Leveraging genetically simple traits to identify small-effect variants for complex phenotypes.

BACKGROUND: Polymorphisms underlying complex traits often explain a small part (less than 1 %) of the phenotypic variance (σ2P). This makes identification of mutations underling complex traits difficult and usually only a subset of large-effect loci are identified. One approach to identify more loci is to increase sample size of experiments but here we propose an alternative. The aim of this paper is to use secondary phenotypes for genetically simple traits during the QTL discovery phase for complex traits. We demonstrate this approach in a dairy cattle data set where the complex traits were milk production phenotypes (fat, milk and protein yield; fat and protein percentage in milk) measured on thousands of individuals while secondary (potentially genetically simpler) traits are detailed milk composition traits (measurements of individual protein abundance, mineral and sugar concentrations; and gene expression). RESULTS: Quantitative trait loci (QTL) were identified using 11,527 Holstein cattle with milk production records and up to 444 cows with milk composition traits. There were eight regions that contained QTL for both milk production and a composition trait, including four novel regions. One region on BTAU1 affected both milk yield and phosphorous concentration in milk. The QTL interval included the gene SLC37A1, a phosphorous antiporter. The most significant imputed sequence variants in this region explained 0.001 σ2P for milk yield, and 0.11 σ2P for phosphorus concentration. Since the polymorphisms were non-coding, association mapping for SLC37A1 gene expression was performed using high depth mammary RNAseq data from a separate group of 371 lactating cows. This confirmed a strong eQTL for SLC37A1, with peak association at the same imputed sequence variants that were most significant for phosphorus concentration. Fitting any of these variants as covariables in the association analysis removed the QTL signal for milk production traits. Plausible causative mutations in the casein complex region were also identified using a similar strategy. CONCLUSIONS: Milk production traits in dairy cows are typical complex traits where polymorphisms explain only a small portion of the phenotypic variance. However, here we show that these mutations can have larger effects on secondary traits, such as concentrations of minerals, proteins and sugars in the milk, and expression levels of genes in mammary tissue. These larger effects were used to successfully map variants for milk production traits. Genetically simple traits also provide a direct biological link between possible causal mutations and the effect of these mutations on milk production.

Bioprospecting keratinous materials.

The concept of bioprospecting for bioactive peptides from keratin-containing materials such as wool, hair, skin and feathers presents an exciting opportunity for discovery of novel functional food ingredients and nutraceuticals, while value-adding to cheap and plentiful natural sources. The published literature reports multiple examples of proline-rich peptides with productive bio-activity in models of human disease including tumour formation, hypertension control and Alzheimer's disease. Bioactive peptides have been identified from food and other protein sources however the bioactivity of keratin-related proteins and peptides is largely unknown. Considering the high representation of proline-rich peptides among proven bioactive peptides, the proline-rich character of keratinous proteins supports current research. A selection of mammalian (cow epidermis, sheep wool) and avian (chicken feather) keratinous materials were subjected to enzymatic hydrolysis using established processing methods. A bio-assay of determining inhibition of early stage amyloid aggregation involved using a model fibril-forming protein - reduced and carboxymethylated bovine K-casein (RCMk-CN) and quantitation of fibril development with the amyloid-specific fluorophore, Thioflavin T (ThT). The assay was fully validated for analytical repeatability and used together with appropriate positive controls. Peptide library products derived from chicken feather (n=9), sheep wool (n=9) and bovine epidermis (n=9) were screened in the fibril inhibition assay based on K-casein. 3 of 27 products exhibited interesting levels of bio-activity with regard to fibril inhibition. HPLC profiles provide an indication of the complexity of the assemblage of peptides in the three active products. We conclude the bioprospecting research using keratinous materials shows promise for discovery of useful bioactive peptides.

Effect of temperature on heart rate in diploid and triploid brook charr, Salvelinus fontinalis, embryos and larvae.

