Chimeric Genes Causing 11ß-Hydroxylase Deficiency: Implications in Clinical and Molecular Diagnosis.

Deficiency of 11ß-hydroxylase (11ß-OHD) is the second most common cause of congenital adrenal hyperplasia (CAH), accounting for 0.2-8% of all cases. The disease is transmitted as an autosomal recessive trait and the underlying genetic causes of 11ß-OHD are primarily small pathogenic variants affecting the CYP11B1 gene coding the 11ß-hydroxylase enzyme. However, special events complicate the molecular diagnosis of 11ß-OHD such as an unequal crossing over between the CYP11B2 (coding aldosterone synthase enzyme) and CYP11B1 genes. The resulting allele contains a hybrid gene, with a CYP11B2 5'-end and a CYP11B1 3'-end, where the CYP11B1 gene is under the control of the CYP11B2 promoter and thus not responding to the adrenocorticotropin (ACTH) but to angiotensin II and K+. This leads a reduction of cortisol production in 11ß-OHD. In particular, CYP11B2/CYP11B1 chimeric genes can be distinguished into two groups depending on the breakpoint site: chimeras with breakpoint after the exon 5 of CYP11B2 preserve the aldosterone synthase activity, the others with breakpoint before exon 5 lose this function. In the last case, a more severe phenotype is expected. The aim of this review was to explore the setting of CYP11B2/CYP11B1 chimeras in 11ß-OHD, performing a careful review of clinical literature cases.

Next-Generation Sequencing for Screening Analysis of Cystic Fibrosis: Spectrum and Novel Variants in a South-Central Italian Cohort.

The incidence of cystic fibrosis (CF) and the spectrum of cystic fibrosis transmembrane conductance regulator (CFTR) gene variants differ among geographic regions. Differences in CF carrier distribution are also reported among Italian regions. We described the spectrum of the CFTR variants observed in a large group of subjects belonging from central-southern Italy. We also provide a predictive evaluation of the novel variants identified. CFTR screening was performed in a south-central Italian cohort of 770 subjects. We adopted a next-generation sequencing (NGS) approach using the Devyser CFTR NGS kit on the Illumina MiSeq System coupled with Amplicon Suite data analysis. Bioinformatics evaluation of the impact of novel variants was described. Overall, the presence of at least one alternative allele in the CFTR gene was recorded for 23% of the subjects, with a carrier frequency of CF pathogenic variants of 1:12. The largest sub-group corresponded to the heterozygous carriers of a variant with a conflicting interpretation of pathogenicity. The common CFTR p.(Phe508del) pathogenic variants were identified in 37% of mutated subjects. Bioinformatics prediction supported a potential damaging effect for the three novel CFTR variants identified: p.(Leu1187Phe), p.(Pro22Thr), and c.744-3C > G. NGS applied to CF screening had the benefit of: effectively identifying asymptomatic carriers. It lies in a wide overview of CFTR variants and gives a comprehensive picture of the carrier prevalence. The identification of a high number of unclassified variants may represent a challenge whilst at the same time being of interest and relevance for clinicians.

Genetic Characterization of a Cohort of Italian Patients with Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency.

INTRODUCTION: Defects in the steroid 21-hydroxylase gene (CYP21A2) cause 21-hydroxylase deficiency (21OHD), the main cause of congenital adrenal hyperplasia (CAH). The disease shows a broad spectrum of clinical forms, ranging from severe or classical (salt wasting, SW, and simple virilizing, SV), to mild late onset or nonclassical (NC). 21OHD affects 1 in 15,000 in its severe classic form and 1 in 200-1000 in its mild NC form. There are many studies reporting the frequency of CYP21A2 pathogenic variants in different populations; however, few of them provide comprehensive information about Italian patients. Here, we present genetic results from a cohort of 245 unrelated Italian individuals with clinical diagnosis of CAH due to 21OHD. METHODS: A specific polymerase chain reaction (PCR) protocol combined with Sanger sequencing was used for CYP21A2 analysis. The multiplex ligation-dependent probe amplification (MLPA) assay was employed for copy number variation (CNV) determination. RESULTS: One hundred fourteen (46.5%) of the index cases had the NC form, 57 (23.3%) had the SV form, and 74 (30.2%) presented the SW form of the disease. The most prevalent variant found in NC patients was the p.Val282Leu (51.3%), while the most frequent variants in the classical form were p.Ile173Asn (8.6%) and c.293-13C>G (26.0%). In our study, the frequency of large rearrangements was 15.3%, with CAH-X alleles representing 40% of all DEL/CONV. In addition, 12 alleles carried rare variants, and 1 had a novel variant p.(Arg342Gln). We observed phenotype-genotype correlation in 94.7% of cases. A complete concordance was observed in Groups 0 (enzyme activity completely impaired) where all patients had the SW form as expected. In Group A (0-1% residual enzyme activity), 78.4% of patients had the anticipated SW form while 21.6% were diagnosed with the SV form. Within Group B (~ 2% residual enzyme activity), 93.4% of patients exhibited SV form and 6.5% SW disease. Finally, 92.6% and 7.4% of patients belonging to Group C (enzyme partially impaired to ~ 20-60% residual activity) exhibited NC and SV phenotypes, respectively. CONCLUSION: This work, representing a comprehensive genetic study, expanded the CYP21A2 variants spectrum of Italian patients with 21OHD and could be helpful in prenatal diagnosis and genetic counseling.

