Rachel Lorier

An Autoinflammatory Disease Due to Homozygous Deletion of the IL1RN Locus

We describe a patient with an autoinflammatory disease in which the main clinical features are pustular rash, marked osteopenia, lytic bone lesions, respiratory insufficiency, and thrombosis. Genetic studies revealed a 175-kb homozygous deletion at chromosome 2q13, which encompasses several interleukin-1 family members, including the gene encoding the interleukin-1-receptor antagonist (IL1RN). Mononuclear cells, obtained from the patient and cultured, produced large amounts of inflammatory cytokines, with increasing amounts secreted after stimulation with lipopolysaccharide. A similar increase was not observed in peripheral-blood mononuclear cells from a patient with neonatal-onset multisystem inflammatory disorder (NOMID). Treatment with anakinra completely resolved the symptoms and lesions.

A Clinician-Driven Automated System for Integration of Pharmacogenetic Interpretations Into an Electronic Medical Record

Advances in pharmacogenetic testing will expand the number of clinically important pharmacogenetic variants. Communication and interpretation of these test results are critical steps in implementation of pharmacogenetics into the clinic. Computational tools that integrate directly into the electronic medical record (EMR) are needed to translate the growing number of genetic variants into interpretive consultations to facilitate gene‐based drug prescribing. Herein, we describe processes for incorporating pharmacogenetic tests and interpretations into the EMR for clinical practice.

IL-10 Produced by Induced Regulatory T Cells (iTregs) Controls Colitis and Pathogenic Ex-iTregs during Immunotherapy

“Natural” regulatory T cells (nTregs) that express the transcription factor Foxp3 and produce IL-10 are required for systemic immunological tolerance. “Induced” regulatory T cells (iTregs) are nonredundant and essential for tolerance at mucosal surfaces, yet their mechanisms of suppression and stability are unknown. We investigated the role of iTreg-produced IL-10 and iTreg fate in a treatment model of inflammatory bowel disease. Colitis was induced in Rag1−/− mice by the adoptive transfer of naive CD4+ T cells carrying a nonfunctional Foxp3 allele. At the onset of weight loss, mice were treated with both iTregs and nTregs where one marked subset was selectively IL-10 deficient. Body weight assessment, histological scoring, cytokine analysis, and flow cytometry were used to monitor disease activity. Transcriptional profiling and TCR repertoire analysis were used to track cell fate. When nTregs were present but IL-10 deficient, iTreg-produced IL-10 was necessary and sufficient for the treatment of disease, and vice versa. Invariably, ∼85% of the transferred iTregs lost Foxp3 expression (ex-iTregs) but retained a portion of the iTreg transcriptome, which failed to limit their pathogenic potential upon retransfer. TCR repertoire analysis revealed no clonal relationships between iTregs and ex-iTregs, either within mice or between mice treated with the same cells. These data identify a dynamic IL-10–dependent functional reciprocity between regulatory T cell subsets that maintains mucosal tolerance. The niche supporting stable iTregs is limited and readily saturated, which promotes a large population of ex-iTregs with pathogenic potential during immunotherapy.

Signal transducer of inflammation gp130 modulates atherosclerosis in mice and man.

Liver-derived acute phase proteins (APPs) emerged as powerful predictors of cardiovascular disease and cardiovascular events, but their functional role in atherosclerosis remains enigmatic. We report that the gp130 receptor, which is a key component of the inflammatory signaling pathway within hepatocytes, influences the risk of atherosclerosis in a hepatocyte-specific gp130 knockout. Mice on an atherosclerosis-prone genetic background exhibit less aortic atherosclerosis (P < 0.05) with decreased plaque macrophages (P < 0.01). Translating these findings into humans, we show that genetic variation within the human gp130 homologue, interleukin 6 signal transducer (IL6ST), is significantly associated with coronary artery disease (CAD; P < 0.05). We further show a significant association of atherosclerotic disease at the ostium of the coronary arteries (P < 0.005) as a clinically important and heritable subphenotype in a large sample of families with myocardial infarction (MI) and a second independent population–based cohort. Our results reveal a central role of a hepatocyte-specific, gp130-dependent acute phase reaction for plaque development in a murine model of atherosclerosis, and further implicate IL6ST as a genetic susceptibility factor for CAD and MI in humans. Thus, the acute phase reaction should be considered an important target for future drug development in the management of CAD.

