Absence of Neutropenia in Patients With Early Exon Nonsense Mutations in ELANE : Clinical Evidence to Support Gene Therapy Approaches for Severe Congenital Neutropenia.

Severe congenital neutropenia is an inherited bone marrow failure disorder characterized by profoundly low neutrophil counts and promyelocytic maturation arrest in bone marrow. Severe congenital neutropenia is most often caused by heterozygous ELANE mutations. In vitro and mouse xenograft studies using CRISPR/Cas9 have shown that introduction of frameshift/nonsense mutations in mutant ELANE may restore neutrophil counts, providing a model for gene therapy. Here, we present 2 children with inherited nonsense mutations in ELANE analogous to those proposed for gene therapy. Their normal peripheral blood neutrophil counts provide support for this approach through human "experiments of nature."

CRISPR-Cas9n-mediated ELANE promoter editing for gene therapy of severe congenital neutropenia.

Severe congenital neutropenia (CN) is an inherited pre-leukemia bone marrow failure syndrome commonly caused by autosomal-dominant ELANE mutations (ELANE-CN). ELANE-CN patients are treated with daily injections of recombinant human granulocyte colony-stimulating factor (rhG-CSF). However, some patients do not respond to rhG-CSF, and approximately 15% of ELANE-CN patients develop myelodysplasia or acute myeloid leukemia. Here, we report the development of a curative therapy for ELANE-CN through inhibition of ELANE mRNA expression by introducing two single-strand DNA breaks at the opposing DNA strands of the ELANE promoter TATA box using CRISPR-Cas9D10A nickases-termed MILESTONE. This editing effectively restored defective neutrophil differentiation of ELANE-CN CD34+ hematopoietic stem and progenitor cells (HSPCs) in vitro and in vivo, without affecting the functions of the edited neutrophils. CRISPResso analysis of the edited ELANE-CN CD34+ HSPCs revealed on-target efficiencies of over 90%. Simultaneously, GUIDE-seq, CAST-Seq, and rhAmpSeq indicated a safe off-target profile with no off-target sites or chromosomal translocations. Taken together, ex vivo gene editing of ELANE-CN HSPCs using MILESTONE in the setting of autologous stem cell transplantation could be a universal, safe, and efficient gene therapy approach for ELANE-CN patients.

Genetics of severe congenital neutropenia as a gateway to personalized therapy.

Severe congenital neutropenias (SCNs) are rare diseases, and to date about 30 subtypes have been described according to their genetic causes. Standard care aims to prevent infections and limit the risk of leukemic transformation; however, several subtypes may have additional organ dysfunction(s), requiring specialized care. Granulocyte colony-stimulating factor and hematopoietic stem cell transplantation are now the bedrock of standard care. Better understanding of SCN mechanisms now offers the possibility of adapted therapy for some entities. An inhibitor of sodium glucose cotransporter, an antidiabetic drug, may attenuate glycogen storage disease type Ib and glucose-6-phosphatase catalytic subunit 3 neutropenias by clearing 1,5-anhydroglucitol, the precursor of the phosphate ester responsible for these SCNs. Chemokine receptor CXCR4 inhibitors contribute to reversing the leukocyte defect in warts, hypoglobulinemia, infections, and myelokathexis syndrome. All these new approaches use oral drugs, which notably improve quality of life. Additionally, improved research into clonal evolution has highlighted some ways to potentially prevent leukemia, such as stimulating somatic genetic rescue, a physiological process that might limit the risk of leukemic transformation.

Pediatric single large-scale mtDNA deletion syndromes: The power of patient reported outcomes.

There is a limited understanding of system-level clinical outcomes and interventions associated with single large-scale mitochondrial DNA deletion syndromes (SLSMDS). Additionally, no research exists that describes patient reported outcomes (PROs) of children with SLSMDS. A global and observational registry was established to understand the multi-systemic course of SLSMDS and track clinical outcomes. The development and design of the registry is described. Demographic characteristics, history and diagnoses, and system level prevalence of problems and interventions are reported for 42 children. System level problems and interventions include information on the following body systems: audiology, cardiac, endocrine, gastrointestinal (including pancreatic and hepatobiliary system), hematological, metabolic, neurological (including autonomic, mobility, & learning), ophthalmic, psychiatric, renal, and respiratory. Results emphasize the need of patient registries and suggest that the diagnostic odyssey and burden of disease for children with SLSMDS is significant. System-level findings may help families and clinical providers with diagnosis, prognostication, and treatment. A multidisciplinary team of clinical experts with a central coordinating specialist for children with SLSMDS is recommended.

