Melanie E. Garrett

Relationship Between Methylome and Transcriptome in Patients With Nonalcoholic Fatty Liver Disease

BACKGROUND & AIMS Cirrhosis and liver cancer are potential outcomes of advanced nonalcoholic fatty liver disease (NAFLD). It is not clear what factors determine whether patients will develop advanced or mild NAFLD, limiting noninvasive diagnosis and treatment before clinical sequelae emerge. We investigated whether DNA methylation profiles can distinguish patients with mild disease from those with advanced NAFLD, and how these patterns are functionally related to hepatic gene expression. METHODS We collected frozen liver biopsies and clinical data from patients with biopsy-proven NAFLD (56 in the discovery cohort and 34 in the replication cohort). Samples were divided into groups based on histologic severity of fibrosis: F0-1 (mild) and F3-4 (advanced). DNA methylation profiles were determined and coupled with gene expression data from the same biopsies; differential methylation was validated in subsets of the discovery and replication cohorts. We then analyzed interactions between the methylome and transcriptome. RESULTS Clinical features did not differ between patients known to have mild or advanced fibrosis based on biopsy analysis. There were 69,247 differentially methylated CpG sites (76% hypomethylated, 24% hypermethylated) in patients with advanced vs mild NAFLD (P < .05). Methylation at fibroblast growth factor receptor 2, methionine adenosyl methyltransferase 1A, and caspase 1 was validated by bisulfite pyrosequencing and the findings were reproduced in the replication cohort. Methylation correlated with gene transcript levels for 7% of differentially methylated CpG sites, indicating that differential methylation contributes to differences in expression. In samples with advanced NAFLD, many tissue repair genes were hypomethylated and overexpressed, and genes in certain metabolic pathways, including 1-carbon metabolism, were hypermethylated and underexpressed. CONCLUSIONS Functionally relevant differences in methylation can distinguish patients with advanced vs mild NAFLD. Altered methylation of genes that regulate processes such as steatohepatitis, fibrosis, and carcinogenesis indicate the role of DNA methylation in progression of NAFLD.

Self-Regulation of Emotion, Functional Impairment, and Comorbidity Among Children With AD/HD

Objective: This study investigated the role of self-regulation of emotion in relation to functional impairment and comorbidity among children with and without AD/HD. Method: A total of 358 probands and their siblings participated in the study, with 74% of the sample participants affected by AD/HD. Parent-rated levels of emotional lability served as a marker for self-regulation of emotion. Results: Nearly half of the children affected by AD/HD displayed significantly elevated levels of emotional lability versus 15% of those without this disorder. Children with AD/HD also displayed significantly higher rates of functional impairment, comorbidity, and treatment service utilization. Emotional lability partially mediated the association between AD/HD status and these outcomes. Conclusion: Findings lent support to the notion that deficits in the self-regulation of emotion are evident in a substantial number of children with AD/HD and that these deficits play an important role in determining functional impairment and comorbidity outcomes.

Fetal hemoglobin in sickle cell anemia: genome-wide association studies suggest a regulatory region in the 5′ olfactory receptor gene cluster

In a genome-wide association study of 848 blacks with sickle cell anemia, we identified single nucleotide polymorphisms (SNPs) associated with fetal hemoglobin concentration. The most significant SNPs in a discovery sample were tested in a replication set of 305 blacks with sickle cell anemia and in subjects with hemoglobin E or beta thalassemia trait from Thailand and Hong Kong. A novel region on chromosome 11 containing olfactory receptor genes OR51B5 and OR51B6 was identified by 6 SNPs (lowest P = 4.7E-08) and validated in the replication set. An additional olfactory receptor gene, OR51B2, was identified by a novel SNP set enrichment analysis. Genome-wide association studies also validated a previously identified SNP (rs766432) in BCL11A, a gene known to affect fetal hemoglobin levels (P = 2.6E-21) and in Thailand and Hong Kong subjects. Elements within the olfactory receptor gene cluster might play a regulatory role in gamma-globin gene expression.

