MW Polyomavirus in diarrheal Italian infants.

BACKGROUND: MXPyV, like MWPyV, was identified in stool samples from children suffering diarrhea in Mexico. In this study, we used a home-made real time PCR to investigate the presence of this novel viruses in stool specimen collected from under-five-year-old children with gastroenteritis. METHODS: A total of 192 fecal specimens previously screened for RV, ADV, NoV, HPeV and SaV, were tested for MWPyV with Taqman real time PCR. RESULTS: The most detected virus was NoV GII (33.8%), followed by RV (21.3%), SaV (10.9%), HPeV (8%), NoV GI (6.7%) and Adv (1%). Real time PCR detected MWPyV in 1/192 (0.5%) patients. CONCLUSIONS: We detected MWPyV in 0.5% of fecal specimens collected from pediatric patients suffering gastroenteritis which is smaller than the previously reported in literature (4.4% in Australia and 12% Mexico).

Prevalence of human Cosavirus and Saffold virus in young children with gastroenteritis in Northern Italy.

BACKGROUND: Gastroenteritis is a common disease in children, characterized by diarrhea, vomiting, abdominal pain, and fever. Human Cosavirus (HCoSV) and Saffold virus (SAFV) both have a worldwide distribution. Both viruses have been detected in the stools of patients with acute gastroenteritis in several countries. METHODS: In order to provide more insights into the epidemiology of enteric viruses that are not included usually in routine diagnostic tests, cases of childhood sporadic gastroenteritis of unknown etiology requiring hospital admission in Turin, Italy, during December 2014 to November 2015, were screened for HCoSV and SAFV. RESULTS: A total of 1 out of 164 (0.6%) episodes of acute gastroenteritis were associated with SAFV genomic detection. Among the 1 SAFV-positive cases, 1 were also positive for Adenovirus. The patient positive for SAFV do not present diarrheal episodes but vomiting. HCoSV was not detected in any of the samples. CONCLUSIONS: In conclusion, this study presents the current epidemiological data regarding the two viruses, HCoSV and SAFV, circulating in pediatric patients admitted to hospital with acute gastroenteritis in Turin, Italy.

Quantification of fecal adenovirus viral load and correlation with Vesikari Score in children with acute gastroenteritis.

BACKGROUND: Human adenoviruses (HAdVs) are an important cause of acute respiratory tract infections, conjunctivitis, hemorrhagic cystitis, and gastroenteritis. In addition to enteric serotypes 40 and 41, some serotypes belonging to subgroups A, B, and C have also been implicated to be etiological agents of gastroenteritis among infants and young children. The Vesikari Scoring System (VSS) is the severity scale that was originally developed to evaluate the effectiveness and efficacy of rotavirus vaccines on 20 points. The aim of this study was to evaluate and compare the diagnostic value of the VSS with HAdVs genome quantification in fecal samples collected from hospitalized children with acute gastroenteritis. METHODS: A total of 137 fecal specimens (69 male and 68 female) were tested for HAdVs. The samples were collected from under-five-year-old children with acute gastroenteritis in pediatric Hospital Regina Margherita of Turin in Italy. RESULTS: A total of 69 out of 137 (50.3%) samples were associated with HAdV genomic detection with a mean viral load of 1.08×1011±9.02×1011 genomes/mg fecal specimens. The samples were grouped on the basis of Mild VSS and Moderate VSS and the HAdV viral load was calculated in the two groups. No statistical differences were observed between two groups (P=0.6123 calculated by Mann-Whitney Test). CONCLUSIONS: Our results did not show a difference in mean viral load between the group with mild VVS and moderate VVS.

Mir-155 expression is downregulated in hematopoietic stem cell transplantation patients with Epstein-Barr virus infection.

