Arya Mani

LRP6 Mutation in a Family with Early Coronary Disease and Metabolic Risk Factors

Coronary artery disease (CAD) is the leading cause of death worldwide and is commonly caused by a constellation of risk factors called the metabolic syndrome. We characterized a family with autosomal dominant early CAD, features of the metabolic syndrome (hyperlipidemia, hypertension, and diabetes), and osteoporosis. These traits showed genetic linkage to a short segment of chromosome 12p, in which we identified a missense mutation in LRP6, which encodes a co-receptor in the Wnt signaling pathway. The mutation, which substitutes cysteine for arginine at a highly conserved residue of an epidermal growth factor–like domain, impairs Wnt signaling in vitro. These results link a single gene defect in Wnt signaling to CAD and multiple cardiovascular risk factors.

Finding genetic contributions to sporadic disease: A recessive locus at 12q24 commonly contributes to patent ductus arteriosus

The causes of many sporadic diseases are unexplained; the contribution of recessive loci with reduced penetrance is one possibility that has been difficult to explore. We describe an approach to this problem by first searching for diseases with higher prevalence in populations with high rates of consanguinity, then determining whether disease cases are more commonly the product of consanguinous union than controls in such populations, followed by analysis of genetic linkage in consanguinous cases. We demonstrate the utility of this approach by investigation of congenital heart disease in Iran. We found that patent ductus arteriosus (PDA), a common congenital heart disease, accounts for a higher fraction of congenital heart disease in Iran (15%) than in the United States (2–7%). Moreover, Iranian PDA cases demonstrated a marked increase of parental consanguinity (63%), compared with the general Iranian population (25%) or control cases with tetralogy of Fallot (30%). The recurrence of PDA among siblings was 5%. A genomewide analysis of linkage in 21 unrelated consanguinous PDA cases demonstrated a multipoint logarithm of odds score of 6.27 in favor of linkage of PDA to a 3-centimorgan interval of chromosome 12q24, with 53% of kindreds linked. These findings together establish a recessive component to PDA and implicate a single locus, PDA1, in one third or more of all PDA cases in Iran; they further suggest a role for this locus in PDA worldwide. Finally, these results suggest a general approach to the identification of recessive contributions to sporadic diseases.

Bicuspid aortic valve: clinical approach and scientific review of a common clinical entity.

Bicuspid aortic valve (BAV) disease is becoming increasingly respected by clinicians. This enhanced appreciation is based on recognition of how common and virulent this disease really is. This disease is now known to be the most common congenital lesion affecting the human heart (with potential competition in frequency posed by mitral valve prolapse). It is estimated that the BAV lesion alone accounts for more morbidity and mortality than all the other congenital cardiac lesions combined. Many authorities feel that this disease is so virulent that every individual with a BAV will, given enough time, develop aortic stenosis, aortic insufficiency or aortic aneurysm/dissection related to the bicuspid valve disease. This review looks in detail at clinical issues related to BAV disease, including genetics, pathophysiology, diagnosis, management and surgical decision making. The picture emerging from basic and clinical studies is of a defect in collagen metabolism affecting not only the aortic valve, but also the wall of the aorta itself. Timely intervention for bicuspid-related aortic valve disease or aneurysm can preserve both duration of life and quality of life in affected individuals.

A Form of the Metabolic Syndrome Associated with Mutations in DYRK1B

BACKGROUND Genetic analysis has been successful in identifying causative mutations for individual cardiovascular risk factors. Success has been more limited in mapping susceptibility genes for clusters of cardiovascular risk traits, such as those in the metabolic syndrome. METHODS We identified three large families with coinheritance of early-onset coronary artery disease, central obesity, hypertension, and diabetes. We used linkage analysis and whole-exome sequencing to identify the disease-causing gene. RESULTS A founder mutation was identified in DYRK1B, substituting cysteine for arginine at position 102 in the highly conserved kinase-like domain. The mutation precisely cosegregated with the clinical syndrome in all the affected family members and was absent in unaffected family members and unrelated controls. Functional characterization of the disease gene revealed that nonmutant protein encoded by DYRK1B inhibits the SHH (sonic hedgehog) and Wnt signaling pathways and consequently enhances adipogenesis. Furthermore, DYRK1B promoted the expression of the key gluconeogenic enzyme glucose-6-phosphatase. The R102C allele showed gain-of-function activities by potentiating these effects. A second mutation, substituting proline for histidine 90, was found to cosegregate with a similar clinical syndrome in an ethnically distinct family. CONCLUSIONS These findings indicate a role for DYRK1B in adipogenesis and glucose homeostasis and associate its altered function with an inherited form of the metabolic syndrome. (Funded by the National Institutes of Health.).

