT-related variant reclassifications, and the proportion of patients who would likely be affected by a concurrent DGT and RGT multigene panel testing approach.

Results: In total, 93 if 909 eligible families (10.2%) submitted samples for RGT. Evidence from RGT clarified the interpretation of 49 of 56 inconclusive cases (88%) studied; 26 (47%) were reclassified as clinically actionable and 23 (41%) were clarified as benign. Variant reclassifications based on RGT results changed clinical management recommendations for 8 of 18 patients (44%) and 14 of 18 families (78%), based on responses from 18 of 45 clinicians (40%) surveyed. A total of 7265 of 307 812 patients who underwent DGT had likely pathogenic variants or variants of uncertain significance potentially affecting splicing, indicating that approximately 1 in 43 individuals could benefit from RGT.

Conclusions And Relevance: In this diagnostic study, conducting RNA testing resolved a substantial proportion of variants of uncertain significance in a cohort of individuals previously tested for cancer predisposition by DGT. Performing RGT might change the diagnostic outcome of at least 1 in 43 patients if performed in all individuals undergoing genetic evaluation for hereditary cancer.
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http://dx.doi.org/10.1001/jamanetworkopen.2019.13900DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6820040PMC
October 2019

Correction: DNA breakpoint assay reveals a majority of gross duplications occur in tandem reducing VUS classifications in breast cancer predisposition genes.

Genet Med 2019 Jul;21(7):1669

Department of Clinical Genomics, Ambry Genetics, 15 Argonaut Drive, Aliso Viejo, California 92656, USA.

The PDF and HTML versions of the article have been updated to include the Creative Commons Attribution 4.0 International License information.
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http://dx.doi.org/10.1038/s41436-018-0276-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7609259PMC
July 2019

DNA breakpoint assay reveals a majority of gross duplications occur in tandem reducing VUS classifications in breast cancer predisposition genes.

Genet Med 2019 03 28;21(3):683-693. Epub 2018 Jul 28.

Department of Clinical Genomics, Ambry Genetics, 15 Argonaut Drive, Aliso Viejo, California, 92656, USA.

Purpose: Gross duplications are ambiguous in terms of clinical interpretation due to the limitations of the detection methods that cannot infer their context, namely, whether they occur in tandem or are duplicated and inserted elsewhere in the genome. We investigated the proportion of gross duplications occurring in tandem in breast cancer predisposition genes with the intent of informing their classifications.

Methods: The DNA breakpoint assay (DBA) is a custom, paired-end, next-generation sequencing (NGS) method designed to capture and detect deep-intronic DNA breakpoints in gross duplications in BRCA1, BRCA2, ATM, CDH1, PALB2, and CHEK2.

Results: DBA allowed us to ascertain breakpoints for 44 unique gross duplications from 147 probands. We determined that the duplications occurred in tandem in 114 (78%) carriers from this cohort, while the remainder have unknown tandem status. Among the tandem gross duplications that were eligible for reclassification, 95% of them were upgraded to pathogenic.

Conclusion: DBA is a novel, high-throughput, NGS-based method that informs the tandem status, and thereby the classification of, gross duplications. This method revealed that most gross duplications in the investigated genes occurred in tandem and resulted in a pathogenic classification, which helps to secure the necessary treatment options for their carriers.
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http://dx.doi.org/10.1038/s41436-018-0092-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6752314PMC
March 2019

Efforts Toward Consensus Variant Interpretation by Commercial Laboratories.

J Clin Oncol 2017 04 30;35(11):1261-1262. Epub 2017 Jan 30.

Jill S. Dolinsky, Ambry Genetics, Aliso Viejo, CA; Kathleen S. Hruska, GeneDx, Gaithersburg, MD; Tina Pesaran and Marcy E. Richardson, Ambry Genetics, Aliso Viejo, CA; Rachel T. Klein and Benjamin D. Solomon, GeneDx, Gaithersburg, MD; and Chia-Ling Gau, Ambry Genetics, Aliso Viejo, CA.

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http://dx.doi.org/10.1200/JCO.2016.71.2505DOI Listing
April 2017

The Homeobox Transcription Factor RHOX10 Drives Mouse Spermatogonial Stem Cell Establishment.

Cell Rep 2016 09;17(1):149-164

School of Medicine, Department of Reproductive Medicine, University of California at San Diego, La Jolla, CA 92093, USA; Institute of Genomic Medicine, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address:

The developmental origins of most adult stem cells are poorly understood. Here, we report the identification of a transcription factor-RHOX10-critical for the initial establishment of spermatogonial stem cells (SSCs). Conditional loss of the entire 33-gene X-linked homeobox gene cluster that includes Rhox10 causes progressive spermatogenic decline, a phenotype indistinguishable from that caused by loss of only Rhox10. We demonstrate that this phenotype results from dramatically reduced SSC generation. By using a battery of approaches, including single-cell-RNA sequencing (scRNA-seq) analysis, we show that Rhox10 drives SSC generation by promoting pro-spermatogonia differentiation. Rhox10 also regulates batteries of migration genes and promotes the migration of pro-spermatogonia into the SSC niche. The identification of an X-linked homeobox gene that drives the initial generation of SSCs has implications for the evolution of X-linked gene clusters and sheds light on regulatory mechanisms influencing adult stem cell generation in general.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5063083PMC
http://dx.doi.org/10.1016/j.celrep.2016.08.090DOI Listing
September 2016

Hormone-induced and DNA demethylation-induced relief of a tissue-specific and developmentally regulated block in transcriptional elongation.

