Publications by authors named "Luca Brunelli"

25 Publications

  • Page 1 of 1

Healthcare and human rights: a reflection.

Pediatr Res 2021 Mar 31. Epub 2021 Mar 31.

Division of Endocrinology, Boston Children's Hospital, Boston, MA, USA.

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http://dx.doi.org/10.1038/s41390-021-01486-7DOI Listing
March 2021

Congenital diaphragmatic hernia and maternal dietary nutrient pathways and diet quality.

Birth Defects Res 2020 11 3;112(18):1475-1483. Epub 2020 Aug 3.

Division of Medical Genetics, Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, Utah, USA.

Introduction: We examined the association of congenital diaphragmatic hernia (CDH) with maternal dietary intake, using semi-Bayes hierarchical models and principal components analysis to consider intake of nutrients that contribute to one-carbon metabolism and oxidative stress pathways, and a diet quality index.

Methods: We included data on 825 cases and 11,108 nonmalformed controls born from 1997-2011 whose mother participated in the National Birth Defects Prevention Study (NBDPS), a multisite, population-based case-control study. Exposure data were from maternal telephone interviews, which included a food frequency questionnaire. Adjusted odds ratios (aOR) and 95% confidence intervals (CI) were generated from logistic regression models that included nutritional factors as continuous variables and were adjusted for maternal energy intake, race-ethnicity, parity, and vitamin supplement intake.

Results: In the semi-Bayes hierarchical model that included all nutrients and confounders, riboflavin was the only nutrient for which the 95% CI excluded 1.0; the aOR for a 1 SD increase was 0.83. The aORs were 0.79 (95% CI 0.69-0.91) for the one-carbon metabolism pathway score, 0.90 (95% CI 0.80-1.01) for oxidative stress, and 0.85 (95% CI 0.77-0.93) for diet quality (the aORs correspond to a 1 SD increase).

Conclusions: The findings from this study provide some support for the hypothesis that better prepregnancy nutrition is associated with reduced risk for CDH. These results provide etiologic clues but should be interpreted with caution given the novelty of the investigation.
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http://dx.doi.org/10.1002/bdr2.1770DOI Listing
November 2020

DNA gap repair in Escherichia coli for multiplex site-directed mutagenesis.

FASEB J 2020 05 13;34(5):6351-6368. Epub 2020 Mar 13.

Department of Pediatrics, University of Nebraska Medical Center, Omaha, NE, USA.

Site-directed mutagenesis allows the generation of novel DNA sequences that can be used for a variety of important applications such as the functional analysis of genetic variants. To overcome the limitations of existing site-directed mutagenesis approaches, we explored in vivo DNA gap repair. We found that site-specific mutations in plasmid DNA can be generated in Escherichia coli using mutant single-stranded oligonucleotides to target PCR-derived linear double-stranded plasmid DNA. We called this method DeGeRing, and we characterized its advantages, including non-biased multiplex mutagenesis, over existing site-directed mutagenesis methods such as recombineering (recombination-mediated genetic engineering), single DNA break repair (SDBR, introduced by W. Mandecki), and QuikChange (Agilent Technologies, La Jolla, CA). We determined the efficiency of DeGeRing to induce site-directed mutations with and without a phenotype in three K-12 E coli strains using multiple single-stranded oligonucleotides containing homological and heterological parts of various sizes. Virtual lack of background made the isolation of mutants with frequencies up to 10 unnecessary. Our data show that endogenous DNA gap repair in E coli supports efficient multiplex site-directed mutagenesis. DeGeRing might facilitate the generation of mutant DNA sequences for protein engineering and the functional analysis of genetic variants in reverse genetics.
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http://dx.doi.org/10.1096/fj.201902260RDOI Listing
May 2020

Live-cell PCR and one-step purification streamline DNA engineering.

FASEB J 2020 03 15;34(3):3448-3460. Epub 2020 Jan 15.

Department of Pediatrics, University of Nebraska Medical Center, Omaha, NE, USA.

In vivo DNA engineering such as recombineering (recombination-mediated genetic engineering) and DNA gap repair typically involve growing Escherichia coli (E coli) containing plasmids, followed by plasmid DNA extraction and purification prior to downstream PCR-mediated DNA modifications and DNA sequencing. We previously demonstrated that crude cell lysates could be used for some limited downstream DNA applications. Here, we show how live E coli cell PCR and one-step LiCl-isopropanol purification can streamline DNA engineering. In DNA gap repair, live-cell PCR allowed the convenient elimination of clones containing background plasmids prior to DNA sequencing. Live-cell PCR also enabled the generation of specific DNA sequences for DNA engineering up to 11 kilo base pairs in length and with up to 80 base pair terminal non-homology. Using gel electrophoresis and DNA melting curve analysis, we showed that LiCl-isopropanol DNA precipitation removed primers and small, nonspecific PCR products from live-cell PCR products in only ~10-minutes. DNA sequencing of purified products yielded Phred quality scores values of ~55%. These data indicate that live-cell PCR and LiCl-isopropanol DNA precipitation are ideal to prepare DNA for sequencing and other downstream DNA applications, and might therefore accelerate high-throughput DNA engineering pipelines.
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http://dx.doi.org/10.1096/fj.201902261RDOI Listing
March 2020

Targeted gene panel sequencing for the rapid diagnosis of acutely ill infants.

