Genetic Basis of Breast and Ovarian Cancer: Approaches and Lessons Learnt from Three Decades of Inherited Predisposition Testing.

Germline variants occurring in BRCA1 and BRCA2 give rise to hereditary breast and ovarian cancer (HBOC) syndrome, predisposing to breast, ovarian, fallopian tube, and peritoneal cancers marked by elevated incidences of genomic aberrations that correspond to poor prognoses. These genes are in fact involved in genetic integrity, particularly in the process of homologous recombination (HR) DNA repair, a high-fidelity repair system for mending DNA double-strand breaks. In addition to its implication in HBOC pathogenesis, the impairment of HR has become a prime target for therapeutic intervention utilizing poly (ADP-ribose) polymerase (PARP) inhibitors. In the present review, we introduce the molecular roles of HR orchestrated by BRCA1 and BRCA2 within the framework of sensitivity to PARP inhibitors. We examine the genetic architecture underneath breast and ovarian cancer ranging from high- and mid- to low-penetrant predisposing genes and taking into account both germline and somatic variations. Finally, we consider higher levels of complexity of the genomic landscape such as polygenic risk scores and other approaches aiming to optimize therapeutic and preventive strategies for breast and ovarian cancer.

Reassessment of the NF1 variants of unknown significance found during the 20-year activity of a genetics diagnostic laboratory.

The finding of variants of uncertain significance (VUS) in the activity of a diagnostic genetic laboratory is a common issue, which is however provisional and needs to be periodically re-evaluated, due to the continuous advancements in our knowledge of the genetic diseases. Neurofibromatosis type 1, caused by the occurrence of heterozygous pathogenic NF1 variants, is a good model for studying the evolution of VUS, due to the widespread use of genetic testing for the disease, the constant enrichment of the international databases with NF1 variants and the full adult penetrance of the disease, which makes genotyping the parents a crucial step in the diagnostic workflow. The present study retrospectively reviewed and reinterpreted the genetic test results of NF1 in a diagnostic genetic laboratory in the period from January 1, 2000 to December 31, 2020. All the VUS were reinterpreted using the 2015 consensus standards and guidelines for the interpretation. Out of 589 NF1 genetic tests which were performed in the period, a total of 85 VUS were found and reinterpreted in 72 cases (84.7%): 21 (29.2%) were reclassified as benign/likely benign, whereas 51 (70.8%) were recoded as pathogenic/likely pathogenic with a significant trend distribution (Chi square test for trend p = 0.005). Synonymous VUS have mainly been reclassified as class 1 and 2 (7/8, 87.5%), whereas missense variants have been attributed to class 4 and 5 in 38 out of the 58 cases (65.5%). These findings underline an improvement in the classification of variants over time, suggesting that a reinterpretation of the genetic tests should be routinely performed to support the physicians in the clinical diagnosis of genetic diseases.

Success and Pitfalls of Genetic Testing in Undiagnosed Diseases: Whole Exome Sequencing and Beyond.

Novel approaches to uncover the molecular etiology of neurodevelopmental disorders (NDD) are highly needed. Even using a powerful tool such as whole exome sequencing (WES), the diagnostic process may still prove long and arduous due to the high clinical and genetic heterogeneity of these conditions. The main strategies to improve the diagnostic rate are based on family segregation, re-evaluation of the clinical features by reverse-phenotyping, re-analysis of unsolved NGS-based cases and epigenetic functional studies. In this article, we described three selected cases from a cohort of patients with NDD in which trio WES was applied, in order to underline the typical challenges encountered during the diagnostic process: (1) an ultra-rare condition caused by a missense variant in MEIS2, identified through the updated Solve-RD re-analysis; (2) a patient with Noonan-like features in which the NGS analysis revealed a novel variant in NIPBL causing Cornelia de Lange syndrome; and (3) a case with de novo variants in genes involved in the chromatin-remodeling complex, for which the study of the epigenetic signature excluded a pathogenic role. In this perspective, we aimed to (i) provide an example of the relevance of the genetic re-analysis of all unsolved cases through network projects on rare diseases; (ii) point out the role and the uncertainties of the reverse phenotyping in the interpretation of the genetic results; and (iii) describe the use of methylation signatures in neurodevelopmental syndromes for the validation of the variants of uncertain significance.