BACKGROUND: MicroRNAs (miRNAs) are a class of short length double strand genome encoded RNAs that are produced to repress post-transcriptionally the expression of cellular mRNAs. 2578 unique mature miRNAs are currently annotated in the human genome and participate in the regulation of multiple events, such as cellular proliferation or apoptosis. The over-expression of miR-155 of cellular origin might play a key role in the life cycle of EBV. In this study 24 pediatric patients undergoing HSCT seropositive and seronegative to EBV were enrolled. Thirty-one peripheral blood samples were collected from these patients. The mir-155 expression profile has been evaluated by a stem-loop Real Time PCR in all these conditions. METHODS: Of 24 patients, 4 were seronegative to EBV and EBV negative to PCR (Group I), 10 were seropositive to EBV and EBV negative to PCR (Group II) and 10 were seropositive to EBV and EBV positive to PCR (Group III). RESULTS: Based on relative quantification, the mir-155 expression was compared among the groups. The comparison between HSCT patients without EBV infection seronegative to EBV (Group I) showed higher levels of mir-155 expression than patients seropositive to EBV (P=0.1419). The mir-155 expression levels in seronegative to EBV were not significantly different compared with the patients seropositive to EBV (P=0.6504). The mir-155 expression levels in seropositive to EBV without and with EBV infection (positive viral load), were not significantly (P=0.7667). Also, when we evaluated the mir-155 expression levels comparing all EBV negative patients with an active EBV infection, we did not observe a statistically significant difference (P=0.9782). CONCLUSIONS: Our results are controversial, showing a higher production of mir-155 levels during EBV infection.

Lack of detection of HPyV12 DNA using real-time PCR in Italian infants with diarrhea.

BACKGROUND: HPyV12 was found in organs of the digestive tract, in particular the liver but also in colon, rectum and feces. Until now, the prevalence of HPyV12 is not well characterized. METHODS: In this study, we investigate the presence of this novel polyomavirus DNA in stool specimens collected from under-five-year-old children with gastroenteritis compared to healthy infants. A total of 190 fecal specimens previously screened for rotavirus (RV) and adenovirus (ADV) and 80 fecal samples from healthy infants, were tested for HPyV12 DNA using a home-made real time PCR. All fecal specimens were tested for the presence of HPyV12 with specific primers and probes. RESULTS: None of 190 (0%) episodes of acute gastroenteritis was associated with HPyV12. We did not detect HPyV12 DNA in any of 80 control subjects, as well. CONCLUSIONS: Our study represents a pilot study aiming to clarify the current epidemiological pattern in pediatric Italian patients regarding the novel and rare HPyV12. Based on our negative data and the recent observations reported in literature, doubts remain on human tropism of the HPyV12 and epidemiology: these issues need further investigations.

Rotavirus quantification in children with acute gastroenteritis.

INTRODUCTION: Most intractable diarrheas are treated with antibiotics, irrespective of the causative agent. This study aims to quantified Rotavirus with taqman real-time PCR in fecal samples of children with acute gastroenteritis in accordance with the program of reduction of drug-resistance and use of antibiotics. METHODS: A total of 190 fecal specimens were collected from under-five-year-old children with acute gastroenteritis in pediatric Hospital Regina Margherita of Turin in Italy in 2017. A total of 38 out of 67 (56.7%) episodes of acute gastroenteritis were associated with Rotavirus genomic detection with multiplex commercial kit. RESULTS: All fecal specimens were tested for the presence of RV and other GE viruses. The most commonly detected virus was norovirus (41%), astrovirus (15.8%), human bocavirus (8.9%), sapovirus (7.9%), human parechovirus (5.8%), rhinovirus (4.2%), and adenovirus (1%). In 66 out of 190 (34.7%) Rotavirus were detectable with the median viral load 7.2E+11±60E+11genomes/mg fecal specimens. DISCUSSION/CONCLUSIONS: Our results showed that RV was present in around 34.7% of children hospitalized for AGE, a rate similar to those reported in previous studies conducted elsewhere which were in the range of 33-75%. Our protocol are able to quantify the absolute number of viral particle/mg of feces. The clinical utility of quantitative molecular assays, such as Rotavirus viral load, will be markedly improved.

Enhanced Expression of Human Endogenous Retroviruses, TRIM28 and SETDB1 in Autism Spectrum Disorder.

Human endogenous retroviruses (HERVs) are relics of ancestral infections and represent 8% of the human genome. They are no longer infectious, but their activation has been associated with several disorders, including neuropsychiatric conditions. Enhanced expression of HERV-K and HERV-H envelope genes has been found in the blood of autism spectrum disorder (ASD) patients, but no information is available on syncytin 1 (SYN1), SYN2, and multiple sclerosis-associated retrovirus (MSRV), which are thought to be implicated in brain development and immune responses. HERV activation is regulated by TRIM28 and SETDB1, which are part of the epigenetic mechanisms that organize the chromatin architecture in response to external stimuli and are involved in neural cell differentiation and brain inflammation. We assessed, through a PCR realtime Taqman amplification assay, the transcription levels of pol genes of HERV-H, -K, and -W families, of env genes of SYN1, SYN2, and MSRV, as well as of TRIM28 and SETDB1 in the blood of 33 ASD children (28 males, median 3.8 years, 25-75% interquartile range 3.0-6.0 y) and healthy controls (HC). Significantly higher expressions of TRIM28 and SETDB1, as well as of all the HERV genes tested, except for HERV-W-pol, were found in ASD, as compared with HC. Positive correlations were observed between the mRNA levels of TRIM28 or SETDB1 and every HERV gene in ASD patients, but not in HC. Overexpression of TRIM28/SETDB1 and several HERVs in children with ASD and the positive correlations between their transcriptional levels suggest that these may be main players in pathogenetic mechanisms leading to ASD.

