Identification of an ultra-rare Alu insertion in the CFTR gene: Pitfalls and challenges in genetic test interpretation.

Cystic fibrosis (CF) is a life-limiting genetic disorder characterized by defective chloride ion transport due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Early detection through newborn screening programs significantly improves outcomes for individuals with CF by enabling timely intervention. Here, we report the identification of an Alu element insertion within the exon 15 of CFTR gene, initially overlooked in standard next-generation sequencing analyses. However, using traditional molecular techniques, based on polymerase chain reaction and Sanger sequencing, allowed the identification of the Alu element and the reporting of a correct diagnosis. Our analysis, based on bioinformatics tools and molecular techniques, revealed that the Alu element insertion severely affects the gene expression, splicing patterns, and structure of CFTR protein. In conclusion, this study emphasizes the importance of how the integration of human expertise and modern technologies represents a pivotal step forward in genomic medicine, ensuring the delivery of precision healthcare to individuals affected by genetic diseases.

One-step NGS molecular analysis of the CFTR gene on newborn dried blood spots gives a higher diagnostic sensitivity in affected and carrier subjects: A pilot study.

BACKGROUND: Cystic fibrosis is the most common hereditary recessive disease with an incidence of about 1:2500/3000. It has long been known that the disease is caused by deleterious mutations in the CFTR gene. Conventionally, the disease is diagnosed in several phases. The analysis of all the possible disease-causing molecular alterations is time consuming and may not lead to a definitive diagnosis in several cases. Consequently, we propose, in this paper, a rapid sequencing method that, in a single procedural asset, reveals the presence of small mutations and also the copy number variants (CNVs) from the DNA extracted from the Guthrie Spot. MATERIALS AND METHODS: We first sequenced 30 blood spots, then we validated the method on 100 spots that underwent both traditional analyses and this complete NGS sequencing, and lastly, we tested the strategy on patients who normally do not reach the molecular sequencing step because of low level of Immune-Reactive Trypsinogen. RESULTS: Using this procedure, we identified 97 variants in the CFTR gene of our samples and 6 CNVs. Notably, the significant data were obtained in the group of patients with borderline or negative IRT who routinely would not undergo molecular testing. We also identified 6 carriers of "disease-causing" variants. CONCLUSION: This method is very robust. Indeed, there was a 100% concordance with Sanger sequencing validation, and 6 mutation carriers were identified who normally escaped molecular testing with actual conventional procedure. There were also 3 duplications of almost the entire gene in heterozygosity, which were not seen with traditional methods. Being quick and easy to perform, we suggest that complete sequencing of the CFTR gene, as in this study be considered for all newborns.

Next-Generation Sequencing Gene Panels in Inheritable Cardiomyopathies and Channelopathies: Prevalence of Pathogenic Variants and Variants of Unknown Significance in Uncommon Genes.

The diffusion of next-generation sequencing (NGS)-based approaches allows for the identification of pathogenic mutations of cardiomyopathies and channelopathies in more than 200 different genes. Since genes considered uncommon for a clinical phenotype are also now included in molecular testing, the detection rate of disease-causing variants has increased. Here, we report the prevalence of genetic variants detected by using a NGS custom panel in a cohort of 133 patients with inherited cardiomyopathies (n = 77) or channelopathies (n = 56). We identified 82 variants, of which 50 (61%) were identified in genes without a strong or definitive evidence of disease association according to the NIH-funded Clinical Genome Resource (ClinGen; "uncommon genes"). Among these, 35 (70%) were variants of unknown significance (VUSs), 13 (26%) were pathogenic (P) or likely pathogenic (LP) mutations, and 2 (4%) benign (B) or likely benign (LB) variants according to American College of Medical Genetics (ACMG) classifications. These data reinforce the need for the screening of uncommon genes in order to increase the diagnostic sensitivity of the genetic testing of inherited cardiomyopathies and channelopathies by allowing for the identification of mutations in genes that are not usually explored due to a currently poor association with the clinical phenotype.

MIS-C: A COVID-19-as sociated condition between hypoimmunity and hyperimmunity.

