Application of droplet digital PCR in minimal residual disease monitoring of rare fusion transcripts and mutations in haematological malignancies.

Leukaemia of various subtypes are driven by distinct chromosomal rearrangement or genetic abnormalities. The leukaemogenic fusion transcripts or genetic mutations serve as molecular markers for minimal residual disease (MRD) monitoring. The current study evaluated the applicability of several droplet digital PCR assays for the detection of these targets at RNA and DNA levels (atypical BCR::ABL1 e19a2, e23a2ins52, e13a2ins74, rare types of CBFB::MYH11 (G and I), PCM1::JAK2, KMT2A::ELL2, PICALM::MLLT10 fusion transcripts and CEBPA frame-shift and insertion/duplication mutations) with high sensitivity. The analytical performances were assessed by the limit of blanks, limit of detection, limit of quantification and linear regression. Our data demonstrated serial MRD monitoring for patients at molecular level could become "digitalized", which was deemed important to guide clinicians in treatment decision for better patient care.

Distinct karyotypic and mutational landscape in trisomy AML.

Trisomy karyotype occurs in 5%-10% of AML. Its mutational landscape and prognostic significance are not well defined. A cohort of 156 trisomy AML patients was analysed, with reference to 615 cytogenetically normal (CN) AML patients. Trisomy AML showed distinct mutational landscape with more prevalent SMC1A, N/KRAS, ASXL1 and BCOR but fewer CEBPAbZIP and NPM1 mutations in patients ≤60, and fewer NPM1 mutations in those >60. NRAS mutations were associated with poor outcome in trisomy AML, whereas DNMT3A and FLT3-ITD mutations had neutral effect. Trisomy AML appeared biologically distinct from CN-AML.

Discrete single-cell microRNA analysis for phenotyping the heterogeneity of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a highly heterogenous cancer in hematopoiesis, and its subtype specification is greatly important in the clinical practice for AML diagnosis and prognosis. Increasing evidence has shown the association between microRNA (miRNA) phenotype and AML therapeutic outcomes, emphasizing the need for novel techniques for convenient, sensitive, and efficient miRNA profiling in clinical practices. Here, we describe a nanoneedle-based discrete single-cell microRNA profiling technique for multiplexed phenotyping of AML heterogeneity without the requirement of sequencing or polymerase chain reaction (PCR). In virtue of a biochip-based and non-destructive nature of the assay, the expression of nine miRNAs in large number of living AML cells can be simultaneously analyzed with discrete single-cell level information, thus providing a proof-of-concept demonstration of an AML subtype classifier based on the multidimensional miRNA data. We showed successful analysis of subtype-specific cellular composition with over 90% accuracy and identified drug-responsive leukemia subpopulations among a mixed suspension of cells modeling different AML subtypes. The adoption of machine learning algorithms for processing the large-scale nanoneedle-based miRNA data shows the potential for powerful prediction capability in clinical applications to assist therapeutic decisions. We believe that this platform provides an efficient and cost-effective solution to move forward the translational prognostic usage of miRNAs in AML treatment and can be readily and advantageously applied in analyzing rare patient-derived clinical samples.

Pharmacogenomic Profiling of Pediatric Acute Myeloid Leukemia to Identify Therapeutic Vulnerabilities and Inform Functional Precision Medicine.

Despite the expanding portfolio of targeted therapies for adults with acute myeloid leukemia (AML), direct implementation in children is challenging due to inherent differences in underlying genetics. Here we established the pharmacologic profile of pediatric AML by screening myeloblast sensitivity to approved and investigational agents, revealing candidates of immediate clinical relevance. Drug responses ex vivo correlated with patient characteristics, exhibited age-specific alterations, and concorded with activities in xenograft models. Integration with genomic data uncovered new gene-drug associations, suggesting actionable therapeutic vulnerabilities. Transcriptome profiling further identified gene-expression signatures associated with on- and off-target drug responses. We also demonstrated the feasibility of drug screening-guided treatment for children with high-risk AML, with two evaluable cases achieving remission. Collectively, this study offers a high-dimensional gene-drug clinical data set that could be leveraged to research the unique biology of pediatric AML and sets the stage for realizing functional precision medicine for the clinical management of the disease. SIGNIFICANCE: We conducted integrated drug and genomic profiling of patient biopsies to build the functional genomic landscape of pediatric AML. Age-specific differences in drug response and new gene-drug interactions were identified. The feasibility of functional precision medicine-guided management of children with high-risk AML was successfully demonstrated in two evaluable clinical cases. This article is highlighted in the In This Issue feature, p. 476.

