Research ArticleGenetic Medicine

Integrated allelic, transcriptional, and phenomic dissection of the cardiac effects of titin truncations in health and disease

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Science Translational Medicine  14 Jan 2015:
Vol. 7, Issue 270, pp. 270ra6
DOI: 10.1126/scitranslmed.3010134
  • Fig. 1. Distribution of TTNtv in healthy individuals and DCM patients, and TTN exon usage in the heart.

    A schematic of the TTN meta-transcript, with sarcomere regions demarcated. The meta-transcript (LRG_391_t1/ENST00000589042) is a manually curated, inferred complete transcript, incorporating all exons of all known TTN isoforms (including fetal and noncardiac isoforms) with the exception of the large alternative terminal exon 48 (dark green) that is unique to the novex-3 transcript (LRG_391_t2/ENST00000360870). Exon usage for the two principal adult cardiac isoforms, N2BA and N2B (ENST00000591111 and ENST00000460472), is shown, although exon usage in vivo is variable (see below). Novex-1 and novex-2 are rare cardiac isoforms that differ from N2B by the inclusion of a single unique exon each (red and blue, respectively, within the N2B track). Exon usage in human LV is depicted as the proportion spliced-in (PSI) (range, 0 to 1; gray bars): the proportion of transcripts that include a given exon. TTNtv are located more distally in cases compared with controls, with A-band and distal I-band enrichment in end-stage (n = 155) and unselected DCM patients (n = 374), and corresponding depletion in the population (n = 3603) and healthy volunteer (n = 308) cohorts.

  • Fig. 2. Factors that discriminate TTNtv in health and disease.

    (A) Usage of TTN exons containing TTNtv in all cohorts. Exon usage is represented as PSI, which is an estimate of the proportion of transcripts that incorporate each exon. Each plotted data point represents the estimated PSI of an exon identified to have a TTNtv, grouped by cohort. There was a strong relationship between the PSI of exons containing TTNtv and disease status (P = 4.9 × 10−3, Kruskal-Wallis), with TTNtv in DCM cases found in more highly used exons (P = 4.7 × 10−4, Mann-Whitney). A similar difference was observed between the replication cohorts (P = 7.5 × 10−4). (B) Relationships between TTNtv location, PSI, and disease status. The positions of TTNtv (amino acid coordinates, reference transcript LRG_391_t1) are shown for constitutively expressed exons only (PSI = 1).

  • Fig. 3. TTNtv and survival in DCM.

    Outcomes in unselected DCM patients with (red) and without (blue) TTNtv. (Left) Age censored at adverse event [death, cardiac transplant, or left-ventricular assist device (LVAD)] or at age 70 years. (Right) Adverse events after enrollment, to control for ascertainment (interval censored from time of enrollment to age 70 years or adverse event). Event-free survival is reduced in TTNtv-positive DCM (P = 0.015) as a result of faster disease progression. A trend to younger presentation (Table 2) and worse outcomes after enrollment (P = 0.05) combine to give reduced survival overall.

  • Fig. 4. Allelic dissection of the impact of TTNtv position on cardiac morphology and function.

    The relationships between TTNtv location and cardiac morphology and function assessed by CMR imaging in an allelic series of DCM cases. Genotype-phenotype relationships are shown for 43 TTNtv in unselected DCM patients. The TTNtv location (x axis) is plotted from the amino (N) to the carboxyl (C) end of the protein. Distal (C-terminal) TTNtv were associated with worse cardiac contractile performance, specifically diminished indexed stroke volume (SVi) and EF of both LV and RV as compared to proximal truncations. A regression line is shown for each variable (tables S14 and S15). EDVi, indexed end-diastolic stroke volume (ml/m2); ESVi, indexed end-systolic volume (ml/m2); SVi, indexed stroke volume (ml/m2); EF, ejection fraction (%).

  • Fig. 5. TTN mRNA and protein expression in LV tissues from DCM patients with and without TTNtv.

