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. Author manuscript; available in PMC: 2014 Nov 15.
Published in final edited form as: N Engl J Med. 2014 May 15;370(20):1909–1919. doi: 10.1056/NEJMoa1301824

A Form of the Metabolic Syndrome Associated with Mutations in DYRK1B

Ali R Keramati 1,#, Mohsen Fathzadeh 1,#, Gwang-Woong Go 1,#, Rajvir Singh 1,#, Murim Choi 1, Saeed Faramarzi 1, Shrikant Mane 1, Mohammad Kasaei 1, Kazem Sarajzadeh-Fard 1, John Hwa 1, Kenneth K Kidd 1, Mohammad A Babaee Bigi 1, Reza Malekzadeh 1, Adallat Hosseinian 1, Masoud Babaei 1, Richard P Lifton 1, Arya Mani 1
PMCID: PMC4069260  NIHMSID: NIHMS599525  PMID: 24827035

Abstract

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-offunction 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.)

Coronary artery disease is the leading cause of death in men and women worldwide.1,2 A cluster of highly heritable risk factors known as the metabolic syndrome is an increasingly prevalent predisposing factor for coronary artery disease.3-5 Although substantial progress has been made in the identification of genetic causes of the individual risk factors,6-11 the underlying genetic factors that unify their association in the metabolic syndrome are not known. We have previously shown in a family with extreme genotypes that single-gene mutations with large effects can occasionally produce features of this syndrome.12

The advent of next-generation sequencing has provided an unprecedented opportunity for the identification of rare variants with moderate-to-large effects.13 One application of this technique is the identification of rare mutations that account for extreme phenotypes in outlier populations. In such populations, the possibility of identifying founder mutations that segregate with extreme phenotypes is dramatically increased.14-19 We used linkage analysis and whole-exome sequencing to investigate three large families, each of which had a recurring familial pattern of central (or abdominal) obesity associated with earlyonset coronary artery disease, severe hypertension, and type 2 diabetes mellitus.

METHODS

STUDY OVERSIGHT

The study was conducted in compliance with the provisions of the Declaration of Helsinki.20 The study protocol was approved by the institutional review board of Shiraz University of Medical Sciences and the ethics committee at Yale University. The adherence of the study to the protocol was monitored by authorities at Shiraz University of Medical Sciences. Written informed consent was obtained from all study participants.

STUDY PARTICIPANTS

We identified three families (with members of one family not known to be closely related to members of another family) from a community in south-west Iran on the basis of their unusual constellation of juvenile-onset central obesity (Fig. S1 in the Supplementary Appendix, available with the full text of this article at NEJM.org) associated with early-onset coronary artery disease, severe hypertension, type 2 diabetes, and modestly elevated fasting serum triglyceride levels (Fig. 1). These families were considered to be outliers because of the low prevalence of early-onset coronary artery disease and obesity in the local community.21

Figure 1. Pedigrees of Three Families with Autosomal Dominant Inheritance of Early-Onset Coronary Artery Disease and Features of the Metabolic Syndrome.

Figure 1

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Family members for whom clinical and laboratory data were available for this study are designated by numbers in each generation. Circles indicate female family members, and squares male family members; slashes indicate that the family member is deceased. Family members with early coronary artery disease are indicated by solid symbols, and those without coronary artery disease are indicated by open symbols. All family members with coronary artery disease also had clinical features of the metabolic syndrome (Tables S1 and S2 in the Supplementary Appendix). The index patients (Patient IV-8 in Family 1 and Patient III-3 in Family 2) are indicated by arrows. Two family members with clinical features of the metabolic syndrome but with unknown status with respect to coronary artery disease are indicated by half-solid symbols.

Each family was notable for having many affected members with the same syndrome (Table S1 in the Supplementary Appendix) and other family members who did not have these traits (Table S2 in the Supplementary Appendix). In each family, affected members could trace their descent from a common ancestor. The familial clustering and pattern of inheritance of these clinical features were consistent with the effect of a highly penetrant autosomal dominant trait and suggested that the affected family members might share a common founder mutation.

