Pedigree analysis exploring the inconsistency between diverse phenotypes and testing criteria for germline TP53 mutations in Chinese women with breast cancer.

PURPOSE: In the present study, we addressed the inconsistency between the testing criteria and diverse phenotypes for germline TP53 mutation in patients with breast cancer in the Chinese population. METHOD: We proposed a new added item (synchronous or metachronous bilateral breast cancer) as one of the testing criteria (aimed at high-penetrance breast cancer susceptibility genes) and applied it for determining TP53 germline mutation status in 420 female patients with breast cancer using multigene panel-based next-generation sequencing, Sanger sequencing, and mass spectrometry. RESULTS: We found that 1.4% of patients carried a pathogenic or likely pathogenic germline TP53 mutation. Compared with BRCA mutation carriers (8.0%) and non-carriers (7.1%), TP53 mutation carriers (33.3%) developed breast cancer earlier. The majority of TP53 mutation carriers (66.7%) developed breast cancer after age 30 and had bilateral breast cancer (33.3%). Pedigree investigation of four TP53 carriers and a patient with a TP53 variant of unknown significance revealed that neither of their parents harbored the same mutations as the probands, indicating that the mutations might occur de novo. CONCLUSION: Our study revealed distinguishing features of TP53 carriers among Chinese women with breast cancer, which is inconsistent with the currently used testing criteria; therefore, the newly proposed testing criteria may be more appropriate.

Effects of centrifugation treatment before electroporation on gene editing in pig embryos.

Genetic mosaicism, characterized by multiple genotypes within an individual, is considered an obstacle to CRISPR/Cas9 genome editing in animal models. Despite the various strategies for minimizing mosaic mutations, no definitive methods exist to eliminate them. This study aimed to enhance gene editing efficiency in porcine zygotes using CRISPR/Cas9, which targets specific genes through centrifugation and zona pellucida removal before electroporation. Centrifugation at 2000 × g did not adversely affect blastocyst formation rates in zygotes electroporated with gRNA targeting the GGTA1 gene; instead, it led to increased total and monoallelic mutation rates compared with control zygotes without centrifugation. However, the groups had no significant differences in biallelic mutation rates. In zygotes electroporated with gRNA targeting the CMAH gene, centrifugation treatments exceeding 1000 × g significantly increased both biallelic mutation rates and mutation efficiency. The combination of centrifugation and zona pellucida removal did not have a detrimental effect on blastocyst formation rates. It led to a higher rate of double biallelic mutations in embryos targeting both GGTA1 and CMAH compared to embryos without centrifugation treatment. In summary, our results demonstrate that pre-electroporation treatments, including centrifugation and zona pellucida removal, positively influenced the reduction of mosaic mutations, with the effectiveness of centrifugation depending on the specific gRNA used.

Mosaic GJB2 mutations in widespread porokeratotic adnexal ostial nevus: Report of two patients.

Porokeratotic adnexal ostial nevus (PAON) is a rare adnexal hamartoma characterized by keratotic papules following Blaschko's lines, typically located on the unilateral distal extremities. Cutaneous somatic GJB2 mutations have been linked to the pathogenesis of PAON. However, the genetic mechanism underlying bilateral or extended forms, which are less documented, remains unknown. In this study, we presented two cases of PAON with widespread cutaneous lesions and scalp involvement, and demonstrated the presence of GJB2 mosaic mutations in both patients. We further investigated the mosaic frequency in different tissues to gain insights into the mutation events contributing to the phenotype of widespread PAON. Our findings suggest that early postzygotic mutation causing mosaic GJB2 mutations may contribute to the widespread phenotype of PAON, thereby enriching the disease spectrum and mutation profile of PAON.

Sporadic gastric juvenile polyposis with a novel SMAD4 nonsense mutation in a mosaic pattern.