Genetics of ANCA-associated vasculitis: role in pathogenesis, classification and management.

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) comprises granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA) and eosinophilic granulomatosis with polyangiitis (EGPA), that share features of pauci-immune small-vessel vasculitis and the positivity of ANCA targeting proteinase-3 (PR3-ANCA) or myeloperoxidase (MPO-ANCA). AAV syndromes are rare, complex diseases and their aetio-pathogenesis is mainly driven by the interaction between environmental and genetic factors. In patients with GPA and MPA, the genetic associations are stronger with ANCA specificity (PR3- versus MPO-ANCA) than with the clinical diagnosis, which, in keeping with the known clinical and prognostic differences between PR3-ANCA-positive and MPO-ANCA-positive patients, supports an ANCA-based re-classification of these disorders. EGPA is also made up of genetically distinct subsets, which can be stratified on ANCA-status (MPO ANCA-positive versus ANCA-negative); these subsets differ in clinical phenotype and possibly in their response to treatment. Interestingly, MPO-ANCA-positive patients with either MPA or EGPA have overlapping genetic determinants, thus strengthening the concept that this EGPA subset is closely related to the other AAV syndromes. The genetics of AAV provides us with essential information to understand its varied phenotype. This Review discusses the main findings of genetic association studies in AAV, their pathogenic implications and their potential effect on classification, management and prognosis.

Recurrent NF1 gene variants and their genotype/phenotype correlations in patients with Neurofibromatosis type I.

Neurofibromatosis type I, a genetic condition due to pathogenic variants in the NF1 gene, is burdened by a high rate of complications, including neoplasms, which increase morbidity and mortality for the disease. We retrospectively re-evaluated the NF1 gene variants found in the period 2000-2019 and we studied for genotype/phenotype correlations of disease complications and neoplasms 34 variants, which were shared by at least two unrelated families (range 2-11) for a total 141 of probands and 21 relatives affected by Neurofibromatosis type I. Recurrent variants could be ascribed to the most common mutational mechanisms (C to T transition, microsatellite slippage, non-homologous recombination). In genotype/phenotype correlations, the variants p.Arg440*, p.Tyr489Cys, and p.Arg1947*, together with the gross gene deletions, displayed the highest rates of complications. When considering neoplasms, carriers of variants falling in the extradomain region at the 5' end of NF1 had a lower age-related cancer frequency than the rest of the gene sequence, showing a borderline significance (p = 0.045), which was not conserved after correction with covariates. We conclude that (1) hotspots in NF1 occur via different mutational mechanisms, (2) several variants are associated with high rates of complications and cancers, and (3) there is an initial evidence toward a lower cancer risk for carriers of variants in the 5' end of the NF1 gene although not significant at the multivariate analysis.

Clinical and Genetic Findings in a Series of Eight Families with Arthrogryposis.

The term "arthrogryposis" is used to indicate multiple congenital contractures affecting two or more areas of the body. Arthrogryposis is the consequence of an impairment of embryofetal neuromuscular function and development. The causes of arthrogryposis are multiple, and in newborns, it is difficult to predict the molecular defect as well as the clinical evolution just based on clinical findings. We studied a consecutive series of 13 participants who had amyoplasia, distal arthrogryposis (DA), or syndromic forms of arthrogryposis with normal intellectual development and other motor abilities. The underlying pathogenic variants were identified in 11 out of 13 participants. Correlating the genotype with the clinical features indicated that prenatal findings were specific for DA; this was helpful to identify familial cases, but features were non-specific for the involved gene. Perinatal clinical findings were similar among the participants, except for amyoplasia. Dilatation of the aortic root led to the diagnosis of Loeys-Dietz syndrome (LDS) in one case. The phenotype of DA type 5D (DA5D) and Escobar syndrome became more characteristic at later ages due to more pronounced pterygia. Follow-up indicated that DA type 1 (DA1)/DA type 2B (DA2B) spectrum and LDS had a more favorable course than the other forms. Hand clenching and talipes equinovarus/rocker bottom foot showed an improvement in all participants, and adducted thumb resolved in all forms except in amyoplasia. The combination of clinical evaluation with Next Generation Sequencing (NGS) analysis in the newborn may allow for an early diagnosis and, particularly in the DAs, suggests a favorable prognosis.

