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Enhanced fetal hemoglobin (Hb F) production can partially compensate for the lack of adult hemoglobin (Hb A) in patients with β-thalassemia major or intermedia, and ameliorate the clinical severity of these diseases. To further elucidate factors governing Hb F levels, we evaluated demographic, clinical, laboratory, and genetic characteristics in 241 unrelated adult β-thalassemia carriers in Hong Kong. They had wide variations in Hb F and F-cell numbers skewing toward higher levels. Individuals who coinherited the Xmn IT-allele in the Gγ-globin gene promoter had higher Hb F and more F-cells compared with those lacking the Xmn I T-allele. However, both groups exhibited a similarly wide spread of Hb F and F-cells. The correlation of Hb F and F-cells corresponded well to both linear and exponential models, suggesting multiple mechanisms for Hb F augmentation. The heritabilities of Hb F and F-cells were calculated in 66 families (111 parents who were β-thalassemia carriers and 82 asymptomatic offspring) to be 0.7 to 0.9. The Xmn I polymorphism accounted for 9% of the Hb F and 13% of the F-cell heritabilities. These results suggest that these family members are well suited for genome wide association studies that will identify genetic loci regulating Hb F production, and likely novel pharmacological targets for reactivating Hb F production in adults. Am. J. Hematol., 2008. © 2008 Wiley-Liss, Inc.

Objective: To present preliminary data on the health-related consequences for people with spinal cord injury (SCI) of participation in a prepaid, risk-based, managed care plan. Design: Baseline data from a longitudinal interview survey. Setting: Massachusetts. Participants: Subjects were 114 independently living adults with SCI recruited from 6 independent living centers and 2 managed care plans; 45 received care from a risk-based prepaid managed care plan and 69 from other sources. Main Outcomes: Self-reports of use of health services; self-assessments of health, health at interview versus 12 months earlier; hospital admissions; numbers of secondary conditions, and experiences of specific secondary conditions (eg, pressure ulcers, depression, fatigue, and chronic pain). Results: Persons in the managed care plan do not differ from their counterparts in terms of age, gender, education, level of SCI, number of comorbid conditions, activity of daily living profiles, household composition, and reliance upon health maintenance programs and routines (eg, bowel and bladder care). In terms of processes of care, they appear generally better-served. There also are a few differences in outcome that are statistically significant, or approach significance, and generally favor the managed care plan. Conclusion: Preliminary data suggest that thoughtfully and sensitively designed and implemented risk-based managed care may improve both access and outcomes.

In patients with sickle cell disease, clinical complications including osteonecrosis can vary in frequency and severity, presumably due to the effects of genes that modify the pathophysiology initiated by the sickle mutation. Here, we examined the association of single nucleotide polymorphisms (SNPs) in candidate genes (cytokines, inflammation, oxidant stress, bone metabolism) with osteonecrosis in patients with sickle cell disease. Genotype distributions were compared between cases and controls using multiple logistic regression techniques. An initial screen and follow-up studies showed that individual SNPs and haplotypes composed of several SNPs in bone morphogenic protein 6, annexin A2, and klotho were associated with sickle cell osteonecrosis. These genes are important in bone morphology, metabolism, and vascular disease. Our results may provide insight into the pathogenesis of osteonecrosis in sickle cell disease, help identify individuals who are at high risk for osteonecrosis, and thus allow earlier and more effective therapeutic intervention.

