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Type 2 diabetes (T2D) genome-wide association studies (GWASs) often overlook rare variants as a result of previous imputation panels’ limitations and scarce whole-genome sequencing (WGS) data. We used TOPMed imputation and WGS to conduct the largest T2D GWAS meta-analysis involving 51,256 cases of T2D and 370,487 controls, targeting variants with a minor allele frequency as low as 5 × 10−5. We identified 12 new variants, including a rare African/African American-enriched enhancer variant near the LEPgene (rs147287548), associated with fourfold increased T2D risk. We also identified a rare missense variant in HNF4A(p.Arg114Trp), associated with eightfold increased T2D risk, previously reported in maturity-onset diabetes of the young with reduced penetrance, but observed here in a T2D GWAS. We further leveraged these data to analyze 1,634 ClinVar variants in 22 genes related to monogenic diabetes, identifying two additional rare variants in HNF1Aand GCKassociated with fivefold and eightfold increased T2D risk, respectively, the effects of which were modified by the individual’s polygenic risk score. For 21% of the variants with conflicting interpretations or uncertain significance in ClinVar, we provided support of being benign based on their lack of association with T2D. Our work provides a framework for using rare variant GWASs to identify large-effect variants and assess variant pathogenicity in monogenic diabetes genes.

It is estimated that 10% of individuals with a myeloid malignancy carry a germline susceptibility. Using the genome-first approach, in which individuals were ascertained on genotype alone, rather than clinical phenotype, we quantified the prevalence and penetrance of pathogenic germline variants in eight myeloid malignancy predisposition (gMMP) genes. ANKRD26, CEBPA, DDX41, MECOM, SRP72, ETV6, RUNX1and GATA2, were analyzed from the Geisinger MyCode DiscovEHR (n= 170,503) and the United Kingdom Biobank (UKBB, n= 469,595). We identified a high risk of myeloid malignancies (MM) (odds ratio[OR] all genes: DiscovEHR, 4.6 [95% confidential interval (CI) 2.1–9.7], p< 0.0001; UKBB, 6.0 [95% CI 4.3–8.2], p= 3.1 × 10-27), and decreased overall survival (hazard ratio [HR] DiscovEHR, 1.8 [95% CI 1.3–2.6], p= 0.00049; UKBB, 1.4 [95% CI 1.2–1.8], p= 8.4 × 10-5) amongst heterozygotes. Pathogenic DDX41variants were the most commonly identified, and in UKBB showed a significantly increased risk of MM (OR 5.7 [95% CI 3.9–8.3], p= 6.0 × 10-20) and increased all-cause mortality (HR 1.35 [95% CI 1.1–1.7], p= 0.0063). Through a genome-first approach, this study genetically ascertained individuals with a gMMP and determined their MM risk and survival.

Coronavirus disease 2019 (COVID-19) and influenza are respiratory illnesses caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses, respectively. Both diseases share symptoms and clinical risk factors1, but the extent to which these conditions have a common genetic etiology is unknown. This is partly because host genetic risk factors are well characterized for COVID-19 but not for influenza, with the largest published genome-wide association studies for these conditions including >2 million individuals2and about 1,000 individuals3–6, respectively. Shared genetic risk factors could point to targets to prevent or treat both infections. Through a genetic study of 18,334 cases with a positive test for influenza and 276,295 controls, we show that published COVID-19 risk variants are not associated with influenza. Furthermore, we discovered and replicated an association between influenza infection and noncoding variants in B3GALT5and ST6GAL1, neither of which was associated with COVID-19. In vitro small interfering RNA knockdown of ST6GAL1—an enzyme that adds sialic acid to the cell surface, which is used for viral entry—reduced influenza infectivity by 57%. These results mirror the observation that variants that downregulate ACE2, the SARS-CoV-2 receptor, protect against COVID-19 (ref. 7). Collectively, these findings highlight downregulation of key cell surface receptors used for viral entry as treatment opportunities to prevent COVID-19 and influenza.

We report a multi-ancestry genome-wide association study on liver cirrhosis and its associated endophenotypes, alanine aminotransferase (ALT) and γ-glutamyl transferase. Using data from 12 cohorts, including 18,265 cases with cirrhosis, 1,782,047 controls, up to 1 million individuals with liver function tests and a validation cohort of 21,689 cases and 617,729 controls, we identify and validate 14 risk associations for cirrhosis. Many variants are located near genes involved in hepatic lipid metabolism. One of these, PNPLA3p.Ile148Met, interacts with alcohol intake, obesity and diabetes on the risk of cirrhosis and hepatocellular carcinoma (HCC). We develop a polygenic risk score that associates with the progression from cirrhosis to HCC. By focusing on prioritized genes from common variant analyses, we find that rare coding variants in GPAMassociate with lower ALT, supporting GPAMas a potential target for therapeutic inhibition. In conclusion, this study provides insights into the genetic underpinnings of cirrhosis.

