Next Generation Sequencing (NGS) Target Approach for Undiagnosed Dysglycaemia.

Next-generation sequencing (NGS) has revolutionized the field of genomics and created new opportunities for basic research. We described the strategy for the NGS validation of the "dysglycaemia panel" composed by 44 genes related to glucose metabolism disorders (MODY, Wolfram syndrome) and familial renal glycosuria using Ion AmpliSeq technology combined with Ion-PGM. Anonymized DNA of 32 previously genotyped cases with 33 different variants were used to optimize the methodology. Standard protocol was used to generate the primer design, library, template preparation, and sequencing. Ion Reporter tool was used for data analysis. In all the runs, the mean coverage was over 200×. Twenty-nine out of thirty three variants (96.5%) were detected; four frameshift variants were missed. All point mutations were detected with high sensitivity. We identified three further variants of unknown significance in addition to pathogenic mutations previously identified by Sanger sequencing. The NGS panel allowed us to identify pathogenic variants in multiple genes in a short time. This could help to identify several defects in children and young adults that have to receive the genetic diagnosis necessary for optimal treatment. In order not to lose any pathogenic variants, Sanger sequencing is included in our analytical protocol to avoid missing frameshift variants.

Clinical Consequences and Functional Impact of the Rare S737F CFTR Variant and Its Responsiveness to CFTR Modulators.

S737F is a Cystic Fibrosis (CF) transmembrane conductance regulator (CFTR) missense variant. The aim of our study was to describe the clinical features of a cohort of individuals carrying this variant. In parallel, by exploiting ex vivo functional and molecular analyses on nasal epithelia derived from a subset of S737F carriers, we evaluated its functional impact on CFTR protein as well as its responsiveness to CFTR modulators. We retrospectively collected clinical data of all individuals bearing at least one S737F CFTR variant and followed at the CF Centre of Tuscany region (Italy). Nasal brushing was performed in cooperating individuals. At study end clinical data were available for 10 subjects (mean age: 14 years; range 1-44 years; 3 adult individuals). Five asymptomatic subjects had CF, 2 were CRMS/CFSPID and 3 had an inconclusive diagnosis. Ex vivo analysis on nasal epithelia demonstrated different levels of CF activity. In particular, epithelia derived from asymptomatic CF subjects and from one of the subjects with inconclusive diagnosis showed reduced CFTR activity that could be rescued by treatment with CFTR modulators. On the contrary, in the epithelia derived from the other two individuals with an inconclusive diagnosis, the CFTR-mediated current was similar to that observed in epithelia derived from healthy donors. In vitro functional and biochemical analysis on S737F-CFTR expressed in immortalized bronchial cells highlighted a modest impairment of the channel activity, that was improved by treatment with ivacaftor alone or in combination with tezacaftor/elexacaftor. Our study provide evidence towards the evaluation of CFTR function on ex vivo nasal epithelial cell models as a new assay to help clinicians to classify individuals, in presence of discordance between clinical picture, sweat test and genetic profile.

"Pesto" Mutation: Phenotypic and Genotypic Characteristics of Eight GCK/MODY Ligurian Patients.

Maturity Onset Diabetes of the Young (MODY) is a monogenic form of diabetes mellitus (DM) that accounts for around 2-5% of all types of diabetes. Autosomal dominant inheritance in pathogenic variations of 14 genes related to ß-cell functions can lead to monogenic types of diabetes. In Italy, GCK/MODY is the most frequent form and it is caused by mutations of the glucokinase (GCK). Patients with GCK/MODY usually have stable mild fasting hyperglycaemia with mildly elevated HbA1c levels and rarely need pharmacological treatment. Molecular analysis of the GCK coding exons was carried out by Sanger sequencing in eight Italian patients. All the probands were found to be heterozygous carriers of a pathogenic gross insertion/deletion c.1279_1358delinsTTACA; p.Ser426_Ala454delinsLeuGln. It was previously described for the first time by our group in a large cohort of Italian GCK/MODY patients. The higher levels of HbA1c (6.57% vs. 6.1%), and the higher percentage of patients requiring insulin therapy (25% vs. 2%) compared to the previously studied Italian patients with GCK/MODY, suggest that the mutation discovered could be responsible for a clinically worse form of GCK/MODY. Moreover, as all the patients carrying this variant share an origin from the same geographic area (Liguria), we postulate a possible founder effect and we propose to name it the "pesto" mutation.

