[Show abstract][Hide abstract] ABSTRACT: The first critical stage in salamander or teleost appendage regeneration is creation of a specialized epidermis that instructs growth from underlying stump tissue. Here, we performed a forward genetic screen for mutations that impair this process in amputated zebrafish fins. Positional cloning and complementation assays identified a temperature-sensitive allele of the ECM component laminin beta 1a (lamb1a) that blocks fin regeneration. lamb1a, but not its paralog lamb1b, is sharply induced in a subset of epithelial cells after fin amputation, where it is required to establish and maintain a polarized basal epithelial cell layer. These events facilitate expression of the morphogenetic factors shha and lef1, basolateral positioning of phosphorylated Igf1r, patterning of new osteoblasts, and regeneration of bone. By contrast, lamb1a function is dispensable for juvenile body growth, homeostatic adult tissue maintenance, repair of split fins, or renewal of genetically ablated osteoblasts. fgf20a mutations or transgenic Fgf receptor inhibition disrupt lamb1a expression, linking a central growth factor to epithelial maturation during regeneration. Our findings reveal transient induction of lamb1a in epithelial cells as a key, growth factor-guided step in formation of a signaling-competent regeneration epidermis.
[Show abstract][Hide abstract] ABSTRACT: The genetic disorder Kabuki syndrome (KS) is characterized by developmental delay and congenital anomalies. Dominant mutations in the chromatin regulators lysine (K)-specific methyltransferase 2D (KMT2D) (also known as MLL2) and lysine (K)-specific demethylase 6A (KDM6A) underlie the majority of cases. Although the functions of these chromatin-modifying proteins have been studied extensively, the physiological systems regulated by them are largely unknown. Using whole-exome sequencing, we identified a mutation in RAP1A that was converted to homozygosity as the result of uniparental isodisomy (UPD) in a patient with KS and a de novo, dominant mutation in RAP1B in a second individual with a KS-like phenotype. We elucidated a genetic and functional interaction between the respective KS-associated genes and their products in zebrafish models and patient cell lines. Specifically, we determined that dysfunction of known KS genes and the genes identified in this study results in aberrant MEK/ERK signaling as well as disruption of F-actin polymerization and cell intercalation. Moreover, these phenotypes could be rescued in zebrafish models by rebalancing MEK/ERK signaling via administration of small molecule inhibitors of MEK. Taken together, our studies suggest that the KS pathophysiology overlaps with the RASopathies and provide a potential direction for treatment design.
The Journal of clinical investigation 08/2015; DOI:10.1172/JCI80102 · 13.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Intellectual disability (ID) affects approximately 1%–3% of humans with a gender bias toward males. Previous studies have identified mutations in more than 100 genes on the X chromosome in males with ID, but there is less evidence for de novo mutations on the X chromosome causing ID in females. In this study we present 35 unique deleterious de novo mutations in DDX3X identified by Whole exome sequencing in 38 females with ID and various other features including hypotonia, movement disorders, behavior problems,corpus callosum hypoplasia, and epilepsy. Based on our findings, mutations in DDX3X are one of the more common causes of ID, accounting for 1%–3% of unexplained ID in females. Although no de novo DDX3X mutations were identified in males, we present three families with segregating missense mutations in DDX3X, suggestive of an X-linked recessive inheritance pattern. In these families, all males with the DDX3X variant had ID, whereas carrier females were unaffected. To explore the pathogenic mechanisms Accounting for the differences in disease transmission and phenotype between affected females and affected males with DDX3X missense variants,
we used canonical Wnt defects in zebrafish as a surrogate measure of DDX3X function in vivo.We demonstrate a consistent loss-of-function effect of all tested de novo mutations on the Wnt pathway, and we further show a differential effect by gender. The differential activity possibly reflects a dose-dependent effect of DDX3X expression in the context of functional mosaic females versus one-copy males, which reflects the complex biological nature of DDX3X mutations.
