Recurrent Heterozygous Mutations Research Articles

Necessity of Smc3 in Embryonic and Adult Hematopoiesis with an Emphasis on Myeloid Compartment

SMC3 encodes a subunit of cohesin associated with recurrent heterozygous mutations in acute myeloid leukemia (AML) and other myeloid malignancies. About one-third of these mutations are nonsense and splice-site mutations and the rest are missense. To understand whether these missense mutations might have dominant-negative effects or phenocopy loss-of-function effects, we compared the consequences of Smc3 deficient and haploinsufficient mouse models.We found that homozygous deletion of Smc3 during embryogenesis (Smc3fl/fl/Vav1-Cre+/-) is embryonic lethal. Grossly, the E13.5 Smc3fl/fl/Vav1-Cre+/- embryos were indistinguishable in size and appearance from other genotypes, except the lack of obvious fetal livers and near complete absence of CD45+Gr1+CD11b+ fetal liver cells, consistent with myeloid biased hematopoietic failure.Likewise, somatic deletion of homozygous Smc3 alleles in adult mice led to complete hematopoietic failure. In Smc3fl/fl/ERT2-Cre+/- mice, tamoxifen-induced Smc3 deletion led to rapid bone marrow (BM) failure and death. In competitive transplantation, the Smc3fl/fl/ERT2-Cre+/- BM cells were quickly outcompeted after tamoxifen treatment, indicating complete loss of hematopoietic stem/progenitor cell (HSPC) functions. Again, the effect was most pronounced within the myeloid compartment. Thus, Smc3 is necessary for both embryonic and adult hematopoiesis.We examined the consequences of Smc3 haploinsufficiency using three mouse models: Smc3fl/+/CMV-Cre+/-, Smc3fl/+/Vav1-Cre+/-, and Smc3fl/+/ERT2-Cre+/- . We found that Smc3 haploinsufficiency led to a normal number of female pups in X-linked CMV-Cre intercrosses, and the female pups had no obvious defects in growth or blood counts, implying tolerance of embryonic heterozygous deletion of Smc3 . In ex vivo methylcellulose assays, Smc3 haploinsufficiency did not result in increased number of colonies, and the Smc3 haploinsufficient BM cells did not replate beyond two weeks. In competitive transplantations, we observed a significant competitive disadvantage in the Smc3fl/+/ERT2-Cre+/- BM cells, most pronounced in the Gr1+ myeloid cells.The patterns of missense and nonsense mutations observed in AML patients suggested that SMC3 likely acts as a tumor suppressor, andthat haploinsufficiency would augment pre-leukemic self-renewal. However, we observed a competitive disadvantage across multiple experiments. This contradicts the competitive advantage observed when Smc3 haploinsufficiency was induced using Mx1-Cre (Viny et al . JEM 212 (11): 1819-1832). Therefore, we examined whether Smc3 haploinsufficiency might augment self-renewal under specific conditions of hematopoietic stress, including pIpC. We again observed a marked competitive disadvantage in the Smc3fl/+/ERT2-Cre+/- BM cells following tamoxifen induction, although the myeloid competitive disadvantage was partially ameliorated in the pIpC-treated group, whereas 5-FU treatment augmented the myeloid competitive disadvantage.In AML patients, SMC3 mutations frequently co-occurred with DNMT3A mutations. We, therefore, asked whether Smc3 haploinsufficiency might lead to a competitive advantage if it occurred in the background of Dnmt3a haploinsufficiency. With the addition of Dnmt3a haploinsufficiency, the severe myeloid competitive disadvantage was partially abrogated, but the significant competitive disadvantage in other lineages remained intact; even with constitutive Dnmt3a haploinsufficiency, Smc3 haploinsufficiency did not result in competitive growth of BM cells.In summary, our data demonstrate that homozygous Smc3 deletion was incompatible with embryonic or adult hematopoiesis, with the greatest defects noted within myeloid compartment, indicating that AML-associated SMC3 mutations are unlikely to have dominant-negative effects. Smc3 haploinsufficiency resulted in a myeloid competitive disadvantage, which was partially ameliorated by pIpC treatment, and accelerated by the stem cell stress of 5-FU. In the presence of concurrent Dnmt3a haploinsufficiency, the myeloid disadvantage was partially abrogated, while the disadvantage in other lineages remained unperturbed, suggesting interactions between the mutations. DisclosuresNo relevant conflicts of interest to declare.

