Genomic instability in congenital lung malformations in children.

PURPOSE: To study the biological relationship between congenital lung malformations (CLMs) and malignancy. METHODS: Biopsies of 12 CPAMs, 6 intralobar sequestrations and 2 extralobar sequestrations were analyzed through whole-genome sequencing. Blood samples from 10 patients were used to confirm or exclude somatic mosaicism. Putative somatic Single Nucleotide Variants (SNVs) were called for each malformed sample with a Panel of Normals built with control DNA samples extracted from blood. The variants were subsequently confirmed by Sanger sequencing and searched, whenever possible, in the blood samples of patients. RESULTS: All CLMs but one presented a signature of genomic instability by means of multiple clusters of cells with gene mutations. Seven tumor transformation-related SNVs were detected in 6/20 congenital lung malformations. Four very rare in the general population SNVs were found in a region previously linked to lung cancer in 5p15.33, upstream of TERT oncogene. Furthermore, we identified missense genetic variants, whose tumorigenic role is well known, in the RET, FANCA and MET genes. CONCLUSIONS: Genomic instability in 95% of CLMs and genetic variants linked to tumor development in 30% of them, regardless of histopathology, are predisposing factors to malignancy, that combined with exposure to carcinogens, might trigger the development of malignancy and explain the association between CLMs and lung cancer.

A loss-of-function NCSTN mutation associated with familial Dowling Degos disease and hidradenitis suppurativa.

Dowling Degos disease (DDD) is a rare autosomal dominant genodermatosis characterized by acquired, slowly progressive reticulated pigmented lesions primarily involving flexural skin areas. Mutations in KRT5, POGLUT-1 and POFUT-1 genes have been associated with DDD, and loss-of-function mutations in PSENEN, a subunit of the gamma-secretase complex, were found in patients presenting with DDD or DDD comorbid with hidradenitis suppurativa (HS). A nonsense mutation in NCSTN, another subunit of the gamma-secretase, was already described in a patient suffering from HS and DDD but whether NCSTN could be considered a novel gene for DDD is still debated. Here, we enrolled a four-generation family with HS and DDD. Through Whole Exome Sequencing (WES) we identified a novel nonsense mutation in the NCSTN gene in all the affected family members. To study the impact of this variant, we isolated outer root sheath cells from patients' hair follicles. We showed that this variant leads to a premature stop codon, activates a nonsense-mediated mRNA decay, and causes NCSTN haploinsufficiency in affected individuals. In fact, cells treated with gentamicin, a readthrough agent, had the NCSTN levels corrected. Moreover, we observed that this haploinsufficiency also affects other subunits of the gamma-secretase complex, possibly causing DDD. Our findings clearly support NCSTN as a novel DDD gene and suggest carefully investigating this co-occurrence in HS patients carrying a mutation in the NCSTN gene.

A rare loss-of-function genetic mutation suggest a role of dermcidin deficiency in hidradenitis suppurativa pathogenesis.

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease with a multifactorial aetiology that involves a strict interplay between genetic factors, immune dysregulation and lifestyle. Familial forms represent around 40% of total HS cases and show an autosomal dominant mode of inheritance of the disease. In this study, we conducted a whole-exome sequence analysis on an Italian family of 4 members encompassing a vertical transmission of HS. Focusing on rare damaging variants, we identified a rare insertion of one nucleotide (c.225dupA:p.A76Sfs*21) in the DCD gene encoding for the antimicrobial peptide dermcidin (DCD) that was shared by the proband, his affected father and his 11-years old daughter. Since several transcriptome studies have shown a significantly decreased expression of DCD in HS skin, we hypothesised that the identified frameshift insertion was a loss-of-function mutation that might be associated with HS susceptibility in this family. We thus confirmed by mass spectrometry that DCD levels were diminished in the affected members and showed that the antimicrobial activity of a synthetic DCD peptide resulting from the frameshift mutation was impaired. In order to define the consequences related to a decrease in DCD activity, skin microbiome analyses of different body sites were performed by comparing DCD mutant and wild type samples, and results highlighted significant differences between the groins of mutated and wild type groups. Starting from genetic analysis conducted on an HS family, our findings showed, confirming previous transcriptome results, the potential role of the antimicrobial DCD peptide as an actor playing a crucial part in the etio-pathogenesis of HS and in the maintenance of the skin's physiological microbiome composition; so, we can hypothesise that DCD could be used as a novel target for personalised therapeutic approach.