We evaluated family members individually and obtained detailed clinical and laboratory data, including anthropometric and neurohormonal data, for all available living members of the three families who were older than 30 years of age (for details, see the Supplementary Appendix). We obtained blood samples, and genomic DNA was prepared by means of phenol–chloroform extraction.

GENOTYPING AND ANALYSIS OF LINKAGE

DNA samples from 21 living family members from the three study families were available for genetic studies, including from 14 family members with angiographically diagnosed early-onset coronary artery disease, 5 unaffected family members, and 2 family members with central obesity, hypertension, and diabetes but with unknown status with respect to coronary artery disease. In addition, we collected 2000 DNA samples from ethnically matched controls.

We performed genomewide analysis of linkage using HumanOmni1-Quad BeadChips (Illumina), containing more than 1.1 million single-nucleotide-polymorphism (SNP) markers. After linkage to a segment of chromosome 19q13 was identified, additional markers were typed in selected intervals to generate a dense map of the linked interval. We performed the analysis of linkage using Genehunter 2.1 software. We estimated the allele frequencies on the basis of mean frequencies from a group of 10 ethnically matched controls.

TARGETED SEQUENCE CAPTURE AND SEQUENCING

The index patients from the two largest families were screened for mutations by means of whole-exome sequencing. Genomic DNA was captured on exomes with the use of the Sequence Capture Human Exome 2.1M Array (Roche NimbleGen). The captured libraries were sequenced on the Illumina Genome Analyzer, after which image analysis and base calling were performed. The resulting sequence data were processed with the use of MAQ22 software. SAMtools software was used to detect single-nucleotide variants. The raw output was further filtered, as described previously, to remove common variants reported in reference genomes.23 Filters were also applied against published databases. Variants were annotated on the basis of the effect on the protein, novelty, conservation, and tissue expression with the use of an automated pipeline for genome annotation.19

A novel variant was identified in DYRK1B, the gene encoding dual-specificity tyrosine-phosphorylation-regulated kinase 1B. In this variant, cysteine is substituted for arginine at position 102 in DYRK1B (Fig. S2 in the Supplementary Appendix).

FUNCTIONAL ANALYSIS OF DYRK1B R102C

We examined the effects of expression of nonmutated DYRK1B and the novel variant DYRK1B R102C, as well as the effects of DYRK1B knockdown using short hairpin RNA (shRNA), on adipogenic differentiation in permanent 3T3-L1 preadipocyte cell lines. We also examined the effects of DYRK1B variants on the expression of glucose-6-phosphatase in HepG2 cells. Details of the functional analyses are provided in the Methods section in the Supplementary Appendix.

STATISTICAL ANALYSIS

We performed all in vitro experiments in groups of four. Data are expressed as means and standard errors. We used the Mann–Whitney U test to perform between-group comparisons (two-tailed). For multiple comparisons, we performed Tukey’s test in conjunction with analysis of variance using GraphPad Prism software. We used analysis of variance to compare the metabolic traits with adjustment and without adjustment for age and sex as covariates. A P value of less than 0.05 was considered to indicate statistical significance. All P values are two-sided.

RESULTS

STUDY POPULATION

The clinically characterized members of the three study families included 25 affected family members (Table S1 in the Supplementary Appendix), 12 unaffected family members (Table S2 in the Supplementary Appendix), and 2 family members with clinical features of the metabolic syndrome but with unknown status with respect to coronary artery disease (Table S1 in the Supplementary Appendix). All 25 affected family members had early-onset myocardial infarction or coronary artery disease at a mean (±SE) age of 44.8±2.6 years in men and 44.2±1.8 years in women. Additional clinical features included central obesity (Table S3 in the Supplementary Appendix), type 2 diabetes, and hypertension; the presence of all three conditions was not explained by neurohormonal activation (Table S4 in the Supplementary Appendix). The constellation of medical conditions in all the affected family members (and none of the unaffected family members) met the standard definition of the metabolic syndrome, according to the criteria of the National Cholesterol Education Program of the National Institutes of Health.24