IMPORTANCE: Knowledge about the prevalence and tumor types of CDKN2A-related melanoma-astrocytoma syndrome (MAS) is limited and could improve disease recognition. OBJECTIVE: To estimate the prevalence and describe the tumor types of MAS. DESIGN, SETTING, AND PARTICIPANTS: This retrospective cohort study analyzed all available MAS cases from medical centers in the US (2 sites) and Europe (2 sites) and from biomedical population genomic databases (UK Biobank [United Kingdom], Geisinger MyCode [US]) between January 1, 1976, and December 31, 2020. Patients with MAS with CDKN2A germline pathogenic variants and 1 or more neural tumors were included. Data were analyzed from June 1, 2022, to January 31, 2023. MAIN OUTCOMES AND MEASURES: Disease prevalence and tumor frequency. RESULTS: Prevalence of MAS ranged from 1 in 170 503 (n = 1 case; 95% CI, 1:30 098-1:965 887) in Geisinger MyCode (n = 170 503; mean [SD] age, 58.9 [19.1] years; 60.6% women; 96.2% White) to 1 in 39 149 (n = 12 cases; 95% CI, 1:22 396-1:68 434) in UK Biobank (n = 469 789; mean [SD] age, 70.0 [8.0] years; 54.2% women; 94.8% White). Among UK Biobank patients with MAS (n = 12) identified using an unbiased genomic ascertainment approach, brain neoplasms (4 of 12, 33%; 1 glioblastoma, 1 gliosarcoma, 1 astrocytoma, 1 unspecified type) and schwannomas (3 of 12, 25%) were the most common malignant and benign neural tumors, while cutaneous melanoma (2 of 12, 17%) and head and neck squamous cell carcinoma (2 of 12, 17%) were the most common nonneural malignant neoplasms. In a separate case series of 14 patients with MAS from the US and Europe, brain neoplasms (4 of 14, 29%; 2 glioblastomas, 2 unspecified type) and malignant peripheral nerve sheath tumor (2 of 14, 14%) were the most common neural cancers, while cutaneous melanoma (4 of 14, 29%) and sarcomas (2 of 14, 14%; 1 liposarcoma, 1 unspecified type) were the most common nonneural cancers. Cutaneous neurofibromas (7 of 14, 50%) and schwannomas (2 of 14, 14%) were also common. In 1 US family, a father and son with MAS had clinical diagnoses of neurofibromatosis type 1 (NF1). Genetic testing of the son detected a pathogenic CDKN2A splicing variant (c.151-1G>C) and was negative for NF1 genetic alterations. In UK Biobank, 2 in 150 (1.3%) individuals with clinical NF1 diagnoses had likely pathogenic variants in CDKN2A, including 1 individual with no detected variants in the NF1 gene. CONCLUSIONS AND RELEVANCE: This cohort study estimates the prevalence and describes the tumors of MAS. Additional studies are needed in genetically diverse populations to further define population prevalence and disease phenotypes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters human host cells via angiotensin-converting enzyme 2 (ACE2) and causes coronavirus disease 2019 (COVID-19). Here, through a genome-wide association study, we identify a variant (rs190509934, minor allele frequency 0.2–2%) that downregulates ACE2expression by 37% (P= 2.7 × 10−8) and reduces the risk of SARS-CoV-2 infection by 40% (odds ratio = 0.60, P= 4.5 × 10−13), providing human genetic evidence that ACE2 expression levels influence COVID-19 risk. We also replicate the associations of six previously reported risk variants, of which four were further associated with worse outcomes in individuals infected with the virus (in/near LZTFL1, MHC, DPP9and IFNAR2). Lastly, we show that common variants define a risk score that is strongly associated with severe disease among cases and modestly improves the prediction of disease severity relative to demographic and clinical factors alone.

Fanconi anemia (FA) is a bone marrow failure and cancer predisposition syndrome caused primarily by biallelic pathogenic variants in 1 of 22 genes involved in DNA interstrand cross-link repair. An enduring question concerns cancer risk of those with a single pathogenic FA gene variant. To investigate all FA genes, this study utilized the DiscovEHR cohort of 170,503 individuals with exome sequencing and electronic health data.

Background/Objectives: Melanocortin-4 receptor (MC4R) plays an essential role in food intake and energy homeostasis. More than 170 MC4Rvariants have been described over the past two decades, with conflicting reports regarding the prevalence and phenotypic effects of these variants in diverse cohorts. To determine the frequency of MC4Rvariants in large cohort of different ancestries, we evaluated the MC4Rcoding region for 20,537 eMERGE participants with sequencing data plus additional 77,454 independent individuals with genome-wide genotyping data at this locus. Subjects/Methods: The sequencing data were obtained from the eMERGE phase III study, in which multisample variant call format calls have been generated, curated, and annotated. In addition to penetrance estimation using body mass index (BMI) as a binary outcome, GWAS and PheWAS were performed using median BMI in linear regression analyses. All results were adjusted for principal components, age, sex, and sites of genotyping. Results: Targeted sequencing data of MC4Rrevealed 125 coding variants in 1839 eMERGE participants including 30 unreported coding variants that were predicted to be functionally damaging. Highly penetrant unreported variants included (L325I, E308K, D298N, S270F, F261L, T248A, D111V, and Y80F) in which seven participants had obesity class III defined as BMI?=?40?kg/m2. In GWAS analysis, in addition to known risk haplotype upstream of MC4R(best variant rs6567160 (P?=?5.36?×?10-25, Beta?=?0.37), a novel rare haplotype was detected which was protective against obesity and encompassed the V103I variant with known gain-of-function properties (P?=?6.23?×?10-08, Beta?=?-0.62). PheWAS analyses extended this protective effect of V103I to type 2 diabetes, diabetic nephropathy, and chronic renal failure independent of BMI. Conclusions: MC4Rscreening in a large eMERGE cohort confirmed many previous findings, extend the MC4Rpleotropic effects, and discovered additional MC4Rrare alleles that probably contribute to obesity.

The UK Biobank is a prospective study of 502,543 individuals, combining extensive phenotypic and genotypic data with streamlined access for researchers around the world1. Here we describe the release of exome-sequence data for the first 49,960 study participants, revealing approximately 4 million coding variants (of which around 98.6% have a frequency of less than 1%). The data include 198,269 autosomal predicted loss-of-function (LOF) variants, a more than 14-fold increase compared to the imputed sequence. Nearly all genes (more than 97%) had at least one carrier with a LOF variant, and most genes (more than 69%) had at least ten carriers with a LOF variant. We illustrate the power of characterizing LOF variants in this population through association analyses across 1,730 phenotypes. In addition to replicating established associations, we found novel LOF variants with large effects on disease traits, including PIEZO1on varicose veins, COL6A1on corneal resistance, MEPEon bone density, and IQGAP2and GMPRon blood cell traits. We further demonstrate the value of exome sequencing by surveying the prevalence of pathogenic variants of clinical importance, and show that 2% of this population has a medically actionable variant. Furthermore, we characterize the penetrance of cancer in carriers of pathogenic BRCA1and BRCA2variants. Exome sequences from the first 49,960 participants highlight the promise of genome sequencing in large population-based studies and are now accessible to the scientific community.

Supplemental Digital Content is available in the text.

Protein-coding genetic variants that strongly affect disease risk can yield relevant clues to disease pathogenesis. Here we report exome-sequencing analyses of 20,791 individuals with type 2 diabetes (T2D) and 24,440 non-diabetic control participants from 5 ancestries. We identify gene-level associations of rare variants (with minor allele frequencies of less than 0.5%) in 4 genes at exome-wide significance, including a series of more than 30 SLC30A8alleles that conveys protection against T2D, and in 12 gene sets, including those corresponding to T2D drug targets (P= 6.1 × 10−3) and candidate genes from knockout mice (P= 5.2 × 10−3). Within our study, the strongest T2D gene-level signals for rare variants explain at most 25% of the heritability of the strongest common single-variant signals, and the gene-level effect sizes of the rare variants that we observed in established T2D drug targets will require 75,000–185,000 sequenced cases to achieve exome-wide significance. We propose a method to interpret these modest rare-variant associations and to incorporate these associations into future target or gene prioritization efforts.

