The BAG3 gene variants in Polish patients with dilated cardiomyopathy: four novel mutations and a genotype-phenotype correlation

The study cohort was drawn from all index patients referred for clinical DCM genetic testing from 2010 to 2013 to the Unit for Screening Studies in Inherited Cardiovascular Diseases and involved 90 unrelated probands with DCM (67 or 74.4% male). The pedigrees of families are shown in Figure 1. DCM was diagnosed according to the ESC criteria [12] with left ventricular ejection fraction below 45% and left ventricular end-diastolic diameter exceeding 117% of value appropriate to age and body surface area. In all probands coronary angiography, or more recently coronary computed tomography angiography (CTA) was performed. Data concerning the heart transplant recipients were reviewed to confirm the diagnosis of DCM prior to heart transplantation. DCM was considered familial when more than one member was affected after clinical, electrocardiographic and echocardiographic evaluation of all the informed and consenting relatives. Creatine phosphokinase (CPK) level was obtained whenever possible. In addition, medical records of hospitalized patients were reviewed, in particular we reviewed: (1) histopathologic data of endomyocardial biopsy in two DCM patients - in one patient biopsy was performed based on clinical indications (acute onset heart failure) and in the other two pieces of endomyocardial tissue were obtained during ventricular assist device implantation due to fulminant heart failure, (2) cardiovascular magnetic resonance (CMR) data of one patient (CMR performed due to clinical suspicion of myocarditis). All patients and relatives gave written informed consent to participate in the study in accordance with the Declaration of Helsinki and study protocol was approved by the local Bioethics Committee. Once a mutation was identified adult first-degree relatives of the mutation carriers were offered mutation screening. The clinical description of the DCM-1 family was reported previously [13, 14]. A phenotypic characteristic was updated to include additional family members. In one subject (III-5) from the DCM-15 family an additional permission from the Bioethics Committee was obtained to confirm the presence of mutation in the DNA extracted from myocardial tissue taken at the time of ventricular assist device (VAD) implantation.

DNA was extracted from the peripheral blood by phenol extraction. We screened the entire coding region together with splice sites of BAG3 by direct Sanger sequencing. For all BAG3 exons PCR was carried out with primers listed in Additional file 1: Table S1. The PCR conditions were: 5 min of initial denaturation at 95°C, followed by 32 cycles of 30 sec at 95°C, 55 sec at 60-68°C, 1 min at 72°C and final extension of 10 min at 72°C. PCR products were examined on 2.5% agarose gels and then sequenced using a 3500×L Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) and BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer’s instructions. The results were analyzed with Variant Reporter 1.1 Software (Applied Biosystems).

The screening for large deletions in BAG3 was performed by quantitative PCR (qPCR) using Applied Biosystems 7500 Real Time PCR System and MESA GREEN MasterMix Plus, Low ROX (Eurogentec, Belgium). The PCR conditions were: 10 min of initial denaturation at 95°C, followed by 40 cycles of 15 sec at 95°C and 1:45 min at 60°C. Albumin gene (ALB) was used as a reference. All qPCR analyses were run in duplicates. Copy number was calculated by delta delta C_t method i.e. ∆∆C_t = (C_{t BAG3} - C_{t ALB})_test - (C_{t BAG3} - C_{t ALB})_{wild type} where C_t denotes mean cycle number in which threshold value of fluorescence was recorded for the BAG3 or ALB primers for the reference (a sample without deletion, i.e. ‘wild type’) and tested (‘test’) sample, respectively. For C_t determination the default method available on the instrument was used. ∆∆C_t > 0.8 was regarded as indicative of deletion. Subsequent fine mapping of the detected deletion was performed in a similar way using a stepwise approach. The sequences of BAG3 and ALB primers used at both stages of analysis are listed in Additional file 2: Table S2. Primers used for final PCR-amplification and sequencing of the breakpoint region were: BAG3intron2/3 5′TGC TCT CAA TTT CGA GGT GA 3′ and BAG3del 5′ CGG GAG AAT CAT GAG GTC AG 3′ (the forward and the reverse primer, respectively). The same primers as those used for sequencing of the breakpoint region were also used for screening of family members for the presence of the deletion.

Direct sequencing of the BAG3 coding sequence and splice sites showed four different mutations in five DCM families: known missense Glu455Lys mutation in 2 families (DCM-16 and DCM-71), two novel small deletions predicted to cause frameshifts: Gln353ArgfsX10 (c.1055delC) (DCM-18), Gly379AlafsX45 (c.1135delG) (DCM-15), and a novel Tyr451X (c.1353C>A) mutation predicted to insert a premature stop codon (DCM-84). Each of the truncating mutations occurred in a single family. Chromatograms illustrating the novel mutations are shown in Figure 2. None of the detected BAG3 mutations were found in population databases.

To search for large deletions in BAG3 we performed qPCR comparing dosage of DNA within BAG3 exons with that a reference gene (albumin). In one sample (proband from the DCM-1 family) using primers RT_Exon3 and RT_Exon4.1 located in exons 3 and 4 respectively we observed shifts in amplification curves consistent with presence of a heterozygous deletion (∆∆C_t = 0.92).