Identification and Molecular Characterization of a Novel Large-Scale Variant (Exons 4_18 Loss) in the LDLR Gene as a Cause of Familial Hypercholesterolaemia in an Italian Family.

Next-generation sequencing (NGS) is nowadays commonly used for clinical purposes, and represents an efficient approach for the molecular diagnosis of familial hypercholesterolemia (FH). Although the dominant form of the disease is mostly due to the low-density lipoprotein receptor (LDLR) small-scale pathogenic variants, the copy number variations (CNVs) represent the underlying molecular defects in approximately 10% of FH cases. Here, we reported a novel large deletion in the LDLR gene involving exons 4-18, identified by the bioinformatic analysis of NGS data in an Italian family. A long PCR strategy was employed for the breakpoint region analysis where an insertion of six nucleotides (TTCACT) was found. Two Alu sequences, identified within intron 3 and exon 18, could underlie the identified rearrangement by a nonallelic homologous recombination (NAHR) mechanism. NGS proved to be an effective tool suitable for the identification of CNVs, together with small-scale alterations in the FH-related genes. For this purpose, the use and implementation of this cost-effective, efficient molecular approach meets the clinical need for personalized diagnosis in FH cases.

Clinical Reasoning: A Young Man With Subacute Onset of Spastic Paraparesis.

Leukodystrophies are a group of rare neurodegenerative disorders, usually presenting in infancy with a variable combination of cognitive, motor, and coordination impairment. Adult-onset cases are even more rare, often representing a diagnostic challenge even for experienced neurologists. Here, we present a case of a 44-year-old man with subacute and rapidly progressive spastic paraplegia, whose brain MRI revealed white matter abnormalities compatible with a diagnosis of leukodystrophy. We discuss how to apply a simplified diagnostic algorithm to distinguish acquired leukoencephalopathies from leukodystrophies and how to delve into the maze of genetic testing for white matter diseases. In our patient, we reached the diagnosis of a treatable disorder, whose early recognition is essential to prevent severe neurologic deterioration.

Triple Genetic Diagnosis in a Patient with Late-Onset Leukodystrophy and Mild Intellectual Disability.

We describe the complex case of a 44-year-old man with polycystic kidney disease, mild cognitive impairment, and tremors in the upper limbs. Brain MRI showed lesions compatible with leukodystrophy. The diagnostic process, which included clinical exome sequencing (CES) and chromosomal microarray analysis (CMA), revealed a triple diagnosis: autosomal dominant polycystic kidney disease (ADPKD) due to a pathogenic variant, c.2152C>T-p.(Gln718Ter), in the PKD1 gene; late-onset phenylketonuria due to the presence of two missense variants, c.842C>T-p.(Pro281Leu) and c.143T>C-p.(Leu48Ser) in the PAH gene; and a 915 Kb duplication on chromosome 15. Few patients with multiple concurrent genetic diagnoses are reported in the literature; in this ADPKD patient, genome-wide analysis allowed for the diagnosis of adult-onset phenylketonuria (which would have otherwise gone unnoticed) and a 15q11.2 duplication responsible for cognitive and behavioral impairment with incomplete penetrance. This case underlines the importance of clinical genetics for interpreting complex results obtained by genome-wide techniques, and for diagnosing concurrent late-onset monogenic conditions.

A Novel GCK Large Genomic Rearrangement in a Patient with MODY-2 Detected by Clinical Exome Sequencing.

