Search

Background: Homologous recombination deficiency (HRD)-causing alterations have been reported in triple-negative breast cancer (TNBC). We hypothesized that TNBCs with HRD alterations might be more sensitive to anthracycline plus cyclophosphamide-based chemotherapy and report on HRD status and BRCA1 promoter methylation (PM) as prognostic markers in TNBC patients treated with adjuvant doxorubicin (A) and cyclophosphamide (C) in SWOG9313., Patients and Methods: In total, 425 TNBC patients were identified from S9313. HRD score, tumor BRCA1/2 sequencing, and BRCA1 PM were carried out on DNA isolated from formalin-fixed paraffin-embedded tissue. Positive HRD status was defined as either a deleterious tumor BRCA1/2 (tBRCA) mutation or a pre-defined HRD score ≥42. Markers were tested for prognostic value on disease-free survival (DFS) and overall survival (OS) using Cox regression models adjusted for treatment assignment and nodal status., Results: HRD status was determined in 89% (379/425) of cases. Of these, 67% were HRD positive (27% with tBRCA mutation, 40% tBRCA-negative but HRD score ≥42). HRD-positive status was associated with a better DFS [hazard ratio (HR) 0.72; 95% confidence interval (CI) 0.51-1.00; P = 0.049] and non-significant trend toward better OS (HR = 0.71; 95% CI 0.48-1.03; P = 0.073). High HRD score (≥42) in tBRCA-negative patients (n = 274) was also associated with better DFS (HR = 0.64; 95% CI 0.43-0.94; P = 0.023) and OS (HR = 0.65; 95% CI 0.42-1.00; P = 0.049). BRCA1 PM was evaluated successfully in 82% (348/425) and detected in 32% of cases. The DFS HR for BRCA1 PM was similar to that for HRD but did not reach statistical significance (HR = 0.79; 95% CI 0.54-1.17; P = 0.25)., Conclusions: HRD positivity was observed in two-thirds of TNBC patients receiving adjuvant AC and was associated with better DFS. HRD status may identify TNBC patients who receive greater benefit from AC-based chemotherapy and should be evaluated further in prospective studies., Clinical Trials Number: Int0137 (The trial pre-dates Clinicaltrial.Gov website establishment).

Background & Aims: Lynch syndrome is a genetic disorder that greatly increases risk for colorectal and other cancers, although it is underdiagnosed. Prediction of MLH1, MSH2, and MSH6 (PREMM 1,2,6 ) is a web-based tool that analyzes individuals' personal/family histories of cancer to quantify their likelihood of carrying a germline mutation associated with Lynch syndrome. We investigated the feasibility of systematic risk assessment for Lynch syndrome in a community gastroenterology practice using a patient-completed version of PREMM 1,2,6 ., Methods: PREMM 1,2,6 was adapted into a computer tablet version designed for self-administration by patients. Individuals presenting to a community gastroenterology office and endoscopy facility in California completed the PREMM 1,2,6 assessment before their visit (n = 3134). The total study duration (8 months) comprised a 2-month initiation period (May 1-June 30, 2013) and a 6-month study period (July 1-December 31, 2013). Genetic counseling and germline analysis for mutations in genes associated with Lynch syndrome (MLH1, MSH2, MSH6, PMS2, and EPCAM) were offered to individuals with PREMM 1,2,6 scores of 5% or higher. Patients and providers completed surveys to evaluate the feasibility and satisfaction with the process., Results: Of the 3134 individuals assessed by PREMM 1,2,6 during the 6-month study period, 177 individuals (5.6%) had scores of 5% or higher. Of these, 146 individuals underwent genetic testing, along with 28 additional participants recruited nonconsecutively during the initiation period. Mutations associated with Lynch syndrome were detected in 3 of the 146 individuals (2.1%) with PREMM 1,2,6 scores of 5% or higher who underwent germline testing, and 3 of the 28 patients (10.7%) recruited during study initiation with PREMM 1,2,6 scores of 5% or higher. Of the participants who underwent genetic analysis, 98.6% stated that they understood the information provided to them. All of the surveyed providers stated that they were satisfied with the incorporation of PREMM 1,2,6 into their clinical practice, and that they would continue using it to assess risk for Lynch syndrome., Conclusions: A patient self-administered version of the PREMM 1,2,6 Lynch syndrome risk assessment model can be used systematically in community-based gastroenterology and endoscopy practices., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)

PurposePanel-based genetic testing has identified increasing numbers of patients with pancreatic ductal adenocarcinoma (PDAC) who carry germ-line mutations. However, small sample sizes or number of genes evaluated limit prevalence estimates of these mutations. We estimated prevalence of mutations in PDAC patients with positive family history.MethodsWe sequenced 25 cancer susceptibility genes in lymphocyte DNA from 302 PDAC patients in the Mayo Clinic Biospecimen Resource for Pancreatic Research Registry. Kindreds containing at least two first-degree relatives with PDAC met criteria for familial pancreatic cancer (FPC), while the remaining were familial, but not FPC.ResultsThirty-six patients (12%) carried at least one deleterious mutation in one of 11 genes. Of FPC patients, 25/185 (14%) were carriers, while 11/117 (9%) non-FPC patients with family history were carriers. Deleterious mutations (n) identified in PDAC patients were BRCA2 (11), ATM (8), CDKN2A (4), CHEK2 (4), MUTYH/MYH (3 heterozygotes, not biallelic), BRCA1 (2), and 1 each in BARD1, MSH2, NBN, PALB2, and PMS2. Novel mutations were found in ATM, BARD1, and PMS2.ConclusionMultiple susceptibility gene testing in PDAC patients with family history of pancreatic cancer is warranted regardless of FPC status and will inform genetic risk counseling for families.

Background: Histopathologic examination alone can be inadequate for diagnosis of certain melanocytic neoplasms. Recently, a 23-gene expression signature was clinically validated as an ancillary diagnostic test to differentiate benign nevi from melanoma. The current study assessed the performance of this test in an independent cohort of melanocytic lesions against clinically proven outcomes. Methods: Archival tissue from primary cutaneous melanomas and melanocytic nevi was obtained from four independent institutions and tested with the gene signature. Cases were selected according to pre-defined clinical outcome measures. Malignant lesions were defined as stage I-III primary cutaneous melanomas that produced distant metastases (metastatic to sites other than proximal sentinel lymph node(s)) following diagnosis of the primary lesion. Melanomas that were metastatic at the time of diagnosis, all re-excisions, and lesions with <10% tumor volume were excluded. Benign lesions were defined as cutaneous melanocytic lesions with no adverse long-term events reported. Results: Of 239 submitted samples, 182 met inclusion criteria and produced a valid gene expression result. This included 99 primary cutaneous melanomas with proven distant metastases and 83 melanocytic nevi. Median time to melanoma metastasis was 18 months. Median follow-up time for nevi was 74.9 months. The gene expression score differentiated melanoma from nevi with a sensitivity of 93.8% and a specificity of 96.2%. Conclusions: The results of gene expression testing closely correlate with long-term clinical outcomes of patients with melanocytic neoplasms. Impact: Collectively, this provides strong evidence that the gene signature adds valuable adjunctive information to aid in the accurate diagnosis of melanoma. Cancer Epidemiol Biomarkers Prev; 26(7); 1107-13. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Missense variants represent a significant proportion of variants identified in clinical genetic testing. In the absence of strong clinical or functional evidence, the American College of Medical Genetics recommends that these findings be classified as variants of uncertain significance (VUS). VUSs may be reclassified to better inform patient care when new evidence is available. It is critical that the methods used for reclassification are robust in order to prevent inappropriate medical management strategies and unnecessary, life-altering surgeries. In an effort to provide evidence for classification, several in silico algorithms have been developed that attempt to predict the functional impact of missense variants through amino acid sequence conservation analysis. We report an analysis comparing internally derived, evidence-based classifications with the results obtained from six commonly used algorithms. We compiled a dataset of 1118 variants in BRCA1, BRCA2, MLH1, and MSH2 previously classified by our laboratory's evidence-based variant classification program. We compared internally derived classifications with those obtained from the following in silico tools: Align-GVGD, CONDEL, Grantham Analysis, MAPP-MMR, PolyPhen-2, and SIFT. Despite being based on similar underlying principles, all algorithms displayed marked divergence in accuracy, specificity, and sensitivity. Overall, accuracy ranged from 58.7 to 90.8% while the Matthews Correlation Coefficient ranged from 0.26-0.65. CONDEL, a weighted average of multiple algorithms, did not perform significantly better than its individual components evaluated here. These results suggest that the in silico algorithms evaluated here do not provide reliable evidence regarding the clinical significance of missense variants in genes associated with hereditary cancer.