A 50-year-old female was diagnosed with gastric hyperplastic polyps 7 years before and was followed up at another hospital. She was referred to our hospital because of the growth of gastric polyps and progression of anemia. She had no family history of polyposis. The polyps were observed only in the stomach, increased in size and number, and the erythematous edema got worse. Endoscopic mucosal resection (EMR) of the gastric polyp was performed. Pathologically, the gastric polyp was hamartomatous polyp, and the intervening mucosa between polyps showed no atypical structure without inflammation. Given that gastric juvenile polyposis (GJP) was clinically suspected, a genetic test using peripheral blood was performed. Target resequencing and Sanger sequencing analysis revealed a nonsense mutation in the SMAD4 gene at codon 169. The mutation was detected at a low frequency of 11%, and considered a mosaic mutation. Therefore, she was diagnosed with a sporadic GJP, and total gastrectomy was performed. Immunostaining of SMAD4 for the resected specimen showed a mixture of stained and unstained area in the epithelium of the polyp, indicating partial loss of SMAD4 expression. To our knowledge, this is the first reported case of GJP with a nonsense SMAD4 mutation at codon 169 in a mosaic pattern.

First Report of a Low-Frequency Mosaic Mutation in the Hydroxymethylbilane Synthase Gene Causing Acute Intermittent Porphyria.

Acute porphyrias are a group of monogenetic inborn errors of heme biosynthesis, characterized by acute and potentially life-threatening neurovisceral attacks upon exposure to certain triggering factors. Biochemical analyses can determine the type of acute porphyria, and subsequent genetic analysis allows for the identification of pathogenic variants in the specific gene, which provides information for family counselling. In 2017, a male Swiss patient was diagnosed with an acute porphyria while suffering from an acute attack. The pattern of porphyrin metabolite excretion in urine, faeces, and plasma was typical for an acute intermittent porphyria (AIP), which is caused by inherited autosomal dominant mutations in the gene for hydroxymethylbilane synthase (HMBS), the third enzyme in the heme biosynthetic pathway. However, the measurement of HMBS enzymatic activity in the erythrocytes was within the normal range and Sanger sequencing of the HMBS gene failed to detect any pathogenic variants. To explore the molecular basis of the apparent AIP in this patient, we performed third-generation long-read single-molecule sequencing (nanopore sequencing) on a PCR product spanning the entire HMBS gene, including the intronic sequences. We identified a known pathogenic variant, c.77G>A, p.(Arg26His), in exon 3 at an allelic frequency of ~22% in the patient's blood. The absence of the pathogenic variant in the DNA of the parents and the results of additional confirmatory studies supported the presence of a de novo mosaic mutation. To our knowledge, such a mutation has not been previously described in any acute porphyria. Therefore, de novo mosaic mutations should be considered as potential causes of acute porphyrias when no pathogenic genetic variant can be identified through routine molecular diagnostics.

Genetic and phenotypic diversities of nevus spilus phenotypes: Case series and a proposed diagnostic algorithm.

Nevus spilus (NS) is composed of multiple types that characterized by a congenital hyperpigmented patch within variable even superimposed lesions originating from melanocytic lineage cells. The molecular mechanism and classification of diverse NS phenotypes remain unclear. Five children with a phenotype of NS were genotyped by the panel based on next-generation sequencing in this study. DNA from biopsies, blood samples and hair follicle were sequenced to confirm the presence of a somatic mutation. Sequencing results indicated somatic mutation in the gene of NRAS or HRAS in all biopsies from the nevi, and the pathogenic variants were not detected in the samples of blood and hair follicle. This study successfully identified the somatic mutation in five unrelated children with diverse NS phenotypes. Moreover, it provided typical images and differential diagnoses between variable NS phenotypes in clinical, pathological, and genetic features, and first proposed a clinical diagnostic algorithm that contributed to simplifying and optimizing the diagnoses and management of these overlapped diseases.

Early Detection of Relapse by ctDNA Sequencing in a Patient with Metastatic Thymic Tumor and MEN1 Mosaicism.

CONTEXT: Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disease caused by inactivating mutations in the MEN1 gene. In the literature, few cases of MEN1 have been reported because of mosaic MEN1 mutations. OBJECTIVE: We performed an extensive molecular characterization in several lesions and blood samples, including plasmatic circulating cell-free DNA (ccfDNA) in an exceptional case of a patient with MEN1 mosaicism causing primary hyperparathyroidism, multiple pancreatic neuroendocrine tumors (NETs), and a metastatic thymic NET. METHODS: Blood, ccfDNA and multiple tissue analysis were performed by next-generation sequencing. RESULTS: MEN1 mosaicism was confirmed by multiple tissue analysis. Somatic analysis of the largest pancreatic NET revealed the same MEN1 second-hit mutation as found in the thymic lesion, demonstrating its metastatic origin from the thymic lesion. Moreover, in ccfDNA we found the mosaic MEN1 mutation but also the somatic second-hit mutation found in the thymic primary tumor, revealing the presence of circulating tumor DNA (ctDNA). After surgical removal of the pancreatic metastasis, the mutated fraction of both mutations decreased, before increasing again several weeks before a new clinical relapse, suggesting that thymic ctDNA may be used as an early tumor biomarker. CONCLUSION: This exceptional MEN1 case highlighted (1) the importance of looking for MEN1 mosaicism, (2) that MEN1 mosaicism can cause very aggressive disease, and (3) the interest in analyzing ccfDNA for confirming MEN1 mosaicism but also as a potential tumor biomarker for NET.