Results and Clinical Interpretation of Germline RET Analysis in a Series of Patients with Medullary Thyroid Carcinoma: The Challenge of the Variants of Uncertain Significance.

Germline RET variants are responsible for approximately 25% of medullary thyroid carcinoma (MTC) cases. Identification of RET variant carriers allows for the adoption of preventative measures which are dependent on the risk associated with the specific alteration. From 2002 to 2020, at our cancer genetics clinic, RET genetic testing was performed in 163 subjects (102 complete gene analyses and 61 targeted analyses), 72 of whom presented with MTC. A germline RET variant was identified in 31.9% of patients affected by MTC (93.8% of those having positive family history and 14.3% of clinically sporadic cases). Subsequent target testing in relatives allowed us to identify 22 asymptomatic carriers, who could undertake appropriate screening. Overall, patients with germline RET variants differed significantly from those who tested negative by family history (p < 0.001) and mean age at MTC diagnosis (44.45 vs. 56.42 years; p = 0.010), but the difference was not significant when only carriers of moderate risk variants were considered (51.78 vs. 56.42 years; p = 0.281). Out of 12 different variants detected in 49 patients, five (41.7%) were of uncertain significance (VUS). For two of these, p.Ser904Phe and p.Asp631_Leu633delinsGlu, co-segregation and genotype/phenotype analysis, matched with data from the literature, provided evidence supporting their classification in the moderate and the highest/high risk class (with a MEN2B phenotype), respectively.

Genome-wide association study of eosinophilic granulomatosis with polyangiitis reveals genomic loci stratified by ANCA status.

Eosinophilic granulomatosis with polyangiitis (EGPA) is a rare inflammatory disease of unknown cause. 30% of patients have anti-neutrophil cytoplasmic antibodies (ANCA) specific for myeloperoxidase (MPO). Here, we describe a genome-wide association study in 676 EGPA cases and 6809 controls, that identifies 4 EGPA-associated loci through conventional case-control analysis, and 4 additional associations through a conditional false discovery rate approach. Many variants are also associated with asthma and six are associated with eosinophil count in the general population. Through Mendelian randomisation, we show that a primary tendency to eosinophilia contributes to EGPA susceptibility. Stratification by ANCA reveals that EGPA comprises two genetically and clinically distinct syndromes. MPO+ ANCA EGPA is an eosinophilic autoimmune disease sharing certain clinical features and an HLA-DQ association with MPO+ ANCA-associated vasculitis, while ANCA-negative EGPA may instead have a mucosal/barrier dysfunction origin. Four candidate genes are targets of therapies in development, supporting their exploration in EGPA.

Spectrum of X-linked intellectual disabilities and psychiatric symptoms in a family harbouring a Xp22.12 microduplication encompassing the RPS6KA3 gene.

Microduplications of the X chromosome are a rare cause of X-linked intellectual disability (XLID), a clinically and genetically heterogeneous spectrum of disorders. In the present study, a 950-kb Xp22.12 microduplication including the RPS6KA3 gene was detected in affected members of a family, including the proband (male), his mother and one maternal uncle. Four female carriers had major depression and one of them also had mild intellectual disability. The present and previous cases with overlapping microduplications suggest that Xp22.12 microduplications can be included in the neuropsychiatric copy number variations.

Association of a polymorphism of the Fcγ-receptor 2A (FCGR2A) gene with chronic periaortitis.

OBJECTIVES: Chronic periaortitis (CP) is an inflammatory disease associated in 20-60% of the cases with IgG4 related disease. Current evidence supports an autoimmune nature for CP. Fc gamma receptors (FcγRs) are involved in several immune system activities and are associated with autoimmunity in general. We explored the influence of genetic variants within this region on susceptibility to CP. METHODS: Genotyping of 4 candidate single nucleotide polymorphisms (SNPs) of the FCGR region was performed in CP patients and controls. RESULTS: One hundred and eighty-three cases and 181 controls were included. An association between the SNP rs1801274 of the FCGR2A and CP was detected (OR 1.6, 95%CI 1.18-2.16;corrected p-value, pcorr=0.0085). After stratification of the population according to clinical characteristics, the association was restricted to cases of idiopathic retroperitoneal fibrosis (OR 1.66, 95%CI 1.21-2.29;pcorr=0.028), without involvement of the thoracic aorta (OR 1.77, 95%CI 1.21-2.57;pcorr=0.043), with deep vein thrombosis at onset (OR 3.96, 95%CI 1.81-8.66;pcorr=0.0021) and with normal IgG4 levels (OR 2.67, 95%CI 1.39-5.12;pcorr=0.031). CONCLUSIONS: In the largest candidate gene approach study performed so far in CP, we demonstrated an association for CP with a gene hallmark of autoimmunity. The association appears restricted to typical cases of CP without increase of IgG4 levels.

