Prasuna Paluru

Microdeletions and Microduplications in Patients with Congenital Heart Disease and Multiple Congenital Anomalies

OBJECTIVE Multiple genetic syndromes are caused by recurrent chromosomal microdeletions or microduplications. The increasing use of high-resolution microarrays in clinical analysis has allowed the identification of previously undetectable submicroscopic copy number variants (CNVs) associated with genetic disorders. We hypothesized that patients with congenital heart disease and additional dysmorphic features or other anomalies would be likely to harbor previously undetected CNVs, which might identify new disease loci or disease-related genes for various cardiac defects. DESIGN Copy number analysis with single nucleotide polymorphism-based, oligonucleotide microarrays was performed on 58 patients with congenital heart disease and other dysmorphic features and/or other anomalies. The observed CNVs were validated using independent techniques and validated CNVs were further analyzed using computational algorithms and comparison with available control CNV datasets in order to assess their pathogenic potential. RESULTS Potentially pathogenic CNVs were detected in twelve of 58 patients (20.7%), ranging in size from 240 Kb to 9.6 Mb. These CNVs contained between 1 and 55 genes, including NRP1, NTRK3, MESP1, ADAM19, and HAND1, all of which are known to participate in cardiac development. CONCLUSIONS Genome-wide analysis in patients with congenital heart disease and additional phenotypes has identified potentially pathogenic CNVs affecting genes involved in cardiac development. The identified variant loci and the genes within them warrant further evaluation in similarly syndromic and nonsyndromic cardiac cohorts.

Patient-Derived Induced Pluripotent Stem Cells Recapitulate Hematopoietic Abnormalities of Juvenile Myelomonocytic Leukemia

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of young children initiated by mutations that deregulate cytokine receptor signaling. Studies of JMML are constrained by limited access to patient tissues. We generated induced pluripotent stem cells (iPSCs) from malignant cells of two JMML patients with somatic heterozygous p.E76K missense mutations in PTPN11, which encodes SHP-2, a nonreceptor tyrosine phosphatase. In vitro differentiation of JMML iPSCs produced myeloid cells with increased proliferative capacity, constitutive activation of granulocyte macrophage colony-stimulating factor (GM-CSF), and enhanced STAT5/ERK phosphorylation, similar to primary JMML cells from patients. Pharmacological inhibition of MEK kinase in iPSC-derived JMML cells reduced their GM-CSF independence, providing rationale for a potential targeted therapy. Our studies offer renewable sources of biologically relevant human cells in which to explore the pathophysiology and treatment of JMML. More generally, we illustrate the utility of iPSCs for in vitro modeling of a human malignancy.

High-level transgene expression in induced pluripotent stem cell–derived megakaryocytes: correction of Glanzmann thrombasthenia

Megakaryocyte-specific transgene expression in patient-derived induced pluripotent stem cells (iPSCs) offers a new approach to study and potentially treat disorders affecting megakaryocytes and platelets. By using a Gp1ba promoter, we developed a strategy for achieving a high level of protein expression in human megakaryocytes. The feasibility of this approach was demonstrated in iPSCs derived from two patients with Glanzmann thrombasthenia (GT), an inherited platelet disorder caused by mutations in integrin αIIbβ3. Hemizygous insertion of Gp1ba promoter-driven human αIIb complementary DNA into the AAVS1 locus of iPSCs led to high αIIb messenger RNA and protein expression and correction of surface αIIbβ3 in megakaryocytes. Agonist stimulation of these cells displayed recovery of integrin αIIbβ3 activation. Our findings demonstrate a novel approach to studying human megakaryocyte biology as well as functional correction of the GT defect, offering a potential therapeutic strategy for patients with diseases that affect platelet function.