Multisystem inflammatory syndrome in children (MIS-C) is a rare, severe complication of COVID-19. A better knowledge of immunological, cellular, and genetic characteristics of MIS-C could help better understand the pathogenesis of the disease and contribute to identifying specific diagnostic biomarkers and develop targeted therapies. We studied 37 MIS-C children at hospital admission and 24 healthy controls analyzing serum cytokines (IFN-α, IFN-ß, IFN-γ, IL-6, IL-10, IL-17A, IL-12p70 and TNF), lymphocyte populations by flow cytometry and 386 genes related to autoimmune diseases, autoinflammation and primary immunodeficiencies by NGS. MIS-C patients showed a significant increase of serum IFNγ (despite a significant reduction of activated Th1) and ILs, even if with a great heterogeneity among patients, revealing different pathways involved in MIS-C pathogenesis and suggesting that serum cytokines at admission may help to select the inflammatory pathways to target in each patient. Flow cytometry demonstrated a relevant reduction of T populations while the percentage of B cell was increased in agreement with an autoimmune pathogenesis of MIS-C. Genetic analysis identified variants in 34 genes and 83.3% of patients had at least one gene variant. Among these, 9 were mutated in more patients. Most genes are related to autoimmune diseases like ATM, NCF1, MCM4, FCN3, and DOCK8 or to autoinflammatory diseases associated to the release of IFNγ like PRF1, NOD2, and MEF. Thus, an incomplete clearance of the Sars-CoV2 during the acute phase may induce tissue damage and self-antigen exposure and genetic variants can predispose to hyper-reactive immune dysregulation events of MIS-C-syndrome. Type II IFN activation and cytokine responses (mainly IL-6 and IL-10) may cause a cytokine storm in some patients with a more severe acute phase of the disease, lymphopenia and multisystemic organ involvement. The timely identification of such patients with an immunocytometric panel might be critical for targeted therapeutic management.

Multi-gene panel testing increases germline predisposing mutations' detection in a cohort of breast/ovarian cancer patients from Southern Italy.

Breast cancer is the most common neoplasia in females worldwide, about 10% being hereditary/familial and due to DNA variants in cancer-predisposing genes, such as the highly penetrant BRCA1/BRCA2 genes. However, their variants explain up to 25% of the suspected hereditary/familial cases. The availability of NGS methodologies has prompted research in this field. With the aim to improve the diagnostic sensitivity of molecular testing, a custom designed panel of 44 genes, including also non-coding regions and 5' and 3' UTR regions, was set up. Here, are reported the results obtained in a cohort of 64 patients, including also few males, from Southern Italy. All patients had a positive personal and/or familial history for breast and other cancers, but tested negative to routine BRCA analysis. After obtaining their written informed consent, a genomic DNA sample/patient was used to obtain an enriched DNA library, then analyzed by NGS. Sequencing data analysis allowed the identification of pathogenic variants in 12 of tested patients (19%). Interestingly, MUTYH was the most frequently altered gene, followed by RNASEL, ATM, MSH6, MRE11A, and PALB2 genes. The reported resultsreinforce the need for enlarged molecular testing beyond BRCA genes, at least in patients with a personal and familial history, strongly suggestive for a hereditary/familial form. This gives also a hint to pursue more specific precision oncology therapy.

Microbiome composition indicate dysbiosis and lower richness in tumor breast tissues compared to healthy adjacent paired tissue, within the same women.

BACKGROUND: Breast cancer (BC) is the most common malignancy in women, in whom it reaches 20% of the total neoplasia incidence. Most BCs are considered sporadic and a number of factors, including familiarity, age, hormonal cycles and diet, have been reported to be BC risk factors. Also the gut microbiota plays a role in breast cancer development. In fact, its imbalance has been associated to various human diseases including cancer although a consequential cause-effect phenomenon has never been proven. METHODS: The aim of this work was to characterize the breast tissue microbiome in 34 women affected by BC using an NGS-based method, and analyzing the tumoral and the adjacent non-tumoral tissue of each patient. RESULTS: The healthy and tumor tissues differed in bacterial composition and richness: the number of Amplicon Sequence Variants (ASVs) was higher in healthy tissues than in tumor tissues (p = 0.001). Moreover, our analyses, able to investigate from phylum down to species taxa for each sample, revealed major differences in the two richest phyla, namely, Proteobacteria and Actinobacteria. Notably, the levels of Actinobacteria and Proteobacteria were, respectively, higher and lower in healthy with respect to tumor tissues. CONCLUSIONS: Our study provides information about the breast tissue microbial composition, as compared with very closely adjacent healthy tissue (paired samples within the same woman); the differences found are such to have possible diagnostic and therapeutic implications; further studies are necessary to clarify if the differences found in the breast tissue microbiome are simply an association or a concausative pathogenetic effect in BC. A comparison of different results on similar studies seems not to assess a universal microbiome signature, but single ones depending on the environmental cohorts' locations.

