Jacqueline N. Milton

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.

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.

Whole Genome Sequences of a Male and Female Supercentenarian, Ages Greater than 114 Years

Supercentenarians (age 110+ years old) generally delay or escape age-related diseases and disability well beyond the age of 100 and this exceptional survival is likely to be influenced by a genetic predisposition that includes both common and rare genetic variants. In this report, we describe the complete genomic sequences of male and female supercentenarians, both age >114 years old. We show that: (1) the sequence variant spectrum of these two individuals’ DNA sequences is largely comparable to existing non-supercentenarian genomes; (2) the two individuals do not appear to carry most of the well-established human longevity enabling variants already reported in the literature; (3) they have a comparable number of known disease-associated variants relative to most human genomes sequenced to-date; (4) approximately 1% of the variants these individuals possess are novel and may point to new genes involved in exceptional longevity; and (5) both individuals are enriched for coding variants near longevity-associated variants that we discovered through a large genome-wide association study. These analyses suggest that there are both common and rare longevity-associated variants that may counter the effects of disease-predisposing variants and extend lifespan. The continued analysis of the genomes of these and other rare individuals who have survived to extremely old ages should provide insight into the processes that contribute to the maintenance of health during extreme aging.

A Genome-Wide Association Study of Total Bilirubin and Cholelithiasis Risk in Sickle Cell Anemia

Serum bilirubin levels have been associated with polymorphisms in the UGT1A1 promoter in normal populations and in patients with hemolytic anemias, including sickle cell anemia. When hemolysis occurs circulating heme increases, leading to elevated bilirubin levels and an increased incidence of cholelithiasis. We performed the first genome-wide association study (GWAS) of bilirubin levels and cholelithiasis risk in a discovery cohort of 1,117 sickle cell anemia patients. We found 15 single nucleotide polymorphisms (SNPs) associated with total bilirubin levels at the genome-wide significance level (p value <5×10−8). SNPs in UGT1A1, UGT1A3, UGT1A6, UGT1A8 and UGT1A10, different isoforms within the UGT1A locus, were identified (most significant rs887829, p = 9.08×10−25). All of these associations were validated in 4 independent sets of sickle cell anemia patients. We tested the association of the 15 SNPs with cholelithiasis in the discovery cohort and found a significant association (most significant p value 1.15×10−4). These results confirm that the UGT1A region is the major regulator of bilirubin metabolism in African Americans with sickle cell anemia, similar to what is observed in other ethnicities.

The impact of conflicts of interest in plastic surgery: an analysis of acellular dermal matrix, implant-based breast reconstruction.

Background: Although conflicts of interest in biomedical research have received significant attention, the impact of conflicts of interest on surgical outcomes has not been fully explored. Methods: A systematic electronic search of the literature was performed for studies that evaluated surgical outcomes in acellular dermal matrix and non–acellular dermal matrix implant-based breast reconstruction. Surgical complications, including infection, seroma, hematoma, necrosis, and explantation, were used as outcome metrics and extracted from studies. Surgical outcomes were then pooled and compared between studies that disclosed conflicts of interest and those that did not disclose conflicts of interest. Results: A total of 776 abstracts were identified, of which only 35 fulfilled the authors’ inclusion criteria. Conflicts of interest were reported in 14 of these abstracts (40 percent). The pooled data from studies that reported no conflicts of interest and studies that reported conflicts of interest included a total of 8241 and 5384 breasts and 2852 and 1864 patients, respectively. Considered collectively, surgical complications were less common in studies that reported a conflict of interest than in studies that reported no conflicts of interest. When surgical outcome data were further stratified by acellular dermal matrix use, surgical complications were less common in studies with conflicts of interest when acellular dermal matrix was used. However, when acellular dermal matrix was not used, surgical complications were similar between authors that reported a conflict of interest and those that did not report a conflict of interest. Conclusions: Self-reported conflicts of interest are common in implant-based breast reconstruction research. Studies authored by groups with conflicts of interest are significantly associated with reporting lower surgical complications and therefore describing positive research findings, especially when industry-marketed products are being used in the study. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.

Genetic determinants of haemolysis in sickle cell anaemia

Haemolytic anaemia is variable among patients with sickle cell anaemia and can be estimated by reticulocyte count, lactate dehydrogenase, aspartate aminotransferase and bilirubin levels. Using principal component analysis of these measurements we computed a haemolytic score that we used as a subphenotype in a genome‐wide association study. We identified in one cohort and replicated in two additional cohorts the association of a single nucleotide polymorphism in NPRL3 (rs7203560; chr16p13·3) (P = 6·04 × 10−07). This association was validated by targeted genotyping in a fourth independent cohort. The HBA1/HBA2 regulatory elements, hypersensitive sites (HS)‐33, HS‐40 and HS‐48 are located in introns of NPRL3. Rs7203560 was in perfect linkage disequilibrium (LD) with rs9926112 (r2 = 1) and in strong LD with rs7197554 (r2 = 0·75) and rs13336641 (r2 = 0·77); the latter is located between HS‐33 and HS‐40 sites and next to a CTCF binding site. The minor allele for rs7203560 was associated with the −∝3·7thalassaemia gene deletion. When adjusting for HbF and ∝ thalassaemia, the association of NPRL3 with the haemolytic score was significant (P = 0·00375) and remained significant when examining only cases without gene deletion∝ thalassaemia (P = 0·02463). Perhaps by independently down‐regulating expression of the HBA1/HBA2 genes, variants of the HBA1/HBA2 gene regulatory loci, tagged by rs7203560, reduce haemolysis in sickle cell anaemia.

Severe sickle cell anemia is associated with increased plasma levels of TNF-R1 and VCAM-1.