Mol Genet Genomic Med 2019 07 13;7(7):e00796. Epub 2019 Jun 13.

University of Utah School of Medicine, Salt Lake City, Utah.

Background: Exome/genome sequencing (ES/GS) have been recently used in neonatal and pediatric/cardiac intensive care units (NICU and PICU/CICU) to diagnose and care for acutely ill infants, but the effectiveness of targeted gene panels for these purposes remains unknown.

Methods: RapSeq, a newly developed panel targeting 4,503 disease-causing genes, was employed on selected patients in our NICU/PICU/CICU. Twenty trios were sequenced from October 2015 to March 2017. We assessed diagnostic yield, turnaround times, and clinical consequences.

Results: A diagnosis was made in 10/20 neonates (50%); eight had de novo variants (ASXL1, CHD, FBN1, KMT2D, FANCB, FLNA, PAX3), one was a compound heterozygote for CHAT, and one had a maternally inherited GNAS variant. Preliminary reports were generated by 9.6 days (mean); final reports after Sanger sequencing at 16.3 days (mean). In all positive infants, the diagnosis changed management. In a case with congenital myasthenia, diagnosis and treatment occurred at 17 days versus 7 months in a historical control.

Conclusions: This study shows that a gene panel that includes the majority of known disease-causing genes can rapidly identify a diagnosis in a large number of tested infants. Due to simpler deployment and interpretation and lower costs, this approach might represent an alternative to ES/GS in the NICU/PICU/CICU.
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http://dx.doi.org/10.1002/mgg3.796DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6625092PMC
July 2019

Many newborns in level IV NICUs are eligible for rapid DNA sequencing.

Am J Med Genet A 2019 02 19;179(2):280-284. Epub 2018 Dec 19.

Children's Hospital & Medical Center and Department of Pediatrics, University of Nebraska Medical Center, Omaha, Nebraska.

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http://dx.doi.org/10.1002/ajmg.a.61011DOI Listing
February 2019

Germline but not somatic de novo mutations are common in human congenital diaphragmatic hernia.

Birth Defects Res 2018 04 23;110(7):610-617. Epub 2018 Mar 23.

Department of Pediatrics (Neonatology), University of Utah School of Medicine, Salt Lake City, Utah.

Objectives: Congenital diaphragmatic hernia (CDH) is a developmental defect of the diaphragm that causes high newborn morbidity and mortality. CDH is considered to be a multifactorial disease, with strong evidence implicating genetic factors. Although recent studies suggest the biological role of deleterious germline de novo variants, the effect of gene variants specific to the diaphragm remains unclear, and few single genes have been definitively implicated in human disease.

Methods: We performed genome sequencing on 16 individuals with CDH and their unaffected parents, including 10 diaphragmatic samples.

Results: We did not detect damaging somatic mutations in diaphragms, but identified germline heterozygous de novo functional mutations of 14 genes in nine patients. Although the majority of these genes are not known to be associated with CDH, one patient with CDH and cardiac anomalies harbored a frameshift mutation in NR2F2 (aka COUP-TFII), generating a premature truncation of the protein. This patient also carried a missense variant predicted to be damaging in XIRP2 (aka Myomaxin), a transcriptional target of MEF2A. Both NR2F2 and MEF2A map to chromosome 15q26, where recurring de novo deletions and unbalanced translocations have been observed in CDH.

Conclusions: Somatic variants are not common in CDH. To our knowledge, this is the second case of a germline de novo frameshift mutation in NR2F2 in CDH. Since NR2F2 null mice exhibit a diaphragmatic defect, and XIRP2 is implicated in cardiac development, our data suggest the role of these two variants in the etiology of CDH, and possibly cardiac anomalies.
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http://dx.doi.org/10.1002/bdr2.1223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5903934PMC
April 2018

Epidemiology and Prognosis of Congenital Diaphragmatic Hernia: A Population-Based Cohort Study in Utah.

Birth Defects Res 2017 Nov 19;109(18):1451-1459. Epub 2017 Sep 19.

Division of Medical Genetics, Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, Utah.

Background: Congenital diaphragmatic hernia (CDH) is a relatively frequent and severe malformation. Population-based data on clinical presentation and associated mortality are scarce. We examined a state-wide cohort of infants with a clinically validated diagnosis of CDH to assess their clinical profile, sociodemographic patterns, and infant mortality.