Familial neurohypophyseal diabetes insipidus: clinical, genetic and functional studies of novel mutations in the arginine vasopressin gene.

Familial neurohypophyseal diabetes insipidus (FNDI) is a rare disorder characterized by childhood-onset progressive polyuria and polydipsia due to mutations in the arginine vasopressin (AVP) gene. The aim of the study was to describe the clinical and molecular characteristics of families with neurohypophyseal diabetes insipidus. Five Portuguese families with autosomal dominant FNDI underwent sequencing of the AVP gene and the identified mutations were functionally characterized by in vitro studies. Three novel and two recurrent heterozygous mutations were identified in the AVP gene. These consisted of one initiation codon mutation in the signal peptide coding region (c.2T > C, p.Met1?), three missense mutations in the neurophysin II (NPII) coding region (c.154T > C, p.Cys52Arg; c.289C > G, p.Arg97Gly; and c.293G > C, p.Cys98Ser), and one nonsense mutation in the NPII coding region (c.343G > T, p.Glu115Ter). In vitro transfection of neuronal cells with expression vectors containing each mutation showed that the mutations resulted in intracellular retention of the vasopressin prohormone. Patients showed progressive symptoms of polyuria and polydipsia, but with wide variability in severity and age at onset. No clear genotype-phenotype correlation was observed. The intracellular accumulation of mutant vasopressin precursors supports the role of cellular toxicity of the mutant proteins in the etiology of the disorder and explains the progressive onset of the disorder. These findings further expand the AVP mutational spectrum in FNDI and contribute to the understanding of the molecular pathogenic mechanisms involved in FNDI.

DIPG-18. IDENTIFICATION OF TARGETABLE PATHWAY DEPENDENCIES IN DIFFUSE INTRINSIC PONTINE GLIOMA

Diffuse Intrinsic Pontine Glioma (DIPG) is a highly aggressive paediatric brainstem tumour with a dismal prognosis. Recurrent heterozygous mutations (p.K27M) in Histone H3 variant genes have been identified in the majority of DIPG cases. While the exact mechanism of H3K27M’s function is poorly understood, evidence suggests a role for epigenetic dysregulation in disease pathogenesis. This study aims to use functional genomics to identify novel therapeutic dependencies in H3K27M DIPG. DIPG drug sensitivity screening was carried out in twelve established and validated patient derived cell lines (10 H3.3K27M and 2 Wt) using an FDA approved drug library containing 1480 compounds. Highly prevalent targets identified from this screen include HDAC, microtubule, proteasome and CDK inhibitors. Additionally, a custom pooled CRISPR knockout library of druggable targets (300 genes, 1200 guide RNAs) was used to identify key DIPG cell survival pathways. To date five DIPG cell lines (1 Wt; 1 H3.1; 3 H3.3) have undergone screening. Knockdown of known DIPG driver genes (TP53; PDGFRA; PIK3CA and PIK3CR1) resulted in reduced cell viability, consistent with their proposed function and validating knockout screen utility. Preliminary data demonstrates Wt and H3K27M DIPGs cluster independently based on genes required for survival, suggesting differing tumorigenesis mechanisms and the potential for therapeutically targeting genotype specific pathways. Correlation of parallel drug screen and RNA-seq data will potentially reveal H3-dependent pathways for therapeutic exploitation. Collectively, we show a functional genomics approach is able to identify genotype-specific pathway dependencies in DIPG, paving the way for molecularly informed personalized therapies for patients.

Smc3 is required for mouse embryonic and adult hematopoiesis

SMC3 encodes a subunit of the cohesin complex that has canonical roles in regulating sister chromatids segregation during mitosis and meiosis. Recurrent heterozygous mutations in SMC3 have been reported in acute myeloid leukemia (AML) and other myeloid malignancies. In this study, we investigated whether the missense mutations in SMC3 might have dominant-negative effects or phenocopy loss-of-function effects by comparing the consequences of Smc3-deficient and -haploinsufficient mouse models. We found that homozygous deletion of Smc3 during embryogenesis or in adult mice led to hematopoietic failure, suggesting that SMC3 missense mutations are unlikely to be associated with simple dominant-negative phenotypes. In contrast, haploinsufficiency was tolerated during embryonic and adult hematopoiesis. Under steady-state conditions, Smc3 haploinsufficiency did not alter colony forming in methylcellulose, only modestly decreased mature myeloid cell populations, and led to limited expression changes and chromatin alteration in Lin-cKit+ bone marrow cells. However, following transplantation, engraftment, and subsequent deletion, we observed a hematopoietic competitive disadvantage across myeloid and lymphoid lineages and within the stem/progenitor compartments. This disadvantage was not affected by hematopoietic stresses, but was partially abrogated by concurrent Dnmt3a haploinsufficiency, suggesting that antecedent mutations may be required to optimize the leukemogenic potential of Smc3 mutations.

