Mary J. Emond

Exome sequencing as a tool for Mendelian disease gene discovery

Exome sequencing — the targeted sequencing of the subset of the human genome that is protein coding — is a powerful and cost-effective new tool for dissecting the genetic basis of diseases and traits that have proved to be intractable to conventional gene-discovery strategies. Over the past 2 years, experimental and analytical approaches relating to exome sequencing have established a rich framework for discovering the genes underlying unsolved Mendelian disorders. Additionally, exome sequencing is being adapted to explore the extent to which rare alleles explain the heritability of complex diseases and health-related traits. These advances also set the stage for applying exome and whole-genome sequencing to facilitate clinical diagnosis and personalized disease-risk profiling.

Long-term survival of medically treated patients in the Coronary Artery Surgery Study (CASS) Registry.

BackgroundThis study describes the impact of clinical, angiographic, and demographic characteristics on the long-term survival of Coronary Artery Surgery Study (CASS) patients while they were under medical treatment. Revascularization rates for the population are also provided. Methods and ResultsAll CASS patients who had not received heart surgery before enrollment (23467 patients) were included in this survival analysis while they were under medical treatment or surveillance. Follow-up time ranged from 0 to 17 years (median, 12 years). Long-term vital status is known for 95.8% of these patients. Log-rank tests, Kaplan-Meier survival curves, and Cox proportional- hazards regression are used to describe and assess the impact of patient characteristics on survival. Characteristics that had a significant impact on survival, in order of observed explanatory power, are age, number of diseased vessels, congestive heart failure score, smoking history, ejection fraction, sex, presence of left main coronary artery disease, presence of diabetes, left ventricular wall motion score, presence of other illnesses, history of myocardial infarction, and presence of left main equivalent disease. Overall, 12-year survival for patients with zero-, one-, two- and three-vessel disease is 88%, 74%, 59%, and 40%, respectively. Twelve-year survival for patients with at least one diseased vessel and ejection fractions in the ranges of 50% to 100%, 35% to 49%, and 0% to 34% is 73%, 54%, and 21%, respectively. High myocardial jeopardy, high anginal class, and two or three proximal diseased vessels characterize the profile of patients most likely to have received surgical treatment during follow-up. ConclusionsThese results contribute to the understanding of the natural history of coronary artery disease and are also of historical interest. The poor survival of patients with three-vessel disease and low ejection fractions continues to emphasize the importance of considering revascularization for these patients.

Ursodiol Use Is Associated with Lower Prevalence of Colonic Neoplasia in Patients with Ulcerative Colitis and Primary Sclerosing Cholangitis