The phenotypic spectrum of ZIC3 mutations includes isolated d‐transposition of the great arteries and double outlet right ventricle

Disease causing mutations for heterotaxy syndrome were first identified in the X‐linked laterality gene, ZIC3. Mutations typically result in males with situs ambiguus and complex congenital heart disease; however affected females and one male with isolated d‐transposition of the great arteries (d‐TGA) have been reported. We hypothesized that a subset of patients with heart defects common to heterotaxy but without laterality defects would have ZIC3 mutations. We also sought to estimate the prevalence of ZIC3 mutations in sporadic heterotaxy. Patients with TGA (n = 169), double outlet right ventricle (DORV; n = 89), common atrioventricular canal (CAVC; n = 41), and heterotaxy (n = 54) underwent sequencing of ZIC3 exons. We tested 90 patients with tetralogy of Fallot (TOF) to correlate genotype with phenotype. Three potentially disease‐related missense mutations were detected: c.49G > T (Gly17Cys) in a female with isolated DORV, c.98C > T (Ala33Val) in a male with isolated d‐TGA, and c.841C > T (His281Tyr) in a female with sporadic heterotaxy. We also identified a novel insertion (CPFP333ins) in a family with heterotaxy. All were absent in 200 control patients and the 1000 Genomes Project (n = 629). No significant mutations were found in patients with TOF. Functional studies demonstrated reduced transcriptional activity of the ZIC3 His281Tyr mutant protein. ZIC3 mutations were rarely identified in isolated DORV and d‐TGA suggesting that a subset of DORV and d‐TGA may fall within the spectrum of laterality defects. ZIC3 mutations were found in 3.7% of patients with sporadic heterotaxy; therefore testing should be considered in patients with heterotaxy.

The negative impact of Wnt signaling on megakaryocyte and primitive erythroid progenitors derived from human embryonic stem cells

The Wnt gene family consists of structurally related genes encoding secreted signaling molecules that have been implicated in many developmental processes, including regulation of cell fate and patterning during embryogenesis. Previously, we found that Wnt signaling is required for primitive or yolk sac-derived-erythropoiesis using the murine embryonic stem cell (ESC) system. Here, we examine the effect of Wnt signaling on the formation of early hematopoietic progenitors derived from human ESCs. The first hematopoietic progenitor cells in the human ESC system express the pan-hematopoietic marker CD41 and the erythrocyte marker, glycophorin A or CD235. We have developed a novel serum-free, feeder-free, adherent differentiation system that can efficiently generate large numbers of CD41+CD235+ cells. We demonstrate that this cell population contains progenitors not just for primitive erythroid and megakaryocyte cells but for the myeloid lineage as well and term this population the primitive common myeloid progenitor (CMP). Treatment of mesoderm-specified cells with Wnt3a led to a loss of hematopoietic colony-forming ability while the inhibition of canonical Wnt signaling with DKK1 led to an increase in the number of primitive CMPs. Canonical Wnt signaling also inhibits the expansion and/or survival of primitive erythrocytes and megakaryocytes, but not myeloid cells, derived from this progenitor population. These findings are in contrast to the role of Wnt signaling during mouse ESC differentiation and demonstrate the importance of the human ESC system in studying species-specific differences in development.

MicroRNA Screen of Human Embryonic Stem Cell Differentiation Reveals miR‐105 as an Enhancer of Megakaryopoiesis from Adult CD34+ Cells