The Million Veteran Program (MVP) was established in 2011 as a national research initiative to determine how genetic variation influences the health of US military veterans. Here we genotyped 312,571 MVP participants using a custom biobank array and linked the genetic data to laboratory and clinical phenotypes extracted from electronic health records covering a median of 10.0 years of follow-up. Among 297,626 veterans with at least one blood lipid measurement, including 57,332 black and 24,743 Hispanic participants, we tested up to around 32 million variants for association with lipid levels and identified 118 novel genome-wide significant loci after meta-analysis with data from the Global Lipids Genetics Consortium (total n> 600,000). Through a focus on mutations predicted to result in a loss of gene function and a phenome-wide association study, we propose novel indications for pharmaceutical inhibitors targeting PCSK9 (abdominal aortic aneurysm), ANGPTL4 (type 2 diabetes) and PDE3B (triglycerides and coronary disease).

To identify genetic variation underlying atrial fibrillation, the most common cardiac arrhythmia, we performed a genome-wide association study of >1,000,000 people, including 60,620 atrial fibrillation cases and 970,216 controls. We identified 142 independent risk variants at 111 loci and prioritized 151 functional candidate genes likely to be involved in atrial fibrillation. Many of the identified risk variants fall near genes where more deleterious mutations have been reported to cause serious heart defects in humans (GATA4, MYH6, NKX2-5, PITX2, TBX5)1, or near genes important for striated muscle function and integrity (for example, CFL2, MYH7, PKP2, RBM20, SGCG, SSPN). Pathway and functional enrichment analyses also suggested that many of the putative atrial fibrillation genes act via cardiac structural remodeling, potentially in the form of an ‘atrial cardiomyopathy’2, either during fetal heart development or as a response to stress in the adult heart.

PurposeThe clinical utility of screening unselected individuals for pathogenic BRCA1/2 variants has not been established. Data on cancer risk management behaviors and diagnoses of BRCA1/2-associated cancers can help inform assessments of clinical utility.MethodsWhole-exome sequences of participants in the MyCode Community Health Initiative were reviewed for pathogenic/likely pathogenic BRCA1/2 variants. Clinically confirmed variants were disclosed to patient–participants and their clinicians. We queried patient–participants’ electronic health records for BRCA1/2-associated cancer diagnoses and risk management that occurred within 12 months after results disclosure, and calculated the percentage of patient–participants of eligible age who had begun risk management.ResultsThirty-seven MyCode patient–participants were unaware of their pathogenic/likely pathogenic BRCA1/2 variant, had not had a BRCA1/2-associated cancer, and had 12 months of follow-up. Of the 33 who were of an age to begin BRCA1/2-associated risk management, 26 (79%) had performed at least one such procedure. Three were diagnosed with an early-stage, BRCA1/2-associated cancer—including a stage 1C fallopian tube cancer—via these procedures.ConclusionScreening for pathogenic BRCA1/2 variants among unselected individuals can lead to occult cancer detection shortly after disclosure. Comprehensive outcomes data generated within our learning healthcare system will aid in determining whether population-wide BRCA1/2 genomic screening programs offer clinical utility.

IMPORTANCE: Risk factors for abdominal aortic aneurysm (AAA) are largely unknown, which has hampered the development of nonsurgical treatments to alter the natural history of disease. OBJECTIVE: To investigate the association between lipid-associated single-nucleotide polymorphisms (SNPs) and AAA risk. DESIGN, SETTING, AND PARTICIPANTS: Genetic risk scores, composed of lipid trait–associated SNPs, were constructed and tested for their association with AAA using conventional (inverse-variance weighted) mendelian randomization (MR) and data from international AAA genome-wide association studies. Sensitivity analyses to account for potential genetic pleiotropy included MR-Egger and weighted median MR, and multivariable MR method was used to test the independent association of lipids with AAA risk. The association between AAA and SNPs in loci that can act as proxies for drug targets was also assessed. Data collection took place between January 9, 2015, and January 4, 2016. Data analysis was conducted between January 4, 2015, and December 31, 2016. EXPOSURES: Genetic elevation of low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TG). MAIN OUTCOMES AND MEASURES: The association between genetic risk scores of lipid-associated SNPs and AAA risk, as well as the association between SNPs in lipid drug targets (HMGCR, CETP, and PCSK9) and AAA risk. RESULTS: Up to 4914 cases and 48 002 controls were included in our analysis. A 1-SD genetic elevation of LDL-C was associated with increased AAA risk (odds ratio [OR], 1.66; 95% CI, 1.41-1.96; P = 1.1 × 10−9). For HDL-C, a 1-SD increase was associated with reduced AAA risk (OR, 0.67; 95% CI, 0.55-0.82; P = 8.3 × 10−5), whereas a 1-SD increase in triglycerides was associated with increased AAA risk (OR, 1.69; 95% CI, 1.38-2.07; P = 5.2 × 10−7). In multivariable MR analysis and both MR-Egger and weighted median MR methods, the association of each lipid fraction with AAA risk remained largely unchanged. The LDL-C–reducing allele of rs12916 in HMGCR was associated with AAA risk (OR, 0.93; 95% CI, 0.89-0.98; P = .009). The HDL-C–raising allele of rs3764261 in CETP was associated with lower AAA risk (OR, 0.89; 95% CI, 0.85-0.94; P = 3.7 × 10−7). Finally, the LDL-C–lowering allele of rs11206510 in PCSK9 was weakly associated with a lower AAA risk (OR, 0.94; 95% CI, 0.88-1.00; P = .04), but a second independent LDL-C–lowering variant in PCSK9 (rs2479409) was not associated with AAA risk (OR, 0.97; 95% CI, 0.92-1.02; P = .28). CONCLUSIONS AND RELEVANCE: The MR analyses in this study lend support to the hypothesis that lipids play an important role in the etiology of AAA. Analyses of individual genetic variants used as proxies for drug targets support LDL-C lowering as a potential effective treatment strategy for preventing and managing AAA.