Abstract 5095: Concurrent Dnmt3a haploinsufficiency partially abrogates competitive disadvantage in Smc3 haploinsufficient myeloid cells

Abstract SMC3 encodes a subunit of cohesin associated with recurrent heterozygous mutations in acute myeloid leukemia (AML) and other myeloid malignancies. About one-third of these mutations are predicted loss-of-function (nonsense and splice-site mutations) and the rest are missense with unclear function. To understand whether these missense mutations might have dominant-negative effects or phenocopy loss-of-function effects, we compared the consequences of Smc3 deficient and haploinsufficient mouse models. We found that both embryonic and somatic deletion of homozygous Smc3 alleles led to complete hematopoietic failure. Furthermore, in competitive transplantation, following engraftment and subsequent tamoxifen treatment, the Smc3fl/fl/ERT2-Cre+/- BM cells were rapidly outcompeted, with earliest cell loss in the Gr1+ myeloid compartment. Hence, Smc3 is indispensable for hematopoiesis and AML-associated SMC3 missense mutations are unlikely to have dominant-negative effects. We examined the consequences of somatic Smc3 haploinsufficiency using Smc3fl/+/ERT2-Cre+/-. We found that Smc3 haploinsufficiency did not lead to increased number of initial methylcellulose colonies, nor did these cells replate beyond two weeks. In competitive transplantations, we again observed a significant competitive disadvantage in the Smc3fl/+/ERT2-Cre+/- BM cells, most pronounced in the Gr1+ myeloid cells. Competitive disadvantage is counter-intuitive for a leukemia-associated mutation. SMC3 mutations frequently co-occur with DNMT3A mutations. We, therefore, asked whether Smc3 haploinsufficiency might lead to a competitive advantage if it occurred in the background of Dnmt3a haploinsufficiency. With the addition of Dnmt3a haploinsufficiency, the severe myeloid competitive disadvantage was partially ameliorated, but the significant competitive disadvantage in other lineages remained intact. In summary, our data demonstrate that homozygous Smc3 deletion was incompatible with embryonic or adult hematopoiesis, with the greatest defects noted within myeloid compartment, indicating that AML-associated SMC3 mutations are unlikely to have dominant-negative effects. Smc3 haploinsufficiency resulted in a myeloid competitive disadvantage, which was partially abrogated in the presence of concurrent Dnmt3a haploinsufficiency, suggesting lineage-specific interactions between the mutations. Citation Format: Tianjiao Wang, John S. Welch. Concurrent Dnmt3a haploinsufficiency partially abrogates competitive disadvantage in Smc3 haploinsufficient myeloid cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5095.

Phenotypic diversity of the recurrent p.Val379Leu missense mutation of the TGM1 gene

Abstract Autosomal recessive congenital ichthyosis type 1 (ARCI1), a clinically heterogeneous group of keratinization disorders, develops due to mutations in the transglutaminase 1 (TGM1) gene. Here we report a Hungarian pedigree affected by the lamellar ichthyosis clinical form of the ARCI1 phenotype. Direct sequencing revealed two recurrent heterozygous mutations: a splice site (c.877-2A > G) and a missense (c.1135G > C, p.Val379Leu) mutation. This splice site mutation is the most frequently observed in ARCI1 worldwide. The missense mutation is relatively rare and has been reported in only 13 Scandinavian patients. Comparison of the clinical phenotypes of our Hungarian patients and the Scandinavian patients demonstrates great phenotypic diversity associated with the p.Val379Leu genotype.