Patients with ulcerative colitis have an increased risk for colorectal neoplasia; this risk approaches 0.5% to 1% per year of disease. The extent and duration of colitis, as well as age at onset of disease, are important risk factors for neoplastic progression (1, 2). Primary sclerosing cholangitis is a chronic inflammatory disease of the biliary tract that occurs in 2% to 4% of patients with ulcerative colitis (3-5). Although the data are still controversial, most studies have shown that patients with ulcerative colitis and primary sclerosing cholangitis are at even higher risk for colonic neoplasia than are those with ulcerative colitis alone (6-11). The risk for colonic dysplasia or cancer in patients with ulcerative colitis and primary sclerosing cholangitis approaches 50% after 25 years of colitis (6-8). Epidemiologic studies suggest that environmental and dietary factors are important in the development of colorectal cancer (12). High-fat diets predispose to colorectal cancer, in part because they generate tumor-promoting secondary bile acids (13, 14). Conversely, the use of aspirin and other nonsteroidal anti-inflammatory drugs is associated with reduced risk for colorectal cancer and adenomatous polyps (15-17). Controversy surrounds the potential chemoprotective role of sulfasalazine in patients with ulcerative colitis (18, 19). Ursodiol, the 7--epimer of chenodeoxycholic acid, has been used in patients with primary sclerosing cholangitis because it substantially improves biochemical indices of liver function, although overall disease progression does not appear to be affected (20). Ursodiol use reduces the colonic concentration of the secondary bile acid deoxycholic acid (21). Although experimental evidence in animal models suggests that ursodiol administration inhibits colonic carcinogenesis, no clear evidence of inhibitory activity has been demonstrated in human studies (7, 22, 23). We sought to determine whether ursodiol use is associated with lower risk for colonic dysplasia, the precursor to colon cancer, in patients with ulcerative colitis and primary sclerosing cholangitis. Methods Patients We studied 59 patients with ulcerative colitis and primary sclerosing cholangitis who were enrolled in the colonoscopic surveillance program at the University of Washington, Seattle, Washington. These patients were involved in a research study of colonic surveillance in ulcerative colitis according to a protocol approved by the Human Subjects Division of the University of Washington. Forty-three patients were male and 16 were female. The diagnosis of primary sclerosing cholangitis was based on conventional cholangiographic criteria. Confirmatory histologic findings in the 35 patients who underwent liver biopsy included concentric periductal fibrosis, destructive inflammatory lesions of ducts, changes of ductal obstruction, or ductopenia ( 50% of at least 20 portal tracts without a duct). The diagnosis of ulcerative colitis was made according to standard histologic criteria: presence of basal lymphoplasmacytosis and distortion of crypt architecture, defined by the presence of two or more branched or irregularly shaped crypts in a single mucosal biopsy specimen. Clinical Variables Clinical variables were abstracted from the medical record and patient interview. Study groups were defined according to whether patients had ever used ursodiol. For all patients whose medical records failed to mention ursodiol use, confirmation was made by direct patient interview. All medications were carefully noted: specifically, the dates of use of ursodiol, sulfasalazine, other 5-aminosalicylic acid preparations, prednisone, azathioprine, cyclosporine, and methotrexate. Estrogen use and menstrual status were assessed in women. Age at onset of symptomatic colitis and date of diagnosis of sclerosing cholangitis were also noted. To estimate the severity of liver disease, ChildPugh class was calculated at the time of each surveillance colonoscopy. For patients who had previously undergone orthotopic liver transplantation, a separate classification was created to reflect the fact that they had had severe liver disease despite a normal current ChildPugh score. Surveillance Colonoscopy for Diagnosis of Dysplasia Colonoscopy was performed every 3 years unless indefinite or low-grade dysplasia or aneuploidy was detected, in which case-patients underwent yearly colonoscopy. Patients who had high-grade dysplasia were referred for colectomy. At colonoscopy, biopsy samples were obtained from flat mucosa (by using a large cupped biopsy forceps) from four quadrants at 10-cm intervals from the cecum through the descending colon and at 5-cm intervals in the sigmoid colon and rectum. Any mucosal irregularity or polyp was sampled separately. The mean number of biopsy samples obtained per colonoscopy was 46.8 1.5. The biopsy samples were oriented submucosal-side-down on monofilament plastic mesh and placed in Hollande fixative. All biopsy samples