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Rochester, MN, United States

Larson D.R.,Mayo Medical School | Maas M.L.,Mayo Medical School | Jen J.,Advanced Genomics Technology Center
Journal of Orthopaedic Research | Year: 2012

This study investigated the comparative ability of bone marrow and skeletal muscle derived stromal cells (BMSCs and SMSCs) to express a tenocyte phenotype, and whether this expression could be augmented by growth and differentiation factor-5 (GDF-5). Tissue harvest was performed on the hind limbs of seven dogs. Stromal cells were isolated via serial expansion in culture. After four passages, tenogenesis was induced using either ascorbic acid alone or in conjunction with GDF-5. CD44, tenomodulin, collagen I, and collagen III expression levels were compared for each culture condition at 7 and 14 days following induction. Immunohistochemistry (IHC) was performed to evaluate cell morphology and production of tenomodulin and collagen I. SMSCs and BMSCs were successfully isolated in culture. Following tenocytic induction, SMSCs demonstrated an increased mean relative expression of tenomodulin, collagen I, and collagen III at 14 days. BMSCs only showed increased mean relative expression of collagen I, and collagen III at 14 days. IHC revealed positive staining for tenomodulin and collagen I at 14 days for both cell types. The morphology of skeletal muscle derived stromal cells at 14 days had an organized appearance in contrast to the haphazard arrangement of the bone marrow derived cells. GDF-5 did not affect gene expression, cell staining, or cell morphology significantly. Stromal cells from either bone marrow or skeletal muscle can be induced to increase expression of matrix genes; however, based on expression of tenomodulin and cell culture morphology SMSCs may be a more ideal candidate for tenocytic differentiation. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1710-1718, 2012. Source


Hopp K.,Mayo Medical School | Heyer C.M.,Mayo Medical School | Hommerding C.J.,Mayo Medical School | Henke S.A.,Advanced Genomics Technology Center | And 9 more authors.
Human Molecular Genetics | Year: 2011

Meckel syndrome (MKS) is an embryonic lethal, autosomal recessive disorder characterized by polycystic kidney disease, central nervous system defects, polydactyly and liver fibrosis. This disorder is thought to be associated with defects in primary cilia; therefore, it is classed as a ciliopathy. To date, six genes have been commonly associated with MKS (MKS1, TMEM67, TMEM216, CEP290, CC2D2A and RPGRIP1L). However, mutation screening of these genes revealed two mutated alleles in only just over half of our MKS cohort (46 families), suggesting an even greater level of genetic heterogeneity. To explore the full genetic complexity of MKS, we performed exon-enriched next-generation sequencing of 31 ciliopathy genes in 12 MKS pedigrees using RainDance microdroplet-PCR enrichment and IlluminaGAIIx next-generation sequencing. In family M456, we detected a splice-donor site change in a novel MKS gene, B9D1. The B9D1 protein is structurally similar to MKS1 and has been shown to be of importance for ciliogenesis in Caenorhabditis elegans. Reverse transcriptase-PCR analysis of fetal RNA revealed, hemizygously, a single smaller mRNA product with a frameshifting exclusion of B9D1 exon 4. ArrayCGH showed that the second mutation was a 1.713 Mb de novo deletion completely deleting the B9D1 allele. Immunofluorescence analysis highlighted a significantly lower level of ciliated patient cells compared to controls, confirming a role for B9D1 in ciliogenesis. The fetus inherited an additional likely pathogenic novel missense change to a second MKS gene, CEP290; p.R2210C, suggesting oligogenic inheritance in this disorder. © The Author 2011. Published by Oxford University Press. All rights reserved. Source


Vasmatzis G.,Center for Individualized Medicine | Johnson S.H.,Center for Individualized Medicine | Knudson R.A.,Mayo Medical School | Ketterling R.P.,Mayo Medical School | And 20 more authors.
Blood | Year: 2012