LINKAGE ANALYSIS

We performed a genomewide multipoint parametric analysis of linkage, using samples obtained from affected family members and specifying coronary artery disease as an autosomal dominant trait. Each family was analyzed independently. Two different prespecified models of the trait locus were applied, with disease allele frequencies of 10−4 and 10−5 and phenocopy rates of 0.001 and 0.0001, respectively. Under the two models, the analysis showed significant evidence of linkage of coronary artery disease to a small segment of chromosome 19q13. Under the stringent model, the maximum multipoint logarithm of odds (LOD) score was 5.27 in the analysis of affected family members (Fig. 2A) and 6.34 (odds ratio, 2,187,761:1 in favor of linkage) after the inclusion of unaffected family members in the analysis. All three families had LOD scores close to the theoretical maximum. No other interval showed a multipoint LOD score greater than 1.0. Separate linkage analyses of the 19q13 region with central obesity, hypertension, and type 2 diabetes showed similar results (Fig. 2A).

Figure 2. Identification of a DYRK1B Mutation Cosegregating with the Metabolic Syndrome in the Three Study Families.

Figure 2

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Panel A shows multipoint heterogeneity LOD (HLOD) scores for linkage of early coronary artery disease, obesity, type 2 diabetes, and high blood pressure to chromosome 19q13. Shown are single-nucleotide polymorphisms and microsatellites (short tandem repeats of two to five nucleotides) that are tightly linked to the location of DYRK1B in the LOD–1 support interval (in which the LOD score is greater than or equal to the maximum minus 1.0, as measured in centimorgans [cM]). The peak of the LOD score occurs at zero recombination with marker D19S881, and the LOD–1 support interval spans 6 cM for all four traits. Panel B shows the minimum shared haplotypes of the three affected families. Listed are two nonsense mutations (DYRK1B R102C and ZNF780 S257F), two intronic (int) mutations, and two synonymous mutations (MEGF8 R1811R and CNFN N33N) that were shared by affected family members within the shared haplotype. The only novel mutation was DYRK1B R102C. The uppercase and lowercase letters in bold below the mutations and SNPs indicate major and minor alleles, respectively, and the numbers below the microsatellites in bold indicate arbitrary allele numbers. Panel C shows the DNA sequence of a segment flanking R102 in DYRK1B from an unaffected family member (at left) and a heterozygous mutation carrier (at right). A single base substitution (indicated by an asterisk) changes the nonmutant cytosine to thymine, leading to the substitution of cysteine (C) for arginine (R) at codon 102. N denotes a heterozygote C→T nucleotide substitution. Panel D shows a portion of the amino acid sequence of DYRK1B from diverse vertebrate species. This segment is highly conserved, and arginine 102 is completely conserved in orthologues in species ranging from lizards to humans.

Scrutiny of the linked haplotypes showed that all three families shared identical markers spanning a 6.1-Mbp haplotype flanked by the markers rs833917 and rs4801770 (Fig. 2B). Haplotype sharing among the families was indicative of their common ancestral origin and suggested the presence of a founder mutation (Fig. S3 in the Supplementary Appendix). No family member was homozygous for this haplotype.

WHOLE-EXOME SEQUENCING

We filtered data from whole-exome sequencing in the two index patients by removing common SNPs. We excluded variants that were found in the NHLBI ESP5400 exome database and the Yale Center for Genome Analysis exome database, and we identified rare protein-altering variants in each family member. Among these variants, only 18 rare variants were shared by the two index patients (Table S5 in the Supplementary Appendix), and only 1 was found in the linkage interval on chromosome 19. Genotyping resulted in complete cosegregation of this variant with coronary artery disease in all three families.

The mutation substituted cysteine for arginine at position 102 of DYRK1B (Fig. 2C). None of the other 17 variants were found in more than five of the affected members, and some were also found in unaffected family members (Table S5 in the Supplementary Appendix).