Familial amyloid polyneuropathy, a lethal genetic disease caused by aggregation of variant transthyretin, induces progressive peripheral nerve deficits and disability. Diflunisal, a nonsteroidal anti-inflammatory agent, stabilizes transthyretin tetramers and prevents amyloid fibril formation in vitro.To determine the effect of diflunisal on polyneuropathy progression in patients with familial amyloid polyneuropathy.International randomized, double-blind, placebo-controlled study conducted among 130 patients with familial amyloid polyneuropathy exhibiting clinically detectable peripheral or autonomic neuropathy at amyloid centers in Sweden (Umeå), Italy (Pavia), Japan (Matsumoto and Kumamoto), England (London), and the United States (Boston, Massachusetts; New York, New York; and Rochester, Minnesota) from 2006 through 2012.Participants were randomly assigned to receive diflunisal, 250 mg (n=64), or placebo (n=66) twice daily for 2 years.The primary end point, the difference in polyneuropathy progression between treatments, was measured by the Neuropathy Impairment Score plus 7 nerve tests (NIS+7) which ranges from 0 (no neurological deficits) to 270 points (no detectable peripheral nerve function). Secondary outcomes included a quality-of-life questionnaire (36-Item Short-Form Health Survey [SF-36]) and modified body mass index. Because of attrition, we used likelihood-based modeling and multiple imputation analysis of baseline to 2-year data.By multiple imputation, the NIS+7 score increased by 25.0 (95% CI, 18.4-31.6) points in the placebo group and by 8.7 (95% CI, 3.3-14.1) points in the diflunisal group, a difference of 16.3 points (95% CI, 8.1-24.5 points; P .001). Mean SF-36 physical scores decreased by 4.9 (95% CI, -7.6 to -2.2) points in the placebo group and increased by 1.5 (95% CI, -0.8 to 3.7) points in the diflunisal group (P .001). Mean SF-36 mental scores declined by 1.1 (95% CI, -4.3 to 2.0) points in the placebo group while increasing by 3.7 (95% CI, 1.0-6.4) points in the diflunisal group (P = .02). By responder analysis, 29.7% of the diflunisal group and 9.4% of the placebo group exhibited neurological stability at 2 years (2-point increase in NIS+7 score; P = .007).Among patients with familial amyloid polyneuropathy, the use of diflunisal compared with placebo for 2 years reduced the rate of progression of neurological impairment and preserved quality of life. Although longer-term follow-up studies are needed, these findings suggest benefit of this treatment for familial amyloid polyneuropathy.clinicaltrials.gov Identifier: NCT00294671.

Sickle cell disease patients have a high risk of developing leg ulcers. In the CSSCD database we found 378 patients with a confirmed history of leg ulcers and chose 920 patients without ulcers to serve as controls. α Thalassemia reduces hemolysis in sickle cell anemia which is consistent with our finding using an age-adjusted comparison, that sickle cell anemia-α thalassemia was more frequent among controls than cases (OR: 0.7, 95% CI: 0.5–0.9). This suggests that sickle cell anemia-α thalassemia patients are significantly less likely to have leg ulcers. Also, leg ulcer patients had lower hemoglobin levels, higher LDH, bilirubin, AST, reticulocyte and white blood cell count than controls. These differences were highly statistically significant (p < 0.001, except for AST, whose p was

Patients with sickle cell disease have an increased risk of bacteremia. To study the genetic basis for this increased susceptibility we studied the association of single nucleotide polymorphisms (SNPs) in candidate genes that might affect the risk of infection and therefore bacteremia. The Cooperative Study of Sickle Cell Disease enrolled 4,082 patients who were observed for about 5 years. Blood from these patients was used for globin gene analysis and SNP genotyping. We limited the present studies to patients with sickle cell anemia, with or without coincident α thalassemia, yielding a pool of 1473 patients with genotype, demographic and clinical data for which DNA samples were available. A case was defined as a patient seen in a clinic, emergency department, or hospital for which a positive blood culture not associated with a known source of infection such as osteomyelitis, septic arthritis, pneumonia, or meningitis, was found. Control patients had no history of bacteremia and no incident bacteremic events. For genetic studies, the samples were genotyped for informative SNPs in candidate genes selected from public and proprietary databases using the Sequenom mass spectrometry SNP genotyping system. For quality control purposes about 3% of the DNA samples were regenotyped and Hardy-Weinberg equilibrium was assessed for each SNP among controls. Genotypic counts were compared between sickle cell patients with bacteremia and patients using multiple logistic regression adjusting for leukocyte count, penicillin prophylaxis, fetal hemoglobin and total hemoglobin levels. In our initial screen, we considered a SNP to have an association with a phenotype when the p-value was equal to or less than 0.01, or if this and other SNPs in the same gene were significant at the 0.05 level. If a SNP met these criteria, a second phase of genotyping was done to study additional haplotype tagging (ht) SNPs in the gene. Because of the importance of the TGF-β beta pathway in the immune response, additional genes in this pathway were also studied. The ABI SNPlex system was utilized for the second phase of genotyping. Among the 201 subjects with bacteremia and 1238 controls, there was no significant difference in age, sex, HbF concentration, distribution of β-globin gene cluster haplotypes or the presence of coincident α thalassemia. Patients with bacteremia had a slightly lower hemoglobin concentration and higher leukocyte count. Subjects who received antibiotic prophylaxis (p < 0.0001) or H. influenza vaccination (p

