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Single Genetic Defect Causes Early Heart Disease
  From the News Archive - Mar. 01, 2007
CHEVY CHASE, Md., March 1, 2007-A team of researchers from the United States and Iran has identified a genetic mutation that causes early onset coronary artery disease in members of a large Iranian family. The genetic mutation leads to heart disease by causing high blood pressure, high blood levels of "bad cholesterol" and diabetes, all risk factors for heart disease. Coronary artery disease is the leading cause of death worldwide.
"Unfortunately, most of the individuals in this family who carried the mutation died in their early fifties from coronary artery disease that resulted in heart attacks and heart failure," said the research team leader, Richard P. Lifton, a Howard Hughes Medical Institute investigator at Yale University School of Medicine. "Our studies identify a single mutation that has quite a large effect on many of the metabolic risk factors for coronary artery disease."
Lifton and his colleagues published their research article in the March 2, 2007, issue of the journal Science. Arya Mani, a cardiologist and member of Lifton's laboratory at Yale, was first author of the article. The Yale group collaborated on the studies with researchers at Amir Kabir University of Technology, Azad University and The Social Welfare and Rehabilitation Sciences University, all of which are in Tehran, Iran.
The genetic defect is rare, so the discovery by itself will not provide an explanation for the more common forms of coronary artery disease, which are caused by a constellation of factors, said Lifton. However, he said, understanding the molecular nature of this single genetic defect, which is at the root of a familial form of such a complex disease, offers invaluable clues. Researchers may be able to apply that knowledge to improve understanding of what causes the body's metabolic machinery to malfunction in the more "garden variety" forms of heart disease.
According to Lifton, physicians in Tehran had long been aware of the family's tragic struggle with early coronary artery disease. In fact, 23 of 28 blood relatives of the first patient identified with this genetic mutation died from coronary artery disease by an average age of 52. The research team obtained medical records and blood samples from all surviving members of the family. The medical records showed that the family members had a characteristic cluster of symptoms called metabolic syndrome. People with this syndrome have hypertension, high blood lipid levels and diabetes, and they are at much higher risk of developing heart disease.
"Dr. Mani identified this family as an example of an extreme outlier of a common disease," said Lifton. "Studying extreme forms of common disease has been a longtime theme in our laboratory." Lifton's strategy is to pinpoint and analyze the rare genetic traits that cause complex disorders. By understanding the causes of those familial forms of complex diseases such as hypertension, Lifton and others have laid the foundation for identifying the mechanisms that underlie more common forms of the disease.
One of the important features of the Iranian family, Lifton noted, was that affected family members exhibited metabolic syndrome and early coronary artery disease, but unaffected family members were normal. "So this told us that the coronary disease was traveling with this cluster of metabolic risk factors," he said. "Those risk factors were behaving as though they were being transmitted through the family as a single factor."
When the researchers performed a detailed genetic comparison of affected and disease-free family members, they found that a specific segment of chromosome 12 was the most likely genomic hiding place for this unknown factor. In mapping the six genes present on this small chunk of chromosome 12, they found that the gene for LDL receptor-related protein 6 (LRP6) was present in that region of the chromosome.
They immediately focused on LRP6, tipped off in part by an earlier discovery by HHMI investigator Matthew Warman at Children's Hospital Boston. In 2001, Warman had shown that members of the LRP gene family were important in bone development. "What caught our attention was that multiple people in the Iranian family we were studying had coronary artery disease and unexplained hip fractures at young ages. We had not made much of the hip fractures at the outset, but once we knew that LRP6 was in this small genomic interval that contained the coronary-artery-disease-causing gene, we immediately thought of LRP6 as the likely culprit," said Lifton.