Based on current consensus guidelines and standard practice, many genetic variants detected in clinical testing are classified as disease causing based on their predicted impact on the normal expression or function of the gene in the absence of additional data. However, our laboratory has identified a subset of such variants in hereditary cancer genes for which compelling contradictory evidence emerged after the initial evaluation following the first observation of the variant. Three representative examples of variants in BRCA1, BRCA2 and MSH2 that are predicted to disrupt splicing, prematurely truncate the protein, or remove the start codon were evaluated for pathogenicity by analyzing clinical data with multiple classification algorithms. Available clinical data for all three variants contradicts the expected pathogenic classification. These variants illustrate potential pitfalls associated with standard approaches to variant classification as well as the challenges associated with monitoring data, updating classifications, and reporting potentially contradictory interpretations to the clinicians responsible for translating test outcomes to appropriate clinical action. It is important to address these challenges now as the model for clinical testing moves toward the use of large multi-gene panels and whole exome/genome analysis, which will dramatically increase the number of genetic variants identified., (© 2015 The Authors. Clinical Genetics published by John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Genetic variants of uncertain clinical significance (VUSs) are a common outcome of clinical genetic testing. Locus-specific variant databases (LSDBs) have been established for numerous disease-associated genes as a research tool for the interpretation of genetic sequence variants to facilitate variant interpretation via aggregated data. If LSDBs are to be used for clinical practice, consistent and transparent criteria regarding the deposition and interpretation of variants are vital, as variant classifications are often used to make important and irreversible clinical decisions. In this study, we performed a retrospective analysis of 2017 consecutive BRCA1 and BRCA2 genetic variants identified from 24,650 consecutive patient samples referred to our laboratory to establish an unbiased dataset representative of the types of variants seen in the US patient population, submitted by clinicians and researchers for BRCA1 and BRCA2 testing. We compared the clinical classifications of these variants among five publicly accessible BRCA1 and BRCA2 variant databases: BIC, ClinVar, HGMD (paid version), LOVD, and the UMD databases. Our results show substantial disparity of variant classifications among publicly accessible databases. Furthermore, it appears that discrepant classifications are not the result of a single outlier but widespread disagreement among databases. This study also shows that databases sometimes favor a clinical classification when current best practice guidelines (ACMG/AMP/CAP) would suggest an uncertain classification. Although LSDBs have been well established for research applications, our results suggest several challenges preclude their wider use in clinical practice.

Background & Aims: Multigene panels are commercially available tools for hereditary cancer risk assessment that allow for next-generation sequencing of numerous genes in parallel. However, it is not clear if these panels offer advantages over traditional genetic testing. We investigated the number of cancer predisposition gene mutations identified by parallel sequencing in individuals with suspected Lynch syndrome., Methods: We performed germline analysis with a 25-gene, next-generation sequencing panel using DNA from 1260 individuals who underwent clinical genetic testing for Lynch syndrome from 2012 through 2013. All patients had a history of Lynch syndrome-associated cancer and/or polyps. We classified all identified germline alterations for pathogenicity and calculated the frequencies of pathogenic mutations and variants of uncertain clinical significance (VUS). We also analyzed data on patients' personal and family history of cancer, including fulfillment of clinical guidelines for genetic testing., Results: Of the 1260 patients, 1112 met National Comprehensive Cancer Network (NCCN) criteria for Lynch syndrome testing (88%; 95% confidence interval [CI], 86%-90%). Multigene panel testing identified 114 probands with Lynch syndrome mutations (9.0%; 95% CI, 7.6%-10.8%) and 71 with mutations in other cancer predisposition genes (5.6%; 95% CI, 4.4%-7.1%). Fifteen individuals had mutations in BRCA1 or BRCA2; 93% of these met the NCCN criteria for Lynch syndrome testing and 33% met NCCN criteria for BRCA1 and BRCA2 analysis (P = .0017). An additional 9 individuals carried mutations in other genes linked to high lifetime risks of cancer (5 had mutations in APC, 3 had bi-allelic mutations in MUTYH, and 1 had a mutation in STK11); all of these patients met NCCN criteria for Lynch syndrome testing. A total of 479 individuals had 1 or more VUS (38%; 95% CI, 35%-41%)., Conclusions: In individuals with suspected Lynch syndrome, multigene panel testing identified high-penetrance mutations in cancer predisposition genes, many of which were unexpected based on patients' histories. Parallel sequencing also detected a high number of potentially uninformative germline findings, including VUS., (Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Recent research efforts have progressively shifted towards preventative psychiatry and prognostic identification of individuals before disease onset. We describe the development of a serum biomarker test for the identification of individuals at risk of developing schizophrenia based on multiplex immunoassay profiling analysis of 957 serum samples. First, we conducted a meta-analysis of five independent cohorts of 127 first-onset drug-naive schizophrenia patients and 204 controls. Using least absolute shrinkage and selection operator regression, we identified an optimal panel of 26 biomarkers that best discriminated patients and controls. Next, we successfully validated this biomarker panel using two independent validation cohorts of 93 patients and 88 controls, which yielded an area under the curve (AUC) of 0.97 (0.95-1.00) for schizophrenia detection. Finally, we tested its predictive performance for identifying patients before onset of psychosis using two cohorts of 445 pre-onset or at-risk individuals. The predictive performance achieved by the panel was excellent for identifying USA military personnel (AUC: 0.90 (0.86-0.95)) and help-seeking prodromal individuals (AUC: 0.82 (0.71-0.93)) who developed schizophrenia up to 2 years after baseline sampling. The performance increased further using the latter cohort following the incorporation of CAARMS (Comprehensive Assessment of At-Risk Mental State) positive subscale symptom scores into the model (AUC: 0.90 (0.82-0.98)). The current findings may represent the first successful step towards a test that could address the clinical need for early intervention in psychiatry. Further developments of a combined molecular/symptom-based test will aid clinicians in the identification of vulnerable patients early in the disease process, allowing more effective therapeutic intervention before overt disease onset.

Purpose: Familial pancreatic cancer kindreds contain at least two affected first-degree relatives. Comprehensive data are needed to assist clinical risk assessment and genetic testing., Methods: Germ-line DNA samples from 727 unrelated probands with positive family history (521 met criteria for familial pancreatic cancer) were tested in compliance with the Clinical Laboratory Improvement Amendments for mutations in BRCA1 and BRCA2 (including analysis of deletions and rearrangements), PALB2, and CDKN2A. We compared prevalence of deleterious mutations between familial pancreatic cancer probands and nonfamilial pancreatic cancer probands (kindreds containing at least two affected biological relatives, but not first-degree relatives). We also examined the impact of family history on breast and ovarian cancers and melanoma., Results: Prevalence of deleterious mutations (excluding variants of unknown significance) among familial pancreatic cancer probands was: BRCA1, 1.2%; BRCA2, 3.7%; PALB2, 0.6%; and CDKN2A, 2.5%. Four novel deleterious mutations were detected. Familial pancreatic cancer probands carry more mutations in the four genes (8.0%) than nonfamilial pancreatic cancer probands (3.5%) (odds ratio: 2.40; 95% confidence interval: 1.06-5.44; P = 0.03). The probability of testing positive for deleterious mutations in any of the four genes ranges up to 10.4%, depending on family history of cancers. BRCA2 and CDKN2A account for the majority of mutations in familial pancreatic cancer., Conclusion: Genetic testing of multiple relevant genes in probands with a positive family history is warranted, particularly for familial pancreatic cancer.