were step-serial sectioned, stained with hematoxylin and eosin, and evaluated by an experienced gastrointestinal pathologist who had no knowledge of the clinical history or colonoscopic findings. The histologic criteria for the diagnosis and grading of dysplasia established by the Inflammatory Bowel Disease/Dysplasia Morphology Study Group were applied, except that the category indefinite for dysplasia was not subdivided (24). Statistical Analysis Study groups were defined according to whether the patient had ever used ursodiol. The primary outcome studied was occurrence of colonic dysplasia. A patient was defined as having achieved the outcome if he or she was known to have dysplasia at any point up to the last surveillance. Hence, the fraction of patients with the dysplasia outcome represented the prevalence of ever having had a diagnosis of dysplasia. For the primary analysis, patients whose biopsies were read as indefinite for dysplasia were combined with those whose biopsies were negative for dysplasia. Two secondary analyses were performed. In the first, all patients classified as indefinite for dysplasia were excluded from the analysis. In the second, the outcome was changed to high-grade dysplasia rather than low-grade dysplasia and high-grade dysplasia. For all analyses, ursodiol use and other variables were assessed up to the time of the outcome or last surveillance. Other variables assessed were use of sulfasalazine, other 5-aminosalicylic acid preparations, prednisone, cyclosporine, azathioprine, or methotrexate; duration of sclerosing cholangitis; duration of ulcerative colitis; ChildPugh class; duration of ursodiol use; age at onset of colitis; and sex. Unpaired t-tests and the Fisher exact test were used to compare means and proportions, respectively. Unadjusted odds ratios were estimated directly from 2 2 tables for ursodiol use versus outcome status, and adjusted odds ratios were estimated by using logistic regression. MathSoft S-Plus 3.4 (MathSoft, Inc., Seattle, Washington) and GraphPad Prism 2.01 (GraphPad Software, San Diego, California) statistical software packages were used. Role of the Funding Source The funding source had no role in the collection, analysis, or interpretation of the data or in the decision to submit the study for publication. Results Fifty-nine patients with ulcerative colitis and primary sclerosing cholangitis were enrolled in the colonoscopic surveillance program. Forty-one patients (69%) had used ursodiol, and 18 (31%) had never used ursodiol. Characteristics of ursodiol users and nonusers are shown in Table 1. Twenty-six patients (44%) had a diagnosis of dysplasia at some time during surveillance; no patient developed cancer. Table 1. Patient Characteristics Of the 26 patients who had dysplasia, 50% (13 of 26) had used ursodiol compared with 85% (28 of 33) of patients with no dysplasia. This difference in ursodiol use was highly significant, with an odds ratio of 0.18 (CI, 0.05 to 0.61; P=0.005) (Table 2). The negative association between dysplasia and ursodiol use remained after adjustment for sex, age at onset of ulcerative colitis, duration of ulcerative colitis, duration of sclerosing cholangitis, ChildPugh class, and use of sulfasalazine (adjusted odds ratio, 0.14 [CI, 0.03 to 0.64]; P=0.01) (Table 3). Results were similar when treatment with 5-aminosalicylic acid, prednisone, cyclosporine, azathioprine, or methotrexate and number of surveillance colonoscopies were included in the model. Because a large number of covariates were included in the final logistic regression model relative to the sample size of 59, computer simulations were performed to check the stability of the modeling procedure. Both the Pvalue and the 95% confidence interval were reliable when the true odds ratio was assumed to be 0.14 to 1.0. Table 2. Medication Use and Development of Dysplasia Table 3. Multivariate Analysis of Risk Factors for Dysplasia Among patients who used ursodiol, the mean duration of use was 3.5 years for those who progressed to dysplasia compared with 4.2 years for those who did not progress to dysplasia (P>0.2). The mean dosage was 9.9 0.7 mg/kg per day in those who progressed to dysplasia and 8.9 0.5 mg/kg per day in those who did not progress to dysplasia (P>0.2). The relationship between total accumulated ursodiol dose (dose duration of use) and progression to dysplasia was not significant (P>0.2). Sixteen of 59 patients (27%) were indefinite for dysplasia at their most advanced colonic histologic stage. In a secondary analysis that excluded these 16 patients, ursodiol use was still negatively associated with colonic dysplasia (odds ratio, 0.21 [CI, 0.05 to 0.99]; P=0.05). After controlling for sex, age at onset of colitis, duration of colitis, duration of sclerosing cholangitis, ChildPugh class, and use of sulfasalazine, the adjusted odds ratio was 0.20 (CI, 0.03 to 1.5; P=0.12). Eleven of the 59 patients (19%) had a diagnosis of high-grade dysplasia duri

