198 research outputs found
COL4A1 mutations cause ocular dysgenesis, neuronal localization defects, and myopathy in mice and Walker-Warburg syndrome in humans.
No full textMuscle-eye-brain disease (MEB) and Walker Warburg Syndrome (WWS) belong to a spectrum of autosomal recessive diseases characterized by ocular dysgenesis, neuronal migration defects, and congenital muscular dystrophy. Until now, the pathophysiology of MEB/WWS has been attributed to alteration in dystroglycan post-translational modification. Here, we provide evidence that mutations in a gene coding for a major basement membrane protein, collagen IV alpha 1 (COL4A1), are a novel cause of MEB/WWS. Using a combination of histological, molecular, and biochemical approaches, we show that heterozygous Col4a1 mutant mice have ocular dysgenesis, neuronal localization defects, and myopathy characteristic of MEB/WWS. Importantly, we identified putative heterozygous mutations in COL4A1 in two MEB/WWS patients. Both mutations occur within conserved amino acids of the triple-helix-forming domain of the protein, and at least one mutation interferes with secretion of the mutant proteins, resulting instead in intracellular accumulation. Expression and posttranslational modification of dystroglycan is unaltered in Col4a1 mutant mice indicating that COL4A1 mutations represent a distinct pathogenic mechanism underlying MEB/WWS. These findings implicate a novel gene and a novel mechanism in the etiology of MEB/WWS and expand the clinical spectrum of COL4A1-associated disorders
Evidence from human and zebrafish that GPC1 is a biliary atresia susceptibility gene.
No full textBackground & Aims: Biliary atresia (BA) is a progressive fibroinflammatory disorder of infants involving the extrahepatic and intrahepatic biliary tree. Its etiology is unclear but is believed to involve exposure of a genetically susceptible individual to certain environmental factors. BA occurs exclusively in the neonatal liver, so variants of genes expressed during hepatobiliary development could affect susceptibility. Genome-wide association studies previously identified a potential region of interest at 2q37. We continued these studies to narrow the region and identify BA susceptibility genes. Methods: We searched for copy number variants that were increased among patients with BA (n = 61) compared with healthy individuals (controls; n = 5088). After identifying a candidate gene, we investigated expression patterns of orthologues in zebrafish liver and the effects of reducing expression, with morpholino antisense oligonucleotides, on biliary development, gene expression, and signal transduction. Results: We observed a statistically significant increase in deletions at 2q37.3 in patients with BA that resulted in deletion of one copy of GPC1, which encodes glypican 1, a heparan sulfate proteoglycan that regulates Hedgehog signaling and inflammation. Knockdown of gpc1 in zebrafish led to developmental biliary defects. Exposure of the gpc1 morphants to cyclopamine, a Hedgehog antagonist, partially rescued the gpc1-knockdown phenotype. Injection of zebrafish with recombinant Sonic Hedgehog led to biliary defects similar to those of the gpc1 morphants. Liver samples from patients with BA had reduced levels of apical GPC1 in cholangiocytes compared with samples from controls. Conclusions: Based on genetic analysis of patients with BA and zebrafish, GPC1 appears to be a BA susceptibility gene. These findings also support a role for Hedgehog signaling in the pathogenesis of BA. © 2013 AGA Institute
Stem cell expansion and bioreactor development
No full textPhDA major challenge to the clinical success of cell-based tissue engineering strategies is the ability to obtain sufficient numbers of cells within an acceptable time frame. The expansion of cells on microcarriers within spinner flask bioreactor has shown promise in meeting that challenge. Spinner flask microcarrier technology is space-saving and media utilisation efficient. However, further optimisation in terms of, for example, seeding efficiency, expansion rates and harvest efficiency is necessary to realise the clinical potential of this technology. The present work is designed to improve cell expansion rates. It involves investigation of microcarrier composition and surface structure and spinner flask shear stress on cell growth.
BMSC growth on PHBV microcarriers was superior to PCL and PLGA microcarriers and comparable to Cytodex 1 microcarriers. Lower density PHBV microcarriers showed promise as a superior alternative to Cytodex 1. Two different impeller designs employed in the w/o/w method of microcarrier synthesis resulted in smoother and rougher PCL microcarriers with Ra = 1.77 ± 0.42 μm to 6.4 ± 1.48 μm respectively. Superior BMSC growth was observed on the rougher PCL microcarriers. Differentiation potential along the osteogenic and adipogenic lineages of BMSCs expanded on the microcarrier types was retained.