A Novel Xeno-Free Method to Isolate Human Endometrial Mesenchymal Stromal Cells (E-MSCs) in Good Manufacturing Practice (GMP) Conditions.

The cyclic regeneration of human endometrium is guaranteed by the proliferative capacity of endometrial mesenchymal stromal cells (E-MSCs). Due to this, the autologous infusion of E-MSCs has been proposed to support endometrial growth in a wide range of gynecological diseases. We aimed to compare two different endometrial sampling methods, surgical curettage and vacuum aspiration biopsy random assay (VABRA), and to validate a novel xeno-free method to culture human E-MSCs. Six E-MSCs cell samples were isolated after mechanical tissue homogenization and cultured using human platelet lysate. E-MSCs were characterized for the colony formation capacity, proliferative potential, and multilineage differentiation. The expression of mesenchymal and stemness markers were tested by FACS analysis and real-time PCR, respectively. Chromosomal alterations were evaluated by karyotype analysis, whereas tumorigenic capacity and invasiveness were tested by soft agar assay. Both endometrial sampling techniques allowed efficient isolation and expansion of E-MSCs using a xeno-free method, preserving their mesenchymal and stemness phenotype, proliferative potential, and limited multi-lineage differentiation ability during the culture. No chromosomal alterations and invasive/tumorigenic capacity were observed. Herein, we report the first evidence of efficient E-MSCs isolation and culture in Good Manufacturing Practice compliance conditions, suggesting VABRA endometrial sampling as alternative to surgical curettage.

HERV-K and W expression in peripheral mononuclear cells of children with Henoch-Schönlein purpura and relation with TLR activation.

BACKGROUND: HERVs expression seems impaired in several diseases, ranging from autoimmune to neoplastic disorders. However, various stimuli may re-activate HERVs transcription. Henoch-Schönlein purpura is the most common cause of vasculitis in children. The role of microbial antigens in the pathogenesis of HSP remains elusive. Toll-like receptors (TLRs) are the first line of defense for the host to initiate an immune and inflammatory response. We aimed to investigate HERV, K and W expression in peripheral mononuclear cells of children with HSP and relation with TLRs activation. METHODS: The study enrolled 63 children affected by HSP. We used RT-PCR to detect GAPDH in the peripheral blood mononuclear cells samples to normalize the data. Subsequently, the viral pol gene HERVs and TLRs transcripts in the same samples were assessed. RESULTS: HERV K and W was expressed in all samples analyzed but the level of expression was much lower in the HSP that in HC P=0.0006 and P=0.0004 for HERV-K and -W respectively. TLR2 was hyper-expressed in 39 vs. 63 (62%) of the HSP, TLR3 in 23 vs. 63 (42%), TLR4 in 42 vs. 63 (66%) and TLR9 in 32 vs. 63 (52%). The different expression was statistically significant only for TLR4 (P=0.0276) no for TLR2,3 and 9 (P=0.1251; 0.3964 and 0.1882 respectively). Spearman's test demonstrated no correlation between the low expression of HERV-K and HERV-W and high expression of TLRs. CONCLUSIONS: The results of the present study demonstrated that mRNA expression of HERV-K and -W and TLRs did not directly correlated.

Evaluation of two RNA extraction methods for human Parechovirus detection on pediatric stool specimens.