Efficacy of canakinumab on AA amyloidosis in late-onset NLRP3-associated autoinflammatory disease with an I574F somatic mosaic mutation.

There have been hundreds of reports on mutations in the NLRP3 gene related to NLRP3-associated autoinflammatory disease, but few of these mutations have occurred as both germline and somatic mosaic mutations. In this case-based review, we report a 68-year-old man with an NLRP3-associated autoinflammatory disease. He developed secondary amyloidosis, including a renal and colorectal presentation in his 50 s. Sequencing of the NLRP3 gene revealed an I574F somatic mosaic mutation, which has up to now only been reported in germline mutations. The patient was treated with canakinumab, which had great efficacy not only on the NLRP3-mediated inflammation, but also on the chronic renal failure and proteinuria provoked by secondary renal amyloidosis. To evaluate the effectiveness of canakinumab, we conducted a literature research on renal amyloidosis related to NLRP3-associated autoinflammatory disease treated with canakinumab. Although our patient had a relatively long medical history and greater amounts of proteinuria than other reported cases, canakinumab had great efficacy on renal impairment, in similar to other reported cases. Along with the first report of a late-onset I574F somatic mosaic mutation in NLRP3-associated autoinflammatory disease, this report demonstrates the effectiveness of canakinumab on renal amyloidosis, probably through the way that IL-1ß blockade minimizes podocyte injury.

Prenatal diagnosis of monogenic skeletal disorders in 22 pedigrees / 中华内分泌代谢杂志

Objective:To evaluate the clinical value of prenatal molecular diagnostic technology in preventing hereditary diseases through analysis of prenatal diagnostic characteristics in 22 monogenic skeletal disorders pedigrees.Methods:This study retrospectively analyzed prenatal molecular diagnostic results of 22 pedigrees with monogenic skeletal disorders who were admitted to Department of Osteoporosis and Bone Diseases in our hospital from January 2014 to July 2021.Results:Among 22 pedigrees, there were 10 pedigrees with X-linked hypophosphatemic rickets due to PHEX gene mutations, in which 8 fetuses were found to carry pathogenic variants; 6 pedigrees with osteopetrosis, including 3 cases of CLCN7 gene mutation, 2 TCIRG1 gene mutation, and 1 CTSK gene mutation, were detected to have 2 affected fetuses and 1 carrier. There were 4 cases of osteogenesis imperfecta, including 2 cases of COL1A1 gene mutation, 1 case of COL1A2 gene mutation, and 1 case of SERPINF1 gene mutation, in which 1 affected fetus and 1 carrier were found; only one case of osteoarthritis with mild chondrodysplasia caused by COL2A1 gene mutation was found to harbor pathogenic variant in fetus; 1 case of hypophosphatasia due to ALPL gene mutation was not detected to carry pathogenic variant in fetus. By the time of follow-up, all 12 affected fetuses were terminated, and the remaining 10 fetuses except for one case still in pregnancy were born in good condition.Conclusion:Prenatal molecular diagnosis may confirm whether the fetus carries pathogenic variants at the first and second trimesters. For monogenic skeletal disorders that comply with Mendel′s law of separation, prenatal diagnosis can be determined by calculating the probability of recurrence of offspring. In addition, for families with de novo mutations in the offspring, it is necessary to pay attention to whether there are mosaic mutations in the parents.

