Impact of Race on Classification of Atherosclerotic Risk Using a National Cardiovascular Risk Prediction Tool.

Introduction: The use of race in clinical risk prediction tools may exacerbate racial disparities in healthcare access and outcomes. This study quantified the number of individuals reclassified for primary prevention of cardiovascular disease owing to a change in their race alone on the basis of a commonly used risk prediction tool. Methods: This is a cross-sectional analysis of individuals aged 40-75 years without a history of cardiovascular events, diabetes, or other high-risk features using the 2005-2018 National Health and Nutritional Examination Survey. Authors compared atherosclerotic cardiovascular disease risk scores using the American Heart Association/American College of Cardiology equation recommended for White individuals or individuals of other races with that recommended for Black individuals. Results: A total of 2,946 White individuals; 1,361 Black individuals; and 2,495 individuals of other races were included in the analysis. Using the American Heart Association/American College of Cardiology equation, the mean 10-year atherosclerotic cardiovascular disease risk was 5.80% (95% CI=5.54, 6.06) for White individuals, 7.04% (956% CI=6.69, 7.39) for Black individuals, and 4.93% (95% CI=4.61, 5.24) for individuals of other races. When using the American Heart Association/American College of Cardiology equation designated for the opposite race (White/other race versus Black), the mean atherosclerotic cardiovascular disease risk score increased by 1.02% (95% CI=0.90, 1.13) for White individuals, decreased by 1.82% (95% CI= -1.67, -1.96) for Black individuals, and increased by 0.98% (95% CI=0.85, 1.10) for individuals of other races. When using clinical atherosclerotic cardiovascular disease categories of <7.5%, 7.5%-10%, and >10%, 16.93% of all individuals were reclassified when using the American Heart Association/American College of Cardiology's equation designated for the opposite race. Conclusions: Changing race within a commonly used cardiovascular risk prediction tool results in significant changes in risk classification among eligible White and Black individuals in the U.S.

Assessment of the U.S. Food and Drug Administration's risk evaluation and mitigation strategy (REMS) for prasugrel (EFFIENT): A narrative review.

Background: Prasugrel, first approved in 2009, was subject to a US Food and Drug Administration (FDA) Risk Evaluation and Mitigation Strategy (REMS) to mitigate the risk of bleeding associated with its use. Methods: We performed a narrative review of FDA documents obtained through a Freedom of Information Act request. Document classification and primary evidence extraction was performed by three authors (TM, JC, and SL). Results: The prasugrel REMS consists of a medication guide and a communication plan. Assessment of the REMS was via patient and clinician surveys. 1560 patients were invited to participate and 212 individuals (13.6 %) completed the survey. Rates of awareness among respondents varied across key messages and were highest for those examining the risks of premature discontinuation (96 % and 88 % of respondents), while lower for those regarding the importance of perioperative discontinuation (66 %) and contraindications posed by a history of stroke (16 %) or transient ischemic attack (17 %). Of the 6000 clinicians invited to participate in the survey, 201 (3.4 %) agreed to take part. Four of 11 key risk messages did not meet prespecified acceptable levels of comprehension. No prespecified levels of patient or provider knowledge were required for the retirement of the REMS, which took place on March 23, 2012 based on the sponsor's request. Conclusions: The prasugrel REMS consisted of passive educational materials whose adequacy was evaluated using highly limited, one-time, cross-sectional surveys. Our assessment adds to evidence suggesting the importance of improving the quality and impact of the FDA's post-approval activities to maximize drug safety.

Characteristics of Prescription Drug Fills Using Pharmacy-Pharmacy Benefit Manager Discount Programs: The "GoodRx" Model.

