Physicochemical aspects of the tumour microenvironment as drivers of vasculogenic mimicry.

Tumour vascularisation is vital for cancer sustainment representing not only the main source of nutrients and oxygen supply but also an escape route for single or clustered cancer cells that, once detached from the primary mass, enter the blood circulation and disseminate to distant organs. Among the mechanisms identified to contribute to tumour vascularisation, vasculogenic mimicry (VM) is gaining increasing interest in the scientific community representing an intriguing target for cancer treatment. VM indeed associates with highly aggressive tumour phenotypes and strongly impairs patient outcomes. Differently from vessels of healthy tissues, tumour vasculature is extremely heterogeneous and tortuous, impeding efficient chemotherapy delivery, and at the meantime hyperpermeable and thus extremely accessible to metastasising cancer cells. Moreover, tumour vessel disorganisation creates a self-reinforcing vicious circle fuelling cancer malignancy and progression. Because of the inefficient oxygen delivery and metabolic waste removal from tumour vessels, many cells within the tumour mass indeed experience hypoxia and acidosis, now considered hallmarks of cancer. Being strong inducers of vascularisation, therapy resistance, inflammation and metastasis, hypoxia and acidosis create a permissive microenvironment for cancer progression and dissemination. Along with these considerations, we decided to focus our attention on the relationship between hypoxia/acidosis and VM. Indeed, besides tumour angiogenesis, VM is strongly influenced by both hypoxia and acidosis, which could potentiate each other and fuel this vicious circle. Thus, targeting hypoxia and acidosis may represent a potential target to treat VM to impair tumour perfusion and cancer cell sustainment.

Evidence for a Pathogenic Role of CSMD1 in Childhood Apraxia of Speech.

Childhood apraxia of speech (CAS) is a pediatric motor speech disorder. The genetic etiology of this complex neurological condition is not yet well understood, although some genes have been linked to it. We describe the case of a boy with a severe and persistent motor speech disorder, consistent with CAS, and a coexisting language impairment.Whole exome sequencing in our case revealed a de novo and splicing mutation in the CSMD1 gene.

Nintedanib-αVß6 Integrin Ligand Conjugates Reduce TGFß-Induced EMT in Human Non-Small Cell Lung Cancer.

Growth factors and cytokines released in the lung cancer microenvironment promote an epithelial-to-mesenchymal transition (EMT) that sustains the progression of neoplastic diseases. TGFß is one of the most powerful inducers of this transition, as it induces overexpression of the fibronectin receptor, αvß6 integrin, in cancer cells which, in turn, is strongly associated with EMT. Thus, αvß6 integrin receptors may be exploited as a target for the selective delivery of anti-tumor agents. We introduce three novel synthesized conjugates, in which a selective αvß6 receptor ligand is linked to nintedanib, a potent kinase inhibitor used to treat advanced adenocarcinoma lung cancer in clinics. The αvß6 integrin ligand directs nintedanib activity to the target cells of the tumor microenvironment, avoiding the onset of negative side effects in normal cells. We found that the three conjugates inhibit the adhesion of cancer cells to fibronectin in a concentration-dependent manner and that αvß6-expressing cells internalized the conjugated compounds, thus permitting nintedanib to inhibit 2D and 3D cancer cell growth and suppress the clonogenic ability of the EMT phenotype as well as intervening in other aspects associated with the EMT transition. These results highlight αvß6 receptors as privileged access points for dual-targeting molecular conjugates engaged in an efficient and precise strategy against non-small cell lung cancer.

uPAR-expressing melanoma exosomes promote angiogenesis by VE-Cadherin, EGFR and uPAR overexpression and rise of ERK1,2 signaling in endothelial cells.

Exosomes (Exos) have been reported to promote pre-metastatic niche formation, proliferation, angiogenesis and metastasis. We have investigated the role of uPAR in melanoma cell lines-derived Exos and their pro-angiogenic effects on human microvascular endothelial cells (HMVECs) and endothelial colony-forming cells (ECFCs). Melanoma Exos were isolated from conditioned media of A375 and M6 cells by differential centrifugation and filtration. Tunable Resistive Pulse Sensing (TRPS) and Nanoparticle tracking analysis were performed to analyze dimension and concentration of Exos. The CRISPR-Cas 9 technology was exploited to obtain a robust uPAR knockout. uPAR is expressed in melanoma Exos that are internalized by HMVECs and ECFCs, enhancing VE-Cadherin, EGFR and uPAR expression in endothelial cells that undergo a complete angiogenic program, including proliferation, migration and tube formation. uPAR loss reduced the pro-angiogenic effects of melanoma Exos in vitro and in vivo by inhibition of VE-Cadherin, EGFR and uPAR expression and of ERK1,2 signaling in endothelial cells. A similar effect was obtained with a peptide that inhibits uPAR-EGFR interaction and with the EGFR inhibitor Gefitinib, which also inhibited melanoma Exos-dependent EGFR phosphorylation. This study suggests that uPAR is required for the pro-angiogenic activity of melanoma Exos. We propose the identification of uPAR-expressing Exos as a potentially useful biomarker for assessing pro-angiogenic propensity and eventually monitoring the response to treatment in metastatic melanoma patients.