Genetic analysis of the SOD1 and C9ORF72 genes in Hungarian patients with amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the death of motor neurons. To date, more than 20 genes have been implicated in ALS, and of these, the 2 most frequently mutated are the superoxide dismutase 1 (SOD1) gene and the chromosome 9 open reading frame 72 (C9ORF72) gene. In this study, we aimed to investigate the contribution of these 2 Mendelian genes to the development of the disease in Hungarian ALS patients (n = 66). Direct sequencing of the SOD1 gene revealed a novel (p.Lys91ArgfsTer8) and 3 recurrent heterozygous mutations (p.Val14Met, p.Asp90Ala, and p.Leu144Phe) in 5 patients. The novel p.Lys91ArgfsTer8 mutation led to a frameshift causing the addition of 8 new amino acids, including a premature stop codon at position 99. The GGGGCC hexanucleotide repeat expansion of the C9ORF72 gene was present in 1 ALS patient. This study represents the first genetic analysis of 2 major ALS causative genes in a cohort of Hungarian ALS patients and contributes to the further understanding of the genetic and phenotypic diversity of ALS.

Targeting Splicing in the Treatment of Myelodysplastic Syndromes and Other Myeloid Neoplasms.

Genome sequencing of primary cells from patients with myelodysplastic syndromes (MDS) led to the identification of recurrent heterozygous mutations in gene encoding components of the spliceosome, the cellular machinery which processes pre-messenger RNA (mRNA) to mature mRNA during gene transcription. Splicing mutations are mutually exclusive with one another and collectively represent the most common mutation class in MDS, occurring in approximately 60% of patients overall and more than 80% of those with ring sideroblasts. Evidence from animal models suggests that homozygous splicing mutations are lethal, and that in heterozygously mutated models, any further disruption of splicing triggers apoptosis and cell death. MDS cells with spliceosome mutations are thus uniquely vulnerable to therapies targeting splicing, which may be tolerated by healthy cells. The spliceosome is emerging as a novel therapeutic target in MDS and related myeloid neoplasms, with the first clinical trial of a splicing modulator opening in 2016.

PO27IDH1 MUTATIONS COMPROMISE THE ADAPTIVE RESPONSE TO HYPOXIA

INTRODUCTION: The recent identification of recurrent heterozygous somatic mutations in the isocitrate dehydrogenase (IDH1) gene in low-grade gliomas and secondary glioblastoma, provides a much-needed advance in our understanding of the drivers of these tumours. Patients with this mutation are generally younger and have improved prognosis. Mutations in IDH1 occur at an arginine residue at codon 132 (R132) and result in production of the putative oncometabolite - (R)-2-hydroxyglutarate (2HG). (R)-2HG production has subsequently been shown to induce changes in DNA and histone methylation. However, there are conflicting reports regarding the metabolic role of IDH1 mutations and subsequent production of (R)-2HG in hypoxia, which are yet to be reconciled. METHOD: To define the hypoxic metabolic phenotype of IDH1 mutations, we applied complementary techniques - gas chromatography mass spectrometry (GC-MS), and nuclear magnetic resonance (NMR) spectroscopy to probe differences in the metabolic adaptation to hypoxia using [13C6]-glucose and [13C5]-glutamine tracers. RESULTS: Employing a human oligodendroglioma (HOG) cell line model, infected with mutant IDH1 by lentivirus, we show that IDH1 mutations metabolically compromise the adaptation to hypoxia, despite observable HIF1alpha stabilisation. CONCLUSION: We hypothesise that the adaptive transcriptional responses of hypoxia is blunted in IDH1 mutants resulting in the inability ofHIF1alpha to fully regulate downstream targets and ultimately cell metabolism. These results may account for the improved prognosis associated with this subset of glial tumours. Increasing our understanding of metabolic redundancies in tumour cells will aid the development of more effective treatments.

Abstract PR06: Mutant U2AF1 alters hematopoiesis and pre-mRNA splicing in transgenic mice.