Genome-wide association studies (GWAS) have identified >250 loci for body mass index (BMI), implicating pathways related to neuronal biology. Most GWAS loci represent clusters of common, noncoding variants from which pinpointing causal genes remains challenging. Here we combined data from 718,734 individuals to discover rare and low-frequency (minor allele frequency (MAF) < 5%) coding variants associated with BMI. We identified 14 coding variants in 13 genes, of which 8 variants were in genes (ZBTB7B, ACHE, RAPGEF3, RAB21, ZFHX3, ENTPD6, ZFR2and ZNF169) newly implicated in human obesity, 2 variants were in genes (MC4Rand KSR2) previously observed to be mutated in extreme obesity and 2 variants were in GIPR. The effect sizes of rare variants are ~10 times larger than those of common variants, with the largest effect observed in carriers of an MC4Rmutation introducing a stop codon (p.Tyr35Ter, MAF = 0.01%), who weighed ~7 kg more than non-carriers. Pathway analyses based on the variants associated with BMI confirm enrichment of neuronal genes and provide new evidence for adipocyte and energy expenditure biology, widening the potential of genetically supported therapeutic targets in obesity.

PurposeArrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart disease. Clinical follow-up of incidental findings in ARVC-associated genes is recommended. We aimed to determine the prevalence of disease thus ascertained.MethodsIndividuals (n = 30,716) underwent exome sequencing. Variants in PKP2, DSG2, DSC2, DSP, JUP, TMEM43, or TGFβ3 that were database-listed as pathogenic or likely pathogenic were identified and evidence-reviewed. For subjects with putative loss-of-function (pLOF) variants or variants of uncertain significance (VUS), electronic health records (EHR) were reviewed for ARVC diagnosis, diagnostic criteria, and International Classification of Diseases (ICD-9) codes.ResultsEighteen subjects had pLOF variants; none of these had an EHR diagnosis of ARVC. Of 14 patients with an electrocardiogram, one had a minor diagnostic criterion; the rest were normal. A total of 184 subjects had VUS, none of whom had an ARVC diagnosis. The proportion of subjects with VUS with major (4%) or minor (13%) electrocardiogram diagnostic criteria did not differ from that of variant-negative controls. ICD-9 codes showed no difference in defibrillator use, electrophysiologic abnormalities or nonischemic cardiomyopathies in patients with pLOF or VUSs compared with controls.ConclusionpLOF variants in an unselected cohort were not associated with ARVC phenotypes based on EHR review. The negative predictive value of EHR review remains uncertain.

Chronic obstructive pulmonary disease (COPD) is characterized by reduced lung function and is the third leading cause of death globally. Through genome-wide association discovery in 48,943 individuals, selected from extremes of the lung function distribution in UK Biobank, and follow-up in 95,375 individuals, we increased the yield of independent signals for lung function from 54 to 97. A genetic risk score was associated with COPD susceptibility (odds ratio per 1 s.d. of the risk score (∼6 alleles) (95% confidence interval) = 1.24 (1.20–1.27), P = 5.05 × 10−49), and we observed a 3.7-fold difference in COPD risk between individuals in the highest and lowest genetic risk score deciles in UK Biobank. The 97 signals show enrichment in genes for development, elastic fibers and epigenetic regulation pathways. We highlight targets for drugs and compounds in development for COPD and asthma (genes in the inositol phosphate metabolism pathway and CHRM3) and describe targets for potential drug repositioning from other clinical indications.

Height is a highly heritable, classic polygenic trait with approximately 700 common associated variants identified through genome-wide association studies so far. Here, we report 83 height-associated coding variants with lower minor-allele frequencies (in the range of 0.1–4.8%) and effects of up to 2 centimetres per allele (such as those in IHH, STC2, ARand CRISPLD2), greater than ten times the average effect of common variants. In functional follow-up studies, rare height-increasing alleles of STC2(giving an increase of 1–2 centimetres per allele) compromised proteolytic inhibition of PAPP-A and increased cleavage of IGFBP-4 in vitro, resulting in higher bioavailability of insulin-like growth factors. These 83 height-associated variants overlap genes that are mutated in monogenic growth disorders and highlight new biological candidates (such as ADAMTS3, IL11RAand NOX4) and pathways (such as proteoglycan and glycosaminoglycan synthesis) involved in growth. Our results demonstrate that sufficiently large sample sizes can uncover rare and low-frequency variants of moderate-to-large effect associated with polygenic human phenotypes, and that these variants implicate relevant genes and pathways.

Purpose:Geisinger Health System (GHS) provides an ideal platform for Precision Medicine. Key elements are the integrated health system, stable patient population, and electronic health record (EHR) infrastructure. In 2007, Geisinger launched MyCode, a system-wide biobanking program to link samples and EHR data for broad research use.Methods:Patient-centered input into MyCode was obtained using participant focus groups. Participation in MyCode is based on opt-in informed consent and allows recontact, which facilitates collection of data not in the EHR and, since 2013, the return of clinically actionable results to participants. MyCode leverages Geisinger’s technology and clinical infrastructure for participant tracking and sample collection.Results:MyCode has a consent rate of >85%, with more than 90,000 participants currently and with ongoing enrollment of ~4,000 per month. MyCode samples have been used to generate molecular data, including high-density genotype and exome sequence data. Genotype and EHR-derived phenotype data replicate previously reported genetic associations.Conclusion:The MyCode project has created resources that enable a new model for translational research that is faster, more flexible, and more cost-effective than traditional clinical research approaches. The new model is scalable and will increase in value as these resources grow and are adopted across multiple research platforms.Genet Med 18 9, 906–913.

The Electronic Medical Records and Genomics Network is a National Human Genome Research Institute–funded consortium engaged in the development of methods and best practices for using the electronic medical record as a tool for genomic research. Now in its sixth year and second funding cycle, and comprising nine research groups and a coordinating center, the network has played a major role in validating the concept that clinical data derived from electronic medical records can be used successfully for genomic research. Current work is advancing knowledge in multiple disciplines at the intersection of genomics and health-care informatics, particularly for electronic phenotyping, genome-wide association studies, genomic medicine implementation, and the ethical and regulatory issues associated with genomics research and returning results to study participants. Here, we describe the evolution, accomplishments, opportunities, and challenges of the network from its inception as a five-group consortium focused on genotype–phenotype associations for genomic discovery to its current form as a nine-group consortium pivoting toward the implementation of genomic medicine.Genet Med 15 10, 761–771.Genetics in Medicine (2013); 15 10, 761–771. doi:10.1038/gim.2013.72

The Electronic Medical Records and Genomics Network is a National Human Genome Research Institute–funded consortium engaged in the development of methods and best practices for using the electronic medical record as a tool for genomic research. Now in its sixth year and second funding cycle, and comprising nine research groups and a coordinating center, the network has played a major role in validating the concept that clinical data derived from electronic medical records can be used successfully for genomic research. Current work is advancing knowledge in multiple disciplines at the intersection of genomics and health-care informatics, particularly for electronic phenotyping, genome-wide association studies, genomic medicine implementation, and the ethical and regulatory issues associated with genomics research and returning results to study participants. Here, we describe the evolution, accomplishments, opportunities, and challenges of the network from its inception as a five-group consortium focused on genotype–phenotype associations for genomic discovery to its current form as a nine-group consortium pivoting toward the implementation of genomic medicine.