Sickle cell anemia (SCA, HBB glu6val) is characterized by multiple complications and a high degree of phenotypic variability: some subjects have only sporadic pain crises and few acute hospitalizations, while others experience multiple serious complications, high levels of morbidity, and accelerated mortality [1]. The tumor necrosis factor-α (TNF-α) signaling pathway plays important roles in inflammation and the immune response; variation in this pathway might be expected to modify the overall severity of SCA through the pathways effects on the vascular endothelium [2,3]. We examined plasma biomarkers of TNF-α activity and endothelial cell activation for associations with SCA severity in 24 adults (12 mild, 12 severe). Two biomarkers, tumor necrosis factor-α receptor-1 (TNF-R1) and vascular cell adhesion molecule-1 (VCAM-1) were significantly higher in subjects with severe SCA. Along with these biomarker differences, we also examined data from a genome-wide association study (GWAS) using SCA severity as a disease phenotype, and found evidence of genetic association between disease severity and a single nucleotide polymorphism (SNP) in VCAM1, which codes for VCAM-1, and several SNPs in ARFGEF2, a gene involved in TNF-R1 release [4].

A GCH1 haplotype confers sex-specific susceptibility to pain crises and altered endothelial function in adults with sickle cell anemia.

GTP cyclohydrolase (GCH1) is rate limiting for tetrahydrobiopterin (BH4) synthesis, where BH4 is a cofactor for nitric oxide (NO) synthases and aromatic hydroxylases. GCH1 polymorphisms are implicated in the pathophysiology of pain, but have not been investigated in African populations. We examined GCH1 and pain in sickle cell anemia where GCH1 rs8007267 was a risk factor for pain crises in discovery (n = 228; odds ratio [OR] 2.26; P = 0.009) and replication (n = 513; OR 2.23; P = 0.004) cohorts. In vitro, cells from sickle cell anemia subjects homozygous for the risk allele produced higher BH4. In vivo physiological studies of traits likely to be modulated by GCH1 showed rs8007267 is associated with altered endothelial dependent blood flow in females with SCA (8.42% of variation; P = 0.002). The GCH1 pain association is attributable to an African haplotype with where its sickle cell anemia pain association is limited to females (OR 2.69; 95% CI 1.21–5.94; P = 0.01) and has the opposite directional association described in Europeans independent of global admixture. The presence of a GCH1 haplotype with high BH4 in populations of African ancestry could explain the association of rs8007267 with sickle cell anemia pain crises. The vascular effects of GCH1 and BH4 may also have broader implications for cardiovascular disease in populations of African ancestry. Am. J. Hematol. 89:187–193, 2014.

Financial Conflicts of Interest: An Association between Funding and Findings in Plastic Surgery.

Background: Despite a growing interest in examining the effects of industry funding on research in plastic surgery, no study to date has comprehensively examined the effects of financial conflicts of interest on publication outcomes. The authors investigated the association between financial conflicts of interest and reported study findings in plastic surgery research. Methods: The authors reviewed all entries in Plastic and Reconstructive Surgery, Annals of Plastic Surgery, and Journal of Plastic, Reconstructive & Aesthetic Surgery from January 1, 2012, to December 31, 2012. All clinical and basic science articles were analyzed. The following article characteristics were extracted: self-reported financial conflicts of interest, sample size, level of evidence, study design, and prospectiveness. The findings reported in each abstract were blindly graded as not applicable, negative, or positive. Results: Of the 1650 abstracts that resulted from the authors’ initial search, 568 fulfilled the inclusion criteria. The majority of the included articles covered breast (20.8 percent), experimental (19.7 percent), and general reconstruction (31.69 percent). Financial conflicts of interest were disclosed in only 17.6 percent of the articles. Of the total studies that met inclusion criteria, 66.2 percent were reviewed as having positive outcomes, and 33.8 percent were reviewed as having negative or not applicable results. Studies that disclosed a financial conflict of interest were 7.12 times more likely (p < 0.0001) to present a positive outcome over a negative outcome compared with studies with no financial conflict of interest. Conclusion: Investigators with a financial conflict of interest are significantly more likely to publish plastic surgery studies with a positive conclusion compared with investigators with no conflicts of interest.

Prediction of Fetal Hemoglobin in Sickle Cell Anemia Using an Ensemble of Genetic Risk Prediction Models

Background—Fetal hemoglobin (HbF) is the major modifier of the clinical course of sickle cell anemia. Its levels are highly heritable, and its interpersonal variability is modulated in part by 3 quantitative trait loci that affect HbF gene expression. Genome-wide association studies have identified single-nucleotide polymorphisms (SNPs) in these quantitative trait loci that are highly associated with HbF but explain only 10% to 12% of the variance of HbF. Combining SNPs into a genetic risk score can help to explain a larger amount of the variability of HbF level, but the challenge of this approach is to select the optimal number of SNPs to be included in the genetic risk score. Methods and Results—We developed a collection of 14 models with genetic risk score composed of different numbers of SNPs and used the ensemble of these models to predict HbF in patients with sickle cell anemia. The models were trained in 841 patients with sickle cell anemia and were tested in 3 independent cohorts. The ensemble of 14 models explained 23.4% of the variability in HbF in the discovery cohort, whereas the correlation between predicted and observed HbF in the 3 independent cohorts ranged between 0.28 and 0.44. The models included SNPs in BCL11A, the HBS1L-MYB intergenic region, and the site of the HBB gene cluster, quantitative trait loci previously associated with HbF. Conclusions—An ensemble of 14 genetic risk models can predict HbF levels with accuracy between 0.28 and 0.44, and the approach may also prove useful in other applications.