CD33-targeting extracellular vesicles deliver antisense oligonucleotides against FLT3-ITD and miR-125b for specific treatment of acute myeloid leukaemia.

INTRODUCTION: Acute Myeloid Leukaemia (AML) is the most common blood cancer in adults. Although 2 out of 3 AML patients go into total remission after chemotherapies and targeted therapies, the disease recurs in 60%-65% of younger adult patients within 3 years after diagnosis with a dramatically decreased survival rate. Therapeutic oligonucleotides are promising treatments under development for AML as they can be designed to silence oncogenes with high specificity and flexibility. However, there are not many well validated approaches for safely and efficiently delivering oligonucleotide drugs. This issue could be resolved by utilizing a new generation of delivery vehicles such as extracellular vesicles (EVs). METHODS: In this study, we harness red blood cell-derived EVs (RBCEVs) and engineer them via exogenous drug loading and surface functionalization to develop an efficient drug delivery system for AML. Particularly, EVs are designed to target CD33, a common surface marker with elevated expression in AML cells via the conjugation of a CD33-binding monoclonal antibody onto the EV surface. RESULTS: The conjugation of RBCEVs with the CD33-binding antibody significantly increases the uptake of RBCEVs by CD33-positive AML cells, but not by CD33-negative cells. We also load CD33-targeting RBCEVs with antisense oligonucleotides (ASOs) targeting FLT3-ITD or miR-125b, 2 common oncogenes in AML, and demonstrate that the engineered EVs improve leukaemia suppression in in vitro and in vivo models of AML. CONCLUSION: Targeted RBCEVs represent an innovative, efficient, and versatile delivery platform for therapeutic ASOs and can expedite the clinical translation of oligonucleotide drugs for AML treatments by overcoming current obstacles in oligonucleotide delivery.

Surface-engineered extracellular vesicles for targeted delivery of therapeutic RNAs and peptides for cancer therapy.

The advent of novel therapeutics in recent years has urged the need for a safe, non-immunogenic drug delivery vector capable of delivering therapeutic payloads specifically to diseased cells, thereby increasing therapeutic efficacy and reducing side effects. Extracellular vesicles (EVs) have garnered attention in recent years as a potentially ideal vector for drug delivery, taking into account their intrinsic ability to transfer bioactive cargo to recipient cells and their biocompatible nature. However, natural EVs are limited in their therapeutic potential and many challenges need to be overcome before engineered EVs satisfy the levels of efficiency, stability, safety and biocompatibility required for therapeutic use. Here, we demonstrate that an enzyme-mediated surface functionalization method in combination with streptavidin-mediated conjugation results in efficient surface functionalization of EVs. Surface functionalization using the above methods permits the stable and biocompatible conjugation of peptides, single domain antibodies and monoclonal antibodies at high copy number on the EV surface. Functionalized EVs demonstrated increased accumulation in target cells expressing common cancer associated markers such as CXCR4, EGFR and EpCAM both in vitro and in vivo. The functionality of this approach was further highlighted by the ability of targeting EVs to specifically deliver therapeutic antisense oligonucleotides to a metastatic breast tumor model, resulting in increased knockdown of a targeted oncogenic microRNA and improved metastasis suppression. The method was also used to equip EVs with a bifunctional peptide that targets EVs to leukemia cells and induces apoptosis, leading to leukemia suppression. Moreover, we conducted extensive testing to verify the biocompatibility, and safety of engineered EVs for therapeutic use, suggesting that surface modified EVs can be used for repeated dose treatment with no detectable adverse effects. This modular, biocompatible method of EV engineering offers a promising avenue for the targeted delivery of a range of therapeutics while addressing some of the safety concerns associated with EV-based drug delivery.

Regulation of proton partitioning in kinase-activating acute myeloid leukemia and its therapeutic implication.

