Sarcomere Mutation-Specific Expression Patterns in Human Hypertrophic Cardiomyopathy Adam S. Helms, Frank M. Davis, David Coleman, Sarah N. Bartolone, Amelia A. Glazier, Francis Pagani, Jaime M. Yob, Sakthivel Sadayappan, Ellen Pedersen, Robert Lyons, Margaret V. Westfall, Richard Jones, Mark W. Russell and Sharlene M. Day Circ Cardiovasc Genet. 2014;7:434-443; originally published online July 16, 2014; doi: 10.1161/CIRCGENETICS.113.000448 Circulation: Cardiovascular Genetics is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2014 American Heart Association, Inc. All rights reserved. Print ISSN: 1942-325X. Online ISSN: 1942-3268
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Original Article Sarcomere Mutation-Specific Expression Patterns in Human Hypertrophic Cardiomyopathy Adam S. Helms, MD*; Frank M. Davis, BS*; David Coleman, BS; Sarah N. Bartolone, BS; Amelia A. Glazier, BS; Francis Pagani, MD, PhD; Jaime M. Yob, MS; Sakthivel Sadayappan, PhD; Ellen Pedersen, BS; Robert Lyons, PhD; Margaret V. Westfall, PhD; Richard Jones, PhD; Mark W. Russell, MD; Sharlene M. Day, MD Background—Heterozygous mutations in sarcomere genes in hypertrophic cardiomyopathy (HCM) are proposed to exert their effect through gain of function for missense mutations or loss of function for truncating mutations. However, allelic expression from individual mutations has not been sufficiently characterized to support this exclusive distinction in human HCM. Methods and Results—Sarcomere transcript and protein levels were analyzed in septal myectomy and transplant specimens from 46 genotyped HCM patients with or without sarcomere gene mutations and 10 control hearts. For truncating mutations in MYBPC3, the average ratio of mutant:wild-type transcripts was ≈1:5, in contrast to ≈1:1 for all sarcomere missense mutations, confirming that nonsense transcripts are uniquely unstable. However, total MYBPC3 mRNA was significantly increased by 9-fold in HCM samples with MYBPC3 mutations compared with control hearts and with HCM samples without sarcomere gene mutations. Full-length MYBPC3 protein content was not different between MYBPC3 mutant HCM and control samples, and no truncated proteins were detected. By absolute quantification of abundance with multiple reaction monitoring, stoichiometric ratios of mutant sarcomere proteins relative to wild type were strikingly variable in a mutation-specific manner, with the fraction of mutant protein ranging from 30% to 84%. Conclusions—These results challenge the concept that haploinsufficiency is a unifying mechanism for HCM caused by MYBPC3 truncating mutations. The range of allelic imbalance for several missense sarcomere mutations suggests that certain mutant proteins may be more or less stable or incorporate more or less efficiently into the sarcomere than wildtype proteins. These mutation-specific properties may distinctly influence disease phenotypes. (Circ Cardiovasc Genet. 2014;7:434-443.) Key Words: cardiomyopathy, hypertrophic ◼ gene expression ◼ gene expression regulation ◼ humans ◼ proteomics ◼ sarcomeres
T
he most common Mendelian cardiovascular condition, hypertrophic cardiomyopathy (HCM), is defined by unexplained cardiac hypertrophy and can be complicated by left ventricular outflow tract obstruction, atrial and ventricular arrhythmias, sudden cardiac death, and heart failure.1 Sarcomere gene mutations account for 50% to 75% of the genetic basis of HCM, the majority found in the 2 largest genes, MYBPC3 (myosin binding protein C) and MYH7 (β-myosin heavy chain).2 Despite identification of >1000 sarcomere gene mutations, molecular mechanisms that elicit disease phenotypes are incompletely defined.3 One fundamental question pertains to the nature of the gene product derived from the mutant allele. Most sarcomere mutations result in a single amino acid substitution that
encodes a full-length protein. The exception is MYBPC3 in which >50% of mutations create a premature termination codon (PTC).4 The widely accepted hypothesis is that truncating MYBPC3 mutations cause haploinsufficiency, as opposed to missense mutations which incorporate into the sarcomere and act in a dominant-negative fashion.3 Previous studies in human HCM addressing this hypothesis have been constrained by small numbers of samples with unique mutations. Here, we comprehensively analyze sarcomere gene and protein levels from a large number of cardiac specimens from patients with HCM of known genotype. We hypothesized that allelic balance between wild-type and
Clinical Perspective on p 443
Received October 10, 2013; accepted May 29, 2014. From the Departments of Internal Medicine (A.S.H., F.D., D.C., S.B., J.M.Y., S.M.D.), Molecular and Integrative Physiology (A.A.G., M.V.W.), Cardiac Surgery (F.P., M.V.W.), Sequencing Core (E.P., R.L.), and Pediatrics (M.W.R.), University of Michigan, Ann Arbor; Department of Cell and Molecular Physiology, Health Sciences Division, Loyola University Chicago, Maywood, IL (S.S.); and MS Bioworks, Ann Arbor, MI (R.J.). *Dr Helms and F. M. Davis contributed equally to this work The Data Supplement is available at http://circgenetics.ahajournals.org/lookup/suppl/doi:10.1161/CIRCGENETICS.113.000448/-/DC1. Correspondence to Sharlene M. Day, MD, 1150 W Medical Center Dr, 7301 MSRB III, Ann Arbor, MI 48109. E-mail [email protected] © 2014 American Heart Association, Inc. Circ Cardiovasc Genet is available at http://circgenetics.ahajournals.org
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DOI: 10.1161/CIRCGENETICS.113.000448
Helms et al Sarcomere Gene Expression in Human HCM 435 mutant sarcomere proteins is variable and mutation specific, reflecting differential stability or efficiency of sarcomere incorporation compared with the wild-type protein. We further proposed that haploinsufficiency may not be the primary driver of disease progression in HCM associated with MYBPC3 truncating mutations.
Methods An expanded Methods section is available in the Data Supplement.
Human Heart Tissue Procurement Ventricular myocardial tissue was snap-frozen in liquid N2 or placed in formalin at the time of collection. This study had the approval of the University of Michigan Institutional Review Board, and subjects gave informed consent.
Transcript Analysis Reverse transcription polymerase chain reaction (PCR), cDNA sequencing, and quantitative reverse transcription PCR were performed by standard techniques (see Data Supplement). A quantitative reverse transcription PCR assay was used to determine allelic specific expression in the samples containing splice site mutations, whereas the single-base extension method was used in samples containing single-nucleotide variants that resulted in premature stop codons (see Methods and Table I in the Data Supplement).
Protein Preparation, Quantification, and Immunolocalization Immunoblotting, immunofluorescent imaging, myofilament fractionation, and extraction of insoluble proteins were performed by established methods (see Data Supplement).
Absolute Quantification of Abundance Myofilament proteins were separated using SDS-PAGE and Coomassie stained. The protein of interest was gel excised. After in-gel enzymatic digestion, samples were analyzed by nano liquid chromatography tandem mass spectrometry performed in the Orbitrap at 70 000 FWHM and 17 500 full width half maximum resolution, respectively. In some cases, peptides were post-translationally modified (eg, Met oxidation) or contained missed cleavages. The abundance of mutant and wild-type sarcomere proteins within each sample was then determined using isotopically labeled synthetic absolute quantification of abundance (AQUA) peptides corresponding to each form of wild-type and mutant peptides for each individual sample. Samples were analyzed by liquid chromatography selected reaction monitoring mass spectrometry with a Waters NanoAcquity HPLC System interfaced to a ThermoFisher TSQ Quantum Ultra. Peak areas for the wild-type or mutant endogenous peptide were expressed as a ratio to their corresponding AQUA peptides allowing the mole ratio of wild-type versus mutant peptide to be calculated. Molar amounts from all wild-type and mutant peptides were summed and expressed as a percentage of total protein. More details can be found in the Data Supplement.