Abstract Our group and others discovered recurrent heterozygous mutations in U2AF1, a gene encoding a splicing factor involved in intronic 3'-splice site recognition, in 11% of patients with myelodysplastic syndromes (MDS), suggesting that perturbations in pre-mRNA splicing play a role in MDS pathogenesis. The most common U2AF1 mutation causes a serine to phenylalanine substitution at amino acid 34 (S34F). To study the effects of U2AF1(S34F) expression on hematopoiesis and pre-mRNA splicing in vivo, we created site-specific, single-copy, doxycycline-inducible U2AF1(WT) and U2AF1(S34F) transgenic mice. To examine the cell-autonomous effects of mutant U2AF1(S34F), we transplanted transgenic donor bone marrow into wild type mouse recipients prior to induction of transgene expression. Following 4 weeks of transgene induction, U2AF1(S34F)-recipient mice have reduced total WBCs in the peripheral blood compared to U2AF1(WT)- and rtTA only-recipient controls (4.3 vs 7.11 and 7.13 K/µl, respectively, p≤0.01), but no significant changes in bone marrow cellularity or spleen size (n=9-11). U2AF1(S34F)-recipient mice have a perturbed mature cell lineage distribution, including reduced monocytes and B cells in both peripheral blood (p≤0.05) and bone marrow (p≤0.01) when compared to control mice (n=9-11). Reduction of bone marrow monocytes occurs as early as 5 days and is associated with increased frequency of Annexin V+ staining (p≤0.05) and phospho-H2AX (p≤0.05) compared to controls, suggesting loss of these cells may be due to apoptosis. In addition, U2AF1(S34F) expression results in alterations of progenitor and stem cell frequency in bone marrow and spleen of mice. U2AF1(S34F)-recipient mice have increased numbers of progenitors in both bone marrow and spleen by CFU-C methylcellulose assay and flow cytometry for c-Kit+ Lineage - (KL) cells, as well as common myeloid progenitors (CMPs), when compared to U2AF1(WT) and rtTA only controls (p≤0.05, n=5-10). U2AF1(S34F)-recipient mice also have an increase in the frequency of bone marrow hematopoietic stem cells (HSCs) measured by flow cytometry for KLS (c-Kit+ Lineage- Sca-1+) cells, short-term HSCs, and dormant long-term HSCs (p≤0.05), as well as an increase in spleen dormant long-term HSCs (p≤0.03) compared to U2AF1(WT)- and rtTA only-recipient controls (n=5-6). The increase in bone marrow KLS cells in U2AF1(S34F)-recipient mice at 5 days is associated with higher levels of intracellular Ki67 (a marker of cell proliferation) in KLS cells compared to controls (p<0.05, n=8-13). Competitive repopulation studies show a disadvantage for bone marrow cells expressing mutant U2AF1(S34F) compared to controls (p≤0.05, n=6-12), suggesting that the increase in KLS cell cycling following U2AF1(S34F) expression may lead to stem cell exhaustion. Collectively, these data indicate U2AF1(S34F) expression alters hematopoiesis in vivo. Next, we performed unbiased RNA sequencing on sorted bone marrow CMPs following 5 days transgene induction in U2AF1(S34F)- and U2AF1(WT)-transplanted mice. We identified 402 splicing junctions that were differentially expressed in U2AF1(S34F) samples compared to U2AF1(WT) (FDR <10%, >2 |log2| fold change). We also observed a preference of the mutant U2AF1(S34F) to skip exons (n=72) and alternative splice sites (n=45) with a T in the -3 position relative to the AG splice acceptor site. We intersected our junction results with RNA sequencing from AML patient samples with U2AF1 mutations and human CD34+ cells expressing U2AF1(S34F), and we identified homologous dysregulated junctions in all 3 datasets that are currently being tested for their functional significance. Together, these results suggest that mutant U2AF1 expression contributes to the altered pre-mRNA splicing and hematopoiesis observed in patients with U2AF1 mutations. Citation Format: Cara Lunn Shirai, James N. Ley, Brian White, Justin Tibbitts, Jin Shao, Matthew Ndonwi, Sanghyun Kim, Theresa Okeyo-Owuor, Timothy A. Graubert, Matthew J. Walter. Mutant U2AF1 alters hematopoiesis and pre-mRNA splicing in transgenic mice. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr PR06.