BACKGROUND: Human Parechovirus (HPeVs), along with human enteroviruses is associated with gastrointestinal and respiratory infections. The aim of this study was to assess the performance characteristics of two nucleic acid extraction for HPeV-RNA quantitation, the RNAlev Extraction Kit associated with Maxwell automated nucleic acid extractor (Promega, Milan, Italy) and RNAzol manually protocol. METHODS: A total of 137 fecal specimens previously routinely screened for Rotavirus and Adenovirus were tested for HPeV virus. RESULTS: Methods 1 and 2 detected HPeV-RNA in 11 and 10 samples, respectively, with a 96.2% concordance. In particular, 124/137 (90.5%) were concordantly negative by both methods; 8/137 (5.8%) concordantly positive. For the 8 specimens that were positive by both tests, the population mean (SD) was 320 (601) copies/mg with method 1 and 1216 (2338) copies/mg with method 2. By referring to the 8 specimens that were concordantly positive, the correlation value between the two methods was not statistically significant (r=0.381 and P=0.3599). Bland-Altman analysis showed that differences between the two methods were within ±1000 copies/mg of the averaged results for all of the tested specimens. CONCLUSIONS: Method 2, being a semi-automated method, provides benefits over a manual method, in terms of turnaround time, less errors and reliability.

CD27 mRNA expression in mycosis fungoides.

BACKGROUND: The etiopathogenesis of MF remains obscure. CD27 is a member of the tumor necrosis factor receptor superfamily (TNFRS) that regulates lymphocyte function. Expression of CD27 protein and mRNA has been reported in B-cell lymphomas and adult T-cell leukemia/lymphoma. In this study, we examined the expression of CD27 in the skin of MF patients by real time PCR. The amount of CD27 was measured in MF patients and healthy controls. METHODS: A total of 98 skin biopsies were analyzed: 12 obtained from healthy donors and 86 obtained cryostatic sections OCT-embedded affected by MF. Relative quantification of mRNA CD27 expression was achieved by means of TaqMan (Thermo Fisher Scientific, Waltham, MA, USA) amplification and normalization to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). RESULTS: Housekeeping gene was detectable in all skin samples and there is no difference between healthy control and MF P value =0.1564. CD27 mRNA sequences were found in 3 of 12 (25%) of skin obtained from healthy donors and in 59 of 86 (68%) of skin obtained from cryostatic sections OCT-embedded affected by MF. The χ2 statistic with Yates correction is 6.8413 and the P value is 0.0089. When we compared the CD27 expression in MF and controls the RQ analysis showed a value of 9.12±14.13. A RQ of 9.12 means that this gene is 9.12 times more expressed in MF skin samples then in the healthy skin samples. No difference was observed in the MF clustered by stages. CONCLUSIONS: Our findings indicates that CD27 can be used as diagnostic/prognostic markers, and whether anti-CD27 antibodies can be used in therapy.

Functional study of TNF-α as a promoter of polymorphisms in psoriasis.

BACKGROUND: TNF-α is an important mediator in the pathogenesis of psoriasis and polymorphisms influence its transcription and could be implicated in psoriasis risk and modify certain aspects of disease, such as age at onset of psoriasis vulgaris and disease severity. Six TNF-α single nucleotide polymorphisms (SNPs) in promoter region has been identified and studied but with discordant results. The aim of this study was to evaluate whether the polymorphisms in TNF-α (-238 [rs361525], -308 [rs1800629], -857 [rs1799724], -1031 [rs1799964]) are associated with severity, itch, early onset or response to drug therapy in psoriasis in Caucasian Italian patients. METHODS: Fifty-eight psoriasis patients from Turin PSOCARE, 23 with psoriasis vulgaris and 35 with psoriatic arthritis were studied. Ready-to-use master mix for allelic discrimination of rs1800629, rs361525 and rs1799964 respectively. RESULTS: Our data showed a significant association between the -857(G) variant and both VAS-itch (P=0.03) and VAS-pain index (P=0.006), OR=0.2 (0.04-0.98) and OR=0.12 (0.02-0.59). No significant association between the genotypes or alleles of TNF-α SNPs has been observed with other clinic-pathologic parameters or etanercept response. CONCLUSIONS: Our data suggest that -857 CC genotype could be involved in pain and itch severity in psoriasis patients.

Impaired transcription of human endogenous retroviruses in the sperm with exception of syncytin 1: short communication.