Gain-of-function kinase mutations are common in AML and usually portend an inferior prognosis. We reported a novel mechanism whereby kinase mutants induced intracellular alkalization characteristic in oncogenesis. Thirteen kinases were found to activate sodium/hydrogen exchanger (NHE1) in normal hematopoietic progenitors, of which FLT3-ITD, KRASG12D, and BTK phosphorylated NHE1 maintained alkaline intracellular pH (pHi) and supported survival of AML cells. Primary AML samples with kinase mutations also showed increased NHE1 phosphorylation and evidence of NHE1 addiction. Amiloride enhanced anti-leukemic effects and intracellular distribution of kinase inhibitors and chemotherapy. Co-inhibition of NHE1 and kinase synergistically acidified pHi in leukemia and inhibited its growth in vivo. Plasma from patients taking amiloride for diuresis reduced pHi of leukemia and enhanced cytotoxic effects of kinase inhibitors and chemotherapy in vitro. NHE1-mediated intracellular alkalization played a key pathogenetic role in transmitting the proliferative signal from mutated-kinase and could be exploited for therapeutic intervention in AML.

High-fidelity KKH variant of Staphylococcus aureus Cas9 nucleases with improved base mismatch discrimination.

The Cas9 nuclease from Staphylococcus aureus (SaCas9) holds great potential for use in gene therapy, and variants with increased fidelity have been engineered. However, we find that existing variants have not reached the greatest accuracy to discriminate base mismatches and exhibited much reduced activity when their mutations were grafted onto the KKH mutant of SaCas9 for editing an expanded set of DNA targets. We performed structure-guided combinatorial mutagenesis to re-engineer KKH-SaCas9 with enhanced accuracy. We uncover that introducing a Y239H mutation on KKH-SaCas9's REC domain substantially reduces off-target edits while retaining high on-target activity when added to a set of mutations on REC and RuvC domains that lessen its interactions with the target DNA strand. The Y239H mutation is modelled to have removed an interaction from the REC domain with the guide RNA backbone in the guide RNA-DNA heteroduplex structure. We further confirmed the greatly improved genome-wide editing accuracy and single-base mismatch discrimination of our engineered variants, named KKH-SaCas9-SAV1 and SAV2, in human cells. In addition to generating broadly useful KKH-SaCas9 variants with unprecedented accuracy, our findings demonstrate the feasibility for multi-domain combinatorial mutagenesis on SaCas9's DNA- and guide RNA- interacting residues to optimize its editing fidelity.

Proton export alkalinizes intracellular pH and reprograms carbon metabolism to drive normal and malignant cell growth.

Proton export is often considered a detoxifying process in animal cells, with monocarboxylate symporters coexporting excessive lactate and protons during glycolysis or the Warburg effect. We report a novel mechanism by which lactate/H+ export is sufficient to induce cell growth. Increased intracellular pH selectively activates catalysis by key metabolic gatekeeper enzymes HK1/PKM2/G6PDH, thereby enhancing glycolytic and pentose phosphate pathway carbon flux. The result is increased nucleotide levels, NADPH/NADP+ ratio, and cell proliferation. Simply increasing the lactate/proton symporter monocarboxylate transporter 4 (MCT4) or the sodium-proton antiporter NHE1 was sufficient to increase intracellular pH and give normal hematopoietic cells a significant competitive growth advantage in vivo. This process does not require additional cytokine triggers and is exploited in malignancy, where leukemogenic mutations epigenetically increase MCT4. Inhibiting MCT4 decreased intracellular pH and carbon flux and eliminated acute myeloid leukemia-initiating cells in mice without cytotoxic chemotherapy. Intracellular alkalization is a primitive mechanism by which proton partitioning can directly reprogram carbon metabolism for cell growth.

Functional reconstruction of human AML reveals stem cell origin and vulnerability of treatment-resistant MLL-rearranged leukemia.

Chemoresistance remains the major challenge for successful treatment of acute myeloid leukemia (AML). Although recent mouse studies suggest that treatment response of genetically and immunophenotypically indistinguishable AML can be influenced by their different cells of origin, corresponding evidence in human disease is still largely lacking. By combining prospective disease modeling using highly purified human hematopoietic stem or progenitor cells with retrospective deconvolution study of leukemia stem cells (LSCs) from primary patient samples, we identified human hematopoietic stem cells (HSCs) and common myeloid progenitors (CMPs) as two distinctive origins of human AML driven by Mixed Lineage Leukemia (MLL) gene fusions (MLL-AML). Despite LSCs from either MLL-rearranged HSCs or MLL-rearranged CMPs having a mature CD34-/lo/CD38+ immunophenotype in both a humanized mouse model and primary patient samples, the resulting AML cells exhibited contrasting responses to chemotherapy. HSC-derived MLL-AML was highly resistant to chemotherapy and expressed elevated amounts of the multispecific anion transporter ABCC3. Inhibition of ABCC3 by shRNA-mediated knockdown or with small-molecule inhibitor fidaxomicin, currently used for diarrhea associated with Clostridium difficile infection, effectively resensitized HSC-derived MLL-AML toward standard chemotherapeutic drugs. This study not only functionally established two distinctive origins of human LSCs for MLL-AML and their role in mediating chemoresistance but also identified a potential therapeutic avenue for stem cell-associated treatment resistance by repurposing a well-tolerated antidiarrhea drug already used in the clinic.