Case Report: Discovery a Novel SARS-CoV-2 Variant in a Six-Months Long-Term Swab Positive Female Suffering From Non-Hodgkin Lymphoma.

BACKGROUND: We report the case of a woman with non-Hodgkin lymphoma who remained positive on the molecular assay for SARS-CoV-2 for six months: she has never experienced a severe form of COVID-19 although in absence of seroconversion. METHODS: The whole SARS-CoV-2 genome analysis was performed by the CleanPlex SARS-CoV-2 Research and Surveillance NGS Panel (PARAGON GENOMICS, Hayward, USA). RESULTS: We found twenty-two mutations in SARS-CoV-2 genome and a novel deleterious ORF3a frameshift c.766_769del corresponding to a unique and novel lineage. The region affected by this frameshift variant is reported as being important in determining SARS-CoV-2 immunogenicity. Patient's immunophenotype showed the absence of B lymphocytes and significantly reduced T-cell count. Only after the treatment with hyperimmune plasma she finally became negative on the swab. CONCLUSIONS: Our findings could be helpful in the management of patients with immunodeficiency, particularly when novel variants, potentially altering the virus immune response, are present.

NGS Gene Panel Analysis Revealed Novel Mutations in Patients with Rare Congenital Diarrheal Disorders.

Congenital diarrheal disorders (CDDs) are early-onset enteropathies generally inherited as autosomal recessive traits. Most patients with CDDs require rapid diagnosis as they need immediate and specific therapy to avoid a poor prognosis, but their clinical picture is often overlapping with a myriad of nongenetic diarrheal diseases. We developed a next-generation sequencing (NGS) panel for the analysis of 92 CDD-related genes, by which we analyzed patients suspect for CDD, among which were (i) three patients with sucrose-isomaltase deficiency; (ii) four patients with microvillous inclusion disease; (iii) five patients with congenital tufting enteropathy; (iv) eight patients with glucose-galactose malabsorption; (v) five patients with congenital chloride diarrhea. In all cases, we identified the mutations in the disease-gene, among which were several novel mutations for which we defined pathogenicity using a combination of bioinformatic tools. Although CDDs are rare, all together, they have an incidence of about 1%. Considering that the clinical picture of these disorders is often confusing, a CDD-related multigene NGS panel contributes to unequivocal and rapid diagnosis, which also reduces the need for invasive procedures.

Step-Up Approach for Sodium Butyrate Treatment in Children With Congenital Chloride Diarrhea.

OBJECTIVES: Oral salt substitutive therapy is pivotal for the survival of patients with congenital chloride diarrhea (CLD), however this therapy is unable to influence the symptoms severity. Butyrate has been proposed to limit diarrhea severity in CLD. Unfortunately, the optimal dose schedule is still largely undefined. In addition, butyrate seems not to be well-tolerated by all patients, with some subjects reporting diarrhea worsening. We investigated the efficacy of a step-up therapeutic approach with sodium butyrate in patients who experienced a diarrhea worsening or an absent improvement after the direct administration of 100 mg/kg/day of sodium butyrate. METHODS: The efficacy of a step-up therapeutic approach starting from 50 mg/Kg/day with a subsequent 25 mg/kg/day weekly increase up to 100 mg/kg/day of oral sodium butyrate was investigated in previously three unresponsive CLD children. RESULTS: The step-up therapeutic approach resulted effective in limiting diarrhea severity in all our three previously unresponsive CLD patients. CONCLUSIONS: Our results suggest the efficacy of the step-up therapeutic approach in CLD children.

Congenital chloride diarrhea clinical features and management: a systematic review.

INTRODUCTION: Congenital chloride diarrhea (CLD) is a rare autosomal recessive disorder characterized by watery diarrhea with a high level of fecal Cl-, metabolic alkalosis, and electrolyte alterations. Several intestinal and extraintestinal complications and even death can occur. An optimal knowledge of the clinical features and best therapeutic strategies is mandatory for an effective management. METHODS: Articles published between 1 January 1965 and 31 December 2019, reported in PUBMED and EMBASE, were evaluated for a systematic review analyzing four categories: anamnestic features, clinical features, management, and follow-up strategies. RESULTS: Fifty-seven papers reporting information on 193 CLD patients were included. The most common anamnestic features were positive family anamnesis for chronic diarrhea (44.4%), consanguinity (75%), polyhydramnios (98.3%), preterm delivery (78.6%), and failure to pass meconium (60.7%). Mean age at diarrhea onset was 6.63 days. Median diagnostic delay was 60 days. Prenatal diagnosis, based on molecular analysis, was described in 40/172 (23.3%). All patients received NaCl/KCl-substitutive therapy. An improvement of diarrhea during adulthood was reported in 91.3% of cases. Failure to thrive (21.6%) and chronic kidney disease (17.7%) were the most common complications. CONCLUSIONS: This analysis of a large population suggests the necessity of better strategies for the management of CLD. A close follow-up and a multidisciplinary approach is mandatory to manage this condition characterized by heterogeneous and multisystemic complications. IMPACT: In this systematic review, we describe data regarding anamnestic features, clinical features, management, and follow-up of CLD patients obtained from the largest population of patients ever described to date. The results of our investigation could provide useful insights for the diagnostic approach and the management of this condition.