Whole Exome Sequencing (“mutatome”) Of Deletion 5q

Interstitial deletion of chromosome 5q (del(5q)) is one of the most common karyotypic abnormalities in MDS. While a relatively small fraction of patients with del(5q) and 5q- syndrome show a relatively uniform clinical phenotype and a low rate of progression, the majority of del(5q) myeloid neoplasms are more heterogeneous. Prognosis correlates with the size and location of the deletion, with large deletions spanning subtelomeric and/or subcentromeric region correlating with a poor prognosis. Survival differences may relate to still undefined pathogenetic mechanisms underlying del(5q), which may involve hemizygous mutations or haploinsufficiency. With the latter scenario, it is possible that heterozygous mutations of genes located on 5q may phenocopy the deletion. To further elucidate the molecular mechanisms underlying del(5q), we preformed a comprehensive analysis of myeloid neoplasms using single nucleotide polymorphism array (SNP-A) and next generation whole exome sequencing (NGS) of paired DNA samples (germline/tumor) from 55 cases characterized by del(5q) among a total 428 patients with MDS and related disorders; we focused on mutations located on 5q in both diploid and deletion cases.In the total cohort, we identified 243 somatic mutations in 158 genes on chr5q, including well-known NPM1 or novel recurrent DDX41 mutations; 147 mutations were heterozygous, 11 hemizygous (in del(5q)). No homozygous mutations were found. Applying SNP-A-based karyotyping, we defined the commonly deleted region (CDR) as between 5q32 and 5q33.2 (145299747-153828955). In patients with 5q- syndrome, the proximal and terminal regions of chr5q were always retained; therefore we defined commonly retained regions (CRR) as CRR1 (proximal, 5q11.1 to 5q14.2, 48400001-81634579) and CRR2 (distal, 5q34 to 5q35.3, 164213764-180915260). The deletions of CRRs consequently contributed to worse prognosis in the aggressive types of MDS with longer del(5q).First we focused our study on the genes located on CRRs. We identified 120 heterozygous alterations in CRRs, including CWC27 (5q12.3), MAP1B (n=2, 5q13.2), NPM (n=50, 5q35.1), C5orf25 (n=4, 5q35.2) and DDX41 (n=4, 5q35.3); these mutations occurred only in a heterozygous configuration. Interestingly, spliceosome-associated gene CWC27 and RNA helicase DDX41 showed haploinsufficient expression in haploid cases without mutation, suggesting that mutated genes located on CRRs can be pathogenic due to both haploinsufficiency of WT genes and heterozygous mutations. Furthermore, patients with decreased expression of these genes had a poor survival (CWC27; HR=2.48, DDX41; HR=1.98).In positions corresponding to CDR and its proximal regions, we found 123 heterozygous alterations in 97 genes (50% of all alterations on 5q found), including recurrently mutated genes (FAT2: n=4; G3BP1: n=2) and hemizygous mutations of KDM3B (n=3, 5q31.2) and MCC (n=1, 5q22.2). In GPR98, associated with Usher syndrome, we detected both recurrent heterozygous and hemizygous mutations (each n=1). Also, minor alleles (frequencies were .002 and .004) of non-synonymous variants of GPR98 were selectively retained and wild-type alleles were deleted in del(5q) cases (n=2).We also searched accessory genetic events observed on other chromosomes in del(5q) cases. By SNP-A, deletions of CRRs (longer del(5q)) were significantly more associated with additional chromosomal defects. Similarly, some specific genes, including the splicing machinery genes and IDH family genes, were uniquely observed in the longer del(5q) cohort.In conclusion, we detected multiple pathogenic mutations in whole chr5q which might stratify del(5q) patients at risk for disease progression, though no single mutations could explain a majority of cases. Decreased expression or mutation of CWC27 and DDX41, located on CRRs, may exemplify the common pathophysiology shared by heterozygous mutations and haploinsufficient expressions on chr5q. Consequently, it is possible that deletion alone, through decreased expression, may be pathogenic. Disclosures:Makishima:AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Polprasert:MDS foundation: Research Funding. Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.