Clinical features and gene mutation analysis of a male case of subcortical band heterotopia caused by mosaic mutation of DCX gene / 中华神经科杂志

Objective:To investigate the clinical phenotype and genotype of a male case of subcortical band heterotopia caused by mosaic mutation of DCX gene.Methods:The clinical data and magnetic resonance imaging (MRI) features of a male case of subcortical band heterotopia diagnosed in the Department of Neurology, Children′s Hospital Affiliated to Zhengzhou University in August 2020 were analyzed retrospectively. At the same time, the whole exon sequencing of the families was performed by next generation sequencing method, the suspicious mutation was verified by polymerase chain reaction Sanger sequencing, and their genetic mutation characteristics were analyzed.Results:The proband, one male, aged 5 years and 1 month, was hospitalized in August 2020 with the complaint of intermittent convulsions for 4 years and six months. Clinical features included that limb muscle tension was slightly high, intellectual and motor development was backward, and head circumference was 48 cm. MRI of his head showed diffuse thick subcortical band heterotopia. The detection of whole exon sequencing in his family showed that there was hemizygous mosaic mutation in DCX gene (mosaic ratio 44%), c.148A>G (p.k50E). The mosaic ratios of oral mucosa and urinalysis were 38.2% and 44.8% respectively. His parents were wild-type, The mutation found in this patient has not been reported at home and abroad.Conclusions:The mosaic variation of DCX gene can cause subcortical band heterotopia in males. The variation of DCX gene c.148A>G (p.k50E) may be the possible cause of the proband, which expands the variation spectrum of subcortical band heterotopia.

TSC2 somatic mosaic mutation, including extra-tumor tissue, may be the developmental cause of solitary subependymal giant cell astrocytoma.

PURPOSE: Subependymal giant cell astrocytomas (SEGAs) are tumors that usually arise in the wall of one or the other lateral ventricle near a foramen of Monro, most often on a background of tuberous sclerosis complex (TSC). TSC has a variety of clinical manifestations caused by germline mutations of the TSC complex subunit 1 or 2 (TSC1, TSC2) genes. SEGAs without clinical manifestations of TSC are termed solitary SEGAs, which are hypothesized to be caused by tumor-only TSC1/2 mutations, or "forme fruste" of TSC with somatic mosaic mutations. However, it is difficult to distinguish between the two. Here, we report three patients with genetically investigated solitary SEGAs and review this rare manifestation. METHODS: SEGA was completely removed in two patients and partially removed in one. Genetic analyses were performed on the tumor tissue and on peripheral blood via DNA microarray, reverse-transcriptase polymerase chain reaction, and next-generation sequencing with ultra-deep sequencing of mutation points. RESULTS: All three patients had tumors with TSC2 somatic mutations and loss of heterozygosity (LOH). In one patient, the same TSC2 mutation was also detected in 1% of leukocytes in his blood. The tumors did not recur, and clinical manifestations of TSC did not develop during the 4-year follow-up. CONCLUSIONS: The genetic cause of solitary SEGAs may be a TSC2 mutation with LOH. In patients with solitary SEGA, mosaic mutations may present in other organs, and TSC may clinically manifest later in life; therefore, patients should be followed up for prolonged periods.

Dual Deep Sequencing Improves the Accuracy of Low-Frequency Somatic Mutation Detection in Cancer Gene Panel Testing.

Cancer gene panel testing requires accurate detection of somatic mosaic mutations, as the test sample consists of a mixture of cancer cells and normal cells; each minor clone in the tumor also has different somatic mutations. Several studies have shown that the different types of software used for variant calling for next generation sequencing (NGS) can detect low-frequency somatic mutations. However, the accuracy of these somatic variant callers is unknown. We performed cancer gene panel testing in duplicate experiments using three different high-fidelity DNA polymerases in pre-capture amplification steps and analyzed by three different variant callers, Strelka2, Mutect2, and LoFreq. We selected six somatic variants that were detected in both experiments with more than two polymerases and by at least one variant caller. Among them, five single nucleotide variants were verified by CEL nuclease-mediated heteroduplex incision with polyacrylamide gel electrophoresis and silver staining (CHIPS) and Sanger sequencing. In silico analysis indicated that the FBXW7 and MAP3K1 missense mutations cause damage at the protein level. Comparing three somatic variant callers, we found that Strelka2 detected more variants than Mutect2 and LoFreq. We conclude that dual sequencing with Strelka2 analysis is useful for detection of accurate somatic mutations in cancer gene panel testing.

The rate and spectrum of mosaic mutations during embryogenesis revealed by RNA sequencing of 49 tissues.