Rescue by elexacaftor-tezacaftor-ivacaftor of the G1244E cystic fibrosis mutation's stability and gating defects are dependent on cell background.

BACKGROUND: Cystic fibrosis is caused by mutations impairing expression, trafficking, stability and/or activity of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. The G1244E mutation causes a severe gating defect that it is not completely rescued by ivacaftor but requires the use of a second compound (a co-potentiator). Recently, it has been proposed that the corrector elexacaftor may act also as a co-potentiator. METHODS: By using molecular, biochemical and functional analyses we performed an in-depth characterization of the G1244E-CFTR mutant in heterologous and native cell models. RESULTS: Our studies demonstrate that processing and function of the mutant protein, as well as its pharmacological sensitivity, are markedly dependent on cell background. In heterologous expression systems, elexacaftor mainly acted on G1244E-CFTR as a co-potentiator, thus ameliorating the gating defect. On the contrary, in the native nasal epithelial cell model, elexacaftor did not act as a co-potentiator, but it increased mature CFTR expression possibly by improving mutant's defective stability at the plasma membrane. CONCLUSIONS: Our study highlights the importance of the cell background in the evaluation of CFTR modulator effects. Further, our results draw attention to the need for the development of novel potentiators having different mechanisms with respect to ivacaftor to improve channel activity for mutants with severe gating defect.

An example of parenchymal renal sparing in the context of complex malformations due to a novel mutation in the PBX1 gene.

INTRODUCTION: PBX1 encodes the pre-B cell leukemia factor 1, a Three Amino acid Loop Extension (TALE) transcription factor crucial to regulate basic developmental processes. PBX1 loss-of-function variants have been initially described in association with renal malformations in both isolated and syndromic forms. CASE REPORT: Herein, we report a male infant presenting multiple organ malformations (cleidosternal dysostosis, micrognathia, left lung hypoplasia, wide interatrial defect, pulmonary hypertension, total anomalous pulmonary venous return, intestinal malrotation) and carrying the heterozygous de novo c.868C > T (p.Arg290Trp) variant in PBX1. This novel variant affects the highly conserved homeodomain of the protein, leading to a non-conservative substitution and consequently altering its tridimensional structure and DNA-binding capacity. CONCLUSION: So far, PBX1 has been reported in association with a broad spectrum of renal anomalies. However, given the role of this gene in many different developing processes, whole-exome sequencing can detect mutations in PBX1 even in patients with different phenotypes, not necessarily involving the renal primordium. This report presents a novel PBX1 variant with a predicted strong deleterious effect. The mutation leads to a non-conservative substitution in a very highly conserved domain of the protein, thus altering its tertiary structure and DNA-binding capacity.

The L467F-F508del Complex Allele Hampers Pharmacological Rescue of Mutant CFTR by Elexacaftor/Tezacaftor/Ivacaftor in Cystic Fibrosis Patients: The Value of the Ex Vivo Nasal Epithelial Model to Address Non-Responders to CFTR-Modulating Drugs.