Mutant U2AF1 Expression Alters Hematopoiesis In Transgenic Mice

Mutations in 8 spliceosome genes have been identified in up to half of patients with myelodysplastic syndromes (MDS), suggesting that perturbations in pre-mRNA splicing play a role in MDS pathogenesis. Our group and others discovered recurrent heterozygous mutations in U2AF1, a gene encoding a splicing factor involved in intronic 3'-splice site recognition, in 11% of MDS patients. The most common U2AF1 mutation causes a serine to phenylalanine substitution at amino acid 34 (S34F). To study the effects of U2AF1(S34F) expression on hematopoiesis, we created site-specific, single-copy, doxycycline-inducible (controlled by the reverse tetracycline-transactivator [rtTA] transgene) U2AF1(WT) and U2AF1(S34F) transgenic mice. To examine the cell-autonomous effects of mutant U2AF1(S34F), we transplanted transgenic donor bone marrow into wild type mouse recipients prior to doxycycline induction of transgene expression.We observed a doxycycline dose-dependent increase in expression of U2AF1(S34F) and U2AF1(WT) transgenes by RT-PCR and pyrosequencing analysis, and we subsequently chose a dose that induces equivalent levels of exogenous U2AF1 transgene to endogenous mouse U2af1 transcripts. Following 4 weeks of doxycycline induction of transgene expression in vivo, U2AF1(S34F)-recipient mouse bone marrow cells showed increased alternative splicing of endogenous Fmr1 pre-mRNA, a transcript previously shown to be alternatively-spliced in U2AF1 mutant MDS patient samples, when compared to U2AF1(WT)- and rtTA only-recipient mouse bone marrow cells (p<0.02, n=9-11). This effect was seen as early as 5 days (p<0.001, n=3-7).Following 4 weeks of transgene induction, U2AF1(S34F)-recipient mice display a decrease in WBCs in the peripheral blood compared to U2AF1(WT)- and rtTA only-recipient controls (average 4.5 vs 6.7 and 7.1 K/µl, respectively; p≤0.01). However, there are no significant differences in bone marrow cellularity or spleen size in these mice (n=9-11). U2AF1(S34F)-recipient mice have reduced frequency of peripheral blood monocytes (average 1.2 vs 3.2 and 2.6%, p<0.05), as well as bone marrow monocytes (3.9 vs 7 and 6.1%, p<0.001) and B cells (average 10.1 vs 20.7 and 20.6%, p<0.001) when compared to U2AF1(WT)- and rtTA only-recipient mice, respectively (n=9-11). Moreover, bone marrow monocytes in U2AF1(S34F)-recipient mice have an increased frequency of Annexin V+ staining compared to controls (average 11.2 vs 6.9 and 7.5%, p<0.05, n=5-6), indicating that loss of these cells may be due to apoptosis. U2AF1(S34F)-recipient mice also have an increase in the frequency of bone marrow neutrophils compared to U2AF1(WT)- and rtTA only-recipient mice (average 65 vs 53 and 50%, respectively, p<0.001, n=9-11). Taken together, these data indicate U2AF1(S34F) expression alters hematopoietic lineage distribution in vivo.In addition, 4 weeks of U2AF1(S34F) expression results in alterations of progenitor and stem cell frequency. U2AF1(S34F)-recipient mice have approximately a 1.5-fold increase in the frequency of progenitor cells in their bone marrow measured by flow cytometry (linlow/c-kit+/Sca-1-) (average 4.1 vs 2.6 and 2.9%; p<0.001, n=5-6) and methylcellulose progenitor colony forming assays (average 85 vs 55 and 59 colonies/plate; p<0.02, n=9-11) when compared to U2AF1(WT)- and rtTA only-recipient mice, respectively. U2AF1(S34F)-recipient mice also display a 1.5-fold increase in the frequency of common myeloid progenitors compared to U2AF1(WT) and rtTA only controls in both bone marrow and spleen (p≤0.02), along with a 2-fold increase of the granulocyte-macrophage progenitors in the spleen (p≤0.03) (n=5-6). In addition to changes in progenitors, U2AF1(S34F)-recipient mice have a 1.4-fold increase in the frequency of bone marrow KLS cells, short-term HSCs, and dormant long-term HSCs (p≤0.05), as well as a 2-fold increase in spleen dormant long-term HSCs (p≤0.03) compared to U2AF1(WT)- and rtTA only-recipient controls (n=5-6). Competitive repopulation studies are ongoing.Collectively, these data indicate that mutant U2AF1(S34F) expression in mouse bone marrow cells results in altered pre-mRNA splicing and changes in hematopoietic cell lineage distribution, as well as increased bone marrow and spleen progenitor and stem cell populations. Furthermore, these results suggest that mutant U2AF1 may contribute to altered hematopoiesis in patients. Disclosures:No relevant conflicts of interest to declare.

Rapp-Hodgkin and Hay-Wells ectodermal dysplasia syndromes represent a variable spectrum of the same genetic disorder

