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. 2013 Jul 11;93(1):67-77.
doi: 10.1016/j.ajhg.2013.05.015. Epub 2013 Jun 13.

Fine Mapping of the 1p36 Deletion Syndrome Identifies Mutation of PRDM16 as a Cause of Cardiomyopathy

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Fine Mapping of the 1p36 Deletion Syndrome Identifies Mutation of PRDM16 as a Cause of Cardiomyopathy

Anne-Karin Arndt et al. Am J Hum Genet. .
Free PMC article

Abstract

Deletion 1p36 syndrome is recognized as the most common terminal deletion syndrome. Here, we describe the loss of a gene within the deletion that is responsible for the cardiomyopathy associated with monosomy 1p36, and we confirm its role in nonsyndromic left ventricular noncompaction cardiomyopathy (LVNC) and dilated cardiomyopathy (DCM). With our own data and publically available data from array comparative genomic hybridization (aCGH), we identified a minimal deletion for the cardiomyopathy associated with 1p36del syndrome that included only the terminal 14 exons of the transcription factor PRDM16 (PR domain containing 16), a gene that had previously been shown to direct brown fat determination and differentiation. Resequencing of PRDM16 in a cohort of 75 nonsyndromic individuals with LVNC detected three mutations, including one truncation mutant, one frameshift null mutation, and a single missense mutant. In addition, in a series of cardiac biopsies from 131 individuals with DCM, we found 5 individuals with 4 previously unreported nonsynonymous variants in the coding region of PRDM16. None of the PRDM16 mutations identified were observed in more than 6,400 controls. PRDM16 has not previously been associated with cardiac disease but is localized in the nuclei of cardiomyocytes throughout murine and human development and in the adult heart. Modeling of PRDM16 haploinsufficiency and a human truncation mutant in zebrafish resulted in both contractile dysfunction and partial uncoupling of cardiomyocytes and also revealed evidence of impaired cardiomyocyte proliferative capacity. In conclusion, mutation of PRDM16 causes the cardiomyopathy in 1p36 deletion syndrome as well as a proportion of nonsyndromic LVNC and DCM.