Impaired myelopoiesis in congenital neutropenia: insights into clonal and malignant hematopoiesis.

A common feature of both congenital and acquired forms of bone marrow failure is an increased risk of developing acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Indeed, the development of MDS or AML is now the major cause of mortality in patients with congenital neutropenia. Thus, there is a pressing clinical need to develop better strategies to prevent, diagnose early, and treat MDS/AML in patients with congenital neutropenia and other bone marrow failure syndromes. Here, we discuss recent data characterizing clonal hematopoiesis and progression to myeloid malignancy in congenital neutropenia, focusing on severe congenital neutropenia (SCN) and Shwachman-Diamond syndrome. We summarize recent studies showing excellent outcomes after allogenic hematopoietic stem cell transplantation for many (but not all) patients with congenital neutropenia, including patients with SCN with active myeloid malignancy who underwent transplantation. Finally, we discuss how these new data inform the current clinical management of patients with congenital neutropenia.

Complicated peripartum course in a patient with very long-chain acyl-coenzyme A dehydrogenase (VLCAD) deficiency.

Very long-chain acyl-coenzyme A (CoA) dehydrogenase (VLCAD) deficiency is an autosomal recessive fatty acid oxidation disorder characterized by rhabdomyolysis, hypoglycemia and cardiomyopathy. The general treatment approach in adult patients is based on the prevention of catabolism. High carbohydrate, low fat diet and supplementation of medium-chain triglycerides are essential in the treatment. There is little experience with pregnancy follow-up in this patient group. We present a complicated peripartum course and successful management in a patient with VLCAD deficiency. Although high-dose glucose infusion was initiated, creatine kinase levels significantly increased in the immediate postpartum period, but the patient remained asymptomatic and rhabdomyolysis resolved rapidly after increasing the glucose infusion rate.

Hematopoietic stem cell transplantation for inherited bone marrow failure syndromes: alternative donor and disease-specific conditioning regimen with unmanipulated grafts.

Objective: The outcomes of alternative donor hematopoietic stem cell transplantation (HSCT) with unmanipulated grafts for Inherited bone marrow failure syndromes (IBMFS) are discouraging. Our study is to demonstrate that IBMFS with disease-specific characteristics requires a tailored conditioning regimens to enhance engraftment and reduce regimen related toxicities. Methods: We retrospectively analyzed 42 patients diagnosed with IBMFS and transplanted with an alternative donor graft at our center from November 2012 to August 2018. Twenty-seven patients had Fanconi anemia (FA), 7 had dyskeratosis congenita (DC), and 8 had severe congenital neutropenia (SCN). Patients received ex-vivo unmanipulated alternative donor grafts from a matched unrelated donor (MUD) (n = 22), haploidentical donor (HID) (n = 17) and unrelated cord blood donor (UCBD) (n = 3). FA and DC patient subgroups received reduce intensified conditioning (RIC), while SCN patients received a myeloablative conditioning (MAC) regimen. Results: The median follow-up time for the surviving patients was 38 months (range: 9-63 months). The failure-free survival (FFS) for entire cohort was 76.1%, and was 72.4%, 100% and 56.2% for patients with FA, DC and SCN, respectively. There were no primary graft failures. The cumulative incidence of aGVHD at day 100 was 48.1%. The cumulative incidence of cGVHD at 1 and 3 years was 35.0% and 69.3%, respectively. Conclusion: HSCT using alternative donors with unmanipulated grafts and disease-specific conditioning regimens for IBMFS patients shows promising survival.

T-Cell Acute Lymphoblastic Leukemia in a Young Adult With Thrombocytopenia-absent Radius Syndrome: A Case Report and Review of the Literature.