Things come in threes: A new complex allele and a novel deletion within the CFTR gene complicate an accurate diagnosis of cystic fibrosis.

BACKGROUND: Despite consolidated guidelines, the clinical diagnosis and prognosis of cystic fibrosis (CF) is still challenging mainly because of the extensive phenotypic heterogeneity and the high number of CFTR variants, including their combinations as complex alleles. RESULTS: We report a family with a complicated syndromic phenotype, which led to the suspicion not only of CF, but of a dominantly inherited skeletal dysplasia (SD). Whereas the molecular basis of the SD was not clarified, segregation analysis was central to make a correct molecular diagnosis of CF, as it allowed to identify three CFTR variants encompassing two known maternal mutations and a novel paternal microdeletion. CONCLUSION: This case well illustrates possible pitfalls in the clinical and molecular diagnosis of CF; presence of complex phenotypes deflecting clinicians from appropriate CF recognition, and/or identification of two mutations assumed to be in trans but with an unconfirmed status, which underline the importance of an in-depth molecular CFTR analysis.

MYO5B Gene Mutations: A Not Negligible Cause of Intrahepatic Cholestasis of Infancy With Normal Gamma-Glutamyl Transferase Phenotype.

OBJECTIVES: Progressive familial intrahepatic cholestasis is an expanding group of autosomal recessive intrahepatic cholestatic disorders. Recently, next-generation sequencing allowed identifying new genes responsible for new specific disorders. Two biochemical phenotypes have been identified according to gamma-glutamyltransferase (GGT) activity. Mutations of the myosin 5B gene (MYO5B) are known to cause microvillus inclusion disease. Recently, different mutations in MYO5B gene have been reported in patients with low-GGT cholestasis. METHODS: A multicenter retrospective and prospective study was conducted in 32 children with cryptogenic intrahepatic cholestasis. Clinical, biochemical, histological, and treatment data were analyzed in these patients. DNA from peripheral blood was extracted, and all patients were studied by whole exome sequencing followed by Sanger sequencing. RESULTS: Six patients out of 32 had mutations in the MYO5B gene. Of these six patients, the median age at disease onset was 0.8 years, and the median length of follow-up was 4.2 years. The most common signs were pruritus, poor growth, hepatomegaly, jaundice, and hypocholic stools. Two patients also showed intestinal involvement. Transaminases and conjugated bilirubin were moderately increased, serum bile acids elevated, and GGT persistently normal. At anti-Myo5B immunostaining, performed in liver biopsy of two patients, coarse granules were evident within the cytoplasm of hepatocytes while bile salt export pump was normally expressed at the canalicular membrane. Six variants in homozygosity or compound heterozygosity in the MYO5B gene were identified, and three of them have never been described before. All nucleotide alterations were located on the myosin motor domain except one missense variant found in the isoleucine-glutamine calmodulin-binding motif. CONCLUSIONS: We identified causative mutations in MYO5B in 18.7% of a selected cohort of patients with intrahepatic cholestasis confirming a relevant role for the MYO5B gene in low-GGT cholestasis.

Whole-Genome Methylation Study of Congenital Lung Malformations in Children.