A global estimation of sickle cell disease severity has been difficult to establish making the integration of clinical and laboratory abnormalities of into a predictive model of disease complications and death, an unrealized goal. A useful model might help us to understand the interactions among common clinical and laboratory abnormalities and allow patients at risk of certain complications and early death to be identified early so that targeted treatment, if available, could be provided. To approach this problem we first integrated clinical and laboratory data from nearly 3500 individuals from the Cooperative Study of Sickle Cell Disease and applied to this data advanced statistical machine learning techniques to identify the significant associations between selected complications of disease and laboratory variables. We looked for predictors of the risk for early death in three separate age groups. The resulting model revealed that complex networks of interactions between clinical and laboratory variables underlie common disease complications and ultimately death. Predictors of risk in this model were the HBA1, HBA2 genotype, stroke, sepsis and acute chest syndrome. While this model can predict the risk for early death, given the presence of other disease complications and variations among common laboratory variables, it did not provide an understanding of the genetic basis for our observations. Accordingly, in over 1000 patients with sickle cell disease we genotyped SNPs in genes chosen because of their possible link to the pathophysiology of disease. We then used our estimate of global disease severity to find associations of genotypes with severity. In the initial screening studies we identified several genes in the TGF-ß/BMP pathway that were associated with selected disease subphenotypes (Sebastiani et al Nature Genet37: 435, 2005; Baldwin et al. Blood106: 372, 2005). The TGF-b/BMP pathway regulates a wide range of biological functions including cell proliferation, cellular differentiation, extracellular matrix production, cell death, tissue repair and immune regulation. To expand our initial findings, we typed haplotype tagging SNPS in a more comprehensive fashion in 21 genes that are key members of this pathway. The genes included BMP6, BMP receptors, TGF-b receptors, SMADs, MAP kinases and their associated co-factors such as SARA, CDH1 and SMURF1. We used a Bayesian significance test to model the associations between the score of disease severity and individual SNPs, as well as a traditional association test with p-values computed using permutation tests. To reduce the problem of multiple comparisons, we selected as significantly associated with disease severity, only those SNPs that passed both tests with strong evidence of association. The most striking associations were found for BMP6, its receptors BMPR1 and BMPR2, several SMAD proteins and SARA1. Our results confirm the importance of the TGF-ß signaling pathway in sickle cell disease pathophysiology and suggest that subtle variation in this cell signaling network can be associated with the severity of disease.

The variable pattern of complications seen among sickle cell disease patients is unlikely to be the result of solely the HBB glu6val mutation. Conventional case-control analyses using single nucleotide polymorphisms (SNPs) are useful in looking for single gene associations, however studying gene interactions becomes increasingly inefficient as the number of SNPs increase. Application of more complex statistical methods such as classification and regression trees (CART), stochastic gradient boosting (SGB) and Bayesian networks (Sebastiani et al, Nature Genet37: 435, 2005) allows for the analysis of many SNPs and covariates simultaneously. CART is a recursive partitioning method that develops a single tree-based model which is grown by creating “if-then” splitting rules which stratify patients into risk groups. The resulting over-fitted tree is then pruned to optimize size and classification and its predictive ability assessed using a test set or cross validation. SGB is a similar method, however, instead of one large tree many small trees are grown sequentially. Each new tree improves the quality of the model based upon the prior stage and is weighted based on its predictive ability. The final predicted value is computed by adding the weighted contribution of each small sub-tree and based on that value, a classification assigned. From the over 4,000 patients in the CSSCD, a subset of 1,353 were genotyped for 353 SNPs in over 160 genes that might impact the disease pathophysiology. Clinical data for these patients were merged with genotype data and 490 patients were identified who had at least one of the following vasoocclusive events: stroke, osteonecrosis of the humeral or femoral head and/or priapism. With the remaining patients serving as controls, CART and SGB was run on a random sample of 80% of the patients to identify genes and covariates whose interactions characterize patients with these vasoocclusive events and the accuracy assessed using the remaining patients as a test set. Along with age, sex and HbF, CART identified TGFBR3, SMAD9, CISH, KL and MAP3K71P1 as being associated with the vasoocclusive event phenotype and classified patients with a sensitivity of 34% and a specificity of 82%. SGB however, while identifying the same pattern of genes and covariates as being associated with vasoocclusive events, was able to classify patients with a sensitivity of 80% and a specificity of 68%. While CART provides a simple initial screening of genes and covariates that may be simultaneously associated with the phenotype, SGB provides a more accurate method of classification with higher sensitivity and similar specificity. Neither method requires the extensive model building of Bayesian networks. A more thorough understanding of the molecular mechanisms will lead to the ability to predict subphenotypes and improve their management.

