Cellular Senescence: The Driving Force of Musculoskeletal Diseases.

The aging of the world population is closely associated with an increased prevalence of musculoskeletal disorders, such as osteoporosis, sarcopenia, and osteoarthritis, due to common genetic, endocrine, and mechanical risk factors. These conditions are characterized by degeneration of bone, muscle, and cartilage tissue, resulting in an increased risk of fractures and reduced mobility. Importantly, a crucial role in the pathophysiology of these diseases has been proposed for cellular senescence, a state of irreversible cell cycle arrest induced by factors such as DNA damage, telomere shortening, and mitochondrial dysfunction. In addition, senescent cells secrete pro-inflammatory molecules, called senescence-associated secretory phenotype (SASP), which can alter tissue homeostasis and promote disease progression. Undoubtedly, targeting senescent cells and their secretory profiles could promote the development of integrated strategies, including regular exercise and a balanced diet or the use of senolytics and senomorphs, to improve the quality of life of the aging population. Therefore, our review aimed to highlight the role of cellular senescence in age-related musculoskeletal diseases, summarizing the main underlying mechanisms and potential anti-senescence strategies for the treatment of osteoporosis, sarcopenia, and osteoarthritis.

Different neuropsychological and brain volumetric profiles in a pair of identical twins with myotonic dystrophy type 1 indicate a non-genetic modulation of clinical phenotype.

We report on genetic and environmental modulation of social cognition abilities and brain volume correlates in two monozygotic twins (Twin1 and Twin2) with genetically confirmed myotonic dystrophy-type1 who grew up in different environmental settings. They both underwent neuropsychological assessment (i.e., Intelligent Quotient [IQ], theory of mind, emotion recognition tests), and MRI scanning to evaluate regional brain volumetrics compared to 10 gender and sex-matched healthy controls. Against a normal IQ level in both patients, Twin1 was more impaired in emotional processing and Twin2 in cognitive aspects of social cognition. Both patients showed grey matter (GM) atrophy in Brodmann Areas 23/31 (BA23/31) and BA7 bilaterally, while Twin2 showed additional GM loss in right BA46. Both patients showed a similar pattern of white matter atrophy involving the thalamus, basal ganglia, and uncinate fasciculus. White matter atrophy appeared to be mostly driven by genetics, while grey matter volumes appeared associated with different impairments in social cognition and possibly modulated by environment.

In Cis Effect of DMPK Expanded Alleles in Myotonic Dystrophy Type 1 Patients Carrying Variant Repeats at 5' and 3' Ends of the CTG Array.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant multisystemic disease caused by a CTG repeat expansion in the 3'-untranslated region (UTR) of DMPK gene. DM1 alleles containing non-CTG variant repeats (VRs) have been described, with uncertain molecular and clinical consequences. The expanded trinucleotide array is flanked by two CpG islands, and the presence of VRs could confer an additional level of epigenetic variability. This study aims to investigate the association between VR-containing DMPK alleles, parental inheritance and methylation pattern of the DM1 locus. The DM1 mutation has been characterized in 20 patients using a combination of SR-PCR, TP-PCR, modified TP-PCR and LR-PCR. Non-CTG motifs have been confirmed by Sanger sequencing. The methylation pattern of the DM1 locus was determined by bisulfite pyrosequencing. We characterized 7 patients with VRs within the CTG tract at 5' end and 13 patients carrying non-CTG sequences at 3' end of the DM1 expansion. DMPK alleles with VRs at 5' end or 3' end were invariably unmethylated upstream of the CTG expansion. Interestingly, DM1 patients with VRs at the 3' end showed higher methylation levels in the downstream island of the CTG repeat tract, preferentially when the disease allele was maternally inherited. Our results suggest a potential correlation between VRs, parental origin of the mutation and methylation pattern of the DMPK expanded alleles. A differential CpG methylation status could play a role in the phenotypic variability of DM1 patients, representing a potentially useful diagnostic tool.

Role of the Vitamin D Receptor (VDR) in the Pathogenesis of Osteoporosis: A Genetic, Epigenetic and Molecular Pilot Study.