Low-dose pembrolizumab and nivolumab were efficacious and safe in relapsed and refractory classical Hodgkin lymphoma: Experience in a resource-constrained setting.

The efficacy and safety of low-dose anti-PD1 antibodies in relapsed/refractory classical Hodgkin lymphoma (cHL) require confirmation. Pembrolizumab (100 mg every 3 weeks, Q3W) or nivolumab (40 mg Q2W) were administered to patients with relapsed/refractory cHL. In the pembrolizumab cohort (N = 11), who had failed a median of three (1-6) therapies (brentuximab vedotin [BV]: 91%; autologous hematopoietic stem cell transplantation [auto-HSCT]: 18%), the overall response rate (ORR) by positron emission tomography-computed tomography was 100% (metabolic complete response [mCR]: 73%; partial response [PR]: 27%). Median cumulative dose for achieving best response was 400 (300-800) mg. Median progression-free survival (PFS) was 35 months. Median overall survival (OS) was not reached. Adverse events (AEs) of grade 1-2 were observed in three patients. In the nivolumab cohort (N = 6), who had failed a median of three (2-6) therapies (BV: 50%; auto-HSCT: 17%; allogeneic HSCT: 34%), the ORR was 100% (mCR: 67%; PR: 17%; indeterminate response: 17%). Median cumulative dose for achieving best response was 160 (160-360) mg. Median PFS was 33 months. Median OS was not reached. AEs of grade 1-2 were observed in four patients, two of whom had pre-existing autoimmune conditions. Five patients with Epstein-Barr virus (EBV) positive Reed-Sternberg cells underwent monitoring of plasma EBV DNA, which became negative in four mCR patients but remained positive in one PR patient who died ultimately from refractory lymphoma. Low-dose pembrolizumab and nivolumab were highly efficacious and safe in relapsed/refractory cHL. These observations have significant financial implications in resource-constrained settings.

Function of Arl4aa in the Initiation of Hematopoiesis in Zebrafish by Maintaining Golgi Complex Integrity in Hemogenic Endothelium.

ADP-ribosylation factor-like 4aa (Arl4aa) is a member of the ADP-ribosylation factor family. It is expressed in hematopoietic tissue during embryonic development, but its function was unknown. Zebrafish arl4aa is preferentially expressed in the ventral wall of the dorsal aorta (VDA) at 24 and 36 hpf and in caudal hematopoietic tissue at 48 hpf. Morpholino knockdown and transcription activator-like effector nuclease (TALEN) knockout of arl4aa significantly reduced expression of genes associated with definitive hematopoietic stem cells (HSCs). Golgi complex integrity in VDA was disrupted as shown by transmission electron microscopy and immunostaining of Golgi membrane Giantin. Mechanistically, arl4aa knockdown reduced Notch signaling in the VDA and its target gene expression. Protein expression of NICD was also reduced. Effects of arl4aa knockdown on definitive hematopoiesis could be restored by NICD expression. This study identified arl4aa as a factor regulating initiation of definitive HSCs by maintaining the integrity of Golgi complex and, secondarily, maturation of the Notch receptor.

Overcoming Resistance to FLT3 Inhibitors in the Treatment of FLT3-Mutated AML.

Acute myeloid leukaemia (AML) carrying internal tandem duplication (ITD) of Fms-Like Tyrosine kinase 3 (FLT3) gene is associated with high risk of relapse and poor clinical outcome upon treatment with conventional chemotherapy. FLT3 inhibitors have been approved for the treatment of this AML subtype but leukaemia relapse remains to be a major cause of treatment failure. Mechanisms of drug resistance have been proposed, including evolution of resistant leukaemic clones; adaptive cellular mechanisms and a protective leukaemic microenvironment. These models have provided important leads that may inform design of clinical trials. Clinically, FLT3 inhibitors in combination with conventional chemotherapy as induction treatment for fit patients; with low-intensity treatment as salvage treatment or induction for unfit patients as well as maintenance treatment with FLT3 inhibitors post HSCT hold promise to improve survival in this AML subtype.