Increased cell size in triploid fish likely affects rates of respiratory gas exchange. Respiratory deficiencies can be addressed in fish by adjustments in cardiac output, through changes in heart rate and stroke volume. The aim of this study was to determine whether heart rate differs between triploid and control (diploid) brook charr, Salvelinus fontinalis, at embryo-larval stages, when the heart is easily visible and the fish are relatively inactive. Heart rate was measured at 6, 9 and 12 degrees C at three developmental stages: eyed-egg, hatch and yolk absorption. Heart rate was unaffected by ploidy, but increased with temperature and age from a low of 43.4+/-2.2 beats/min (6 degrees C, eyed egg) to a high of 73.3+/-1.5 beats/min (12 degrees C, yolk absorption). The Q(10) for heart rate was unaffected by ploidy and age, but decreased with temperature from 1.99+/-0.28 at 6-9 degrees C to 1.72+/-0.17 at 9-12 degrees C. Triploid brook charr thus do not use adjustments in heart rate as a mechanism to deal with the physiological consequences of altered haematology at embryo-larval stages.

Mycophenolate mofetil versus azathioprine in patients surviving the initial cardiac transplant hospitalization: an analysis of the Joint UNOS/ISHLT Thoracic Registry.

BACKGROUND: Although the randomized mycophenolate mofetil- (MMF) azathioprine (AZA) trial is likely applicable to cardiac transplantation in general, it was limited to select and usually larger cardiac transplant centers and suffered from substantial cross-over and failure of many patients to receive assigned treatment drug. METHODS: The Joint ISHLT/UNOS Thoracic Registry was analyzed for the effects of MMF versus AZA in patients 1) on a cyclosporine- (CsA) based immunosuppression protocol; 2) having survived long enough to be discharged from the transplant hospitalization. RESULTS: A total of 5599 patients (4942 CsA/AZA and 657 CsA/MMF) were included with no significant differences between the MMF and AZA groups in baseline characteristics with the exception of recipient age (50 vs. 47 years), donor age (29 vs. 28 years), ischemic time (3.0 vs. 2.9 hr), and pretransplant medical condition (more AZA patients in ICU, more MMF patients on VAD). Actuarial survival was greater in the MMF group compared to the AZA group in patients surviving the initial transplant hospitalization (1 year 96 vs. 93%, 3 years 91 vs. 86%, P=0.0012). This difference was confirmed in the logistic regression analysis of 3-year mortality showing a relative risk of 0.62 (P=0.011). CONCLUSIONS: These data provide independent support for the broad applicability of the positive results from the randomized MMF-AZA clinical trial in a substantially larger patient population and confirm improved survival in patients using mycophenolate mofetil compared to azathioprine late after cardiac transplantation.

Single vs bilateral, sequential lung transplantation for end-stage emphysema: influence of recipient age on survival and secondary end-points.

BACKGROUND: The appropriate age to perform bilateral, sequential lung transplants (BSLT) in patients with chronic obstructive pulmonary disease (COPD) remains controversial. Although single lung transplant (SLT) offers an advantage in terms of organ availability, the long-term survival may not warrant this strategy in all age groups. METHODS: We analyzed 2,260 lung transplant recipients (1835 SLT, 425 BSLT) with COPD recorded in the International Society for Heart and Lung Transplantation/United Network for Organ Sharing thoracic registry between January 1991 and December 1997. To assess mortality, we performed univariate (Kaplan-Meier method and the chi-square statistic) and multivariate analyses (proportional hazards method). Because of incomplete morbidity data in the international registry, only data from U.S. centers (n = 1778, 1467 SLT, 311 BSLT) were used in the morbidity analysis. RESULTS: Survival rates (%) computed using the Kaplan-Meier method at 30 days, 1 year, and 5 years for the patients aged < 50 years were 93.6, 80.2, and 43.6, respectively, for the SLT patients, and 94.9, 84.7, and 68.2, respectively, for the BSLT patients. For patients aged 50 to 60 years, survival rates (%) were 93.5, 79.4, and 39.8 for the SLT patients compared with 93.0, 79.7, and 60.5 for the BSLT patients. For those aged > 60 years, SLT survival (%) was 93.0, 72.9, and 36.4, compared with 77.8 and 66.0 for the BSLT group (a 5-year rate could not be completed in this group). The multivariate model showed a higher risk ratio for mortality in patients aged 40 to 57 years who received SLT vs BSLT. Recipient age and procedure type did not appear to affect the development of rejection, bronchiolitis obliterans, bronchial stricture, or lung infection. CONCLUSIONS: Single lung transplant may offer acceptable early survival for patients with end-stage respiratory failure. However, long-term survival data favors BSLT in recipients until approximately age 60 years. These data suggest that a BSLT approach offers a significant survival advantage to recipients younger than 60 years of age.