Neurofibromatosis type I and multiple myeloma coexistence: A possible link?

The association between Neurofibromatosis type I (NF1) and multiple myeloma (MM), a plasma cell, dyscrasia is very rare. Here we put to the attention of the scientific community two new cases. The first one is a patient with active MM whereas the second with smoldering MM. Both patients present typical features of NF1 but skeletal alterations were present only in the second case including dysplasia, marked scoliosis and osteoporosis. MM osteolytic lesions were absent in both patients. In addition to the clinical diagnosis of NF1, a molecular testing for NF1 gene mutations has been performed finding that patient one was heterozygous for the c.6855C>A (Tyr2285Ter) mutation, while patient two was heterozygous for the c.7838dupC (Lys2614GlufsTer20) mutation. The two mutations were diagnosed both in genomic DNA from peripheral blood and from MM cells. The potential link between NF1 mutation and the increased risk of MM is discussed in the report.

FCGR2A single nucleotide polymorphism confers susceptibility to childhood-onset idiopathic nephrotic syndrome.

Childhood-onset idiopathic nephrotic syndrome affects 1.15-3.4 children/100,000 children/year in Western Countries. Immune-mediated mechanisms, particularly T cell-mediated, are thought to play a key pathogenic role. The genetic basis of the disease is still poorly understood. We tested the association between single nucleotide polymorphisms (SNPs) of four genes encoding Fc gamma receptors (FCGR2A, FCGR2B, FCGR3A, FCGR3B) and idiopathic nephrotic syndrome in a case-control study of paediatric patients. Children with idiopathic nephrotic syndrome (aged 1-16 years) were included. FCGR2A rs1801274 and FCGR3A rs396991 SNPs were genotyped using real-time PCR with the TaqMan method, while FCGR2B rs1050501 and FCGR3B NA1/NA2 were genotyped using Sanger sequencing. Fisher's exact test was used to explore genetic association. We enrolled 103 idiopathic nephrotic syndrome patients and 181 healthy controls. A significant association was found between idiopathic nephrotic syndrome and FCGR2A rs1801274 SNP (both with the T allele and the TT genotype, p value=0.0009, OR 1.81, 95% CI 1.27-2.59 and p value=0.0007, OR 2.39, 95% CI 1.44-3.99, respectively). No associations were found for the remaining SNPs. Fc gamma receptors might modulate response to rituximab; since 60 of the enrolled patients were treated with rituximab, we also tested the association between the studied SNPs and rituximab efficacy in this patient subgroup, but found only a weak association with FCGR2A CC genotype (p value=0.03). The FCGR2A rs1801274 SNP in the gene encoding the activating receptor CD32A confers susceptibility to idiopathic nephrotic syndrome.

Patterns of Novel Alleles and Genotype/Phenotype Correlations Resulting from the Analysis of 108 Previously Undetected Mutations in Patients Affected by Neurofibromatosis Type I.

Neurofibromatosis type I, a genetic disorder due to mutations in the NF1 gene, is characterized by a high mutation rate (about 50% of the cases are de novo) but, with the exception of whole gene deletions associated with a more severe phenotype, no specific hotspots and few solid genotype/phenotype correlations. After retrospectively re-evaluating all NF1 gene variants found in the diagnostic activity, we studied 108 patients affected by neurofibromatosis type I who harbored mutations that had not been previously reported in the international databases, with the aim of analyzing their type and distribution along the gene and of correlating them with the phenotypic features of the affected patients. Out of the 108 previously unreported variants, 14 were inherited by one of the affected parents and 94 were de novo. Twenty-nine (26.9%) mutations were of uncertain significance, whereas 79 (73.2%) were predicted as pathogenic or probably pathogenic. No differential distribution in the exons or in the protein domains was observed and no statistically significant genotype/phenotype correlation was found, confirming previous evidences.