Concordance of DMET plus genotyping results with those of orthogonal genotyping methods.

There are several hurdles to the clinical implementation of pharmacogenetics. One approach is to employ pre‐prescription genotyping, involving interrogation of multiple pharmacogenetic variants using a high‐throughput platform. We compared the performance of the Drug Metabolizing Enzymes and Transporters (DMET) Plus array (1,931 variants in 225 genes) with that of orthogonal genotyping methods in 220 pediatric patients. A total of 1,692 variants had call rates >98% and were in Hardy–Weinberg equilibrium. Of these, 259 were genotyped by at least one independent method, and a total of 19,942 single‐nucleotide polymorphism (SNP)–patient sample pairs were evaluated. The concordance rate was 99.9%, with only 28 genotype discordances observed. For the genes deemed most likely to be clinically relevant (TPMT, CYP2D6, CYP2C19, CYP2C9, VKORC1, DPYD, UGT1A1, and SLCO1B1), a total of 3,799 SNP–patient sample pairs were evaluable and had a concordance rate of 99.96%. We conclude that the DMET Plus array performs well with primary patient samples, with the results in good concordance with those of several lower‐throughput genotyping methods.

RNA expression profiling of human iPSC-derived cardiomyocytes in a cardiac hypertrophy model.

Cardiac hypertrophy is an independent risk factor for cardiovascular disease and heart failure. There is increasing evidence that microRNAs (miRNAs) play an important role in the regulation of messenger RNA (mRNA) and the pathogenesis of various cardiovascular diseases. However, the ability to comprehensively study cardiac hypertrophy on a gene regulatory level is impacted by the limited availability of human cardiomyocytes. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer the opportunity for disease modeling. Here we utilize a previously established in vitro model of cardiac hypertrophy to interrogate the regulatory mechanism associated with the cardiac disease process. We perform miRNA sequencing and mRNA expression analysis on endothelin 1 (ET-1) stimulated hiPSC-CMs to describe associated RNA expression profiles. MicroRNA sequencing revealed over 250 known and 34 predicted novel miRNAs to be differentially expressed between ET-1 stimulated and unstimulated control hiPSC-CMs. Messenger RNA expression analysis identified 731 probe sets with significant differential expression. Computational target prediction on significant differentially expressed miRNAs and mRNAs identified nearly 2000 target pairs. A principal component analysis approach comparing the in vitro data with human myocardial biopsies detected overlapping expression changes between the in vitro samples and myocardial biopsies with Left Ventricular Hypertrophy. These results provide further insights into the complex RNA regulatory mechanism associated with cardiac hypertrophy.

Copy Number Variation Analysis in 98 Individuals with PHACE Syndrome

PHACE syndrome is the association of large segmental facial hemangiomas and congenital anomalies, such as posterior fossa malformations, cerebral arterial anomalies, coarctation of the aorta, eye anomalies and sternal defects. To date, the reported cases of PHACE syndrome have been sporadic suggesting that PHACE may have a complex pathogenesis. We report here genomic copy number variation (CNV) analysis in 98 individuals with PHACE syndrome as a first step in deciphering a potential genetic basis of PHACE syndrome. A total of 3,772 CNVs (2,507 duplications and 1,265 deletions) were detected in 98 individuals with PHACE syndrome. CNVs were then eliminated if they failed to meet established criteria for quality, spanned centromeres or did not contain genes. CNVs were defined as “rare” if not documented in the Database of Genomic Variants. Ten rare CNVs were discovered (size range: 134–406 kb), located at 1q32.1, 1q43, 3q26.32-3q26.33, 3p11.1, 7q33, 10q24.32, 12q24.13, 17q11.2, 18p11.31, and Xq28. There were no rare CNV events that occurred in greater than one subject. Therefore, further study is needed to determine the significance of these CNVs in the pathogenesis of PHACE syndrome.

