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.

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.

Lung cancer multi-omics digital human avatars for integrating precision medicine into clinical practice: the LANTERN study.

BACKGROUND: The current management of lung cancer patients has reached a high level of complexity. Indeed, besides the traditional clinical variables (e.g., age, sex, TNM stage), new omics data have recently been introduced in clinical practice, thereby making more complex the decision-making process. With the advent of Artificial intelligence (AI) techniques, various omics datasets may be used to create more accurate predictive models paving the way for a better care in lung cancer patients. METHODS: The LANTERN study is a multi-center observational clinical trial involving a multidisciplinary consortium of five institutions from different European countries. The aim of this trial is to develop accurate several predictive models for lung cancer patients, through the creation of Digital Human Avatars (DHA), defined as digital representations of patients using various omics-based variables and integrating well-established clinical factors with genomic data, quantitative imaging data etc. A total of 600 lung cancer patients will be prospectively enrolled by the recruiting centers and multi-omics data will be collected. Data will then be modelled and parameterized in an experimental context of cutting-edge big data analysis. All data variables will be recorded according to a shared common ontology based on variable-specific domains in order to enhance their direct actionability. An exploratory analysis will then initiate the biomarker identification process. The second phase of the project will focus on creating multiple multivariate models trained though advanced machine learning (ML) and AI techniques for the specific areas of interest. Finally, the developed models will be validated in order to test their robustness, transferability and generalizability, leading to the development of the DHA. All the potential clinical and scientific stakeholders will be involved in the DHA development process. The main goals aim of LANTERN project are: i) To develop predictive models for lung cancer diagnosis and histological characterization; (ii) to set up personalized predictive models for individual-specific treatments; iii) to enable feedback data loops for preventive healthcare strategies and quality of life management. DISCUSSION: The LANTERN project will develop a predictive platform based on integration of multi-omics data. This will enhance the generation of important and valuable information assets, in order to identify new biomarkers that can be used for early detection, improved tumor diagnosis and personalization of treatment protocols. ETHICS COMMITTEE APPROVAL NUMBER: 5420 - 0002485/23 from Fondazione Policlinico Universitario Agostino Gemelli IRCCS - Università Cattolica del Sacro Cuore Ethics Committee. TRIAL REGISTRATION: clinicaltrial.gov - NCT05802771.

Unexpected finding of a rare pathogenic germline BRCA1 variant in an intrahepatic cholangiocarcinoma using the Oncomine Focus DNA assay: clinical and diagnostic implications.

BACKGROUND: Cholangiocarcinoma (CCA) is a malignant tumor arising from the epithelial cells of the bile ducts and is the second most common liver cancer after hepatocellular carcinoma. Recently, our Institution launched a Comprehensive Genomic Profiling (CGP) program (named FPG500 program), set up to provide a complete molecular characterization through the TruSight Oncology 500 High Throughput (TSO500HT) solution and samples that do not reach pre-set sample quantity and/or quality thresholds required for TSO500HT, are addressed to Oncomine Focus DNA Assay (OFA) and the Archer's FusionPlex Lung Panel (AFL). METHODS AND RESULTS: Here we report the case of a patient with iCCA enrolled in the FPG500 program and screened by the orthogonal workflow (OFA/AFL). Although BRCA1 is not among the genes declared in the OFA panel, we unexpectedly detected a pathogenic variant in this gene (c.5278-2del, rs878853285). CONCLUSIONS: This case highlights the diagnostic capabilities of CGP, now widely used in both clinical practice and academic setting. The incidental involvement of BRCA1 focuses attention on the role of BRCA genes in biliary tract cancers. Finally, as an orthogonal test confirmed the germline origin of BRCA1 c.5278-2del variant, the germline implications of CGP need to be considered.

A Computational Framework for Comprehensive Genomic Profiling in Solid Cancers: The Analytical Performance of a High-Throughput Assay for Small and Copy Number Variants.

In January 2022, our institution launched a comprehensive cancer genome profiling program on 10 cancer types using a non-IVD solution named the TruSight Oncology 500 Assay provided by Illumina®. The assay analyzes both DNA and RNA, identifying Single-Nucleotide Variants (SNV)s and Insertion-Deletion (InDel) in 523 genes, as well as known and unknown fusions and splicing variants in 55 genes and Copy Number Alterations (CNVs), Mutational Tumor Burden (MTB) and Microsatellite Instability (MSI). According to the current European IVD Directive 98/79/EC, an internal validation was performed before running the test. A dedicated open-source bioinformatics pipeline was developed for data postprocessing, panel assessment and embedding in high-performance computing framework using the container technology to ensure scalability and reproducibility. Our protocols, applied to 71 DNA and 64 RNA samples, showed full agreement between the TruSight Oncology 500 assay and standard approaches, with only minor limitations, allowing to routinely perform our protocol in patient screening.

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 testing on buccal swab to improve access to healthcare and cancer prevention: a performance evaluation.