BACKGROUND: Human endogenous retroviruses (HERVs), remnants of ancestral infections, represent 8% of the human genome. HERVs are co-opted for important physiological functions during embryogenesis; however, little is known about their expression in human gametes. We evaluated the transcriptional levels of several retroviral sequences in human spermatozoa. METHODS AND RESULTS: We assessed, through a Real-Time PCR assay, the transcription levels of the pol genes of HERV-H, -K and -W families and of env genes of syncytin (Syn)1 and Syn2 in the spermatozoa from 8 normospermic subjects. The entity and distribution of their expressions were compared to values found in white blood cells (WBCs) from 16 healthy volunteers. The level of HERV transcripts was significantly lower in spermatozoa than in WBCs for HERV-H-pol, HERV-K-pol, HERV-W-pol, and Syn2.In contrast, the level of expression of Syn1 in the sperm was similar to that found in WBCs and it was significantly higher than the mRNA concentrations of other HERV genes in spermatozoa. CONCLUSIONS: Our findings show, for the first time, the presence of several retroviral mRNAs in the sperm, although in low amounts. The higher concentration of Syn1 suggests that it could play a key role in the fusion process between gametes during fertilization and, perhaps, be involved in embryo development. Further studies could clarify whether aberrant HERV expressions, in particular of Syn1, negatively affect fertilization and embryo growth and whether sperm manipulation procedures, such as cryopreservation, may potentially influence HERV transcription in the human male gamete.

Bufavirus, Cosavirus, and Salivirus in Diarrheal Italian Infants.

Three newly discovered viruses have been recently described in diarrheal patients: Cosavirus (CosV) and Salivirus (SalV), 2 picornaviruses, and bufavirus (BuV), a parvovirus. The detection rate and the role of these viruses remain to be established in acute gastroenteritis (AGE) in diarrheal Italian infants. From November 2016 to November 2017, stool samples were collected from 160 children <5 years old suffering from AGE and attending the Children's Hospital in Turin, Italy. During the study period, 1 (0.5%) sample was positive for 1 of the 3 investigated viruses: 0 (0%) CosV, 1 (0.5%) SalV, and 0 (0%) BuV, whereas 42 (26.0%) children were infected with rotavirus and 2 (1%) with adenovirus. No mixed infections involving the 3 viruses were found. Although these viruses are suspected to be responsible for AGE in children, our data showed that this association was uncertain. Therefore, further studies with large cohorts of healthy and diarrheal children will be needed to evaluate their clinical role in AGE.

Chronic HCV Infection Is Associated with Overexpression of Human Endogenous Retroviruses that Persists after Drug-Induced Viral Clearance.

Chronic hepatitis C virus (HCV) infection is associated with several hepatic and extrahepatic complications, including cancers and autoimmune disorders, whose frequency is reduced but not abolished after drug-induced viral clearance. The causes of these complications and of their persistence are ill-defined. Human endogenous retroviruses (HERVs) are remnants of ancestral infections and constitute 8% of the human genome. Most HERV elements are inactive, but some are transcribed. HERV overexpression is associated with many cancers and autoimmune diseases with a putative pathogenetic role. Several viral infections trigger HERV activation, but there are no studies on HCV-infected subjects. We assessed, through a PCR real-time amplification assay, the transcription levels of the pol genes of HERV-H, -K, and -W, and of their repressor TRIM28 in white blood cells (WBCs) of vertically infected children, both before and after therapy with direct-acting antivirals (DAAs). The results documented significantly higher expressions of HERV-H-pol and HERV-K-pol, not of HERV-W-pol, in HCV-infected subjects as compared to age-matched controls. HERV RNA levels remained unchanged after DAA-driven viral clearance. No significant variations in transcription levels of TRIM28 were observed in infected subjects. Our findings demonstrate HERV-H-pol and HERV-K-pol overexpression in subjects with chronic HCV infection, without variations after a positive response to DAAs; this might justify their predisposition to cancers and autoimmune disorders that persist after a DAA-induced resolution of viremia.

Modulation of human endogenous retroviruses -H, -W and -K transcription by microbes.

The human endogenous retroviruses (HERVs) are endogenous retroviruses that are inserted into the germ cell DNA of humans over 30 million years ago. Using real-time RT-PCR we describe HERV modulation by commensal microbes in the human gut. Infants, exclusively or predominant breast milk feeding, less than 12 weeks of age, during bacteria gut colonization, were assessed for eligibility. Our data demonstrate that the colonization with commensal microbes, in particular, Bifidobacterium spp., of the gut causes modulation of HERVs.

Lack of detection of Cutavirus DNA using PCR real time in cutaneous T-cell lymphomas.