Statistical Analysis All values are expressed as mean±SEM unless otherwise indicated. Normality was determined by the Shapiro–Wilk test. Western blot data for troponin T type 2, alpha actin type 1, troponin I type 3, and myosin light chain 2 were normally distributed and analyzed by 1-way ANOVA. For non-normally distributed data, analyses were performed on ranks using the Mann–Whitney rank-sum test for 2 group comparisons (PTC versus non-PTC containing transcripts) or the Kruskal–Wallis test for multiple group comparisons. Race and sex comparisons among patient groups were analyzed by χ2 test. All
statistical analyses were performed using Sigma Stat 12.5 or IBM SPSS Statistics with a 2-sided P value G (a)), and 3 at the time of transplant (MYBPC3 Trp890*(a), MYH7 Thr1377Met, and MYH7 Gly708Ala). Controls and patients with HCM were comparable in age and sex (Table). Racial background was white, except for 2 control hearts from black donors. Ethnicity was Hispanic for 2 hearts (1 control and 1 HCM) and Middle Eastern for 1 heart (HCM). Wall thickness was greater in all HCM groups compared with controls (PA for which a mutant transcript was not detectable), whereas mutations in intron splice acceptor sites (n=2) resulted in inclusion of the intron containing the mutation (Figure I in the Data Supplement). Table. Patient Demographics and Echocardiographic Measurements Septal LVOT Thickness, Gradient, Age, y % Male mm EF, % mm Hg Controls (n=10)
52±5
40
11.8±0.7 60±2
... 79±16
Patients with HCM No sarcomere mutation (n=14) 51±3
64
19.9±1.2* 70±2
Non-MYBPC3 mutations (n=10) 40±5
50
19.3±0.8* 72±3* 78±13
MYBPC3 mutations (n=22)
50
24.2±1.7* 70±3* 78±10
40±3
Gradient measured at rest in myectomy patients only. HCM indicates hypertrophic cardiomyopathy; EF, indicates ejection fraction; and LVOT, Left ventricular outflow tract. *PC (Glu542Gln) was present, in addition to the mutant splice variant transcript, and constituted 13% and 16% of total full-length MYBPC3 transcripts for each sample respectively (Figure 1B). However, no missense transcript was detected for either c.772G>A (Glu258Lys) sample, indicating that 100% of the transcripts from the mutant allele exist as truncating mutant transcripts. The Sequenom software was not
able to design primers for the MYBPC3 mutation c.2308G>A (Asp770Asn). However, amplification of cDNA using primers in exons 22 and 24 yielded a single band containing only wild-type sequence (Figure IC in the Data Supplement). Since the most likely resultant splice error in this transcript would be skipping of exon 23, we designed a primer that spanned a conjectured junction of exon 22 and exon 24. No band was detected, suggesting that this PTC-containing transcript is probably very susceptible to NMD. Less likely is the possibility that splicing is altered at a distant splice site not detected using these primers. The ratio of mutant to wild-type transcript for MYBPC3 c.3742_3759dup (Gly1248_Cys1253dup) could not be precisely quantified because the wild-type transcript could not be uniquely amplified (addition of adenosine after extension primer could be either wild type or second repeat of mutant transcript). However, the mutant transcript was identified in a standard sequencing reaction (Figure IIA in the Data Supplement) and constituted ≥30% of the total by single-base extension (ie, 30% of the extension product contained the first nucleotide of the duplicated sequence). Using single-base extension, ratios of all true missense mutations in MYBPC3, MYH7, MYL2, and TNNT2 were present at the expected ≈1:1 ratio with wild-type transcripts Figure 1. Sarcomere gene expression in human hypertrophic cardiomyopathy (HCM) samples. A Left, Allele-specific expression from wild-type and MYBPC3 splice-error premature termination codon (PTC)–containing transcripts quantified by quantitative reverse transcription polymerase chain reaction with custom-designed primers (Table V in the Data Supplement). Right, Allele-specific expression from wild-type and MYBPC3 PTC-containing transcripts (without splice errors) quantified by customdesigned single-base extension reactions. These mutations are either single-nucleotide substitutions that encode for a termination codon or an insertion that causes a frameshift. B, Quantification of wild-type and single-nucleotide substitution mutant sarcomere gene transcripts without PTCs by custom-designed single-base extension reactions. For the MYBPC3 exon splice site mutations, the mutant fraction is the proportion of detectable missense transcript. The remainder of the samples contain true missense mutations. In instances where 2 samples carried the same mutation, these are designated as (a) and (b) respectively in this figure and maintained throughout the article. C, Total MYBPC3 transcript abundance (left) and total MYH7 transcript abundance (right) in carriers of MYBPC3 (n=18) or MYH7 (n=6) mutations, compared with HCM samples in which no sarcomere mutation (n=7) was identified and control (nonHCM donor, n=7) hearts. *P=0.02 vs control hearts; †P=0.003 vs HCM with no sarcomere mutations. Open circles above the box plots indicate statistical outliers. D, Reverse transcription polymerase chain reaction analysis showing uniformly increased intensity of amplified MYBPC3 sequence in samples containing MYBPC3 mutations compared with the other 3 groups.
