Numerous studies have shown that the risk of Alzheimer's disease (AD) is associated with the dose of the epsilon 4 allele of apolipoprotein E (ApoE). However, more than one third of AD patients lack ...epsilon 4 and many persons having epsilon 4 survive cognitively intact to old age. We evaluated the lifetime risk of disease in 3,999 first-degree relatives of 549 probands who met the criteria for probable or definite AD and whose ApoE genotypes were known. ApoE genotypes for relatives were not determined. After age 65 the risk among relatives was proportional, as much as 7 to 10% at age 85, to the number of epsilon 4 alleles present in the proband. Risks to relatives of ApoE 2/2 and 2/3 probands were nearly identical at all ages to risks for relatives of ApoE 3/3 probands. The expected proportion of relatives having at least one epsilon 4 allele was calculated for each genotype group based on the distribution of parents, sibs, and offspring in the sample. Among relatives in the ApoE 3/3 group, the lifetime risk for AD by age 90 was three times greater than the expected proportion of epsilon 4 carriers, suggesting that factors other than ApoE contribute to AD susceptibility. Furthermore, the 44% risk of AD by age 93 among relatives of ApoE 4/4 probands indicates that as many as 50% of people having at least one epsilon 4 allele do not develop AD. We also found that among male relatives, risk of AD in the ApoE 3/4 group was similar to that for the ApoE 3/3 group but significantly less than the risk for the ApoE 4/4 group. In contrast, among female relatives the risk for the ApoE 3/4 group was nearly twice that for the ApoE 3/3 group and identical to the risk for the ApoE 4/4 group. These findings are consistent with a sex-modification effect of the E4 isoform on disease susceptibility.
Although sickle cell anemia is a single gene (
HBB) disorder, the variable pattern of complications seen among patients is unlikely the result of solely the
HBB 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 allows for the analysis of many SNPs and covariates simultaneously.
From the over 4000 patients in the Cooperative Study for Sickle Cell Disease, a subset of 1584 were genotyped for 180 SNPs in 92 genes. Clinical data for these patients were merged with genotype data and 576 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 was run to identify genes and covariates whose interactions characterize patients with vasoocclusive events.
The final tree, after pruning, shows five terminal nodes that predict vasoocclusive event. Along with age, the genes
ANXA2, BMP6, SELP, TGFBR2 and
TGFBR3 were found to be associated with the vasoocclusive event phenotype; each of these genes have been found to be associated with at least one of the individual phenotypes of stroke, osteonecrosis and/or priapism previously.
These results support our parallel work using Bayesian networks to understand the interactions among genes underlying the clinical heterogeneity of sickle cell anemia and studies of individual phenotypes. While Bayesian networks have superior predictive power, CART provides a simple initial screening of genes and covariates that may be simultaneously associated with the phenotype.
Cutaneous leg ulcers are common in sickle cell anaemia and their risk might be genetically determined. We studied sickle cell anaemia patients to examine the relationship of leg ulcers with ...haemolysis and with SNPs in candidate genes that could affect sickle vasoocclusion. Leg ulcer patients had lower haemoglobin levels and higher levels of lactate dehydrogenase, bilirubin, aspartate aminotransferase and reticulocytes than did control patients with sickle cell anaemia but without leg ulcers. Age-adjusted comparisons showed that sickle cell anaemia-α thalassaemia was more frequent among controls than cases. These results strongly suggested that the likelihood of having leg ulcers was related to the intensity of haemolysis. Two-hundred fifteen SNPs in more than 100 candidate genes were studied. Associations were found with SNPs in Klotho,
TEK
and several genes in the TGF-β/BMP signaling pathway by genotypic association analyses.
KL
directly or indirectly promotes endothelial NO production and the
TEK
receptor tyrosine kinase is involved in angiogenesis. The TGF-β/BMP signaling pathway modulates wound healing and angiogenesis, among its other functions. Haemolysis-driven phenotypes like leg ulcers could be improved by agents that reduce sickle erythrocyte density or increase NO bioavailability.