Extracellular Acidosis Differentially Regulates Estrogen Receptor ß-Dependent EMT Reprogramming in Female and Male Melanoma Cells.

Clinical outcomes of melanoma patients pointed out a gender disparity that supports a correlation between sex hormone activity on estrogen receptors (ER) and melanoma development and progression. Here, we found that the epithelial-to-mesenchymal transition (EMT) of melanoma cells induced by extracellular acidosis, which is a crucial hallmark of solid cancers, correlates with the expression of ERß, the most representative ER on melanoma cells. Extracellular acidosis induces an enhanced expression of ERß in female cells and EMT markers remain unchanged, while extracellular acidosis did not induce the expression of ERß in male cells and EMT was strongly promoted. An inverse relationship between ERß expression and EMT markers in melanoma cells of different sex exposed to extracellular acidosis was revealed by two different technical approaches: florescence-activated cell sorting of high ERß expressing cell subpopulations and ERß receptor silencing. Finally, we found that ERß regulates EMT through NF-κB activation. These results demonstrate that extracellular acidosis drives a differential ERß regulation in male and female melanoma cells and that this gender disparity might open new perspectives for personalized therapeutic approaches.

When to test fetuses for RASopathies? Proposition from a systematic analysis of 352 multicenter cases and a postnatal cohort.

PURPOSE: Recent studies have identified suggestive prenatal features of RASopathies (e.g., increased nuchal translucency [NT], cystic hygroma [CH], hydrops, effusions, congenital heart diseases [CHD], polyhydramnios, renal anomalies). Our objective is to clarify indications for RASopathy prenatal testing. We compare genotype distributions between pre- and postnatal populations and propose genotype-phenotype correlations. METHODS: Three hundred fifty-two chromosomal microarray-negative cases sent for prenatal RASopathy testing between 2012 and 2019 were collected. For most, 11 RASopathy genes were tested. Postnatal cohorts (25 patients with available prenatal information and 108 institutional database genotypes) and the NSeuroNet database were used for genotypic comparisons. RESULTS: The overall diagnostic yield was 14% (50/352), with rates >20% for effusions, hydrops, and CHD. Diagnostic yield was significantly improved in presence of hypertrophic cardiomyopathy (HCM), persistent or associated CH, any suggestive finding combined with renal anomaly or polyhydramnios, or ≥2 ultrasound findings. Largest prenatal contributors of pathogenic variants were PTPN11 (30%), RIT1 (16%), RAF1 (14%), and HRAS (12%), which considerably differ from their prevalence in postnatal populations. HRAS, LZTR1, and RAF1 variants correlated with hydrops/effusions, and RIT1 with prenatal onset HCM. CONCLUSION: After normal chromosomal microarray, RASopathies should be considered when any ultrasound finding of lymphatic dysplasia or suggestive CHD is found alone or in association.

Glycolysis-derived acidic microenvironment as a driver of endothelial dysfunction in systemic sclerosis.

