Christopher T. Johansen

Exome sequencing identifies rare LDLR and APOA5 alleles conferring risk for myocardial infarction

Summary Myocardial infarction (MI), a leading cause of death around the world, displays a complex pattern of inheritance1,2. When MI occurs early in life, the role of inheritance is substantially greater1. Previously, rare mutations in low-density lipoprotein (LDL) genes have been shown to contribute to MI risk in individual families3–8 whereas common variants at more than 45 loci have been associated with MI risk in the population9–15. Here, we evaluate the contribution of rare mutations to MI risk in the population. We sequenced the protein-coding regions of 9,793 genomes from patients with MI at an early age (≤50 years in males and ≤60 years in females) along with MI-free controls. We identified two genes where rare coding-sequence mutations were more frequent in cases versus controls at exome-wide significance. At low-density lipoprotein receptor (LDLR), carriers of rare, damaging mutations (3.1% of cases versus 1.3% of controls) were at 2.4-fold increased risk for MI; carriers of null alleles at LDLR were at even higher risk (13-fold difference). This sequence-based estimate of the proportion of early MI cases due to LDLR mutations is remarkably similar to an estimate made more than 40 years ago using total cholesterol16. At apolipoprotein A-V (APOA5), carriers of rare nonsynonymous mutations (1.4% of cases versus 0.6% of controls) were at 2.2-fold increased risk for MI. When compared with non-carriers, LDLR mutation carriers had higher plasma LDL cholesterol whereas APOA5 mutation carriers had higher plasma triglycerides. Recent evidence has connected MI risk with coding sequence mutations at two genes functionally related to APOA5, namely lipoprotein lipase15,17 and apolipoprotein C318,19. When combined, these observations suggest that, beyond LDL cholesterol, disordered metabolism of triglyceride-rich lipoproteins contributes to MI risk.

Excess of rare variants in genes identified by genome-wide association study of hypertriglyceridemia

Genome-wide association studies (GWAS) have identified multiple loci associated with plasma lipid concentrations. Common variants at these loci together explain <10% of variation in each lipid trait. Rare variants with large individual effects may also contribute to the heritability of lipid traits; however, the extent to which rare variants affect lipid phenotypes remains to be determined. Here we show an accumulation of rare variants, or a mutation skew, in GWAS-identified genes in individuals with hypertriglyceridemia (HTG). Through GWAS, we identified common variants in APOA5, GCKR, LPL and APOB associated with HTG. Resequencing of these genes revealed a significant burden of 154 rare missense or nonsense variants in 438 individuals with HTG, compared to 53 variants in 327 controls (P = 6.2 × 10−8), corresponding to a carrier frequency of 28.1% of affected individuals and 15.3% of controls (P = 2.6 × 10−5). Considering rare variants in these genes incrementally increased the proportion of genetic variation contributing to HTG.

Genetic determinants of plasma triglycerides

Plasma triglyceride (TG) concentration is reemerging as an important cardiovascular disease risk factor. More complete understanding of the genes and variants that modulate plasma TG should enable development of markers for risk prediction, diagnosis, prognosis, and response to therapies and might help specify new directions for therapeutic interventions. Recent genome-wide association studies (GWAS) have identified both known and novel loci associated with plasma TG concentration. However, genetic variation at these loci explains only ∼10% of overall TG variation within the population. As the GWAS approach may be reaching its limit for discovering genetic determinants of TG, alternative genetic strategies, such as rare variant sequencing studies and evaluation of animal models, may provide complementary information to flesh out knowledge of clinically and biologically important pathways in TG metabolism. Herein, we review genes recently implicated in TG metabolism and describe how some of these genes likely modulate plasma TG concentration. We also discuss lessons regarding plasma TG metabolism learned from various genomic and genetic experimental approaches. Treatment of patients with moderate to severe hypertriglyceridemia with existing therapies is often challenging; thus, gene products and pathways found in recent genetic research studies provide hope for development of more effective clinical strategies.