The vitamin D receptor (VDR) regulates bone development and calcium homeostasis, suggesting a central role in musculoskeletal diseases such as osteoporosis (OP). Several studies have examined the contribution of VDR polymorphisms and epigenetic signatures in bone metabolism and OP risk, with sometimes inconclusive results. Our study aimed to explore the association between genetic variability, expression and the methylation pattern of VDR with the risk of OP in a cohort of Caucasian patients. Genomic DNA from 139 OP, 54 osteopenic (Ope) and 73 healthy (CTR) subjects were used for genotyping the rs731236 (TaqI), rs2228570 (FokI) and rs11568820 (Cdx2) polymorphisms of the VDR gene by an allelic discrimination assay. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of VDR expression levels and pyrosequencing analysis of a VDR promoter CpG island were carried out in a subcohort (25 OP and 25 CTR) of subjects. Data obtained showed a significantly higher OP risk for rs11568820 G/A and A/A genotypes (p = 0.05). qRT-PCR revealed lower VDR gene expression levels in the OP group compared to CTR subjects (p = 0.0009), also associated with both the rs11568820 A/A genotype (p = 0.03) and femoral fragility fractures (p = 0.05). No association was found between the methylation pattern of the region analyzed of the VDR promoter and its expression levels. Our results identify a significative association between Cdx2 rs11568820 polymorphism and OP risk. In addition, the VDR transcriptomic profile suggests a putative interconnection with OP progression, providing a useful tool to stratify OP phenotype and fragility fracture risk.

Methylation of the Vitamin D Receptor Gene in Human Disorders.

The Vitamin D Receptor (VDR) mediates the actions of 1,25-Dihydroxvitamin D3 (1,25(OH)2D3), which has important roles in bone homeostasis, growth/differentiation of cells, immune functions, and reduction of inflammation. Emerging evidences suggest that epigenetic modifications of the VDR gene, particularly DNA methylation, may contribute to the onset and progression of many human disorders. This review aims to summarize the available information on the role of VDR methylation signatures in different pathological contexts, including autoimmune diseases, infectious diseases, cancer, and others. The reversible nature of DNA methylation could enable the development of therapeutic strategies, offering new avenues for the management of these worldwide diseases.

Characterization of full-length CNBP expanded alleles in myotonic dystrophy type 2 patients by Cas9-mediated enrichment and nanopore sequencing.

Myotonic dystrophy type 2 (DM2) is caused by CCTG repeat expansions in the CNBP gene, comprising 75 to >11,000 units and featuring extensive mosaicism, making it challenging to sequence fully expanded alleles. To overcome these limitations, we used PCR-free Cas9-mediated nanopore sequencing to characterize CNBP repeat expansions at the single-nucleotide level in nine DM2 patients. The length of normal and expanded alleles can be assessed precisely using this strategy, agreeing with traditional methods, and revealing the degree of mosaicism. We also sequenced an entire ~50 kbp expansion, which has not been achieved previously for DM2 or any other repeat-expansion disorders. Our approach precisely counted the repeats and identified the repeat pattern for both short interrupted and uninterrupted alleles. Interestingly, in the expanded alleles, only two DM2 samples featured the expected pure CCTG repeat pattern, while the other seven presented also TCTG blocks at the 3' end, which have not been reported before in DM2 patients, but confirmed hereby with orthogonal methods. The demonstrated approach simultaneously determines repeat length, structure/motif, and the extent of somatic mosaicism, promising to improve the molecular diagnosis of DM2 and achieve more accurate genotype-phenotype correlations for the better stratification of DM2 patients in clinical trials.

Circulating MicroRNAs as Biomarkers of Osteoporosis and Fragility Fractures.