Thrombocytopenia-absent radius (TAR) syndrome is a rare inherited bone marrow failure syndrome not generally associated with acute leukemia. The authors report a case of T-cell acute lymphoblastic leukemia in an adult female individual newly diagnosed with TAR syndrome. A 347-kb microdeletion of chromosome 1q21.1 involving the RBM8A gene was detected within a gain of whole chromosome 1. Next-generation sequencing on fibroblasts confirmed germline heterozygous deletion of RBM8A but on the other allele, noncoding low-frequency regulatory single-nucleotide polymorphism of RBM8A (rs139428292; rs201779890) were not found. The tolerance of the treatment was unusual and mostly marked by a slow hematopoietic recovery leading to a 6-month delay at the beginning of the maintenance phase. Only 5 cases of acute leukemia were reported in patients with TAR syndrome in the literature: 4 acute myeloid leukemia and one B-cell acute lymphoblastic leukemia. This is the first report of T-cell acute lymphoid leukemia occurring in the context of TAR syndrome.

Acquisition of monosomy 7 and a RUNX1 mutation in Pearson syndrome.

Pearson syndrome (PS) is a very rare and often fatal multisystem disease caused by deletions in mitochondrial DNA that result in sideroblastic anemia, vacuolization of marrow precursors, and pancreatic dysfunction. Spontaneous recovery from anemia is often observed within several years of diagnosis. We present the case of a 4-month-old male diagnosed with PS who experienced prolonged severe pancytopenia preceding the emergence of monosomy 7. Whole-exome sequencing identified two somatic mutations, including RUNX1 p.S100F that was previously reported as associated with myeloid malignancies. The molecular defects associated with PS may have the potential to progress to advanced myelodysplastic syndrome .

Special pre- and posttransplant considerations in inherited bone marrow failure and hematopoietic malignancy predisposition syndromes.

Advances in the diagnosis and treatment of inherited bone marrow failure syndromes (IBMFS) have provided insight into the complexity of these diseases. The diseases are heterogeneous and characterized by developmental abnormalities, progressive marrow failure, and predisposition to cancer. A correct diagnosis allows for appropriate treatment, genetic counseling, and cancer surveillance. The common IBMFSs are Fanconi anemia, dyskeratosis congenita, and Diamond-Blackfan anemia. Hematopoietic cell transplantation (HCT) offers curative treatment of the hematologic complications of IBMFS. Because of the systemic nature of these diseases, transplant strategies are modified to decrease immediate and late toxicities. HCT from HLA-matched related or unrelated donors offers excellent survival for young patients in aplasia. Challenges include the treatment of adults with marrow aplasia, presentation with myeloid malignancy regardless of age, and early detection or treatment of cancer. In this article, I will describe our approach and evaluation of patients transplanted with IBMFS and review most frequent complications before and after transplant.

Effects of fasting, feeding and exercise on plasma acylcarnitines among subjects with CPT2D, VLCADD and LCHADD/TFPD.

BACKGROUND: The plasma acylcarnitine profile is frequently used as a biochemical assessment for follow-up in diagnosed patients with fatty acid oxidation disorders (FAODs). Disease specific acylcarnitine species are elevated during metabolic decompensation but there is clinical and biochemical heterogeneity among patients and limited data on the utility of an acylcarnitine profile for routine clinical monitoring. METHODS: We evaluated plasma acylcarnitine profiles from 30 diagnosed patients with long-chain FAODs (carnitine palmitoyltransferase-2 (CPT2), very long-chain acyl-CoA dehydrogenase (VLCAD), and long-chain 3-hydroxy acyl-CoA dehydrogenase or mitochondrial trifunctional protein (LCHAD/TFP) deficiencies) collected after an overnight fast, after feeding a controlled low-fat diet, and before and after moderate exercise. Our purpose was to describe the variability in this biomarker and how various physiologic states effect the acylcarnitine concentrations in circulation. RESULTS: Disease specific acylcarnitine species were higher after an overnight fast and decreased by approximately 60% two hours after a controlled breakfast meal. Moderate-intensity exercise increased the acylcarnitine species but it varied by diagnosis. When analyzed for a genotype/phenotype correlation, the presence of the common LCHADD mutation (c.1528G > C) was associated with higher levels of 3-hydroxyacylcarnitines than in patients with other mutations. CONCLUSIONS: We found that feeding consistently suppressed and that moderate intensity exercise increased disease specific acylcarnitine species, but the response to exercise was highly variable across subjects and diagnoses. The clinical utility of routine plasma acylcarnitine analysis for outpatient treatment monitoring remains questionable; however, if acylcarnitine profiles are measured in the clinical setting, standardized procedures are required for sample collection to be of value.