OBJECTIVES: This study aimed to characterize products using pharmacy-pharmacy benefit manager (PBM) discounts and to estimate the association among such discounts, prescription utilization, and out-of-pocket costs. METHODS: This is a retrospective cohort study using IQVIA's Formulary Impact Analyzer, which contains anonymized, individual-level pharmacy claims representing US retail pharmacy transactions. We focused on 20 products with the greatest number of transactions using a pharmacy-PBM discount. Our unit of analysis was a treatment episode, defined as the length of time from an incident fill to no continuous use for 60 consecutive days after allowing for indefinite stockpiling. Outcome measures included products with greatest pharmacy-PBM discount use, characteristics of treatment episodes, and out-of-pocket costs with and without pharmacy-PBM discount. RESULTS: Across all products, 3.82% of transactions and 7.69% of treatment episodes were accompanied by a pharmacy-PBM discount. Commonly discounted products included generic treatments for chronic disease (lisinopril, levothyroxine, metformin) and neuropsychiatric conditions (alprazolam, amphetamine, buprenorphine, hydrocodone). The median postdiscount out-of-pocket cost was >2.5-fold higher during treatment episodes with a discount than those without ($15.15, interquartile range [IQR] $8.53-32.00, vs $5.88, IQR $1.40-15.00). Median treatment episode duration was 249 days (IQR 132-418) with discount use compared with 236 days (IQR 121-396) without discount use, although treatment episodes that began with a discount had fewer transactions per treatment episode and were shorter (median 212 days, IQR 114-360) than those that did not (313 days, IQR 178-500). CONCLUSIONS: Pharmacy-PBM discounts may foster market competition and improve access for under- and uninsured individuals; however, these programs may not generate savings for many insured individuals.

Potential drug-drug interactions among U.S. adults treated with nirmatrelvir/ritonavir: A cross-sectional study of the National Covid Cohort Collaborative (N3C).

STUDY OBJECTIVE: To estimate the prevalence of potential moderate to severe drug-drug interactions (DDIs) involving nirmatrelvir/ritonavir, identify interacting medications, and evaluate risk factors associated with potential DDIs. DESIGN: Cross-sectional study. DATA SOURCE: Electronic health records from the National COVID Cohort Collaborative Enclave, one of the largest COVID-19 data resources in the United States. PATIENTS: Outpatients aged ≥18 years and started nirmatrelvir/ritonavir between December 23, 2021 and March 31, 2022. INTERVENTION: Nirmatrelvir/ritonavir. MEASUREMENTS: The outcome is potential moderate to severe DDIs, defined as starting interacting medications reported by National Institutes of Health 30 days before or 10 days after starting nirmatrelvir/ritonavir. MAIN RESULTS: Of 3214 outpatients who started nirmatrelvir/ritonavir, the mean age was 56.8 ± 17.1 years, 39.5% were male, and 65.8% were non-Hispanic white. Overall, 521 (16.2%) were potentially exposed to at least one moderate to severe DDI, most commonly to atorvastatin (19.2% of all DDIs), hydrocodone (14.0%), or oxycodone (14.0%). After adjustment for covariates, potential DDIs were more likely among individuals who were older (odds ratio [OR] 1.16 per 10-year increase, 95% confidence interval [CI] 1.08-1.25), male (OR 1.36, CI 1.09-1.71), smokers (OR 1.38, CI 1.10-1.73), on more co-medications (OR 1.35, CI 1.31-1.39), and with a history of solid organ transplant (OR 3.63, CI 2.05-6.45). CONCLUSIONS: One in six of individuals receiving nirmatrelvir/ritonavir were at risk of a potential moderate or severe DDI, underscoring the importance of clinical and pharmacy systems to mitigate such risks.

Estimated Use of Prescription Medications Among Individuals Incarcerated in Jails and State Prisons in the US.