    (A) TTN mRNA in TTNtv-positive (n = 18) and TTNtv-negative (n = 66) patients (quantile-normalized read counts). (B) Allelic balance of TTNtv compared to nontruncating TTN SNPs, a surrogate for the proportion of transcripts with variant alleles (TTNtv or SNPs) among DCM patients with and without TTNtvs. The comparable allelic expression of TTNtv and SNPs does not support substantial nonsense-mediated decay (see also fig. S9). Bars indicate median and quartiles. (C) Protein electrophoresis from a healthy LV (lane 1) and LV from DCM patients (lanes 2 to 12: +, TTNtv-positive; −, TTNtv-negative). Sample IDs are shown for subjects with TTNtv: variant details are shown in table S4. Truncated protein was not seen in TTNtv-positive samples. Arrowheads, approximate expected sizes of the truncated N2B and N2BA isoforms; T2, a TTN degradation product; MDa, megadaltons. Semiquantitative analysis of TTN protein relative to myosin (MHC, myosin heavy chain) showed no reduction of TTN in TTNtv-positive samples.

  • Table 1. Number of TTNtv in DCM patients and controls.

    Numbers of subjects with a TTNtv are shown for each group. TTNtv are classified by type, the affected transcript, and expression level of the variant-encoding exon. Comparisons between groups were assessed by Fisher’s exact test. Cohort ethnicity: Caucasian: healthy volunteer, 75%; FHS, 100%; JHS, 0%; unselected DCM, 88%; end-stage, DCM 85%; African American: healthy volunteer, 2%; FHS, 0%; JHS, 100%; unselected DCM, 4%; end-stage DCM, 6%.

    Discovery cohortsReplication cohorts
    Healthy
    volunteers
    (n = 308)
    FHS
    (n = 1623)
    JHS
    (n = 1980)
    Unselected
    DCM
    (n = 371)*
    End-stage
    DCM
    (n = 155)
    P value DCM
    versus controls
    WHI
    (n = 667)
    Familial DCM
    (n = 163)
    P value DCM
    versus
    controls
    Unselected
    DCM
    All DCM
    Transcript affected by truncation
    N2BA and N2B4112042341.7 × 10−255.5 × 10−462313.8 × 10−21
    N2BA only428700.00140.0083201
    Neither N2BA or
    N2B (novex-3
    terminal exon
    only)
    1 (0)8 (5)4 (1)1 (1)1 (1)10.75 (4)0 (0)1
      Totals921325035931
    Sarcomere domain
    A-band (18,235
    amino acids)
    271232290.0110.000141250.015
    Non–A-band
    (17,756
    amino acids)
    791918846
      Totals916314934531
    Usage§ of exon containing truncation
    Low (PSI <0.15) 144200.250.38101
    Intermediate
    (PSI 0.15 to 0.9)
    417500.0120.042201
    High (PSI >0.9) 4112042341.7 × 10−255.5 × 10−462313.8 × 10−21
      Totals916314934531
    Variant type
    Frameshift
    variant
    23722154.9 × 10−164.0 × 10−251151.9 × 10−10
    Stop gained37615127.3 × 10−092.3 × 10−152162.8 × 10−10
    Canonical splice
    sites
    237952.9 × 10−052.3 × 10−07101
    Splice
    variant
    predictions||
    2311450.0890.0015101
      Totals9163149341.4 × 10−252.8 × 10−435311.6 × 10−18

    *Three of 374 subjects were excluded from these analyses due to relatedness to other subjects.

    †Variants that only affect the alternative terminal exon of novex-3 are excluded elsewhere.

    ‡Total number of individuals with TTNtv; four individuals with DCM (one unselected, three end-stage) carry a second TTNtv, which is a splice variant prediction in three cases.

    §Exon usage levels are displayed categorically on the basis of PSI.

    ¶Canonical splice sites refer to the two intronic base pairs at the 5′ and 3′ splice junctions.

    ||Variants close to canonical splice sites that are predicted in silico to alter splicing.