Optimal Unified Approach for Rare-Variant Association Testing with Application to Small-Sample Case-Control Whole-Exome Sequencing Studies

We propose in this paper a unified approach for testing the association between rare variants and phenotypes in sequencing association studies. This approach maximizes power by adaptively using the data to optimally combine the burden test and the nonburden sequence kernel association test (SKAT). Burden tests are more powerful when most variants in a region are causal and the effects are in the same direction, whereas SKAT is more powerful when a large fraction of the variants in a region are noncausal or the effects of causal variants are in different directions. The proposed unified test maintains the power in both scenarios. We show that the unified test corresponds to the optimal test in an extended family of SKAT tests, which we refer to as SKAT-O. The second goal of this paper is to develop a small-sample adjustment procedure for the proposed methods for the correction of conservative type I error rates of SKAT family tests when the trait of interest is dichotomous and the sample size is small. Both small-sample-adjusted SKAT and the optimal unified test (SKAT-O) are computationally efficient and can easily be applied to genome-wide sequencing association studies. We evaluate the finite sample performance of the proposed methods using extensive simulation studies and illustrate their application using the acute-lung-injury exome-sequencing data of the National Heart, Lung, and Blood Institute Exome Sequencing Project.

Chromosomal instability in ulcerative colitis is related to telomere shortening

Ulcerative colitis, a chronic inflammatory disease of the colon, is associated with a high risk of colorectal carcinoma that is thought to develop through genomic instability. We considered that the rapid cell turnover and oxidative injury observed in ulcerative colitis might accelerate telomere shortening, thereby increasing the potential of chromosomal ends to fuse, resulting in cycles of chromatin bridge breakage and fusion and chromosomal instability associated with tumor cell progression. Here we have used quantitative fluorescence in situ hybridization to compare chromosomal aberrations and telomere shortening in non-dysplastic mucosa taken from individuals affected by ulcerative colitis, either with (UC progressors) or without (UC non-progressors) dysplasia or cancer. Losses, but not gains, of chromosomal arms and centromeres are highly correlated with telomere shortening. Chromosomal losses are greater and telomeres are shorter in biopsy samples from UC progressors than in those from UC non-progressors or control individuals without ulcerative colitis. A mechanistic link between telomere shortening and chromosomal instability is supported by a higher frequency of anaphase bridges—an intermediate in the breakage and fusion of chromatin bridges—in UC progressors than in UC non-progressors or control individuals. Our study shows that telomere length is correlated with chromosomal instability in a precursor of human cancer.

Overexpression of Catalase Targeted to Mitochondria Attenuates Murine Cardiac Aging

Background— Age is a major risk for cardiovascular diseases. Although mitochondrial reactive oxygen species have been proposed as one of the causes of aging, their role in cardiac aging remains unclear. We have previously shown that overexpression of catalase targeted to mitochondria (mCAT) prolongs murine median lifespan by 17% to 21%. Methods and Results— We used echocardiography to study cardiac function in aging cohorts of wild-type and mCAT mice. Changes found in wild-type mice recapitulate human aging: age-dependent increases in left ventricular mass index and left atrial dimension, worsening of the myocardial performance index, and a decline in diastolic function. Cardiac aging in mice is accompanied by accumulation of mitochondrial protein oxidation, increased mitochondrial DNA mutations and deletions and mitochondrial biogenesis, increased ventricular fibrosis, enlarged myocardial fiber size, decreased cardiac SERCA2 protein, and activation of the calcineurin–nuclear factor of activated T-cell pathway. All of these age-related changes were significantly attenuated in mCAT mice. Analysis of survival of 130 mice demonstrated that echocardiographic cardiac aging risk scores were significant predictors of mortality. The estimated attributable risk to mortality for these 2 parameters was 55%. Conclusions— This study shows that cardiac aging in the mouse closely recapitulates human aging and demonstrates the critical role of mitochondrial reactive oxygen species in cardiac aging and the impact of cardiac aging on survival. These findings also support the potential application of mitochondrial antioxidants in reactive oxygen species–related cardiovascular diseases.

Homologous Recombination Resolution Defect in Werner Syndrome

ABSTRACT Werner syndrome (WRN) is an uncommon autosomal recessive disease whose phenotype includes features of premature aging, genetic instability, and an elevated risk of cancer. We used three different experimental strategies to show that WRN cellular phenotypes of limited cell division potential, DNA damage hypersensitivity, and defective homologous recombination (HR) are interrelated. WRN cell survival and the generation of viable mitotic recombinant progeny could be rescued by expressing wild-type WRN protein or by expressing the bacterial resolvase protein RusA. The dependence of WRN cellular phenotypes on RAD51-dependent HR pathways was demonstrated by using a dominant-negative RAD51 protein to suppress mitotic recombination in WRN and control cells: the suppression of RAD51-dependent recombination led to significantly improved survival of WRN cells following DNA damage. These results define a physiological role for the WRN RecQ helicase protein in RAD51-dependent HR and identify a mechanistic link between defective recombination resolution and limited cell division potential, DNA damage hypersensitivity, and genetic instability in human somatic cells.