By sequencing the LRP6 gene in affected family members, the scientists found that the mutation caused the substitution of a single amino acid in the protein the gene produced. In contrast, the researchers did not find the mutation when they analyzed large comparison populations of Iranians and Americans. However, the researchers' statistical analysis of the family members revealed a high correlation between the presence of the mutation, metabolic syndrome, and osteoporosis. In cell culture studies, Dan Wu, a Yale biochemist, found that the mutation compromised how the LRP6 protein functions in the Wnt signaling pathway. Wnt is at the heart of a key metabolic signaling pathway that is important in embryonic development and contributes to an array of normal physiological processes in adults. Lifton emphasized that the compromised Wnt pathway is intriguing and the pathway will likely become an important research target for understanding coronary artery disease.
"For example, now that we know that altered Wnt signaling can lead to multiple components of the metabolic syndrome, this raises the question of whether other mutations in the Wnt signaling pathway-or acquired defects that cause alteration of Wnt pathway activity-might commonly contribute to metabolic syndrome and coronary artery disease," he said.
Basic understanding of how the Wnt pathway malfunctions could lead to new treatments for coronary artery disease. "Although it's a long way off, we might ultimately develop ways to either disrupt or increase the activity of particular components of the pathway, to prevent development of metabolic syndrome and coronary artery disease," he said.
The discovery that the LRP6 mutation also causes osteoporosis raises the possibility of a linkage with coronary artery disease, said Lifton. "There is emerging recognition that osteoporosis and coronary artery disease occur together more often than expected by chance," he said. "We have now implicated altered activity of the Wnt pathway in development of both coronary artery disease and osteoporosis. So, one can imagine identifying patients with osteoporosis and coronary artery disease as a way of selecting those who might be particularly interesting to investigate for altered Wnt signaling."
LRP6 Mutation in a Family with Early Coronary Disease and Metabolic Risk Factors

Arya Mani,1* Jayaram Radhakrishnan,1 He Wang,2 Alaleh Mani,3 Mohammad-Ali Mani,4 Carol Nelson-Williams,1 Khary S. Carew,1 Shrikant Mane,1 Hossein Najmabadi,5 Dan Wu,2 Richard P. Lifton1*
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.
Coronary artery disease (CAD) due to atherosclerosis results in myocardial infarction (MI) and is the leading cause of death worldwide (1). Epidemiologic studies and clinical intervention trials have established the key roles of specific risk factors for CAD, including smoking, hypertension, high low-density lipoprotein (LDL) cholesterol, high triglycerides, low high-density lipoprotein (HDL) cholesterol, and diabetes mellitus (2-4). Surprisingly, many of these risk factors cluster with one another more often than expected by chance (5, 6). This metabolic syndrome is recognized to be a common cause of CAD; however, the molecular mechanisms that unify their association have been obscure.
The marked increase in risk of early cardiovascular mortality to a second monozygotic twin when the first has died from early CAD provides evidence for a strong genetic effect and supports investigation of families with early disease (7). Such studies have the capacity to identify genes and pathways whose altered function impart large effects on CAD outcome; these may provide insight into basic mechanisms that are also involved in common forms of disease and that may be manipulated for health benefit.
From a screen of patients and families with CAD, we identified one extreme outlier kindred with an extraordinary prevalence of early CAD. Kindred CAD-100 is of Iranian ancestry, ascertained via Subject II-7 (table S1), who presented with MI at age 48. CAD risk factors included hypertension, hyperlipidemia, and diabetes mellitus; he had never smoked and his body mass index (BMI) was 24. Evaluation revealed critical stenosis of all three major coronary arteries, which led to coronary artery bypass grafting. His course was complicated by progressive atherosclerosis of the grafts and internal carotid arteries. At age 62, he suffered a low-impact hip fracture and was found to have very low bone mineral density of unknown cause (z score of -3.4 at the femoral neck of his intact hip). He died from a stroke at age 72.
Among 58 blood relatives of the index case, 28 were diagnosed with early CAD (MI, angina, or sudden cardiac death) at or before age 50 (men) or 55 (women) (Fig. 1). Of these, 23 have died from CAD (mean age of death, 52 years). In contrast, kindred members without early CAD died at a mean age of 81. This familial clustering is noteworthy given that early CAD and early CAD death are uncommon in the general population (8, 9).