Collagen V mutations underlie classic Ehlers-Danlos syndrome, and joint hypermobility is an important clinical manifestation. We define the function of collagen V in tendons and ligaments, as well as the role of alterations in collagen V expression in the pathobiology in classic Ehlers-Danlos syndrome. A conditional Col5a1(flox/flox) mouse model was bred with Scleraxis-Cre mice to create a targeted tendon and ligament Col5a1-null mouse model, Col5a1(Δten/Δten). Targeting was specific, resulting in collagen V-null tendons and ligaments. Col5a1(Δten/Δten) mice demonstrated decreased body size, grip weakness, abnormal gait, joint laxity, and early-onset osteoarthritis. These gross changes were associated with abnormal fiber organization, as well as altered collagen fibril structure with increased fibril diameters and decreased fibril number that was more severe in a major joint stabilizing ligament, the anterior cruciate ligament (ACL), than in the flexor digitorum longus tendon. The ACL also had a higher collagen V content than did the flexor digitorum longus tendon. The collagen V-null ACL and flexor digitorum longus tendon both had significant alterations in mechanical properties, with ACL exhibiting more severe changes. The data demonstrate critical differential regulatory roles for collagen V in tendon and ligament structure and function and suggest that collagen V regulatory dysfunction is associated with an abnormal joint phenotype, similar to the hypermobility phenotype in classic Ehlers-Danlos syndrome., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Aim: These studies were to validate the analytical performance of a gene expression signature that differentiates melanoma and nevi, using RNA expression from 14 signature genes and nine normalization genes that generates a melanoma diagnostic score (MDS)., Materials & Methods: Formalin-fixed paraffin-embedded melanocytic lesions were evaluated in these studies., Results: The overall SD of the assay was determined to be 0.69 MDS units. Individual amplicons within the signature had an average amplification efficiency of 92% and a SD less than 0.5 CT. The MDS was reproducible across a 2000-fold dilution range of input RNA. Melanin, an inhibitor of PCR, does not interfere with the signature., Conclusion: These studies indicate this signature is robust and reproducible and is analytically validated on formalin-fixed paraffin-embedded melanocytic lesions.

Introduction: The aim of this study was to validate a molecular expression signature [cell cycle progression (CCP) score] that identifies patients with a higher risk of cancer-related death after surgical resection of early stage (I-II) lung adenocarcinoma in a large patient cohort and evaluate the effectiveness of combining CCP score and pathological stage for predicting lung cancer mortality., Methods: Formalin-fixed paraffin-embedded surgical tumor samples from 650 patients diagnosed with stage I and II adenocarcinoma who underwent definitive surgical treatment without adjuvant chemotherapy were analyzed for 31 proliferation genes by quantitative real-time polymerase chain reaction. The prognostic discrimination of the expression score was assessed by Cox proportional hazards analysis using 5-year lung cancer-specific death as primary outcome., Results: The CCP score was a significant predictor of lung cancer-specific mortality above clinical covariates [hazard ratio (HR) = 1.46 per interquartile range (95% confidence interval = 1.12-1.90; p = 0.0050)]. The prognostic score, a combination of CCP score and pathological stage, was a more significant indicator of lung cancer mortality risk than pathological stage in the full cohort (HR = 2.01; p = 2.8 × 10) and in stage I patients (HR = 1.67; p = 0.00027). Using the 85th percentile of the prognostic score as a threshold, there was a significant difference in lung cancer survival between low-risk and high-risk patient groups (p = 3.8 × 10)., Conclusions: This study validates the CCP score and the prognostic score as independent predictors of lung cancer death in patients with early stage lung adenocarcinoma treated with surgery alone. Patients with resected stage I lung adenocarcinoma and a high prognostic score may be candidates for adjuvant therapy to reduce cancer-related mortality.

Genetic testing has the potential to guide the prevention and treatment of disease in a variety of settings, and recent technical advances have greatly increased our ability to acquire large amounts of genetic data. The interpretation of this data remains challenging, as the clinical significance of genetic variation detected in the laboratory is not always clear. Although regulatory agencies and professional societies provide some guidance regarding the classification, reporting, and long-term follow-up of variants, few protocols for the implementation of these guidelines have been described. Because the primary aim of clinical testing is to provide results to inform medical management, a variant classification program that offers timely, accurate, confident and cost-effective interpretation of variants should be an integral component of the laboratory process. Here we describe the components of our laboratory's current variant classification program (VCP), based on 20 years of experience and over one million samples tested, using the BRCA1/2 genes as a model. Our VCP has lowered the percentage of tests in which one or more BRCA1/2 variants of uncertain significance (VUSs) are detected to 2.1% in the absence of a pathogenic mutation, demonstrating how the coordinated application of resources toward classification and reclassification significantly impacts the clinical utility of testing., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Collagen V is a regulatory fibril-forming collagen that forms heterotypic fibrils with collagen I. Deletion of collagen V in the mouse is associated with a lack of fibril assembly in the embryonic mesenchyme, with a resultant lethal phenotype. The current work elucidates the regulatory roles of collagen V during development and growth of tissues. A conditional mouse model with a mutation in Col5a1 was developed using a Cre-loxP approach. Col5a1 was ablated in Col5a1(flox/flox) mice using a cornea stroma-specific Kera-Cre driver mouse to produce a bitransgenic Col5a1(Δst/Δst) line that is null for collagen V. This permits analyses of the corneal stroma, a widely used model for studies of collagen V. The collagen-V-knockout stroma demonstrated severe dysfunctional regulation of fibrillogenesis. Fibril diameters were significantly increased, with an abnormal, heterogeneous distribution; fibril structure was abnormal, fibril number was decreased and lamellae were disorganized with decreased stroma thickness. The phenotype was more severe in the anterior versus posterior stroma. Opacity was demonstrated throughout the Col5a1(Δst/Δst) stroma, with significantly increased haze intensity compared with control mice. These data indicate central regulatory roles for collagen V in fibril and matrix assembly during tissue development, with dysfunctional regulation resulting in a functional loss of transparency.

Collagens V and XI comprise a single regulatory type of fibril-forming collagen with multiple isoforms. Both co-assemble with collagen I or II to form heterotypic fibrils and have been implicated in regulation of fibril assembly. The objective of this study was to determine the roles of collagens V and XI in the regulation of tendon fibrillogenesis. Flexor digitorum longus tendons from a haplo-insufficient collagen V mouse model of classic Ehlers Danlos syndrome (EDS) had decreased biomechanical stiffness compared with controls consistent with joint laxity in EDS patients. However, fibril structure was relatively normal, an unexpected finding given the altered fibrils observed in dermis and cornea from this model. This suggested roles for other related molecules, i.e. collagen XI, and compound Col5a1(+/-),Col11a1(+/-) tendons had altered fibril structures, supporting a role for collagen XI. To further evaluate this, transcript expression was analyzed in wild type tendons. During development (E18-P10) both collagen V and XI were comparably expressed; however, collagen V predominated in mature (P30) tendons. The collagens had a similar expression pattern. Tendons with altered collagen V and/or XI expression (Col5a1(+/-); Col11a1(+/-); Col5a1(+/-),Col11a1(+/-); Col11a1(-/-); Col5a1(+/-),Col11a1(-/-)) were analyzed at E18. All genotypes demonstrated a reduced fibril number and altered structure. This phenotype was more severe with a reduction in collagen XI. However, the absence of collagen XI with a reduction in collagen V was associated with the most severe fibril phenotype. The data demonstrate coordinate roles for collagens V and XI in the regulation of fibril nucleation and assembly during tendon development.

