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This study evaluates specific BRCA1 and BRCA2 intronic splicing variants by minigene assays to clarify splice-site consequences. Four candidate variants were selected; exon skipping and intron retention were observed for selected constructs. Aberrant splicing patterns enabled reclassification, including LP for BRCA1:c.80 + 3_80 + 5del and VUS for BRCA1:c.548-15G >A and BRCA2:c.8755–19 A >G.",{"@graph":69,"@context":123},[70,84,106],{"@type":71,"itemListElement":72},"BreadcrumbList",[73,77,79,82],{"item":74,"name":75,"@type":76,"position":8},"https://docshare.wps.com","Home","ListItem",{"item":78,"name":9,"@type":76,"position":14},"https://docshare.wps.com/document/",{"item":80,"name":35,"@type":76,"position":81},"https://docshare.wps.com/document/healthcare/",3,{"item":83,"name":65,"@type":76,"position":19},"https://docshare.wps.com/document/functional-analysis-of-brca1-and-brca2-splicing-variants-using-a-minigene-assay/442644/",{"url":83,"name":65,"@type":85,"image":86,"author":91,"headline":65,"publisher":94,"fileFormat":97,"inLanguage":63,"description":67,"dateModified":98,"datePublished":99,"encodingFormat":97,"isAccessibleForFree":100,"interactionStatistic":101},"DigitalDocument",{"url":87,"@type":88,"width":89,"height":90},"https://docshare.wps.com/thumbnails/functional-analysis-of-brca1-and-brca2-splicing-variants-using-a-minigene-assay/442644.png","ImageObject",300,407,{"name":92,"@type":93},"A glass of water","Person",{"url":74,"name":95,"@type":96},"DocShare","Organization","application/pdf","2026-10-30","2026-09-29",true,{"@type":102,"interactionType":103,"userInteractionCount":105},"InteractionCounter",{"@type":104},"ViewAction",14,{"@type":107,"mainEntity":108},"FAQPage",[109,115,119],{"name":110,"@type":111,"acceptedAnswer":112},"Why are intronic BRCA1 and BRCA2 variants often classified as VUS?","Question",{"text":113,"@type":114},"Intronic variants may cause aberrant splicing events, but their functional impact is not always predictable or confirmed. Without clear experimental evidence, they remain classified as variant uncertain significance (VUS).","Answer",{"name":116,"@type":111,"acceptedAnswer":117},"Which BRCA1/BRCA2 variants were selected for functional testing?",{"text":118,"@type":114},"The study selected BRCA1:c.80 + 3_80 + 5del, BRCA1:c.548-15G >A, BRCA2:c.8755–19 A >G, and BRCA2:c.317–10 A >G for minigene assay evaluation.",{"name":120,"@type":111,"acceptedAnswer":121},"What splicing outcomes and reclassification results were reported?",{"text":122,"@type":114},"Minigene assays showed exon skipping for BRCA1:c.80 + 3_80 + 5del and intron retention for BRCA1:c.548-15G >A and BRCA2:c.8755–19 A >G. Three of four variants showed aberrant splicing, leading to reclassification: BRCA1:c.80 + 3_80 + 5del as LP, while BRCA1:c.548-15G >A and BRCA2:c.8755–19 A >G were classified as VUS.","https://schema.org",{"og:url":83,"og:type":125,"og:title":65,"og:site_name":95,"og:description":67},"article",{"robots":127,"canonical":83},"index,follow",{"doc_id":129,"site_id":62},442644,1790741748,{"code":4,"msg":5,"data":132},{"doc_id":129,"user_id":133,"nickname":92,"user_avatar":134,"doc_module":4,"category_id":34,"category_name":35,"doc_title":65,"doc_description":67,"doc_content":135,"file_id":136,"file_url":137,"file_type":138,"file_size":139,"view_count":105,"is_deleted":4,"is_public":8,"is_downloadable":8,"audit_status":8,"page_count":52,"language":140,"language_code":63,"site_id":62,"html_lang":63,"table_of_contents":141,"faqs":142,"seo_title":143,"seo_description":67,"update_tm":144,"read_time":145,"preview_page_count":4},962090760832,"https://ap-avatar.wpscdn.com/davatar_155a257f0dc6eb9ab79c44ca47cae57d","Yim etal. Human Genomics (2026) 20:1 [https://doi.org/10.1186/s40246-025-00853-3](https://doi.org/10.1186/s40246-025-00853-3)  \nHuman Genomics  \nRESEARCH Open Access  \nFunctional analysis of BRCA1  \nand BRCA2  \nsplicing variants using a minigene assay  \nHaraYim,1,2, Seonhoo Youn,1,2, Seung Won Chae 1,2, Yeseul Kim 1, Joowon Jang 1, Sung Im Cho 1, Jee-Soo Lee 1* and Moon-woo Seong 1,2*  \nAbstract  \nBackground BRCA1 and BRCA2, known as tumor suppressor genes, have been shown to increase the risk of developing breast and ovarian cancer. Intronic variants that can result in aberrant splicing events are classified as Variant uncertain significance until the functional impact is clearly predicted or confirmed. Thus, the purpose of this study is to assist in the interpretation of splicing variants and to reclassify the clinical significance of non-coding region variants that are classified as VUS.  \nResults The variants BRCA1:c.80 + 3_80 + 5del, BRCA1:c. 548-15G >A, BRCA2:c. 8755–19 A >G, and  \nBRCA2:c. 317–10 A >G were ultimately chosen. Through the minigene assay, we can observe exon skipping in BRCA1:c.80 + 3_80 + 5del, intron retention in BRCA1:c. 548-15G >A and BRCA2:c. 8755–19 A >G. This study performed aminigene assay using the remaining samples from the patients tested at Seoul National University Hospital. Wild-type and mutant type minigene constructs were designed respectively for each variant sample.  \nConclusion Finally aberrant splicing patterns were found in three of the four variants: BRCA1:c.80 + 3_80 + 5del, BRCA1:c. 548-15G >A, BRCA2:c. 8755–19 A >G that were previously classified as VUS through experimental analysis. Asa result, we reclassify BRCA1;c.80 + 3_80 + 5del as LP and we classify BRCA1;c. 548-15G >A and BRCA2;c.8755–19 A >Gas VUS.  \nKeywords Minigene assay, BRCA gene, Intronic variant, Splicing effect, Pathogenicity, Clinical significance  \nBackground  \nThe tumor suppressor genes BRCA1 and BRCA2 are associated with a significantly increased risk of breast cancer and ovarian cancer. Individuals carrying a BRCA mutation have an estimated 60–80% lifetime risk of developing the disease [1]. The germline mutations in  \n*Correspondence: Jee-Soo Lee [leciel85@snu.ac.kr](leciel85@snu.ac.kr)[ ](leciel85@snu.ac.kr)Moon-woo Seong [mwseong@snu.ac.kr](mwseong@snu.ac.kr)  \n1Department of Laboratory Medicine, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Korea  \n2Cancer Research Institute, Seoul National University, Seoul, Korea  \nBRCA1 and BRCA2 have a strong effect on cancer risk and are inherited in an autosomal dominant pattern [2].  \nBreast cancer, which is ranked as the number one cancer in women around the world, has a high incidence rate in most of age groups especially between ages 20–50 [3] and is noteworthy because it has a family history and heredity. Among the various factors that cause breast cancer like radiation, age and hormonal, hereditary breast cancer accounts for about 5–10%[4], but when a mutation occurs in the BRCA gene, there is a 72% and 69% in BRCA1 or BRCA2 mutation carrier chance that women will develop breast cancer or ovarian cancer [5, 6] and men will develop prostate cancer [7]. The cumulative risk of breast cancer by age 80 for BRCA1 gene carriers is  \n© The Author(s) 2025. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line t","cbCaildKCgvMeyDa","https://ap.wps.com/l/cbCaildKCgvMeyDa","pdf",2792607,"English","# Abstract\n# Background\n# Results\n# Conclusion\n# Keywords","[{\"question\":\"Why are intronic BRCA1 and BRCA2 variants often classified as VUS?\",\"answer\":\"Intronic variants may cause aberrant splicing events, but their functional impact is not always predictable or confirmed. Without clear experimental evidence, they remain classified as variant uncertain significance (VUS).\"},{\"question\":\"Which BRCA1/BRCA2 variants were selected for functional testing?\",\"answer\":\"The study selected BRCA1:c.80 + 3_80 + 5del, BRCA1:c.548-15G \\u003eA, BRCA2:c.8755–19 A \\u003eG, and BRCA2:c.317–10 A \\u003eG for minigene assay evaluation.\"},{\"question\":\"What splicing outcomes and reclassification results were reported?\",\"answer\":\"Minigene assays showed exon skipping for BRCA1:c.80 + 3_80 + 5del and intron retention for BRCA1:c.548-15G \\u003eA and BRCA2:c.8755–19 A \\u003eG. Three of four variants showed aberrant splicing, leading to reclassification: BRCA1:c.80 + 3_80 + 5del as LP, while BRCA1:c.548-15G \\u003eA and BRCA2:c.8755–19 A \\u003eG were classified as VUS.\"}]","Functional analysis of BRCA1 and BRCA2 splicing variants using a minigene assay | PDF",1790701077,25]