Maturity-onset diabetes of the young (MODY) is a rare form of non-autoimmune diabetes with an autosomal dominant inheritance. To date, 14 genes have been reported as genetic basis of MODY. GCK gene, encoding the glucokinase enzyme, was the first MODY gene to be identified. GCK heterozygous inactivating variants cause the GCK-MODY or MODY2 subtype. However, partial or whole gene deletions have been rarely identified, showing it to be a rare cause of GCK-MODY. We reported the molecular evaluation of a Ukrainian patient with clinical diagnosis of MODY2. We performed the Next generation sequencing of the clinical exome using the Clinical Exome Solution® kit (SOPHiA Genetics), followed by the design of a 14 genes virtual panel related to the suggestive diagnosis of MODY. Bioinformatics analysis was performed using the SOPHiA DDM platform (SOPHiA Genetics). The SALSA MLPA kit for MODY (MRC-Holland) was used for relative quantification of GCK exons. From the molecular evaluation, no pathogenic sequence variants were detected in the investigated genes. Copy Number Variation analysis was able to identify a large deletion involving the last three exons of the GCK gene. This result was confirmed by MLPA. To the best of our knowledge, the identified rearrangement has never been reported in the literature.

BRCA Mutation Status in Triple-Negative Breast Cancer Patients Treated with Neoadjuvant Chemotherapy: A Pivotal Role for Treatment Decision-Making.

Triple-negative breast cancer (TNBC) is characterized by earlier recurrence and shorter survival compared with other types of breast cancer. Moreover, approximately 15 to 25% of all TNBC patients harbor germline BRCA (gBRCA) 1/2 mutations, which confer a more aggressive phenotype. However, TNBC seems to be particularly sensitive to chemotherapy, the so-called 'triple negative paradox'. Therefore, Neoadjuvant chemotherapy (NACT) is currently considered the preferred approach for early-stage TNBC. BRCA status has also been studied as a predictive biomarker of response to platinum compounds. Although several randomized trials investigated the addition of carboplatin to standard NACT in early-stage TNBC, the role of BRCA status remains unclear. In this retrospective analysis, we evaluated data from 136 consecutive patients with Stage I-III TNBC who received standard NACT with or without the addition of carboplatin, in order to define clinical features and outcomes in BRCA 1/2 mutation carriers and non-carrier controls. Between January 2013 and February 2021, 67 (51.3%) out of 136 patients received a standard anthracyclines/taxane regimen and 69 (50.7%) patients received a platinum-containing chemotherapy regimen. Deleterious germline BRCA1 or BRCA2 mutations were identified in 39 (28.7%) patients. Overall, patients with deleterious gBRCA1/2 mutation have significantly higher pCR rate than non-carrier patients (23 [59%] of 39 vs. 33 [34%] of 97; p = 0.008). The benefit of harboring a gBRCA mutation was confirmed only in the subset of patients who received a platinum-based NACT (17 [65.4%] of 26 vs. 13 [30.2%] of 43; p = 0.005) while no differences were found in the platinum-free subgroup. Patients who achieved pCR after NACT had significantly better EFS (OR 4.5; 95% CI 1.9-10.7; p = 0.001) and OS (OR 3.3; 95% CI 1.3-8.9; p = 0.01) than patients who did not, regardless of BRCA1/2 mutation status and type of NACT received. Our results based on real-world evidence show that TNBC patients with the gBRCA1/2 mutation who received platinum-based NACT have a higher pCR rate than non-carrier patients, supporting the use of this chemotherapy regimen in this patient population. Long-term follow-up analyses are needed to further define the role of gBRCA mutation status on clinical outcomes in patients with early-TNBC.

Pregnancy and Prenatal Management of Congenital Adrenal Hyperplasia.

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive diseases that may cause cortisol insufficiency together with other hormonal alterations. The most common form is 21-hydroxylase deficiency, in which the lack of pituitary negative feedback causes an increase in ACTH and adrenal androgens. Classical forms of CAHs can lead to severe adrenal failure and female virilization. To date, the appropriate management of pregnant CAH patients is still debated regarding appropriate maternal therapy modifications during pregnancy and the risks and benefits of prenatal treatment of the fetus. We conducted a literature search of relevant papers to collect current evidence and experiences on the topic. The most recent and significant articles were selected, and current international guidelines were consulted to update current recommendations and guide clinical practice. Given the lack of randomized clinical trials and other high-quality scientific evidence, the issue is still debated, and great heterogeneity exists in current practice in terms of risk/benefit evaluation and pharmacological choices for pregnancy and prenatal treatment. Glucocorticoid therapy is advised not only in classical CAH patients but also in non-classical, milder forms. The choice of which glucocorticoid to use, and the safety and benefits of dexamethasone therapy aimed at preventing genital virilization are still debated issues. Several advances, however, have been made, especially in terms of fertility and reproduction. This review aims to present the most recent scientific and real-world updates on pregnancy and prenatal management of CAH, with the presentation of various clinical scenarios and specific case-by-case recommendations.