Helms et al Sarcomere Gene Expression in Human HCM 437 (Figure 1B). The Sequenom software could not design primers for TPM1 c.850A>G (Ile284Val), perhaps because it is in the terminal codon. However, cDNA sequencing showed polymorphism at that site (Figure IIB in the Data Supplement). Overall, PTC-containing transcripts in MYBPC3 are significantly less abundant than transcripts from non–PTC-containing mutant alleles from either MYBPC3 or other sarcomere genes (mean, 16±3% versus 47±1% of the total respectively; PC
ACGTTGGATGGATGC TCTGGTACACCTCC
ACGTTGGATGTATGC ACTGTGCACTAGCGG
CTGAGCTCATTGTGCAG
MYBPC3 c.2308G>A
ACGTTGGATGGCTG CCCGCCATCGTAGG
ACGTTGGATGAGGTC AACCTCACAGTCAAG
TCACAGTCAAGGTCATC
MYBPC3 c.3225_3227insT
ACGTTGGATGCTCCA GAGCCACATTAAGAC
ACGTTGGATGTGTTG ACAAGCCAAGTCCTC
GGATCTCCGGGTGACTG
MYBPC3 c.3288G>A
ACGTTGGATGATGTG GCTCTGGAGTGGAAG
ACGTTGGATGTTGTC GGCTTTCTGCACTGT
TCTGCACTGTGTACCCC
MYBPC3 c.3742_3759dup
ACGTTGGATGCCTCG CCCTGTAAGTTGGT
ACGTTGGATGAAAGC CCTGCCCCTTTGAC
GGGGCATCTATGTCTGC
MYBPC3 c.3697C>T
ACGTTGGATGATCTC CAGAGTCAACACTCC
ACGTTGGATGCCTGG TTCAAGAATGGCCTG
GCTGAACATGCGGAA
MYBPC3 c.2670G>A
ACGTTGGATGTAGAG GACGTCTCTGACACC
ACGTTGGATGTACTC CACGCTGTAGCCATC
ACGGTCTCCCTCAAGTG
MYBPC3 c.3233G>A
ACGTTGGATGTCCACTC CAGAGCCACATTA
ACGTTGGATGACAAG CCAAGTCCTCCCCAG
AGCCACATTAAGACCC
MYH7 c.2389G>A
ACGTTGGATGGCATC ATCACGCGTATCCAG
ACGTTGGATGCCAGC AGCTTTTTGTACTCC
TTTTTGTACTCCATTCTGG
MYH7 c.3286G>T
ACGTTGGATGGAGCT CCTTGAGCTTCTTCTG
ACGTTGGATGTGAGC TGAATGCTCTCAACG
CGAGGGCCTGTTCAT
MYH7 c.4816C>T
ACGTTGGATGTGGTG GACTCGCTGCAGAC
ACGTTGGATGTTCTT CTTCACCCTCAGGGC
CTGGACGCAGAGACA
MYH7 c.4817G>A
ACGTTGGATGTGGTG GACTCGCTGCAGAC
ACGTTGGATGTTCTT CTTCACCCTCAGGGC
GGGCCTCGTTGCGGCTG
MYH7 c.4130C>T
ACGTTGGATGGTCCT TTCCAAGGCCAACTC
ACGTTGGATGAGCTC CTCAGTCCGCTGAAT
GGAGGACCAAGTATGAGA
MYH7 c.968T>A
ACGTTGGATGATCAG TGGCCATGAGCTCCT
ACGTTGGATGTTCAT CTCCCAAGGAGAGAC
TCCTCAGCGTCATCA
MYH7 c.2123G>C
ACGTTGGATGGAAGTC CCCGTAGAGGATG
ACGTTGGATGAGCTGC GCTGCAATGGTGT
ATGCGGTTGGGGAAG
TNNT2 c.257A>C
ACGTTGGATGATTCA GGTCCTTCTCCATGC
ACGTTGGATGGTTCA TGCCCAACTTGGTGC
CGGTGGATGTCATCAAAG
MYL2 c.482A>G
ACGTTGGATGACAAG AACCTGGTGCACATC
ACGTTGGATGAGGGA CCACTCTGCAAAGAC
AGTCCTTCTCTTCTCCG
8
Supplementary Table 2. HCM gene mutation and corresponding protein designation
Gene
Mutation
Protein
Gene location
Mutation type
MYBPC3
c.2670 G>A
p.Trp890*
Exon 26
Truncating
MYBPC3
c.3226_3227insT
p.Asp1076Valfs*6
Exon 30
Truncating
MYBPC3
c.3233 G>A
p.Trp1078*
Exon 30
Truncating
MYBPC3
c.3294 G>A
p.Trp1098*
Exon 30
Truncating
MYBPC3
c.3697 C>T
p.Gln1233*
Exon 33
Truncating
MYBPC3
c.927-9 G>A
-
Intron 11
Truncating
MYBPC3
c.1624+4 A>T
-
Intron 17
Truncating
MYBPC3
c.1928-2 A>G
-
Intron 20
Truncating
MYBPC3
c.2905+1 G>A
-
Intron 27
Truncating
MYBPC3
c.3330+2 T>G
-
Intron 30
Truncating
MYBPC3
c.772 G>A
p.Glu258Lys
Exon 6
Truncating
MYBPC3
c.1624 G>C
p.Glu542Gln
Exon 17
Truncating and missense
MYBPC3
c.2308 G>A
p.Asp770Asn
Exon 23
Truncating
MYBPC3
c.3742_3759dup
p.Gly1248_Cys1253dup
Exon 33
In frame duplication
MYBPC3
c.1484 G>A
p.Arg495Gln
Exon 17
Missense
MYH7
c.968 T>A
p.Ile323Asn
Exon 11
Missense
MYH7
c.2123 G>C
p.Gly708Ala
Exon 19
Missense
MYH7
c.2389 G>A
p.Ala797Thr
Exon 21
Missense
MYH7
c.3286 G>T
p.Asp1096Tyr
Exon 26
Missense
MYH7
c.4130 C>T
p.Thr1377Met
Exon 30
Missense
MYH7
c.4816C>T
p.Arg1606Cys
Exon 34
Missense
Exon 34
Missense
1
MYH7
c.4817 G>A
p.Arg1606His
MYL2
c.482 A>G
p.His161Arg
Exon 7
Missense
TNNT2
c.257 A>C
p.Asp86Ala
Exon 8
Missense
Exon 9
Missense
TPM1
c.850 A>G
p.Ile284Val
2
Unless otherwise designated, all mutations studied were classified as pathogenic or likely pathogenic based on standard criteria7: published or found in affected individuals in testing laboratory with proven segregation in family members, premature truncation, species conservation, absent or very low population frequency, in silico prediction algorithms. 1 MYH7 Arg1606His is designated as a variant of unknown significance. Pathogenicity is supported by very low population frequency (1:8600) in NHLBI Exome Sequencing Project, species conservation, and in silico model predicting a probably damaging effect on the protein. 2 TPM1 Ile284Val is designated as a variant of unknown significance. Pathogenicity is supported by absence in the general population, species conservation, and in silico model predicting a probably damaging effect on the protein. The variant also segregated with 2 individuals in the family (proband and mother).
9
Supplementary Table 3. cDNA sequencing for intron and exon splice site MYBPC3 mutations.
Mutation
Mutation location
Mutant transcript sequence
Location of PTC
c.1624+4 A>T
Intron 17 (donor)
Skips exon 17
Junction of exon 16/18
c.2905+1 G>A
Intron 27 (donor)
Skips exon 27
Junction of exon 26/28
c.3330+2 T>G
Intron 30 (donor)
Skips exon 30
Exon 31
c.927-9 G>A
Intron 11 (acceptor)
Inclusion of intron 11
Intron 11
c.1928-2 A>G
Intron 20 (acceptor)
Inclusion of intron 20
Intron 20
c.772 G>A
Exon 6 (donor)
Skips exon 6
Exon 9
c.1624 G>C
Exon 17 (donor)
Skips exon 17
Junction of exon 16/18
c.2308 G>A
Exon 23 (donor)
Not detectable
10
Supplementary Table 4. Allele-specific protein expression using absolute quantification of abundance Gene
Mutation
Enzyme
Wild-type (Wt) AQUA peptide(s)
Wt peptide fmol (%)
Mutant AQUA peptide(s)
Mutant peptide fmol (%)
MYH7
Ala797Thr
Trypsin
GVLAR
107.91 (51.2) 310.52 (48.2)
GVLTR
102.71 (48.8) 333.02 (51.8)
MYH7
Arg1606His
Trypsin
VVDSLQTSLDAETR
77.41 (63.6) 1031.62 (52.1)
VVDSLQTSLDAETHSR