Sorafenib and omacetaxine mepesuccinate as a safe and effective treatment for acute myeloid leukemia carrying internal tandem duplication of Fms-like tyrosine kinase 3.

BACKGROUND: Omacetaxine mepesuccinate (OME) has antileukemic effects against acute myeloid leukemia (AML) carrying an internal tandem duplication of Fms-like tyrosine kinase 3 (FLT3-ITD). A phase 2 clinical trial was conducted to evaluate a combination treatment of sorafenib and omacetaxine mepesuccinate (SOME). METHODS: Relapsed or refractory (R/R) or newly diagnosed patients were treated with sorafenib (200-400 mg twice daily) and OME (2 mg daily) for 7 (first course) or 5 days (second course onward) every 21 days until disease progression or allogeneic hematopoietic stem cell transplantation (HSCT). The primary endpoint was composite complete remission, which was defined as complete remission (CR) plus complete remission with incomplete hematologic recovery (CRi). Secondary endpoints were leukemia-free survival (LFS) and overall survival (OS). RESULTS: Thirty-nine R/R patients and 5 newly diagnosed patients were recruited. Among the R/R patients, 28 achieved CR or CRi. Two patients showed partial remission, and 9 patients did not respond. Among the 5 newly diagnosed patients, 4 achieved CR, and 1 achieved CRi. The median LFS and OS were 5.6 and 10.9 months, respectively. Prior Fms-like tyrosine kinase 3 (FLT3) inhibitor exposure (P = .007), 2 or more inductions (P = .001), and coexisting IDH2 (P = .008) and RUNX1 mutations (P = .003) were associated with lower CR/CRi rates. HSCT consolidation and deep molecular responses (defined as an FLT3-ITD variant allelic frequency [VAF] ≤ 0.1% or a nucleophosmin 1 [NPM1] mutant VAF ≤ 0.01%) were associated with better OS and LFS. Prior FLT3 inhibitor exposure and 2 or more inductions were associated with inferior LFS. CONCLUSIONS: SOME was safe and effective for R/R and newly diagnosed FLT3-ITD AML.

Diverse pathogenetic roles of SOX genes in acute myeloid leukaemia and their therapeutic implications.

Acute myeloid leukaemia (AML) is a heterogeneous group of diseases with diverse pathogenetic pathways. When treated uniformly with conventional chemotherapy and allogeneic haematopoietic stem cell transplantation (HSCT), it showed variable clinical outcome and prognosis. Members of the SOX [Sry-related high-mobility group (HMG) box] gene family are involved in diverse embryonic and oncogenic processes. The roles of SOX genes in AML are not entirely clear but emerging evidence, including that arising from studies in solid-cancers, showed that SOX genes can function as tumour suppressors or oncogenes and may be involved in key pathogenetic pathways in AML involving C/EBPα mutations, activation of ß-catenin/Wnt and Hedgehog pathways and aberrant TP53 signals. Recent data based on genomics and proteomics have identified key interactions between SOX genes and partnering proteins of pathogenetic significance. The observations illustrated the principles and feasibilities of developing lead molecules of potential therapeutic values. Studying the diverse pathogenetic roles of SOX genes in AML may shed lights to the heterogeneity of AML and generate information that can be translated into novel therapeutic strategies.

Distinct mutation spectrum, clinical outcome and therapeutic responses of typical complex/monosomy karyotype acute myeloid leukemia carrying TP53 mutations.

The present study aimed to define a subtype of complex/monosomal karyotype (CK/MK) acute myeloid leukemia (AML) by its distinct clinical features, p53 signaling and responses to p53 targeting agents. Ninety-eight young adults (range: 21-60 years; median: 49 years) with CK/MK AML were studied. They received standard induction, consolidation and allogeneic hematopoietic stem cell transplantation from siblings or matched unrelated donors if available. Chromosomal abnormalities most commonly affected chromosome 5 (30%), 7 (22%) and 17 (21%). Next generation sequencing of a 54-myeloid gene panel were available in 76 patients. Tumor protein 53 (TP53) mutations were most common (49%) and associated with the presence of -5/5q- (P < .001) and -17/17p- (P < .001), but not -7/7q- (P = .370). This "typical" CK/MK AML subtype was associated with significantly lower presenting white cell counts, higher number of karyotypic abnormalities, and inferior leukemia-free and overall survivals, compared with CK/MK AML without the typical features. Blood or bone marrow samples from typical CK/MK AML patients showed defective p53 signaling upon induction by etoposide. In vitro drug sensitivity analysis showed that they were sensitive to APR-246 that targeted mutant p53, but resistant to MDM2 antagonist MI-77301. Novel therapeutic strategies targeting TP53 mutations in CK/MK AML should be developed and tested in clinical trials.