OBJECTIVES: SSc is an autoimmune disease characterized by peripheral vasculopathy and skin and internal organ fibrosis. Accumulating evidence underlines a close association between a metabolic reprogramming of activated fibroblasts and fibrosis. This prompted us to determine the metabolism of SSc dermal fibroblasts and the effect on the vasculopathy characterizing the disease. METHODS: A Seahorse XF96 Extracellular Flux Analyzer was used to evaluate SSc fibroblast metabolism. In vitro invasion and capillary morphogenesis assays were used to determine the angiogenic ability of endothelial cells (ECs). Immunofluorescence, flow cytometry and real-time PCR techniques provided evidence of the molecular mechanism behind the impaired vascularization that characterizes SSc patients. RESULTS: SSc fibroblasts, compared with controls, showed a boosted glycolytic metabolism with increased lactic acid release and subsequent extracellular acidification that in turn was found to impair EC invasion and organization in capillary-like networks without altering cell viability. A molecular link between extracellular acidosis and endothelial dysfunction was identified as acidic ECs upregulated MMP-12, which cleaves and inactivates urokinase-type plasminogen activator receptor, impairing angiogenesis in SSc. Moreover, the acidic environment was found to induce the loss of endothelial markers and the acquisition of mesenchymal-like features in ECs, thus promoting the endothelial-to-mesenchymal transition process that contributes to both capillary rarefaction and tissue fibrosis in SSc. CONCLUSION: This study showed the relationship of the metabolic reprogramming of SSc dermal fibroblasts, extracellular acidosis and endothelial dysfunction that may contribute to the impairment and loss of peripheral capillary networks in SSc disease.

Enhanced Vasculogenic Capacity Induced by 5-Fluorouracil Chemoresistance in a Gastric Cancer Cell Line.

Chemotherapy is still widely used as a coadjutant in gastric cancer when surgery is not possible or in presence of metastasis. During tumor evolution, gatekeeper mutations provide a selective growth advantage to a subpopulation of cancer cells that become resistant to chemotherapy. When this phenomenon happens, patients experience tumor recurrence and treatment failure. Even if many chemoresistance mechanisms are known, such as expression of ATP-binding cassette (ABC) transporters, aldehyde dehydrogenase (ALDH1) activity and activation of peculiar intracellular signaling pathways, a common and universal marker for chemoresistant cancer cells has not been identified yet. In this study we subjected the gastric cancer cell line AGS to chronic exposure of 5-fluorouracil, cisplatin or paclitaxel, thus selecting cell subpopulations showing resistance to the different drugs. Such cells showed biological changes; among them, we observed that the acquired chemoresistance to 5-fluorouracil induced an endothelial-like phenotype and increased the capacity to form vessel-like structures. We identified the upregulation of thymidine phosphorylase (TYMP), which is one of the most commonly reported mutated genes leading to 5-fluorouracil resistance, as the cause of such enhanced vasculogenic ability.

Expanding the phenotype of Wiedemann-Steiner syndrome: Craniovertebral junction anomalies.

Wiedemann-Steiner syndrome (WDSTS) is a rare autosomal dominant condition caused by heterozygous loss of function variants in the KMT2A (MLL) gene, encoding a lysine N-methyltransferase that mediates a histone methylation pattern specific for epigenetic transcriptional activation. WDSTS is characterized by a distinctive facial phenotype, hypertrichosis, short stature, developmental delay, intellectual disability, congenital malformations, and skeletal anomalies. Recently, a few patients have been reported having abnormal skeletal development of the cervical spine. Here we describe 11 such individuals, all with KMT2A de novo loss-of-function variants: 10 showed craniovertebral junction anomalies, while an 11th patient had a cervical abnormality in C7. By evaluating clinical and diagnostic imaging data we characterized these anomalies, which consist primarily of fused cervical vertebrae, C1 and C2 abnormalities, small foramen magnum and Chiari malformation type I. Craniovertebral anomalies in WDSTS patients have been largely disregarded so far, but the increasing number of reports suggests that they may be an intrinsic feature of this syndrome. Specific investigation strategies should be considered for early identification and prevention of craniovertebral junction complications in WDSTS patients.

The Carbonic Anhydrase IX inhibitor SLC-0111 as emerging agent against the mesenchymal stem cell-derived pro-survival effects on melanoma cells.

Mesenchymal stem cells (MSC) take part to solid tumour-associated stroma and critically influence progression of malignancy. Our study represents a striking example of melanoma progression to a more malignant and resistant phenotype promoted by MSC and the possibility to contrast this diabolic liaison using CAIX inhibitors. In particular, we demonstrated that melanoma cells exposed to a MSC-conditioned medium switch to a more malignant phenotype, characterised by resistance to programmed cell death and endowed with an epithelial-to-mesenchymal transition and stem cell characteristics. These effects were reversed abrogating MSC CAIX activity using SLC-0111, a CAIX inhibitor. Moreover, the acquisition by melanoma cells of a Vemurafenib-resistant phenotype upon MSC-conditioned medium exposure was removed when MSC were treated with SLC-0111. Therefore, MSC may profoundly reprogramme melanoma cells towards a wide resistant phenotype through CAIX involvement, as the use of SLC-0111 is able to contrast the development of this highly risky adaptation for disease progression.