BACKGROUND: Mosaic mutations acquired during early embryogenesis can lead to severe early-onset genetic disorders and cancer predisposition, but are often undetectable in blood samples. The rate and mutational spectrum of embryonic mosaic mutations (EMMs) have only been studied in few tissues, and their contribution to genetic disorders is unknown. Therefore, we investigated how frequent mosaic mutations occur during embryogenesis across all germ layers and tissues. METHODS: Mosaic mutation detection in 49 normal tissues from 570 individuals (Genotype-Tissue Expression (GTEx) cohort) was performed using a newly developed multi-tissue, multi-individual variant calling approach for RNA-seq data. Our method allows for reliable identification of EMMs and the developmental stage during which they appeared. RESULTS: The analysis of EMMs in 570 individuals revealed that newborns on average harbor 0.5-1 EMMs in the exome affecting multiple organs (1.3230 × 10-8 per nucleotide per individual), a similar frequency as reported for germline de novo mutations. Our multi-tissue, multi-individual study design allowed us to distinguish mosaic mutations acquired during different stages of embryogenesis and adult life, as well as to provide insights into the rate and spectrum of mosaic mutations. We observed that EMMs are dominated by a mutational signature associated with spontaneous deamination of methylated cytosines and the number of cell divisions. After birth, cells continue to accumulate somatic mutations, which can lead to the development of cancer. Investigation of the mutational spectrum of the gastrointestinal tract revealed a mutational pattern associated with the food-borne carcinogen aflatoxin, a signature that has so far only been reported in liver cancer. CONCLUSIONS: In summary, our multi-tissue, multi-individual study reveals a surprisingly high number of embryonic mosaic mutations in coding regions, implying novel hypotheses and diagnostic procedures for investigating genetic causes of disease and cancer predisposition.

RASA1 mosaic mutations in patients with capillary malformation-arteriovenous malformation.

BACKGROUND: Capillary malformation-arteriovenous malformation is an autosomal dominant disorder, characterised by capillary malformations and increased risk of fast-flow vascular malformations, caused by loss-of-function mutations in the RASA1 or EPHB4 genes. Around 25% of the patients do not seem to carry a germline mutation in either one of these two genes. Even if other genes could be involved, some individuals may have mutations in the known genes that escaped detection by less sensitive techniques. We tested the hypothesis that mosaic mutations could explain some of previously negative cases. METHODS: DNA was extracted from peripheral blood lymphocytes, saliva or vascular malformation tissues from four patients. RASA1 and EPHB4 coding regions and exon/intron boundaries were analysed by targeted custom gene panel sequencing. A second panel and/or Sanger sequencing were used to confirm the identified mutations. RESULTS: Four distinct mosaic RASA1 mutations, with an allele frequency ranging from 3% to 25%, were identified in four index patients with classical capillary malformation-arteriovenous malformation phenotype. Three mutations were known, one was novel. In one patient, a somatic second hit was also identified. One index case had three affected children, illustrating that the mosaicism was also present in the germline. CONCLUSION: This study shows that RASA1 mosaic mutations can cause capillary malformation-arteriovenous malformation. Thus, highly sensitive sequencing techniques should be considered as diagnostic tools, especially for patients with no family history. Even low-level mosaicism can cause the classical phenotype and increased risk for offspring. In addition, our study further supports the second-hit pathophysiological mechanism to explain the multifocality of vascular lesions in this disorder.

A novel mutation of MSX1 inherited from maternal mosaicism causes a severely affected child with nonsyndromic oligodontia.

Mutations of MSX1 have been associated with nonsyndromic hypodontia. To seek the causal gene mutation sites in a family with nonsyndromic oligodontia, whole-exome sequencing (WES) was performed to seek the causative locus of the family. The candidate mutation was further identified by Sanger sequencing afterward. Two mutations of MSX1 were found both in the proband and her mother. One novel heterozygous missense mutation (c.C667G, p.R223G) of MSX1 inherited from the asymptomatic mother with mosaic mutation was located in the highly conserved fragment of exon 2. The other was a synonymous mutation (c.C348T, p.G116G) in exon 1, which had been reported. The novel maternal heterozygous missense mutation (c.C667G, p.R223G) was likely to be the major reason for nonsyndromic oligodontia in the family. This is the first mosaic variant that has been recorded of the MSX1 gene. Our study expands the phenotype-genotype correlation associated with MSX1 variants. Our study also suggests that the determination of the mosaicism is essential for precise genetic counseling if a disease appears to arise de novo.

