A study in Polish patients with cardiomyopathy emphasizes pathogenicity of phospholamban (PLN) mutations at amino acid position 9 and low penetrance of heterozygous null PLN mutations

Abstract

Background: In humans mutations in the PLN gene, encoding phospholamban - a regulator of sarcoplasmic reticulum calcium ATPase (SERCA), cause cardiomyopathy with prevalence depending on the population. Our purpose was to identify PLN mutations in Polish cardiomyopathy patients.

Methods: We studied 161 unrelated subjects referred for genetic testing for cardiomyopathies: 135 with dilated cardiomyopathy, 22 with hypertrophic cardiomyopathy and 4 with other cardiomyopathies. In 23 subjects multiple genes were sequenced by next generation sequencing and in all subjects PLN exons were analyzed by Sanger sequencing. Control group included 200 healthy subjects matched with patients for ethnicity, sex and age. Large deletions/insertions were screened by real time polymerase chain reaction.

Results: We detected three different heterozygous mutations in the PLN gene: a novel null c.9_10insA:(p.Val4Serfs*15) variant and two missense variants: c.25C > T:(p.Arg9Cys) and c.26G > T:(p.Arg9Leu). The (p.Val4Serfs*15) variant occurred in the patient with Wolff-Parkinson-White syndrome in whom the diagnosis of cardiomyopathy was not confirmed and his mother who had concentric left ventricular remodeling but normal left ventricular mass and function. We did not detect large deletions/insertions in PLN in cohort studied.

Conclusions: In Poland, similar to most populations, PLN mutations rarely cause cardiomyopathy. The 9(th) PLN residue is apparently a mutation hot spot whereas a single dose of c.9_10insA, and likely other null PLN mutations, cause the disease only with low penetrance or are not pathogenic.

Figure 1

Chromatograms of PLN c.9_10insA:(p.Val4Serfs*15) and c.25C > T:p.Arg9Cys variants. Chromatograms from direct sequencing by the Sanger method showing the c.9_10insA:(p.Val4Serfs*15) (A) and c.25C > T:p.Arg9Cys (B) variants. Below chromatogram A predicted effect of c.9_10insA:(p.Val4Serfs*15) variant on PLN protein sequence. Black font color denotes unaffected amino acid residues, green font original PLN amino acid sequence, red font altered amino acid residues of PLN in c.9_10insA:(p.Val4Serfs*15) variant.

Figure 2

PLN c.26G > T :p.Arg9Leu and DES :c.665G > A:(p.Arg222His) variants IGV view of PLN c.26G > T :p.Arg9Leu variant found by whole exome seguencing (A), pedigree of the family (B) and chromatograms from direct sequencing by the Sanger method showing the PLN p.Arg9Leu variant (C) and DES Arg222His variant (D) in the proband. Pedigree: squares represent males and circles represent females. An arrowhead denotes the proband. A diagonal line marks deceased individuals. Solid black symbols denote dilated cardiomyopathy and grey shading sudden death. Open symbols with asterisk denote unaffected individuals. The presence or absence of a mutation is indicated by a +/− symbol, respectively (top PLN Arg9Leu variant, bottom DES Arg222His variant).

Figure 3

Standard 12-lead electrocardiogram in the proband (A) and his daughter (B). Regular sinus rhythm in both subjects, low QRS voltage in limb leads (A), and in all leads (B). In addition, in the proband (A) ST-T changes in inferolateral leads as well as left atrial enlargement. Diffuse ST-T changes were identified in the probands’s daughter (B).

Figure 4

Two-dimensional echocardiographic study of proband III-1. A: Parasternal long axis view in systole with color flow Doppler. Severe mitral valve regurgitation (vena contracta of 8 mm) due to restriction of the mitral valve leaflets. B: Apical four-chamber view, speckle tracking method. Enlarged left ventricle, left ventricular end-diastolic volume (LVEDV 154 ml) with low ejection fraction (LVEF 37%). Enlarged left atrium chamber.

Figure 5

Cardiac magnetic resonance cine image of proband’s daughter IV-2, 4-chamber image (A) demonstrating dilated left ventricle in end-diastole, late gadolinium enhancement images in 2-chamber view (B) and mid-ventricular short axis view (C) showing mid wall enhancement of the anterior and inferior wall (B) and interventricular septum (C).