Next, we carried out qPCR experiments aimed at stepwise narrowing of the deleted region. Eight such experiments were performed and they eventually allowed to narrow down the deletion to a region sufficiently short to design PCR primers likely to amplify the deletion breakpoint. The PCR product was obtained and its identity was verified by sequencing which also demonstrated that the deletion encompassed a region of 17,990 bp including 3 and 4 exons of BAG3 and extending into the telomeric end in the direction of the INPP5F gene (Figure 2D). We noted that the 5 bp of the DNA sequence directly adjacent to the centromeric end of the deleted fragment was identical to the sequence at the telomeric end of the deleted fragment (AGTGG in both cases, Figure 2D).

Among the six probands with BAG3 mutations one had an acute onset with a fulminant course of the disease (DCM-15 III-7) and died awaiting heart transplantation (HTX) while on ventricular assist devices within three months from the diagnosis. Three probands (DCM-18 III-2, DCM-1 IV-1, DCM-71 III-4) received HTX after 60, 108 or 196 months from the diagnosis, respectively. Two probands experienced partial recovery: one remains stable after 108 months with LVEF 36% and another one with 2 months follow-up’ time experienced significant improvement in LV systolic function (LVEF 51%) while on standard treatment of heart failure. Clinical characteristics of probands are given in Additional file 3: Table S3.

Histopathological analysis in patients with the Glu455Lys variant (DCM-16 III-1) was performed within one month after the onset of acute heart failure. The findings were non specific with features of cellular hypertrophy, myocardial cell degeneration and interstitial fibrosis as usually found in DCM. In the specimen from the patient with the Gly379AlafsX45 variant (DCM-15 III-5) tissue severe myocytolysis with scarce inflammatory infiltrate was found.

In addition to six probands we have identified 21 relatives with BAG3 mutations. Eleven (52.4%) of these subjects had DCM with the mean age at onset of 36 years (range 15–53). There were two deaths: one female died suddenly at 58 years after 60 months from the diagnosis and there was one heart failure death at 42 years after 120 months from the diagnosis in a male patient. Significant left ventricular dysfunction at the end of follow-up with LVEF ≤ 45% was present in 9 relatives (42.9%). In two mutation carriers (9.5%) an improvement in LV function was observed over the follow-up while on standard treatment for heart failure. Persistent normal LV function was found in 10 (47.6%) carriers whose mean age was 29.2 years (range 20–53). Clinical characteristics of relatives with BAG3 mutations are given in Additional file 3: Table S3.

CMR study in DCM-15 III-2 (Gly379AlafsX45), performed within one month after the onset of symptoms showed dilated hypocontractile left ventricle and the presence of CMR diagnostic criteria for myocarditis [15]. In particular, T2-weighted images demonstrated global myocardial signal intensity (SI) increase (>2) in comparison to skeletal muscle indicating global edema (Additional file 4: Figure S1A). There was also an increased global myocardial early gadolinium enhancement ratio (>4) in comparison to skeletal muscle in* gadolinium-enhanced T1-weighted images indicating global myocardial hyperemia. Late gadolinium enhancement (LGE) revealed intramyocardial foci in the interventricular septum and at the low junction point of the right and left ventricle indicating myocardial fibrosis (Additional file 4: Figure S1B). At the time of CMR study, serological examinations showed positive serum IgM Lyme titres and borderline IgG, positive IgG antyParvovirus B19 titres and detectable human herpes virus 6 (HHV6) genome in the serum. The patient received gammaglobulin 0.1 g/kg i.v. along with doxycycline 100 mg PO bid for 21 days leading to substantial clinical improvement (a rise in LVEF up to 36%).

All subjects with BAG3 mutations who were DCM free had truncating mutations whereas all those with missense mutations were affected (Table 1). A trend for higher DCM prevalence among those with missense BAG3 mutations was also found on reanalysis of data previously reported for mutation positive family members of probands with BAG3 related DCM [3, 4]. When our data were combined with data reported previously [3, 4] there was a statistically significant difference indicating higher prevalence of DCM among those with non-truncating vs. truncating BAG3 mutations (OR = 8.33, P = 0.045, Table 1). The association was even stronger (P = 0.0058) when a healthy child aged 7 reported by Villard et al. was excluded from analysis as uninformative due to young age (Table 1).

These results suggested that missense vs. truncating BAG3 mutations could be associated with earlier disease onset and/or lower penetrance or just age difference between the groups. To study this further we analyzed age at DCM onset in the two groups by Kaplan-Meier survival analysis. Figure 3 shows time to DCM onset in family members of the probands stratified by the mutation category (subjects from the present study were pooled with those reported previously [3, 4]). As can be seen, whereas by the age of 70 the disease penetrance is apparently 100%, by the age of 50 years the prevalence of DCM among those with non-truncating vs. truncating BAG3 mutations differs (~90% vs. ~55%, respectively, Figure 3). The difference between the two groups in DCM free survival was statistically significant (P = 0.006, Cox’s F-Test).