Low Density Lipoprotein (LDL) Receptor-related Protein 6 (LRP6) Regulates Body Fat and Glucose Homeostasis by Modulating Nutrient Sensing Pathways and Mitochondrial Energy Expenditure

Background: The link between obesity and diabetes is poorly understood. Wnt signaling is implicated in adipogenesis and glucose metabolism. Results: LRP6+/− mice are protected against diet-induced obesity and insulin resistance by regulation of genes involved in adipogenesis, metabolism, and insulin signaling. Conclusion: This study identifies novel pathways that regulate metabolism. Significance: LRP6 is a potential target for novel therapeutics for diabetes and obesity. Body fat, insulin resistance, and type 2 diabetes are often linked together, but the molecular mechanisms that unify their association are poorly understood. Wnt signaling regulates adipogenesis, and its altered activity has been implicated in the pathogenesis of type 2 diabetes and metabolic syndrome. LRP6+/− mice on a high fat diet were protected against diet-induced obesity and hepatic and adipose tissue insulin resistance compared with their wild-type (WT) littermates. Brown adipose tissue insulin sensitivity and reduced adiposity of LRP6+/− mice were accounted for by diminished Wnt-dependent mTORC1 activity and enhanced expression of brown adipose tissue PGC1-α and UCP1. LRP6+/− mice also exhibited reduced endogenous hepatic glucose output, which was due to diminished FoxO1-dependent expression of the key gluconeogenic enzyme glucose-6-phosphatase (G6pase). In addition, in vivo and in vitro studies showed that loss of LRP6 allele is associated with increased leptin receptor expression, which is a likely cause of hepatic insulin sensitivity in LRP6+/− mice. Our study identifies LRP6 as a nutrient-sensitive regulator of body weight and glucose metabolism and as a potential target for pharmacological interventions in obesity and diabetes.

LRP6 enhances glucose metabolism by promoting TCF7L2-dependent insulin receptor expression and IGF receptor stabilization in humans.

Common genetic variations in Wnt signaling genes have been associated with metabolic syndrome and diabetes by mechanisms that are poorly understood. A rare nonconservative mutation in Wnt coreceptor LRP6 (LRP6(R611C)) has been shown to underlie autosomal dominant early onset coronary artery disease, type 2 diabetes, and metabolic syndrome. We examined the interplay between Wnt and insulin signaling pathways in skeletal muscle and skin fibroblasts of healthy nondiabetic LRP6(R611C) mutation carriers. LRP6 mutation carriers exhibited hyperinsulinemia and reduced insulin sensitivity compared to noncarrier relatives in response to oral glucose ingestion, which correlated with a significant decline in tissue expression of the insulin receptor and insulin signaling activity. Further investigations showed that the LRP6(R611C) mutation diminishes TCF7L2-dependent transcription of the IR while it increases the stability of IGFR and enhances mTORC1 activity. These findings identify the Wnt/LRP6/TCF7L2 axis as a regulator of glucose metabolism and a potential therapeutic target for insulin resistance.

Wild-type LRP6 inhibits, whereas atherosclerosis-linked LRP6R611C increases PDGF-dependent vascular smooth muscle cell proliferation

Vascular smooth muscle cell (VSMC) proliferation is an important event in atherosclerosis and other vasculopathies. PDGF signaling is a key mediator of SMC proliferation, but the mechanisms that control its activity remain unclear. We previously identified a mutation in LDL receptor-related protein 6 (LRP6), LRP6R611C, that causes early atherosclerosis. Examination of human atherosclerotic coronary arteries showed markedly increased expression of LRP6 and colocalization with PDGF receptor β (PDGFR-β). Further investigation showed that wild-type LRP6 inhibits but LRP6R611C promotes VSMC proliferation in response to PDGF. We found that wild-type LRP6 forms a complex with PDGFR-β and enhances its lysosomal degradation, functions that are severely impaired in LRP6R611C. Further, we observed that wild-type and mutant LRP6 regulate cell-cycle activity by triggering differential effects on PDGF-dependent pathways. These findings implicate LRP6 as a critical modulator of PDGF-dependent regulation of cell cycle in smooth muscle and indicate that loss of this function contributes to development of early atherosclerosis in humans.