Worldwide thoracic organ transplantation: a report from the UNOS/ISHLT international registry for thoracic organ transplantation.

Based on data reported to the UNOS/ISHLT Thoracic and International Registry for Thoracic Organ Transplantation: 1. The number of heart transplant operations performed in the United States decreased between 1998-1999 and 17 (1%) more procedures were performed in 1999 (2,181) than in 2000 (2,198). Sixty-nine more lung transplants (an 8% increase) were reported in 2000 than in 1999. 2. Coronary artery disease and cardiomyopathy were the most frequently cited indications for heart transplantation in the US and have been reported at similar rates during the past 10 years. Combined, these diagnoses account for approximately 85% of all heart transplants. In 2000, half of all lung transplants were performed for emphysema/COPD or alpha-1 antitrypsin deficiency. The most frequently reported diagnoses for thoracic transplantation outside the US were: cardiomyopathy (49%) for heart, cystic fibrosis (30%) for double lung, emphysema/COPD (34%) for single lung and primary pulmonary hypertension (21%) for heart-lung transplants. 3. US heart transplant recipients were predominately male (76%), aged 50-64 (51%) and white (81%). US lung transplant recipients were also predominately between ages 50-64 (47%) and white (90%), but unlike heart recipients were more likely to be female (51%). No meaningful variance from the US recipient demographic profile was noted for the non-US recipients during the same time period. 4. Pediatric recipients (< 18 years of age) received 11% of the reported heart transplants and 6% of the reported lung transplants in the US. 5. Among US thoracic transplant recipients during 1999, the one-year survival rates were 84% for heart, 59% for heart-lung and 77% for lung. The 5-year survival rates for transplants performed during 1995 were: 71% for heart, 56% for heart-lung and 44% for lung transplants. 6. The long-term patient survival rates were: 23% at 19 years for heart, 16% at 11 years for lung and 23% at 14 years for heart-lung recipients. 7. During the first year after transplantation, 66% of heart recipients and 44% of lung recipients did not require rehospitalization. Among those recipients who were rehospitalized, the major cause was infection.

Effect of donor age and ischemic time on intermediate survival and morbidity after lung transplantation.

BACKGROUND: Pressure to expand the donor pool has required the use of lungs from older donors or from more-distant procurement areas. The long-term consequences of this policy have not yet been fully addressed. The effect of donor age and donor ischemic time on intermediate survival and important secondary end points after lung transplantation was therefore examined. METHODS: A cohort of 1,800 lung transplant recipients with complete 2-year follow-up, operated on in the United States between April 1, 1993, and March 31, 1996, was studied to assess survival. For analysis of secondary end points, the cohort was limited to 1,450 patients. RESULTS: Donor age when analyzed independently did not significantly affect intermediate survival (p = 0.4). Secondary end points were also not affected by age, with the exception of the incidence of hospitalization for rejection in the univariate analysis (p = 0.02) and in the multivariate analysis (p = 0.04). Moreover, there was not a significant impact of donor age or ischemic time independently on survival in the multivariate analysis. Similarly, when the interaction between ischemic time and donor age was examined in all of the multivariate models, none of the secondary end points were found to be significantly influenced. However, the combined interaction between donor age and ischemia time demonstrated a significantly worse survival at 2 years (p = 0.02) with donor age of > 50 years and donor ischemic time > 7 h. CONCLUSIONS: Donor age and donor ischemic time did not independently influence survival or important secondary end points after lung transplantation. However, intermediate-term survival was affected by the use of older donors when combined with a prolonged ischemic time. The impact of this combination should be considered when attempting to expand the donor pool.

The results of cardiac retransplantation: an analysis of the Joint International Society for Heart and Lung Transplantation/United Network for Organ Sharing Thoracic Registry.