CRISPR-Cas9-Mediated ELANE Mutation Correction in Hematopoietic Stem and Progenitor Cells to Treat Severe Congenital Neutropenia.

Severe congenital neutropenia (SCN) is a monogenic disorder. SCN patients are prone to recurrent life-threatening infections. The main causes of SCN are autosomal dominant mutations in the ELANE gene that lead to a block in neutrophil differentiation. In this study, we use CRISPR-Cas9 ribonucleoproteins and adeno-associated virus (AAV)6 as a donor template delivery system to repair the ELANEL172P mutation in SCN patient-derived hematopoietic stem and progenitor cells (HSPCs). We used a single guide RNA (sgRNA) specifically targeting the mutant allele, and an sgRNA targeting exon 4 of ELANE. Using the latter sgRNA, ∼34% of the known ELANE mutations can in principle be repaired. We achieved gene correction efficiencies of up to 40% (with sgELANE-ex4) and 56% (with sgELANE-L172P) in the SCN patient-derived HSPCs. Gene repair restored neutrophil differentiation in vitro and in vivo upon HSPC transplantation into humanized mice. Mature edited neutrophils expressed normal elastase levels and behaved normally in functional assays. Thus, we provide a proof of principle for using CRISPR-Cas9 to correct ELANE mutations in patient-derived HSPCs, which may translate into gene therapy for SCN.

Nonsense Suppression Therapy: New Hypothesis for the Treatment of Inherited Bone Marrow Failure Syndromes.

Inherited bone marrow failure syndromes (IBMFS) are a group of cancer-prone genetic diseases characterized by hypocellular bone marrow with impairment in one or more hematopoietic lineages. The pathogenesis of IBMFS involves mutations in several genes which encode for proteins involved in DNA repair, telomere biology and ribosome biogenesis. The classical IBMFS include Shwachman-Diamond syndrome (SDS), Diamond-Blackfan anemia (DBA), Fanconi anemia (FA), dyskeratosis congenita (DC), and severe congenital neutropenia (SCN). IBMFS are associated with high risk of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and solid tumors. Unfortunately, no specific pharmacological therapies have been highly effective for IBMFS. Hematopoietic stem cell transplantation provides a cure for aplastic or myeloid neoplastic complications. However, it does not affect the risk of solid tumors. Since approximately 28% of FA, 24% of SCN, 21% of DBA, 20% of SDS, and 17% of DC patients harbor nonsense mutations in the respective IBMFS-related genes, we discuss the use of the nonsense suppression therapy in these diseases. We recently described the beneficial effect of ataluren, a nonsense suppressor drug, in SDS bone marrow hematopoietic cells ex vivo. A similar approach could be therefore designed for treating other IBMFS. In this review we explain in detail the new generation of nonsense suppressor molecules and their mechanistic roles. Furthermore, we will discuss strengths and limitations of these molecules which are emerging from preclinical and clinical studies. Finally we discuss the state-of-the-art of preclinical and clinical therapeutic studies carried out for IBMFS.

Adult-onset Repeat Rhabdomyolysis with a Very Long-chain Acyl-CoA Dehydrogenase Deficiency Due to Compound Heterozygous ACADVL Mutations.

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is a genetic disorder of fatty acid beta oxidation that is caused by a defect in ACADVL, which encodes VLCAD. The clinical presentation of VLCAD deficiency is heterogeneous, and either a delayed diagnosis or a misdiagnosis may sometimes occur. We herein describe a difficult-to-diagnose case of the muscle form of adult-onset VLCAD deficiency with compound heterozygous ACADVL mutations including c.790A>G (p.K264E) and c.1246G>A (p.A416T).

One potential hotspot ACADVL mutation in Chinese patients with very-long-chain acyl-coenzyme A dehydrogenase deficiency.