The transcription factor cyclic AMP-responsive element-binding protein H regulates triglyceride metabolism

Here we report that the transcription factor cyclic AMP–responsive element–binding protein H (CREB-H, encoded by CREB3L3) is required for the maintenance of normal plasma triglyceride concentrations. CREB-H–deficient mice showed hypertriglyceridemia secondary to inefficient triglyceride clearance catalyzed by lipoprotein lipase (Lpl), partly due to defective expression of the Lpl coactivators Apoc2, Apoa4 and Apoa5 (encoding apolipoproteins C2, A4 and A5, respectively) and concurrent augmentation of the Lpl inhibitor Apoc3. We identified multiple nonsynonymous mutations in CREB3L3 that produced hypomorphic or nonfunctional CREB-H protein in humans with extreme hypertriglyceridemia, implying a crucial role for CREB-H in human triglyceride metabolism.

Genetic bases of hypertriglyceridemic phenotypes

Purpose of review Hypertriglyceridemia (HTG) is a common diagnosis. Although secondary factors are important for clinical expression, susceptibility to HTG has a strong genetic component, which we review here. Recent findings Severe HTG in a few families follows Mendelian – typically autosomal recessive – inheritance of rare loss-of-function mutations in genes such as LPL, APOC2, APOA5, LMF1, and GPIHBP1. In contrast, common complex HTG results from the cumulative influence of small-effect variants (single nucleotide polymorphisms) in genes such as APOA5, GCKR, LPL, and APOB. Intensive resequencing of these four genes has also shown accumulated heterozygous rare variants in HTG patients. Together, more than 20% of the susceptibility to HTG is now accounted for by common and rare variants. Further, classical Fredrickson HTG phenotypes, which were once considered to be distinct based on biochemical features, have a shared genetic architecture. Summary Compared to other complex traits, genetic variants account for a high proportion of HTG diagnoses. By tallying the number of HTG risk alleles, it is possible to discriminate between individuals with HTG and normolipidemia, particularly in those with extreme scores. Future directions include finding the missing genetic component and determining whether genetic profiling can help with diagnosis or personalized treatment advice.

Conservation of Lotus and Arabidopsis Basic Helix-Loop-Helix Proteins Reveals New Players in Root Hair Development

Basic helix-loop-helix (bHLH) proteins constitute a large family of transcriptional regulators in plants. Although they have been shown to play important roles in a wide variety of developmental processes, relatively few have been functionally characterized. Here, we describe the map-based cloning of the Lotus japonicus ROOTHAIRLESS1 (LjRHL1) locus. Deleterious mutations in this locus prevent root hair development, which also aborts root hair-dependent colonization of the host root by nitrogen-fixing bacteria. We show that the LjRHL1 gene encodes a presumed bHLH transcription factor that functions in a nonredundant manner to control root hair development in L. japonicus. Homology search and cross-species complementation experiments defined three members of the Arabidopsis (Arabidopsis thaliana) bHLH protein family, At2g24260, At4g30980, and At5g58010, as functionally equivalent to LjRHL1. Curiously, At2g24260 and At4g30980 mRNA species accumulate independently from the known positive regulators of root hair cell fate, while all three genes act in a partially redundant manner to regulate root hair development in Arabidopsis.

Lotus japonicus symRK‐14 uncouples the cortical and epidermal symbiotic program

SYMRK is a leucine-rich-repeat (LRR)-receptor kinase that mediates intracellular symbioses of legumes with rhizobia and arbuscular mycorrhizal fungi. It participates in signalling events that lead to epidermal calcium spiking, an early cellular response that is typically considered as central for intracellular accommodation and nodule organogenesis. Here, we describe the Lotus japonicus symRK-14 mutation that alters a conserved GDPC amino-acid sequence in the SYMRK extracellular domain. Normal infection of the epidermis by fungal or bacterial symbionts was aborted in symRK-14. Likewise, epidermal responses of symRK-14 to bacterial signalling, including calcium spiking, NIN gene expression and infection thread formation, were significantly reduced. In contrast, no major negative effects on the formation of nodule primordia and cortical infection were detected. Cumulatively, our data show that the symRK-14 mutation uncouples the epidermal and cortical symbiotic program, while indicating that the SYMRK extracellular domain participates in transduction of non-equivalent signalling events. The GDPC sequence was found to be highly conserved in LRR-receptor kinases in legumes and non-legumes, including the evolutionarily distant bryophytes. Conservation of the GDPC sequence in nearly one-fourth of LRR-receptor-like kinases in the genome of Arabidopsis thaliana suggests, however, that this sequence might also play an important non-symbiotic function in this plant.