CONTEXT: Measurement of circulating microRNAs (miRNAs) as potential biomarkers of fragility fracture risk has recently become a subject of investigation. OBJECTIVE: Measure by next-generation sequencing (NGS), global miRNA expression in serum samples of osteoporotic subjects vs individuals with normal bone mineral density (BMD). DESIGN: Samples were collected from patients with different bone phenotypes and/or fragility fractures who did not receive any antiresorptive and/or bone-forming drug at the time of blood collection. SETTING: Samples and data were collected at 7 medical centers in Italy. PATIENTS: NGS prescreening: 50 osteoporotic patients vs 30 individuals with normal BMD. Droplet digital polymerase chain reaction (ddPCR) validation: 213 patients with different bone phenotypes, including the NGS-analyzed cohort. RESULTS: NGS identified 5 miRNAs (miR-8085, miR-320a-3p, miR-23a-3p, miR-4497, miR-145-5p) differentially expressed in osteoporosis cases without fractures vs controls. ddPCR validation confirmed lower c-miR-23a-3p expression in osteoporotic patients, with or without fracture, than in osteopenic and normal subjects and increased c-miR-320a-3p expression in osteoporotic patients with fracture and lower expression in osteoporotic patients without fracture. ddPCR analysis showed a significantly increased expression of miR-21-5p in osteoporotic patients, with or without fracture, than in osteopenic and normal subjects, not evidenced by the NGS prescreening. DISCUSSION: Our study confirmed levels of c-miR-23a-3p and c-miR-21-5p as able to distinguish osteoporotic patients and subjects with normal BMD. Increased levels of c-miR-320a-3p specifically associated with fractures, independently by BMD, suggesting c-miR-320a-3p as a prognostic indicator of fracture risk in osteoporotic patients, to be confirmed in prospective studies on incident fractures.

Association Between DRD2 and DRD4 Polymorphisms and Eating Disorders in an Italian Population.

Anorexia nervosa (AN), bulimia nervosa (BN), and binge eating disorder (BED) are the three most common eating disorders (EDs). Their etiopathogenesis is multifactorial where both the environmental and genetic factors contribute to the disease outcome and severity. Several polymorphisms in genes involved in the dopaminergic pathways seem to be relevant in the susceptibility to EDs, but their role has not been fully elucidated yet. In this study, we have analyzed the association between selected common polymorphisms in the DRD2 and DRD4 genes in a large cohort of Italian patients affected by AN (n = 332), BN (n = 122), and BED (n = 132) compared to healthy controls (CTRs) (n = 172). Allelic and genotypic frequencies have been also correlated with the main psychopathological and clinical comorbidities often observed in patients. Our results showed significant associations of the DRD2-rs6277 single nucleotide polymorphism (SNP) with AN and BN, of the DRD4-rs936461 SNP with BN and BED and of DRD4 120-bp tandem repeat (TR) polymorphism (SS plus LS genotypes) with BED susceptibility. Moreover, genotyping of DRD4 48-bp variable number TR (VNTR) identified the presence of ≥7R alleles as risk factors to develop each type of EDs. The study also showed that ED subjects with a history of drugs abuse were characterized by a significantly higher frequency of the DRD4 rs1800955 TT genotype and DRD4 120-bp TR short-allele. Our findings suggest that specific combinations of variants in the DRD2 and DRD4 genes are predisposing factors not only for EDs but also for some psychopathological features often coupled specifically to AN, BN, and BED. Further functional research studies are needed to better clarify the complex role of these proteins and to develop novel therapeutic compounds based on dopamine modulation.

Deregulated Clusterin as a Marker of Bone Fragility: New Insights into the Pathophysiology of Osteoporosis.

Clusterin (CLU) is a secreted heterodimeric glycoprotein expressed in all organism fluids as well as in the intracellular matrix that plays key roles in several pathological processes. Its recent involvement in muscle degeneration of osteoporotic patients led to investigation of the role of CLU in bone metabolism, given the biochemical and biomechanical crosstalk of the bone-muscle unit. Quantitative real time-polymerase chain reaction (qRT-PCR) analysis of CLU expression was performed in both osteoblasts and Peripheral Blood Mononuclear Cells (PBMCs) from osteoporotic patients (OP) and healthy individuals (CTR). Furthermore, immunohistochemical analysis on femoral head tissues and enzyme-linked immunosorbent assay (ELISA) in plasma samples were performed to investigate CLU expression pattern. Finally, genotyping of CLU rs11136000 polymorphism has also been performed by qRT-PCR assays to explore a possible association with CLU expression levels. Data obtained showed a significantly increased expression level of secreted CLU isoform in PBMCs and osteoblasts from OP patients. Immunohistochemical analysis confirms the increased expression of CLU in OP patients, both in osteocytes and osteoblasts, while plasma analysis reveals a statistically significant decrease of CLU levels. Unfortunately, no functional association between CLU expression levels and the presence of CLU rs11136000 polymorphism in OP patients was found. These data suggest a potential role played by CLU as a potential biomarker for the diagnosis and prognosis of OP progression.

Two Different Therapeutic Approaches for SARS-CoV-2 in hiPSCs-Derived Lung Organoids.