44.41 (36.4) 949.12 (47.9)
MYH7
Arg1606Cys
Trypsin
VVDSLQTSLDAETR
387.9 (63)
VVDSLQTSLDAETCSR*
228.6 (37)
MYH7
Asp1096Tyr
Trypsin
IEDEQALGSQLQK IEDEQALGSQLQKK Total
87.5 248.9 336.4 (53.1)
IEYEQALGSQLQK IEYEQALGSQLQKK Total
192.7 104.4 297.1 (46.9)
MYH7
Thr1377Met (LV septum)
Trypsin
YETDAIQR TKYETDAIQR TKYETDAIQRTEELEEAK Total
1.6 50.4 6.3 58.3 (51)
YEMDAIQR YEM(ox)DAIQR TKYEMDAIQR TKYEM(ox)DAIQR TKYEMDAIQRTEELEEAK TKYEM(ox)DAIQRTEELEEAK Total
1.0 0 47.1 8.6 0 0 56.7 (49)
MYH7
Thr1377Met (LV Lateral)
Trypsin
YETDAIQR TKYETDAIQR TKYETDAIQRTEELEEAK Total
3.3 68.5 7.6 79.4 (46)
YEMDAIQR YEM(ox)DAIQR TKYEMDAIQR TKYEM(ox)DAIQR TKYEMDAIQRTEELEEAK TKYEM(ox)DAIQRTEELEEAK Total
2.5 0.5 62.7 20.3 7.6 0 93.6 (54)
MYH7
Thr1377Met (RV)
Trypsin
YETDAIQR TKYETDAIQR TKYETDAIQRTEELEEAK Total
2.8 108.5 17.4 128.7 (50)
YEMDAIQR YEM(ox)DAIQR TKYEMDAIQR TKYEM(ox)DAIQR TKYEMDAIQRTEELEEAK TKYEM(ox)DAIQRTEELEEAK Total
1.4 0 86.7 20 14.4 8.6 131.1 (50)
MYH7
Ile323Asn
Asp-N
DYAFISQGETTVASI DYAFISQGETTVASID Total
100.6 37.9 138.5 (31%)
DYAFISQGETTVASN DYAFISQGETTVASND Total
301.73 0 301.73 (69%)
11
MYH7
Gly708Ala (LV septum)
Trypsin
KGFPNR
250.1 (91)
KAFPNR
24.1 (9)
MYH7
Gly708Ala (LV lateral)
Trypsin
KGFPNR
677.1 (90)
KAFPNR
74.8 (10)
MYH7
Gly708Ala (RV)
Trypsin
KGFPNR
277.5 (89)
KAFPNR
33.3 (11)
TNNT2
Asp86Ala
Trypsin
TPM1
Ile284Val
Trypsin
VDFDDIHR
4.61 (19.2) 9.722 (12.3)
AISEELDHALNDMTSI AISEELDHALNDM(ox)TSI Totals
VHIITHGEEK KNLVHIITHGEEK Total
VAFDDIHR
19.41 (80.8) 69.22 (87.7)
1.41 536.22 57 603.8 58.41 (59) 11402 (52.1)
AISEELDHALNDMTSV
133.7 66.1 199.8 (69.2)
VHIITRGEEK KNLVHIITRGEEK Total
67 21.8 88.8 (30.8)
AISEELDHALNDM(ox)TSV
4.21 449.92 36.31 596.32 40.51 (41) 1046.22 (47.9)
MYL2
His161Arg
Chymotrypsin
MYBPC3
Arg495Gln (a)
Lys-C
DGVELTREETFK
52.91 (25.6) 23.12 (36.6)
DGVELTQEETFK
153.41 (74.4) 40.02 (63.4)
MYBPC3
Arg495Gln (b)
Lys-C
DGVELTREETFK
36.91 (36.3) 35.32 (39.7)
DGVELTQEETFK
64.81 (63.7) 53.72 (60.3)
MYBPC3
Glu542Gln (a)
Chymotrypsin
IVQEKKLEVY
2.10 (95.2)
IVQQKKLEVY
0.11 (4.8)
MYBPC3
Glu542Gln (b)
Chymotrypsin
IVQEKKLEVY
16.3 (98.4)
IVQQKKLEVY
0.26 (1.6)
Values with subscripts of 1 and 2 indicate samples in which 2 separate LC/MS/MS runs were performed. In samples where more than one AQUA peptide was used (missed cleavage, post-translational modifications), the total provided is the sum of the absolute amounts of each peptide (wild-type or mutant) and % of total (i.e. wild-type/mutant + wild-type peptides). Variation in absolute peptide quantities is primarily due to differences in the amount of starting material, but relative amounts of wild-type and mutant peptides within the same sample were similar between replicates (CV 4-14%). Amino acids in red indicate variant. * Cysteine is carbamidomethylated to correspond to the modification of endogenous peptide and Ox = oxidized modification of Met.
12
Supplementary Table 5. Taqman hydrolysis probes used to examine allele-specific expression in samples containing MYBPC3 splice site mutations
Mutation
Taqman Hydrolysis Probe
Exon Border
Transcript Recognized
c.1624+4 A>T
Hs01076199_g1
16-17
Wild Type
Hs01076202_m1
20-21
Both
Hs01076206_m1
26-27
Wild Type
Hs01076202_m1
20-21
Both
Hs01076209_g1
30-31
Wild Type
Hs01076211_g1
32-33
Both
Hs01079709_g1
11-12
Wild Type
Hs00165232_m1
12-13
Both
Hs01076202_m1
20-21
Wild Type
Hs01076206_m1
26-27
Both
Hs01076217_m1
5-6
Wild Type
Hs01076212_m1
2-3
Both
Hs01076199_g1
16-17
Wild Type
Hs01076202_m1
20-21
Both
Hs01076203_m1
22-23
Wild Type
Hs01076202_m1
20-21
Both
c.2905+1 A>G
c.3330+2 T>G
c.927-9 A>G
c.1928-2 A>G
c.772 G>A
c.1624 G>C
c.2308 G>A
13
Supplementary Figure 1. A, B and C. PCR amplification of wild-type and splice site mutant cDNA. Corresponding sequencing results for each band from each sample are found in Supplementary Table 2. * For c.1928-2 A>G, only a single band corresponding to wild-type sequence was identified using primers complementary to adjacent exon sequence (exons 19 and 21). Therefore, an alternative forward primer was designed to be complementary to intron 20 and uniquely amplified cDNA from the mutant allele that included intron 20. Primer sequences for each sample are as follows: c.1624 G>C, 1624+4 A>T: GCC CCC TGT GCT CAT CA (forward); CAC CTT TAT GCG GCT GTC G (reverse) c.2905+a G>A: ATG CGG CTG AAC TTC GAC CTG ATT (forward); CTG TGG GGC TGT TGC GGA TGC TC (reverse) c.3330+2 T>G: GGC CTC AGG TGA CCT GG (forward); AGA CGG GCT CCT TGG TGG TG (reverse) c.772 G>A: CTC AGC AGC TCT CAA TGG TCC TAC (forward); CTG ATC CGC CGA CCA CCT C (reverse) c.1928-2 A>G: CGA CGT CAC ACC TGC CG (forward, exon 19); ACT GAG GGC AGA TTC CTG ATT (forward, intron 20); CTG CGT GAT AGC CTT CTG C (reverse) c.927-9 G>A: GGC TGG AGG TGG TCG G (forward); GCC TCA TGC CCT TGA GCC (reverse) c.2308 G>A: GCC CAG GCC GCA TAC CAG AC (forward); AGG TCG AAG TTC AGC CGC ATC C (reverse) D. PCR amplification of MYH7 in the sample containing the missense mutation c.2123 G>C in MYH7. Only a single band was amplified at the expected size (857 bp) and sequence-confirmed to contain only wild-type and missense sequence. Primer sequences for this samples were CTGAAGCCCACTTCTCCCTG (forward) and GCGTGTGAACTCCTCCTTCA (reverse). 14
Supplementary Figure 2. Sequence chromatograms of cDNA from human HCM samples with sarcomere gene mutations. Heterozygous expression of mutant and wild-type sequences are shown for A. MYBPC3 3742_3759dup (Gly1248_Cys1253dup), and B. TPM1 c.850A>G (Ile284Val).