J Biol Chem 2014 Dec 20;289(51):35087-101. Epub 2014 Oct 20.

From the Department of Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, the Department of Reproductive Medicine, University of California at San Diego, La Jolla, California 92037, the Institute of Genomic Medicine, University of California at San Diego, La Jolla, California 92093, and

Genome-wide studies have revealed that genes commonly have a high density of RNA polymerase II just downstream of the transcription start site. This has raised the possibility that genes are commonly regulated by transcriptional elongation, but this remains largely untested in vivo, particularly in vertebrates. Here, we show that the proximal promoter from the Rhox5 homeobox gene recruits polymerase II and begins elongating in all tissues and cell lines that we tested, but it only completes elongation in a tissue-specific and developmentally regulated manner. Relief of the elongation block is associated with recruitment of the elongation factor P-TEFb, the co-activator GRIP1, the chromatin remodeling factor BRG1, and specific histone modifications. We provide evidence that two mechanisms relieve the elongation block at the proximal promoter: demethylation and recruitment of androgen receptor. Together, our findings support a model in which promoter proximal pausing helps confer tissue-specific and developmental gene expression through a mechanism regulated by DNA demethylation-dependent nuclear hormone receptor recruitment.
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http://dx.doi.org/10.1074/jbc.M114.615435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4271199PMC
December 2014

Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins.

Nat Biotechnol 2013 Dec 9;31(12):1137-42. Epub 2013 Oct 9.

1] Department of Pathology and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA. [2] Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA. [3] Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, Massachusetts, USA. [4].

Genome-wide studies have defined cell type-specific patterns of DNA methylation that are important for regulating gene expression in both normal development and disease. However, determining the functional significance of specific methylation events remains challenging, owing to the lack of methods for removing such modifications in a targeted manner. Here we describe an approach for efficient targeted demethylation of specific CpGs in human cells using fusions of engineered transcription activator-like effector (TALE) repeat arrays and the TET1 hydroxylase catalytic domain. Using these TALE-TET1 fusions, we demonstrate that modification of critical methylated promoter CpG positions can lead to substantial increases in the expression of endogenous human genes. Our results delineate a strategy for understanding the functional significance of specific CpG methylation marks in the context of endogenous gene loci and validate programmable DNA demethylation reagents with potential utility for research and therapeutic applications.
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http://dx.doi.org/10.1038/nbt.2726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3858462PMC
December 2013

Epigenetic regulation of the RHOX homeobox gene cluster and its association with human male infertility.

Hum Mol Genet 2014 Jan 13;23(1):12-23. Epub 2013 Aug 13.

School of Medicine, Department of Reproductive Medicine, University of California, San Diego, 9500 Gilman Drive, MC 0695, La Jolla, CA 92093-0864, USA.

The X-linked RHOX cluster encodes a set of homeobox genes that are selectively expressed in the reproductive tract. Members of the RHOX cluster regulate target genes important for spermatogenesis promote male fertility in mice. Studies show that demethylating agents strongly upregulate the expression of mouse Rhox genes, suggesting that they are regulated by DNA methylation. However, whether this extends to human RHOX genes, whether DNA methylation directly regulates RHOX gene transcription and how this relates to human male infertility are unknown. To address these issues, we first defined the promoter regions of human RHOX genes and performed gain- and loss-of-function experiments to determine whether human RHOX gene transcription is regulated by DNA methylation. Our results indicated that DNA methylation is necessary and sufficient to silence human RHOX gene expression. To determine whether RHOX cluster methylation associates with male infertility, we evaluated the methylation status of RHOX genes in sperm from a large cohort of infertility patients. Linear regression analysis revealed a strong association between RHOX gene cluster hypermethylation and three independent types of semen abnormalities. Hypermethylation was restricted specifically to the RHOX cluster; we did not observe it in genes immediately adjacent to it on the X chromosome. Our results strongly suggest that human RHOX homeobox genes are under an epigenetic control mechanism that is aberrantly regulated in infertility patients. We propose that hypermethylation of the RHOX gene cluster serves as a marker for idiopathic infertility and that it is a candidate to exert a causal role in male infertility.
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http://dx.doi.org/10.1093/hmg/ddt392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3857941PMC
January 2014
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