Methods: We identified CDH cases from Utah's statewide population-based surveillance program among the cohort of all pregnancy outcomes (live births, stillbirths, and pregnancy terminations) delivered from 1999 to 2011. Clinical geneticists reviewed all cases and classified them based on etiology (known, unknown), and whether they were isolated, multiple (additional unrelated major malformations or unique minor malformation), or syndromic (genetic, chromosomal).

Results: CDH occurred in 1 in 3156 births (227/718,990, or 3.17 per 10,000), with no time trend during the 13 years (p = 0.85). CDH was much more common in males (male to female ratio, 1.72:1; p < 0.01). Clinically, 64% of the cases were isolated, 23% were multiples, and 13% were syndromic. Most cases were live born (90%), with fewer stillbirths (7%) and pregnancy terminations (3%). Overall infant mortality was 32.5%, and varied considerably by underlying etiology (isolated 21%; multiple 44%; syndromic 82%). Prognosis was related to specific clinical findings within each etiologic group (e.g., prematurity, low Apgar score, and intrathoracic liver).

Conclusion: This information on specific clinical and etiologic factors associated with prognosis can help clinicians and parents in the complex discussions about care planning and management that often occur in a crisis situation, following the diagnosis of CDH, whether prior or after delivery. Birth Defects Research 109:1451-1459, 2017.© 2017 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/bdr2.1106DOI Listing
November 2017

Early results for treatment of two- and three-part fractures of the proximal humerus using Contours PHP (proximal humeral plate).

Acta Biomed 2017 04 28;88(1):65-73. Epub 2017 Apr 28.

Gaetano Pini-CTO, Milano.

Background And Aim Of The Work: The management of displaced 2- and 3-part fractures of the proximal humerus is controversial, both in younger and in elderly patients. The purpose of this paper is to evaluate the functional results of the Contours Proximal Humerus Plate (OrthofixR, Bussolengo,Verona, Italy), for the treatment of displaced 2- and 3-part fractures of the proximal humerus.

Methods: We retrospectively reviewed 55 patients with proximal humerus fractures, who underwent osteosynthesis with Contours Proximal Humerus Plate from December 2011 to March 2015. We had 21 patients with 2-part fractures and with an average age of 67.1 years and 34 patients with 3-part fractures, with average age of 63.6 years.

Results: The average union time was 3 months. The mean Constant score was 67 for 2-part fracture group and 64.9 for 3-part fracture group. The difference was not statistically significant (p = 0.18). The overall complication rate was 14.5 %. Six patients underwent additional surgery (10.9%).

Conclusions: The most frequent major complication was secondary loss of reduction following varus collapse of the fracture (2 cases). In these patients, there was loss of medial hinge integrity due to impaction and osteoporosis. The placement of the main locking screw in the calcar area to provide inferomedial support is the rational of the Contours Proximal Humerus Plate. Osteosynthesis with Contours Proximal Humerus Plate is a safe system for treating displaced 2- and 3-part fractures of the proximal humerus, with good functional results and complication rates comparable to those reported in the literature.
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http://dx.doi.org/10.23750/abm.v88i1.5193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166187PMC
April 2017

Direct Isolation of Seamless Mutant Bacterial Artificial Chromosomes.

Curr Protoc Mol Biol 2017 04 3;118:8.6.1-8.6.29. Epub 2017 Apr 3.

Eccles Institute of Human Genetics, University of Utah School of Medicine, Salt Lake City, Utah.

Seamless (i.e., without unwanted DNA sequences) mutant bacterial artificial chromosomes (BACs) generated via recombination-mediated genetic engineering (recombineering) are better suited to study gene function compared to complementary DNA (cDNA) because they contain only the specific mutation and provide all the regulatory sequences required for in vivo gene expression. However, precisely mutated BACs are typically rare (∼1:1,000 to 1:100,000), making their isolation quite challenging. Although these BACs have been classically isolated by linking the mutation to additional genes, i.e., selectable markers, this approach is prone to false positives and is labor-intensive because it requires the subsequent removal of the selectable marker. We created Founder Principle-driven Enrichment (FPE), a method based on the population genetics "founder principle," to directly isolate rare mutant BACs, without any selectable marker, from liquid cultures via the polymerase chain reaction (PCR). Here, we provide a detailed description of FPE, including protocols for BAC recombineering and PCR screening. © 2017 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpmb.34DOI Listing
April 2017

14-3-3epsilon controls multiple developmental processes in the mouse heart.

Dev Dyn 2016 11 18;245(11):1107-1123. Epub 2016 Sep 18.

Department of Pediatrics (Neonatology), University of Utah School of Medicine, Salt Lake City, Utah.

Background: 14-3-3ε plays an important role in the maturation of the compact ventricular myocardium by modulating the cardiomyocyte cell cycle via p27 . However, additional cardiac defects are possible given the ubiquitous expression pattern of this protein.