BACKGROUND: A novel human protoparvovirus named Cutavirus has been discovered. We investigated the presence of Cutavirus in a sample of Cutaneous T-cell lymphomas by using PCR real time TaqMan® (Thermo Fisher Scientific, Waltham, MA, USA). METHODS: In total, 55 CTCL samples were analyzed using a TaqMan® Real time PCR on a 7500 ABI instrument. All of these shown internal control amplification. RESULTS: The presence of Cutavirus DNA corresponding was examined. CuV DNA sequences were not detected in any skin specimen. CONCLUSIONS: The role of Cutaviruses in cutaneous cancers remains to be investigated.

Is HERV-K and HERV-W expression regulated by mir-155 in Sézary Syndrome?

BACKGROUND: According to the latest update, 2578 unique mature miRNAs are currently annotated in the human genome and participate in the regulation of multiple events, such as cellular proliferation or apoptosis. A previous study analyzing global miRNA expression patterns in GH cells (high HERV-K versus low) showed that two miRNAs (miR-663 and miR-638) are differentially regulated and exhibit expression parallel to that of HERV-K. The aim of this study was to evaluate HERV-K and -W pol gene and mir-155 expression in SS patients and possible relationship between them. METHODS: The comparison between SS patients and healthy donor showed a significant difference in terms of mir-155 expression P=0.0003 as previously reported by our groups. RESULTS: We demonstrated that HERV-K and -W pol gene expression was significantly higher in SS patients vs. healthy donor as previously reported by our groups. Our correlation data suggest that miR-155 are not directly involved in regulating the HERVs. CONCLUSIONS: Furthermore, further studies including other cohorts of pathology with mir-155 and HERVs involvement such as inflammatory diseases are needed to investigate the role of mir-155 in the cross-activations of HERVs.

Interferon signature in immunosuppressed patients with lower respiratory tract infections: dosage on bronchoalveolar lavage.

BACKGROUND: Interferon signature (IS) is the measure of transcripts belonging to pathways of interferon activation. Viral infections can interfere with the interferon pathway, in particular herpesvirus present in immunocompromised hosts. The aim of our study was to evaluate if herpesvirus infections in immunocompromised patients with lower respiratory tract infections (LRTI) could lead to IS alterations. METHODS: We measured IS transcription of six genes on bronchoalveolar lavage of immunocompromised patients with LRTI (IFI27, IFI44, IFIT1, ISG15, RSAD2, SIGLEC1). Patients were divided in three groups based on Epstein-Barr virus (EBV) and other herpesviruses coinfections. RESULTS: We included 56 patients, 10 without and 17 with only EBV reactivation (respectively N and E groups) and 29 with EBV and other herpesviruses (group C). IS was higher in group C (P=0.01) compared to other ones, but single gene expressions were different among groups: IFI27 was higher whereas IFIT1 and ISG15 were lower in group C (P<0.05). CONCLUSIONS: The continuous stimulation of interferon cascade by herpesviruses enhances IS. The analysis of IS in immunocompromised population is possible by limiting the use of IFI27, IFIT1, ISG15 genes. Our preliminary results seem to indicate that IS is a useful biomarker of cellular response to herpesvirus infection in immunocompromised patients.

Improved Molecular Diagnosis of McCune-Albright Syndrome and Bone Fibrous Dysplasia by Digital PCR.

McCune-Albright syndrome (MAS) is a rare congenital disorder characterized by the association of endocrine and nonendocrine anomalies caused by somatic activating variants of GNAS. The mosaic state of variants makes the clinical presentation extremely heterogeneous depending on involved tissues. Biological samples bearing a low level of mosaicism frequently lead to false-negative results with an underestimation of causative molecular alterations, and the analysis of biopsies is often needed to obtain a molecular diagnosis. To date, no reliable analytical method for the noninvasive testing of blood is available. This study was aimed at validating a novel and highly sensitive technique, the digital PCR (dPCR), to increase the detection rate of GNAS alterations in patients with a clinical suspicion of MAS and, in particular, in blood. We screened different tissues (blood, bone, cutis, ovary, and ovarian cyst) collected from 54 MAS patients by different technical approaches. Considering blood, Sanger was unable to detect mutations, the allele-specific PCR and the co-amplification at lower denaturation temperature had a 9.1% and 18.1% detection rate, respectively, whereas the dPCR reached a 37.8% detection rate. In conclusion, the dPCR resulted in a cost-effective, reliable, and rapid method allowing the selective amplification of low-frequency variants and able to improve GNAS mutant allele detection, especially in the blood.