OBJECTIVE: BRCA1/2 (BRCA) genetic testing allows patients with high-grade serous ovarian cancer to receive appropriate medical management with molecular target therapy and prevention strategies. Most of the BRCA sequencing methods use blood as the primary source of germline DNA. Buccal swab emerged as an alternative collection device due to its convenient and non-invasive characteristics. This study assessed the suitability of buccal swabs as the DNA source in next-generation sequencing-based BRCA genotyping. METHODS: Matched buccal swabs and blood samples were collected from 51 patients with high-grade serous ovarian cancer, including 29 BRCA-mutated patients, from June to December 2021. Buccal swabs were self-collected using COPAN FLOQSwabs hDNA Free. BRCA genes were amplified using Devyser's BRCA next-generation sequencing kit and sequenced on the Illumina MiSeq platform. We evaluated collection and extraction procedures, amplification and sequencing performances, coverage data, blood/swab variant calling concordance, and interpretation. RESULTS: Comparable sequencing parameters were observed between the two sample types in term of mean total number of reads passing filter for indexed sample (p>0.05) and sequencing coverage distribution, with a widespread overlap of mean depth of coverage/target region between blood and swab samples. An overall concordance of 100% in both polymorphisms and pathogenic variants calling between the two DNA sources were observed, including the copy number variation prediction. CONCLUSIONS: Data from this study support the use of buccal swabs as an alternative source of DNA for BRCA evaluation. The use of this alternative delivery mode of BRCA testing may facilitate access to care without compromising patient outcomes.

Role of Homologous Recombination Repair (HRR) Genes in Uterine Leiomyosarcomas: A Retrospective Analysis.

Uterine leiomyosarcomas (uLMS) is a very rare disease, and patients experience a dismal prognosis even when treated with chemotherapy. Therefore, a more in-depth molecular characterization of this disease could provide suitable data for the identification of potential target-based drugs. This retrospective, single institutional study aimed to define the frequencies of gene alterations in uLMS, especially regarding the somatic mutations of BRCA and Homologous Recombination Repair (HRR) genes, and the impact of molecular alterations on clinical outcomes. The 16-genes Next-Generation Sequencing (NGS) panel, Homologous Recombination Solution TM (HRS, Sophia Genetics, Saint Sulpice, Switzerland), was used for the molecular evaluation of samples. The majority of patients (66/105, 63%) carried at least one sequence alteration, with a prevalence of TP53 involvement followed by RAD51B, BRCA1/2, and FANCL. Patients with TP53 gene alterations experienced a significantly worse prognosis for progression free survival (PFS) and overall survival (OS) versus wild-type patients. Given the number of patients with the BRCA1/2 mutation (N = 12), we included them in the HRR patient group; there was no difference in clinical outcomes with HRR versus non-HRR. The Cox's multivariate analysis showed that stage and TP53 gene alterations resulted in a significantly worse OS. The integration of gene networking data, such as tumor mutation burdens and cancer driver gene identification, could show a clearer discrimination of gene distribution patterns, and lead to the implementation of therapeutic targets.

Assessing the pathogenicity of BRCA1/2 variants of unknown significance: Relevance and challenges for breast cancer precision medicine.

Introduction: Breast cancer (BC) is the leading cause of cancer-related death in women worldwide. Pathogenic variants in BRCA1 and BRCA2 genes account for approximately 50% of all hereditary BC, with 60-80% of patients characterized by Triple Negative Breast Cancer (TNBC) at an early stage phenotype. The identification of a pathogenic BRCA1/2 variant has important and expanding roles in risk-reducing surgeries, treatment planning, and familial surveillance. Otherwise, finding unclassified Variants of Unknown Significance (VUS) limits the clinical utility of the molecular test, leading to an "imprecise medicine". Methods: We reported the explanatory example of the BRCA1 c.5057A>C, p.(His1686Pro) VUS identified in a patient with TNBC. We integrated data from family history and clinic-pathological evaluations, genetic analyses, and bioinformatics in silico investigations to evaluate the VUS classification. Results: Our evaluation posed evidences for the pathogenicity significance of the investigated VUS: 1) association of the BRCA1 variant to cancer-affected members of the family; 2) absence of another high-risk mutation; 3) multiple indirect evidences derived from gene and protein structural analysis. Discussion: In line with the ongoing efforts to uncertain variants classification, we speculated about the relevance of an in-depth assessment of pathogenicity of BRCA1/2 VUS for a personalized management of patients with BC. We underlined that the efficient integration of clinical data with the widest number of supporting molecular evidences should be adopted for the proper management of patients, with the final aim of effectively guide the best prognostic and therapeutic paths.

A novel low-density lipoprotein receptor variant in a Ukrainian patient: a case report and overview of the disease-causing low-density lipoprotein receptor variants associated to familial hypercholesterolemia.