    • Table 2. Clinical characteristics of DCM patients with and without TTNtv.

      Unselected DCM cohort. Values are means ± SD. Measurements are indexed to body surface area where indicated. LV, left ventricle; RV, right ventricle; EDVi/ESVi, indexed end-diastolic/systolic volume; SVi, indexed stroke volume; EF, ejection fraction; LVMi, indexed LV mass; WTi, indexed wall thickness; VT, ventricular tachycardia; NYHA, New York Heart Association functional class. Groups were compared using Wilcoxon-Mann-Whitney test for continuous variables, and Fisher’s exact test for categorical. P values not corrected for multiple testing, as variables were not independent.

      CMR and clinical dataTTNtv-negative (n = 277)TTNtv-positive (n = 42)P
      LVEDVi136 ± 38137 ± 34.10.677
      ESVi87.5 ± 39.193.7 ± 36.40.205
      SVi48.2 ± 12.943.4 ± 14.10.041
      EF37.5 ± 12.233.3 ± 13.30.047
      RVEDVi89.4 ± 24.789.6 ± 26.10.972
      ESVi45.2 ± 22.350.4 ± 25.90.234
      SVi44.4 ± 12.639.2 ± 15.80.031
      EF51.6 ± 14.145.2 ± 160.036
      LVMi95.4 ± 27.687.1 ± 18.30.106
      Lateral WTi3.13 ± 0.732.77 ± 0.7130.003
      Midwall fibrosis94/270 (35%)13/41 (32%)0.869
      Age at diagnosis (years)53.4 ± 13.449.3 ± 13.70.115
      NYHA status 1/2/3/4116/104/36/119/16/4/10.692
      Sustained VT20/97 (21%)9/14 (64%)0.001
      Conduction disease82/227 (36%)8/36 (22%)0.130
      Family history of DCM24/218 (11%)9/37 (24%)0.034

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/7/270/270ra6/DC1

      Phenotype ascertainment methods for study cohorts

      Fig. S1. Schematic representation of the unselected DCM cohort recruitment pathway and analyses.

      Fig. S2. TTN sequencing coverage for each cohort.

      Fig. S3. Sites susceptible to truncating events are non-uniformly distributed within the TTN gene but do not influence clustering effects in the A-band.

      Fig. S4. Alternative splicing of TTN in the human heart.

      Fig. S5. Time to events in FHS individuals grouped by TTNtv presence.

      Fig. S6. Truncated transcript length is correlated with indices of cardiac impairment severity in DCM.

      Fig. S7. FHS exam 7 CMR.

      Fig. S8. FHS and JHS additional CMR and echocardiography exams.

      Fig. S9. mRNA transcripts encoding truncated TTN proteins are expressed in human LV.

      Table S1. TTNtv identified in UK prospective DCM cohort.

      Table S2. TTNtv identified in the FHS offspring cohort.

      Table S3. TTNtv identified in the JHS cohort.

      Table S4. TTNtv identified in end-stage DCM.

      Table S5. TTNtv identified in healthy volunteers.

      Table S6. Titin reference transcript and protein identifiers.

      Table S7. Overview of TTN transcripts and exon usage.

      Table S8. TTNtv in publicly available control populations.

      Table S9. Burden, type, and distribution of TTNtv in publicly available control populations.

      Table S10. FHS exam 7 CMR phenotype grouped by TTNtv presence.

      Table S11. Prevalence of DCM in FHS and JHS participants, grouped by TTNtv presence.

      Table S12. Time to event empirical Cox proportional hazard models for the FHS cohort.

      Table S13. TTNtv identified in replication cohorts.

      Table S14. Linear modeling of the relationship between TTN genotype and phenotype for 14 continuous variables in the unselected DCM cohort.

      Table S15. Full linear model describes impact of multivariate TTN genotype on phenotype for 14 continuous variables in the unselected DCM cohort.

      Table S16. Linear models for FHS exam 7 CMR.