The arginine residue at position 102 of DYRK1B is found in a kinase-like domain of the protein (Fig. S2 in the Supplementary Appendix) and is highly conserved among orthologues and paralogues in species ranging from lizards to humans (Fig. 2D). The R102C mutation was absent in chromosome samples obtained from 2000 ethnically matched Iranians and 3600 white controls in the United States. The mutation was also absent in samples obtained from 2500 persons of diverse ethnic backgrounds in the Allele Frequency Database (ALFRED) (Table S6 in the Supplementary Appendix), 5000 exomes from the Yale Center for Genome Analysis database, and 5400 exomes in the NHLBI ESP5400 database. Both the PolyPhen-1 and PolyPhen-2 poly-morphism-phenotyping programs predicted the variant to be deleterious.

We performed parametric analysis of linkage in affected family members using body-mass index, blood pressure, or type 2 diabetes as the phenotype. This analysis confirmed linkage to the DYRK1B R102C mutation, with LOD scores of 5.63, 5.86, and 5.70 for the three variables, respectively. After adjustment for age and sex, differences in fasting blood glucose levels, blood-pressure levels, and body-mass index between mutation carriers and noncarriers were highly significant (Table 1). There was a trend toward higher levels of serum low-density lipoprotein cholesterol and triglycerides in mutation carriers, as compared with noncarriers.

Table 1. Comparison of Metabolic Traits in DYRK1B R102C Carriers and Noncarriers in the Three Study Families*.

Trait Nonmutated
DYRK1B 		DYRK1B
R102C		 P Value
Unadjusted Adjusted†
Fasting blood glucose (mg/dl) 94.6±1.7 175.8±12.5 0.001 0.003
Low-density lipoprotein cholesterol (mg/dl) 84.0±10.6 109.1±9.8 0.17 0.09
Triglycerides (mg/dl) 111.4±12.2 168.1±16.9 0.07 0.09
Blood pressure (mm Hg)
 Systolic 110.0±4.4 175.3±5.5 <0.001 <0.001
 Diastolic 78.0±2.6 99.5±2.2 <0.001 <0.001
Body-mass index‡ 23.6±0.5 33.0±0.4 <0.001 <0.001
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*

Plus–minus values are means ±SE. Included in this analysis were 16 affected family members (including 2 with clinical features of the metabolic syndrome but with unknown status with respect to coronary artery disease) and 5 unaffected family members for whom genotype data were available. All analyses were performed with the use of analysis of variance. To convert the values for glucose to millimoles per liter, multiply by 0.05551. To convert the values for cholesterol to millimoles per liter, multiply by 0.02586. To convert the values for triglycerides to millimoles per liter, multiply by 0.01129.

†

P values were adjusted for age and sex.

‡

The body-mass index is the weight in kilograms divided by the square of the height in meters.

EFFECT OF DYRK1B R102C ON ADIPOGENIC TRANSFORMATION

DYRK1B belongs to the Dyrk family of proteins, a group of evolutionarily conserved protein kinases that are involved in cell differentiation, survival, and proliferation.25-27 The DYRK1B protein is an arginine-directed serine–threonine kinase, which is ubiquitously expressed in mice28 and humans (see the Methods section and Fig. S4 in the Supplementary Appendix).

The expression of DYRK1B increases dramatically during adipogenic differentiation.28 Adipogenesis is a process of maturation of undifferentiated mesenchymal stem cells toward the adipocyte lineage. The inhibition of sonic hedgehog (SHH) pathways results in decreased expression of Wnt proteins29 and is associated with increased expression of the adipogenic proteins CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ)30 and adipogenic transformation.31 Since DYRK1B inhibits SHH signaling,32 we hypothesized that it promotes adipogenesis.