BACKGROUND: It is well established that repeat heart transplantation has a significantly worse outcome when compared with primary (first time) transplantation. Defining the risk factors for mortality within this group has been difficult due to small numbers of patients at individual centers. METHODS: All cardiac retransplants performed in the United States and registered in the Joint International Society for Heart and Lung Transplantation (ISHLT)/United Network for Organ Sharing (UNOS) Thoracic Registry were analyzed for demographics, morbidity posttransplantation, immunosuppression, and risk factors for mortality. RESULTS: The study cohort included 514 patients of which 81% were male with a mean age of 47+/-12 years. Time from primary transplant to retransplantation ranged from 1 day to 15.5 years and more than 50% of the patients underwent retransplantation for chronic rejection. More than 60% of patients were in the intensive care unit at the time of retransplantation and more than 40% of the patients were reported to be on some form of life support (ventricular assist device, ventilator, and/or inotropic therapy). Survival for the entire retransplant cohort was 65, 59, and 55% for 1, 2, and 3 years, respectively, but was substantially lower when the intertransplant interval was short. Conversely, when the interval between primary and retransplantation was more than 2 years, 1 year survival postretransplantation approached that of primary transplantation. Additional independent risk factors for mortality for the retransplant cohort included overall cardiac transplant center volume, the use of a ventricular assist device or ventilator, the patient being in the intensive care unit, and recipient age. The four most common causes of death were infection, primary/nonspecific graft failure, chronic rejection (allograft vasculopathy), and acute rejection. CONCLUSIONS: The data confirm that repeat heart transplantation is a higher risk procedure than primary transplantation, especially early after the primary heart transplant.

Does human leukocyte antigen matching influence the outcome of lung transplantation? An analysis of 3,549 lung transplantations.

BACKGROUND AND OBJECTIVE: Human leukocyte antigen (HLA) compatibility has been shown to improve the outcome of renal and cardiac transplantation. However, its impact on outcome following lung transplantation is not clear, with several single-center studies reporting inconsistent results. We studied the influence of HLA matching on survival and the development of rejection and obliterative bronchiolitis after lung transplantation, using data from the United Network for Organ Sharing/International Society for Heart and Lung Transplantation registry. METHODS: The study population included adult patients who received cadaveric lung transplants between October 1987 and June 1997 for whom HLA data were available. Two cohorts were examined, depending on the era of transplantation: (1) October 1987 to June 1997 (n = 3,549): Differences in actuarial survival as stratified by either the total number of HLA mismatches or the number of mismatches at each HLA locus were determined using a log-rank test. Multivariate logistic regression models were developed to determine independent predictors of survival at 1, 3, and 5 years following lung transplantation. (2) April 1994 to June 1997 (n = 1,796): The association of HLA mismatching with acute rejection and obliterative bronchiolitis was determined using a chi-squared analysis. RESULTS: Only 164 patients (4.6%) received lung grafts with 2 or fewer HLA mismatches. Univariate analyses demonstrated a significant difference in post-transplant survival by mismatch level, with the total number of HLA mismatches (p = 0.0008) and mismatching at the HLA-A locus (p = 0.002) associated with worse survival. Multivariate logistic regression demonstrated that the number of mismatches at the HLA-A and HLA-DR loci predicted 1-year mortality (incremental odds ratios 1.18, p = 0.01, and 1.15, p = 0. 03, respectively). The total number of HLA mismatches predicted 3- and 5-year mortality (incremental odds ratios 1.13 at 3 years, p = 0. 0004, and 1.14 at 5 years, p = 0.0002). However, other covariates such as repeat transplantation, transplantation for congenital heart disease, advanced recipient age, and an early era of transplantation were stronger predictors of mortality. We found no significant association between HLA mismatching and the development of obliterative bronchiolitis, although there was an association between mismatching at the HLA-A locus and acute rejection episodes requiring hospital admission (p = 0.008). We also found no association between mismatching at the HLA-B locus and rejection episodes requiring either hospitalization or the alteration of anti-rejection medications (p = 0.034). CONCLUSION: Although the number of HLA mismatches at the HLA-A and HLA-DR loci predicted 1-year mortality and the total number of mismatches predicted 3- and 5-year mortality following lung transplantation, the effect of each covariate was small in this multicenter study of 3,549 patients. Further close follow-up of registry patients is necessary to determine the effect of HLA matching on long-term survival and freedom from obliterative bronchiolitis and rejection following lung transplantation. A prospective study of HLA matching for lung transplantation should not yet be considered in view of the small number of grafts with 2 or fewer mismatches and the modest effect of HLA matching on outcome.