Importance: Although incarcerated individuals experience higher rates of chronic conditions, little is known regarding the use of prescription medications in jails and prisons in the US. Objective: To characterize treatment with prescription medications in jails and state prisons relative to noncorrectional settings in the US. Design, Setting, and Participants: This cross-sectional analysis using 2018 to 2020 data from the National Survey on Drug Use and Health (NSDUH) estimated the prevalence of disease among recently incarcerated and nonincarcerated adults in the US. The study used 2018 to 2020 IQVIA's National Sales Perspective (NSP) to quantify the distribution of medications to incarcerated and nonincarcerated populations. The NSP provides national dollar and unit sales of prescription medications across multiple distribution channels, including prisons and jails. The study population included incarcerated and nonincarcerated individuals from NSDUH. Seven common chronic conditions were assessed. Data were analyzed in May 2022. Exposures: Medications being sent to correctional facilities vs all other settings in the US. Main Outcomes and Measures: The main outcomes were distribution of medications to treat diabetes, asthma, hypertension, hepatitis B and C, human immunodeficiency virus (HIV), depression, and severe mental illness to incarcerated and nonincarcerated populations. Results: The proportion of pharmaceuticals distributed to jails and state prisons to treat type 2 diabetes (0.15%), asthma (0.15%), hypertension (0.18%), hepatitis B or C (1.68%), HIV (0.73%), depression (0.36%), and severe mental illness (0.48%) was much lower compared with the relative burden of disease among this population. The incarcerated population in state prisons and jails accounted for 0.44% (95% CI, 0.34%-0.56%) of estimated individuals with diabetes, 0.85% (95% CI, 0.67%-1.06%) of individuals with asthma, 0.42% (95% CI, 0.35%-0.51%) of hypertension, 3.13% (95% CI, 2.53%-3.84%) of hepatitis B or C, 2.20% (95% CI, 1.51%-3.19%) of HIV, 1.46% (95% CI, 1.33%-1.59%) of depression, and 1.97% (95% CI, 1.81%-2.14%) of severe mental illness. After adjusting for disease prevalence, the relative disparity was 2.9-fold for diabetes, 5.5-fold for asthma, 2.4-fold for hypertension, 1.9-fold for hepatitis B or C, 3.0-fold for HIV, 4.1-fold for depression, and 4.1-fold for severe mental illness. Conclusions and Relevance: In this cross-sectional, descriptive study of the distribution of prescription medications for chronic conditions in jails and state prisons, the findings suggest that there may be underuse of pharmacological treatment in correctional facilities relative to the nonincarcerated population. These findings, which require further investigation, may reflect inadequate care in jails and prisons and represent a critical public health issue.

Medical use and combination drug therapy among US adult users of central nervous system stimulants: a cross-sectional analysis.

OBJECTIVE: Examine patterns of adult medical use of amphetamine and methylphenidate stimulant drugs, classified in the USA as Schedule II controlled substances with a high potential for psychological or physical dependence. DESIGN: Cross-sectional study. SETTING AND PARTICIPANTS: Prescription drug claims for US adults, age 19-64 years, included in a commercial insurance claims database with 9.1 million continuously enrolled adults from 1 October 2019, through 31 December 2020. Stimulant use was defined as adults filling one or more stimulant prescriptions during calendar 2020. OUTCOME MEASURES: The primary outcome was an outpatient prescription claim, service date and days' supply for central nervous system (CNS)-active drugs. Combination-2 was defined as 60 days or more of combination treatment with a Schedule II stimulant and one or more additional CNS-active drugs. Combination-3 therapy was defined as the addition of 2 or more additional CNS-active drugs. Using service date and days' supply, we examined the number of stimulant and other CNS-active drugs for each of the 366 days of 2020. RESULTS: Among 9 141 877 continuously enrolled adults, the study identified 276 223 individuals (3.0%) using Schedule II stimulants during 2020. They filled a median of 8 (IQR, 4-11) prescriptions for these stimulant drugs that provided 227 (IQR, 110-322) treatment days of exposure. Among this group, 125 781 (45.5%) combined use of one or more additional CNS active drugs for a median of 213 (IQR, 126-301) treatment days. Also, 66 996 (24.3%) stimulant users used two or more additional CNS-active drugs for a median of 182 (IQR, 108-276) days. Among stimulants users, 131 485 (47.6%) were exposed to an antidepressant, 85 166 (30.8%) filled prescriptions for anxiety/sedative/hypnotic medications and 54 035 (19.6%) received opioid prescriptions. CONCLUSION: A large proportion of adults using Schedule II stimulants are simultaneously exposed to one or more other CNS-active drugs, many with tolerance, withdrawal effects or potential for non-medical use. There are no approved indications and limited clinical trial testing of these multi-drug combinations, and discontinuation may be challenging.

