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Variants affecting the critical intronic +1 and +2 GT nucleotides of the 5\u2019 splice sites (5'ss) are generally strong indicators of pathogenicity. However, some +2\u2009T variants create functional noncanonical 5'ss that generate wildtype transcripts, hampering accurate variant interpretation and genetic counseling. We previously showed that variants\n                    <jats:italic>PALB2<\/jats:italic>\n                    c.108+2T &gt; C and\n                    <jats:italic>ATM<\/jats:italic>\n                    c.1898+2T &gt; G generated significant levels of full\u2010length (FL) transcripts by creating functional atypical GC and GG donor sites, respectively. In this study, we aimed to investigate the splicing impact of +2T variants in the breast cancer susceptibility genes\n                    <jats:italic>ATM<\/jats:italic>\n                    ,\n                    <jats:italic>BRCA1<\/jats:italic>\n                    , and\n                    <jats:italic>PALB2<\/jats:italic>\n                    . For this purpose, five minigenes encompassing 29 exons of\n                    <jats:italic>ATM<\/jats:italic>\n                    ,\n                    <jats:italic>BRCA1<\/jats:italic>\n                    , and\n                    <jats:italic>PALB2<\/jats:italic>\n                    were employed. A total of 30 +2T &gt; C\/G\/A variants were introduced into these constructs by site\u2010directed mutagenesis and analyzed in MCF\u20107 cells. Four +2T &gt; C variants (\n                    <jats:italic>ATM<\/jats:italic>\n                    c.6347+2T &gt; C,\n                    <jats:italic>BRCA1<\/jats:italic>\n                    c.5193+2T &gt; C and c.5277+2T &gt; C, and\n                    <jats:italic>PALB2<\/jats:italic>\n                    c.2748+2T &gt; C) and\n                    <jats:italic>ATM<\/jats:italic>\n                    variants c.6347+2T &gt; A\/G produced FL\u2010transcripts (4%\u201381% of the overall expression). All +2T &gt; C leaky variants conserved a central core of 6 nucleotides (AGgcaa). Variants were assessed according to the ClinGen specifications of the American College of Medical Genetics and Genomics\/Association for Molecular Pathology (ACMG\/AMP) interpretation guidelines. Two variants (\n                    <jats:italic>ATM<\/jats:italic>\n                    c.6347+2T &gt; C and\n                    <jats:italic>BRCA1<\/jats:italic>\n                    c.5193+2T &gt; C) were classified as likely benign, consistent with predictions based on their respective ACMG\/AMP\u2010based gene specifications. Conversely, two variants (\n                    <jats:italic>ATM<\/jats:italic>\n                    c.6347+2T &gt; G and\n                    <jats:italic>BRCA1<\/jats:italic>\n                    c.4675+2T &gt; C), initially predicted as likely pathogenic, were reclassified as variant of uncertain significance (VUS). In conclusion, a significant proportion of +2T variants can create functional noncanonical 5'ss, resulting in the production of FL\u2010transcripts that may preserve gene function. Variant\u2010splicing assays provide essential data for accurate clinical classification and for the development of effective clinical management strategies for patients and their families. \u00a9 2025 The Author(s).\n                    <jats:italic>The Journal of Pathology<\/jats:italic>\n                    published by John Wiley &amp; Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.\n                  <\/jats:p>","DOI":"10.1002\/path.6497","type":"journal-article","created":{"date-parts":[[2025,11,13]],"date-time":"2025-11-13T11:23:43Z","timestamp":1763033023000},"page":"150-163","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Generation of functional noncanonical donor splice sites by +\n                    <scp>2T<\/scp>\n                    variants in breast cancer susceptibility genes: impact on clinical interpretation"],"prefix":"10.1002","volume":"268","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7838-0838","authenticated-orcid":false,"given":"In\u00e9s","family":"Llinares\u2010Burguet","sequence":"first","affiliation":[{"name":"Splicing and genetic susceptibility to cancer. Instituto de Biomedicina y Gen\u00e9tica Molecular de Valladolid (IBGM). Consejo Superior de Investigaciones Cient\u00edficas \u2010 Universidad de Valladolid (CSIC\u2010UVa)  Valladolid Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lara","family":"Sanoguera\u2010Miralles","sequence":"additional","affiliation":[{"name":"Splicing and genetic susceptibility to cancer. Instituto de Biomedicina y Gen\u00e9tica Molecular de Valladolid (IBGM). Consejo Superior de Investigaciones Cient\u00edficas \u2010 Universidad de Valladolid (CSIC\u2010UVa)  Valladolid Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elena","family":"Bueno\u2010Mart\u00ednez","sequence":"additional","affiliation":[{"name":"Splicing and genetic susceptibility to cancer. Instituto de Biomedicina y Gen\u00e9tica Molecular de Valladolid (IBGM). Consejo Superior de Investigaciones Cient\u00edficas \u2010 Universidad de Valladolid (CSIC\u2010UVa)  Valladolid Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alicia","family":"Garc\u00eda\u2010\u00c1lvarez","sequence":"additional","affiliation":[{"name":"Splicing and genetic susceptibility to cancer. Instituto de Biomedicina y Gen\u00e9tica Molecular de Valladolid (IBGM). Consejo Superior de Investigaciones Cient\u00edficas \u2010 Universidad de Valladolid (CSIC\u2010UVa)  Valladolid Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alberto","family":"Valenzuela\u2010Palomo","sequence":"additional","affiliation":[{"name":"Splicing and genetic susceptibility to cancer. Instituto de Biomedicina y Gen\u00e9tica Molecular de Valladolid (IBGM). Consejo Superior de Investigaciones Cient\u00edficas \u2010 Universidad de Valladolid (CSIC\u2010UVa)  Valladolid Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Pedro","family":"P\u00e9rez\u2010Segura","sequence":"additional","affiliation":[{"name":"Molecular Oncology Laboratory, Hospital Cl\u00ednico San Carlos, IdISSC (Instituto de Investigaci\u00f3n Sanitaria del Hospital Cl\u00ednico San Carlos)  Madrid Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Miguel","family":"de la Hoya","sequence":"additional","affiliation":[{"name":"Molecular Oncology Laboratory, Hospital Cl\u00ednico San Carlos, IdISSC (Instituto de Investigaci\u00f3n Sanitaria del Hospital Cl\u00ednico San Carlos)  Madrid Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9682-5589","authenticated-orcid":false,"given":"Eladio A","family":"Velasco\u2010Sampedro","sequence":"additional","affiliation":[{"name":"Splicing and genetic susceptibility to cancer. 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