Loss-of-function mutations of the CFTR gene cause cystic fibrosis (CF) through a variety of molecular mechanisms involving altered expression, trafficking, and/or activity of the CFTR chloride channel. The most frequent mutation among CF patients, F508del, causes multiple defects that can be, however, overcome by a combination of three pharmacological agents that improve CFTR channel trafficking and gating, namely, elexacaftor, tezacaftor, and ivacaftor. This study was prompted by the evidence of two CF patients, compound heterozygous for F508del and a minimal function variant, who failed to obtain any beneficial effects following treatment with the triple drug combination. Functional studies on nasal epithelia generated in vitro from these patients confirmed the lack of response to pharmacological treatment. Molecular characterization highlighted the presence of an additional amino acid substitution, L467F, in cis with the F508del variant, demonstrating that both patients were carriers of a complex allele. Functional and biochemical assays in heterologous expression systems demonstrated that the double mutant L467F-F508del has a severely reduced activity, with negligible rescue by CFTR modulators. While further studies are needed to investigate the actual prevalence of the L467F-F508del allele, our results suggest that this complex allele should be taken into consideration as plausible cause in CF patients not responding to CFTR modulators.

Fibrodysplasia Ossificans Progressiva: What Have We Achieved and Where Are We Now? Follow-up to the 2015 Lorentz Workshop.

Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare progressive genetic disease effecting one in a million individuals. During their life, patients with FOP progressively develop bone in the soft tissues resulting in increasing immobility and early death. A mutation in the ACVR1 gene was identified as the causative mutation of FOP in 2006. After this, the pathophysiology of FOP has been further elucidated through the efforts of research groups worldwide. In 2015, a workshop was held to gather these groups and discuss the new challenges in FOP research. Here we present an overview and update on these topics.

A case of Fibrodysplasia Ossificans Progressiva associated with a novel variant of the ACVR1 gene.

BACKGROUND: Fibrodysplasia Ossificans Progressiva (FOP) is a rare autosomal dominant disease characterized by congenital malformation of the great toes and progressive heterotopic ossification of soft tissues leading to cumulative disability. The genetic cause of FOP are mutations in the ACVR1 gene that encodes a type I receptor of Bone Morphogenetic Proteins. The most recurrent mutation in FOP patients is R206H affecting the Glycine-Serine rich domain and causing the hyper-activation of the receptor and the responsivity to the non-canonical ligand, Activin A. In the present study, we described a 3-years old child with early and highly suggestive clinical features of FOP who was found negative for the recurrent p.R206H substitution. METHODS: Molecular screening of the whole ACVR1 coding sequence and functional characterization in transfection-based assays. RESULTS AND CONCLUSIONS: We identified a novel, de novo variant in the fifth ACVR1 coding exon (NM_001111067.4:c.772A>T; NP_001104537.1:p.(R258W)). This substitution, never reported in association with FOP, affects a conserved arginine residue in the kinase domain of the protein. In silico analysis predicted the pathogenicity of this substitution, demonstrated by in vitro assays showing that the p.R258W ACVR1 mutated receptor acquires the ability to transduce the aberrant Activin A-mediated signaling, as observed for the gene variants associated with FOP.

Genotype-Phenotype Correlations in Neurofibromatosis Type 1: A Single-Center Cohort Study.

Neurofibromatosis type 1 (NF1) is a proteiform genetic condition caused by pathogenic variants in NF1 and characterized by a heterogeneous phenotypic presentation. Relevant genotype-phenotype correlations have recently emerged, but only few pertinent studies are available. We retrospectively reviewed clinical, instrumental, and genetic data from a cohort of 583 individuals meeting at least 1 diagnostic National Institutes of Health (NIH) criterion for NF1. Of these, 365 subjects fulfilled ≥2 NIH criteria, including 235 pediatric patients. Genetic testing was performed through cDNA-based sequencing, Next Generation Sequencing (NGS), and Multiplex Ligation-dependent Probe Amplification (MLPA). Uni- and multivariate statistical analysis was used to investigate genotype-phenotype correlations. Among patients fulfilling ≥ 2 NIH criteria, causative single nucleotide variants (SNVs) and copy number variations (CNVs) were detected in 267/365 (73.2%) and 20/365 (5.5%) cases. Missense variants negatively correlated with neurofibromas (p = 0.005). Skeletal abnormalities were associated with whole gene deletions (p = 0.05) and frameshift variants (p = 0.006). The c.3721C>T; p.(R1241*) variant positively correlated with structural brain alterations (p = 0.031), whereas Lisch nodules (p = 0.05) and endocrinological disorders (p = 0.043) were associated with the c.6855C>A; p.(Y2285*) variant. We identified novel NF1 genotype-phenotype correlations and provided an overview of known associations, supporting their potential relevance in the implementation of patient management.