A potentiated cooperation of carbonic anhydrase IX and histone deacetylase inhibitors against cancer.

The emergence of tumour recurrence and resistance limits the survival rate for most tumour-bearing patients. Only, combination therapies targeting pathways involved in the induction and in the maintenance of cancer growth and progression might potentially result in an enhanced therapeutic efficacy. Herein, we provided a prospective combination treatment that includes suberoylanilide hydroxamic acid (SAHA), a well-known inhibitor of histone deacetylases (HDACs), and SLC-0111, a novel inhibitor of carbonic anhydrase (CA) IX. We proved that HDAC inhibition with SAHA in combination with SLC-0111 affects cell viability and colony forming capability to greater extent than either treatment alone of breast, colorectal and melanoma cancer cells. At the molecular level, this therapeutic regimen resulted in a synergistically increase of histone H4 and p53 acetylation in all tested cell lines. Overall, our findings showed that SAHA and SLC-0111 can be regarded as very attractive combination providing a potential therapeutic strategy against different cancer models.

The carbonic anhydrase IX inhibitor SLC-0111 sensitises cancer cells to conventional chemotherapy.

Drug combination represents one of the most accredited strategies of cancer therapy able to improve drug efficacy and possibly overcome drug resistance. Among the agents used to complement conventional chemotherapy, carbonic anhydrase IX (CAIX) inhibitors appear as one of the most suitable, as markers of hypoxic and acidic cancer cells which do not respond to chemo- and radiotherapy. We performed preclinical in vitro assays to evaluate whether the SLC-0111 CAIX inhibitor co-operates and potentiates the cytotoxic effects of conventional chemotherapeutic drugs in A375-M6 melanoma cells, MCF7 breast cancer cells, and HCT116 colorectal cancer cells. Here, we demonstrate that the SLC-0111 CAIX inhibitor potentiates cytotoxicity of Dacarbazine and Temozolomide currently used for advanced melanoma treatment. SLC-0111 also increases breast cancer cell response to Doxorubicin and enhances 5-Fluorouracil cytostatic activity on colon cancer cells. These findings disclose the possibility to extend the use of CAIX inhibitors in the combination therapy of various cancer histotypes.

SOX2 as a novel contributor of oxidative metabolism in melanoma cells.

BACKGROUND: Deregulated metabolism is a hallmark of cancer and recent evidence underlines that targeting tumor energetics may improve therapy response and patient outcome. Despite the general attitude of cancer cells to exploit the glycolytic pathway even in the presence of oxygen (aerobic glycolysis or "Warburg effect"), tumor metabolism is extremely plastic, and such ability to switch from glycolysis to oxidative phosphorylation (OxPhos) allows cancer cells to survive under hostile microenvironments. Recently, OxPhos has been related with malignant progression, chemo-resistance and metastasis. OxPhos is induced under extracellular acidosis, a well-known characteristic of most solid tumors, included melanoma. METHODS: To evaluate whether SOX2 modulation is correlated with metabolic changes under standard or acidic conditions, SOX2 was silenced and overexpressed in several melanoma cell lines. To demonstrate that SOX2 directly represses HIF1A expression we used chromatin immunoprecipitation (ChIP) and luciferase assay. RESULTS: In A375-M6 melanoma cells, extracellular acidosis increases SOX2 expression, that sustains the oxidative cancer metabolism exploited under acidic conditions. By studying non-acidic SSM2c and 501-Mel melanoma cells (high- and very low-SOX2 expressing cells, respectively), we confirmed the metabolic role of SOX2, attributing SOX2-driven OxPhos reprogramming to HIF1α pathway disruption. CONCLUSIONS: SOX2 contributes to the acquisition of an aggressive oxidative tumor phenotype, endowed with enhanced drug resistance and metastatic ability.

The acidic microenvironment as a possible niche of dormant tumor cells.

Although surgical excision, chemo-, and radio-therapy are clearly advanced, tumors may relapse due to cells of the so-called "minimal residual disease". Indeed, small clusters of tumor cells persist in host tissues after treatment of the primary tumor elaborating strategies to survive and escape from immunological attacks before their relapse: this variable period of remission is known as "cancer dormancy". Therefore, it is crucial to understand and consider the major concepts addressing dormancy, to identify new targets and disclose potential clinical strategies. Here, we have particularly focused the relationships between tumor microenvironment and cancer dormancy, looking at a re-appreciated aspect of this compartment that is the low extracellular pH. Accumulating evidences indicate that acidity of tumor microenvironment is associated with a poor prognosis of tumor-bearing patients, stimulates a chemo- and radio-therapy resistant phenotype, and suppresses the tumoricidal activity of cytotoxic lymphocytes and natural killer cells, and all these aspects are useful for dormancy. Therefore, this review discusses the possibility that acidity of tumor microenvironment may provide a new, not previously suggested, adequate milieu for "dormancy" of tumor cells.