Hepatic Gene Expression Profiles Differentiate Presymptomatic Patients With Mild Versus Severe Nonalcoholic Fatty Liver Disease

Clinicians rely upon the severity of liver fibrosis to segregate patients with well‐compensated nonalcoholic fatty liver disease (NAFLD) into subpopulations at high‐ versus low‐risk for eventual liver‐related morbidity and mortality. We compared hepatic gene expression profiles in high‐ and low‐risk NAFLD patients to identify processes that distinguish the two groups and hence might be novel biomarkers or treatment targets. Microarray analysis was used to characterize gene expression in percutaneous liver biopsies from low‐risk, “mild” NAFLD patients (fibrosis stage 0‐1; n = 40) and high‐risk, “severe” NAFLD patients (fibrosis stage 3‐4; n = 32). Findings were validated in a second, independent cohort and confirmed by real‐time polymerase chain reaction and immunohistochemistry (IHC). As a group, patients at risk for bad NAFLD outcomes had significantly worse liver injury and more advanced fibrosis (severe NAFLD) than clinically indistinguishable NAFLD patients with a good prognosis (mild NAFLD). A 64‐gene profile reproducibly differentiated severe NAFLD from mild NAFLD, and a 20‐gene subset within this profile correlated with NAFLD severity, independent of other factors known to influence NAFLD progression. Multiple genes involved with tissue repair/regeneration and certain metabolism‐related genes were induced in severe NAFLD. Ingenuity Pathway Analysis and IHC confirmed deregulation of metabolic and regenerative pathways in severe NAFLD and revealed overlap among the gene expression patterns of severe NAFLD, cardiovascular disease, and cancer. Conclusion: By demonstrating specific metabolic and repair pathways that are differentially activated in livers with severe NAFLD, gene profiling identified novel targets that can be exploited to improve diagnosis and treatment of patients who are at greatest risk for NAFLD‐related morbidity and mortality. (Hepatology 2014;59:471–482)

Factors associated with survival in a contemporary adult sickle cell disease cohort.

In this study, the relationship of clinical differences among patients with sickle cell disease (SCD) was examined to understand the major contributors to early mortality in a contemporary cohort. Survival data were obtained for 542 adult subjects who were enrolled since 2002 at three university hospitals in the southeast United States. Subjects were followed up for a median of 9.3 years. At enrollment, clinical parameters were collected, including hemoglobin (Hb) genotype, baseline laboratory values, comorbidities, and medication usage. Levels of soluble adhesion molecules were measured for a subset of 87 subjects. The relationship of clinical characteristics to survival was determined using regression analysis. Median age at enrollment was 32 years. Median survival was 61 years for all subjects. Median survival for Hb SS and Sβ0 was 58 years and for Hb SC and Sβ+ was 66 years. Elevated white blood count, lower estimated glomerular filtration rate, proteinuria, frequency of pain crises, pulmonary hypertension, cerebrovascular events, seizures, stroke, sVCAM‐1, and short‐acting narcotics use were significantly associated with decreased survival. Forty‐two percent of subjects were on hydroxyurea therapy, which was not associated with survival. SCD continues to reduce life expectancy for affected individuals, particularly those with Hb Sβ0 and SS. Not only were comorbidities individually associated with decreased survival but also an additive effect was observed, thus, those with a greater number of negative endpoints had worse survival (P < 0.0001). The association of higher sVCAM‐1 levels with decreased survival suggests that targeted therapies to reduce endothelial damage and inflammation may also be beneficial. Am. J. Hematol. 89:530–535, 2014.

MYH9 and APOL1 are both associated with sickle cell disease nephropathy

Renal failure occurs in 5–18% of sickle cell disease (SCD) patients and is associated with early mortality. At‐risk SCD patients cannot be identified prior to the appearance of proteinuria and the pathobiology is not well understood. The myosin, heavy chain 9, non‐muscle (MYH9) and apolipoprotein L1 (APOL1) genes have been associated with risk for focal segmental glomerulosclerosis and end‐stage renal disease in African Americans. We genotyped 26 single nucleotide polymorphisms (SNPs) in MYH9 and 2 SNPs in APOL1 (representing the G1 and G2 tags) in 521 unrelated adult (18–83 years) SCD patients screened for proteinuria. Using logistic regression, SNPs were evaluated for association with proteinuria. Seven SNPs in MYH9 and one in APOL1 remained significantly associated with proteinuria after multiple testing correction (P < 0·0025). An MYH9 risk haplotype (P = 0·001) and the APOL1 G1/G2 recessive model (P < 0·0001) were strongly associated with proteinuria, even when accounting for the other. Glomerular filtration rate was negatively correlated with proteinuria (P < 0·0001), and was significantly predicted by an interaction between MYH9 and APOL1 in age‐adjusted analyses. Our data provide insight into the pathobiology of renal dysfunction in SCD, suggesting that MYH9 and APOL1 are both associated with risk.