Evaluating the Influence of a G-Quadruplex Prone Sequence on the Transactivation Potential by Wild-Type and/or Mutant P53 Family Proteins through a Yeast-Based Functional Assay.

P53, P63, and P73 proteins belong to the P53 family of transcription factors, sharing a common gene organization that, from the P1 and P2 promoters, produces two groups of mRNAs encoding proteins with different N-terminal regions; moreover, alternative splicing events at C-terminus further contribute to the generation of multiple isoforms. P53 family proteins can influence a plethora of cellular pathways mainly through the direct binding to specific DNA sequences known as response elements (REs), and the transactivation of the corresponding target genes. However, the transcriptional activation by P53 family members can be regulated at multiple levels, including the DNA topology at responsive promoters. Here, by using a yeast-based functional assay, we evaluated the influence that a G-quadruplex (G4) prone sequence adjacent to the p53 RE derived from the apoptotic PUMA target gene can exert on the transactivation potential of full-length and N-terminal truncated P53 family α isoforms (wild-type and mutant). Our results show that the presence of a G4 prone sequence upstream or downstream of the P53 RE leads to significant changes in the relative activity of P53 family proteins, emphasizing the potential role of structural DNA features as modifiers of P53 family functions at target promoter sites.

Genomic Context and Mechanisms of the ACVR1 Mutation in Fibrodysplasia Ossificans Progressiva.

Basic research in Fibrodysplasia Ossificans Progressiva (FOP) was carried out in the various fields involved in the disease pathophysiology and was important for designing therapeutic approaches, some of which were already developed as ongoing or planned clinical trials. Genetic research was fundamental in identifying the FOP causative mutation, and the astonishing progress in technologies for genomic analysis, coupled to related computational methods, now make possible further research in this field. We present here a review of molecular and cellular factors which could explain why a single mutation, the R206H in the ACVR1 gene, is absolutely prevalent in FOP patients. We also address the mechanisms by which FOP expressivity could be modulated by cis-acting variants in the ACVR1 genomic region in human chromosome 2q. Finally, we also discuss the general issue of genetic modifiers in FOP.

Genetic and Acquired Heterotopic Ossification: A Translational Tale of Mice and Men.

Heterotopic ossification is defined as an aberrant formation of bone in extraskeletal soft tissue, for which both genetic and acquired conditions are known. This pathologic process may occur in many different sites such as the skin, subcutaneous tissue, skeletal muscle and fibrous tissue adjacent to joints, ligaments, walls of blood vessels, mesentery and other. The clinical spectrum of this disorder is wide: lesions may range from small foci of ossification to massive deposits of bone throughout the body, typical of the progressive genetically determined conditions such as fibrodysplasia ossificans progressiva, to mention one of the most severe and disabling forms. The ectopic bone formation may be regarded as a failed tissue repair process in response to a variety of triggers and evolving towards bone formation through a multistage differentiation program, with several steps common to different clinical presentations and distinctive features. In this review, we aim at providing a comprehensive view of the genetic and acquired heterotopic ossification disorders by detailing the clinical and molecular features underlying the different human conditions in comparison with the corresponding, currently available mouse models.

P63 modulates the expression of the WDFY2 gene which is implicated in cancer regulation and limb development.