15
Supplementary Figure 3. A. Sarcomere protein expression for MYH7, MYL2, TNNT2, TNNI3 and ACTC1 analyzed by western blot and normalized to GAPDH, expressed as a % of a single control heart run on each gel. There were no significant differences among the groups. B. MYBPC3 protein expression in control (n=6) and non-HCM failing hearts (n=14) of non-ischemic and ischemic etiologies. There was no significant difference in full-length MYBPC3 protein between the 2 groups. Degradation products were not visualized in either group.
16
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17
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Data Supplement (unedited) at: http://circgenetics.ahajournals.org/content/suppl/2014/07/16/CIRCGENETICS.113.000448.DC1.html
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Original Article Sarcomere Mutation-Specific Expression Patterns in Human Hypertrophic Cardiomyopathy Adam S. Helms, MD*; Frank M. Davis, BS*; David Coleman, BS; Sarah N. Bartolone, BS; Amelia A. Glazier, BS; Francis Pagani, MD, PhD; Jaime M. Yob, MS; Sakthivel Sadayappan, PhD; Ellen Pedersen, BS; Robert Lyons, PhD; Margaret V. Westfall, PhD; Richard Jones, PhD; Mark W. Russell, MD; Sharlene M. Day, MD Background—Heterozygous mutations in sarcomere genes in hypertrophic cardiomyopathy (HCM) are proposed to exert their effect through gain of function for missense mutations or loss of function for truncating mutations. However, allelic expression from individual mutations has not been sufficiently characterized to support this exclusive distinction in human HCM. Methods and Results—Sarcomere transcript and protein levels were analyzed in septal myectomy and transplant specimens from 46 genotyped HCM patients with or without sarcomere gene mutations and 10 control hearts. For truncating mutations in MYBPC3, the average ratio of mutant:wild-type transcripts was ≈1:5, in contrast to ≈1:1 for all sarcomere missense mutations, confirming that nonsense transcripts are uniquely unstable. However, total MYBPC3 mRNA was significantly increased by 9-fold in HCM samples with MYBPC3 mutations compared with control hearts and with HCM samples without sarcomere gene mutations. Full-length MYBPC3 protein content was not different between MYBPC3 mutant HCM and control samples, and no truncated proteins were detected. By absolute quantification of abundance with multiple reaction monitoring, stoichiometric ratios of mutant sarcomere proteins relative to wild type were strikingly variable in a mutation-specific manner, with the fraction of mutant protein ranging from 30% to 84%. Conclusions—These results challenge the concept that haploinsufficiency is a unifying mechanism for HCM caused by MYBPC3 truncating mutations. The range of allelic imbalance for several missense sarcomere mutations suggests that certain mutant proteins may be more or less stable or incorporate more or less efficiently into the sarcomere than wildtype proteins. These mutation-specific properties may distinctly influence disease phenotypes. (Circ Cardiovasc Genet. 2014;7:434-443.) Key Words: cardiomyopathy, hypertrophic ◼ gene expression ◼ gene expression regulation ◼ humans ◼ proteomics ◼ sarcomeres
T
he most common Mendelian cardiovascular condition, hypertrophic cardiomyopathy (HCM), is defined by unexplained cardiac hypertrophy and can be complicated by left ventricular outflow tract obstruction, atrial and ventricular arrhythmias, sudden cardiac death, and heart failure.1 Sarcomere gene mutations account for 50% to 75% of the genetic basis of HCM, the majority found in the 2 largest genes, MYBPC3 (myosin binding protein C) and MYH7 (β-myosin heavy chain).2 Despite identification of >1000 sarcomere gene mutations, molecular mechanisms that elicit disease phenotypes are incompletely defined.3 One fundamental question pertains to the nature of the gene product derived from the mutant allele. Most sarcomere mutations result in a single amino acid substitution that
encodes a full-length protein. The exception is MYBPC3 in which >50% of mutations create a premature termination codon (PTC).4 The widely accepted hypothesis is that truncating MYBPC3 mutations cause haploinsufficiency, as opposed to missense mutations which incorporate into the sarcomere and act in a dominant-negative fashion.3 Previous studies in human HCM addressing this hypothesis have been constrained by small numbers of samples with unique mutations. Here, we comprehensively analyze sarcomere gene and protein levels from a large number of cardiac specimens from patients with HCM of known genotype. We hypothesized that allelic balance between wild-type and
Clinical Perspective on p 443
Received October 10, 2013; accepted May 29, 2014. From the Departments of Internal Medicine (A.S.H., F.D., D.C., S.B., J.M.Y., S.M.D.), Molecular and Integrative Physiology (A.A.G., M.V.W.), Cardiac Surgery (F.P., M.V.W.), Sequencing Core (E.P., R.L.), and Pediatrics (M.W.R.), University of Michigan, Ann Arbor; Department of Cell and Molecular Physiology, Health Sciences Division, Loyola University Chicago, Maywood, IL (S.S.); and MS Bioworks, Ann Arbor, MI (R.J.). *Dr Helms and F. M. Davis contributed equally to this work The Data Supplement is available at http://circgenetics.ahajournals.org/lookup/suppl/doi:10.1161/CIRCGENETICS.113.000448/-/DC1. Correspondence to Sharlene M. Day, MD, 1150 W Medical Center Dr, 7301 MSRB III, Ann Arbor, MI 48109. E-mail [email protected] © 2014 American Heart Association, Inc. Circ Cardiovasc Genet is available at http://circgenetics.ahajournals.org
434
DOI: 10.1161/CIRCGENETICS.113.000448
Helms et al Sarcomere Gene Expression in Human HCM 435 mutant sarcomere proteins is variable and mutation specific, reflecting differential stability or efficiency of sarcomere incorporation compared with the wild-type protein. We further proposed that haploinsufficiency may not be the primary driver of disease progression in HCM associated with MYBPC3 truncating mutations.
Methods An expanded Methods section is available in the Data Supplement.
Human Heart Tissue Procurement Ventricular myocardial tissue was snap-frozen in liquid N2 or placed in formalin at the time of collection. This study had the approval of the University of Michigan Institutional Review Board, and subjects gave informed consent.
Transcript Analysis Reverse transcription polymerase chain reaction (PCR), cDNA sequencing, and quantitative reverse transcription PCR were performed by standard techniques (see Data Supplement). A quantitative reverse transcription PCR assay was used to determine allelic specific expression in the samples containing splice site mutations, whereas the single-base extension method was used in samples containing single-nucleotide variants that resulted in premature stop codons (see Methods and Table I in the Data Supplement).
Protein Preparation, Quantification, and Immunolocalization Immunoblotting, immunofluorescent imaging, myofilament fractionation, and extraction of insoluble proteins were performed by established methods (see Data Supplement).
Absolute Quantification of Abundance Myofilament proteins were separated using SDS-PAGE and Coomassie stained. The protein of interest was gel excised. After in-gel enzymatic digestion, samples were analyzed by nano liquid chromatography tandem mass spectrometry performed in the Orbitrap at 70 000 FWHM and 17 500 full width half maximum resolution, respectively. In some cases, peptides were post-translationally modified (eg, Met oxidation) or contained missed cleavages. The abundance of mutant and wild-type sarcomere proteins within each sample was then determined using isotopically labeled synthetic absolute quantification of abundance (AQUA) peptides corresponding to each form of wild-type and mutant peptides for each individual sample. Samples were analyzed by liquid chromatography selected reaction monitoring mass spectrometry with a Waters NanoAcquity HPLC System interfaced to a ThermoFisher TSQ Quantum Ultra. Peak areas for the wild-type or mutant endogenous peptide were expressed as a ratio to their corresponding AQUA peptides allowing the mole ratio of wild-type versus mutant peptide to be calculated. Molar amounts from all wild-type and mutant peptides were summed and expressed as a percentage of total protein. More details can be found in the Data Supplement.