Background & Aims: We developed and validated a model to estimate the risks of mutations in the mismatch repair (MMR) genes MLH1, MSH2, and MSH6 based on personal and family history of cancer., Methods: Data were analyzed from 4539 probands tested for mutations in MLH1, MSH2, and MSH6. A multivariable polytomous logistic regression model (PREMM(1,2,6)) was developed to predict the overall risk of MMR gene mutations and the risk of mutation in each of the 3 genes. The discriminative ability of the model was validated in 1827 population-based colorectal cancer (CRC) cases., Results: Twelve percent of the original cohort carried pathogenic mutations (204 in MLH1, 250 in MSH2, and 71 in MSH6). The PREMM(1,2,6) model incorporated the following factors from the probands and first- and second-degree relatives (odds ratio; 95% confidence intervals [CIs]): male sex (1.9; 1.5-2.4), a CRC (4.3; 3.3-5.6), multiple CRCs (13.7; 8.5-22), endometrial cancer (6.1; 4.6-8.2), and extracolonic cancers (3.3; 2.4-4.6). The areas under the receiver operating characteristic curves were 0.86 (95% CI, 0.82-0.91) for MLH1 mutation carriers, 0.87 (95% CI, 0.83-0.92) for MSH2, and 0.81 (95% CI, 0.69-0.93) for MSH6; in validation, they were 0.88 for the overall cohort (95% CI, 0.86-0.90) and the population-based cases (95% CI, 0.83-0.92)., Conclusions: We developed the PREMM(1,2,6) model, which incorporates information on cancer history from probands and their relatives to estimate an individual's risk of mutations in the MMR genes MLH1, MSH2, and MSH6. This Web-based decision making tool can be used to assess risk of hereditary CRC and guide clinical management., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Ductal and lobular carcinoma in situ (CIS) accounted for 62,280 (24.5%) of all new breast cancer diagnoses in 2009. BRCA1/2 mutations confer an extremely high risk of breast cancer, and management guidelines for BRCA1/2 mutation carriers advise close follow-up, intensive screening, and consideration of prophylactic surgery to lower this risk. The limited relevant previous data are not definitive in establishing the prevalence of BRCA1/2 mutations in breast CIS patients, creating uncertainty as to whether referral for cancer risk assessment and genetic testing is appropriate for this group. Therefore, we conducted a cross-sectional analysis of the Myriad Genetics BRCA1/2 database to determine the prevalence of these mutations in breast CIS patients. All statistical tests were 2-sided, and confidence intervals (CI) are reported at the 95% level (α = 0.05). The source population was 64,717 consecutive women who were not Ashkenazi Jewish, underwent BRCA1/2 testing, and provided a personal and family history of invasive breast and ovarian cancer; 7,295 (11.3%) reported a diagnosis of CIS (ductal or lobular) and had an overall 5.9% prevalence of mutated BRCA1/2 (mBRCA). Subgrouped by history (personal or family) of invasive breast and/or ovarian cancer, these CIS patients had the following prevalences of mBRCA: (1) no personal or family history, 2.3%; (2) personal history, 5.2%; (3) family history, 5%; and (4) personal and family history, 10.3%. mBRCA risk was significantly higher in women with early-onset (<50 years old) CIS than with late-onset (≥ 50 years old) CIS [odds ratio (OR) = 1.5; 95% CI = 1.1-2.1). Disease onset at less than 40 years age was associated with an even higher mBRCA risk (OR = 1.8; 95% CI = 1.3-2.3). By far the largest analysis of BRCA1/2 mutation prevalence in non-Ashkenazi Jewish breast CIS patients, this study shows that early-onset CIS is associated with mBRCA1/2 in patients referred for genetic testing. When a family history of breast and/or ovarian cancer are also present, testing women with early-onset CIS may increase both the likelihood of detecting BRCA1/2 mutations and opportunities for carriers to consider additional cancer prevention strategies., (©2010 AACR.)

Classic Ehlers-Danlos syndrome is a heritable connective tissue disorder characterized by skin hyperextensibility, fragile and soft skin, delayed wound healing with formation of atrophic scars, easy bruising, and generalized joint hypermobility. It comprises Ehlers-Danlos syndrome type I and Ehlers-Danlos syndrome type II, but it is now apparent that these form a continuum of clinical findings and differ only in phenotypic severity. It is currently estimated that approximately 50% of patients with a clinical diagnosis of classic Ehlers-Danlos syndrome harbor mutations in the COL5A1 and the COL5A2 gene, encoding the α1 and the α2-chain of type V collagen, respectively. However, because no prospective molecular studies of COL5A1 and COL5A2 have been performed in a clinically well-defined patient group, this number may underestimate the real proportion of patients with classic Ehlers-Danlos syndrome harboring a mutation in one of these genes. In the majority of patients with molecularly characterized classic Ehlers-Danlos syndrome, the disease is caused by a mutation leading to a nonfunctional COL5A1 allele and resulting in haploinsufficiency of type V collagen. A smaller proportion of patients harbor a structural mutation in COL5A1 or COL5A2, causing the production of a functionally defective type V collagen protein. Most mutations identified so far result in a reduced amount of type V collagen in the connective tissues available for collagen fibrillogenesis. Inter- and intrafamilial phenotypic variability is observed, but no genotype-phenotype correlations have been observed. No treatment for the underlying defect is presently available for Ehlers-Danlos syndrome. However, a series of preventive guidelines are applicable.

Mutation screening of the breast and ovarian cancer-predisposition genes BRCA1 and BRCA2 is becoming an increasingly important part of clinical practice. Classification of rare nontruncating sequence variants in these genes is problematic, because it is not known whether these subtle changes alter function sufficiently to predispose cells to cancer development. Using data from the Myriad Genetic Laboratories database of nearly 70,000 full-sequence tests, we assessed the clinical significance of 1,433 sequence variants of unknown significance (VUSs) in the BRCA genes. Three independent measures were employed in the assessment: co-occurrence in trans of a VUS with known deleterious mutations; detailed analysis, by logistic regression, of personal and family history of cancer in VUS-carrying probands; and, in a subset of probands, an analysis of cosegregation with disease in pedigrees. For each of these factors, a likelihood ratio was computed under the hypothesis that the VUSs were equivalent to an "average" deleterious mutation, compared with neutral, with respect to risk. The likelihood ratios derived from each component were combined to provide an overall assessment for each VUS. A total of 133 VUSs had odds of at least 100 : 1 in favor of neutrality with respect to risk, whereas 43 had odds of at least 20 : 1 in favor of being deleterious. VUSs with evidence in favor of causality were those that were predicted to affect splicing, fell at positions that are highly conserved among BRCA orthologs, and were more likely to be located in specific domains of the proteins. In addition to their utility for improved genetics counseling of patients and their families, the global assessment reported here will be invaluable for validation of functional assays, structural models, and in silico analyses.