The global health emergency for SARS-CoV-2 (COVID-19) created an urgent need to develop new treatments and therapeutic drugs. In this study, we tested, for the first time on human cells, a new tetravalent neutralizing antibody (15033-7) targeting Spike protein and a synthetic peptide homologous to dipeptidyl peptidase-4 (DPP4) receptor on host cells. Both could represent powerful immunotherapeutic candidates for COVID-19 treatment. The infection begins in the proximal airways, namely the alveolar type 2 (AT2) cells of the distal lung, which express both ACE2 and DPP4 receptors. Thus, to evaluate the efficacy of both approaches, we developed three-dimensional (3D) complex lung organoid structures (hLORGs) derived from human-induced pluripotent stem cells (iPSCs) and resembling the in vivo organ. Afterward, hLORGs were infected by different SARS-CoV-2 S pseudovirus variants and treated by the Ab15033-7 or DPP4 peptide. Using both approaches, we observed a significant reduction of viral entry and a modulation of the expression of genes implicated in innate immunity and inflammatory response. These data demonstrate the efficacy of such approaches in strongly reducing the infection efficiency in vitro and, importantly, provide proof-of-principle evidence that hiPSC-derived hLORGs represent an ideal in vitro system for testing both therapeutic and preventive modalities against COVID-19.

Epigenetics of Myotonic Dystrophies: A Minireview.

Myotonic dystrophy type 1 and 2 (DM1 and DM2) are two multisystemic autosomal dominant disorders with clinical and genetic similarities. The prevailing paradigm for DMs is that they are mediated by an in trans toxic RNA mechanism, triggered by untranslated CTG and CCTG repeat expansions in the DMPK and CNBP genes for DM1 and DM2, respectively. Nevertheless, increasing evidences suggest that epigenetics can also play a role in the pathogenesis of both diseases. In this review, we discuss the available information on epigenetic mechanisms that could contribute to the DMs outcome and progression. Changes in DNA cytosine methylation, chromatin remodeling and expression of regulatory noncoding RNAs are described, with the intent of depicting an epigenetic signature of DMs. Epigenetic biomarkers have a strong potential for clinical application since they could be used as targets for therapeutic interventions avoiding changes in DNA sequences. Moreover, understanding their clinical significance may serve as a diagnostic indicator in genetic counselling in order to improve genotype-phenotype correlations in DM patients.

Characterization of FMR1 Repeat Expansion and Intragenic Variants by Indirect Sequence Capture.

Traditional methods for the analysis of repeat expansions, which underlie genetic disorders, such as fragile X syndrome (FXS), lack single-nucleotide resolution in repeat analysis and the ability to characterize causative variants outside the repeat array. These drawbacks can be overcome by long-read and short-read sequencing, respectively. However, the routine application of next-generation sequencing in the clinic requires target enrichment, and none of the available methods allows parallel analysis of long-DNA fragments using both sequencing technologies. In this study, we investigated the use of indirect sequence capture (Xdrop technology) coupled to Nanopore and Illumina sequencing to characterize FMR1, the gene responsible of FXS. We achieved the efficient enrichment (> 200×) of large target DNA fragments (~60-80 kbp) encompassing the entire FMR1 gene. The analysis of Xdrop-enriched samples by Nanopore long-read sequencing allowed the complete characterization of repeat lengths in samples with normal, pre-mutation, and full mutation status (> 1 kbp), and correctly identified repeat interruptions relevant for disease prognosis and transmission. Single-nucleotide variants (SNVs) and small insertions/deletions (indels) could be detected in the same samples by Illumina short-read sequencing, completing the mutational testing through the identification of pathogenic variants within the FMR1 gene, when no typical CGG repeat expansion is detected. The study successfully demonstrated the parallel analysis of repeat expansions and SNVs/indels in the FMR1 gene at single-nucleotide resolution by combining Xdrop enrichment with two next-generation sequencing approaches. With the appropriate optimization necessary for the clinical settings, the system could facilitate both the study of genotype-phenotype correlation in FXS and enable a more efficient diagnosis and genetic counseling for patients and their relatives.

A 14-Year Italian Experience in DM2 Genetic Testing: Frequency and Distribution of Normal and Premutated CNBP Alleles.