Extensive CFTR Gene Analysis Revealed a Higher Occurrence of Cystic Fibrosis Transmembrane Regulator-Related Disorders (CFTR-RD) among CF Carriers.

Background: A wide range of cystic fibrosis (CF)-related conditions are reported in CF carriers, but no study has explored the possibility that such subjects may be affected by cystic fibrosis transmembrane regulator-related disorders (CFTR-RD). No data are available so far on the occurrence of CFTR-RD among CF carriers. Methods: We studied 706 CF carriers-first- and second-degree relatives of CF patients that carried the parental mutation; such subjects were divided in two groups: a first group (353 subjects, group A) performed at first only the analysis of the CFTR proband mutation; we retrospectively evaluated the number of cases that had been diagnosed as CFTR-RD based on subsequent symptoms; a second group (353 subjects, group B) performed extensive CFTR molecular analysis in absence of any reported symptoms, followed by a clinical evaluation in cases that carry a second CFTR mutation; we evaluated the number of cases that prospectively were diagnosed as CFTR-RD. Results: We found seven (2.0%) out of 353 subjects of group A and 24 (6.8%) out of 353 subjects of group B as affected by CFTR-RD (chi square, p = 0.002). Conclusions: A percentage of CF carriers are affected by undiagnosed CFTR-RD. Genetic tasting scanning analysis helps to identify CFTR-RD, some of which may benefit from follow-up and specific therapies improving their outcome.

Genotype-Phenotype Correlation: A Triple DNA Mutational Event in a Boy Entering Sport Conveys an Additional Pathogenicity Risk.

The purpose of this paper is to present a clinical and laboratory study of a family, in which a 12-year-old boy was examined to assess his health status before starting competitive sports. A variety of clinical and instrumental tests were used to evaluate the status of the heart and its functions. Using Sanger sequencing (SS), we sequenced six related genes to verify suspected arrhythmogenic right ventricular cardiomyopathy (ARVC) hypothesized at the cardiac assessment and, subsequently, by a next-generation sequencing (NGS)-based multi-gene panel for more paramount genetic risk of sudden cardiac death (SCD) assessment. SS revealed two variants in the PKP2 gene, one was inherited from the father and the other from the mother. The analysis on a large panel of genes (n = 138), putatively associated with sudden cardiac death, revealed, in the proband, a third variant in a different gene (DES) that encodes the protein desmin. Our results indicate that: i) NGS revealed a mutational event in a gene not conventionally screened as a first-line test in the presence of clinical suspicion of the arrhythmic disease; ii) a plurality of variants in different genes in the same subject (the proband) may increase the risk of heart disease; iii) in silico analysis with various methodological software and bioinformatic prediction tools indicates that the cumulative effects of the three variants in the same subject constitute an additional risk factor. This case report indicates that more pathogenic variants or likely pathogenic variants can contribute to the clinical phenotype of an individual, thereby contributing to the diagnosis and prognosis of inherited heart diseases.

A Functional Analysis of the Unclassified Pro2767Ser BRCA2 Variant Reveals Its Potential Pathogenicity that Acts by Hampering DNA Binding and Homology-Mediated DNA Repair.