TP63 is a member of the TP53 gene family, sharing a common gene structure that produces two groups of mRNAs' encoding proteins with different N-terminal regions (ΔN and TA isoforms); both transcripts are also subjected to alternative splicing mechanisms at C-terminus, generating a variety of isoforms. p63 is a master regulator of epidermal development and homoeostasis as well as an important player in tumorigenesis and cancer progression with both oncogenic and tumour suppressive roles. A number of studies have aimed at the identification of p63 target genes, allowing the dissection of the molecular pathways orchestrated by the different isoforms. In the present study we investigated in more detail the p63 responsiveness of the WDFY2 (WD repeat and FYVE domain containing 2) gene, encoding for an endosomal protein identified as a binding partner of the PI-3K/AKT signalling pathway. We showed that overexpression of different p63 isoforms was able to induce WDFY2 expression in TP53-null cells. The p63-dependent transcriptional activation was associated with specific response elements (REs) that have been identified by a bioinformatics tool and validated by yeast- and mammal-based assays. Interestingly, to confirm that WDFY2 belongs to the p63 network of cancer regulation, we analysed the impact of WDFY2 alterations, by showing its frequent deletion in different types of tumours and suggesting its expression level as a prognostic biomarker. Lastly, we identified a chromosomal translocation involving the WDFY2 locus in a patient affected by a rare congenital limb anomaly, indicating WDFY2 as a possible susceptibility gene placed downstream p63 in the network of limb development.

Severe Heterotopic Ossification in the Skeletal Muscle and Endothelial Cells Recruitment to Chondrogenesis Are Enhanced by Monocyte/Macrophage Depletion.

Altered macrophage infiltration upon tissue damage results in inadequate healing due to inappropriate remodeling and stem cell recruitment and differentiation. We investigated in vivo whether cells of endothelial origin phenotypically change upon heterotopic ossification induction and whether infiltration of innate immunity cells influences their commitment and alters the ectopic bone formation. Liposome-encapsulated clodronate was used to assess macrophage impact on endothelial cells in the skeletal muscle upon acute damage in the ECs specific lineage-tracing Cdh5CreERT2:R26REYFP/dtTomato transgenic mice. Macrophage depletion in the injured skeletal muscle partially shifts the fate of ECs toward endochondral differentiation. Upon ectopic stimulation of BMP signaling, monocyte depletion leads to an enhanced contribution of ECs chondrogenesis and to ectopic bone formation, with increased bone volume and density, that is reversed by ACVR1/SMAD pathway inhibitor dipyridamole. This suggests that macrophages contribute to preserve endothelial fate and to limit the bone lesion in a BMP/injury-induced mouse model of heterotopic ossification. Therefore, alterations of the macrophage-endothelial axis may represent a novel target for molecular intervention in heterotopic ossification.

Identification of reference genes for quantitative PCR during C3H10T1/2 chondrogenic differentiation.

C3H10T1/2, a mouse mesenchymal stem cell line, is a well-known in vitro model of chondrogenesis that can be easily employed to recapitulate some of the mechanisms intervening in this process. Moreover, these cells can be used to validate the effect of candidate molecules identified by high throughput screening approaches applied to the development of targeted therapy for human disorders in which chondrogenic differentiation may be involved, as in conditions characterized by heterotopic endochondral bone formation. Chondrogenic differentiation of C3H10T1/2 cells can be monitored by applying quantitative polymerase chain reaction (qPCR), one of the most sensitive methods that allows detection of small dynamic changes in gene expression between samples obtained under different experimental conditions. In this work, we have used qPCR to monitor the expression of specific markers during chondrogenic differentiation of C3H10T1/2 cells in micromass cultures. Then we have applied the geNorm approach to identify the most stable reference genes suitable to get a robust normalization of the obtained expression data. Among 12 candidate reference genes (Ap3d1, Csnk2a2, Cdc40, Fbxw2, Fbxo38, Htatsf1, Mon2, Pak1ip1, Zfp91, 18S, ActB, GAPDH) we identified Mon2 and Ap3d1 as the most stable ones during chondrogenesis. ActB, GAPDH and 18S, the most commonly used in the literature, resulted to have an expression level too high compared to the differentiation markers (Sox9, Collagen type 2a1, Collagen type 10a1 and Collagen type 1a1), therefore are actually less recommended for these experimental conditions. In conclusion, we identified nine reference genes that can be equally used to obtain a robust normalization of the gene expression variation during the C3H10T1/2 chondrogenic differentiation.