Purpose: The aim of the study was to determine the actual GJB2 and GJB6 mutation frequencies in North America after several years of generalized testing for autosomal recessive nonsyndromic sensorineural hearing loss to help guide diagnostic testing algorithms, especially in light of molecular diagnostic follow-up to universal newborn hearing screening., Methods: Mutation types, frequencies, ethnic distributions, and genotype-phenotype correlations for GJB2 and GJB6 were assessed in a very large North American cohort., Results: GJB2 variants were identified in 1796 (24.3%) of the 7401 individuals examined, with 399 (5.4%) homozygous and 429 (5.8%) compound heterozygous. GJB6 deletion testing was performed in 12.0% (888/7401) of all cases. The >300-kb deletion was identified in only nine individuals (1.0%), all of whom were compound heterozygous for mutations in GJB2 and GJB6. Among a total of 139 GJB2 variants identified, 53 (38.1%) were previously unreported, presumably representing novel pathogenic or benign variants., Conclusions: The frequency and distribution of sequence changes in GJB2 and GJB6 in North America differ from those previously reported, suggesting a considerable role for loci other than GJB2 and GJB6 in the etiology of autosomal recessive nonsyndromic sensorineural hearing loss, with minimal prevalence of the GJB6 deletion.

Unlabelled: The effect of ERT with imiglucerase on BMD in type 1 GD was studied using BMD data from the International Collaborative Gaucher Group Gaucher Registry. Data were analyzed for 160 untreated patients and 342 ERT-treated patients. Imiglucerase significantly improves BMD in patients with GD, with 8 years of ERT leading to normal BMD., Introduction: The objective was to determine the effect of enzyme replacement therapy (ERT; Cerezyme, imiglucerase) on BMD in type 1 Gaucher disease (GD)., Materials and Methods: The study population included all adults (men, 18-70 years; women, 18-50 years) enrolled in the International Collaborative Gaucher Group (ICGG) Gaucher Registry for whom lumbar spine BMD measurements were available. BMD data with up to 8 years of follow-up were analyzed for 160 patients who received no ERT and 342 patients treated with ERT alone. BMD was assessed by DXA of the lumbar spine. Z scores for patients with GD were compared with a reference population. From the model's estimate, percent of patients by age and sex with osteoporosis (T score < or = -2.5) were calculated., Results: DXA Z scores for patients with GD in the no ERT (untreated) group were significantly below normal (y intercept = -0.80 Z score units, p < 0.001) and remained approximately 1 SD below the reference population over time (slope = -0.010 Z score units per year, p = 0.68). The DXA Z scores for patients with GD who received ERT at a dose of 60 U/kg/2 weeks were significantly lower than the reference population at baseline (y-intercept = -1.17 Z score units, p < 0.001), but improved significantly over time (slope = +0.132 Z score units per year, p < 0.001). A significant dose-response relationship was noted for the ERT group, with the slopes for the three main dosing groups of 15, 30, and 60 U/kg/2 weeks of +0.064, +0.086, and +0.132 Z score units per year, respectively. The BMD of patients with GD treated with ERT increased to -0.12 (60 U/kg/2 weeks), -0.48 (30 U/kg/2 weeks), and -0.66 (15 U/kg/2 weeks) SD of the mean of the reference population after 8 years of ERT, approaching the reference population. Estimated risk of osteoporosis of this GD population, if left untreated, ranged from approximately 10 to 30% in women and 10% to 25% in men., Conclusions: ERT with imiglucerase (Cerezyme) may increase BMD in patients with GD. Response to treatment with imiglucerase is slower for BMD than for hematologic and visceral aspects of GD. A normal (age- and sex-adjusted) BMD should be a therapeutic goal for patients with type 1 GD.

The most commonly identified mutations causing Ehlers-Danlos syndrome (EDS) classic type result in haploinsufficiency of proalpha1(V) chains of type V collagen, a quantitatively minor collagen that co-assembles with type I collagen as heterotypic fibrils. To determine the role(s) of type I/V collagen interactions in fibrillogenesis and elucidate the mechanism whereby half-reduction of type V collagen causes abnormal connective tissue biogenesis observed in EDS, we analyzed mice heterozygous for a targeted inactivating mutation in col5a1 that caused 50% reduction in col5a1 mRNA and collagen V. Comparable with EDS patients, they had decreased aortic stiffness and tensile strength and hyperextensible skin with decreased tensile strength of both normal and wounded skin. In dermis, 50% fewer fibrils were assembled with two subpopulations: relatively normal fibrils with periodic immunoreactivity for collagen V where type I/V interactions regulate nucleation of fibril assembly and abnormal fibrils, lacking collagen V, generated by unregulated sequestration of type I collagen. The presence of the aberrant fibril subpopulation disrupts the normal linear and lateral growth mediated by fibril fusion. Therefore, abnormal fibril nucleation and dysfunctional fibril growth with potential disruption of cell-directed fibril organization leads to the connective tissue dysfunction associated with EDS.

Purpose: Type V collagen forms heterotypic fibrils with type I collagen and accounts for 10% to 20% of corneal collagen. The purpose of this study was to define the ocular phenotype resulting from mutations in the type V collagen genes COL5A1 and COL5A2 and to study the pathogenesis of anomalies in a Col5a1-deficient mouse., Methods: Seven patients with classic Ehlers-Danlos syndrome (EDS) due to COL5A1 haploinsufficiency and one with an exon-skipping mutation in COL5A2 underwent an ocular examination, corneal topography, pachymetry, and specular microscopy. A Col5a1-haploinsufficient mouse model of classic EDS was used for biochemical and immunochemical analyses of corneas. Light and electron microscopy were used to quantify stromal thickness, fibril density, fibril structure, and diameter., Results: Five males and three females (mean age, 26 +/- 13.57 years; range, 11-52) were studied. All patients had "floppy eyelids." The corneas of all eyes were thinner (mean corneal thickness: 435.75 +/- 12.51 microm) when compared with control corneas (568.89 +/- 28.46 microm; P < 0.0001). In the Col5a1+/- mouse cornea, type V collagen content was reduced by approximately 49%, and stromal thickness was reduced by approximately 26%. Total collagen deposition in Col5a1(+/-) corneas also was reduced. Collagen fibril diameters were increased, but fibril density was decreased throughout the stroma at all developmental stages., Conclusions: In the eye, COL5A1 and COL5A2 mutations manifest as abnormally thin and steep corneas with floppy eyelids. Mechanisms involved in producing the latter anomalies probably involve altered regulation of collagen fibrillogenesis due to abnormalities in heterotypic type I/V collagen interactions similar to those observed in the Col5a1+/- mouse cornea.

Hammerhead ribozymes are small catalytic RNA molecules that can be targeted to any RNA molecule containing a putative cleavage site. We developed a vector (pCOLZ) that uses the COL1A1 promoter to drive expression of a self-cleaving multimeric ribozyme (M8Rz547) and its monomeric counterpart (Rz547). The ribozymes were stably coexpressed in MC3T3-E1 osteoblasts expressing a truncated COL1A1 target transcript. The multimeric ribozyme exhibited self-cleavage to derivative fragments, including monomers. Increased expression of ribozymes was found in cells expressing the multimeric ribozyme. A modest reduction of truncated target transcript and protein was seen in cells expressing the ribozyme monomer, while nearly complete ablation of target transcript and protein occurred in cells expressing the ribozyme multimer. A reversion to a more normal collagen phenotype, measured as an increase in fibril diameter and restored fibrillar architecture, and a decreased rate of collagen turnover were seen in cells expressing the ribozyme multimer.