Prevalence of CAH-X Syndrome in Italian Patients with Congenital Adrenal Hyperplasia (CAH) Due to 21-Hydroxylase Deficiency.

21-hydroxylase deficiency (21OHD), the most common form of congenital adrenal hyperplasia (CAH), is associated with pathogenic variants in CYP21A2 gene. The clinical form of the disease ranges from classic or severe to non-classic (NC) or mild late onset. The CYP21A2 gene is located on the long arm of chromosome 6, within the RCCX region, one of the most complex loci in the human genome. The 3'untranslated sequence of CYP21A2 exon 10 overlap the last exon of TNXB gene (these genes lie on the opposite strands of DNA and have the opposite transcriptional direction) that encodes an extracellular matrix glycoprotein tenascin-X (TNX). A recombination event between TNXB and its pseudogene TNXA causes a 30 kb deletion producing a chimeric TNXA/TNXB gene (CAH-X chimera) where both CYP21A2 and TNXB genes are impaired. This genetic condition characterizes a subset of patients with 21OHD who display the hypermobility phenotype of Ehlers-Danlos syndrome (hEDS) (CAH-X Syndrome). The aim of this study was to assess the prevalence of CAH-X syndrome in an Italian cohort of patients with 21OHD. At this purpose, 196 probands were recruited. Multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing were used to identify the CAH-X genotype. Twenty-one individuals showed the heterozygous continuous deletion involving the CYP21A2 and part of the TNXB gene. EDS-related clinical manifestations were identified in most patients carrying the CAH-X chimera. A CAH-X prevalence of 10.7% was estimated in our population.

CAH-X Syndrome: Genetic and Clinical Profile.

The term CAH-X was coined to describe a subset of patients with 21-hydroxylase deficiency displaying a phenotype compatible with the hypermobility type of Ehlers Danlos syndrome. The genetic defect is due to the monoallelic presence of a CYP21A2 deletion extending into the gene encoding tenascin X (TNXB), a connective tissue extracellular matrix protein. The result is a chimeric TNXA/TNXB gene causing tenascin-X haploinsufficiency. The prevalence of CAH-X was estimated to be around 14-15% in large cohorts of patients with 21-hydroxylase deficiency. However, population studies are still scarce and the clinical picture of the syndrome has yet to be fully defined. In this review, we discuss the current knowledge regarding the genetic and clinical profile of the CAH-X syndrome.

Tumor BRCA testing in ovarian cancer and EQA scheme: our experience of a critical evaluation.

Next generation sequencing (NGS) is a widespread molecular biology method integrated into clinical practice to detect genetic variants, for diagnostic and prognostic purposes. The scheduled external quality assessments (EQA) is integral part of clinical molecular laboratory quality assurance. The EQA provides an efficient system to compare analytic test performances among different laboratories, which is essential to evaluate consistency of molecular test. EQA failures demands targeted corrective action plans. In this context, the complexity of the NGS techniques requires careful and continuous quality control procedures. We report a tumor BRCA1/2 (tBRCA) testing benchmark discrepancy provided by the European Molecular Genetics Quality Network in our laboratory during a round of EQA for somatic mutation testing of BRCA genes in relation to ovarian cancer. The critical analysis emerging from the tBRCA EQA is presented. We underline that harmonization processes are still required for the EQA in the molecular biology field, especially if applied to the evaluation of methods characterized by high complexity.

Genes and Pseudogenes: Complexity of the RCCX Locus and Disease.