Cutaneous leg ulcers are common in sickle cell anaemia and their risk might be genetically determined. Sickle cell anaemia patients were studied to examine the relationship of leg ulcers with ...haemolysis and with single nucleotide polymorphisms (SNPs) in candidate genes that could affect sickle vasoocclusion. Leg ulcer patients had lower haemoglobin levels and higher levels of lactate dehydrogenase, bilirubin, aspartate transaminase and reticulocytes than did control patients with sickle cell anaemia but without leg ulcers. Age-adjusted comparisons showed that sickle cell anaemia- alpha thalassaemia was more frequent among controls than cases. These results strongly suggested that the likelihood of having leg ulcers was related to the intensity of haemolysis. 215 SNPs in more than 100 candidate genes were studied. Associations were found with SNPs in Klotho, TEK and several genes in the TGF- beta /BMP signalling pathway by genotypic association analyses. KL directly or indirectly promotes endothelial nitric oxide (NO) production and the TEK receptor tyrosine kinase is involved in angiogenesis. The TGF- beta /BMP signalling pathway modulates wound healing and angiogenesis, among its other functions. Haemolysis-driven phenotypes, such as leg ulcers, could be improved by agents that reduce sickle erythrocyte density or increase NO bioavailability.
A single point mutation in the β hemoglobin gene causes sickle cell disease (SCD), but patients have extremely variable phenotypes. Hemolysis-related complications include pulmonary hypertension ...(PHT), priapism, stroke and leg ulceration; blood viscosity and sickle vasoocclusion are associated with painful episodes, acute chest syndrome and osteonecrosis. Predicting who is at highest risk of death would be useful therapeutically and prognostically. Applying Bayesian network modeling that describes complex interactions among many variables by factorizing their joint probability distribution into modules, to data from 3380 SCD patients, we constructed a disease severity score (DSS: 0, least severe; 1, most severe), defining severity as risk of death within 5 years. A network of 24 variables described complex associations among clinical and laboratory complications of SCD. The analysis was validated in 140 patients whose SCD severity was assessed by expert clinicians and 210 adults where severity was also assessed by the echocardiographic diagnosis of PHT and death. Information about PHT allowed a comparison of the DSS with the tricuspid regurgitant jet velocity (TRJV), an objective marker of PHT and an independent risk factor for death. DSS and three indices of clinical severity (severity ranking of individuals by expert clinicians; objective measurement of the presence and severity of PHT; risk of prospective death) were correlated. Among living subjects, the median score was 0.57 in 135 patients without PHT, 0.64 in 40 patients with mild PHT and 0.86 in 15 patients with severe PHT. The difference in average score between living patients with and without PHT is significant. The same increasing trend was noticeable in the subjects who died during follow-up: 0.60 in subjects without PHT; 0.68 in subjects with mild PHT; 0.79 in subjects with severe PHT. The utility of the DSS is also supported by the ability to assign a score to subjects for whom the TRJV cannot be measured. Surprisingly, besides known risk factors like renal insufficiency and leukocytosis, we identified the intensity of hemolytic anemia and clinical events associated with hemolytic anemia as contributing to risk for death. Priapism, an excellent reflection of the hemolytic anemia-related complications of SCD, is associated with PHT and its association with death was unexpected. Laboratory variables predictive of disease severity included LDH and reticulocytes that reflect the intensity of hemolytic anemia. Elevated systolic blood pressure increased the odds of death by 3.4, consistent with hypertension as a marker of early death in SCD. Subjects with sickle cell anemia are at greatest risk compared with subjects with sickle cell anemia-α thalassemia and with subjects with HbSC disease. Our model suggests that the intensity of hemolytic anemia, estimated by LDH, reticulocyte count and AST, and shown previously to be associated with PHT, priapism, leg ulceration and possibly stroke, is an important contributor to death. This model can be used to compute a personalized measure of disease severity that might be useful for guiding therapeutic decisions and designing clinical trials.
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 Genet 37: 435, 2005; Baldwin et al. Blood 106: 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 Genet 37: 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.