F7 gene variants modulate protein levels in a large cohort of patients with factor VII deficiency. Results from a genotype-phenotype study.

Congenital factor VII (FVII) deficiency is a rare bleeding disorder caused by mutations in F7 gene with autosomal recessive inheritance. A clinical heterogeneity with poor correlation with FVII:C levels has been described. It was the objective of this study to identify genetic defects and to evaluate their relationships with phenotype in a large cohort of patients with FVII:C<50 %. One hundred twenty-three probands were genotyped for F7 mutations and three polymorphic variants and classified according to recently published clinical scores. Forty out of 123 patients (33 %) were symptomatic (43 bleedings). A severe bleeding tendency was observed only in patients with FVII:C<0.10 %. Epistaxis (11 %) and menorrhagia (32 % of females in fertile age) were the most frequent bleedings. Molecular analysis detected 48 mutations, 20 not reported in the F7 international databases. Most mutations (62 %) were missense, large deletions were 6.2 %. Compound heterozygotes/homozygotes for mutations presented lower FVII:C levels compared to the other classes (Chi2=43.709, p<0,001). The polymorphisms distribution was significantly different among the three F7 genotypic groups (Chi2=72.289, p<0,001). The presence of truncating mutations was associated with lowest FVII:C levels (Chi2=21.351, p=0.002). This study confirms the clinical and molecular variability of the disease and the type of symptoms. It shows a good correlation between the type of F7 mutation and/or polymorphisms and FVII:C levels, without a direct link between FVII:C and bleeding tendency. The results suggest that large deletions are underestimated and that they represent a common mechanism of F7 gene inactivation which should always be investigated in the diagnostic testing for FVII deficiency.

Monogenic Autoinflammatory Diseases with Mendelian Inheritance: Genes, Mutations, and Genotype/Phenotype Correlations.

Autoinflammatory diseases (AIDs) are a genetically heterogeneous group of diseases caused by mutations of genes encoding proteins, which play a pivotal role in the regulation of the inflammatory response. In the pathogenesis of AIDs, the role of the genetic background is triggered by environmental factors through the modulation of the innate immune system. Monogenic AIDs are characterized by Mendelian inheritance and are caused by highly penetrant genetic variants in single genes. During the last years, remarkable progress has been made in the identification of disease-associated genes by using new technologies, such as next-generation sequencing, which has allowed the genetic characterization in undiagnosed patients and in sporadic cases by means of targeted resequencing of a gene panel and whole exome sequencing. In this review, we delineate the genetics of the monogenic AIDs, report the role of the most common gene mutations, and describe the evidences of the most sound genotype/phenotype correlations in AID.

Giant elephantiasis neuromatosa in the setting of neurofibromatosis type 1: A case report.

Elephantiasis neuromatosa (EN) can arise from a plexiform neurofibroma of the superficial and deep nerves developing from a hyperproliferation of the perineural connective tissue infiltrating adjacent fat and muscles. To date, the clinical association between EN and neurofibromatosis type 1 (NF1) has been poorly defined, particularly with regard to the role of lymphatic alterations and the consequent lymphedema. The present study reports the clinical and biomolecular features of EN in a NF1 patient with the clear clinical diagnostic criteria of multiple cafè-au-lait macules, neurofibromas, EN, a positive family history and a novel NF1 germline c.1541_1542del mutation. Lymphoscintigraphy (LS) highlighted marked dermal backflow in the affected limb, hypertrophy of the ipsilateral inguinal and external iliac lymph nodes, and a bilateral lower limb lymph flow delay. These data support the hypothesis that an extensive hyperproliferative process involving perineural connective, limb soft tissues, bones and the lymphatic system can be responsible for EN in NF1 patients, on the basis of adipocyte metaplasia triggered by lymphostasis and lymphedema, and bone overgrowth and gigantism caused by chronic hyperemia. LS and magnetic resonance imaging can be efficacious tools in the diagnosis and clinical characterization of the early onset of the disease.