The genetic and clinical spectrum of a large cohort of patients with distal renal tubular acidosis.

Primary distal renal tubular acidosis is a rare genetic disease. Mutations in SLC4A1, ATP6V0A4, and ATP6V1B1 genes have been described as the cause of the disease, transmitted as either an autosomal dominant or recessive trait. Particular clinical features, such as sensorineural hearing loss, have been mainly described in association with mutations in one gene instead of the others. Nevertheless, the diagnosis of distal renal tubular acidosis is essentially based on clinical and laboratory findings, and the series of patients described so far are usually represented by small cohorts. Therefore, a strict genotype-phenotype correlation is still lacking, and questions about whether clinical and laboratory data should direct the genetic analysis remain open. Here, we applied next-generation sequencing in 89 patients with a clinical diagnosis of distal renal tubular acidosis, analyzing the prevalence of genetic defects in SLC4A1, ATP6V0A4, and ATP6V1B1 genes and the clinical phenotype. A genetic cause was determined in 71.9% of cases. In our group of sporadic cases, clinical features, including sensorineural hearing loss, are not specific indicators of the causal underlying gene. Mutations in the ATP6V0A4 gene are quite as frequent as mutations in ATP6V1B1 in patients with recessive disease. Chronic kidney disease was frequent in patients with a long history of the disease. Thus, our results suggest that when distal renal tubular acidosis is suspected, complete genetic testing could be considered, irrespective of the clinical phenotype of the patient.

Differential uPAR recruitment in caveolar-lipid rafts by GM1 and GM3 gangliosides regulates endothelial progenitor cells angiogenesis.

Gangliosides and the urokinase plasminogen activator receptor (uPAR) tipically partition in specialized membrane microdomains called lipid-rafts. uPAR becomes functionally important in fostering angiogenesis in endothelial progenitor cells (EPCs) upon recruitment in caveolar-lipid rafts. Moreover, cell membrane enrichment with exogenous GM1 ganglioside is pro-angiogenic and opposite to the activity of GM3 ganglioside. On these basis, we first checked the interaction of uPAR with membrane models enriched with GM1 or GM3, relying on the adoption of solid-supported mobile bilayer lipid membranes with raft-like composition formed onto solid hydrophilic surfaces, and evaluated by surface plasmon resonance (SPR) the extent of uPAR recruitment. We estimated the apparent dissociation constants of uPAR-GM1/GM3 complexes. These preliminary observations, indicating that uPAR binds preferentially to GM1-enriched biomimetic membranes, were validated by identifying a pro-angiogenic activity of GM1-enriched EPCs, based on GM1-dependent uPAR recruitment in caveolar rafts. We have observed that addition of GM1 to EPCs culture medium promotes matrigel invasion and capillary morphogenesis, as opposed to the anti-angiogenesis activity of GM3. Moreover, GM1 also stimulates MAPKinases signalling pathways, typically associated with an angiogenesis program. Caveolar-raft isolation and Western blotting of uPAR showed that GM1 promotes caveolar-raft partitioning of uPAR, as opposed to control and GM3-challenged EPCs. By confocal microscopy, we have shown that in EPCs uPAR is present on the surface in at least three compartments, respectively, associated to GM1, GM3 and caveolar rafts. Following GM1 exogenous addition, the GM3 compartment is depleted of uPAR which is recruited within caveolar rafts thereby triggering angiogenesis.

Two novel splicing mutations in the SLC45A2 gene cause Oculocutaneous Albinism Type IV by unmasking cryptic splice sites.