We examined the effects of nonmutant DYRK1B, DYRK1B R102C, and knockdown of DYRK1B on adipogenic differentiation in 3T3-L1 cells. An adipogenic medium that contains the Wnt inhibitor IBMX was used to stimulate differentiation of the 3T3-L1 cells into adipocytes. We examined the expression of C/EBPα and PPARγ isoforms 1 and 2, along with the expression of Gli-2 (a mediator of Shh signaling), PGC1α (a transcriptional coactivator that interacts with PPARγ), and cyclin-dependent kinase inhibitor (p27Kip), which is regulated by DYRK1B and is involved in adipogenic differentiation.33,34

Total levels of DYRK1B expression were similar in cells expressing either nonmutant DYRK1B or DYRK1B R102C (Fig. S5 in the Supplementary Appendix). Adipogenic differentiation (as shown by means of oil red O staining) started approximately 5 days earlier in cells expressing nonmutant DYRK1B or DYRK1B R102C than in those transfected with vector alone, and the accumulation of intracellular lipid (as judged by the intensity of staining) was significantly greater in cells expressing DYRK1B R102C than in cells expressing nonmutant DYRK1B or in cells transfected with vector alone (Fig. 3A). Accordingly, the expression levels of C/EBPα, PPARγ isoforms 1 and 2, and PGC1α were highest, and those of Gli-2 and p27Kip were lowest, in cells expressing the R102C variant, followed by cells expressing the nonmutant form of the protein and those transfected with empty vector (Fig. 3B, and Fig. S6 in the Supplementary Appendix). DYRK1B knockdown diminished the expression of C/EBPα, PPARγ isoforms 1 and 2, and PGC1α and abrogated adipocyte differentiation (Fig. 3A and 3C).

Figure 3. Enhanced Adipogenic Effect Associated with DYRK1B R102C.

Figure 3

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Permanent 3T3-L1 preadipocyte cell lines expressing nonmutant DYRK1B, the DYRK1B R102C mutation, DYRK1B-specific short hairpin RNA (shRNA), or vector alone were created, and the time course of adipocyte differentiation was examined. Panel A shows adipogenic transformations for days 2, 5, and 9. Staining with oil red O shows the presence of intracellular lipid, thus labeling adipocytes. Cells expressing nonmutant (NM) DYRK1B or DYRK1B R102C were transformed into adipocytes considerably earlier than were cells transfected with an empty vector. The accumulation of intracellular lipid (as judged by the intensity of staining) was highest in cells expressing DYRK1B R102C, followed by those expressing nonmutant DYRK1B, and lowest in cells transfected with an empty vector. No significant adipogenic differentiation was observed in cells expressing DYRK1B-specific shRNA (DYRK1B knockdown [KD]). Panel B shows the expression levels of CCAAT/enhancer-binding protein α (C/EBPα), peroxisome proliferator-activated receptor γ (PPARγ) isoforms 1 and 2, and PGC1α (a transcriptional coactivator that interacts with PPARγ), which were highest in adipocytes expressing the R102C variant, followed by those expressing nonmutant DYRK1B, and lowest in cells transfected with vector alone from early stages of adipogenesis. Conversely, the expression levels of Gli-2 (a mediator of sonic hedgehog signaling) and cyclin-dependent kinase inhibitor (p27Kip) were lowest in adipocytes expressing the R102C variant, followed by cells expressing nonmutant DYRK1B, and highest in those transfected with vector alone. β-actin was used as a loading control. Panel C shows that the expression levels of C/EBPα, PPARγ isoforms 1 and 2, and PGC1α were significantly lower in DYRK1B knockdown cells than in cells transfected with vector alone. Panel D shows that cells expressing the R102C variant had substantial adipogenic transformation without the addition of adipogenic medium, as compared with those expressing nonmutant DYRK1B or those transfected with vector alone.

The effect of nonmutant DYRK1B and the R102C variant on Wnt signaling was examined in a well-established dual luciferase reporter assay,35,36 with and without Wnt3a stimulation. Wnt signaling activity was lowest in cells expressing the R102C variant, followed by cells expressing the nonmutant form, and was highest in cells transfected with empty vector (Fig. S7 in the Supplementary Appendix). Correspondingly, cells expressing the R102C variant were able to transform into mature adipocytes without requiring the addition of the adipogenic medium (Fig. 3D).