BACKGROUND AND OBJECTIVES: The treatment of asymptomatic patients with congenital pulmonary malformations (CPMs) remains controversial, partially because the relationship between congenital lung malformations and malignancy is still undefined. Change in methylation pattern is a crucial event in human cancer, including lung cancer. We therefore studied all differentially methylated regions (DMRs) in a series of CPMs in an attempt to find methylation anomalies in genes already described in association with malignancy. METHODS: The DNA extracted from resected congenital lung malformations and control lung tissue was screened using Illumina MethylationEPIC arrays. Comparisons between the group of malformed samples or the malformed samples of same histology or each malformed sample and the controls and between a pleuropulmonary blastoma (PPB) and controls were performed. Moreover, each malformed sample was pairwise compared with its respective control. All differentially methylated regions (DMRs) with an adjusted p-value <0,05 were studied. RESULTS: Every comparison highlighted a number of DMRs closed to genes involved either in cell proliferation or in embryonic development or included in the Cancer Gene Census. Their abnormal methylation had been already described in lung tumors. CONCLUSIONS: Methylation anomalies already described in lung tumors and also shared by the PPB were found in congenital lung malformations, regardless the histology. The presence of methylation abnormalities is suggestive of a correlation between congenital lung malformations and some step of malignant transformation.

Systematic analysis of factors that improve homologous direct repair (HDR) efficiency in CRISPR/Cas9 technique.

The CRISPR/Cas9 bacterial system has proven to be an powerful tool for genetic manipulation in several organisms, but the efficiency of sequence replacement by homologous direct repair (HDR) is substantially lower than random indel creation. Many studies focused on improving HDR efficiency using double sgRNA, cell synchronization cycle, and the delivery of single-stranded oligo DNA nucleotides (ssODN) with a rational design. In this study, we evaluate these three methods' synergistic effects to improve HDR efficiency. For our tests, we have chosen the TNFα gene (NM_000594) for its crucial role in various biological processes and diseases. For the first time, our results showed how the use of two sgRNA with asymmetric donor design and triple transfection events dramatically increase the HDR efficiency from an undetectable HDR event to 39% of HDR efficiency and provide a new strategy to facilitate CRISPR/Cas9-mediated human genome editing. Besides, we demonstrated that the TNFα locus could be edited with CRISPR/Cas9 methodology, an opportunity to safely correct, in the future, the specific mutations of each patient.

Heterogeneity in Circulating Tumor Cells: The Relevance of the Stem-Cell Subset.

The release of circulating tumor cells (CTCs) into vasculature is an early event in the metastatic process. The analysis of CTCs in patients has recently received widespread attention because of its clinical implications, particularly for precision medicine. Accumulated evidence documents a large heterogeneity in CTCs across patients. Currently, the most accepted view is that tumor cells with an intermediate phenotype between epithelial and mesenchymal have the highest plasticity. Indeed, the existence of a meta-stable or partial epithelial⁻mesenchymal transition (EMT) cell state, with both epithelial and mesenchymal features, can be easily reconciled with the concept of a highly plastic stem-like state. A close connection between EMT and cancer stem cells (CSC) traits, with enhanced metastatic competence and drug resistance, has also been described. Accordingly, a subset of CTCs consisting of CSC, present a stemness profile, are able to survive chemotherapy, and generate metastases after xenotransplantation in immunodeficient mice. In the present review, we discuss the current evidence connecting CTCs, EMT, and stemness. An improved understanding of the CTC/EMT/CSC connections may uncover novel therapeutic targets, irrespective of the tumor type, since most cancers seem to harbor a pool of CSCs, and disclose important mechanisms underlying tumorigenicity.

Target sequencing approach intended to discover new mutations in non-syndromic intellectual disability.

The technological improvements over the last years made considerable progresses in the knowledge of the etiology of intellectual Disability (ID). However, at present very little is known about the genetic heterogeneity underlying the non-syndromic form of ID (NS-ID). To investigate the genetic basis of NS-ID we analyzed 43 trios and 22 isolated NS-ID patients using a targeted sequencing (TS) approach. 71 NS-ID genes have been selected and sequenced in all subjects. We found putative pathogenic mutations in 7 out of 65 patients. The pathogenic role of mutations was evaluated through sequence comparison and structural analysis was performed to predict the effect of alterations in a 3D computational model through molecular dynamics simulations. Additionally, a deep patient clinical re-evaluation has been performed after the molecular results. This approach allowed us to find novel pathogenic mutations with a detection rate close to 11% in our cohort of patients. This result supports the hypothesis that many NS-ID related genes still remain to be discovered and that NS-ID is a more complex phenotype compared to syndromic form, likely caused by a complex and broad interaction between genes alterations and environment factors.