How to predict return to work after lumbar discectomy: answers from the NeuroPoint-SD registry.

OBJECTIVE To date, the factors that predict whether a patient returns to work after lumbar discectomy are poorly understood. Information on postoperative work status is important in analyzing the cost-effectiveness of the procedure. METHODS An observational prospective cohort study was completed at 13 academic and community sites (NeuroPoint-Spinal Disorders [NeuroPoint-SD] registry). Patients undergoing single-level lumbar discectomy were included. Variables assessed included age, sex, body mass index (BMI), SF-36 physical function score, Oswestry Disability Index (ODI) score, presence of diabetes, smoking status, systemic illness, workers' compensation status, and preoperative work status. The primary outcome was working status within 3 months after surgery. Stepwise logistic regression analysis was performed to determine which factors were predictive of return to work at 3 months following discectomy. RESULTS There were 127 patients (of 148 total) with data collected 3 months postoperatively. The patients' average age at the time of surgery was 46 ± 1 years, and 66.9% of patients were working 3 months postoperatively. Statistical analyses demonstrated that the patients more likely to return to work were those of younger age (44.5 years vs 50.5 years, p = 0.008), males (55.3% vs 28.6%, p = 0.005), those with higher preoperative SF-36 physical function scores (44.0 vs 30.3, p = 0.002), those with lower preoperative ODI scores (43.8 vs 52.6, p = 0.01), nonsmokers (83.5% vs 66.7%, p = 0.03), and those who were working preoperatively (91.8% vs 26.2%, p < 0.0001). When controlling for patients who were working preoperatively (105 patients), only age was a statistically significant predictor of postoperative return to work (44.1 years vs 51.1 years, p = 0.049). CONCLUSIONS In this cohort of lumbar discectomy patients, preoperative working status was the strongest predictor of postoperative working status 3 months after surgery. Younger age was also a predictor. Factors not influencing return to work in the logistic regression analysis included sex, BMI, SF-36 physical function score, ODI score, presence of diabetes, smoking status, and systemic illness. Clinical trial registration no.: 01220921 ( clinicaltrials.gov ).

Predictive value of 3-month lumbar discectomy outcomes in the NeuroPoint-SD Registry.

OBJECT: The authors have established a multicenter registry to assess the efficacy and costs of common lumbar spinal procedures using prospectively collected outcomes. Collection of these data requires an extensive commitment of resources from each site. The aim of this study was to determine whether outcomes data from shorter-interval follow-up could be used to accurately estimate long-term outcome following lumbar discectomy. METHODS: An observational prospective cohort study was completed at 13 academic and community sites. Patients undergoing single-level lumbar discectomy for treatment of disc herniation were included. SF-36 and Oswestry Disability Index (ODI) data were obtained preoperatively and at 1, 3, 6, and 12 months postoperatively. Quality-adjusted life year (QALY) data were calculated using SF-6D utility scores. Correlations among outcomes at each follow-up time point were tested using the Spearman rank correlation test. RESULTS: One hundred forty-eight patients were enrolled over 1 year. Their mean age was 46 years (49% female). Eleven patients (7.4%) required a reoperation by 1 year postoperatively. The overall 1-year follow-up rate was 80.4%. Lumbar discectomy was associated with significant improvements in ODI and SF-36 scores (p < 0.0001) and with a gain of 0.246 QALYs over the 1-year study period. The greatest gain occurred between baseline and 3-month follow-up and was significantly greater than improvements obtained between 3 and 6 months or 6 months and 1 year(p < 0.001). Correlations between 3-month, 6-month, and 1-year outcomes were similar, suggesting that 3-month data may be used to accurately estimate 1-year outcomes for patients who do not require a reoperation. Patients who underwent reoperation had worse outcomes scores and nonsignificant correlations at all time points. CONCLUSIONS: This national spine registry demonstrated successful collection of high-quality outcomes data for spinal procedures in actual practice. Three-month outcome data may be used to accurately estimate outcome at future time points and may lower costs associated with registry data collection. This registry effort provides a practical foundation for the acquisition of outcome data following lumbar discectomy.