The American Journal of Human Genetics 08/2015; 97(2):343-352. DOI:10.1016/j.ajhg.2015.07.004. · 10.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: African Americans have a disproportionate risk for developing nephropathy. This disparity has been attributed to coding variants (G1 and G2) in apolipoprotein L1 (APOL1); however, there is little functional evidence supporting the role of this protein in renal function. Here, we combined genetics and in vivo modeling to examine the role of apol1 in glomerular development and pronephric filtration and to test the pathogenic potential of APOL1 G1 and G2. Translational suppression or CRISPR/Cas9 genome editing of apol1 in zebrafish embryos results in podocyte loss and glomerular filtration defects. Complementation of apol1 morphants with wild-type human APOL1 mRNA rescues these defects. However, the APOL1 G1 risk allele does not ameliorate defects caused by apol1 suppression and the pathogenicity is conferred by the cis effect of both individual variants of the G1 risk haplotype (I384M/S342G). In vivo complementation studies of the G2 risk allele also indicate that the variant is deleterious to protein function. Moreover, APOL1 G2, but not G1, expression alone promotes developmental kidney defects, suggesting a possible dominant-negative effect of the altered protein. In sickle cell disease (SCD) patients, we reported previously a genetic interaction between APOL1 and MYH9. Testing this interaction in vivo by co-suppressing both transcripts yielded no additive effects. However, upon genetic or chemical induction of anemia, we observed a significantly exacerbated nephropathy phenotype. Furthermore, concordant with the genetic interaction observed in SCD patients, APOL1 G2 reduces myh9 expression in vivo, suggesting a possible interaction between the altered APOL1 and myh9. Our data indicate a critical role for APOL1 in renal function that is compromised by nephropathy-risk encoding variants. Moreover, our interaction studies indicate that the MYH9 locus is also relevant to the phenotype in a stressed microenvironment and suggest that consideration of the context-dependent functions of both proteins will be required to develop therapeutic paradigms.
[Show abstract][Hide abstract] ABSTRACT: To analyze the expansion of CTG18.1 allele associated with Fuchs' corneal dystrophy (FCD) in our large cohort of late-onset FCD cases.
CTG repeats within the CTG18.1 allele were estimated by short tandem repeat (STR) and triplet primed PCR (TP-PCR) assays in our large cohort of 574 late-onset FCD cases and 354 controls and large multigeneration familial cases. The age versus severity relationships were analyzed in FCD genotypes, namely, nonexpanded (N/N), monoallelic expansion (N/X), and biallelic expansion (X/X) with N ≤ 40 CTG monomers. The threshold for causality conferred by an expansion of CTG18.1 was identified by excluding the population of FCD cases who harbored an allele length equivalent to the maximum CTG monomers observed in the controls.
The expanded CTG18.1 for (CTG)n>40 showed a strong association (P = 1.56 × 10-82) with FCD. Importantly, we delineated the threshold of expansion to 103 CTG repeats above which the allele confers causality in 17.8% of FCD cases. Regression analyses demonstrated a significant correlation between disease severity and age in individuals who harbor either a monoallelic expansion or a biallelic expansion at (CTG)n>40. These analyses helped predict FCD in two previously unaffected individuals based on their CTG18.1 expansion genotype.
A monoallelic expansion of CTG18.1 contributes to increased disease severity and is causal at (CTG)n>103, whereas a biallelic expansion is sufficient to be causal for FCD at (CTG)n>40. This study highlights the largest contributory causal allele for FCD.
[Show abstract][Hide abstract] ABSTRACT: Patterns of amino acid conservation have served as a tool for understanding protein evolution. The same principles have also found broad application in human genomics, driven by the need to interpret the pathogenic potential of variants in patients. Here we performed a systematic comparative genomics analysis of human disease-causing missense variants. We found that an appreciable fraction of disease-causing alleles are fixed in the genomes of other species, suggesting a role for genomic context. We developed a model of genetic interactions that predicts most of these to be simple pairwise compensations. Functional testing of this model on two known human disease genes revealed discrete cis amino acid residues that, although benign on their own, could rescue the human mutations in vivo. This approach was also applied to ab initio gene discovery to support the identification of a de novo disease driver in BTG2 that is subject to protective cis-modification in more than 50 species. Finally, on the basis of our data and models, we developed a computational tool to predict candidate residues subject to compensation. Taken together, our data highlight the importance of cis-genomic context as a contributor to protein evolution; they provide an insight into the complexity of allele effect on phenotype; and they are likely to assist methods for predicting allele pathogenicity.