Results: Germ line deletion of 14-3-3ε led to malalignment of both the outflow tract (OFT) and atrioventricular (AV) cushions, with resulting tricuspid stenosis and atresia, mitral valve abnormalities, and perimembranous ventricular septal defects (VSDs). We confirmed myocardial non-compaction and detected a spongy septum with muscular VSDs and blebbing of the epicardium. These defects were associated with abnormal patterning of p27 expression in the subendocardial and possibly the epicardial cell populations. In addition to abnormal pharyngeal arch artery patterning, we found deep endocardial recesses and paucity of intramyocardial coronary vasculature as a result of defective coronary plexus remodeling.

Conclusions: The malalignment of both endocardial cushions provides a new explanation for tricuspid and mitral valve defects, while myocardial non-compaction provides the basis for the abnormal coronary vasculature patterning. These abnormalities might arise from p27 dysregulation and a resulting defect in epithelial-to-mesenchymal transformation. These data suggest that 14-3-3ε, in addition to left ventricular non-compaction (LVNC), might be linked to different forms of congenital heart disease (CHD). Developmental Dynamics 245:1107-1123, 2016. © 2016 Wiley Periodicals, Inc.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5065397PMC
http://dx.doi.org/10.1002/dvdy.24440DOI Listing
November 2016

Micro-Computed Tomography for the Quantitative 3-Dimensional Assessment of the Compact Myocardium in the Mouse Embryo.

Circ J 2016 Jul 15;80(8):1795-803. Epub 2016 Jun 15.

Small Animal Imaging Facility, Department of Bioengineering, University of Utah.

Background: Ventricular non-compaction is characterized by a thin layer of compact ventricular myocardium and it is an important abnormality in the mouse heart. It is reminiscent of left ventricular non-compaction, a fairly common human congenital cardiomyopathy. Non-compaction in transgenic mice has been classically evaluated by measuring the thickness of the compact myocardium through histological techniques involving image analysis of 2-dimensional (D) sections. Given the 3D nature of the heart, the aim of this study was to determine whether a technique for the non-destructive, 3D assessment of the mouse embryonic compact myocardium could be developed.

Methods and results: Micro-computed tomography (micro-CT), in combination with iodine staining, enabled the differentiation of the trabecular from the compact myocardium in wild-type mice. The 3D and digital nature of the micro-CT data allowed computation anatomical techniques to be readily applied, which were demonstrated via construction of group atlases and atlas-based descriptive statistics. Finally, micro-CT was used to identify the presence of non-compaction in mice with a deletion of the cell cycle inhibitor protein, p27(Kip1).

Conclusions: Iodine staining-enhanced micro-CT with computational anatomical analysis represents a valid addition to classical histology for the delineation of compact myocardial wall thickness in the mouse embryo. Given the quantitative 3D resolution of micro-CT, these approaches might provide helpful information for the analysis of non-compaction. (Circ J 2016; 80: 1795-1803).
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http://dx.doi.org/10.1253/circj.CJ-16-0180DOI Listing
July 2016

Isolation of rare recombinants without using selectable markers for one-step seamless BAC mutagenesis.

Nat Methods 2014 Sep 13;11(9):966-970. Epub 2014 Jul 13.

Department of Pediatrics (Neonatology), The University of Utah School of Medicine, Salt Lake City, Utah, USA.

Current methods to isolate rare (1:10,000-1:100,000) bacterial artificial chromosome (BAC) recombinants require selectable markers. For seamless BAC mutagenesis, selectable markers need to be removed after isolation of recombinants through counterselection. Here we illustrate founder principle-driven enrichment (FPE), a simple method to rapidly isolate rare recombinants without using selectable markers, allowing one-step seamless BAC mutagenesis. As proof of principle, we isolated 1:100,000 seamless fluorescent protein-modified Nodal BACs and confirmed BAC functionality by generating fluorescent reporter mice. We also isolated small indel P1 phage-derived artificial chromosome (PAC) and BAC recombinants. Statistical analysis revealed that 1:100,000 recombinants can be isolated with <40 PCRs, and we developed a web-based calculator to optimize FPE.
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http://dx.doi.org/10.1038/nmeth.3030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4149595PMC
September 2014

Family-based studies to identify genetic variants that cause congenital heart defects.

Future Cardiol 2013 Jul;9(4):507-18

Department of Pediatrics (Cardiology) University of Utah School of Medicine, Eccles Institute of Human Genetics, 15 North 2030 East, Room 7110B, Salt Lake City, UT 84112, USA.

Congenital heart defects (CHDs) are the most common congenital abnormalities. Analysis of large multigenerational families has led to the identification of a number of genes for CHDs. However, identifiable variations in these genes are the cause of a small proportion of cases of CHDs, suggesting significant genetic heterogeneity. In addition, large families with CHDs are rare, making the identification of additional genes difficult. Next-generation sequencing technologies will provide an opportunity to identify more genes in the future. However, the significant genetic variation between individuals will present a challenge to distinguish between 'pathogenic' and 'benign' variants. We have demonstrated that the analysis of multiple individuals in small families using combinations of algorithms can reduce the number of candidate variants to a small, manageable number. Thus, the analysis of small nuclear families or even distantly related 'sporadic' cases may begin to uncover the 'dark matter' of CHD genetics.
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http://dx.doi.org/10.2217/fca.13.40DOI Listing
July 2013

14-3-3ε gene variants in a Japanese patient with left ventricular noncompaction and hypoplasia of the corpus callosum.