Detailed clinical data were obtained for all available kindred members, including 13 affected with early CAD, 5 free of early CAD at or beyond the age threshold of 50 years (men) and 55 (women), and 9 younger asymptomatic members (CAD phenotype unknown; mean age 35 years) (table S1). Cardiac risk factors before or at presentation among affected subjects were surprisingly homogeneous, including high fasting LDL cholesterol in all (mean 176.4 mg/dl, nl < 130 mg/dl), high fasting triglycerides in 90% (mean 240 mg/dl, nl < 150 mg/dl), marked hypertension in all (mean 175/103 mm Hg, nl < 140/90, typically diagnosed after age 40), and type II diabetes mellitus in 77% (fasting blood glucose > 126 mg/dl; typically diagnosed after hypertension and hyperlipidemia). Despite high triglycerides, HDL levels were normal in all, and only one had a history of smoking. Nearly all of the affected subjects met criteria of the NIH National Cholesterol Education Program for metabolic syndrome based on the presence of diabetes, high triglycerides, and hypertension (10). Although obesity is strongly associated with metabolic syndrome and each of these risk factors, it is noteworthy that obesity is absent among affected subjects (mean BMI 24.6, none greater than 26). In contrast to these affected subjects, the five unaffected family members all had normal levels of blood pressure (mean 116/81 mm Hg), LDL cholesterol (mean 97.8 mg/dl), and triglycerides (mean 60.75 mg/dl), and type 2 diabetes mellitus was absent. Finally, among younger subjects with CAD phenotype unknown, all nine had high LDL levels and seven had high triglyceride levels, whereas hypertension and glucose intolerance were less frequent (table S1).
The marked clustering of early CAD and risk factors in this kindred suggests a strong genetic component. Among sibships in which all subjects are beyond the age threshold for onset of CAD, the offspring of single affected parents yielded 17 affected and 15 unaffected subjects, and male-to-male transmission is present; in addition, the union of two affected first cousins yielded six affected and one unaffected offspring. Similarly, LDL levels are strongly bimodal in the kindred, with all members having either high levels (157 to 195 mg/dl) or low levels (92 to 105 mg/dl), and these levels cosegregate with early CAD. This extreme familial clustering and segregation of phenotypes within the kindred is unlikely to be explained by chance or multifactorial determination and provides strong evidence that early CAD is transmitted as a highly penetrant autosomal dominant trait.
Nineteen family members were available for genetic studies, including seven with early CAD, five unaffected subjects, and seven with CAD phenotype unknown due to young age. A genome-wide analysis of linkage was performed using Affymetrix 10K Gene Chips. We analyzed linkage using all single-nucleotide polymorphisms (SNPs) by applying two prespecified models of the trait locus-a conservative model that specified 90% penetrance, 1% phenocopies, and disease allele frequency of 0.001 and a stringent model that specified 99% penetrance, 0.1% phenocopies, and allele frequency of 0.0001 (11). Results under both models demonstrated significant evidence of linkage to a segment of chromosome 12p, and no other interval yielded a logarithm (base 10) of the odds ratio (lod score) greater than 1.5. Under the stringent model, the maximum multipoint lod score was 4.4 for linkage of CAD within the 2.7-cM interval flanked by loci rs2213177 and rs747726 (Figs. 1 and 2A; odds ratio 25,000:1 in favor of linkage). Results of linkage under the conservative model were similar. Linkage was confirmed by genotyping 11 highly polymorphic di- and tetranucleotide repeat markers this interval of 12p (Fig. 1), and virtually indistinguishable results were obtained when all markers were combined in the analysis. The observed lod scores approximate the theoretical maximum under the specified models. It is worth noting that the index case, who was the offspring of affected first cousins, was homozygous across this interval, implying that he was homozygous for the underlying disease-causing mutation.