Worldwide thoracic organ transplantation: a report from the UNOS/ISHLT International Registry for Thoracic Organ Transplantation.

Based on data reported to the UNOS/ISHLT International Registry for Thoracic Organ Transplantation, we showed that: 1. The number of heart transplant operations performed in the United States has decreased by 164 procedures between 1998 (2,346) and 1999 (2,182). The number of lung transplants increased by 13 in 1999 to 877. 2. The most frequently reported indication for heart transplantation in the US is coronary artery disease (44.8%). For other thoracic transplants, the most frequently reported indications include cystic fibrosis (35.5%) for double lung, emphysema/COPD (49.7%) for single lung and congenital heart disease (46.6%) for heart-lung. The most frequently reported diagnoses for thoracic transplantation outside the US include cardiomyopathy (43.8%) for heart, cystic fibrosis (33.4%) for double-lung, emphysema/COPD (26.6%) for single-lung and primary pulmonary hypertension (24.8%) for heart-lung transplants. 3. US heart transplant recipients are predominately male (76.7%), between 50 and 64 years of age (51.3%) and white (81.4%). US lung transplant recipients are also predominately between 50 and 64 years of age (44.7%) and white (89.9%), but unlike heart recipients are more likely to be female (51.2%). No meaningful variance from the US recipient demographic profile is noted for the non-US recipients during the same time period. 4. Pediatric recipients (< 18 years of age) received 10.9% of the reported heart transplants and 6.2% of reported lung transplants. 5. One-year survival for thoracic transplants performed in the US is 82.4% for heart, 74.1% for lung and 62.0% for heart-lung. Five-year survival for US thoracic transplants is 66.8% for heart and 43.2% for lung. 6. Long-term patient survival rates are: 22.5% at 17 years for heart, 20.8% at 10 years for lung and 24.3% at 13 years for heart-lung recipients. 7. The most important risk factor for mortality of US heart recipients at one month, one year and conditionally at 5 years after transplantation was receipt of a previous heart transplant. Significant short-term risk factors include donor age, recipient age and ischemic time. Substantial long-term risk factors include older donor age, recipient age, recipient race and diagnosis. 8. The factors having the most significant impact on lung mortality at all time points are related to either the patient's medical condition (e.g., in the ICU prior to transplant, requiring mechanical ventilation) or diagnosis. 9. Mechanical ventilation, recipient race and recipient age have the largest impact on heart-lung mortality. 10. For heart and lung recipients, the major cause of hospitalization during the first year after transplantation is infection alone.

Influence of graft ischemic time and donor age on survival after lung transplantation.

BACKGROUND: Increased graft ischemic time and donor age are risk factors for early death after heart transplantation, but the effect of these variables on survival after lung transplantation has not been determined in a large, multinational study. METHODS: All recipients of cadaveric lung transplantations performed between October 1, 1987 and June 30, 1997 which were reported to the United Network for Organ Sharing/International Society for Heart and Lung Transplantation (UNOS/ISHLT) Registry were analyzed. Patient survival rates were estimated using Kaplan-Meier methods. Multivariate logistic regression was used to determine the impact of donor and recipient characteristics on patient survival after transplantation. To examine whether the impact of donor age varied with ischemic time, interactions between the 2 terms were examined in a separate multivariate logistic regression model. RESULTS: Kaplan-Meier survival did not differ according to the total lung graft ischemia time, but recipient survival was significantly adversely affected by young (-10 years) or old (-51 years) donor age (p = 0.01). On multivariate analysis, neither donor age nor lung graft ischemic time per se were independent predictors of early survival after transplantation, except if quadratic terms of these variables were included in the model. The interaction between donor age and graft ischemia time, however, predicted 1 year mortality after lung transplantation (p = 0.005), especially if donor age was greater than 55 years and ischemic time was greater than 6 to 7 hours. CONCLUSIONS: Graft ischemia time alone is not a risk factor for early death after lung transplantation. Very young or old donor age was associated with decreased early survival, whereas the interaction between donor age and ischemic time was a significant predictor of 1 year mortality after transplantation. Cautious expansion of donor acceptance criteria (especially as regards ischemic time) is advisable, given the critical shortage of donor lung grafts.