Statistical Analysis All values are expressed as mean±SEM unless otherwise indicated. Normality was determined by the Shapiro–Wilk test. Western blot data for troponin T type 2, alpha actin type 1, troponin I type 3, and myosin light chain 2 were normally distributed and analyzed by 1-way ANOVA. For non-normally distributed data, analyses were performed on ranks using the Mann–Whitney rank-sum test for 2 group comparisons (PTC versus non-PTC containing transcripts) or the Kruskal–Wallis test for multiple group comparisons. Race and sex comparisons among patient groups were analyzed by χ2 test. All
statistical analyses were performed using Sigma Stat 12.5 or IBM SPSS Statistics with a 2-sided P value G (a)), and 3 at the time of transplant (MYBPC3 Trp890*(a), MYH7 Thr1377Met, and MYH7 Gly708Ala). Controls and patients with HCM were comparable in age and sex (Table). Racial background was white, except for 2 control hearts from black donors. Ethnicity was Hispanic for 2 hearts (1 control and 1 HCM) and Middle Eastern for 1 heart (HCM). Wall thickness was greater in all HCM groups compared with controls (PA for which a mutant transcript was not detectable), whereas mutations in intron splice acceptor sites (n=2) resulted in inclusion of the intron containing the mutation (Figure I in the Data Supplement). Table. Patient Demographics and Echocardiographic Measurements Septal LVOT Thickness, Gradient, Age, y % Male mm EF, % mm Hg Controls (n=10)
52±5
40
11.8±0.7 60±2
... 79±16
Patients with HCM No sarcomere mutation (n=14) 51±3
64
19.9±1.2* 70±2
Non-MYBPC3 mutations (n=10) 40±5
50
19.3±0.8* 72±3* 78±13
MYBPC3 mutations (n=22)
50
24.2±1.7* 70±3* 78±10
40±3
Gradient measured at rest in myectomy patients only. HCM indicates hypertrophic cardiomyopathy; EF, indicates ejection fraction; and LVOT, Left ventricular outflow tract. *PC (Glu542Gln) was present, in addition to the mutant splice variant transcript, and constituted 13% and 16% of total full-length MYBPC3 transcripts for each sample respectively (Figure 1B). However, no missense transcript was detected for either c.772G>A (Glu258Lys) sample, indicating that 100% of the transcripts from the mutant allele exist as truncating mutant transcripts. The Sequenom software was not
able to design primers for the MYBPC3 mutation c.2308G>A (Asp770Asn). However, amplification of cDNA using primers in exons 22 and 24 yielded a single band containing only wild-type sequence (Figure IC in the Data Supplement). Since the most likely resultant splice error in this transcript would be skipping of exon 23, we designed a primer that spanned a conjectured junction of exon 22 and exon 24. No band was detected, suggesting that this PTC-containing transcript is probably very susceptible to NMD. Less likely is the possibility that splicing is altered at a distant splice site not detected using these primers. The ratio of mutant to wild-type transcript for MYBPC3 c.3742_3759dup (Gly1248_Cys1253dup) could not be precisely quantified because the wild-type transcript could not be uniquely amplified (addition of adenosine after extension primer could be either wild type or second repeat of mutant transcript). However, the mutant transcript was identified in a standard sequencing reaction (Figure IIA in the Data Supplement) and constituted ≥30% of the total by single-base extension (ie, 30% of the extension product contained the first nucleotide of the duplicated sequence). Using single-base extension, ratios of all true missense mutations in MYBPC3, MYH7, MYL2, and TNNT2 were present at the expected ≈1:1 ratio with wild-type transcripts Figure 1. Sarcomere gene expression in human hypertrophic cardiomyopathy (HCM) samples. A Left, Allele-specific expression from wild-type and MYBPC3 splice-error premature termination codon (PTC)–containing transcripts quantified by quantitative reverse transcription polymerase chain reaction with custom-designed primers (Table V in the Data Supplement). Right, Allele-specific expression from wild-type and MYBPC3 PTC-containing transcripts (without splice errors) quantified by customdesigned single-base extension reactions. These mutations are either single-nucleotide substitutions that encode for a termination codon or an insertion that causes a frameshift. B, Quantification of wild-type and single-nucleotide substitution mutant sarcomere gene transcripts without PTCs by custom-designed single-base extension reactions. For the MYBPC3 exon splice site mutations, the mutant fraction is the proportion of detectable missense transcript. The remainder of the samples contain true missense mutations. In instances where 2 samples carried the same mutation, these are designated as (a) and (b) respectively in this figure and maintained throughout the article. C, Total MYBPC3 transcript abundance (left) and total MYH7 transcript abundance (right) in carriers of MYBPC3 (n=18) or MYH7 (n=6) mutations, compared with HCM samples in which no sarcomere mutation (n=7) was identified and control (nonHCM donor, n=7) hearts. *P=0.02 vs control hearts; †P=0.003 vs HCM with no sarcomere mutations. Open circles above the box plots indicate statistical outliers. D, Reverse transcription polymerase chain reaction analysis showing uniformly increased intensity of amplified MYBPC3 sequence in samples containing MYBPC3 mutations compared with the other 3 groups.
Helms et al Sarcomere Gene Expression in Human HCM 437 (Figure 1B). The Sequenom software could not design primers for TPM1 c.850A>G (Ile284Val), perhaps because it is in the terminal codon. However, cDNA sequencing showed polymorphism at that site (Figure IIB in the Data Supplement). Overall, PTC-containing transcripts in MYBPC3 are significantly less abundant than transcripts from non–PTC-containing mutant alleles from either MYBPC3 or other sarcomere genes (mean, 16±3% versus 47±1% of the total respectively; PC