Mammalian isoforms of calcium-permeableDrosophilatransient receptor potential channels (TRPC) are involved in the sustained phase of calcium entry in nonexcitable cells. Erythropoietin (Epo) stimulates a rise in intracellular calcium ([Ca]i) via activation of voltage-independent calcium channel(s) in erythroid cells. Here, involvement of murine orthologs of classical TRPC in the Epo-modulated increase in [Ca]iwas examined. RT-PCR of TRPC 1–6 revealed high expression of only TRPC2 in Epo-dependent cell lines HCD-57 and Ba/F3 Epo-R, in which Epo stimulates a rise in [Ca]i. Using RT-PCR, Western blotting, and immunolocalization, expression of the longest isoform of mTRPC2, clone 14, was demonstrated in HCD-57 cells, Ba/F3 Epo-R cells, and primary murine erythroblasts. To determine whether erythropoietin is capable of modulating calcium influx through TRPC2, CHO cells were cotransfected with Epo-R subcloned into pTracer-CMV and either murine TRPC2 clone 14 or TRPC6, a negative control, into pQBI50. Successful transfection of Epo-R was verified in single cells by detection of green fluorescent protein from pTracer-CMV using digital video imaging, and successful transfection of TRPC was confirmed by detection of blue fluorescent protein fused through a flexible linker to TRPC. [Ca]ichanges were simultaneously monitored in cells loaded with Rhod-2 or Fura Red. Epo stimulation of CHO cells cotransfected with Epo-R and TRPC2 resulted in a rise in [Ca]iabove base line (372 ± 71%), which was significantly greater (p≤ 0.0007) than that seen in cells transfected with TRPC6 or empty pQBI50 vector. This rise in [Ca]irequired Epo and extracellular calcium. These results identify a calcium-permeable channel, TRPC2, in erythroid cells and demonstrate modulation of calcium influx through this channel by erythropoietin.

Heparan sulfate chains of proteoglycans bind to and regulate the function of a wide variety of ligands. In myoblasts, heparan sulfate proteoglycans modulate basic fibroblast growth factor activity and regulate skeletal muscle differentiation. The aim of this study was to identify endogenous extracellular ligands for muscle cell heparan sulfate proteoglycans. [35S]heparin ligand blot assays identified a 33/30 kDa doublet(p33/30) in detergent/high ionic strength extracts and heparin soluble fractions obtained from intact C2C12 myoblasts. p33/30 is localized on the plasma membrane or in the extracellular matrix where its level increases during muscle differentiation. Heparin-agarose-purified p33/30 was identified as histone H1. In vitro binding assays showed that histone H1 binds specifically to perlecan. Immunofluorescence microscopy showed that an extracellular pool of histone H1 colocalizes with perlecan in the extracellular matrix of myotube cultures and in regenerating skeletal muscle. Furthermore, histone H1 incorporated into the extracellular matrix strongly stimulated myoblast proliferation via a heparan-sulfate-dependent mechanism. These results indicate that histone H1 is present in the extracellular matrix of skeletal muscle cells, where it interacts specifically with perlecan and exerts a strong proliferative effect on myoblasts, suggesting a role for histone H1 during skeletal muscle regeneration.

Previously we reported that type V collagen synthesized by Schwann cells inhibits the outgrowth of axons from rat embryo dorsal root ganglion neurons but promotes Schwann cell migration (Chernousov, M. A., Stahl, R. C., and Carey, D. J. (2001) J. Neurosci. 21, 6125–6135). Analysis of Schwann cell adhesion and spreading on dishes coated with various type V collagen domains revealed that Schwann cells adhered effectively only to the non-collagenous N-terminal domain (NTD) of the α4(V) collagen chain. Schwann cell adhesion to α4(V)-NTD induced actin cytoskeleton assembly, tyrosine phosphorylation, and activation of the Erk1/Erk2 protein kinases. Adhesion to α4(V)-NTD is cell type-specific because rat fibroblasts failed to adhere to dishes coated with this polypeptide. Schwann cell adhesion and spreading on α4(V)-NTD was strongly inhibited by soluble heparin (IC50∼30 ng/ml) but not by chondroitin sulfate. Analysis of the heparin binding activities of a series of recombinant α4(V)-NTD fragments and deletion mutants identified a highly basic region (not present in other type V collagen NTD) as the site responsible for high affinity heparin binding. Schwann cells adhered poorly to dishes coated with α4(V)-NTD that lacked the heparin binding site and failed to spread or assemble organized actin-cytoskeletal structures. Soluble α4(V)-NTD polypeptide that contained the heparin binding site inhibited spreading of Schwann cells on dishes coated with α4(V)-NTD. Affinity chromatography of Schwann cell detergent extracts on a column of immobilized α4(V)-NTD resulted in the isolation of syndecan-3, a transmembrane heparan sulfate proteoglycan. Together, these results suggest that Schwann cells bind to collagen type V via syndecan-3-dependent binding to a novel high affinity heparin binding site in the α4(V)-NTD.

: Mounting evidence suggests that nitric oxide (NO) plays an important role in aneurysm pathogenesis. Nitric oxide synthase (NOS) expression, hemodynamic consequences of NO inhibition, and the effect of NO on matrix metalloproteinase (MMP) expression during aneurysm formation are unknown. In this study, a standard intraaortic elastase infusion rat model was used. Control animals received intraaortic elastase infusion and intraperitoneal saline injections. Experimental groups received intraaortic elastase infusion and intraperitoneal injections of aminoguanidine (500 mg/kg) or L-NAME (2 mg/kg or 10 mg/kg). Aortic diameter, blood pressure, and NO metabolites were measured at surgery and postoperative (POD) 7. A second series of rats were randomly infused with intraaortic elastase or saline and aortas were analyzed on POD 1, 3, and 7 with Western blotting for iNOS, eNOS, and MMP-9 expression. Infusion of elastase produced aneurysms (p <0.0001) in all rats. Inhibition of NO with aminoguanidine or L-NAME limited aneurysm expansion in all groups (p <0.05). Nitric oxide metabolites were increased (p <0.003) in control rats on POD 7. Arterial hypertension was present in all treated animals (p <0.05). Early up-regulation on POD 1 of iNOS (p <0.003) was noted in elastase-infused animals, but there was no iNOS expression with saline infusion. MMP-9 expression was present in both groups, with a significant increase in expression for elastase-infused animals noted on POD 7. iNOS expression is up-regulated early in experimental aneurysm formation, followed by increases in MMP-9 expression. Inhibition of NO limits aneurysmal expansion despite production of arterial hypertension.