Gene 2013 Feb 20;515(1):173-80. Epub 2012 Dec 20.

Department of Pediatrics, University of Toyama, Toyama, Japan.

Background: Left ventricular noncompaction (LVNC) is a cardiomyopathy characterized by a prominent trabecular meshwork and deep intertrabecular recesses, and is thought to be due to an arrest of normal endomyocardial morphogenesis. However, the genes contributing to this process remain poorly understood. 14-3-3ε, encoded by YWHAE, is an adapter protein belonging to the 14-3-3 protein family which plays important roles in neuronal development and is involved in Miller-Dieker syndrome. We recently showed that mice lacking this gene develop LVNC. Therefore, we hypothesized that variants in YWHAE may contribute to the pathophysiology of LVNC in humans.

Methods And Results: In 77 Japanese patients with LVNC, including the probands of 29 families, mutation analysis of YWHAE by direct DNA sequencing identified 7 novel variants. One of them, c.-458G>T, in the YWHAE promoter, was identified in a familial patient with LVNC and hypoplasia of the corpus callosum. The -458G>T variant is located within a regulatory CCAAT/enhancer binding protein (C/EBP) response element of the YWHAE promoter, and it reduced promoter activity by approximately 50%. Increased binding of an inhibitory C/EBPβ isoform was implicated in decreasing YWHAE promoter activity. Interestingly, we had previously shown that C/EBPβ is a key regulator of YWHAE.

Conclusions: These data suggest that the -458G>T YWHAE variant contributes to the abnormal myocardial morphogenesis characteristic of LVNC as well as abnormal brain development, and implicate YWHAE as a novel candidate gene in pediatric cardiomyopathies.
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http://dx.doi.org/10.1016/j.gene.2012.12.049DOI Listing
February 2013

A family-based paradigm to identify candidate chromosomal regions for isolated congenital diaphragmatic hernia.

Am J Med Genet A 2012 Dec 19;158A(12):3137-47. Epub 2012 Nov 19.

Department of Pediatrics (Cardiology), University of Utah School of Medicine, Salt Lake City, Utah 84112, USA.

Congenital diaphragmatic hernia (CDH) is a developmental defect of the diaphragm that causes high newborn mortality. Isolated or non-syndromic CDH is considered a multifactorial disease, with strong evidence implicating genetic factors. As low heritability has been reported in isolated CDH, family-based genetic methods have yet to identify the genetic factors associated with the defect. Using the Utah Population Database, we identified distantly related patients from several extended families with a high incidence of isolated CDH. Using high-density genotyping, seven patients were analyzed by homozygosity exclusion rare allele mapping (HERAM) and phased haplotype sharing (HapShare), two methods we developed to map shared chromosome regions. Our patient cohort shared three regions not previously associated with CDH, that is, 2q11.2-q12.1, 4p13 and 7q11.2, and two regions previously involved in CDH, that is, 8p23.1 and 15q26.2. The latter regions contain GATA4 and NR2F2, two genes implicated in diaphragm formation in mice. Interestingly, three patients shared the 8p23.1 locus and one of them also harbored the 15q26.2 segment. No coding variants were identified in GATA4 or NR2F2, but a rare shared variant was found in intron 1 of GATA4. This work shows the role of heritability in isolated CDH. Our family-based strategy uncovers new chromosomal regions possibly associated with disease, and suggests that non-coding variants of GATA4 and NR2F2 may contribute to the development of isolated CDH. This approach could speed up the discovery of the genes and regulatory elements causing multifactorial diseases, such as isolated CDH.
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http://dx.doi.org/10.1002/ajmg.a.35664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3507422PMC
December 2012

14-3-3ε plays a role in cardiac ventricular compaction by regulating the cardiomyocyte cell cycle.

Mol Cell Biol 2012 Dec 15;32(24):5089-102. Epub 2012 Oct 15.

Departments of Pediatrics, The University of Utah School of Medicine, Salt Lake City, Utah, USA.