EFFECT OF R102C VARIANT ON GLUCOSE-6-PHOSPHATASE

Another function of DYRK1B is the induction of the key gluconeogenic enzyme glucose-6-phosphatase.37 Increased expression38,39 and activity 40 of glucose-6-phosphatase are associated with elevated fasting glucose levels in patients with type 2 diabetes.41 In a luciferase reporter assay, DYRK1B R102C significantly potentiated the induction of glucose-6-phosphatase (Fig. 4A). This finding was confirmed by the increased expression of glucose-6-phosphatase messenger RNA and protein in cells expressing the R102C variant, as compared with cells expressing the nonmutant protein and cells transfected with vector alone (Fig. 4B and 4C, and Fig. S8 in the Supplementary Appendix). The analysis further showed that DYRK1B-associated induction of glucose-6-phosphatase is dose-dependent (Fig. 4D, and Fig. S8 in the Supplementary Appendix), which confirms a gain-of-function effect of the R102C variant. This function of DYRK1B is independent of its kinase activity, since the kinase-defective mutant DYRK1B Y271/273F and the nonmutant protein showed similar effects on glucose-6-phosphatase expression (Fig. 4C, and Fig. S8 in the Supplementary Appendix).

Figure 4. Enhanced Kinase-Independent Induction of Glucose-6-Phosphatase by DYRK1B R102C.

Figure 4

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Panel A shows nonmutant DYRK1B and DYRK1B R102C expressed in HepG2 cells. Their transcriptional activities were assayed by glucose-6-phosphatase (G6pase) promoter-mediated expression of luciferase, as measured in refractive index units (RIU). Nonmutant DYRK1B more than doubled the activity of luciferase, as compared with vector alone. The effect of DYRK1B R102C on luciferase activity was more than 1.5 times the effect of nonmutant DYRK1B. The T bars indicate standard errors. Panel B shows that the messenger RNA expression levels of glucose-6-phosphatase were significantly higher in cells expressing DYRK1B R102C than in those expressing nonmutant DYRK1B or vector alone. Panel C shows nonmutant DYRK1B, the R102C variant, and the kinase-defective mutant DYRK1B Y271/273F expressed in HepG2 cells. Their effect on glucose-6-phosphatase expression was assayed with the use of Western blotting. The protein expression level of glucose-6-phosphatase was highest in cells expressing the R102C variant. The next highest levels were in cells expressing nonmutant (NM) DYRK1B and the kinase-defective Y271/273F variant, which were roughly equal and significantly higher than in the vector alone. Panel D shows the dose effect of nonmutant DYRK1B on endogenous glucose-6-phosphatase expression.

ASSOCIATION BETWEEN A SECOND DYRK1B ALLELE AND CENTRAL OBESITY AND DIABETES

We screened 300 morbidly obese white patients with coronary artery disease and multiple metabolic phenotypes for the DYRK1B R102C allele. This led to the identification of a novel DYRK1B allele (H90P) in five unrelated patients. We then evaluated DNA samples obtained from family members of one of the mutation carriers, which showed a consistent pattern of cosegregation of the novel allele with features of the metabolic syndrome in an autosomal dominant pattern. In vitro studies showed that the presence of DYRK1B H90P enhanced glucose-6-phosphatase expression. (See the Methods section and Fig. S8C and S9 in the Supplementary Appendix.)

DISCUSSION

Our study establishes an association between mutations in DYRK1B and a syndrome of central obesity, early-onset coronary artery disease, hypertension, and type 2 diabetes. Our evidence includes the finding of an identical nonconservative mutation on a shared haplotype background in three separate families with this syndrome, the identification of a second nonconservative mutation in an ethnically distinct population with a similar phenotype, and biochemical data indicating that the mutations alter two important functions of the encoded protein. These results show that rare alleles may underlie an association with a cluster of metabolic risk factors of coronary artery disease known as the metabolic syndrome.