Genetic modifiers of the severity of sickle cell anemia identified through a genome-wide association study

We conducted a genome‐wide association study (GWAS) to discover single nucleotide polymorphisms (SNPs) associated with the severity of sickle cell anemia in 1,265 patients with either “severe” or “mild” disease based on a network model of disease severity. We analyzed data using single SNP analysis and a novel SNP set enrichment analysis (SSEA) developed to discover clusters of associated SNPs. Single SNP analysis discovered 40 SNPs that were strongly associated with sickle cell severity (odds for association >1,000); of the 32 that we could analyze in an independent set of 163 patients, five replicated, eight showed consistent effects although failed to reach statistical significance, whereas 19 did not show any convincing association. Among the replicated associations are SNPs in KCNK6 a K+ channel gene. SSEA identified 27 genes with a strong enrichment of significant SNPs (P < 10−6); 20 were replicated with varying degrees of confidence. Among the novel findings identified by SSEA is the telomere length regulator gene TNKS. These studies are the first to use GWAS to understand the genetic diversity that accounts the phenotypic heterogeneity sickle cell anemia as estimated by an integrated model of severity. Additional validation, resequencing, and functional studies to understand the biology and reveal mechanisms by which candidate genes might have their effects are the future goals of this work. Am. J. Hematol., 2010.

SNPs in dopamine D2 receptor gene (DRD2) and norepinephrine transporter gene (NET) are associated with continuous performance task (CPT) phenotypes in ADHD children and their families.

Haplotype‐tagging SNP analyses were conducted to identify molecular genetic substrates of quantitative phenotypes derived from performance on a Continuous Performance Task (CPT). Three hundred sixty‐four individuals were sampled from 152 families ascertained on the basis of at least one child having ADHD. Probands, their affected and unaffected siblings, and parents were administered a CPT. Four different components of performance were analyzed and tested for association with SNPs from 10 candidate genes involved in monoaminergic function. After correcting for multiple comparisons and controlling for multiple individuals from the same family, significant associations were identified between commission errors and SNPs in the DRD2 gene (rs2075654, rs1079596), and between reaction time variability and a SNP in the NET gene (rs3785155). These findings suggest that commission errors and reaction time variability are excellent candidates as ADHD endophenotypes based on previously published criteria. Results also shed light on the molecular genetic basis of specific processes that may underlie the disorder.

Discovery and functional annotation of SIX6 variants in primary open-angle glaucoma.

Glaucoma is a leading cause of blindness worldwide. Primary open-angle glaucoma (POAG) is the most common subtype and is a complex trait with multigenic inheritance. Genome-wide association studies have previously identified a significant association between POAG and the SIX6 locus (rs10483727, odds ratio (OR) = 1.32, p = 3.87×10−11). SIX6 plays a role in ocular development and has been associated with the morphology of the optic nerve. We sequenced the SIX6 coding and regulatory regions in 262 POAG cases and 256 controls and identified six nonsynonymous coding variants, including five rare and one common variant, Asn141His (rs33912345), which was associated significantly with POAG (OR = 1.27, p = 4.2×10−10) in the NEIGHBOR/GLAUGEN datasets. These variants were tested in an in vivo Danio rerio (zebrafish) complementation assay to evaluate ocular metrics such as eye size and optic nerve structure. Five variants, found primarily in POAG cases, were hypomorphic or null, while the sixth variant, found only in controls, was benign. One variant in the SIX6 enhancer increased expression of SIX6 and disrupted its regulation. Finally, to our knowledge for the first time, we have identified a clinical feature in POAG patients that appears to be dependent upon SIX6 genotype: patients who are homozygous for the SIX6 risk allele (His141) have a statistically thinner retinal nerve fiber layer than patients homozygous for the SIX6 non-risk allele (Asn141). Our results, in combination with previous SIX6 work, lead us to hypothesize that SIX6 risk variants disrupt the development of the neural retina, leading to a reduced number of retinal ganglion cells, thereby increasing the risk of glaucoma-associated vision loss.

Meta-analysis of 2040 sickle cell anemia patients: BCL11A and HBS1L-MYB are the major modifiers of HbF in African Americans

To the editor: Fetal hemoglobin (HbF) protects against many but not all of the hematologic and clinical complications of sickle cell anemia.[1][1],[2][2] This protection is dependent on the ability of HbF to hinder deoxyHbS polymerization. HbF level is variable and highly heritable. Previous