Trabecular myocardium accounts for the majority of the ventricles during early cardiogenesis, but compact myocardium is the primary component at later developmental stages. Elucidation of the genes regulating compact myocardium development is essential to increase our understanding of left ventricular noncompaction (LVNC), a cardiomyopathy characterized by increased ratios of trabecular to compact myocardium. 14-3-3ε is an adapter protein expressed in the lateral plate mesoderm, but its in vivo cardiac functions remain to be defined. Here we show that 14-3-3ε is expressed in the developing mouse heart as well as in cardiomyocytes. 14-3-3ε deletion did not appear to induce compensation by other 14-3-3 isoforms but led to ventricular noncompaction, with features similar to LVNC, resulting from a selective reduction in compact myocardium thickness. Abnormal compaction derived from a 50% decrease in cardiac proliferation as a result of a reduced number of cardiomyocytes in G(2)/M and the accumulation of cardiomyocytes in the G(0)/G(1) phase of the cell cycle. These defects originated from downregulation of cyclin E1 and upregulation of p27(Kip1), possibly through both transcriptional and posttranslational mechanisms. Our work shows that 14-3-3ε regulates cardiogenesis and growth of the compact ventricular myocardium by modulating the cardiomyocyte cell cycle via both cyclin E1 and p27(Kip1). These data are consistent with the long-held view that human LVNC may result from compaction arrest, and they implicate 14-3-3ε as a new candidate gene in congenital human cardiomyopathies.
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http://dx.doi.org/10.1128/MCB.00829-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3510533PMC
December 2012

Loss of function of hNav1.5 by a ZASP1 mutation associated with intraventricular conduction disturbances in left ventricular noncompaction.

Circ Arrhythm Electrophysiol 2012 Oct 28;5(5):1017-26. Epub 2012 Aug 28.

Electrophysiology Research Laboratory, Texas Heart Institute/St. Luke's Episcopal Hospital, Houston, TX, USA.

Background: Defects of cytoarchitectural proteins can cause left ventricular noncompaction, which is often associated with conduction system diseases. We have previously identified a p.D117N mutation in the LIM domain-binding protein 3-encoding Z-band alternatively spliced PDZ motif gene (ZASP) in a patient with left ventricular noncompaction and conduction disturbances. We sought to investigate the role of p.D117N mutation in the LBD3 NM_001080114.1 isoform (ZASP1-D117N) for the regulation of cardiac sodium channel (Na(v)1.5) that plays an important role in the cardiac conduction system.

Methods And Results: Effects of ZASP1-wild-type and ZASP1-D117N on Na(v)1.5 were studied in human embryonic kidney-293 cells and neonatal rat cardiomyocytes. Patch-clamp study demonstrated that ZASP1-D117N significantly attenuated I(Na) by 27% in human embryonic kidney-293 cells and by 32% in neonatal rat cardiomyocytes. In addition, ZASP1-D117N rightward shifted the voltage-dependent activation and inactivation in both systems. In silico simulation using Luo-Rudy phase 1 model demonstrated that altered Na(v)1.5 function can reduce cardiac conduction velocity by 28% compared with control. Pull-down assays showed that both wild-type and ZASP1-D117N can complex with Na(v)1.5 and telethonin/T-Cap, which required intact PDZ domains. Immunohistochemical staining in neonatal rat cardiomyocytes demonstrates that ZASP1-D117N did not significantly disturb the Z-line structure. Disruption of cytoskeletal networks with 5-iodonaphthalene-1-sulfonyl homopiperazine and cytochalasin D abolished the effects of ZASP1-D117N on Na(v)1.5.

Conclusions: ZASP1 can form protein complex with telethonin/T-Cap and Na(v)1.5. The left ventricular noncompaction-specific ZASP1 mutation can cause loss of function of Na(v)1.5, without significant alteration of the cytoskeletal protein complex. Our study suggests that electric remodeling can occur in left ventricular noncompaction subject because of a direct effect of mutant ZASP on Na(v)1.5.
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http://dx.doi.org/10.1161/CIRCEP.111.969220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4331025PMC
October 2012

A one-step miniprep for the isolation of plasmid DNA and lambda phage particles.

PLoS One 2011 15;6(8):e23457. Epub 2011 Aug 15.

Division of Neonatology, Department of Pediatrics, The University of Utah School of Medicine, Salt Lake City, Utah, United States of America.

Plasmid DNA minipreps are fundamental techniques in molecular biology. Current plasmid DNA minipreps use alkali and the anionic detergent SDS in a three-solution format. In addition, alkali minipreps usually require additional column-based purification steps and cannot isolate other extra-chromosomal elements, such as bacteriophages. Non-ionic detergents (NIDs) have been used occasionally as components of multiple-solution plasmid DNA minipreps, but a one-step approach has not been developed. Here, we have established a one-tube, one-solution NID plasmid DNA miniprep, and we show that this approach also isolates bacteriophage lambda particles. NID minipreps are more time-efficient than alkali minipreps, and NID plasmid DNA performs better than alkali DNA in many downstream applications. In fact, NID crude lysate DNA is sufficiently pure to be used in digestion and sequencing reactions. Microscopic analysis showed that the NID procedure fragments E. coli cells into small protoplast-like components, which may, at least in part, explain the effectiveness of this approach. This work demonstrates that one-step NID minipreps are a robust method to generate high quality plasmid DNA, and NID approaches can also isolate bacteriophage lambda particles, outperforming current standard alkali-based minipreps.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0023457PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3156146PMC
February 2012

Hofmeister series salts enhance purification of plasmid DNA by non-ionic detergents.