Next-generation sequencing with comprehensive bioinformatics analysis facilitates somatic mosaic APC gene mutation detection in patients with familial adenomatous polyposis.

BACKGROUND: Familial adenomatous polyposis (FAP) is an autosomal dominant colorectal tumor characterized by numerous adenomatous colonic polyps that often lead to colon cancer. Although most patients with FAP harbored germline mutations in APC gene, it was recently recognized that patients with clinical FAP, but without detectable pathogenic mutations, could be associated with somatic mosaic APC mutation. METHODS: We reanalyzed the nest-generation sequencing (NGS) gene panel testing results of patients who were diagnosed with FAP, but did not have APC mutations, at Yonsei Cancer Prevention Center between July 2016 and March 2018. We tested several variant calling algorithms to identify low level mosaic variants. In one patient with a low frequency APC mutation, NGS analysis was performed together with endoscopic biopsy. Variant calling tools HaplotypeCaller, MuTect2, VarScan2, and Pindel were used. We also used 3'-Modified Oligonucleotides (MEMO)-PCR or conventional PCR for confirmation. RESULTS: Among 28 patients with clinical suspicion of FAP but no detectable pathogenic variants of colonic polyposis associated genes, somatic mosaic pathogenic variants were identified in seven patients. The variant allele frequency ranged from 0.3 to 7.7%. These variants were mostly detected through variant caller MuTect2 and Pindel, and were further confirmed using mutant enrichment with MEMO-PCR. CONCLUSIONS: The NGS with an adequate combination of bioinformatics tools is effective to detect low level somatic variants in a single assay. Because mosaic APC mutations are more frequent than previously thought, the presence of mosaic mutations must be considered when analyzing genetic tests of patients with FAP.

Danon disease presenting with early onset of hypertrophic cardiomyopathy and peripheral pigmentary retinal dystrophy in a female with a de novo novel mosaic mutation in the LAMP2 gene.

Purpose: To describe the phenotype and genotype in a young woman with Danon disease. Methods: The patient underwent an ophthalmic examination including best corrected visual acuity (BCVA), fundus photography and fundus autofluorescence (FAF), full-field electroretinography (full-field ERG), multifocal ERG, optical coherence tomography (OCT) and SAP-Humphrey 30-2 at the ages of 20 and 25. Electrooculography, fluorescein angiography (FA), indocyanine angiography and OCT angiography were performed only once. Genetic testing using a Next-Generation Sequencing panel and immunohistochemical analysis of LAMP2 protein expression were performed in the patient's explanted heart, and the patient's cardiologic and ophthalmologic records were retrospectively reviewed. Results: A de novo, novel, mosaic mutation, c.135dupA; p.(Trp46Metfs*10) was identified in exon 2 of the LAMP2 gene. Immunohistochemical investigation of the myocardium in the explanted heart revealed pronounced deficiency of LAMP2 protein in cardiomyocytes. The color photographs, FAF images and FA revealed more extensive peripheral pigmentary retinal dystrophy (PPRD) at the 5-year follow-up examination. No changes were observed in BCVA, OCT, SAP-Humphrey 30-2 or multifocal ERG findings at follow-up. Full-field ERG showed an asymmetric interocular reduction in ERG response at follow-up: the b-wave amplitude of the rod response had decreased by 29% in the right eye, but by only 6 % in the left eye. The a-wave amplitude of single-flash response had decreased by 9 % in the left eye, while it had increased by 3% in the right eye. Conclusions: Although PPRD progressed slowly, it was an important clue in the diagnosis of the life-threatening condition of Danon disease.

Using Digital Droplet Polymerase Chain Reaction to Detect the Mosaic GNAS Mutations in Whole Blood DNA or Circulating Cell-Free DNA in Fibrous Dysplasia and McCune-Albright Syndrome.

The GNAS postzygotic mosaic activating mutations involved in fibrous dysplasia and McCune-Albright syndrome (MAS) are not detectable in leukocytes by Sanger sequencing. Digital droplet polymerase chain reaction detects GNAS mutations in 7 of 12 patients (58.3%) suspected to have fibrous dysplasia/MAS from whole blood DNA, and in 4 of 5 patients (80%) from circulating cell-free DNA.