[Show abstract][Hide abstract] ABSTRACT: -Left ventricular noncompaction (LVNC) is an autosomal dominant, genetically heterogeneous cardiomyopathy with variable severity, which may co-occur with cardiac hypertrophy.
-Here, we generated whole exome sequence (WES) data from multiple members from five families with LVNC. In four out of five families, the candidate causative mutation segregates with disease in known LVNC genes MYH7 and TPM1. Subsequent sequencing of MYH7 in a larger LVNC cohort identified seven novel likely disease causing variants. In the fifth family, we identified a frameshift mutation in NNT, a nuclear encoded mitochondrial protein, not implicated previously in human cardiomyopathies. Resequencing of NNT in additional LVNC families identified a second likely pathogenic missense allele. Suppression of nnt in zebrafish caused early ventricular malformation and contractility defects, likely driven by altered cardiomyocyte proliferation. In vivo complementation studies showed that mutant human NNT failed to rescue nnt morpholino-induced heart dysfunction, indicating a probable haploinsufficiency mechanism.
-Together, our data expand the genetic spectrum of LVNC and demonstrate how the intersection of WES with in vivo functional studies can accelerate the identification of genes that drive human genetic disorders.
[Show abstract][Hide abstract] ABSTRACT: Human peripheral blood and umbilical cord blood represent attractive sources of cells for reprogramming to induced pluripotent stem cells (iPSCs). However, to date, most of the blood-derived iPSCs were generated using either integrating methods or starting from T-lymphocytes that have genomic rearrangements thus bearing uncertain consequences when using iPSC-derived lineages for disease modeling and cell therapies. Recently, both peripheral blood and cord blood cells have been reprogrammed into transgene-free iPSC using the Sendai viral vector. Here we demonstrate that peripheral blood can be utilized for medium-throughput iPSC production without the need to maintain cell culture prior to reprogramming induction. Cell reprogramming can also be accomplished with as little as 3000 previously cryopreserved cord blood cells under feeder-free and chemically defined Xeno-free conditions that are compliant with standard Good Manufacturing Practice (GMP) regulations. The first iPSC colonies appear 2-3 weeks faster in comparison to previous reports. Notably, these peripheral blood- and cord blood-derived iPSCs are free of detectable immunoglobulin heavy chain (IGH) and T cell receptor (TCR) gene rearrangements, suggesting they did not originate from B- or T- lymphoid cells. The iPSCs are pluripotent as evaluated by the scorecard assay and in vitro multi lineage functional cell differentiation. Our data show that small volumes of cryopreserved peripheral blood or cord blood cells can be reprogrammed efficiently at a convenient, cost effective and scalable way. In summary, our method expands the reprogramming potential of limited or archived samples either stored at blood banks or obtained from pediatric populations that cannot easily provide large quantities of peripheral blood or a skin biopsy.
[Show abstract][Hide abstract] ABSTRACT: Autism is a multifactorial neurodevelopmental disorder affecting more males than females; consequently, under a multifactorial genetic hypothesis, females are affected only when they cross a higher biological threshold. We hypothesize that deleterious variants at conserved residues are enriched in severely affected patients arising from female-enriched multiplex families with severe disease, enhancing the detection of key autism genes in modest numbers of cases. Here we show the use of this strategy by identifying missense and dosage sequence variants in the gene encoding the adhesive junction-associated δ-catenin protein (CTNND2) in female-enriched multiplex families and demonstrating their loss-of-function effect by functional analyses in zebrafish embryos and cultured hippocampal neurons from wild-type and Ctnnd2 null mouse embryos. Finally, through gene expression and network analyses, we highlight a critical role for CTNND2 in neuronal development and an intimate connection to chromatin biology. Our data contribute to the understanding of the genetic architecture of autism and suggest that genetic analyses of phenotypic extremes, such as female-enriched multiplex families, are of innate value in multifactorial disorders.