The DYRK1B locus on 19q13 has been linked to type 2 diabetes42,43 and traits associated with the metabolic syndrome44 in genomewide association studies. This raises the question of whether common genetic variants in DYRK1B are also associated with these traits in the general population. Most if not all carriers of the DYRK1B R102C and H90P mutations were obese, had hypertension and diabetes, and met the criteria for the metabolic syndrome. These observations indicate the broad and important effect of the mutated gene and suggest that the clustering of the individual risk factors imparts extremely high cardiovascular risk to mutation carriers.

Very little is known about the function of DYRK1B and its role in human disease. DYRK1B is a nutrient-sensing protein that inhibits the RAS–RAF–MEK pathway,45 which is widely recognized for its regulation of glucose uptake and glycolysis.46 Mice that are deficient in kinase suppressor of ras 2 (KSR2), the molecular scaffold of RAS–RAF–MEK, are obese and have insulin resistance.47 Loss-of-function mutations in KSR2 have been shown to be associated with childhood obesity and insulin resistance.48 These mutations similarly impair the RAS–RAF–MEK pathway, a finding that is consistent with the gain-of-function effects of DYRK1B mutations.

The increased expression of DYRK1B during adipogenic differentiation28 is suggestive of its involvement in adipogenesis. DYRK1B may enhance adipogenesis through inhibition of the SHH pathway or by increasing the turnover of p27Kip.33,34,49 Mice that are deficient in p27Kip are observed to have atherosclerosis,50-52 obesity, and insulin resistance.53 In our study, we found that the DYRK1B R102C mutation augments p27Kip turnover and potentiates the adipogenic effects of DYRK1B. Our findings are consistent with the recent observation that the overexpression of DYRK1B homologue DYRK1A and minibrain protein in mice and drosophila leads to an increase in food intake and body weight, and deficiencies are associated with loss of body weight.54

Previous studies have also shown that DYRK1B promotes in vitro transcription of the key hepatic gluconeogenic enzyme glucose-6-phosphatase.41 We found that this function of DYRK1B is kinase-independent and is augmented by the DYRK1B R102C and H90P mutations. These in vitro studies provide initial insights into some of the potential disease mechanisms through which DYRK1B mutations may contribute to the metabolic syndrome.

In conclusion, we found that mutations in DYRK1B are associated with a clinical phenotype that is characterized by central obesity, hypertension, type 2 diabetes, and early-onset coronary artery disease. Our findings suggest that DYRK1B plays a central role in the biologic pathways that are disrupted in the disorder known as the metabolic syndrome.

Supplementary Material

Supplement1

Acknowledgments

Supported by grants from the National Institutes of Health (NIH) (R01HL094784-01 and R01HL094574-03, to Dr. Mani) and by a grant from the NIH Centers for Mendelian Genomics (5U54HG006504).

We thank Reza Mohammadi and Tahmures Mohammadi, local schoolteachers, and Dr. Mohammad Javad Zibaee Nezhad, director of the Cardiovascular Research Center, Shiraz University of Medical Sciences, for their active role in identification and recruitment of the family members; Dr. Javad Tavakkoly Bazzaz, head of the Department of Medical Genetics, Tehran University of Medical Sciences, and Saeed Pour Ali, deputy for research, Iranian Academic Center for Education, Culture, and Research, for their organizational support for patient recruitment; Dr. Sekar Kathiresan at the Massachusetts General Hospital and Drs. Orhan Buglivar and Murat Gunel at the Yale University School of Medicine for providing us with access to their whole-exome-sequencing database; and Dr. Nicholas Gekakis at the Scripps Institute for providing us with glucose-6-phosphatase-responsive luciferase plasmid.

Footnotes

No potential conflict of interest relevant to this article was reported.

Disclosure forms provided by the authors are available with the full text of this article at NEJM.org.

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