Biotechnol Bioeng 2011 Aug 17;108(8):1872-82. Epub 2011 Mar 17.

Laboratory of Protein Dynamics and Signaling, National Cancer Institute, National Institutes of Health, NCI-Frederick, Frederick, Maryland 21702, USA.

Ion-exchange chromatography is the standard technique used for plasmid DNA purification, an essential molecular biology procedure. Non-ionic detergents (NIDs) have been used for plasmid DNA purification, but it is unclear whether Hofmeister series salts (HSS) change the solubility and phase separation properties of specific NIDs, enhancing plasmid DNA purification. After scaling-up NID-mediated plasmid DNA isolation, we established that NIDs in HSS solutions minimize plasmid DNA contamination with protein. In addition, large-scale NID/HSS solutions eliminated lipopolysaccharides (LPS) contamination of plasmid DNA more effectively than Qiagen ion-exchange columns. Large-scale NID isolation/NID purification generated increased yields of high-quality DNA compared to alkali isolation/column purification. This work characterizes how HSS enhance NID-mediated plasmid DNA purification, and demonstrates that NID phase transition is not necessary for LPS removal from plasmid DNA. Specific NIDs such as IGEPAL CA-520 can be utilized for rapid, inexpensive, and efficient laboratory-based large-scale plasmid DNA purification, outperforming Qiagen-based column procedures.
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http://dx.doi.org/10.1002/bit.23116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3117116PMC
August 2011

A ZASP missense mutation, S196L, leads to cytoskeletal and electrical abnormalities in a mouse model of cardiomyopathy.

Circ Arrhythm Electrophysiol 2010 Dec 18;3(6):646-56. Epub 2010 Sep 18.

Department of Pediatrics, Baylor College of Medicine and Texas Children's Hospital, Houston, Tex, USA.

Background: Dilated cardiomyopathy (DCM) is a primary disease of the heart muscle associated with sudden cardiac death secondary to ventricular tachyarrhythmias and asystole. However, the molecular pathways linking DCM to arrhythmias and sudden cardiac death are unknown. We previously identified a S196L mutation in exon 4 of LBD3-encoded ZASP in a family with DCM and sudden cardiac death. These findings led us to hypothesize that this mutation may precipitate both cytoskeletal and conduction abnormalities in vivo. Therefore, we investigated the role of the ZASP4 mutation S196L in cardiac cytoarchitecture and ion channel biology.

Methods And Results: We generated and analyzed transgenic mice with cardiac-restricted expression of the S196L mutation. We also performed cellular electrophysiological analysis on isolated S196L cardiomyocytes and protein-protein interaction studies. Ten month-old S196L mice developed hemodynamic dysfunction consistent with DCM, whereas 3-month-old S196L mice presented with cardiac conduction defects and atrioventricular block. Electrophysiological analysis on isolated S196L cardiomyocytes demonstrated that the L-type Ca(2+) currents and Na(+) currents were altered. The pull-down assay demonstrated that ZASP4 complexes with both calcium (Ca(v)1.2) and sodium (Na(v)1.5) channels.

Conclusions: Our findings provide new insight into the mechanisms by which mutations of a structural/cytoskeletal protein, such as ZASP, lead to cardiac functional and electric abnormalities. This work represents a novel framework to understand the development of conduction defects and arrhythmias in subjects with cardiomyopathies, including DCM.
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http://dx.doi.org/10.1161/CIRCEP.109.929240DOI Listing
December 2010

Remodeling of dystrophin and sarcomeric Z-band occurs in pediatric cardiomyopathies: a unifying mechanism for force transmission defect.

J Cardiovasc Med (Hagerstown) 2009 Feb;10(2):149-56

Department of Pediatrics (Cardiology), Baylor College of Medicine, Houston, Texas, USA.

Background: Cardiomyopathies (CMPs) lead to associated systolic dysfunction and are the major causes of congestive heart failure and a leading cause for heart transplantation. Although the precise mechanism leading to systolic dysfunction is still elusive, chronic mechanical loading, along with altered calcium (Ca) cellular homeostasis, is believed to impair force transmission and induce cardiac morphological and structural changes, namely cardiac remodeling. Interestingly, dystrophin remodeling has been previously reported to occur in adults with end-stage CMP irrespective of the underlying cause.

Methods: In order to determine the structural culprit associated with pediatric dilated cardiomyopathy (DCM) due to various causes, we investigated the structural continuum connecting dystrophin and the dystrophin-associated glycoprotein complex to the contractile apparatus in heart samples from four children with idiopathic dilated CMP: one with myocarditis, one sporadic DCM child previously identified with a delta-sarcoglycan deletion mutation, and one child with X-linked CMP with a reported splicing site mutation in the dystrophin-coded DYS gene.

