Molecular Biology Reports

, Volume 47, Issue 2, pp 1513–1520 | Cite as

Spliceogenic analysis of BRCA1 c.439T>C (rs794727800) variant by High Resolution Melting Analysis

  • Angelo MinucciEmail author
  • Giorgia Mazzuccato
  • Marco D’Indinosante
  • Lucia Di Nardo
  • Paola Concolino
  • Maria De Bonis
  • Andrea Urbani
  • Giovanni Scambia
  • Anna Fagotti
  • Ettore Capoluongo
Short Communication


Correct classification of genomic variants causing potentially aberrant splicing is of utmost importance for patient management, especially in clinically actionable genes such as BRCA1/2. In this article, we report molecular evaluation of the BRCA1 c.439T>C (rs794727800, p.Leu147=) variant based on RNA of a patient suffering with high-grade serous ovarian cancer syndrome, to add new evidence to the only in silico data available for this variant. High Resolution Melting Analysis (HRMA) was used for the first time to investigate the spliceogenicity of a BRCA1 variant. HRMA with Sanger sequencing provided evidence that the c.439C allele does not cause aberrant splicing of the BRCA1 exon 7. In addition, HRMA with Sanger highlighted a different expression of the naturally occurring BRCA1 r.442_444del (c.442_444delCAG, p.Gln148del, at DNA level) isoform between blood and tumor, in this patient. HRMA is an alternative molecular approach to analyze spliceogenic properties of the c.439T>C variant and potentially for all those BRCA1/2 variants affecting splicing sites. These new evidences allowed to classify definitively the c.439T>C variant as benign. Furthermore, the different BRCA1 r.442_444del expression opens the discussion to consider a wider classification criteria for the splicing variants, including molecular evaluation at tissue level, which is an aspect currently scarcely considered in BRCA1/2 variant classification recommendations.


Spliceogenic analysis BRCA1/2 genes High Resolution Melting Analysis BRCA1 c.439T>C variant BRCA1 r.442_444del isoform 



Hereditary breast and/or ovarian cancer


Pathogenic variants




Poly (ADP-ribose) polymerase


Variants of unknown significance


Evidence-based network for the interpretation of germline mutant alleles


Minor allele frequency


High grade serous ovarian cancer


High-resolution melting analysis


International federation of gynecologists and obstetricians


Fresh tissue


Next generation sequencing


Variant allele frequency



We would like to thank Franziska M. Lohmeyer for her critical review and English revision.

Compliance with ethical standards

Conflict of interest

The authors declared no potential conflicts of interest.

Ethical approval

This study complied with the Ethical Principles for Medical Research Involving Human Subjects according to the World Medical Association Declaration of Helsinki and was certified by the Committee of the Applicable Institution of the Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Rome.

Informed consent

Informed consent was taken from all participants.

Supplementary material

11033_2019_5199_MOESM1_ESM.docx (836 kb)
Supplementary material 1 (DOCX 836 kb)


  1. 1.
    Miller RE, Ledermann JA (2016) The status of poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitors in ovarian cancer, part 1: olaparib. Clin Adv Hematol Oncol 14:619–627PubMedGoogle Scholar
  2. 2.
    Houdayer C, Caux-Moncoutier V, Krieger S et al (2012) Guidelines for splicing analysis in molecular diagnosis derived from a set of 327 combined in silico/in vitro studies on BRCA1 and BRCA2 variants. Hum Mutat 33:1228–1238. CrossRefPubMedGoogle Scholar
  3. 3.
    Minucci A, Concolino P, De Bonis M et al (2018) Preliminary molecular evidence associating a novel BRCA1 synonymous variant with hereditary ovarian cancer syndrome. Hum Genome Var 20(5):2. CrossRefGoogle Scholar
  4. 4.
    Minucci A, Lalle M, De Leo R et al (2019) Additional molecular and clinical evidence open the way to definitive IARC classification of the BRCA1 c.5332G>A (p.Asp1778Asn) variant. Clin Biochem 63:54–58. CrossRefPubMedGoogle Scholar
  5. 5.
    Gaildrat P, Krieger S, Théry JC et al (2010) The BRCA1 c.5434C->G (p.Pro1812Ala) variant induces a deleterious exon 23 skipping by affecting exonic splicing regulatory elements. J Med Genet 47:398–403. CrossRefPubMedGoogle Scholar
  6. 6.
    Lindor NM, Guidugli L, Wang X et al (2012) A review of a multifactorial probability-based model for classification of BRCA1 and BRCA2 variants of uncertain significance (VUS). Hum Mutat 33:8–21. CrossRefPubMedGoogle Scholar
  7. 7.
    Colombo M, Blok MJ, Whiley P, Santamariña M et al (2014) Comprehensive annotation of splice junctions supports pervasive alternative splicing at the BRCA1 locus: a report from the ENIGMA consortium. Hum Mol Genet 23:3666–3680. CrossRefPubMedGoogle Scholar
  8. 8.
    Lindor NM, Goldgar DE, Tavtigian SV et al (2013) BRCA1/2 sequence variants of uncertain significance: a primer for providers to assist in discussions and in medical management. Oncologist 18:518–524. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Concolino P, Rizza R, Mignone F et al (2018) A comprehensive BRCA1/2 NGS pipeline for an immediate Copy Number Variation (CNV) detection in breast and ovarian cancer molecular diagnosis. Clin Chim Acta 480:173–179. CrossRefPubMedGoogle Scholar
  10. 10.
    Minucci A, Scambia G, Santonocito C et al (2015) Clinical impact on ovarian cancer patients of massive parallel sequencing for BRCA mutation detection: the experience at Gemelli hospital and a literature review. Expert Rev Mol Diagn 15:1383–1403. CrossRefPubMedGoogle Scholar
  11. 11.
    Minucci A, De Bonis M, De Paolis E et al (2017) Erratum to: high resolution melting analysis is very useful to identify BRCA1 c.4964_4982del19 (rs80359876) founder calabrian pathogenic variant on peripheral blood and buccal swab DNA. Mol Diagn Ther 21(2):233CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Angelo Minucci
    • 1
    Email author
  • Giorgia Mazzuccato
    • 1
  • Marco D’Indinosante
    • 2
  • Lucia Di Nardo
    • 3
  • Paola Concolino
    • 1
  • Maria De Bonis
    • 1
  • Andrea Urbani
    • 1
    • 3
  • Giovanni Scambia
    • 2
    • 3
  • Anna Fagotti
    • 2
    • 3
  • Ettore Capoluongo
    • 4
  1. 1.Molecular and Genomic Diagnostics LaboratoryFondazione Policlinico Universitario A. Gemelli IRCCSRomeItaly
  2. 2.Gynecologic OncologyFondazione Policlinico Universitario A. Gemelli IRCCSRomeItaly
  3. 3.Università Cattolica del Sacro CuoreRomeItaly
  4. 4.Università Federico II-CEINGEBiotecnologie AvanzateNaplesItaly

Personalised recommendations