BRCA1 and BRCA2 are the genes most frequently associated with hereditary breast and ovarian cancer (HBOC). They are crucial for the maintenance of genome stability, particularly in the homologous recombination-mediated repair pathway of DNA double-strand breaks (HR-DSBR). Widespread BRCA1/2 next-generation sequencing (NGS) screening has revealed numerous variants of uncertain significance. Assessing the clinical significance of these variants is challenging, particularly regarding the clinical management of patients. Here, we report the functional characterization of the unclassified BRCA2 c.8299C > T variant, identified in a young breast cancer patient during BRCA1/2 NGS screening. This variant causes the change of Proline 2767 to Serine in the DNA binding domain (DBD) of the BRCA2 protein, necessary for the loading of RAD51 on ssDNA during the HR-DSBR. Our in silico analysis and 3D-structure modeling predicted that the p.Pro2767Ser substitution is likely to alter the BRCA2 DBD structure and function. Therefore, to evaluate the functional impact of the p.Pro2767Ser variant, we used a minigene encoding a truncated protein that contains the BRCA2 DBD and the nearby nuclear localization sequence. We found that the ectopically expressed truncated protein carrying the normal DBD, which retains the DNA binding function and lacks the central RAD51 binding domain, interferes with endogenous wild-type BRCA2 mediator functions in the HR-DSBR. We also demonstrated that the BRCA2 Pro2767Ser DBD is unable to compete with endogenous BRCA2 DNA binding, thereby suggesting that the p.Pro2767Ser substitution in the full-length protein causes the functional loss of BRCA2. Consequently, our data suggest that the p.Pro2767Ser variant should be considered pathogenic, thus supporting a revision of the ClinVar interpretation. Moreover, our experimental strategy could be a valid method with which to preliminarily evaluate the pathogenicity of the unclassified BRCA2 germline variants in the DBD and their risk of predisposing to HBOC.

A multi-gene panel beyond BRCA1/BRCA2 to identify new breast cancer-predisposing mutations by a picodroplet PCR followed by a next-generation sequencing strategy: a pilot study.

By analyzing multiple gene panels, next-generation sequencing is more effective than conventional procedures in identifying disease-related mutations that are useful for clinical decision-making. Here, we aimed to test the efficacy of an 84 genes customized-panel in BRCA1 and BRCA2 mutation-negative patients. Twenty-four patients were enrolled in this study. DNA libraries were prepared using a picodroplet PCR-based approach and sequenced with the MiSeq System. Highly putative pathogenic mutations were identified in genes other than the commonly tested BRCA1/2: 2 pathogenic mutations one in TP53 and one in MUTYH; 2 missense variants in MSH6 and ATM, respectively; 2 frameshift variants in KLLN, and ATAD2, respectively; an intronic variant in ANPEP, and 3 not functionally known variants (a frameshift variant in ATM a nonsense variant in ATM and a missense variant in NFE2L2). Our results show that this molecular screening will increase diagnostic sensitivity leading to a better risk assessment in breast cancer patients and their families. This strategy could also reveal genes that have a higher penetrance for breast and ovarian cancers by matching gene mutation with familial and clinical data, thereby increasing information about hereditary breast and ovarian cancer genetics and improving cancer prevention measures or therapeutic approaches.

Setup of Quantitative PCR for Oral Neisseria spp. Evaluation in Celiac Disease Diagnosis.

Coeliac disease (CD) is a multifactorial autoimmune disorder and gut dysbiosis contributes to its pathogenesis. We previously profiled by 16S rRNA sequencing duodenal and oropharyngeal microbiomes in active CD (a-CD), gluten-free diet (GFD) patients, and controls (CO) and found significantly higher levels of Neisseria spp., with pro-inflammatory activities, in a-CD patients than in the other two groups. In this study, we developed a fast and simple qPCR-based method to evaluate the abundance of the oral Neisseria spp. and the diagnostic performances of the test in CD diagnosis. The Neisseria spp. abundances detected by quantitative PCR (qPCR) were: CO = 0.14, GFD = 0.15, a-CD = 2.08, showing a similar trend to those previously measured by next generation sequencing (NGS). In particular, Neisseria spp. values obtained by both methods were significantly higher (p < 0.001) in a-CD than in the other two groups GFD and CO-the latter almost overlapping. We calculated by ROC curve analysis the threshold of 1.12 ng/µL of Neisseria spp. to discriminate between CO+GFD and a-CD patients with 100% and 96.7% of diagnostic sensitivity and specificity, respectively. In conclusion, our data, if confirmed in other cohorts, suggest the q-PCR evaluation of oral Neisseria spp. could be a fast and simple method to assess CD-associated dysbiosis for diagnostic purposes.

Oropharyngeal microbiome evaluation highlights Neisseria abundance in active celiac patients.