BACKGROUND: Familial hypercholesterolemia (FH) is characterized by high low-density lipoprotein-cholesterol levels and it is primarily caused by pathogenic/likely pathogenic variants (P/LPVs) in LDLR, APOB or PCSK9 genes. Next generation sequencing (NGS) technology allows the evaluation of more genes simultaneously, rising the diagnostic throughput of genomics laboratories. MATERIALS AND METHODS: We report a Ukrainian 37-year-old woman hypercholesterolemic since 2010. Despite a suggestive family history, FH was suspected only when the patient referred to the Endocrine and Metabolic Diseases Center of the Fondazione Policlinico Universitario A. Gemelli IRCCS in Rome. After specialist advice, genetic testing was offered to the patient at our Molecular and Genomic Diagnostics Unit. RESULTS: A targeted NGS-based pipeline highlighted a novel out-of-frame deletion in the LDLR gene. This variant has a clear deleterious effect on the LDLR protein and it can be classified as PV. CONCLUSIONS: The ideal model of care for FH is an evidence-based system aimed to provide the highest-quality health services to all FH patients. In fact, this study reports that the integrated care pathway adopted in our hospital for FH patients led successfully to the discovery of a novel LDLR PV in an Ukrainian patient. The finding of this LDLR variant allowed the clinical FH diagnosis in this patient and in her family, expanding the knowledge of FH-related genetic variants in the Ukrainian population.

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.

Duplex high resolution melting analysis (dHRMA) to detect two hot spot CYP24A1 pathogenic variants (PVs) associated to idiopathic infantile hypercalcemia (IIH).

Pathogenic variants (PVs) in CYP24A1 gene are associated with Idiopathic Infantile Hypercalcemia disease (IIH). The identification of CYP24A1 PVs can be a useful tool for the improvement of target therapeutic strategies. Aim of this study is to set up a rapid and inexpensive High Resolution Melting Analysis (HRMA)-based method for the simultaneous genotyping of two hot spot PVs in CYP24A1 gene, involved in IIH. A duplex-HRMA (dHRMA) was designed in order to detect simultaneously CYP24A1 c.428_430delAAG, p.(Glu143del) (rs777676129) and c.1186C > T, p.(Arg396Trp) (rs114368325), in peculiar cases addressed to our Laboratory. dHRMA was able to identify clearly and simultaneously both hot spot CYP24A1 PVs evaluating melting curve shape and melting temperature (Tm). This is the first dHRMA approach to rapidly screen the two most frequent CYP24A1 PVs in peculiar case, providing useful information for diagnosis and patient management in IIH disease.

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.

The BRCA1 c.788G > T (NM_007294.4) variant in a high grade serous ovarian cancer (HGSOC) patient: foods for thought.

In this report we described the case of a BRCA1/2 (BRCA) molecular testing performed on tumor sample in a High Grade Serous Ovarian Cancer (HGSOC) patient with two different Next Generation Tumor Sequencing (NGTS) pipelines. The two clinical reports leaded to apparently different BRCA status, providing important foods for thought. After NGTS, the gene sequencing information (i.e., reads) are aligned to the reference gene sequences obtained from public databases, in order to provide an uniform nomenclature for unambiguous variant designation. However, the criteria adopted for variant reporting in tissue test are not always univocal. Particularly, this is the case of rare and unclassified BRCA variants for which the molecular evaluation may be a relevant challenge. Here we described a BRCA1 unclassified variant that may be re-evaluated in the context of alternative BRCA1 transcripts due to its different biological effect. We underlined that an in-depth knowledge of BRCA testing is mandatory for its appropriate use.

Pemphigus vulgaris in two pairs of siblings from two unrelated Italian families: Human leukocyte antigen genotypes, ST18 mutation and immunological profile.

Pemphigus vulgaris (PV) is an autoimmune intraepithelial bullous disease. Associations with the class II human leukocyte antigen (HLA) alleles and pemphigus vulgaris have been described. Furthermore, an association between the single nucleotide polymorphism of the ST18 gene and pemphigus vulgaris has been reported. We report two pairs of siblings from two unrelated Italian families affected by pemphigus vulgaris, characterizing their genetic and immunological profile. In order to assess the genetic background, HLA-DQA1, HLA-DQB1, HLA-DRB1 and a relevant ST18 polymorphism were investigated. As for the immunological profiles, anti-desmoglein antibodies were analyzed. In family A, the two pemphigus vulgaris patients had the same HLA genetic profile: HLA-DQA1 *01:04/*03:01, HLA-DQB1 *03:02/*05:03 and HLA-DRB1 *04:02/*14:01. The male patient was heterozygous for the ST18 mutation while the female patient had a wild genotype. In family B, the two pemphigus vulgaris patients were both wild type for the ST18 mutation and showed the same HLA genotype: HLA-DQA1 *03:01/*05:08, HLA-DQB1 *03:01/*03:03 and HLA-DRB1 *04:02/*11:01. Our data show a relevant relationship between the HLA profile and pemphigus vulgaris in our Italian families. In family A, all six alleles are frequently associated with pemphigus vulgaris and were expressed only in the two pemphigus patients; and in family B, two of the six alleles are frequently associated with pemphigus vulgaris. No relevant relationship was found between ST18 polymorphism and pemphigus disease.