Copy Number Variations (CNVs) account for a large proportion of human genome and are a primary contributor to human phenotypic variation, in addition to being the molecular basis of a wide spectrum of disease. Multiallelic CNVs represent a considerable fraction of large CNVs and are strictly related to segmental duplications according to their prevalent duplicate alleles. RCCX CNV is a complex, multiallelic and tandem CNV located in the major histocompatibility complex (MHC) class III region. RCCX structure is typically defined by the copy number of a DNA segment containing a series of genes - the serine/threonine kinase 19 (STK19), the complement 4 (C4), the steroid 21-hydroxylase (CYP21), and the tenascin-X (TNX) - lie close to each other. In the Caucasian population, the most common RCCX haplotype (69%) consists of two segments containing the genes STK19-C4A-CYP21A1P-TNXA-STK19B-C4B-CYP21A2-TNXB, with a telomere-to-centromere orientation. Nonallelic homologous recombination (NAHR) plays a key role into the RCCX genetic diversity: unequal crossover facilitates large structural rearrangements and copy number changes, whereas gene conversion mediates relatively short sequence transfers. The results of these events increased the RCCX genetic diversity and are responsible of specific human diseases. This review provides an overview on RCCX complexity pointing out the molecular bases of Congenital Adrenal Hyperplasia (CAH) due to CYP21A2 deficiency, CAH-X Syndrome and disorders related to CNV of complement component C4.

Spectrum of DICER1 Germline Pathogenic Variants in Ovarian Sertoli-Leydig Cell Tumor.

Sertoli-Leydig Cell Tumors (SLCTs) are rare ovarian sex cord-stromal neoplasms, which predominantly affect adolescents and young female adults. The SLCTs clinical diagnosis and treatment remains challenging due to the rarity and the varied presentation. A large majority of SLCTs are unilateral, but also bilateral neoplasms have been reported, sometimes in the context of DICER1 syndrome. In fact, the most significant discovery regarding the molecular genetics basis of SLCTs was the finding of somatic and germline pathogenic variants in the DICER1 gene. The DICER1 protein is a key component of the micro-RNA processing pathway. Germline DICER1 pathogenic variants are typically inherited in an autosomal dominant pattern and are most often loss-of-function variants dispersed along the length of the gene. Contrarily, DICER1-related tumors harbor a characteristic missense "RNase IIIb hotspot" mutation occurring in trans, or, less frequently, loss of heterozygosity (LOH) event involving the wild-type allele. While DICER1 mutations have been identified in approximately 60% of SLCTs, especially in the moderately or poorly differentiated types, there are only a few case reports of ovarian SLCT with underlying germline DICER1 mutations. In this review, we focus on the molecular genetic features of SLCT, performing an extensive survey of all germline pathogenic variants modifying the whole sequence of the DICER1 gene. We point out that DICER1 genetic testing, coupled with an accurate variants classification and timely counseling, is of crucial importance in the clinical management of ovarian SLCT-affected patients.

Case Report: Detection of a Novel Germline PALB2 Deletion in a Young Woman With Hereditary Breast Cancer: When the Patient's Phenotype History Doesn't Lie.

The partner and localizer of BRCA2 (PALB2) is a major BRCA2 binding partner that participates in homologous recombination repair in response to DNA double-strand breaks. Germline alterations of the PALB2 gene have recently been associated with a high risk of developing breast cancer. We investigated a 37-year-old Caucasian woman with breast cancer and family history of breast cancer using targeted next generation sequencing. A novel heterozygous deletion involving exons 5 and 6 was found in the PALB2 gene, and resulted in the production of a truncated PALB2 protein. These findings expand the mutational spectra of PALB2-associated breast cancer, and may improve the mutation-based screening and genetic diagnosis of breast cancer.

A Novel ATM Pathogenic Variant in an Italian Woman with Gallbladder Cancer.

Gallbladder carcinoma (GBC) is one of the most aggressive malignancies with poor prognosis and a high fatality rate. The disease presents in advanced stages where the treatment is ineffective. Regarding GBC pathogenesis, as with other neoplasia, this tumor is a multifactorial disorder involving different causative factors such as environmental, microbial, metabolic, and molecular. Genetic alterations can be germline or somatic that involving proto-oncogenes, tumor suppressor genes, cell cycle genes, and growth factors. The ataxia telangiectasia mutated (ATM) gene, coding a serine/threonine kinase involved in the early stages of the homologous recombination (HR) mechanism, is one of the most altered genes in GBC. Here, we present the molecular characterization of a novel germline ATM large genomic rearrangement (LGR) identified by next-generation sequencing (NGS) analysis in an Italian woman diagnosed with metastatic GBC at the age of 55. The results underline the importance of expanding the NGS approach in gallbladder cancer in order to propose new molecular markers of predisposition and prognosis exploitable by novel targeted therapies that may improve the response of patients with ATM-deficient cancers.