Postoperative cervical sagittal imbalance negatively affects outcomes after surgery for cervical spondylotic myelopathy.

STUDY DESIGN: Prospective observational cohort study. OBJECTIVE: To determine if postoperative cervical sagittal balance is an independent predictor of health-related quality of life outcome after surgery for cervical spondylotic myelopathy. SUMMARY OF BACKGROUND DATA: Both ventral and dorsal fusion procedures for CSM are effective at reducing the symptoms of myelopathy. The importance of cervical sagittal balance in predicting overall health-related quality of life outcome after ventral versus dorsal surgery for CSM has not been previously explored. METHODS: A prospective, nonrandomized cohort of 49 patients undergoing dorsal and ventral fusion surgery for CSM was examined. Preoperative and postoperative C2-C7 sagittal vertical axis was measured on standing lateral cervical spine radiographs. Outcome was assessed with 2 disease-specific measures-the modified Japanese Orthopedic Association scale and the Oswestry Neck Disability Index and 2 generalized outcome measures-the Short-Form 36 physical component summary (SF-36 PCS) and Euro-QOL-5D. Assessments were performed preoperatively, and at 3 months, 6 months, and 1 year postoperatively. Statistical analyses were performed using SAS version 9.3 (SAS Institute). RESULTS: Most patients experienced improvement in all outcome measures regardless of approach. Both preoperative and postoperative C2-C7 sagittal vertical axis measurements were independent predictors of clinically significant improvement in SF-36 PCS scores (P = 0.03 and P = 0.02). The majority of patients with C2-C7 sagittal vertical axis values greater than 40 mm did not improve from an overall health-related quality of life perspective (SF-36 PCS) despite improvement in myelopathy. The postoperative sagittal balance value was inversely correlated with a clinically significant improvement of SF-36 PCS scores in patients undergoing dorsal surgery but not ventral surgery (P = 0.03 vs. P = 0.93). CONCLUSION: Preoperative and postoperative sagittal balance measurements independently predict clinical outcomes after surgery for CSM. LEVEL OF EVIDENCE: 2.

Cost-effectiveness of lumbar discectomy and single-level fusion for spondylolisthesis: experience with the NeuroPoint-SD registry.

OBJECT: There is significant practice variation and uncertainty as to the value of surgical treatments for lumbar spine disorders. The authors' aim was to establish a multicenter registry to assess the efficacy and costs of common lumbar spinal procedures by using prospectively collected outcomes. METHODS: An observational prospective cohort study was completed at 13 academic and community sites. Patients undergoing single-level fusion for spondylolisthesis or single-level lumbar discectomy were included. The 36-Item Short Form Health Survey (SF-36) and Oswestry Disability Index (ODI) data were obtained preoperatively and at 1, 3, 6, and 12 months postoperatively. Power analysis estimated a sample size of 160 patients: lumbar disc (125 patients) and lumbar listhesis (35 patients). The quality-adjusted life year (QALY) data were calculated using 6-dimension utility index scores. Direct costs and complication costs were estimated using Medicare reimbursement values from 2011, and indirect costs were estimated using the human capital approach with the 2011 US national wage index. Total costs equaled $14,980 for lumbar discectomy and $43,852 for surgery for lumbar spondylolisthesis. RESULTS: There were 198 patients enrolled over 1 year. The mean age was 46 years (49% female) for lumbar discectomy (n = 148) and 58.1 years (60% female) for lumbar spondylolisthesis (n = 50). Ten patients with disc herniation (6.8%) and 1 with listhesis (2%) required repeat operation at 1 year. The overall 1-year follow-up rate was 88%. At 30 days, both lumbar discectomy and single-level fusion procedures were associated with significant improvements in ODI, visual analog scale, and SF-36 scores (p = 0.0002), which persisted at the 1-year evaluation (p < 0.0001). By 1 year, more than 80% of patients in each cohort who were working preoperatively had returned to work. Lumbar discectomy was associated with a gain of 0.225 QALYs over the 1-year study period ($66,578/QALY gained). Lumbar spinal fusion for Grade I listhesis was associated with a gain of 0.195 QALYs over the 1-year study period ($224,420/QALY gained). CONCLUSIONS: This national spine registry demonstrated successful collection of high-quality outcomes data for spinal procedures in actual practice. These data are useful for demonstrating return to work and cost-effectiveness following surgical treatment of single-level lumbar disc herniation or spondylolisthesis. One-year cost per QALY was obtained, and this cost per QALY is expected to improve further by 2 years. This work sets the stage for real-world analysis of the value of health interventions.