Rapp-Hodgkin syndrome (RHS) and Hay-Wells [also known as ankyloblepharon-ectodermal defects-cleft lip/palate (AEC)] syndrome have been designated as distinct ectodermal dysplasia syndromes despite both disorders having overlapping clinical features and the same mutated gene, TP63. To search for TP63 mutations in two unrelated cases of RHS and two of AEC syndrome and to review the TP63 mutation database and clinical descriptions of affected individuals, the goal being to refine genotype-phenotype correlation and to determine the clinical/molecular justification for RHS and AEC continuing to exist as separate entities. Clinical examination of four affected cases and sequencing of genomic DNA using TP63-specific primers. Literature review of published clinical descriptions of RHS and AEC syndrome cases containing TP63 mutation data. Cases of RHS and AEC show considerable clinical overlap, particularly with regard to hypotrichosis and mid-face hypoplasia, and the clinical feature of ankyloblepharon in AEC is often subtle, transient and a poor distinguishing clinical sign. We identified two new and two recurrent heterozygous mutations in TP63: c.1456insA (p.Leu486fsX52), RHS; c.1537T>G (p.Phe513Val), RHS; c.1787delG (p.Gly596fsX68), AEC; and c.1682G>A (p.Gly561Asp), AEC. Including this study, 42 different mutations in TP63 in RHS and AEC have now been reported, three of which are exactly the same in both syndromes. Our clinicopathological and molecular findings indicate that there is no justification for the continued use of eponyms in referring to these particular ectodermal dysplasia syndromes. We support the view that the terms ‘Hay-Wells’ and ‘Rapp-Hodgkin’ should be abandoned in favour of the all-inclusive diagnosis ‘AEC syndrome’, notwithstanding the inconsistency or often transient nature of the ankyloblepharon.

Novel and Recurrent KIF21A Mutations in Congenital Fibrosis of the Extraocular Muscles Type 1 and 3

To characterize the disease-causing mutations and associated clinical phenotypes in 5 Chinese families with congenital fibrosis of the extraocular muscles (CFEOM). Ophthalmic investigations included visual acuity, levator function, documentation of compensatory head position, ocular motility, and slitlamp and fundus examinations. The kinesin family member 21A gene (KIF21A) was sequenced for mutation detection. Genotyping and linkage analysis were performed for the KIF21A/FEOM1 and FEOM3 loci. Four families were clinically classified as having CFEOM type 1 (CFEOM1) with full expression of severe ptosis and ophthalmoplegia. One family had CFEOM type 3 (CFEOM3) with typically varying expression of phenotypes between individuals. Recurrent heterozygous KIF21A mutations were identified in 2 CFEOM1 families (2860C>T) and the CFEOM3 family (2861G>A). In another CFEOM1 family, a novel missense mutation (84C>G, C28W) was revealed. The novel KIF21A mutation 84C>G demonstrated in a CFEOM1 family affects the kinesin motor domain, supporting that mutations may also occur outside the commonly involved coiled-coil domain. The 2861G>A mutation found in a CFEOM3 family has been previously reported in CFEOM1, further supporting that different phenotypes can arise from identical mutations. Clinical Relevance Clinical and genetic characterization are complementary tools for diagnostic, prognostic, and treatment purposes in CFEOM.

FBN1, TGFBR1, and the Marfan‐craniosynostosis/mental retardation disorders revisited

The recent identification of TGFBR2 mutations in Marfan syndrome II (MFSII) [Mizuguchi et al. (2004); Nat Genet 36:855-860] and of TGFBR1 and TGFBR2 mutations in Loeys-Dietz aortic aneurysm syndrome (LDS) [Loeys et al. (2005); Nat Genet 37:275-281] [OMIM 609192] has provided direct evidence of abnormal signaling in transforming growth factors beta (TGF-beta) in the pathogenesis of Marfan syndrome (MFS). In light of this, we describe the phenotypes and genotypes of five individuals. Patient 1 had MFS and abnormal cranial dura. Patient 2 had severe early onset MFS and an abnormal skull. Patients 3 and 4 had probable Furlong syndrome (FS). Patient 5 had marfanoid (MD) features, mental retardation (MR), and a deletion of chromosome 15q21.1q21.3. All patients had a condition within the MFS, MD-craniosynostosis (CS) or MD-MR spectrum. The names of these entities may become redundant, and instead, come to be considered within the spectrum of TGF-beta signaling pathway disorders. Two recurrent heterozygous FBN1 mutations were found in Patients 1 and 2, and an identical novel heterozygous de novo TGFBR1 mutation was found in Patients 3 and 4, in whom altered fibrillin-1 processing was demonstrated previously [Milewicz et al. (2000); Am J Hum Genet 67:279]. A heterozygous FBN1 deletion was found in Patient 5. These findings support the notion that perturbation of extracellular matrix homeostasis and/or remodeling caused by abnormal TGF-beta signaling is the core pathogenetic mechanism in MFS and related entities including the MD-CS syndromes.