Peripheral T-cell lymphomas (PTCLs) are aggressive malignancies of mature T lymphocytes with 5-year overall survival rates of only ∼ 35%. Improvement in outcomes has been stymied by poor understanding of the genetics and molecular pathogenesis of PTCL, with a resulting paucity of molecular targets for therapy. We developed bioinformatic tools to identify chromosomal rearrangements using genome-wide, next-generation sequencing analysis of mate-pair DNA libraries and applied these tools to 16 PTCL patient tissue samples and 6 PTCL cell lines. Thirteen recurrent abnormalities were identified, of which 5 involved p53-related genes (TP53, TP63, CDKN2A, WWOX, and ANKRD11). Among these abnormalities were novel TP63 rearrangements encoding fusion proteins homologous to ΔNp63, a dominant-negative p63 isoform that inhibits the p53 pathway. TP63 rearrangements were seen in 11 (5.8%) of 190 PTCLs and were associated with inferior overall survival; they also were detected in 2 (1.2%) of 164 diffuse large B-cell lymphomas. As TP53 mutations are rare in PTCL compared with other malignancies, our findings suggest that a constellation of alternate genetic abnormalities may contribute to disruption of p53-associated tumor suppressor function in PTCL. © 2012 by The American Society of Hematology. Source


Shekunov J.,Rochester College | De Groen P.C.,Mayo Medical School | Lindor N.M.,Mayo Medical School | Klee G.G.,Foundation Medicine | And 3 more authors.
Journal of AAPOS | Year: 2011

Purpose: Hereditary hyperferritinemia cataract syndrome (HHCS), an autosomal-dominant disorder characterized by hyperferritinemia and bilateral cataracts, is caused by mutations in the iron-responsive element of the ferritin light chain (FTL) gene. The purpose of this study is to describe the genotypic and phenotypic manifestations of HHCS observed in 2 large sets of unrelated American families. Methods: Forty-five patients were recruited from 2 unrelated families. Each underwent ophthalmological and general physical evaluation as well as laboratory testing of serum ferritin, iron, transferrin saturation, and total iron binding capacity. Serum DNA was evaluated for mutations by DNA amplification and sequencing of the FTL gene. Results: Numerous cortical and nuclear white opacities in a stellate pattern occurred in 22 affected individuals and were the only clinical manifestation of HHCS. Of the 22, 16 (73%) demonstrated >1.00 D of astigmatism. Genetic analysis revealed mutation G32A in Pedigree 1 and mutation G32T in Pedigree 2, both heterozygous and located in the iron-responsive element of the ferritin light chain mRNA. Serum ferritin levels of affected subjects ranged from 555 to 2,453 μg/L (normal range, 24-336 μg/L male, 11-307 μg/L female), with greater ferritin levels and more severe cataracts associated with mutation G32A. Conclusions: Most clinical and genetic findings from these families are consistent with previous reports of HHCS. Astigmatism, previously not associated with HHCS, was present in the majority. Ferritin levels and age of cataract surgery varied among subjects with both FTL gene mutations, suggesting that phenotypic variability is modulated by other genetic or environmental factors. Copyright © 2011 Published by Elsevier Inc. on behalf of American Association for Pediatric Ophthalmology and Strabismus. Source


Rossetti S.,Mayo Medical School | Hopp K.,Mayo Medical School | Sikkink R.A.,Advanced Genomics Technology Center | Sundsbak J.L.,Mayo Medical School | And 7 more authors.
Journal of the American Society of Nephrology | Year: 2012

Mutations in two large multi-exon genes, PKD1 and PKD2, cause autosomal dominant polycystic kidney disease (ADPKD). The duplication of PKD1 exons 1-32 as six pseudogenes on chromosome 16, the high level of allelic heterogeneity, and the cost of Sanger sequencing complicate mutation analysis, which can aid diagnostics of ADPKD. We developed and validated a strategy to analyze both the PKD1 and PKD2 genes using next-generation sequencing by pooling long-range PCR amplicons and multiplexing barcoded libraries. We used this approach to characterize a cohort of 230 patients with ADPKD. This process detected definitely and likely pathogenic variants in 115 (63%) of 183 patients with typical ADPKD. In addition, we identified atypical mutations, a gene conversion, and one missed mutation resulting from allele dropout, andwe characterized the pattern of deep intronic variation for both genes. In summary, this strategy involving next-generation sequencing is a model for future genetic characterization of large ADPKD populations. Copyright © 2012 by the American Society of Nephrology. Source

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