Radiographic predictors of delayed instability following decompression without fusion for degenerative grade I lumbar spondylolisthesis.

OBJECT: It is not known whether adding fusion to lumbar decompression is necessary for all patients undergoing surgery for degenerative lumbar spondylolisthesis with symptomatic stenosis. Determining specific radiographic traits that might predict delayed instability following decompression surgery might guide clinical decision making regarding the utility of up-front fusion in patients with degenerative Grade I spondylolisthesis. METHODS: Patients with Grade I degenerative lumbar spondylolisthesis (3-14 mm) with symptomatic stenosis were prospectively enrolled from a single site between May 2002 and September 2009 and treated with decompressive laminectomy without fusion. Patients with mechanical back pain or with gross motion (> 3 mm) on flexion-extension lumbar radiographs were excluded. The baseline radiographic variables measured included amount of slippage, disc height, facet angle, motion at spondylolisthesis (flexion-extension), and sagittal rotation angle. Data were analyzed using multivariate forward selection stepwise logistic regression, chi-square tests, Student t-test, and ANOVA. RESULTS: Forty patients were enrolled and treated with laminectomy without fusion, and all patients had complete radiographic data sets that were available for analysis. Reoperation was performed in 15 (37.5%) of 40 patients, with a mean follow-up duration of 3.6 years. Reoperation was performed for pain caused by instability at the index level in all 15 cases. Using multivariate stepwise logistic regression with a threshold p value of 0.35, motion at spondylolisthesis, disc height, and facet angle were predictors of reoperation following surgery. Facet angle > 50° was associated with a 39% rate of reoperation, disc height > 6.5 mm was associated with a 45% rate of reoperation, and motion at spondylolisthesis > 1.25 mm was associated with a 54% rate of reoperation. Patients with all 3 risk factors for instability had a 75% rate of reoperation, whereas patients with no risk factors for instability had a 0% rate of reoperation (p = 0.14). CONCLUSIONS: Patients with motion at spondylolisthesis > 1.25 mm, disc height > 6.5 mm, and facet angle > 50° are more likely to experience instability following decompression surgery for Grade I lumbar spondylolisthesis. Identification of key risk factors for instability might improve patient selection for decompression without fusion surgery.

The effect of carotid endarterectomy on cerebral blood flow and cognitive function.