The Horizon of a Therapy for Rare Genetic Diseases: A "Druggable" Future for Fibrodysplasia Ossificans Progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic condition characterized by progressive extra-skeletal ossification leading to cumulative and severe disability. FOP has an extremely variable and episodic course and can be induced by trauma, infections, iatrogenic harms, immunization or can occur in an unpredictable way, without any recognizable trigger. The causative gene is ACVR1, encoding the Alk-2 type I receptor for bone morphogenetic proteins (BMPs). The signaling is initiated by BMP binding to a receptor complex consisting of type I and II molecules and can proceed into the cell through two main pathways, a canonical, SMAD-dependent signaling and a p38-mediated cascade. Most FOP patients carry the recurrent R206H substitution in the receptor Glycine-Serine rich (GS) domain, whereas a few other mutations are responsible for a limited number of cases. Mutations cause a dysregulation of the downstream BMP-dependent pathway and make mutated ACVR1 responsive to a non-canonical ligand, Activin A. There is no etiologic treatment for FOP. However, many efforts are currently ongoing to find specific therapies targeting the receptor activity and the downstream aberrant pathway at different levels or targeting cellular components and/or processes that are important in modifying the local environment leading to bone neo-formation.

Correction to: Rarely occurring mutation of ACVR1 gene in Moroccan patient with fibrodysplasia ossificans progressiva.

One of the author's name on this article was incorrectly spelled as "Renata Borcciadi". The correct spelling is "Renata Bocciardi" and is now presented correctly in this article.

Peripheral Blood Mononuclear Cell Immunophenotyping in Fibrodysplasia Ossificans Progressiva Patients: Evidence for Monocyte DNAM1 Up-regulation.

BACKGROUND: Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder caused by sporadic heterozygous mutations in ACVR1 gene which progressively leads to severe heterotopic ossification. FOP is characterized by episodic flare-ups triggered by different factors such as viral infections, tissue injuries, vaccinations, or occurring without a recognizable cause. The sporadic course of the disease, the documented presence of an important inflammatory reaction in early lesions and the partial response to corticosteroids support the idea that the immune system, and in particular the innate component, may play a role in FOP pathogenesis. However, an extensive expression profile of the peripheral blood mononuclear cells (PBMC) of FOP patients has never been done. METHODS: In this study, we carried out a wide PBMC immunophenotyping on a cohort of FOP patients and matching controls by multiparametric analysis of the expression of a panel of 37 markers associated with migration, adhesion, inhibition, activation, and cell death of circulating immune cells. RESULTS: We observed a statistically significant increase of the expression of DNAM1 receptor in patients' monocytes as compared to controls, and little but significant differences in the expression profile of CXCR1 (CD181), CD62L, CXCR4 (CD184), and HLA-DR molecules. CONCLUSIONS: DNAM1 had been previously shown to play a pivotal role in monocyte migration through the endothelial barrier and the increased expression detected in patients' monocytes might suggest a role of this surface receptor during the early phases of FOP flare-ups in which the activation of the immune response is believed to represent a crucial event. © 2017 International Clinical Cytometry Society.

Hints on transcriptional control of essential players in heterotopic ossification of Fibrodysplasia Ossificans Progressiva.

Signaling of the Bone Morphogenetic Protein (BMP) pathway is influenced by the level of expression of its components, in particular receptors, intracellular molecules and target genes which largely depends on gene transcription. One peculiar aspect of Fibrodysplasia Ossificans Progressiva (FOP) relates to the cell types in which the genetic mutation exerts its effects, then not only those involved in the heterotopic ossification processes but also others that participate in the inflammatory phases preceding and triggering heterotopic ossification. Such effects are in part detectable as variation in gene expression, which is also variably manifesting in term of time of appearance in different phases of the inflammatory or ossification processes.