Myotonic dystrophy type 2 (DM2) is a multisystemic disorder caused by a (CCTG) n in intron 1 of the CNBP gene. The CCTG repeat tract is part of a complex (TG) v (TCTG) w (CCTG) x (NCTG) y (CCTG) z motif generally interrupted in CNBP healthy range alleles. Here we report our 14-year experience of DM2 postnatal genetic testing in a total of 570 individuals. The DM2 locus has been analyzed by a combination of SR-PCR, TP-PCR, LR-PCR, and Sanger sequencing of CNBP alleles. DM2 molecular diagnosis has been confirmed in 187/570 samples analyzed (32.8%) and is mainly associated with the presence of myotonia in patients. This set of CNBP alleles showed unimodal distribution with 25 different alleles ranging from 108 to 168 bp, in accordance with previous studies on European populations. The most frequent CNBP alleles consisted of 138, 134, 140, and 136 bps with an overall locus heterozygosity of 90%. Sequencing of 103 unexpanded CNBP alleles in DM2-positive patients revealed that (CCTG)5(NCTG)3(CCTG)7 and (CCTG)6(NCTG)3(CCTG)7 are the most common interruption motifs. We also characterized five CNBP premutated alleles with (CCTG) n repetitions from n = 36 to n = 53. However, the molecular and clinical consequences in our cohort of samples are not unequivocal. Data that emerged from this study are representative of the Italian population and are useful tools for National and European centers offering DM2 genetic testing and counseling.

The long pentraxin PTX3: a novel serum marker to improve the prediction of osteoporosis and osteoarthritis bone-related phenotypes.

BACKGROUND: The long pentraxin PTX3 is generating great interest given the recent discovery of its involvement in bone metabolism. This study investigates the role of circulating PTX3 as a marker of bone-related phenotypes in patients with osteoporosis (OP) and osteoarthritis (OA). METHODS: Serum PTX3 levels were determined using an enzyme-linked immunosorbent assay (ELISA) in a total of OP (n=32), OA (n=19) patients and healthy controls (CTR; n=25). ROC curve analysis was carried out to evaluate the potential of PTX3 for the diagnosis of bone-related phenotypes. In addition, the association between PTX3 serum levels and biochemical markers was estimated by Spearman correlation analysis. RESULTS: Serum analysis reveals a statistically significant increase of PTX3 levels in OP and OA patients, compared to CTR subjects (**** p < 0.0001, **** p < 0.0001). ROC curve of PTX3 levels exhibits an excellent sensitivity and specificity for OP and OA diseases (**** p < 0.0001 and **** p < 0.0001, respectively). Moreover, serum PTX3 levels are positively associated with ALP (r = - 0.5257, p = 0.0083) and PTH levels (r = 0.4704, p = 0.0203) in OP patients. CONCLUSIONS: These results confirm the pivotal role of PTX3 in bone metabolism and suggest its potential use as a predictor of OP and OA bone-related phenotypes.

DNA Methylation Signatures of Bone Metabolism in Osteoporosis and Osteoarthritis Aging-Related Diseases: An Updated Review.

DNA methylation is one of the most studied epigenetic mechanisms that play a pivotal role in regulating gene expression. The epigenetic component is strongly involved in aging-bone diseases, such as osteoporosis and osteoarthritis. Both are complex multi-factorial late-onset disorders that represent a globally widespread health problem, highlighting a crucial point of investigations in many scientific studies. In recent years, new findings on the role of DNA methylation in the pathogenesis of aging-bone diseases have emerged. The aim of this systematic review is to update knowledge in the field of DNA methylation associated with osteoporosis and osteoarthritis, focusing on the specific tissues involved in both pathological conditions.

Genetic variability in noncoding RNAs: involvement of miRNAs and long noncoding RNAs in osteoporosis pathogenesis.

The pathogenesis of osteoporosis is multifactorial and is the consequence of genetic, hormonal and lifestyle factors. Epigenetics, including noncoding RNA (ncRNA) deregulation, represents a link between susceptibility to develop the disease and environmental influences. The majority of studies investigated the expression of ncRNAs in osteoporosis patients; however, very little information is available on their genetic variability. In this review, we focus on two classes of ncRNAs: miRNAs and long noncoding RNAs (lncRNAs). We summarize recent findings on how polymorphisms in miRNAs and lncRNAs can perturb the lncRNA/miRNA/mRNA axis and may be involved in osteoporosis clinical outcome. We also provide a general overview on databases and bioinformatic tools useful for associating miRNAs and lncRNAs variability with complex genetic diseases.