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 <0.005) and support the impression that cases have a higher rate of hemolysis than controls. Fetal hemoglobin was higher in controls compared with cases (p=0.002). The haplotype of the β-like globin gene cluster, creatinine and ALT were not associated with leg ulcers. Leg ulcers were associated with other clinical manifestations of sickle cell disease like ischemic stroke (OR:1.4, 95% CI: 0.9–2.0) and acute chest syndrome (OR:1.5, 95% CI:1.1–1.9). When analysis of laboratory data was restricted to the 759 patients (243 cases vs. 516 controls) that were also genotyped for SNPs in candidate genes, the results were similar. We studied 132 SNPs in 47 candidate genes for their association with leg ulcers. SNPs were studied by mass spectrometry in a screening phase and by using haplotype tagging (ht) SNPs and the ABI SNPlex for follow-up. Candidate genes included: mediators of inflammation; oxidant injury; NO biology; vasoregulation; cell-cell interaction; blood coagulation; hemostasis; growth factors; cytokines and receptors. The candidate genes having multiple SNPs associated with leg ulcers were KL (rs685417 and rs516306; p values <0.02), TEK (rs603085 and rs671084; p values <0.025), SMAD1 (rs1899784, rs10519733 and rs2068991; p values <0.05), and SARA1 (rs2271690 and rs870801; p values <0.04). KL directly or indirectly promotes endothelial NO production. The TEK receptor tyrosine kinase (TIE2) is expressed almost exclusively in endothelial cells, is involved in angiogenesis and is the ligand for angiopoietin-1 (ANG1). SMAD1 and SARA1 are members of the TGF-β/BMP pathway. The TGF-β receptor signals through the SMAD family of transcriptional regulators that are anchored to the cell membrane by factors like SARA (SMAD Anchor for Receptor Activation). This pathway modulates immunosuppression, cell migration, wound healing and angiogenesis, among its other functions. Hemolysis is likely to be an antecedent of certain vascular complications of sickle cell disease, like pulmonary hypertension This, and the current studies, suggest that there is a hemolysis-driven phenotype in sickle cell disease that now includes leg ulcers. While candidate genes that might modulate the rate of hemolysis have not yet been studied, we find that SNPs in genes that may be pathogenetically important in sickle vasculopathy are associated with the disease subphenotype of leg ulcer. Linking gene polymorphisms with disease subphenotypes, may eventually provide useful means of foretelling the likelihood of complications and allow better individualized treatment.
Previous studies have localized the gene for Waardenburg syndrome (WS) type I to the distal portion of chromosome 2q, near the ALPP locus. We pooled linkage data obtained from 41 WS type I and 3 WS ...type II families which were typed for six polymorphic loci on chromosome 2q in order to refine the location of the WS locus (WS1) and evaluate the extent of genetic heterogeneity. In the course of this work, we developed diagnostic criteria for genetic and phenotypic studies. Our findings, based on two-locus and multilocus analysis using a linkage map established from reference pedigrees, suggest that there are two or more mutations causing WS, one of which (i.e., WS1) is located on chromosome 2q, between the ALPP and FN1 loci, at distances of 7.8 cM and 11.2 cM for each marker, respectively. The results also indicate that WS1 is responsible for the illness in approximately 45% of all families in this sample. However, the odds favoring this position over a location between ALPP and SAG are only 2:1 when alternate assumptions about the proportion of linked families are considered. We conclude that a more saturated map of this region of chromosome 2q, including highly polymorphic markers, will be needed to accurately distinguish linked families and, ultimately, isolate the mutant gene.
Calcium pyrophosphate-deposition disease (CPDD), also called "chondrocalcinosis" or "pseudogout," is a disorder characterized by the deposition of calcium-containing crystals in joint tissue, which ...leads to arthritis-like symptoms. The presence of these crystals in joint tissue is a common finding in the elderly, and, in this population, there is a poor correlation with joint pain. In contrast, early-onset CPDD has been described in several large families in which the disease progresses to severe degenerative osteoarthritis (OA). In these families, an autosomal dominant mode of inheritance is observed, with an age at onset between the 2d and 5th decades of life. In this report, we describe a large New England family with early-onset CPDD and severe degenerative OA. We found genetic linkage between the disease in this family and chromosome 8q, with a multipoint lod score of 4.06. These results suggest that a defective gene at this location causes the disease in this family.
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