Vertebrate collagen fibrils are heterotypically composed of a quantitatively major and minor fibril collagen. In non-cartilaginous tissues, type I collagen accounts for the majority of the collagen mass, and collagen type V, the functions of which are poorly understood, is a minor component. Type V collagen has been implicated in the regulation of fibril diameter, and we reported recently preliminary evidence that type V collagen is required for collagen fibril nucleation (Wenstrup, R. J., Florer, J. B., Cole, W. G., Willing, M. C., and Birk, D. E. (2004) J. Cell. Biochem. 92, 113-124). The purpose of this study was to define the roles of type V collagen in the regulation of collagen fibrillogenesis and matrix assembly. Mouse embryos completely deficient in pro-alpha1(V) chains were created by homologous recombination. The col5a1-/- animals die in early embryogenesis, at approximately embryonic day 10. The type V collagen-deficient mice demonstrate a virtual lack of collagen fibril formation. In contrast, the col5a1+/- animals are viable. The reduced type V collagen content is associated with a 50% reduction in fibril number and dermal collagen content. In addition, relatively normal, cylindrical fibrils are assembled with a second population of large, structurally abnormal collagen fibrils. The structural properties of the abnormal matrix are decreased relative to the wild type control animals. These data indicate a central role for the evolutionary, ancient type V collagen in the regulation of fibrillogenesis. The complete dependence of fibril formation on type V collagen is indicative of the critical role of the latter in early fibril initiation. In addition, this fibril collagen is important in the determination of fibril structure and matrix organization.

Symptomatic patients with Gaucher disease (GD) (acid beta-glucosidase [Gcase] deficiency) are treated with injectable human recombinant GCase. Treatment results in significant decreases in lipid storage in liver, spleen, and bone marrow, but the generalized osteopenia and focal bone lesions present in many adult patients are refractory to treatment. A double-blind, 2-arm, placebo-controlled trial of alendronate (40 mg/d) was performed in adults with GD who had been treated with enzyme for at least 24 months. Primary therapeutic endpoints were improvements in (1) bone mineral density (BMD) and content (BMC) at the lumbar spine, and (2) focal lesions in x-rays of long bones assessed by a blinded reviewer. There were 34 patients with GD type 1 (age range, 18-50 years) receiving enzyme therapy who were randomized for this study. After 18 months, DeltaBMD at the lumbar spine was 0.068 +/- 0.21 and 0.015 +/- 0.034 for alendronate and placebo groups, respectively (P =.001). Long-bone x-rays showed no change in focal lesions or bone deformities in any subject in either arm. Alendronate is a useful adjunctive therapy in combination with enzyme replacement therapy (ERT) for the treatment of GD-related osteopenia in adults, but it cannot be expected to improve focal lesions.

Antisense hammerhead ribozymes have the capability to cleave complementary RNA in a sequence-dependent manner. In osteogenesis imperfecta, a genetic disorder of connective tissue, mutant collagen type I has been shown to participate in but not sustain formation of the triple helix. Selective ablation of mutant collagen gene transcript could potentially remove the mutant gene product and reverse the dominant-negative effect exerted by the abnormal protein. In earlier studies we showed that the hammerhead ribozyme Col1A1Rz547 selectively cleaved a mutant Col1A1 gene transcript in a murine calvarial osteoblast cell line. In order to test the possible therapeutic efficacy of this approach, a dramatic downregulation of the mutant transcript must be achieved, a function directly related to high steady-state level of intracellular ribozyme. We report significantly enhanced expression of Col1A1Rz547 by vaccinia T7 polymerase following infection with an attenuated T7-pol vaccinia virus as shown both by the intracellular level of the ribozyme and the cleavage of the mutant Col1A1 gene transcript. We also describe the engineering of a multimeric ribozyme construct comprising eight subunits, which can self-cleave to monomers. These studies suggest the potential use of multimeric ribozymes expressed by a vaccinia-based system in the therapy of a variety of disorders.

Purpose: To determine the prevalence of proximal aortic abnormalities in patients with Ehlers-Danlos syndrome (EDS)., Methods: In a prospective cohort study, aortic measurements by two-dimensional echocardiography were performed on consecutive EDS patients., Results: Twenty-eight percent (20 of 71) had aortic root dilation (ARD) (> +2 SD above population based norms). Fourteen of 42 individuals with the classical form of EDS (types I/II) and 6 of 29 individuals with the hypermobile form (type III) had ARD, with no gender differences., Conclusion: ARD is a common finding in EDS. Longitudinal studies are indicated to determine progression of ARD and its clinical significance.

We have identified haploinsufficiency of the COL5A1 gene that encodes the proalpha1(V) chain of type V collagen in the classical form of the Ehlers-Danlos syndrome (EDS), a heritable connective-tissue disorder that severely alters the collagen-fibrillar structure of the dermis, joints, eyes, and blood vessels. Eight of 28 probands with classical EDS who were heterozygous for expressed polymorphisms in COL5A1 showed complete or nearly complete loss of expression of one COL5A1 allele. Reduced levels of proalpha1(V) mRNA relative to the levels of another type V collagen mRNA, proalpha2(V), were also observed in the cultured fibroblasts from EDS probands. Products of the two COL5A1 alleles were approximately equal after the addition of cycloheximide to the fibroblast cultures. After harvesting of mRNAs from cycloheximide-treated cultured fibroblasts, heteroduplex analysis of overlapping reverse transcriptase-PCR segments spanning the complete proalpha1(V) cDNA showed anomalies in four of the eight probands that led to identification of causative mutations, and, in the remaining four probands, targeting of CGA-->TGA mutations in genomic DNA revealed a premature stop at codon in one of them. We estimate that approximately one-third of individuals with classical EDS have mutations of COL5A1 that result in haploinsufficiency. These findings indicate that the normal formation of the heterotypic collagen fibrils that contain types I, III, and V collagen requires the expression of both COL5A1 alleles.

Neurofibromatosis type 1 patients develop peripheral nerve tumors (neurofibromas) composed mainly of Schwann cells and fibroblasts, in an abundant collagen matrix produced by fibroblasts. Trauma has been proposed to trigger neurofibroma formation. To test if loss of the neurofibromatosis type 1 gene (Nf1) compromises fibroblast function in vivo following trauma, skin wounding was performed in Nf1 knockout mice. The pattern and amount of collagen-rich granulation bed tissue, manufactured by fibroblasts, was grossly abnormal in 60% of Nf1+/- wounds. Nf1 mutant fibroblasts showed cell autonomous abnormalities in collagen deposition in vitro that were not mimicked by Ras activation in fibroblasts, even though some Nf1 effects are mediated through Ras. Nf1+/- skin wound fibroblasts also proliferated past the normal wound maturation phase; this in vivo effect was potentiated by muscle injury. In vitro, Nf1+/- fibroblasts showed higher proliferation in 10% serum than Nf1+/+ fibroblasts. Macrophage-conditioned media or epidermal growth factor potentiated Nf1+/- fibroblast proliferation in vitro, demonstrating abnormal response of mutant fibroblasts to wound cytokines. Thus Nf1 is a key regulator of fibroblast responses to injury, and Nf1 mutation in mouse fibroblasts causes abnormalities characteristic of human neurofibromas.

We have identified five families in whom individuals affected with the Ehlers Danlos syndrome (EDS) types I, II or III had aortic root dilatation (ARD). All propositi had a low upper/lower segment ratio but no other diagnostic skeletal or ocular features of Marfan syndrome. Their skin had the soft, velvety texture characteristic of EDS and all had significant joint laxity. Probands included a 4-year-old girl with EDS type I, 4- and 8-year-old girls with EDS type III, a 35-year-old male with EDS type II, and a 51-year-old female with EDS type III. Review of these cases suggests the need for multicenter clinical studies in order to determine the prevalence and the rate of progression of ARD in EDS types I, II, and III. Such studies are necessary to determine whether echocardiograms (including measurement of aortic root diameter) should be considered on initial evaluation of all patients with mild forms of EDS.