      Table S17. Linear models for additional FHS and JHS exams.

      Table S18. Allele-specific expression of exons containing TTNtv using RNA sequencing data.

      References (6567)

    • Supplementary Material for:

      Integrated allelic, transcriptional, and phenomic dissection of the cardiac effects of titin truncations in health and disease

      Angharad M. Roberts, James S. Ware, Daniel S. Herman, Sebastian Schafer, John Baksi, Alexander G. Bick, Rachel J. Buchan, Roddy Walsh, Shibu John, Samuel Wilkinson, Francesco Mazzarotto, Leanne E. Felkin, Sungsam Gong, Jacqueline A. L. MacArthur, Fiona Cunningham, Jason Flannick, Stacey B. Gabriel, David M. Altshuler, Peter S. Macdonald, Matthias Heinig, Anne M. Keogh, Christopher S. Hayward, Nicholas R. Banner, Dudley J. Pennell, Declan P. O'Regan, Tan Ru San, Antonio de Marvao, Timothy J. W. Dawes, Ankur Gulati, Emma J. Birks, Magdi H. Yacoub, Michael Radke, Michael Gotthardt, James G. Wilson, Christopher J. O'Donnell, Sanjay K. Prasad, Paul J. R. Barton, Diane Fatkin, Norbert Hubner, Jonathan G. Seidman, Christine E. Seidman,* Stuart A. Cook*

      *Corresponding author. E-mail: stuart.cook{at}nhcs.com.sg (S.A.C.); cseidman{at}genetics.med.harvard.edu (C.E.S.)

      Published 14 January 2015, Sci. Transl. Med. 7, 270ra6 (2015)
      DOI: 10.1126/scitranslmed.3010134

      This PDF file includes:

      • Phenotype ascertainment methods for study cohorts
      • Fig. S1. Schematic representation of the unselected DCM cohort recruitment pathway and analyses.
      • Fig. S2. TTN sequencing coverage for each cohort.
      • Fig. S3. Sites susceptible to truncating events are non-uniformly distributed within the TTN gene but do not influence clustering effects in the A-band.
      • Fig. S4. Alternative splicing of TTN in the human heart.
      • Fig. S5. Time to events in FHS individuals grouped by TTNtv presence.
      • Fig. S6. Truncated transcript length is correlated with indices of cardiac impairment severity in DCM.
      • Fig. S7. FHS exam 7 CMR.
      • Fig. S8. FHS and JHS additional CMR and echocardiography exams.
      • Fig. S9. mRNA transcripts encoding truncated TTN proteins are expressed in human LV.
      • Table S1. TTNtv identified in UK prospective DCM cohort.
      • Table S2. TTNtv identified in the FHS offspring cohort.
      • Table S3. TTNtv identified in the JHS cohort.
      • Table S4. TTNtv identified in end-stage DCM.
      • Table S5. TTNtv identified in healthy volunteers.
      • Table S6. Titin reference transcript and protein identifiers.
      • Table S7. Overview of TTN transcripts and exon usage.
      • Table S8. TTNtv in publicly available control populations.
      • Table S9. Burden, type, and distribution of TTNtv in publicly available control populations.
      • Table S10. FHS exam 7 CMR phenotype grouped by TTNtv presence.
      • Table S11. Prevalence of DCM in FHS and JHS participants, grouped by TTNtv presence.
      • Table S12. Time to event empirical Cox proportional hazard models for the FHS cohort.
      • Table S13. TTNtv identified in replication cohorts.
      • Table S14. Linear modeling of the relationship between TTN genotype and phenotype for 14 continuous variables in the unselected DCM cohort.
      • Table S15. Full linear model describes impact of multivariate TTN genotype on phenotype for 14 continuous variables in the unselected DCM cohort.
      • Table S16. Linear models for FHS exam 7 CMR.
      • Table S17. Linear models for additional FHS and JHS exams.
      • Table S18. Allele-specific expression of exons containing TTNtv using RNA sequencing data.
      • References (6567)

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