Age-dependent cardiomyopathy in mitochondrial mutator mice is attenuated by overexpression of catalase targeted to mitochondria.

Mitochondrial defects have been found in aging and several age‐related diseases. Mice with a homozygous mutation in the exonuclease encoding domain of mitochondrial DNA polymerase gamma (Polgm/m) are prone to age‐dependent accumulation of mitochondrial DNA mutations and have shown a broad spectrum of aging‐like phenotypes. However, the mechanism of cardiac phenotypes in relation to the role of mitochondrial DNA mutations and oxidative stress in this mouse model has not been fully addressed. We demonstrate age‐dependent cardiomyopathy in Polgm/m mice, which by 13–14 months of age displays marked cardiac hypertrophy and dilatation, impairment of systolic and diastolic function, and increased cardiac fibrosis. This age‐dependent cardiomyopathy is associated with increases in mitochondrial DNA (mtDNA) deletions and protein oxidative damage, increased expression of apoptotic and senescence markers, as well as a decline in signaling for mitochondrial biogenesis. The relationship of these changes to mitochondrial reactive oxygen species (ROS) was tested by crossing Polgm/m mice with mice that overexpress mitochondrial targeted catalase (mCAT). All of the above phenotypes were partially rescued in Polgm/m/mCAT mice. These data indicate that accumulation of mitochondrial DNA damage with age can lead to cardiomyopathy and that this phenotype is partly mediated by mitochondrial oxidative stress.

Exome sequencing of extreme phenotypes identifies DCTN4 as a modifier of chronic Pseudomonas aeruginosa infection in cystic fibrosis

Exome sequencing has become a powerful and effective strategy for the discovery of genes underlying Mendelian disorders. However, use of exome sequencing to identify variants associated with complex traits has been more challenging, partly because the sample sizes needed for adequate power may be very large. One strategy to increase efficiency is to sequence individuals who are at both ends of a phenotype distribution (those with extreme phenotypes). Because the frequency of alleles that contribute to the trait are enriched in one or both phenotype extremes, a modest sample size can potentially be used to identify novel candidate genes and/or alleles. As part of the National Heart, Lung, and Blood Institute (NHLBI) Exome Sequencing Project (ESP), we used an extreme phenotype study design to discover that variants in DCTN4, encoding a dynactin protein, are associated with time to first P. aeruginosa airway infection, chronic P. aeruginosa infection and mucoid P. aeruginosa in individuals with cystic fibrosis.

Endoscopic management of biliary strictures in liver transplant recipients: Effect on patient and graft survival

BACKGROUND Biliary strictures in liver transplant recipients cause significant morbidity and can lead to reduced patient and graft survival. METHODS Of 251 liver transplant recipients, 22 patients with biliary strictures were categorized into two groups: donor hepatic duct (n = 12) or anastomotic (n = 10). Strictures were dilated and stented. Endoscopic therapy was considered successful if a patient did not require repeat stenting or dilation for 1 year. RESULTS Patient and graft survival did not differ significantly in the 22 patients compared with patients without strictures (relative risk of death and graft survival 1.8 and 1.3). Donor hepatic duct strictures required significantly longer therapy than anastomotic strictures (median days 185 versus 67, p = 0.02). Twenty-two months after the first endoscopic treatment, 73% of the donor hepatic duct stricture group were stent free compared with 90% of the anastomotic group (p = 0.02). The former group had significantly more (p < 0.05) hepatic artery thrombosis (58.3% versus 10%), cholangitis (58.3% versus 30%), choledocholithiasis (91% versus 10%), and endoscopic interventions. No patient undergoing endoscopic treatment required retransplantation or biliary reconstruction during a median follow-up of 35.7 months. CONCLUSION Endoscopic therapy of biliary strictures after liver transplantation is effective and is not accompanied by reduced patient or graft survival.