To examine the autocrine effects that an organizing extracellular matrix has on osteoblast precursors, we created MC3T3-E1 cell lines that stably expressed pro-alpha1(I) collagen chains with a truncated triple helical domain. Cells that had incorporated the pro-alpha1(I) expression plasmid (pMG155) expressed shortened pro-alpha1(I) transcripts at high levels and efficiently secreted the expression gene products into culture media. Those cells lost over 30% of newly deposited collagenous matrix compared with virtually no loss in control cultures, and media from the abnormal cells had qualitative differences in matrix metalloprotinase production. Electron micrographs strongly suggested that type I collagen molecules containing the truncated pro-alpha1(I) chains dramatically interfered with collagen fibrillogenesis in newly forming osteoblast matrix. Abnormal collagen fibrillogenesis was also associated with altered characteristics of cellular differentiation in that abnormal cells displayed a delayed and attenuated increase in alkaline phosphatase activity. Surprisingly, synthesis of osteocalcin was more than 5-fold higher than control cultures. These findings demonstrate that osteoblasts require a normally structured collagenous matrix for up-regulation of alkaline phosphatase activity. However, in the presence of rapid turnover of osteoblast matrix, osteocalcin gene expression may be up-regulated in response to local signals by an unknown mechanism.

This paper reports the molecular cloning of a novel gene in the mouse that shows structural similarities to the microfibril protein fibrillin and to the latent transforming growth factor-beta (TGF-beta) binding protein (LTBP), a component of the latent TGF-beta complex. The gene was initially isolated during a low stringency polymerase chain reaction screen of a NIH 3T3 cell cDNA library using primers that amplify a human fibrillin-1 epidermal growth factor-like repeat. Three lines of evidence suggest that the mouse gene is a third member of the LTBP gene family, which we designate LTBP-3. First, the deduced polypeptide, which consists of 15 epidermal growth factor-like repeats, 3 TGF binding protein repeats, and 2 proline- and glycine-rich sequences, shows 38.4% identity with LTBP-1 but only 27% identity with fibrillin-1. Second, the gene appears to be co-expressed in developing mouse tissues with TGF-beta. Third, immunoprecipitation studies using mouse preosteoblast MC3T3-E1 cells and a specific anti-peptide polyclonal antiserum reveal that the mouse polypeptide forms a complex with the TGF-beta 1 precursor. Finally, we note that the LTBP-3 gene was recently localized to a distinct genetic locus (Li, X., Yin, W., Perez-Jurado, L., Bonadio, J., and Francke, U. (1995) Mamm. Genome 6, 42-45). Identification of a third binding protein provides further insight into a mechanism by which latent TGF-beta complexes can be targeted to connective tissue matrices and cells.

COL5A1, the gene for the alpha 1 chain of type V collagen, has been considered a candidate gene for certain diseases based on chromosomal location and/or disease phenotype. We have employed 3'-untranslated region RFLPs to exclude COL5A1 as a candidate gene in families with tuberous sclerosis 1, Ehlers-Danlos syndrome type II, and nail-patella syndrome. In addition, we describe a polymorphic simple sequence repeat (SSR) within a COL5A1 intron. This SSR is used to exclude COL5A1 as a candidate gene in hereditary hemorrhagic telangiectasia (Osler-Rendu-Weber disease) and to add COL5A1 to the existing map of "index" markers of chromosome 9 by evaluation of the COL5A1 locus on the CEPH 40-family reference pedigree set. This genetic mapping places COL5A1 between markers D9S66 and D9S67.

Osteogenesis imperfecta (OI) is a heritable disorder of connective tissue associated with fractures, osteopenia, and short stature. OI results from mutations affecting the pro alpha 1 or pro alpha 2 gene of type I collagen. We describe a strain of mice with a nonlethal recessively inherited mutation (oim) that results in phenotypic and biochemical features that simulate moderate to severe human OI. The phenotype of homozygous oim mice includes skeletal fractures, limb deformities, generalized osteopenia, and small body size. Their femurs are smaller and demonstrate marked cortical thinning and fewer medullary trabeculae than those of wild-type mice. Breeding studies show the mutation is inherited in most crosses as a single recessive gene on chromosome 6, near the murine Cola-2 gene. Biochemical analysis of skin and bone, as well as isolated dermal fibroblast cultures, demonstrate that alpha 1(I) homotrimeric collagen accumulates in these tissues and is secreted by fibroblasts. Short labeling studies in fibroblasts demonstrate an absence of pro alpha 2(I) collagen chains. Nucleotide sequencing of the cDNA encoding the COOH-propeptide reveals a G deletion at pro alpha 2(I) nucleotide 3983; this results in an alteration of the sequence of the last 48 amino acids. The oim mouse will facilitate the study of type I collagen-related skeletal disease.

Comparison of the nucleotide sequence and primary structure of murine and human pro alpha 2(I) collagen indicates a high degree of homology: 87% at the nucleotide level and 87% at the amino acid level, with the greatest degree of variability in the amino- and carboxy-pro-peptide domains. The homology is greatest in the triple helical domain, repeating [Gly-X-Y]338, exhibiting 90% homology at the amino acid level, with only X and Y position residue substitutions. The X and Y residues show 86% homology between murine and human pro alpha 2(I) collagen triple helices, with no truly nonconservative substitutions.

We examine clonal murine calvarial MC3T3-E1 cells to determine if they exhibit a developmental sequence similar to osteoblasts in bone tissue, namely, proliferation of undifferentiated osteoblast precursors followed by postmitotic expression of differentiated osteoblast phenotype. During the initial phase of developmental (days 1-9 of culture), MC3T3-E1 cells actively replicate, as evidenced by the high rates of DNA synthesis and progressive increase in cell number, but maintain a fusiform appearance, fail to express alkaline phosphatase, and do not accumulate mineralized extracellular collagenous matrix, consistent with immature osteoblasts. By day 9 the cultures display cuboidal morphology, attain confluence, and undergo growth arrest. Downregulation of replication is associated with expression of osteoblast functions, including production of alkaline phosphatase, processing of procollagens to collagens, and incremental deposition of a collagenous extracellular matrix. Mineralization of extracellular matrix, which begins approximately 16 days after culture, marks the final phase of osteoblast phenotypic development. Expression of alkaline phosphatase and mineralization is time but not density dependent. Type I collagen synthesis and collagen accumulation are uncoupled in the developing osteoblast. Although collagen synthesis and message expression peaks at day 3 in immature cells, extracellular matrix accumulation is minimal. Instead, matrix accumulates maximally after 7 days of culture as collagen biosynthesis is diminishing. Thus, extracellular matrix formation is a function of mature osteoblasts. Ascorbate and beta-glycerol phosphate are both essential for the expression of osteoblast phenotype as assessed by alkaline phosphatase and mineralization of extracellular matrix. Ascorbate does not stimulate type I collagen gene expression in MC3T3-E1 cells, but it is absolutely required for deposition of collagen in the extracellular matrix. Ascorbate also induces alkaline phosphatase activity in mature cells but not in immature cells. beta-glycerol phosphate displays synergistic actions with ascorbate to further stimulate collagen accumulation and alkaline phosphatase activity in postmitotic, differentiated osteoblast-like cells. Mineralization of mature cultures requires the presence of beta-glycerol phosphate. Thus, MC3T3-E1 cells display a time-dependent and sequential expression of osteoblast characteristics analogous to in vivo bone formation. The developmental sequence associated with MC3T3-E1 differentiation should provide a useful model to study the signals that mediate the switch between proliferation and differentiation in bone cells, as well as provide a renewable culture system to examine the molecular mechanism of osteoblast maturation and the formation of bone-like extracellular matrix.