A CASQ1 founder mutation in three Italian families with protein aggregate myopathy and hyperCKaemia.

BACKGROUND: Protein aggregate myopathies are increasingly recognised conditions characterised by a surplus of endogenous proteins. The molecular and mutational background for many protein aggregate myopathies has been clarified with the discovery of several underlying mutations. Familial idiopathic hyperCKaemia is a benign genetically heterogeneous condition with autosomal dominant features in a high proportion of cases. METHODS: In 10 patients from three Italian families with autosomal dominant benign vacuolar myopathy and hyperCKaemia, we performed linkage analysis and exome sequencing as well as morphological and biochemical investigations. RESULTS AND CONCLUSIONS: We show, by Sanger and exome sequencing, that the protein aggregate myopathy with benign evolution and muscle inclusions composed of excess CASQ1, affecting three Italian families, is due to the D244G heterozygous missense mutation in the CASQ1 gene. Investigation of microsatellite markers revealed a common haplotype in the three families indicating consanguinity and a founder effect. Results from immunocytochemistry, electron microscopy, biochemistry and transfected cell line investigations contribute to our understanding of pathogenetic mechanisms underlining this defect. The mutation is common to other Italian patients and is likely to share a founder effect with them. HyperCKaemia in the CASQ1-related myopathy is common and sometimes the sole overt manifestation. It is likely that CASQ1 mutations may remain undiagnosed if a muscle biopsy is not performed, and the condition could be more common than supposed.

DNA mismatch repair gene MSH6 implicated in determining age at natural menopause.

The length of female reproductive lifespan is associated with multiple adverse outcomes, including breast cancer, cardiovascular disease and infertility. The biological processes that govern the timing of the beginning and end of reproductive life are not well understood. Genetic variants are known to contribute to ∼50% of the variation in both age at menarche and menopause, but to date the known genes explain <15% of the genetic component. We have used genome-wide association in a bivariate meta-analysis of both traits to identify genes involved in determining reproductive lifespan. We observed significant genetic correlation between the two traits using genome-wide complex trait analysis. However, we found no robust statistical evidence for individual variants with an effect on both traits. A novel association with age at menopause was detected for a variant rs1800932 in the mismatch repair gene MSH6 (P = 1.9 × 10(-9)), which was also associated with altered expression levels of MSH6 mRNA in multiple tissues. This study contributes to the growing evidence that DNA repair processes play a key role in ovarian ageing and could be an important therapeutic target for infertility.

A novel CRYBB2 missense mutation causing congenital autosomal dominant cataract in an Italian family.

Congenital cataract is a leading cause of visual impairment in children and brings approximately 10% of childhood blindness worldwide. Molecular analysis revealed ~60 loci to be associated with several phenotypes of childhood cataracts. Until now, more than 30 loci and 18 genes on different chromosomes have been associated with autosomal dominant congenital cataract (ADCC). Here, we present a three-generation Italian family with a non syndromic ADCC. A linkage analysis carried out using HumanCytoSNP-12 DNA Analysis BeadChip led us to identify ten genomic regions virtually involved in the disease. All the genes located in these regions were scored for possible relationship with ADCC and, according to a strict clinical and genetic selection, 4 genes have been analyzed. A novel sequence variant was found in the CRYBB2 gene (p.Ser143Phe). This variant affects a conserved aminoacid in the third Greek key motif of the protein, cosegregates with the disease phenotype in all affected individuals and is not present both in the unaffected family members and 100 healthy control subjects. Finally, we identified the first CRYBB2 mutation in an Italian family causing a clinical picture of ADCC.