Purpose:Research investigating abdominal aortic aneurysms (AAAs) commonly uses a rat model dependent on aortic infusion of porcine pancreatic elastase to initiate AAA formation. Unfortunately, the sizes of AAAs generated by this model have varied widely among published studies. This may reflect lot-to-lot variations in commercial elastase preparations. This study was undertaken to investigate the ability of different lots of elastase to induce AAAs and explain the variability identified. Methods:Four lots of elastase were evaluated in the standard rat AAA model. Saline solution was used as a control. Additional groups of rats were treated with higher concentrations of elastase with or without the macrophage activator thioglycollate medium. Aortic diameters were measured in all rats. Inflammation and elastin degradation was examined histologically. Elastase activity and purity were evaluated for all lots. Results:Of the four lots tested, only one was able to consistently generate AAAs at the standard dose (P< .05). Increasing the amount of elastase infused produced AAAs in some ineffective lots. Infusion of thioglycollate medium in combination with otherwise ineffective elastase produced AAAs (P= .02). However, the elastase with the highest purity failed to generate AAAs, even at the highest dose tested or in combination with thyioglycollate medium. Thioglycollate medium alone failed to result in AAA formation. All elastase lots displayed elastolytic activity in vitro and produced elastin degradation in vivo. Elastin degradation did not correlate with AAA size in elastase-treated rats (P= NS). Aneurysm size correlated with extent of inflammation (P= .005). Conclusion:Induction of AAAs does not correlate with elastolytic activity. Infusion of pure elastase alone is not sufficient to induce AAA formation in spite of evidence of elastin degradation. Presumed inflammatory modifiers, which contaminate some elastase preparations, enhance AAA formation. Future use of this rat model will need to take the variability of elastase preparations into account with controls for each new elastase lot. (J Vasc Surg 2001;33:1255-62.)

Purpose:Nitric oxide (NO), frequently cited for its protective role, can also generate toxic metabolites known to degrade elastin. Both abdominal aortic aneurysms (AAAs) and inducible nitric oxide synthase (iNOS) are associated with inflammatory states, yet the relationship between NO production by iNOS and AAA development is unknown. The current study examines iNOS expression, NO production, and the effects of selective inhibition of iNOS by aminoguanidine in experimental AAA. Methods:An intra-aortic elastase infusion model was used. Control rats received intra-aortic saline infusion and postoperative intraperitoneal saline injections (Group 1). In the remaining groups, intra-aortic elastase infusion was used to induce aneurysm formation. These rats were treated with intraperitoneal injections of saline postoperatively (Group 2), aminoguanidine postoperatively (Group 3), or aminoguanidine preoperatively and postoperatively (Group 4). Aortic diameter and plasma nitrite/nitrate levels were measured on the day of surgery and postoperative day 7. Aortas were harvested for biochemical and histologic analysis on postoperative day 7. Results:Infusion of elastase produced AAAs (P<.001) with significant production of iNOS (P<.05) and nitrite/nitrate (P<.003) compared with controls. Selective inhibition of iNOS with aminoguanidine in elastase-infused aortas significantly reduced aneurysm size (P<.01) compared with elastase infusion alone. Aminoguanidine-treated rats displayed suppression of iNOS expression and plasma nitrite/nitrate production not significantly different from the control group. Histologic evaluation revealed equivalent inflammatory infiltrates in elastase-infused groups. Conclusion:Expression of iNOS is induced and plasma nitrite/nitrate levels are increased in experimental AAA. Inhibition of iNOS limits NO production and iNOS expression, resulting in smaller aneurysm size. NO production by iNOS plays an important role with detrimental effects during experimental aneurysm development. (J Vasc Surg 2001;33:579-86.)

The immature rat uterus has been extensively used as an in vivomodel system to study the molecular mechanisms of steroid hormone actions. In this study, we demonstrated the regulated expression of syndecan-3 in the rat uterus by the steroid hormone 17β-estradiol. Administration of a single physiological dose of 17β-estradiol (40 μg/kg) to ovariectomized immature animals induced a rapid and transient increase in uterine syndecan-3 mRNA. Transcript levels reached a peak elevation of 3-fold above saline control tissues 4 h after hormone administration. Inhibition of message up-regulation by actinomycin D but not cycloheximide indicated a hormone response dependent on RNA transcription but not new protein synthesis. The estrogenic ligands estriol and tamoxifen were also effective at raising syndecan-3 mRNA levels; however, nonestrogenic ligands, including progesterone, 5α-dihydrotestosterone, and dexamethasone, failed to stimulate a change in mRNA levels. Hormone-induced changes in mRNA led to transient changes in syndecan-3 protein content and significant alteration in the temporal and spatial expression in endometrial epithelial cells. Collectively, these data show that the steroid hormone 17β-estradiol, regulates transcription of the syndecan-3 gene in the uterus via an estrogen receptor-dependent mechanism. This estrogen-regulated expression of syndecan-3 may play an important role in changes in tissue ultrastructure crucial for proper uterine growth.

We previously identified a phosphatidylinositol-specific phospholipase c (PI-PLC)-releasable heparan sulfate proteoglycan (HSPG) on the surface of rat Schwann cells (D. J. Carey and R. C. Stahl, J. Cell Biol.111, 2053-2062, 1990). The present study was carried out to investigate the localization and processing of this proteoglycan. The HSPG was synthesized as a PI-PLC-releasable form that was shed into the culture medium with a T1/2of 17 h. Degradation of the HSPG was negligible. The HSPG was present on the surface of Schwann cells on small (100-200 nm diameter) cylindrical membrane extensions that resembled filopodia. In neonatal peripheral nerve, brain, heart, and striated muscle the HSPG was found to be localized principally to regions of the cell surface that were in contact with basement membranes. Northern blot analysis with cDNA coding for rat glypican (a previously described human fibroblast HSPG) demonstrated abundant expression of glypican mRNA in Schwann cells. Antibodies made against recombinant rat glypican core protein immunoprecipitated the Schwann cell PI-PLC-releasable HSPG. These data demonstrate that the Schwann cell HSPG is rat glypican and support the hypothesis that this proteoglycan functions in cell-extracellular matrix interactions.