We previously profiled duodenal microbiome in active (a-), gluten-free diet (GFD) celiac disease (CD) patients and controls finding higher levels of the Proteobacterium Neisseria flavescens in a-CD patients than in the other two groups. Here, we investigate the oropharyngeal microbiome in CD patients and controls to evaluate whether this niche share microbial composition with the duodenum. We characterized by 16S rRNA gene sequencing the oropharyngeal microbiome in 14 a-CD, 22 GFD patients and 20 controls. Bacteroidetes, Proteobacteria and Firmicutes differed significantly between the three groups. In particular, Proteobacteria abounded in a-CD and Neisseria species mostly accounted for this abundance (p < 0.001), whereas Bacteroidetes were more present in control and GFD microbiomes. Culture-based oropharyngeal microbiota analysis confirmed the greater abundance of Proteobacteria and of Neisseria species in a-CD. Microbial functions prediction indicated a greater metabolic potential for degradation of aminoacids, lipids and ketone bodies in a-CD microbiome than in control and GFD microbiomes, in which polysaccharide metabolism predominated. Our results suggest a continuum of a-CD microbial composition from mouth to duodenum. We may speculate that microbiome characterization in the oropharynx, which is a less invasive sampling than the duodenum, could contribute to investigate the role of dysbiosis in CD pathogenesis.

The Cause of Death of a Child in the 18th Century Solved by Bone Microbiome Typing Using Laser Microdissection and Next Generation Sequencing.

The history of medicine abounds in cases of mysterious deaths, especially by infectious diseases, which were probably unresolved because of the lack of knowledge and of appropriate technology. The aim of this study was to exploit contemporary technologies to try to identify the cause of death of a young boy who died from a putative "infection" at the end of the 18th century, and for whom an extraordinarily well-preserved minute bone fragment was available. After confirming the nature of the sample, we used laser microdissection to select the most "informative" area to be examined. Tissue genotyping indicated male gender, thereby confirming the notary's report. 16S ribosomal RNA sequencing showed that Proteobacteria and Actinobacteria were more abundant than Firmicutes and Bacteroidetes, and that Pseudomonas was the most abundant bacterial genus in the Pseudomonadaceae family. These data suggest that the patient most likely died from Pseudomonas osteomyelitis. This case is an example of how new technological approaches, like laser microdissection and next-generation sequencing, can resolve ancient cases of uncertain etiopathology. Lastly, medical samples may contain a wealth of information that may not be accessible until more sophisticated technology becomes available. Therefore, one may envisage the possibility of systematically storing medical samples for evaluation by future generations.

A Novel Pathogenic BRCA1 Splicing Variant Produces Partial Intron Retention in the Mature Messenger RNA.

About 10% of all breast cancers arise from hereditary mutations that increase the risk of breast and ovarian cancers; and about 25% of these are associated with the BRCA1 or BRCA2 genes. The identification of BRCA1/BRCA2 mutations can enable physicians to better tailor the clinical management of patients; and to initiate preventive measures in healthy carriers. The pathophysiological significance of newly identified variants poses challenges for genetic counseling. We characterized a new BRCA1 variant discovered in a breast cancer patient during BRCA1/2 screening by next-generation sequencing. Bioinformatic predictions; indicating that the variant is probably pathogenetic; were verified using retro-transcription of the patient's RNA followed by PCR amplifications performed on the resulting cDNA. The variant causes the loss of a canonic donor splice site at position +2 in BRCA1 intron 21; and consequently the partial retention of 156 bp of intron 21 in the patient's transcript; which demonstrates that this novel BRCA1 mutation plays a pathogenetic role in breast cancer. These findings enabled us to initiate appropriate counseling and to tailor the clinical management of this family. Lastly; these data reinforce the importance of studying the effects of sequence variants at the RNA level to verify their potential role in disease onset.

Cracking the Code of Human Diseases Using Next-Generation Sequencing: Applications, Challenges, and Perspectives.

Next-generation sequencing (NGS) technologies have greatly impacted on every field of molecular research mainly because they reduce costs and increase throughput of DNA sequencing. These features, together with the technology's flexibility, have opened the way to a variety of applications including the study of the molecular basis of human diseases. Several analytical approaches have been developed to selectively enrich regions of interest from the whole genome in order to identify germinal and/or somatic sequence variants and to study DNA methylation. These approaches are now widely used in research, and they are already being used in routine molecular diagnostics. However, some issues are still controversial, namely, standardization of methods, data analysis and storage, and ethical aspects. Besides providing an overview of the NGS-based approaches most frequently used to study the molecular basis of human diseases at DNA level, we discuss the principal challenges and applications of NGS in the field of human genomics.