Molecular Analysis of 21-Hydroxylase Deficiency Reveals Two Novel Severe Genotypes in Affected Newborns.

BACKGROUND AND OBJECTIVE: Congenital adrenal hyperplasia involves a series of autosomal recessive disorders where adrenal steroidogenesis is affected. We present a detailed molecular investigation of 13 newborns affected from the severe form of congenital adrenal hyperplasia related to 21-hydroxylase deficiency. METHODS: All patients were diagnosed with classical congenital adrenal hyperplasia in the neonatal period due to adrenal crisis and/or ambiguous genitalia presentation. None of the infants was identified through a congenital adrenal hyperplasia newborn screening program. A molecular analysis of the CYP21A2 gene and a familiar segregation analysis were performed. RESULTS: Adrenal crisis was the most severe manifestation in the male salt-wasting newborns while all female patients presented with atypical genitalia. Newborns were correctly genotyped and no genotype-phenotype divergences were found. Two novel severe genotypes, not previously reported, were identified. The novel CYP21A2 frameshift mutations (c.793delG and c.297dupG) were added to the other 45 variants recently reported in the literature, leading to a total count of 279 pathogenic variants affecting the gene. CONCLUSIONS: We have successfully genotyped 13 infants diagnosed with classical congenital adrenal hyperplasia after birth. Our molecular approach led to the identification of two novel frameshift CYP21A2 pathogenic variants related to the salt-wasting form of congenital adrenal hyperplasia.

BRCA testing delay during the COVID-19 pandemic: How to act?

Recently, our lab, part of a referral center in Italy, reported its experience regarding the execution of germline BRCA1/2 (gBRCA) testing during the first months of the coronavirus disease-2019 (COVID-19) pandemic, which highlights a substantial reduction (about 60%) compared with the first 2 months of the current year. This evidence appeared to be a lockdown effect due to extraordinary restriction measures to slow down the spread of SARS-CoV-2. In this study, we aimed to evaluate the overall effects of the ongoing pandemic on gBRCA testing in our institution and to understand how COVID-19 has influenced testing after the complete lockdown (March 8-May 5, 2020). Additionally, we compared this year's trend with trends of the last 3 years to better monitor gBRCA testing progress. This detailed analysis highlights two important findings: (1) gBRCA testing did not increase significantly after the lockdown period (May-October 2020) compared with the lockdown period (March-April 2020), emphasizing that even after the lockdown period testing remained low. (2) Comparing the total tests per year (January-October 2017, 2018, 2019, with 2020), the impact of COVID-19 on gBRCA testing is apparent, with similarities of trends registered in 2017. These evidences reveal a gBRCA testing delay for cancer patients and healthy patients at this moment, and the new era of gBRCA testing in the management of ovarian, breast, pancreas and prostate cancer patients has been seriously questioned due to the COVID-19 pandemic. As consequence, we underline that measures to guarantee oncogenetic testing (e.g., gBRCA testing) along with new diagnostic/clinic strategies are mandatory. For these reasons, several proposals are presented in this study.

Droplet digital PCR for large genomic rearrangements detection: A promising strategy in tissue BRCA1 testing.

BACKGROUND AND AIMS: With the introduction of Olaparib as target therapy for High Grade Serous Ovarian Cancer (HGSOC) patients with germline and somatic BRCA1/2 mutations, the genetic test performed on tumor tissue has become important like the germline test. In somatic testing the evaluation of Large Genomic Rearrangements (LGRs) represents the main challenge. We describe a droplet digital PCR (ddPCR) assay for the evaluation of target BRCA1 LGRs on blood and formalin-fixed paraffin-embedded (FFPE)/Fresh Frozen Tissue (FFT) samples. MATERIALS AND METHODS: We analyzed blood, FFPE and FFT samples in a validation setting of n = 78 HGSOC patients. We applied the ddPCR to BRCA1 exons 2, 20 and 21 as some of the most common BRCA1 exons involved in LGRs in our cohort of patients. RESULTS: The ddPCR custom assays allowed the identification of LGRs in all sample types, including FFPE specimens. Moreover, we were able to clearly detect LGRs accounted as somatic event. CONCLUSION: The introduction of ddPCR in a comprehensive workflow, encompassing both germline and somatic tests, represents an improvement in BRCA1/2 testing. ddPCR can overcome challenges related to BRCA testing, especially on FFPE analysis. Finally, ddPCR represents a promising alternative strategy to the established standard methods currently used in clinical setting.