Construction of large collagen cDNA has been hindered by the relatively large size and high G-C content of processed mRNA. We describe here the development of a rapid and efficient method for obtaining large full-length collagen cDNA. A full-length (4.3 kb) murine pro alpha 2(I) collagen cDNA was constructed by synthesis of a first-strand cDNA library with use of poly-A RNA (MC3T3-E1) and the oligo-dT17-adapter primer described by Frohman et al (Proc Natl Acad Sci USA 85:8998, 1988). Pro alpha 2(I) collagen cDNA were specifically amplified by the polymerase chain reaction (PCR) with a pro alpha 2(I) specific primer as the 5' primer (20mer; corresponding to nucleotide positions 42-61 in the first exon of the murine pro alpha 2(I) collagen gene, COL1A2), and with the adapter sequence 5' to the dT17 as the 3' primer. The PCR conditions were optimized to allow amplification of the expected 4.0-5.0-kb product; a major 4.3-kb product was visualized by ethidium bromide, identified by in situ gel hybridization, and cloned. DNA sequencing determined that it contained the correct 5' sequence and the 3' end had a 68 basepair (bp) 3' untranslated region. The entire sequence that codes the amino-terminal propeptide domain has been determined and compared to the human sequence. The homology between human and mouse is less in the amino terminal propeptide than in the triple helical domain; exon 5 of murine COL1A2 codes for an additional six amino acids not found in human COL1A2.

Dermatitis herpetiformis (DH) is a blistering autoimmune skin disease associated with a 95-100% incidence of the HLA class II antigen HLA-DQw2. Although the precise role of this antigen in the pathogenesis of DH is unclear, one theory proposes that patients with DH possess a molecularly unique subtype of the HLA-DQw2 antigen that causes immune abnormalities eventuating in the clinical manifestations of DH. To test this hypothesis, we performed DNA sequence analysis on the highly polymorphic HLA-DQB1 and HLA-DQA1 loci of eight patients with dermatitis herpetiformis. All DQB1 alleles sequenced were identical to the previously described HLA-DQB*0201 allele from HLA-DQw2 normal subjects. In addition, DQA1 alleles sequenced were identical to those alleles previously associated with HLA-DQw2 (DQA*0201, DQA*0501). These data document that although HLA-DQw2 appears to be a necessary element in the pathogenesis of DH, the development of DH is not dependent on the presence of a unique HLA-DQw2 antigen. HLA-DQ allelic typing by restriction fragment length polymorphism analysis of PCR-amplified HLA-DQA1 and HLA-DQB1 fragments was also performed in ten patients with DH to determine the allelic distribution among both HLA-DR3 (eight patients) and non-DR3 (two patients) DH patients. At the HLA-DQ beta chain locus, all patients possessed the DQB1*0201 allele. At the HLA-DQ alpha chain locus, all HLA-DR3 patients and one non-DR3 patient displayed a pattern consistent with the DQA1*0501 allele, whereas one non-DR3 patient displayed a pattern consistent with the DQA1*0201 allele. These data document that patients with DH do not express a unique HLA-DQw2 heterodimer, that the HLA-DQw2 molecules present in patients with DH have no DNA sequence differences from those found in normal HLA-DQw2 subjects and therefore that susceptibility to DH is not due to a unique HLA-DQw2 molecule.

Affected individuals from two apparently distinct, mild osteogenesis imperfecta families were heterozygous for a G to T transition in the COL1A2 gene that resulted in cysteine for glycine substitutions at position 646 in the alpha 2(I) chain of type I collagen. A child with a moderately severe form of osteogenesis imperfecta was heterozygous for a G to T transition that resulted in a substitution of cysteine for glycine at position 259 in the COL1A2 gene. Type I collagen molecules containing an alpha 2(I) chain with cysteine at position 259 denaturated at a lower temperature than molecules containing an alpha 2(I) chain with cysteine at position 646. In contrast to cysteine for glycine substitutions in the alpha 1(I) chain, the severity of the osteogenesis imperfecta phenotype is not directly proportional to the distance of the mutation from the amino-terminal end of the triple helix. These findings could be explained if the type I collagen triple helix contains discontinuous domains that differ in their contributions to maintaining helix stability.

A substitution for a highly conserved non-glycine residue in the triple-helical domain of the pro alpha 2(I) collagen molecule was found in an individual with a variant of the Marfan syndrome. A single base change resulted in substitution of arginine618 by glutamine at the Y position of a Gly-X-Y repeat, and is responsible for the decreased migration in SDS-polyacrylamide gels of some pro alpha 2(I) chains of type I collagen synthesized by dermal fibroblasts from this individual. Family studies suggest that this substitution was inherited from the individual's father who also produces abnormally migrating pro alpha 2(I) collagen chains and shares some of the abnormal skeletal features. This single base change creates a new Bsu36 I (Sau I, Mst II) restriction site detectable in genomic DNA by Southern blot analysis when probed with a COL1A2 fragment. The analysis of 52 control individuals (103 chromosomes) was negative for the new Bsu36 I site, suggesting that the substitution is not a common polymorphism.

Dermatitis herpetiformis (DH) is characterized in part by an associated gluten-sensitive enteropathy (GSE), and a strong association with the HLA antigens HLA-A1, -B8, -DR3, and -DQw2, essentially identical to that seen in patients with isolated GSE (celiac disease). A 4.0-kb RsaI RFLP has been identified using a DQ beta-chain cDNA and localized to the HLA-DP beta-chain region. This RFLP has been found more frequently in patients with isolated GSE than in normal HLA matched controls. We have analyzed genomic DNA from 24 patients with DH and 15 HLA-matched controls to determine if this 4.0-kb RsaI RFLP was present in patients with DH. Twenty-one of 24 (87%) of patients with DH were found to have this RFLP as compared to 7 of 10 (70%) HLA-DR3, -DQw2 matched control subjects (p = 0.23). Thus, the 4.0-kb RsaI RFLP detected in patients with isolated GSE is also present in patients with DH; however, its frequency in DH patients does not differ significantly from that of HLA matched controls. Family studies of patients with DH revealed that although the 4.0-kb RsaI RFLP segregated with the HLA-A1, -B8, -DR3, -DQw2 haplotype in one family, it did not segregate with this disease-associated haplotype in two other families. In both patient and control populations, this RFLP was associated with HLA-DPw1 or -DPw3 phenotypes; 25 of 26 (96%) HLA-DPw1 or -DPw3 subjects were found to have this RFLP compared to only 1 of 6 (17%) who did not express HLA-DPw1 or -DPw3 (pc = 0.0009). These population and family data suggest that this 4.0-kb RsaI RFLP is primarily associated with the HLA-DPw1, -DPw3 phenotype, rather than the clinical manifestations of DH. These data further document that the strongest association of DH with HLA antigens remains with HLA-DQw2 and HLA-DR3 antigens.

We reviewed clinical and biochemical findings from 132 probands with nonlethal forms of osteogenesis imperfecta (OI) whose fibroblasts were sent to the University of Washington for diagnostic studies in the years 1981-87. In cells from 86% of probands with nonlethal OI we identified biochemical alterations compatible with heterozygosity for a mutation that affected expression or structure of alpha chains of type I procollagen. We observed two major biochemical phenotypes. Cells from 40 probands (group A) secreted about half the normal amount of normal type I procollagen and no identifiable abnormal molecules; these patients were generally of normal stature, rarely had bone deformity or dentinogenesis imperfecta, and had blue sclerae. Cells from 74 probands (group B) produced and secreted normal and abnormal type I procollagen molecules; these patients were usually short and had bone deformity and dentinogenesis imperfecta, and many had grey or blue-grey sclerae. In cells from an additional 18 probands (group C) we were unable to identify altered type I procollagen synthesis or structure. Detection of these abnormalities has value in the determination of mode of inheritance and in the prediction of clinical severity.