LipidSeq: a next-generation clinical resequencing panel for monogenic dyslipidemias

We report the design of a targeted resequencing panel for monogenic dyslipidemias, LipidSeq, for the purpose of replacing Sanger sequencing in the clinical detection of dyslipidemia-causing variants. We also evaluate the performance of the LipidSeq approach versus Sanger sequencing in 84 patients with a range of phenotypes including extreme blood lipid concentrations as well as additional dyslipidemias and related metabolic disorders. The panel performs well, with high concordance (95.2%) in samples with known mutations based on Sanger sequencing and a high detection rate (57.9%) of mutations likely to be causative for disease in samples not previously sequenced. Clinical implementation of LipidSeq has the potential to aid in the molecular diagnosis of patients with monogenic dyslipidemias with a high degree of speed and accuracy and at lower cost than either Sanger sequencing or whole exome sequencing. Furthermore, LipidSeq will help to provide a more focused picture of monogenic and polygenic contributors that underlie dyslipidemia while excluding the discovery of incidental pathogenic clinically actionable variants in nonmetabolism-related genes, such as oncogenes, that would otherwise be identified by a whole exome approach, thus minimizing potential ethical issues.

An Increased Burden of Common and Rare Lipid-Associated Risk Alleles Contributes to the Phenotypic Spectrum of Hypertriglyceridemia

Objective—Earlier studies have suggested that a common genetic architecture underlies the clinically heterogeneous polygenic Fredrickson hyperlipoproteinemia (HLP) phenotypes defined by hypertriglyceridemia (HTG). Here, we comprehensively analyzed 504 HLP-HTG patients and 1213 normotriglyceridemic controls and confirmed that a spectrum of common and rare lipid-associated variants underlies this heterogeneity. Methods and Results—First, we demonstrated that genetic determinants of plasma lipids and lipoproteins, including common variants associated with plasma triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) from the Global Lipids Genetics Consortium were associated with multiple HLP-HTG phenotypes. Second, we demonstrated that weighted risk scores composed of common TG-associated variants were distinctly increased across all HLP-HTG phenotypes compared with controls; weighted HDL-C and LDL-C risk scores were also increased, although to a less pronounced degree with some HLP-HTG phenotypes. Interestingly, decomposition of HDL-C and LDL-C risk scores revealed that pleiotropic variants (those jointly associated with TG) accounted for the greatest difference in HDL-C and LDL-C risk scores. The APOE E2/E2 genotype was significantly overrepresented in HLP type 3 versus other phenotypes. Finally, rare variants in 4 genes accumulated equally across HLP-HTG phenotypes. Conclusion—HTG susceptibility and phenotypic heterogeneity are both influenced by accumulation of common and rare TG-associated variants.

Excess of Rare Variants in Non–Genome-Wide Association Study Candidate Genes in Patients With Hypertriglyceridemia

Background— Rare variant accumulation studies can implicate genes in disease susceptibility when a significant burden is observed in patients versus control subjects. Such analyses might be particularly useful for candidate genes that are selected based on experiments other than genome-wide association studies (GWAS). We sought to determine whether rare variants in non-GWAS candidate genes identified from mouse models and human mendelian syndromes of hypertriglyceridemia (HTG) accumulate in patients with polygenic adult-onset HTG. Methods and Results— We resequenced protein coding regions of 3 genes with established roles (APOC2, GPIHBP1, LMF1) and 2 genes recently implicated (CREB3L3 and ZHX3) in TG metabolism. We identified 41 distinct heterozygous rare variants, including 29 singleton variants, in the combined sample; in total, we observed 47 rare variants in 413 HTG patients versus 16 in 324 control subjects (odds ratio=2.3; P=0.0050). Post hoc assessment of genetic burden in individual genes using 3 different tests suggested that the genetic burden was most prominent in the established genes LMF1 and APOC2, and also in the recently identified CREB3L3 gene. Conclusions— These extensive resequencing studies show a significant accumulation of rare genetic variants in non-GWAS candidate genes among patients with polygenic HTG, and indicate the importance of testing specific hypotheses in large-scale resequencing studies.