ACGTTGGATGGATGC TCTGGTACACCTCC
ACGTTGGATGTATGC ACTGTGCACTAGCGG
CTGAGCTCATTGTGCAG
MYBPC3 c.2308G>A
ACGTTGGATGGCTG CCCGCCATCGTAGG
ACGTTGGATGAGGTC AACCTCACAGTCAAG
TCACAGTCAAGGTCATC
MYBPC3 c.3225_3227insT
ACGTTGGATGCTCCA GAGCCACATTAAGAC
ACGTTGGATGTGTTG ACAAGCCAAGTCCTC
GGATCTCCGGGTGACTG
MYBPC3 c.3288G>A
ACGTTGGATGATGTG GCTCTGGAGTGGAAG
ACGTTGGATGTTGTC GGCTTTCTGCACTGT
TCTGCACTGTGTACCCC
MYBPC3 c.3742_3759dup
ACGTTGGATGCCTCG CCCTGTAAGTTGGT
ACGTTGGATGAAAGC CCTGCCCCTTTGAC
GGGGCATCTATGTCTGC
MYBPC3 c.3697C>T
ACGTTGGATGATCTC CAGAGTCAACACTCC
ACGTTGGATGCCTGG TTCAAGAATGGCCTG
GCTGAACATGCGGAA
MYBPC3 c.2670G>A
ACGTTGGATGTAGAG GACGTCTCTGACACC
ACGTTGGATGTACTC CACGCTGTAGCCATC
ACGGTCTCCCTCAAGTG
MYBPC3 c.3233G>A
ACGTTGGATGTCCACTC CAGAGCCACATTA
ACGTTGGATGACAAG CCAAGTCCTCCCCAG
AGCCACATTAAGACCC
MYH7 c.2389G>A
ACGTTGGATGGCATC ATCACGCGTATCCAG
ACGTTGGATGCCAGC AGCTTTTTGTACTCC
TTTTTGTACTCCATTCTGG
MYH7 c.3286G>T
ACGTTGGATGGAGCT CCTTGAGCTTCTTCTG
ACGTTGGATGTGAGC TGAATGCTCTCAACG
CGAGGGCCTGTTCAT
MYH7 c.4816C>T
ACGTTGGATGTGGTG GACTCGCTGCAGAC
ACGTTGGATGTTCTT CTTCACCCTCAGGGC
CTGGACGCAGAGACA
MYH7 c.4817G>A
ACGTTGGATGTGGTG GACTCGCTGCAGAC
ACGTTGGATGTTCTT CTTCACCCTCAGGGC
GGGCCTCGTTGCGGCTG
MYH7 c.4130C>T
ACGTTGGATGGTCCT TTCCAAGGCCAACTC
ACGTTGGATGAGCTC CTCAGTCCGCTGAAT
GGAGGACCAAGTATGAGA
MYH7 c.968T>A
ACGTTGGATGATCAG TGGCCATGAGCTCCT
ACGTTGGATGTTCAT CTCCCAAGGAGAGAC
TCCTCAGCGTCATCA
MYH7 c.2123G>C
ACGTTGGATGGAAGTC CCCGTAGAGGATG
ACGTTGGATGAGCTGC GCTGCAATGGTGT
ATGCGGTTGGGGAAG
TNNT2 c.257A>C
ACGTTGGATGATTCA GGTCCTTCTCCATGC
ACGTTGGATGGTTCA TGCCCAACTTGGTGC
CGGTGGATGTCATCAAAG
MYL2 c.482A>G
ACGTTGGATGACAAG AACCTGGTGCACATC
ACGTTGGATGAGGGA CCACTCTGCAAAGAC
AGTCCTTCTCTTCTCCG
8
Supplementary Table 2. HCM gene mutation and corresponding protein designation
Gene
Mutation
Protein
Gene location
Mutation type
MYBPC3
c.2670 G>A
p.Trp890*
Exon 26
Truncating
MYBPC3
c.3226_3227insT
p.Asp1076Valfs*6
Exon 30
Truncating
MYBPC3
c.3233 G>A
p.Trp1078*
Exon 30
Truncating
MYBPC3
c.3294 G>A
p.Trp1098*
Exon 30
Truncating
MYBPC3
c.3697 C>T
p.Gln1233*
Exon 33
Truncating
MYBPC3
c.927-9 G>A
-
Intron 11
Truncating
MYBPC3
c.1624+4 A>T
-
Intron 17
Truncating
MYBPC3
c.1928-2 A>G
-
Intron 20
Truncating
MYBPC3
c.2905+1 G>A
-
Intron 27
Truncating
MYBPC3
c.3330+2 T>G
-
Intron 30
Truncating
MYBPC3
c.772 G>A
p.Glu258Lys
Exon 6
Truncating
MYBPC3
c.1624 G>C
p.Glu542Gln
Exon 17
Truncating and missense
MYBPC3
c.2308 G>A
p.Asp770Asn
Exon 23
Truncating
MYBPC3
c.3742_3759dup
p.Gly1248_Cys1253dup
Exon 33
In frame duplication
MYBPC3
c.1484 G>A
p.Arg495Gln
Exon 17
Missense
MYH7
c.968 T>A
p.Ile323Asn
Exon 11
Missense
MYH7
c.2123 G>C
p.Gly708Ala
Exon 19
Missense
MYH7
c.2389 G>A
p.Ala797Thr
Exon 21
Missense
MYH7
c.3286 G>T
p.Asp1096Tyr
Exon 26
Missense
MYH7
c.4130 C>T
p.Thr1377Met
Exon 30
Missense
MYH7
c.4816C>T
p.Arg1606Cys
Exon 34
Missense
Exon 34
Missense
1
MYH7
c.4817 G>A
p.Arg1606His
MYL2
c.482 A>G
p.His161Arg
Exon 7
Missense
TNNT2
c.257 A>C
p.Asp86Ala
Exon 8
Missense
Exon 9
Missense
TPM1
c.850 A>G
p.Ile284Val
2
Unless otherwise designated, all mutations studied were classified as pathogenic or likely pathogenic based on standard criteria7: published or found in affected individuals in testing laboratory with proven segregation in family members, premature truncation, species conservation, absent or very low population frequency, in silico prediction algorithms. 1 MYH7 Arg1606His is designated as a variant of unknown significance. Pathogenicity is supported by very low population frequency (1:8600) in NHLBI Exome Sequencing Project, species conservation, and in silico model predicting a probably damaging effect on the protein. 2 TPM1 Ile284Val is designated as a variant of unknown significance. Pathogenicity is supported by absence in the general population, species conservation, and in silico model predicting a probably damaging effect on the protein. The variant also segregated with 2 individuals in the family (proband and mother).
9
Supplementary Table 3. cDNA sequencing for intron and exon splice site MYBPC3 mutations.
Mutation
Mutation location
Mutant transcript sequence
Location of PTC
c.1624+4 A>T
Intron 17 (donor)
Skips exon 17
Junction of exon 16/18
c.2905+1 G>A
Intron 27 (donor)
Skips exon 27
Junction of exon 26/28
c.3330+2 T>G
Intron 30 (donor)
Skips exon 30
Exon 31
c.927-9 G>A
Intron 11 (acceptor)
Inclusion of intron 11
Intron 11
c.1928-2 A>G
Intron 20 (acceptor)
Inclusion of intron 20
Intron 20
c.772 G>A
Exon 6 (donor)
Skips exon 6
Exon 9
c.1624 G>C
Exon 17 (donor)
Skips exon 17
Junction of exon 16/18
c.2308 G>A
Exon 23 (donor)
Not detectable
10
Supplementary Table 4. Allele-specific protein expression using absolute quantification of abundance Gene
Mutation
Enzyme
Wild-type (Wt) AQUA peptide(s)
Wt peptide fmol (%)
Mutant AQUA peptide(s)
Mutant peptide fmol (%)
MYH7
Ala797Thr
Trypsin
GVLAR
107.91 (51.2) 310.52 (48.2)
GVLTR
102.71 (48.8) 333.02 (51.8)
MYH7
Arg1606His
Trypsin
VVDSLQTSLDAETR
77.41 (63.6) 1031.62 (52.1)
VVDSLQTSLDAETHSR
44.41 (36.4) 949.12 (47.9)
MYH7
Arg1606Cys
Trypsin
VVDSLQTSLDAETR
387.9 (63)
VVDSLQTSLDAETCSR*
228.6 (37)
MYH7
Asp1096Tyr
Trypsin
IEDEQALGSQLQK IEDEQALGSQLQKK Total
87.5 248.9 336.4 (53.1)
IEYEQALGSQLQK IEYEQALGSQLQKK Total
192.7 104.4 297.1 (46.9)
MYH7
Thr1377Met (LV septum)
Trypsin
YETDAIQR TKYETDAIQR TKYETDAIQRTEELEEAK Total
1.6 50.4 6.3 58.3 (51)
YEMDAIQR YEM(ox)DAIQR TKYEMDAIQR TKYEM(ox)DAIQR TKYEMDAIQRTEELEEAK TKYEM(ox)DAIQRTEELEEAK Total
1.0 0 47.1 8.6 0 0 56.7 (49)
MYH7
Thr1377Met (LV Lateral)