Particle Image Velocimetry was used to quantify shear stress within a spinner flask bioreactor. It was found that 80% of the shear stress was localised within the impeller region which occupied 55% of the bioreactor working volume. Shear stress increased as Cytodex 1 microcarrier concentration and impeller rotational speed increased. Superior BMSC growth rates on microcarriers were observed for the lowest shear stress experimental group (3.4 x 10-3 N/m2 ≤ impeller region mean shear stress ≤ 4.6 x 10-3 N/m2) as compared to the three higher shear stress groups (5.5 x 10-3 N/m2 ≤ mean shear stress ≤ 1.3 x 10-2 N/m2). Expanded BMSCs on the cytodex 1 microcarriers retained multipotentiality for the range of shear stresses investigated
Heterozygous deletion of FOXA2 segregates with disease in a family with heterotaxy, panhypopituitarism, and biliary atresia
No full textBiliary atresia (BA) is a pediatric cholangiopathy with unknown etiology occurring in isolated and syndromic forms. Laterality defects affecting the cardiovascular and gastrointestinal systems are the most common features present in syndromic BA. Most cases are sporadic, although reports of familial cases have led to the hypothesis of genetic susceptibility in some patients. We identified a child with BA, malrotation, and interrupted inferior vena cava whose father presented with situs inversus, polysplenia, panhypopituitarism, and mildly dysmorphic facial features. Chromosomal microarray analysis demonstrated a 277 kb heterozygous deletion on chromosome 20, which included a single gene, FOXA2, in the proband and her father. This deletion was confirmed to be de novo in the father. The proband and her father share a common diagnosis of heterotaxy, but they also each presented with a variety of other issues. Further genetic screening revealed that the proband carried an additional protein-altering polymorphism (rs1904589; p.His165Arg) in the NODAL gene that is not present in the father, and this variant has been shown to decrease expression of the gene. As FOXA2 can be a regulator of NODAL expression, we propose that haploinsufficiency for FOXA2 combined with a decreased expression of NODAL is the likely cause for syndromic BA in this proband
Replication of a GWAS signal in a Caucasian population implicates ADD3 in susceptibility to biliary atresia
No full textIn the United States, biliary atresia (BA) is the most frequent indication for liver transplantation in pediatric patients. BA is a complex disease, with suspected environmental and genetic risk factors. A genome-wide association study in Chinese patients identified association to the 10q24.2 (hg18) genomic region. This signal was upstream of two genes, XPNPEP1 and ADD3, both expressed in intrahepatic bile ducts. We tested association to this region in 171 BA patients and 1,630 controls of European descent and found the strongest signal to be at rs7099604 (p = 2.5 x 10(-3)) in intron 1 of the ADD3 gene. Moreover, expression data suggest that ADD3, but not XPNPEP1, is differentially expressed in BA patients. The role of ADD3 in biliary development is unclear, but our findings suggest that this gene may be functionally relevant for the development of BA
Compound heterozygous mutations in NEK8 in siblings with end-stage renal disease with hepatic and cardiac anomalies
No full textWe studied two brothers who presented in the newborn period with cardiac, renal, and hepatic anomalies that were initially suggestive of ALGS, although no mutations in JAG1 or NOTCH2 were identified. Exome sequencing demonstrated compound heterozygous mutations intheNEK8gene (Never in mitosis A-related Kinase 8), a ciliary kinase indispensable for cardiac and renal development based on murine studies. The mutations included a c.2069_2070insC variant (p.Ter693LeufsTer86), and a c.1043C>T variant (p.Thr348Met) in the highly conserved RCC1 (Regulation of Chromosome Condensation 1) domain. The RCC1 domain is crucial for localization of the NEK8 protein to the centrosomes and cilia. Mutations in NEK8 have been previously reported in three fetuses (from a single family) with renal-hepatic-pancreatic dysplasia 2 (RHPD2), similar to Ivemark syndrome, and in three individuals with nephronophthisis (NPHP9). This is the third report of disease-causing mutations in the NEK8 gene in humans and only the second describing multi-organ involvement. The clinical features we describe differ from those in the previously published report in that (1) a pancreatic phenotype was not observed in the individuals reported here, (2) there were more prominent cardiac findings, (consistent with observations in murine models), and (3) we observed bile duct hypoplasia rather than ductal plate malformation.The patients reported here expand our understanding of the NEK8-associated phenotype. Our findings highlight the variable phenotypic expressivity and the spectrum of clinical manifestations due to mutations in the NEK8 gen
Identification of Novel Exon and Transcript of EHMTI Improves Kleefstra Syndrome Diagnosis and Highlights Limitations of Our Current Knowledge of the Human Genome
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