We next considered the impact of this mutation on CAD risk factors. Analysis revealed complete linkage of LRP6R611C and high LDL, with a lod score of 5.5 (Fig. 2A, odds of 316,000:1 in favor of linkage). The difference in mean LDL levels between mutation carriers and noncarriers is significant (170 ± 12 mg/dl versus 98 ± 5 mg/dl, P = 6 x 10-6; Table 1). Because high LDL levels are found in all mutation carriers, regardless of age, this trait can serve as a bio-marker of the mutation in subjects too young to manifest CAD. Similarly, LRP6R611C imparts significant effects on triglyceride levels, blood pressure, fasting blood glucose, and prevalence of diabetes (Table 1). No significant effects were seen on HDL levels or body mass index. Finally, all five mutation carriers studied have low bone densities, each with values expected in less than 12.5% of the population (P < 0.001).
Table 1. Comparison of phenotypes in carriers and noncarriers of LRPR611C. Means ± standard deviation are shown for quantitative traits. All kindred members with measured values were included for LDL, triglyceride, HDL, and BMI measurements. For blood pressure, fasting blood glucose, and diabetes, results for subjects over age 40 are shown.
Our findings establish a causal link between LRP6 mutation and early CAD with high LDL, high triglycerides, hypertension, diabetes, and low bone density. The evidence includes strong a priori evidence of segregation of the disease as an autosomal dominant trait in this kindred, linkage of this trait and underlying risk factors to a single small genomic interval, identification of a single rare mutation in the linked interval that alters a highly conserved amino acid, biochemical evidence that the mutation impairs function of the encoded protein, and evidence from mouse models that mutations in orthologs and paralogs of the identified gene confer similar effects on risk factors. We anticipate that the mutation we identified is very rare in the population; to date we have found no other suggestive mutations in LRP6 among 400 unrelated subjects with CAD. Nonetheless, as for other Mendelian forms of important medical traits-including cardiovascular risk factors, neuro-degenerative diseases, and pain perception, among many others (18-20)-the findings from this rare kindred may provide key insight into pathways that cause disease and that may be manipulated for health benefit. These findings motivate further investigation of the genes and activity of this pathway to search for inherited or acquired variation in Wnt signaling in common forms of CAD and metabolic syndrome.
The impact of the LRP6 mutation on multiple CAD risk factors is notable. Most mutation carriers over age 45 meet criteria for the metabolic syndrome. Although the observed effects on these risk factors are substantial, they are not individually as large as those seen with many previously defined Mendelian traits. For example, the mean LDL levels among LRP6 mutation carriers are 170 mg/dl, versus a mean level of about 300 mg/dl among carriers of heterozygous loss-of-function mutations in the LDL receptor (21). Similarly, the hypertension and diabetes in this family typically appear in middle age, at ages that could easily be mistaken for "garden variety" forms of these risk factors. These observations suggest that it is the combined effects of these risk factors that account for the very high cardiovascular risk to mutation carriers.
Because loss-of-function mutations in LRP5 (12, 15) and LRP6 (16) result in reduced bone density, the osteoporosis among LRPR611C carriers lends further support to the functional significance of this mutation. Moreover, recent epidemiologic studies have found strong association of osteoporosis and CAD (22). Our observations suggest that osteoporosis and CAD can be pleiotropic consequences of impaired Wnt signaling, raising the question of whether the co-occurrence of osteoporosis and CAD might commonly identify individuals with inherited or acquired impairment in Wnt signaling.
Our findings underscore emerging evidence implicating effects of altered Wnt signaling on cardiovascular risk factors. Common intronic variants in the Wnt-responsive transcription factor TCF7L2 result in altered insulin secretion and type II diabetes mellitus (23). Similarly, rare mutations in other Wnt-related transcription factors cause maturity onset diabetes of youth (24, 25). The LRPR611C mutation confers effects not only on many risk factors but on CAD outcomes as well. The ubiquitous expression of LRP6 (26) supports the possibility of pleiotropic effects in diverse tissues. Further investigation of Wnt signaling in patients with early CAD, metabolic syndrome, and its components may provide new insight into disease pathophysiology and approaches to prevention of these disorders.
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