The expression of cell surface heparan sulfate proteoglycans in rat heart was investigated by Northern blot analysis with specific cDNA probes. In adult heart syndecan-3 and glypican mRNAs were abundantly expressed. Lower levels of syndecan-2 mRNA and very low levels of syndecan-1 mRNA were also detected. Analysis of RNA isolated from hearts of rats of various ages revealed that syndecan-3 and glypican mRNAs levels increased dramatically at birth, and continued to be expressed at high levels in adult animals. To determine which of these proteoglycans was expressed in cardiomyocytes, primary cultures of cardiomyocytes and nonmyocytes isolated from neonatal rat hearts were analyzed for proteoglycan expression. Glypican mRNA was localized almost exclusively to cardiomyocytes. Syndecan-3 mRNA was not detected in myocytes, but was detected in the nonmyocyte cells. Biochemical characterization of cardiomyocyte glypican revealed that it was a phosphatidylinositol-anchored heparan sulfate proteoglycan. Results of immunofluorescent staining of rat hearts with anti-glypican antibodies were consistent with the Northern blot data, and localized glypican to the lateral regions of myocyte plasma membrane that contact the basement membrane, as well as sites of myocyte adhesion junctions. At the latter site glypican colocalized with vinculin. Visualization of basic fibroblast growth factor binding sites by means of a tissue slice overlay assay also revealed colocalization with glypican. These results demonstrate developmental and cell-type-specific expression of membrane heparan sulfate proteoglycans in the heart. They also show that glypican is a major heparan sulfate proteoglycan expressed on the cardiomyocyte plasma membrane.

Expression of the transmembrane proteoglycan syndecan-1 in Schwann cells leads to enhanced spreading and cytoskeletal reorganization, but without an apparent stable association of syndecan-1 with cytoskeletal structures such as focal adhesions. Since cell surface oligomerization may be a mechanism for regulating the activities of transmembrane receptors, we wanted to investigate whether antibody-induced aggregation of the proteoglycan would promote its association with the cytoskeleton. When syndecan-1-expressing cells were incubated with anti-syndecan-1 and anti-IgG antibodies, clustering of proteoglycan on the cell surface was observed by immunofluorescence microscopy. The resulting pattern of syndecan-I distribution was very similar to that of the underlying microfilament network, as visualized by fluorescent-phalloidin staining. In cells that were fixed briefly with paraformaldehyde before addition of the anti-IgG antibodies no such colocalization of syndecan-1 and microfilaments was observed. Additional findings supported the conclusion that this pattern of syndecan-1 distribution reflected an association with microfilaments: aggregated syndecan-1 was resistant to extraction by nonionic detergent; incubation of the cells with cytochalasin b, but not colchicine, altered the pattern of aggregated syndecan-1 distribution; antibody-induced clustering of syndecan-1 led to a reorganization of actin filaments. Syndecan-1 remained on the cell surface following antibody-induced clustering, as revealed by immunogold staining and transmission electron microscopy. A mutant form of syndecan-1 lacking most of the cytoplasmic domain failed to exhibit actin filament association or induce actin reorganization following antibody-mediated aggregation. These results suggest that transient associations of syndecan family proteoglycans with microfilaments may be important aspects of their biological functions.

We expressed domains of the core protein of the transmembrane heparan sulfate proteoglycan N-syndecan (syndecan-3) either individually or as maltose-binding protein fusion proteins. Biochemical characterization of the purified proteins revealed that some of them were capable of self-association and formed stable, noncovalent multimeric complexes. The formation of N-syndecan core protein complexes was also demonstrated in mammalian cells by in situcross-linking. Identification of structural motifs in the core protein of N-syndecan responsible for the formation of these complexes was accomplished by analyzing a series of constructs comprising different regions of the protein as well as site-directed mutants. Self-association was assayed by SDS-polyacrylamide gel electrophoresis, glutaraldehyde cross-linking, and size-exclusion high pressure liquid chromatography. Our results indicated that (i) the transmembrane domain of the N-syndecan core protein was required but not sufficient for the formation of stable complexes; (ii) the minimal amino acid sequence that conferred the ability of the N-syndecan core protein to form multimeric complexes included the last four amino acids (ERKE) of the extracellular domain plus the transmembrane domain; (iii) point mutations that changed the basic residues in this sequence to alanine residues either partially or completely abolished the ability of the N-syndecan core protein to form complexes; and (iv) replacement of conserved glycine residues in the transmembrane domain with leucines abolished complex formation. This property is similar to the oligomerization activity of other transmembrane receptors and suggests that regulated self-association may be important for the biological activity of transmembrane proteoglycans.

Cultured rat Schwann cells were stimulated to deposit fibrillar extracellular matrix by treatment with ascorbic acid in the absence of nerve cells. Immunofluoresence staining of the matrix showed that it contains collagens types I and IV, fibronectin and perlecan but not laminin. Collagen type IV, fibronectin and perlecan co-distributed completely in the matrix fibrils, whereas collagen type I was present in only a subset of these fibrils. Time course studies indicated that collagen type I fibrils appear at late stages of matrix formation. Digestion of Schwann cell extracellular matrix with collagenase effectively disrupted most of the matrix including fibronectin fibrils. This was in contrast with fibroblasts, where collagenase treatment removed collagen with no visible effect on fibronectin fibrils. α5 integrin was expressed on the cell surface of Schwann cells and partially codistributed with fibronectin-containing fibrils. This suggests that the inability of Schwann cells to deposit fibronectin-containing matrix through a conventional, collagen-independent mechanism was not due to the lack of fibronectin-binding integrins on their cell surface. Polyclonal anti-fibronectin antibodies inhibited the deposition of fibronectin into the matrix fibrils, whereas collagen type IV fibrils were generally unaffected. Growth of Schwann cells on collagen type IV-coated substrate in the absence of ascorbate induced deposition of fine fibronectin fibrils. These results suggest that Schwann cells use an apparently novel, collagen type IV-dependent mechanism for the deposition of fibronectin into their extracellular matrix.