LRP6 Protein Regulates Low Density Lipoprotein (LDL) Receptor-mediated LDL Uptake

Background: Elevated serum LDL cholesterol is a major risk factor for atherosclerosis. Mechanisms that regulate LDL homeostasis are not well understood. Results: LRP6 forms a complex with LDLR and other endocytic proteins, and its knockdown or mutation impairs LDLR endocytosis. Conclusion: LRP6 regulates LDLR-dependent LDL uptake. Significance: LRP6 is a potential target for development of novel lipid-lowering drugs. Genetic variations in LRP6 gene are associated with high serum LDL cholesterol levels. We have previously shown that LDL clearance in peripheral B-lymphocytes of the LRP6R611C mutation carriers is significantly impaired. In this study we have examined the role of wild type LRP6 (LRP6WT) and LRP6R611C in LDL receptor (LDLR)-mediated LDL uptake. LDL binding and uptake were increased when LRP6WT was overexpressed and modestly reduced when it was knocked down in LDLR-deficient CHO (ldlA7) cells. These findings implicated LRP6 in LDLR-independent cellular LDL binding and uptake. However, LRP6 knockdown in wild type CHO cells resulted in a much greater decline in LDL binding and uptake compared with CHO-ldlA7 cells, suggesting impaired function of the LDLR. LDLR internalization was severely diminished when LRP6 was knocked down and was restored after LRP6 was reintroduced. Further analysis revealed that LRP6WT forms a complex with LDLR, clathrin, and ARH and undergoes a clathrin-mediated internalization after stimulation with LDL. LDLR and LRP6 internalizations as well as LDL uptake were all impaired in CHO-k1 cells expressing LRP6R611C. These studies identify LRP6 as a critical modulator of receptor-mediated LDL endocytosis and introduce a mechanism by which variation in LRP6 may contribute to high serum LDL levels.

Rare Nonconservative LRP6 Mutations Are Associated with Metabolic Syndrome

A rare mutation in LRP6 has been shown to underlie autosomal dominant coronary artery disease (CAD) and metabolic syndrome in an Iranian kindred. The prevalence and spectrum of LRP6 mutations in the disease population of the United States is not known. Two hundred white Americans with early onset familial CAD and metabolic syndrome and 2,000 healthy Northern European controls were screened for nonconservative mutations in LRP6. Three novel mutations were identified, which cosegregated with the metabolic traits in the kindreds of the affected subjects and none in the controls. All three mutations reside in the second propeller domain, which plays a critical role in ligand binding. Two of the mutations substituted highly conserved arginines in the second YWTD domain and the third substituted a conserved glycosylation site. The functional characterization of one of the variants showed that it impairs Wnt signaling and acts as a loss of function mutation.

Mutation in EGFP Domain of LDL Receptor-Related Protein 6 Impairs Cellular LDL Clearance

Mutation in the EGFP domain of LDL receptor-related protein 6 (LRP6R611C) is associated with hypercholesterolemia and early-onset atherosclerosis, but the mechanism by which it causes disease is not known. Cholesterol uptake was examined in cells from LRP6+/− mice and LRP6R611C mutation carriers. Splenic B cells of LRP6+/− mice have significantly lower LRP6 expression and low-density lipoprotein (LDL) uptake than those of the wild-type littermates. Although similar levels of total LRP6 were found in lymphoblastoid cells (LCLs) of LRP6R611C mutation carriers and those of the unaffected family member, LDL uptake was significantly lower in the mutant cells. Mutant and wild-type receptors show similar affinities for apolipoprotein B at neutral pH. LRP6 colocalized with LDL and was coimmunoprecipitated with NPC1 (Niemann–Pick disease type C1), an endocytic regulator of LDL trafficking. However, the cellular localization of LRP6 in the mutant cells shifted from cell surface to late endosomes/lysosomes. Plasma membrane expression levels of LRP6R611C was lower compared to wild-type receptor and declined to a greater extent in LDL-rich medium. Further examinations revealed lower efficacy of apolipoprotein B dissociation from LRP6R611C compared to wild-type receptor at an acidic pH. These studies identify LRP6 as a receptor for LDL endocytosis and imply that R611C mutation results in reduced LRP6 membrane expression and decreased LDL clearance. Based on our findings, we conclude that the increased affinity of the mutant receptor for LDL in acidic pH leads to their impaired dissociation in late endosomes, which compromises their recycling to the plasma membrane.