Rapid versus gradual lung function decline in bronchiolitis obliterans syndrome after haematopoietic stem cell transplantation is associated with survival outcome.

BACKGROUND AND OBJECTIVE: Bronchiolitis obliterans syndrome (BOS) after haematopoietic stem cell transplantation (HSCT) presents with lung function decline. The pattern of lung function decline after BOS diagnosis could impact prognostication of BOS as a complication after HSCT. The aim of this study was to assess the impact of lung function decline on overall survival (OS) in BOS subjects. METHODS: Subjects with BOS were compared to those without BOS and matched for age, gender, primary diagnoses, conditioning regimes and chronic graft versus host disease. Lung function tests at baseline, at BOS diagnosis and every 3 months after HSCT were evaluated. RESULTS: Of the 1461 subjects undergoing allogeneic HSCT (allo-HSCT) between 1998 and 2015, 95 (6.5%) were diagnosed with BOS. A total of 159 matched HSCT recipients without BOS were identified. A 25% decline in FEV1 within the first 3 months after BOS diagnosis would separate BOS subjects into a subgroup with initial rapid decline and another subgroup with initial gradual decline in lung function. The rapid decline group showed lower subsequent lung function parameters and significantly worse OS compared to the gradual decline group (P = 0.013). CONCLUSION: Post-HSCT BOS subjects with initial rapid lung function decline within 3 months after BOS diagnosis will have significantly poorer lung function and worse OS compared to those with initial gradual decline in lung function after BOS diagnosis. HSCT BOS patients with rapid initial decline in lung function warrant closer monitoring for the development of other post-HSCT complications that could affect their survival.

Efficient RNA drug delivery using red blood cell extracellular vesicles.

Most of the current methods for programmable RNA drug therapies are unsuitable for the clinic due to low uptake efficiency and high cytotoxicity. Extracellular vesicles (EVs) could solve these problems because they represent a natural mode of intercellular communication. However, current cellular sources for EV production are limited in availability and safety in terms of horizontal gene transfer. One potentially ideal source could be human red blood cells (RBCs). Group O-RBCs can be used as universal donors for large-scale EV production since they are readily available in blood banks and they are devoid of DNA. Here, we describe and validate a new strategy to generate large-scale amounts of RBC-derived EVs for the delivery of RNA drugs, including antisense oligonucleotides, Cas9 mRNA, and guide RNAs. RNA drug delivery with RBCEVs shows highly robust microRNA inhibition and CRISPR-Cas9 genome editing in both human cells and xenograft mouse models, with no observable cytotoxicity.

Integrating Functional Analysis in the Next-Generation Sequencing Diagnostic Pipeline of RASopathies.

RASopathies are a group of heterogeneous conditions caused by germline mutations in RAS/MAPK signalling pathway genes. With next-generation sequencing (NGS), sequencing capacity is no longer a limitation to molecular diagnosis. Instead, the rising number of variants of unknown significance (VUSs) poses challenges to clinical interpretation and genetic counselling. We investigated the potential of an integrated pipeline combining NGS and the functional assessment of variants for the diagnosis of RASopathies. We included 63 Chinese patients with RASopathies that had previously tested negative for PTPN11 and HRAS mutations. In these patients, we performed a genetic analysis of genes associated with RASopathies using a multigene NGS panel and Sanger sequencing. For the VUSs, we evaluated evidence from genetic, bioinformatic and functional data. Twenty disease-causing mutations were identified in the 63 patients, providing a primary diagnostic yield of 31.7%. Four VUSs were identified in five patients. The functional assessment supported the pathogenicity of the RAF1 and RIT1 VUSs, while the significance of two VUSs in A2ML1 remained unclear. In summary, functional analysis improved the diagnostic yield from 31.7% to 36.5%. Although technically demanding and time-consuming, a functional genetic diagnostic analysis can ease the clinical translation of these findings to aid bedside interpretation.