Whole-exome sequencing and an iPSC-derived cardiomyocyte model provides a powerful platform for gene discovery in left ventricular hypertrophy

Rationale: Left ventricular hypertrophy (LVH) is a heritable predictor of cardiovascular disease, particularly in blacks. Objective: Determine the feasibility of combining evidence from two distinct but complementary experimental approaches to identify novel genetic predictors of increased LV mass. Methods: Whole-exome sequencing (WES) was conducted in seven African-American sibling trios ascertained on high average familial LV mass indexed to height (LVMHT) using Illumina HiSeq technology. Identified missense or nonsense (MS/NS) mutations were examined for association with LVMHT using linear mixed models adjusted for age, sex, body weight, and familial relationship. To functionally assess WES findings, human induced pluripotent stem cell-derived cardiomyocytes (induced pluripotent stem cell-CM) were stimulated to induce hypertrophy; mRNA sequencing (RNA-seq) was used to determine gene expression differences associated with hypertrophy onset. Statistically significant findings under both experimental approaches identified LVH candidate genes. Candidate genes were further prioritized by seven supportive criteria that included additional association tests (two criteria), regional linkage evidence in the larger HyperGEN cohort (one criterion), and publically available gene and variant based annotations (four criteria). Results: WES reads covered 91% of the target capture region (of size 37.2 MB) with an average coverage of 65×. WES identified 31,426 MS/NS mutations among the 21 individuals. A total of 295 MS/NS variants in 265 genes were associated with LVMHT with q-value <0.25. Of the 265 WES genes, 44 were differentially expressed (P < 0.05) in hypertrophied cells. Among the 44 candidate genes identified, 5, including HLA-B, HTT, MTSS1, SLC5A12, and THBS1, met 3 of 7 supporting criteria. THBS1 encodes an adhesive glycoprotein that promotes matrix preservation in pressure-overload LVH. THBS1 gene expression was 34% higher in hypertrophied cells (P = 0.0003) and a predicted conserved and damaging NS variant in exon 13 (A2099G) was significantly associated with LVHMT (P = 4 × 10−6). Conclusion: Combining evidence from cutting-edge genetic and cellular experiments can enable identification of novel LVH risk loci.

Whole exome sequencing identifies a POLRID mutation segregating in a father and two daughters with findings of Klippel-Feil and Treacher Collins syndromes.

We report on a father and his two daughters diagnosed with Klippel–Feil syndrome (KFS) but with craniofacial differences (zygomatic and mandibular hypoplasia and cleft palate) and external ear abnormalities suggestive of Treacher Collins syndrome (TCS). The diagnosis of KFS was favored, given that the neck anomalies were the predominant manifestations, and that the diagnosis predated later recognition of the association between spinal segmentation abnormalities and TCS. Genetic heterogeneity and the rarity of large families with KFS have limited the ability to identify mutations by traditional methods. Whole exome sequencing identified a nonsynonymous mutation in POLR1D (subunit of RNA polymerase I and II): exon2:c.T332C:p.L111P. Mutations in POLR1D are present in about 5% of individuals diagnosed with TCS. We propose that this mutation is causal in this family, suggesting a pathogenetic link between KFS and TCS.

Characterization of autosomal copy-number variation in African Americans: the HyperGEN Study

African Americans are a genetically diverse population with a high burden of many, common heritable diseases. However, our understanding of genetic variation in African Americans is substandard because of a lack of published population-based genetic studies. We report the distribution of copy-number variation (CNV) in African Americans collected as part of the Hypertension Genetic Epidemiology Network (HyperGEN) using the Affymetrix 6.0 array and the CNV calling algorithms Birdsuite and PennCNV. We present population estimates of CNV from 446 unrelated African-American subjects randomly selected from the 451 families collected within HyperGEN. Although the majority of CNVs discovered were individually rare, we found the frequency of CNVs to be collectively high. We identified a total of 11 070 CNVs greater than 10 kb passing quality control criteria that were called by both algorithms – leading to an average of 24.8 CNVs per person covering 2214 kb (median). We identified 1541 unique copy-number variable regions, 309 of which did not overlap with the Database of Genomic Variants. These results provide further insight into the distribution of CNV in African Americans.