Hydroxyurea (HU) is an effective treatment for most patients with symptomatic sickle cell anemia, yet the fetal hemoglobin (HbF) response to treatment is variable. A capacity to predict an individual’s HbF response to HU would aid the selection of patients for treatment and reduce toxicity from unfruitful dose escalation. Unfortunately, this is presently not possible. We hypothesized that HbF levels and the HbF response to HU are regulated as complex genetic traits and previously showed that 12 single nucleotide polymorphisms (SNPs), associated with a 20% to 30% difference in baseline HbF concentrations, were found in the introns of 4 genes, PDE7B, MAP7, MAP3K5 and PEX7, spanning the genomic region from 136.1 Mb to 137.5 Mb on chromosome 6q (Cell Mol Biol 50:23, 2004). To begin to define the genetic predictors of the HbF response to (HU), we examined SNPs in candidate genes and genetic loci in 214 patients with sickle cell anemia whose HbF levels were available before HU treatment was started and after these patients reached a stable dose of this drug. Forty-six candidate genes were chosen because of their possible role in HbF regulation and HU metabolism and 226 SNPs in these genes were examined by mass spectrometry. A computer application developed in STATA was used to carry out multiple linear regression analysis with simultaneous adjustment for age, sex and the α- and β-globin gene cluster haplotypes for each SNP and combinations of nearby SNPs. Dominant, codominant and recessive models for modulating HbF expression were tested. In this QTL analysis, SNPs in a member of the cytochrome P450 family (CYP2C9), in aquaporin 9 (AQP9) and in the chromosome 6q qtl described above were significantly associated with the HbF response to HU. The effect of genotype on the magnitude of HbF response to HU was examined for selected SNPs in AQP9 and CYP2C9. In AQP9, AA was associated with an average increase of 6% in HbF compared with GG (rs1867380; OR 6.6, p

The phenotypic heterogeneity of sickle cell anemia (HbSS) is likely to be accounted for by multiple genetic modifiers. Priapism, a common vasoocclusive complication of HbSS, may reflect sickle vasculopathy. We hypothesized that the likelihood of developing priapism, and other vascular complications of sickle cell disease, may be influenced by genetic heterogeneity in genes that modulate inflammation, oxidant injury, nitric oxide (NO) biology, vasoregulation, cell-cell interaction and hemostasis. Accordingly, we studied patients with HbSS with or without coincident α thalassemia and examined the association of 129 single nucleotide polymorphisms (SNPs) in 44 candidate genes with priapism. One hundred forty-eight patients had at least one episode of priapism and were compared with 529 controls. Validated SNPs in the candidate genes were first selected from a public database. Genotypic counts were compared between cases and controls using multiple logistic regression. For each SNP, odds ratio (OR) and 95% confidence intervals (CI) were calculated. Pairwise linkage disequilibrium between each pair of SNP loci was evaluated by using a maximum likelihood method to infer phase for dual heterozygotes and was expressed as r2. In our first analysis, we considered a SNP to have an association with a phenotype when the p-value was equal to or less than 0.01, unless there was more than one SNP in a gene showing an association, when the p-value for significance was set at 0.05. When a SNP met these criteria, we further studied the gene with as many informative SNPs as possible. Haplotypes were reconstructed by using Bayesian methods as implemented in the PHASE package. A sliding window approach was used to assess evidence for association between haplotype and priapism. Patients with HbSS-α thalassemia were less likely to have priapism than patients with HbSS (p