Circulating Long Non-Coding RNA GAS5 Is Overexpressed in Serum from Osteoporotic Patients and Is Associated with Increased Risk of Bone Fragility.

Osteoporosis (OP) is a multifactorial disorder in which environmental factors along with genetic variants and epigenetic mechanisms have been implicated. Long non-coding RNAs (lncRNAs) have recently emerged as important regulators of bone metabolism and OP aetiology. In this study, we analyzed the expression level and the genetic association of lncRNA GAS5 in OP patients compared to controls. Quantitative RT-PCR analysis of GAS5 was performed on the serum of 56 OP patients and 28 healthy individuals. OP subjects were divided into three groups of analysis: 29 with fragility fractures of lumbar spine (OP_VF), 14 with fragility fractures of femoral neck (OP_FF) and 13 without fractures (OP_WF). Genotyping of the rs145204276 insertion/deletion polymorphism has also been performed by Restriction fragment length polymorphism (RFLP) and direct sequencing analyses. Expression of circulating GAS5 is significantly increased in OP patients compared to controls (p < 0.01), with a statistically higher significance in fractured OP individuals vs. healthy subjects (p < 0.001). No statistically significant change was found in female OP patients; conversely, GAS5 is upregulated in the subgroup of fractured OP women sera (p < 0.01) and in all OP males (p < 0.05). Furthermore, a direct correlation between GAS5 expression level and parathyroid hormone (PTH) concentration was found in OP patients (r = 0.2930; p = 0.0389). Genetic analysis of rs145204276 revealed that the deletion allele was correlated with a higher expression of GAS5 in OP patients (0.22 ± 0.02 vs. 0.15 ± 0.01, ** p < 0.01). Our results suggest circulating GAS5 as a putative biomarker for the diagnosis and prognosis of OP and OP-related fractures.

Identification, molecular characterization and segregation analysis of a variant DMPK pre-mutation allele in a three-generation Italian family.

DM1 is an autosomal dominant multisystemic disease caused by an unstable CTG repeat expansion in the 3'-untranslated region (UTR) of the DMPK gene. The complex variant DMPK expanded the alleles containing CAG, CCG, CTC and/or GGC interruptions repetition sequences have been reported in 3-8% of DM1 patients. To date, very few information is available about the frequency and clinical consequences of pre-mutated DMPK variant allele. In this study, we describe a three-generation Italian family showing the segregation of an interrupted DMPK allele within the premutation range. TP-PCR with primers complementary to CCG repetitions and direct sequencing allow us to identify a hetero-triplet (CTG)6(CCGCTG)15(CTG)5 repeat structure. The haplotype analysis demonstrated that this variant allele is associated with the European founder DM1 haplotype. The pyrosequencing analysis of the CpG islands contained in the flanking regions of the CTG array, did not show the presence of a cis effect of the CCG interruptions on the methylation profile of the DM1 locus. The analysis of both meiotic transmissions, one maternal and one paternal, revealed the intrafamilial stability of the DM1 premutation among relatives. Our findings further support the hypothesis of a stabilizing effect of CCG interruptions on the mutational dynamics of the DM1 locus, also in intermediate DMPK alleles.

Carrier frequency of CFTR variants in the non-Caucasian populations by genome aggregation database (gnomAD)-based analysis.

The complexity in the molecular diagnosis of Cystic Fibrosis (CF) also depends on the variable prevalence/incidence of the disease associated with the wide CFTR allelic heterogeneity among different populations. In fact, CF incidence in Asian and African countries is underestimated and the few patients reported so far have rare or unique CFTR pathogenic variants. To obtain insights into CF variants profile and frequency, we used the large population sequencing data in the Genome Aggregation Database (gnomAD). We selected 207 CF-causing/varying clinical consequence variants from CFTR2 database and additional 15 variants submitted to the ClinVar database. Only 14 of these variants were found in the East-Asian population, while for South-Asian and African populations we identified 43 and 52 variants, respectively, confirming the peculiarity of the CFTR allelic spectrum with only few population-specific variants. These data could be used to optimize CFTR carrier screening in non-Caucasian subjects, choosing between the full gene sequencing and cost and time-effective targeted panels.