Monoclonal antibody (mAb) therapy directed against CD20 is an important tool in the treatment of B cell disorders. However, variable patient response and acquired resistance remain important clinical challenges. To identify genetic factors that may influence sensitivity to treatment, the cytotoxic activity of three CD20 mAbs: rituximab; ofatumumab; and obinutuzumab, were screened in high-throughput assays using 680 ethnically diverse lymphoblastoid cell lines (LCLs) followed by a pharmacogenomic assessment. GWAS analysis identified several novel gene candidates. The most significant SNP, rs58600101, in the gene MKL1 displayed ethnic stratification, with the variant being significantly more prevalent in the African cohort and resulting in reduced transcript levels as measured by qPCR. Functional validation of MKL1 by shRNA-mediated knockdown of MKL1 resulted in a more resistant phenotype. Gene expression analysis identified the developmentally associated TGFB1I1 as the most significant gene associated with sensitivity. qPCR among a panel of sensitive and resistant LCLs revealed immunoglobulin class-switching as well as differences in the expression of B cell activation markers. Flow cytometry showed heterogeneity within some cell lines relative to surface Ig isotype with a shift to more IgG+ cells among the resistant lines. Pretreatment with prednisolone could partly reverse the resistant phenotype. Results suggest that the efficacy of anti-CD20 mAb therapy may be influenced by B cell developmental status as well as polymorphism in the MKL1 gene. A clinical benefit may be achieved by pretreatment with corticosteroids such as prednisolone followed by mAb therapy.
The nature and organization of polymorphisms, or differences, between genomes of individuals are of great interest, because these variations can be associated with or even underlie phenotypic traits, including disease susceptibility. To gain insight into the genetic and evolutionary factors influencing such biological variation, we have examined the arrangement (haplotype) of single-nucleotide polymorphisms across the genomes of eight inbred strains of mice. These analyses define blocks of high or low diversity, often extending across tens of megabases that are delineated by abrupt transitions. These observations provide a striking contrast to the haplotype structure of the human genome.
Reactive oxygen species contribute to the pathogenesis of many acute and chronic pulmonary disorders, including bronchopulmonary dysplasia (BPD), a respiratory condition affecting preterm infants. BPD treatment often involves respiratory support with high O 2 , and oxidative stress is an adverse effect associated with vascular damage and impaired lung development and function in a subset of infants. Genetic polymorphisms in a few candidate genes have been associated with BPD susceptibility, however the genetic basis of differential susceptibility remains poorly understood. Understanding the role of genetics is critical to improving treatment strategies and reducing oxidant injury in developing lungs. We developed a neonatal mouse model to mimic many features of BPD, and utilized genome‐wide association (GWA) mapping of quantitative phenotypes to identify susceptibility genes for neonatal hyperoxic lung injury. One day after birth, neonatal mice from 29 inbred strains were exposed for 72 hr to normoxia (room air, 20% O 2 ) or hyperoxia (>95% O 2 ). Hyperoxia induced phenotypes (inflammatory cells/histopathology) that are similar to BPD when compared to normal postnatal lung development in normoxia. We also measured hyperoxia‐induced lesions in nuclear (nuc) and mitochondrial (mt) DNA, and mitochondrial copy number from lung homogenates. Significant inter‐strain variation was found for mtDNA and nucDNA lesions after normoxia and hyperoxia. GWA mapping identified significant quantitative trait loci for mtDNA lesions, nucDNA lesions, and mitochondrial copy number after normoxia and for hyperoxia. We ultra‐deep sequenced neonatal lung mtDNA from mice exposed to normoxia and hyperoxia and found 385 sequence variations in wild‐derived PWD/PhJ inbred mice compared to reference C57BL/6J mice. Higher variation frequency (1/32 bases) was found in the D‐loop region compared to the rest of the