Figures

Figure 1
Figure 1
Alignment of Regions of Loss in Individuals with 1p36del Syndrome Associated with Cardiomyopathy and Identification of PRDM16 Mutations in Nonsyndromic LVNC and DCM (A) Mapping in 18 probands (for further information see Tables S1 and S2) with chromosome 1p36 deletion syndrome and cardiomyopathy shows the respective 1p36-deleted intervals (blue, according to the five data sources: NCBI, Genoglyphix, Charite Berlin, Decipher, and Ecaruca) and the common minimal region of deletion (yellow) to contain the PRDM16 gene (GRCh37/hg19). The common minimal region (yellow) in probands with cardiomyopathy comprises of 130,098 bp at 3,224,674–3,354,772 bp in 17/18 probands and contains exon 4–17 of PRDM16. Abbreviations are as follows: ASD, atrial septal defect; VSD, ventricular septal defect; PDA, patent ductus arteriosus; MI, mitral insufficiency. (B) PRDM16 domain structure with conserved motifs and binding domains and location of amino acid changes in three nonsyndromic LVNC and five nonsyndromic DCM probands. The three mutations in LVNC are all located in exon 9 (orange); two DCM probands share the same substitution (p.Val1101Met). The complete PRDM16 1,276 aa containing protein with the N-terminal PR domain (violet), two zinc finger DNA binding domains (black bars), and a PLDLS motif at position 804–808 (red bar) is shown. The PR region corresponds to a SET domain, an 130 amino acid, evolutionarily conserved sequence motif with histone methyltransferase activity. The ten zinc finger domains correspond to the classical C2H2-type, in which the first pair of zinc-coordinating residues are cysteines and the second pair are histidines, conferring zinc-dependent DNA- or RNA-binding properties.
Figure 2
Figure 2
Left Ventricular Morphology and Clinical Description of LVNC Probands with PRDM16 Mutations (A) Echocardiographic apical 4-chamber view of proband 1 showing involvement of apical and lateral segments. Proband 1 carried a frameshift mutation (c.1573dupC [p.Arg525Profs79]) and presented at age 33 years with severe biventricular heart failure with systolic and diastolic dysfunction, secondary pulmonary hypertension, and dilatation of both atria and ventricles. He received a biventricular intracardiac defibrillator. (B) Short axis view of the same proband at the level below the LV papillary muscles showing marked thickening of the inferior noncompacted layer and thinning of the compacted layer. (C) Echocardiographic apical 4-chamber view of proband 2 showing involvement of the LV midventricular lateral wall. Proband 2, with a truncation mutation (c.2104A>T [p.Lys702]), was diagnosed at age 12 years because of arrhythmias and showed mild to moderate left ventricular dysfunction and dilatation in addition to LVNC. (D) Haematoxylin staining of LV myocardium of proband 3. In proband 3 a missense mutation (c.2447A>G [p.Asn816Ser]) was detected. He had been sent to cardiac surgery for the reconstruction of a dysplastic mitral valve at the age of 11 years because of mitral insufficiency grade 3. The left atrium and left ventricle were enlarged with preserved cardiac function. Histology of a left ventricular biopsy taken at cardiac surgery showed increased interstitial fibrosis and myocyte disarray.
Figure 3
Figure 3
PRDM16 Localization in the Human and Mouse Heart (A and B) Immunofluorescence staining of fetal (A) and adult (B) human left ventricular myocardium showing PRDM16 (in red) in the nuclei (in blue) of both cardiomyocytes (positive for Troponin T, in green, arrowheads) and interstitial cells (arrows). (C) Within the ventricular myocardium of 13.5 dpc mouse embryos, PRDM16 localization is detectable in cardiomyocytes of the compact and trabeculated layer as well as in epicardial (arrowheads) and endocardial (arrows) cells. (D) In the adult mouse heart, PRDM16 is located primarily in cardiomyocyte nuclei (identified by WGA membrane staining, in green, see arrowheads), although some nonmyocytes show weak PRDM16 staining as well (arrows). Scale bars represent 25 μm.
Figure 4
Figure 4
PRDM16 Knockdown and Human PRDM16 Truncation Mutant in a Zebrafish Model (A) There is significantly reduced heart rate and cardiac output in PRDM16 MO and PRDM16 mutant animals compared to WT, MO control, and PRDM16 WT. (B) In PRDM16 MO and PRDM16 mutant hearts, there is significant reduction in total cell number and rates of cellular proliferation at 48 hpf that is only partially rescued by PRDM16 WT overexpression. (C) Time-dependent effect of PRDM16 on cell number and proliferation in WT, PRDM16 MO, PRDM16 mutant, and PRDM16 WT embryos. Plotting cell number during cardiac development reveals that both morphant and mutant fish exhibit reduced cell numbers that despite changes in proliferation rates are not fully recovered by 96 hpf. The effects of mutant and wild-type PRDM16 constructs appear to act in opposing directions between 48 or 96 hpf and 72 hpf. The mechanism for this effect is unknown but is not related to changes in the baseline expression of PRDM16. One-way ANOVA test: p < 0.05; ∗∗p < 0.005; ∗∗∗p < 0.0005. The error bars represent the mean ± SEM.
Figure 5
Figure 5
Cell Coupling in a PRDM16 Zebrafish Model and Interaction of PRDM16 with SKI (A) Loss of PRMD16 leads to partial uncoupling of cardiomyocytes in the zebrafish ventricle. Isochronal maps of wild-type (WT), PRDM16 WT transgenic, PRDM16 morphant (MO), and PRMD16 mutant transgenic hearts. The lines represent the positions of the action potential wavefront at 5 ms intervals. The color scale depicts the timing of electrical activation (blue areas activated before red areas). (B) Mean estimated conduction velocities from the outer curvature of the ventricle (OC) confirm a significant reduction in impulse propagation velocities in morphant and mutant hearts when compared with uninjected controls or wild-type. (C) PRDM16 and SKI double morphant embryos have a more profound effect on cardiac output, suggesting a synergistic genetic interaction. One-way ANOVA test: p < 0.05; ∗∗p < 0.005; ∗∗∗p < 0.0005. The error bars represent the mean ± SEM.

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