Synchronizing Allelic Effects of Opposing Quantitative Trait Loci Confirmed a Major Epistatic Interaction Affecting Acute Lung Injury Survival in Mice

Increased oxygen (O2) levels help manage severely injured patients, but too much for too long can cause acute lung injury (ALI), acute respiratory distress syndrome (ARDS) and even death. In fact, continuous hyperoxia has become a prototype in rodents to mimic salient clinical and pathological characteristics of ALI/ARDS. To identify genes affecting hyperoxia-induced ALI (HALI), we previously established a mouse model of differential susceptibility. Genetic analysis of backcross and F2 populations derived from sensitive (C57BL/6J; B) and resistant (129X1/SvJ; X1) inbred strains identified five quantitative trait loci (QTLs; Shali1-5) linked to HALI survival time. Interestingly, analysis of these recombinant populations supported opposite within-strain effects on survival for the two major-effect QTLs. Whereas Shali1 alleles imparted the expected survival time effects (i.e., X1 alleles increased HALI resistance and B alleles increased sensitivity), the allelic effects of Shali2 were reversed (i.e., X1 alleles increased HALI sensitivity and B alleles increased resistance). For in vivo validation of these inverse allelic effects, we constructed reciprocal congenic lines to synchronize the sensitivity or resistance alleles of Shali1 and Shali2 within the same strain. Specifically, B-derived Shali1 or Shali2 QTL regions were transferred to X1 mice and X1-derived QTL segments were transferred to B mice. Our previous QTL results predicted that substituting Shali1 B alleles onto the resistant X1 background would add sensitivity. Surprisingly, not only were these mice more sensitive than the resistant X1 strain, they were more sensitive than the sensitive B strain. In stark contrast, substituting the Shali2 interval from the sensitive B strain onto the X1 background markedly increased the survival time. Reciprocal congenic lines confirmed the opposing allelic effects of Shali1 and Shali2 on HALI survival time and provide unique models to identify their respective quantitative trait genes and to critically assess the apparent bidirectional epistatic interactions between these major-effect loci

Stem Cell-Specific Mechanisms Ensure Genomic Fidelity within HSCs and upon Aging of HSCs

SummaryWhether aged hematopoietic stem and progenitor cells (HSPCs) have impaired DNA damage repair is controversial. Using a combination of DNA mutation indicator assays, we observe a 2- to 3-fold increase in the number of DNA mutations in the hematopoietic system upon aging. Young and aged hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) do not show an increase in mutation upon irradiation-induced DNA damage repair, and young and aged HSPCs respond very similarly to DNA damage with respect to cell-cycle checkpoint activation and apoptosis. Both young and aged HSPCs show impaired activation of the DNA-damage-induced G1-S checkpoint. Induction of chronic DNA double-strand breaks by zinc-finger nucleases suggests that HSPCs undergo apoptosis rather than faulty repair. These data reveal a protective mechanism in both the young and aged hematopoietic system against accumulation of mutations in response to DNA damage

A Model for Transgenerational Imprinting Variation in Complex Traits

Despite the fact that genetic imprinting, i.e., differential expression of the same allele due to its different parental origins, plays a pivotal role in controlling complex traits or diseases, the origin, action and transmission mode of imprinted genes have still remained largely unexplored. We present a new strategy for studying these properties of genetic imprinting with a two-stage reciprocal F mating design, initiated with two contrasting inbred lines. This strategy maps quantitative trait loci that are imprinted (i.e., iQTLs) based on their segregation and transmission across different generations. By incorporating the allelic configuration of an iQTL genotype into a mixture model framework, this strategy provides a path to trace the parental origin of alleles from previous generations. The imprinting effects of iQTLs and their interactions with other traditionally defined genetic effects, expressed in different generations, are estimated and tested by implementing the EM algorithm. The strategy was used to map iQTLs responsible for survival time with four reciprocal F populations and test whether and how the detected iQTLs inherit their imprinting effects into the next generation. The new strategy will provide a tool for quantifying the role of imprinting effects in the creation and maintenance of phenotypic diversity and elucidating a comprehensive picture of the genetic architecture of complex traits and diseases

Multi-site investigation of strategies for the clinical implementation of CYP2D6 genotyping to guide drug prescribing

PURPOSE: A number of institutions have clinically implemented CYP2D6 genotyping to guide drug prescribing. We compared implementation strategies of early adopters of CYP2D6 testing, barriers faced by both early adopters and institutions in the process of implementing CYP2D6 testing, and approaches taken to overcome these barriers. METHODS: We surveyed eight early adopters of CYP2D6 genotyping and eight institutions in the process of adoption. Data were collected on testing approaches, return of results procedures, applications of genotype results, challenges faced, and lessons learned. RESULTS: Among early adopters, CYP2D6 testing was most commonly ordered to assist with opioid and antidepressant prescribing. Key differences among programs included test ordering and genotyping approaches, result reporting, and clinical decision support. However, all sites tested for copy-number variation and nine common variants, and reported results in the medical record. Most sites provided automatic consultation and had designated personnel to assist with genotype-informed therapy recommendations. Primary challenges were related to stakeholder support, CYP2D6 gene complexity, phenotype assignment, and sustainability. CONCLUSION: There are specific challenges unique to CYP2D6 testing given the complexity of the gene and its relevance to multiple medications. Consensus lessons learned may guide those interested in pursuing similar clinical pharmacogenetic programs

Schematic summary of B.X1-4 consomic and reciprocal X1.B-4 and B.X1-4 congenics and subcongenics for <i>Shali2</i>.