Oculocutaneous albinism (OCA) is characterized by hypopigmentation of the skin, hair and eye, and by ophthalmologic abnormalities caused by a deficiency in melanin biosynthesis. OCA type IV (OCA4) is one of the four commonly recognized forms of albinism, and is determined by mutation in the SLC45A2 gene. Here, we investigated the genetic basis of OCA4 in an Italian child. The mutational screening of the SLC45A2 gene identified two novel potentially pathogenic splicing mutations: a synonymous transition (c.888G>A) involving the last nucleotide of exon 3 and a single-nucleotide insertion (c.1156+2dupT) within the consensus sequence of the donor splice site of intron 5. As computer-assisted analysis for mutant splice-site prediction was not conclusive, we investigated the effects on pre-mRNA splicing of these two variants by using an in vitro minigene approach. Production of mutant transcripts in HeLa cells demonstrated that both mutations cause the almost complete abolishment of the physiologic donor splice site, with the concomitant unmasking of cryptic donor splice sites. To our knowledge, this work represents the first in-depth molecular characterization of splicing defects in a OCA4 patient.

Radiosynthesis and micro-SPECT analysis of triazole-based RGD integrin ligands as non-peptide molecular imaging probes for angiogenesis.

Taking advantage of click chemistry, we synthesized triazole-containing RGD peptidomimetics capable of binding to αvß3 integrin with diverse potency, and selected (125)I-labeled compounds proved to interact in vitro and in vivo with αvß3 integrin expressed by melanoma cells. Two (125)I-compounds containing either 2-aminobenzimidazole or 2-aminopyridine groups as the arginine bioisostere with the capacity to selectively bind cells of highly expressing αvß3 melanoma xenografts were found using micro-SPECT imaging studies.

CHARGE-like presentation, craniosynostosis and mild Mowat-Wilson Syndrome diagnosed by recognition of the distinctive facial gestalt in a cohort of 28 new cases.

Mowat-Wilson syndrome (MWS) is characterized by moderate to severe intellectual disability and distinctive facial features in association with variable structural congenital anomalies/clinical features including congenital heart disease, Hirschsprung disease, hypospadias, agenesis of the corpus callosum, short stature, epilepsy, and microcephaly. Less common clinical features include ocular anomalies, craniosynostosis, mild intellectual disability, and choanal atresia. These cases may be more difficult to diagnose. In this report, we add 28 MWS patients with molecular confirmation of ZEB2 mutation, including seven with an uncommon presenting feature. Among the "unusual" patients, two patients had clinical features of charge syndrome including choanal atresia, coloboma, cardiac defects, genitourinary anomaly (1/2), and severe intellectual disability; two patients had craniosynostosis; and three patients had mild intellectual disability. Sixteen patients have previously-unreported mutations in ZEB2. Genotype-phenotype correlations were suggested in those with mild intellectual disability (two had a novel missense mutation in ZEB2, one with novel splice site mutation). This report increases the number of reported patients with MWS with unusual features, and is the first report of MWS in children previously thought to have CHARGE syndrome. These patients highlight the importance of facial gestalt in the accurate identification of MWS when less common features are present.

Loss-of-function mutations in PTPN11 cause metachondromatosis, but not Ollier disease or Maffucci syndrome.

Metachondromatosis (MC) is a rare, autosomal dominant, incompletely penetrant combined exostosis and enchondromatosis tumor syndrome. MC is clinically distinct from other multiple exostosis or multiple enchondromatosis syndromes and is unlinked to EXT1 and EXT2, the genes responsible for autosomal dominant multiple osteochondromas (MO). To identify a gene for MC, we performed linkage analysis with high-density SNP arrays in a single family, used a targeted array to capture exons and promoter sequences from the linked interval in 16 participants from 11 MC families, and sequenced the captured DNA using high-throughput parallel sequencing technologies. DNA capture and parallel sequencing identified heterozygous putative loss-of-function mutations in PTPN11 in 4 of the 11 families. Sanger sequence analysis of PTPN11 coding regions in a total of 17 MC families identified mutations in 10 of them (5 frameshift, 2 nonsense, and 3 splice-site mutations). Copy number analysis of sequencing reads from a second targeted capture that included the entire PTPN11 gene identified an additional family with a 15 kb deletion spanning exon 7 of PTPN11. Microdissected MC lesions from two patients with PTPN11 mutations demonstrated loss-of-heterozygosity for the wild-type allele. We next sequenced PTPN11 in DNA samples from 54 patients with the multiple enchondromatosis disorders Ollier disease or Maffucci syndrome, but found no coding sequence PTPN11 mutations. We conclude that heterozygous loss-of-function mutations in PTPN11 are a frequent cause of MC, that lesions in patients with MC appear to arise following a "second hit," that MC may be locus heterogeneous since 1 familial and 5 sporadically occurring cases lacked obvious disease-causing PTPN11 mutations, and that PTPN11 mutations are not a common cause of Ollier disease or Maffucci syndrome.