MicroRNAs (miRNAs) can control stem cell differentiation by targeting mRNAs. Using 96‐well plate electroporation, we screened 466 human miRNA mimics by four‐color flow cytometry to explore differentiation of common myeloid progenitors (CMP) derived from human embryonic stem cells (hESCs). The transfected cells were then cultured in a cytokine cocktail that supported multiple hematopoietic lineages. At 4–5 days post‐transfection, flow cytometry of erythroid (CD235+CD41−), megakaryocyte (CD41+CD42+), and myeloid (CD18+CD235−) lineages revealed miR‐105 as a novel enhancer of megakaryocyte production during in vitro primitive hematopoiesis. In hESC‐derived CMPs, miR‐105 caused a sixfold enhancement in megakaryocyte production. miR‐513a, miR‐571, and miR‐195 were found to be less potent megakaryocyte enhancers. We confirmed the relevance of miR‐105 in adult megakaryopoiesis by demonstrating increased megakaryocyte yield and megakaryocyte colony forming potential in human adult CD34+ cells derived from peripheral blood. In addition, adult CD34+ cells express endogenous miR‐105 during megakaryocyte differentiation. siRNA knockdown of the hematopoietic transcription factor c‐Myb caused a similar enhancement of megakaryocyte production as miR‐105. Finally, a luciferase/c‐Myb‐3′UTR construct and Western blot analysis demonstrated that the hematopoietic transcription factor c‐Myb mRNA was a target of miR‐105. We report a novel hESC‐based miR screening platform and demonstrate that miR‐105 is an enhancer of megakaryopoiesis in both primitive and definitive hematopoiesis. Stem Cells 2014;32:1337–1346

Refinement of the X-linked nonsyndromic high-grade myopia locus MYP1 on Xq28 and exclusion of 13 known positional candidate genes by direct sequencing.

PURPOSE Myopia is a common vision problem affecting almost one third of the worlds population. It can occur as an isolated genetic condition or be associated with other anomalies and/or syndromes. Seventeen myopia loci have been identified on various chromosomes; however, no specific gene mutations have yet been identified. METHODS Two large multigeneration Asian Indian pedigrees (UR006 and UR077) with isolated, nonsyndromic myopia were studied, in which the condition appeared to segregate as an X-linked recessive trait (MYP1; MIM 310460). The degree of myopia was variable in both families, ranging from -6 to -23 D (mean, -8.48 D) with the majority >7.0 D. To map the myopia locus in these families, polymorphic microsatellite markers covering the entire X chromosome were used in linkage analyses performed on 42 genomic DNA samples (13 affected and 29 normal) from both families. RESULTS Marker DXYS154, which is located within the pseudoautosomal region in distal Xq28 (PAR2; pseudoautosomal region 2), gave a combined maximum LOD score of 5.3 at = 0 under an autosomal recessive model. Other markers in the region (near but not within the PAR2 region) that showed no recombination with the phenotype in both the families included DXS1108, DXS8087, and F8i13. CONCLUSIONS Observation of recombination in family UR006 refined the disease locus to a ∼1.25-Mb region flanked by the proximal marker DXS1073 and distal marker DXYS154. Mutation search in exons and splice junctions of candidate genes CTAG2, GAB3, MPP1, F8Bver, FUNDC2, VBP1, RAB39B, CLIC2, TMLHE, SYBL, IL9R, SPRY3, and CXYorf1 did not detect a pathogenic or predisposing variant.

Variants of folate metabolism genes and risk of left‐sided cardiac defects

BACKGROUND Congenital heart defects (CHDs) are the most common, serious group of birth defects. Although relatively little is known about the causes of these conditions and there are no established prevention strategies, evidence suggests that the risk of CHDs may be related to maternal folate status as well as genetic variants in folate-related genes. Efforts to establish the relationships between these factors and CHD risk have, however, been hampered by a number of factors, including small study sample sizes and phenotypic heterogeneity. METHODS The present study examined the relationship between nine genetic variants in eight folate-related genes and a relatively homogeneous group of left-sided cardiac defects in a cohort of 386 case-parent triads. Log-linear analyses were used to assess both maternal and inherited genetic effects. RESULTS Analyses of the study data provided marginal evidence that the maternal MTR A2756G (unadjusted p = 0.01) and the inherited BHMT G742A (unadjusted p = 0.06) genotypes influence the risk of this subset of CHDs. However, neither association achieved significance when the false-discovery rate was controlled at 0.05. CONCLUSIONS These results, which are based on the largest study sample and most comprehensive assessment of the relationship between left-sided cardiac defects and folate-related genes reported to date, provide little evidence that this subset of CHDs is folate related. However, even larger studies and more comprehensive evaluations of the folate pathway genes are required to fully explore the relationship between folate and left-sided cardiac defects.