<p>Data represent chromosome 4 of each strain, with X1 (solid white bar) and B (solid black bar) representing the recipient background strain of X1.B-4 and B.X1-4 lines, respectively. Chromosome 4 on the far left depicts MIT markers and their genomic positions (Mbp), along with the <i>Shali2</i> interval (black box). The current <i>Shali2</i> interval is also indicated by dashed lines. Crossovers not matching dashed lines indicate SNPs from the Mouse Universal Genotyping Analysis (MUGA; GeneSeek) panel. For X1.B-4 lines (left half), regions in black denote substituted genome, with congenic lines X1.B-4A, X1.B-4B and X1.B-4C. X1.B-4BA and X1.B-4BB are subcongenic lines derived from the X1.B-4B line. For B.X1-4 lines (right half), regions in white represent substituted genome. B.X1-4 represents a consomic line (full chromosome substitution). B.X1-4A and B.X1-4C are congenic lines and B.X1-4BA is a subcongenic line of B.X1-4B (died). Grey regions represent areas not yet tested for parental origin. Tables below each set of congenic lines summarize mean survival time (MST) and sample size (n) for males and females of each line. *significantly different than same-sex background strain (<i>p</i><0.00132; MWW U-test with Bonferroni correction for 38 group comparisons). +, represents a historical control and includes combined data for new and previously reported controls <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038177#pone.0038177-Prows1" target="_blank">[24]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038177#pone.0038177-Prows2" target="_blank">[25]</a>.</p

Box-plots comparing survival times for controls and the major <i>Shali1</i> and <i>Shali2</i> congenic lines.

<p>Boxes represent the 25^th to 75^th percentiles, and horizontal lines within the box represent median survival times; short dashed lines within boxes denote mean survival times. Whiskers (error bars) above and below the boxes indicate the 90^th and 10^th percentiles, respectively. The dashed line across the graph denotes the threshold survival time (153 hrs) for designation of the resistant phenotype <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038177#pone.0038177-Prows1" target="_blank">[24]</a>. Both sexes of all lines (B, X1.B-1A, and X1.B-4BB) differed significantly from the X1 progenitor strain. *, within-strain mean survival times of males (M) and females (F) differed significantly (<i>p</i><0.00132).</p

Percent penetrance of sensitivity or resistance to hyperoxia-induced acute lung injury mortality.

<p>Percent penetrance of sensitivity or resistance to hyperoxia-induced acute lung injury mortality.</p

Genomic makeup of the <i>Shali1</i> (Chr 1) and <i>Shali2</i> (Chr 4) reciprocal congenic lines.

<p>A schematic overview comparing the 19 autosomes and X chromosome for the X1 (white) and B (black) control inbred strains along with the major <i>Shali1</i> and <i>Shali2</i> reciprocal congenic strains generated on the recipient X1 strain with B strain donor (X1.B-1A and X1.B-4BB) or recipient B strain with X1 strain donor (B.X1-1A and X1.B-4A). Small regions of unknown parental origin, which map between known SNP markers, are colored grey. MIT markers representing the peak linkage (LOD) score for the <i>Shali1</i> and <i>Shali2</i> QTLs are shown at the left, with approximate map location relative to chromosome and transferred region.</p

Schematic summary of the reciprocal X1.B-1 and B.X1-1 consomic, congenic and subcongenic lines for <i>Shali1</i>.

<p>Data represent chromosome 1 of each strain, with X1 (solid white bar) and B (solid black bar) representing the recipient background strain of X1.B-1 and B.X1-1 lines, respectively. Chromosome 1 on the far left depicts MIT markers and positions (Mbp), along with the putative <i>Shali1</i> interval (black box). The current validated <i>Shali1</i> and <i>Shali5</i> intervals are indicated by dashed lines. X1.B-1 and B.X1-1 represent reciprocal consomic lines (full chromosome substitution). For X1-B-1 lines (left half), regions in black denote the substituted chromosome region, including the congenic lines X1.B-1A and X1.B-1B. For B.X1-1 lines (right half), regions in white represent the substituted regions. B.X1-1A and B.X1-1C are congenic lines and B.X1-1AA and B.X1-1BB are subcongenic lines of B.X1-1A and B.X1-1B (died), respectively. B.X1-1.303 was derived from a rare double recombinant identified early the screening process and fixed after removing all donor regions outside of the ∼11-Mbp area around marker <i>D1Mit303</i>. Grey regions represent areas not yet tested for parental origin. Tables below each set of congenics summarizes the mean survival time (MST) and sample size (n) for males and females of each line. *significantly different than same-sex background strain (<i>p</i><0.00132; MWW U-test with Bonferroni correction for 38 group comparisons). +, represents a historical control and includes combined data for new and previously reported controls <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038177#pone.0038177-Prows1" target="_blank">[24]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038177#pone.0038177-Prows2" target="_blank">[25]</a>.</p