mitochondrial genome (1/42 bases). There was also more heteroplasmy in PWD/PhJ mice compared to C57BL/6J. We found five haplotype groups among 27 strains that were sequenced, with classical inbred strains in haplotype 1, NZB and NZO mice in haplotype 2, and PWD/PhJ and PWK/PhJ mice in haplotype 3. Wild derived strains had more variation than classical inbred strains. We conclude that exposure to low (20%) and high (>95%) concentrations of oxygen early in life cause lesions in mouse lung mtDNA and nucDNA that are strain dependent. Ultra‐deep sequencing identified mtDNA sequence variation and differences in heteroplasmy and indels across inbred mouse strains that associate with disease phenotypes. Through these combined approaches, we have identified novel candidate susceptibility genes that may improve our understanding of neonatal lung injury and development. Support or Funding Information Support by the NIEHS Intramural Program
Tacrolimus exhibits high inter-patient pharmacokinetics (PK) variability, as well as a narrow therapeutic index, and therefore requires therapeutic drug monitoring. Germline mutations in cytochrome P450 isoforms 4 and 5 genes (CYP3A4/5) and the ATP-binding cassette B1 gene (ABCB1) may contribute to interindividual tacrolimus PK variability, which may impact clinical outcomes among allogeneic hematopoietic stem cell transplantation (HSCT) patients. In this study, 252 adult patients who received tacrolimus for acute graft versus host disease (aGVHD) prophylaxis after allogeneic HSCT were genotyped to evaluate if germline genetic variants associated with tacrolimus PK and pharmacodynamic (PD) variability. Significant associations were detected between germline variants in CYP3A4/5 and ABCB1 and PK endpoints (e.g., median steady-state tacrolimus concentrations and time to goal tacrolimus concentration). However, significant associations were not observed between CYP3A4/5 or ABCB1 germline variants and PD endpoints (e.g., aGVHD and treatment-emergent nephrotoxicity). Decreased age and CYP3A5*1/*1 genotype were independently associated with subtherapeutic tacrolimus trough concentrations while CYP3A5*1*3 or CYP3A5*3/*3 genotypes, myeloablative allogeneic HSCT conditioning regimen (MAC) and increased weight were independently associated with supratherapeutic tacrolimus trough concentrations. Future lines of prospective research inquiry are warranted to use both germline genetic and clinical data to develop precision dosing tools that will optimize both tacrolimus dosing and clinical outcomes among adult HSCT patients.
Hirschsprung disease (HSCR) is a complex disorder that exhibits incomplete penetrance and variable expressivity due to interactions among multiple susceptibility genes. Studies in HSCR families have identified RET-dependent modifiers for short-segment HSCR (S-HSCR), but epistatic effects in long-segment (L-HSCR) and syndromic cases have not been fully explained. SOX10 mutations contribute to syndromic HSCR cases and Sox10 alleles in mice exhibit aganglionosis and pigmentary anomalies typical of a subset of HSCR patients categorized as Waardenburg–Shah syndrome (WS4, OMIM 277580). Sox10 mutant alleles in mice exhibit strain-dependent variation in penetrance and expressivity of aganglionic megacolon analogous to the variation observed in patients with aganglionosis. In this study, we focused on enteric ganglia deficits in Sox10Dom mice and defined aganglionosis as a quantitative trait in Sox10Dom intercross progeny to investigate the contribution of strain background to variation in enteric nervous system deficits. We observe that the phenotype of Sox10Dom/+ mutants ranges over a continuum from severe aganglionosis to no detectable phenotype in the gut. To systematically identify genes that modulate Sox10-dependent aganglionosis, we performed a single nucleotide polymorphism-based genome scan in Sox10Dom/+ F1 intercross progeny. Our analysis reveals modifier loci on mouse chromosomes 3, 5, 8, 11 and 14 with distinct effects on penetrance and severity of aganglionosis. Three of these loci on chromosomes 3, 8 and 11 do not coincide with previously known aganglionosis susceptibility genes or modifier loci and offer new avenues for elucidating the genetic network that modulates this complex neurocristopathy.