BACKGROUND: The effect of carotid endarterectomy on cognitive function is not fully understood. This study aims to characterize changes in cerebral blood flow after carotid endarterectomy and to determine if patients with improvement in cerebral blood flow have improved cognitive function after endarterectomy. METHODS: Cerebral blood flow was measured preoperatively and 1 month postoperatively using phase contrast magnetic resonance angiography. Preoperative flow impairment was defined as ipsilateral flow at least 20% less than contralateral flow. Improvement in flow was defined as an absolute increase of at least 0.10 in flow ratio from pre- to postoperative assessments. Patients underwent cognitive testing preoperatively and at 1, 6, and 12 months postoperatively. RESULTS: Twenty-four patients with unilateral carotid stenosis were enrolled from 3 sites. Preoperative internal carotid artery (ICA) and middle cerebral artery (MCA) flow impairment was observed in 50% and 22% of patients, respectively. Patients with preoperative flow impairment had an average of 0.25 and 0.16 absolute improvement in flow ratio in the ICA and MCA vessels, respectively; this was statistically significant for patients with baseline ICA flow impairment (P < .01). One hundred percent of patients with improvement in MCA flow had a significant improvement in attention compared to 56% of patients without MCA flow improvement (P = .06). Clinically significant improvements in all 4 cognitive domains were observed at 1 year (P < .01). CONCLUSIONS: Patients with baseline impairment of MCA blood flow were more likely to experience improvement in flow after revascularization. Improvement in MCA blood flow was associated with greater cognitive improvement in attention and executive functioning.

Cigarette smoking and the risk of dying from tobacco-related malignancies by race.

BACKGROUND: The aim was to investigate whether differential risks from cigarette smoking contribute to the disproportionate burden of tobacco-related malignancies other than lung cancer (TRM-nonLC) suffered by African Americans (AAs) compared to Caucasians. MATERIALS AND METHODS: Data from two prospective cohort studies (39% AAs) established in 1960 and followed through 1990 and 2000 in the southeastern U.S. were pooled (N=5,363). Each cohort had 30 years minimum follow-up. RESULTS: Compared to Caucasians, the association between cigarette smoking and TRM-nonLC was significantly weaker in the AA men (hazards ratio (HR) 1.0 in AA men versus 3.6 in Caucasian men) and non-significantly weaker in the AA women (HR 1.1 in AA women versus 2.7 in Caucasian women). CONCLUSION: In these study cohorts, differential susceptibility to tobacco-caused carcinogenesis was, by itself, an unlikely contributing factor to the racial disparity in tobacco-related malignancies.

Redundant proteolytic mechanisms process seed storage proteins in the absence of seed-type members of the vacuolar processing enzyme family of cysteine proteases.

Seed-type vacuolar processing enzyme (VPE) activity is predicted to be essential for post-translational proteolysis of seed storage proteins in the protein storage vacuole of developing seeds. To test this hypothesis, we examined the protein profiles of developing and germinating seeds from Arabidopsis plants containing transposon-insertional knockout mutations in the genes that encode the two seed-type VPEs in Arabidopsis, betaVPE, which was identified previously, and deltaVPE, which is described here. The effects of these mutations were studied individually in single mutants and together in a double mutant. Surprisingly, we found that most of the seed protein still was processed proteolytically in seed-type VPE mutants. The minor differences observed in polypeptide accumulation between wild-type and betaVPE mutant seeds were characterized using a two-dimensional gel/mass spectrometric analysis approach. The results showed increased amounts of propolypeptide forms of legumin-type globulins accumulating in mutant seeds. However, the majority of protein (>80%) still was processed to mature alpha- and beta-chains, as observed in wild-type seeds. Furthermore, we identified several legumin-type globulin polypeptides, not corresponding to pro or mature forms, that increased in accumulation in betaVPE mutant seeds compared with wild-type seeds. Together, these results indicate the existence of both redundant and alternative processing activities in seeds. The latter was substantiated by N-terminal sequencing of a napin-type albumin protein, indicating cleavage consistent with previous in vitro studies using purified aspartic protease. Analysis of genome-wide transcript profiling data sets identified six protease genes (including an aspartic protease gene and betaVPE) that shared spatial and temporal expression patterns with seed storage proteins. From these results, we conclude that seed-type VPEs constitute merely one pathway for processing seed storage protein and that other proteolytic enzymes also can process storage proteins into chains capable of stable accumulation in mature seeds.