Very long-chain acyl-coenzyme A dehydrogenase deficiency (VLCAD deficiency), a rare autosomal recessive disorder, is characterized by hypoketotic hypoglycemia, cardiomyopathy, liver damage, and myopathy. VLCAD deficiency is caused by defects of ACADVL gene, which encodes VLCAD protein. The aim of this study was to determine the clinical, biochemical, prognosis and mutation spectrum of patients with VLCAD deficiency in mainland China. A total of Six families visited us, four patients (2 boys and 2 girls) were admitted in hospital due to liver dysfunction, hypoglycemia, and positive newborn screen result. The parents of the other two patients (2 girls) visited us for genetic consultation after their children's death. All the six patients had elevated level of serum tetradecenoylcarnitine (C14:1-carnitine), four of them showed decreased free carnitine (C0) level, and three had dicarboxylic aciduria. Eight types of mutations of the ACADVL gene were detected, three of them are novel, including c.563G > A (p.G188D) c.1387G > A (p.G463R) and c.1582_1586del (p.L529Sfs*31). The p.R450H mutation accounts for 9/52 alleles (5/40 in previous study of 20 unrelated patients, and 4/12 in this study) of genetically diagnosed Chinese VLCAD deficiency cases. The four alive patients (Patient 1-4) responded well to diet prevention and drug therapy with stable hepatic dysfunction condition. In conclusion, we describe three novel mutations of the ACADVL gene among six unrelated families with VLCAD deficiency. Moreover, we suggest that the p.R450H may be a potential hotspot mutation in the Chinese population.

Exosomes derived from mesenchymal stem cells improved function and survival of neutrophils from severe congenital neutropenia patients in vitro.

Severe congenital neutropenia (SCN) is described by the absolute neutrophil counts less than 500 cells/mm3, bacterial infections, and an arrest of neutrophil differentiation. So, effective strategies for improving the function and lifespan of the existing neutrophils in these patients are necessary. Mesenchymal stem cells (MSCs) have supportive effects on neutrophils. Recently, it was determined that MSCs exert their effects, mostly by secreting soluble factors and exosomes. So, in this study, neutrophils were isolated from the bloodstream of healthy donors and SCN patients and cultured with medium, MSC-exosomes or MSC-conditioned media (MSC-CM). Then, the effects of the two treatments on neutrophil respiratory burst, apoptosis and phagocytosis percentage were assessed using nitro blue tetrazolium (NBT) assay, annexin V-propidium iodide (PI) and Giemsa staining, respectively. Both treatments could significantly augment respiratory burst of neutrophils from SCN patients and healthy donors. But, only CM could significantly enhance phagocytosis index. About the lifespan of neutrophils, only exosomes could significantly enhance it in both groups. Based on these results, both exosomes and CM derived from MSCs could be attractive candidates for rescuing SCN patients from serious infections.

The fate of medium-chain fatty acids in very long-chain acyl­CoA dehydrogenase deficiency (VLCADD): A matter of sex?

Medium-chain-triglycerides (MCT) are widely applied in the treatment of long-chain fatty acid oxidation disorders (lcFAOD). Long-term treatment with MCT led to a sexually dimorphic response in the mouse model of very-long-chain-acyl-CoA-dehydrogenase-deficiency (VLCAD-/-) with the subsequent development of a metabolic syndrome in female mice. In order to evaluate the molecular mechanisms responsible for this sex specific response we performed a comprehensive metabolic phenotyping, SILAC-based quantitative proteomics and characterized the involved signaling pathways by western blot analysis and gene expression. WT and VLCAD-/- mice showed strong sex-dependent differences in basal metabolism and expression of proteins involved in the distinct metabolic pathways, even more prominent after treatment with octanoate. The investigation of molecular mechanisms responsible for the sexual dimorphisms delineated the selective activation of the ERK/mTORc1 signaling pathway leading to an increased biosynthesis and elongation of fatty acids in VLCAD-/- females. In contrast, octanoate induced the activation of ERK/PPARγ pathway and the subsequent upregulation of peroxisomal ߭oxidation in males. We here provide first evidence that sex has to be considered as important variable in disease phenotype. These findings may have implications on treatment strategies in the different sexes.