Trypsin
YETDAIQR TKYETDAIQR TKYETDAIQRTEELEEAK Total
3.3 68.5 7.6 79.4 (46)
YEMDAIQR YEM(ox)DAIQR TKYEMDAIQR TKYEM(ox)DAIQR TKYEMDAIQRTEELEEAK TKYEM(ox)DAIQRTEELEEAK Total
2.5 0.5 62.7 20.3 7.6 0 93.6 (54)
MYH7
Thr1377Met (RV)
Trypsin
YETDAIQR TKYETDAIQR TKYETDAIQRTEELEEAK Total
2.8 108.5 17.4 128.7 (50)
YEMDAIQR YEM(ox)DAIQR TKYEMDAIQR TKYEM(ox)DAIQR TKYEMDAIQRTEELEEAK TKYEM(ox)DAIQRTEELEEAK Total
1.4 0 86.7 20 14.4 8.6 131.1 (50)
MYH7
Ile323Asn
Asp-N
DYAFISQGETTVASI DYAFISQGETTVASID Total
100.6 37.9 138.5 (31%)
DYAFISQGETTVASN DYAFISQGETTVASND Total
301.73 0 301.73 (69%)
11
MYH7
Gly708Ala (LV septum)
Trypsin
KGFPNR
250.1 (91)
KAFPNR
24.1 (9)
MYH7
Gly708Ala (LV lateral)
Trypsin
KGFPNR
677.1 (90)
KAFPNR
74.8 (10)
MYH7
Gly708Ala (RV)
Trypsin
KGFPNR
277.5 (89)
KAFPNR
33.3 (11)
TNNT2
Asp86Ala
Trypsin
TPM1
Ile284Val
Trypsin
VDFDDIHR
4.61 (19.2) 9.722 (12.3)
AISEELDHALNDMTSI AISEELDHALNDM(ox)TSI Totals
VHIITHGEEK KNLVHIITHGEEK Total
VAFDDIHR
19.41 (80.8) 69.22 (87.7)
1.41 536.22 57 603.8 58.41 (59) 11402 (52.1)
AISEELDHALNDMTSV
133.7 66.1 199.8 (69.2)
VHIITRGEEK KNLVHIITRGEEK Total
67 21.8 88.8 (30.8)
AISEELDHALNDM(ox)TSV
4.21 449.92 36.31 596.32 40.51 (41) 1046.22 (47.9)
MYL2
His161Arg
Chymotrypsin
MYBPC3
Arg495Gln (a)
Lys-C
DGVELTREETFK
52.91 (25.6) 23.12 (36.6)
DGVELTQEETFK
153.41 (74.4) 40.02 (63.4)
MYBPC3
Arg495Gln (b)
Lys-C
DGVELTREETFK
36.91 (36.3) 35.32 (39.7)
DGVELTQEETFK
64.81 (63.7) 53.72 (60.3)
MYBPC3
Glu542Gln (a)
Chymotrypsin
IVQEKKLEVY
2.10 (95.2)
IVQQKKLEVY
0.11 (4.8)
MYBPC3
Glu542Gln (b)
Chymotrypsin
IVQEKKLEVY
16.3 (98.4)
IVQQKKLEVY
0.26 (1.6)
Values with subscripts of 1 and 2 indicate samples in which 2 separate LC/MS/MS runs were performed. In samples where more than one AQUA peptide was used (missed cleavage, post-translational modifications), the total provided is the sum of the absolute amounts of each peptide (wild-type or mutant) and % of total (i.e. wild-type/mutant + wild-type peptides). Variation in absolute peptide quantities is primarily due to differences in the amount of starting material, but relative amounts of wild-type and mutant peptides within the same sample were similar between replicates (CV 4-14%). Amino acids in red indicate variant. * Cysteine is carbamidomethylated to correspond to the modification of endogenous peptide and Ox = oxidized modification of Met.
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Supplementary Table 5. Taqman hydrolysis probes used to examine allele-specific expression in samples containing MYBPC3 splice site mutations
Mutation
Taqman Hydrolysis Probe
Exon Border
Transcript Recognized
c.1624+4 A>T
Hs01076199_g1
16-17
Wild Type
Hs01076202_m1
20-21
Both
Hs01076206_m1
26-27
Wild Type
Hs01076202_m1
20-21
Both
Hs01076209_g1
30-31
Wild Type
Hs01076211_g1
32-33
Both
Hs01079709_g1
11-12
Wild Type
Hs00165232_m1
12-13
Both
Hs01076202_m1
20-21
Wild Type
Hs01076206_m1
26-27
Both
Hs01076217_m1
5-6
Wild Type
Hs01076212_m1
2-3
Both
Hs01076199_g1
16-17
Wild Type
Hs01076202_m1
20-21
Both
Hs01076203_m1
22-23
Wild Type
Hs01076202_m1
20-21
Both
c.2905+1 A>G
c.3330+2 T>G
c.927-9 A>G
c.1928-2 A>G
c.772 G>A
c.1624 G>C
c.2308 G>A
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Supplementary Figure 1. A, B and C. PCR amplification of wild-type and splice site mutant cDNA. Corresponding sequencing results for each band from each sample are found in Supplementary Table 2. * For c.1928-2 A>G, only a single band corresponding to wild-type sequence was identified using primers complementary to adjacent exon sequence (exons 19 and 21). Therefore, an alternative forward primer was designed to be complementary to intron 20 and uniquely amplified cDNA from the mutant allele that included intron 20. Primer sequences for each sample are as follows: c.1624 G>C, 1624+4 A>T: GCC CCC TGT GCT CAT CA (forward); CAC CTT TAT GCG GCT GTC G (reverse) c.2905+a G>A: ATG CGG CTG AAC TTC GAC CTG ATT (forward); CTG TGG GGC TGT TGC GGA TGC TC (reverse) c.3330+2 T>G: GGC CTC AGG TGA CCT GG (forward); AGA CGG GCT CCT TGG TGG TG (reverse) c.772 G>A: CTC AGC AGC TCT CAA TGG TCC TAC (forward); CTG ATC CGC CGA CCA CCT C (reverse) c.1928-2 A>G: CGA CGT CAC ACC TGC CG (forward, exon 19); ACT GAG GGC AGA TTC CTG ATT (forward, intron 20); CTG CGT GAT AGC CTT CTG C (reverse) c.927-9 G>A: GGC TGG AGG TGG TCG G (forward); GCC TCA TGC CCT TGA GCC (reverse) c.2308 G>A: GCC CAG GCC GCA TAC CAG AC (forward); AGG TCG AAG TTC AGC CGC ATC C (reverse) D. PCR amplification of MYH7 in the sample containing the missense mutation c.2123 G>C in MYH7. Only a single band was amplified at the expected size (857 bp) and sequence-confirmed to contain only wild-type and missense sequence. Primer sequences for this samples were CTGAAGCCCACTTCTCCCTG (forward) and GCGTGTGAACTCCTCCTTCA (reverse). 14
Supplementary Figure 2. Sequence chromatograms of cDNA from human HCM samples with sarcomere gene mutations. Heterozygous expression of mutant and wild-type sequences are shown for A. MYBPC3 3742_3759dup (Gly1248_Cys1253dup), and B. TPM1 c.850A>G (Ile284Val).
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Supplementary Figure 3. A. Sarcomere protein expression for MYH7, MYL2, TNNT2, TNNI3 and ACTC1 analyzed by western blot and normalized to GAPDH, expressed as a % of a single control heart run on each gel. There were no significant differences among the groups. B. MYBPC3 protein expression in control (n=6) and non-HCM failing hearts (n=14) of non-ischemic and ischemic etiologies. There was no significant difference in full-length MYBPC3 protein between the 2 groups. Degradation products were not visualized in either group.
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