Results: Immunohistochemical analysis of cytoskeletal proteins connecting the dystrophin-associated glycoprotein complex to the sarcomere identified that myocarditis, idiopathic, and genetic-based DCM are characterized by disruption of the dystrophin connection to the sarcomere and perturbation of the Z-band.

Conclusion: Our data suggest that both dystrophin remodeling and sarcomeric Z-band/disk derangements may occur in the myocardium of children with DCM irrespective of the cause. This suggests that genetic mutations in the dystrophin-associated glycoprotein complex or any of its partners could result in sarcomere-sarcolemma connection alteration and associated Z-band disturbance, thus leading to force transmission dysfunction.
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http://dx.doi.org/10.2459/JCM.0b013e328318954cDOI Listing
February 2009

Peroxisome proliferator-activated receptor-delta upregulates 14-3-3 epsilon in human endothelial cells via CCAAT/enhancer binding protein-beta.

Circ Res 2007 Mar 15;100(5):e59-71. Epub 2007 Feb 15.

Department of Pediatrics, The University of Texas at Houston Medical School, Houston, TX 77030-1503, USA.

Peroxisome proliferator-activated receptor delta (PPARdelta) agonists are promising new agents for treatment of the metabolic syndrome. Although they possess antiatherosclerotic properties in vivo and promote endothelial cell survival, their mechanism of action is incompletely understood. 14-3-3epsilon is a critical component of the endothelial cell antiapoptotic machinery, which is essential to maintain homeostasis of the vascular wall. To test the hypothesis that PPARdelta targets 14-3-3epsilon in endothelial cells, we studied the response of the gene that encodes 14-3-3epsilon in humans, YWHAE, to PPARdelta ligands (L-165,041 and GW501516). We found that PPARdelta activates YWHAE promoter in a concentration and time-dependent manner. Consistent with these findings, L-165,041 increased 14-3-3epsilon mRNA and protein level, whereas PPARdelta small interfering RNA suppressed both basal and L-165,041-dependent YWHAE transcription and 14-3-3epsilon protein expression. Surprisingly, PPAR response elements in YWHAE promoter were not required for upregulation by PPARdelta, whereas a CCAAT/enhancer binding protein (C/EBP) site located at -160/-151 bp regulated both basal and PPARdelta-dependent promoter activity. Intriguingly, activation or knock down of endogenous PPARdelta regulated C/EBPbeta protein expression. Chromatin immunoprecipitation assays demonstrated that L-165,041 determines the localization of C/EBPbeta to the region spanning this C/EBP response element, whereas sequential chromatin immunoprecipitation analysis revealed that C/EBPbeta and PPARdelta form a transcriptional activating complex on this C/EBP site. Our work uncovers a novel role for C/EBPbeta as a mediator of PPARdelta-dependent 14-3-3epsilon gene regulation in human endothelial cells and provides insight into the mechanism by which PPARdelta agonists may be beneficial in atherosclerosis.
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http://dx.doi.org/10.1161/01.RES.0000260805.99076.22DOI Listing
March 2007

Perfluorochemical liquids enhance delivery of superoxide dismutase to the lungs of juvenile rabbits.

Pediatr Res 2006 Jul 11;60(1):65-70. Epub 2006 May 11.

Division of Neonatology , Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.

Previous studies suggest acute lung injury (ALI) in premature newborns is associated with relative deficiency of antioxidant enzymes that may be ameliorated by recombinant human superoxide dismutase (rhSOD). Perfluorochemicals (PFCs) are distributed homogeneously and support gas exchange in diseased lungs. We investigated whether PFCs could provide an effective delivery system for rhSOD. Juvenile rabbits were lung-lavaged, treated with surfactant, and randomized: group I: fluorescently labeled rhSOD (5 mg/kg in 2 mL/kg saline); group II: fluorescently labeled rhSOD (5 mg/kg in 18 mL/kg PFC). Animals were ventilated with oxygen for 4 h; the lungs were harvested for analysis of SOD distribution and oxidative injury. Cardiopulmonary indices remained stable and similar between groups. Qualitative assessment (QA) showed a more homogeneous lung SOD distribution in group II and a better histologic profile. QA of lung SOD distribution showed significant increase in SOD concentrations in group II (7.37 +/- 1.54 microg/mg protein) compared with group I (1.65 +/- 0.23 microg/mg protein). Oxidative injury as assessed by normalized protein carbonyl was 149.1 +/- 26.8% SEM in group II compared with 200.5 +/- 7.3% SEM in group I. Plasma SOD was significantly higher in group II. Administration of rhSOD with or without PFCs does not compromise cardiovascular function or impede lung recovery after ALI. PFCs enhance rhSOD delivery to the lungs by 400% while decreasing lung oxidative damage by 25% compared with rhSOD alone. These data suggest that PFCs optimize lung rhSOD delivery and might enhance the beneficial effects of rhSOD in preventing acute and chronic lung injury.
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http://dx.doi.org/10.1203/01.pdr.0000219392.73509.70DOI Listing
July 2006