Worldwide thoracic organ transplantation: a report from the UNOS/ISHLT International Registry for Thoracic Organ Transplantation.

1. The number of heart transplant operations performed in the US has increased by 51 procedures between 1997 (2,294) and 1998 (2,345). The number of lung transplants decreased by 67 in 1998 (862). 2. The most frequently reported indication for heart transplantation in the US is coronary artery disease (44.6%). For other thoracic transplants, the most frequently reported indications include other/unknown (43.9%) for double lung, emphysema/COPD (53.5%) for single lung and other/unknown (53.2%) for heart-lung. The most frequently reported diagnoses for thoracic transplantation outside the US include cardiomyopathy (50.5%) for heart, cystic fibrosis (32.0%) for double lung, idiopathic pulmonary fibrosis (32.7%) for single lung and congenital heart disease (24.7%) for heart-lung. 3. US heart transplant recipients were predominately male (77%), between 50-64 years old (51.4%) and White (81.7%). In contrast, US lung transplant recipients are predominantly female (51.3%), between 50-64 years of age (44.7%) and White (89.7%). No meaningful variance from the US recipient demographic profile was noted for the non-US recipients during the same time period. 4. Pediatric recipients (< 18 years of age) received 10.9% of the reported heart transplants and 6.5% of reported lung transplants. 5. One-year survival for thoracic transplants performed in the US was 83.2% for heart, 70.6% for lung and 62.5%. Five-year survival for US thoracic transplants was 70% for heart and 49.1% for lung. 6. Long-term patient survival rates were: 22.3% at 18 years for heart, 20% at 9 years for lung and 25% at 12 years for heart-lung recipients. 7. The most important risk factor for mortality of US heart recipients at one month, one and 5 years after transplantation was receipt of a previous heart transplant. Significant short-term risk factors included donor age, recipient age and ischemic time. Substantial long-term risk factors include older donor age, donor race and recipient race. 8. The factors having the most significant impact on lung mortality at all time points were related to either the patient's medical condition (e.g., in the ICU prior to transplant, requiring mechanical ventilation) or diagnosis. 9. Mechanical ventilation and previous transplant had the largest impact on heart-lung mortality. 10. For heart and lung recipients, the major cause of hospitalization during the first posttransplant year was infection.

Transplantation with older donor hearts for presumed "stable" recipients: an analysis of the Joint International Society for Heart and Lung Transplantation/United Network for Organ Sharing Thoracic Registry.

BACKGROUND: We have previously demonstrated that the use of older donor hearts (>45 years) increases the odds of death nearly twofold in the early posttransplantation period when compared with the use of hearts from younger donors. Before excluding this segment of the donor pool, however, the mortality risk for remaining on the waiting list compared with that of receiving an older donor heart should also be considered. METHODS: We examined all adult status 2 patients added to the United Network for Organ Sharing heart transplant waiting list for primary transplantation between 1992 and 1995 (n = 4681). To account for the transient increased risk after transplantation, we used a time-dependent nonproportional hazards model with an exponential decay component for the analysis. For patients with an equal time since listing, the resulting risk ratios represent the ratio of mortality risk for a patient who receives an older donor heart to the mortality risk for a patient who remains on the waiting list. RESULTS: After 30 days posttransplantation, the risk of death for recipients of 45- to 49-year-old donor hearts was lower than if they had remained on the waiting list, and by 6 months the relative risk was 0.37 (95% confidence interval: 0.22, 0.62). For recipients of hearts from donors 50 years or older, the risk after transplantation was lower after 64 days, and by 6 months the relative risk was 0.48 (95% confidence interval: 0.31, 0.75). CONCLUSION: These results suggest that in spite of a high initial risk resulting from the transplant procedure, there was a clear long-term survival benefit for status 2 recipients of older donor hearts. Thus overall, in spite of the increased risk of death associated with receiving older donor hearts, the risk of death without a transplant was even greater. On the basis of this analysis we cannot support the exclusion of older donors from the donor pool.