Syndecan-1 is an integral membrane proteoglycan involved in the interaction of cells with extracellular matrix proteins and growth factors. It is transiently expressed in several condensing mesenchymal tissues after epithelial induction. In this study we evaluated the expression of syndecan-1 during skeletal muscle differentiation. The expression of syndecan-1 as determined by Northern blot analyses and immunofluorescence microscopy is down-regulated during differentiation. The transcriptional activity of a syndecan-1 promoter construct is also down-regulated in differentiating muscle cells. The decrease in syndecan-1 gene expression is not dependent on the presence of E-boxes, binding sites for the MyoD family of transcription factors in the promoter region, or myogenin expression. Deletion of the region containing the E-boxes or treatment of differentiating cells with sodium butyrate, an inhibitor of myogenin expression, had no effect on syndecan-1 expression. Basic fibroblast growth factor and transforming growth factor type β, which are inhibitors of myogenesis, had little effect on syndecan-1 expression. When added together, however, they induced syndecan-1 expression. Retinoic acid, an inducer of myogenesis, inhibited syndecan-1 expression and abolished the effect of the growth factors. These results indicate that syndecan-1 expression is down-regulated during myogenesis and that growth factors and retinoic acid modulate syndecan-1 expression by a mechanism that is independent of myogenin.

A heparin-binding glycoprotein was purified from conditioned medium of cultured rat Schwann cells. The protein, p200, which has an apparent molecular mass of approximately 200 kDa, was identified by its ability to bind the cell surface heparan sulfate proteoglycan N-syndecan (syndecan-3) in a membrane overlay assay. Soluble heparin but not chondroitin sulfate inhibited the binding, suggesting the involvement of heparan sulfate chains of proteoglycan in the interaction. Purified p200 promoted the attachment and spreading of Schwann cells. Adhesion to p200 was blocked by heparin, suggesting that heparan sulfate proteoglycans are cell surface receptors for p200. The tissue distribution of p200 was determined by immunoblot analysis with anti-p200 antibodies. Among neonatal rat tissues examined p200 was detected only in sciatic nerve and, at lower levels, in skeletal muscle. p200 expression in sciatic nerve was detectable only during the first 2-3 weeks of postnatal development and was not detected in adult rats. Immunofluorescent staining of rat sciatic nerve showed that p200 was localized in the extracellular matrix surrounding individual Schwann cells-axon units. Two tryptic peptides from p200 were purified and sequenced. These contained multiple GXXcollagen-like repeats. Bacterial collagenase digestion of p200 produced a product with an apparent molecular mass of approximately 90 kDa. These data suggest that Schwann cells secrete an apparently novel collagen-like adhesive protein that interacts with cells through cell surface heparan sulfate proteoglycans.

This review will summarize our current state of knowledge of the structure, biochemical properties and functions of syndecans, a family of transmembrane heparan sulphate proteoglycans. Syndecans bind a variety of extracellular ligands via their covalently attached heparan sulphate chains. Syndecans have been proposed to play a role in a variety of cellular functions, including cell proliferation and cell–matrix and cell–cell adhesion. Syndecan expression is highly regulated and is cell-type- and developmental-stage-specific. The main function of syndecans appears to be to modulate the ligand-dependent activation of primary signalling receptors at the cell surface. Principal functions of the syndecan core proteins are to target the heparan sulphate chains to the appropriate plasma-membrane compartment and to interact with components of the actin-based cytoskeleton. Several functions of the syndecans, including syndecan oligomerization and actin cytoskeleton association, have been localized to specific structural domains of syndecan core proteins.

The cytoplasmic domain of the syndecan family of heparan sulfate proteoglycans is punctuated by the presence of four regularly spaced tyrosine residues. In this report, we explore the possibility of whether the four tyrosine residues in the cytoplasmic domain of N-syndecan (Syndecan 3) are potential substrates for phosphorylation by a tyrosine kinase. Bacterially expressedelkkinase was used to phosphorylate a series of bacterially expressed N-syndecan fusion proteins. Our results clearly demonstrate that the tyrosine residues in the cytoplasmic domain of N-syndecan can be phosphorylated by a tyrosine-specific kinase, and that all four tyrosine residues are capable of being phosphorylated.

ABSTRACTA peptide corresponding to the epidermal growth factor homology domain of ß-heregulin stimulated autophosphorylation of the heregulin receptors erbB2 and erbB3 in Schwann cells and activation of the mitogen-activated protein (MAP) kinases ERK1 and ERK2. Heregulin-dependent activation of PAK65, a component of the stress-activated signaling pathway, ribosomal S6 kinase, and a cyclic AMP (cAMP) response element binding protein (CREB) kinase, identified as p95RSK2, was also observed. Receptor phosphorylation and activation of these kinases in response to heregulin occurred in the absence of forskolin stimulation and were not augmented in cells treated with forskolin, a direct activator of adenylyl cyclase. Schwann cell proliferation in response to heregulin was observed only when the cells were also exposed to an agent that elevates cAMP levels. In the absence of heregulin, elevation of cAMP levels failed to stimulate Schwann cell proliferation. Forskolin significantly enhanced heregulin-stimulated expression of cyclin D and phosphorylation of the retinoblastoma gene product. In cells treated with both heregulin and forskolin there was a sustained accumulation of phospho-CREB, which was not observed in cells treated with either agent alone. Heregulin and forskolin synergistically activated transcription of a cyclin D promoter construct. These results demonstrate that heregulin-stimulated activation of MAP kinase is not sufficient to induce maximal Schwann cell proliferation. Expression of critical cell cycle regulatory proteins and cell division require activation of both heregulin and cAMP-dependent processes.

The use of specific inhibitors of proteoglycan synthesis have demonstrated essential functions for these molecules. Proteoglycans do not appear to be essential for cell viability or proliferation but are necessary for stable assembly of ECM and functional cell-ECM interaction. Because of their ability to nearly completely abolish ECM assembly in cell culture systems, proteoglycan synthesis inhibitors are useful tools to examine effects of ECM on the phenotypic behavior of cells.

= 3; donor, 4.5 ± 2.2, n= 6; AAA, 4.0 ± 0.95, n= 15). There was a significant increase in the level of MMP-9 mRNA in AAA specimens (occlusive, 16.8 ± 3, n= 3; donor, 5.7 ± 1.2, n= 6; AAA, 56.7 ± 11, n= 15, p= 0.0069). The levels of mRNA coding for TIMP-1 were not significantly different. There was a small but statistically significant increase in TIMP-2 mRNA in AAA tissue. These data support the hypothesis that increased activity of MMP-9, but not MMP-2, is an important factor in the etiology of AAAs. This enhanced MMP-9 activity could then result in degradation of the ECM, leading to aneurysmal dilatation.