RYR1 and TTN: The challenge of giant genes implicated in Centronuclear Myopathy (CNM)

Heinz Jungbluth MD PhD & Mathias Gautel MD PhD 1Department of Paediatric Neurology, Neuromuscular Service, Evelina Children’s Hospital, London;

2Randall Division for Cell and Molecular Biophysics, Muscle Signalling Section, and3Clinical Neuroscience Division, IoPPN, King’s College London

ZNM Myotubular Trust European Family Conference, London, July 14th , 2018

Repairing an engine…

Striated muscles are formed

from long chains of sarcomeres.

Sarcomeres are giant, regular protein complexes

Sarcomeres: the most precisely assembled machines in biology

skeletal cardiac

Braun & Gautel, Nat Rev Mol Cell Biol 2011

Sarcomeres: smallest contractile units of skeletal and heart muscle

What is Big?

Titin molecules are over 1 µm long and slender

Single titindimersSingle myosin

Single titinmonomers

Anti-Titin Z-disk

Anti-Titin M-band

1 µm Klaus Weber Dieter Fürst

TTN intron-exon structure and domains

Bang M.-L. et al., Circ Res 2001

364 exons, 169 Ig domains, 132 fibronectin domains, 1 kinase, “other stuff”

TTN splice variants Big, and very big.

Chauveau et al., Human Mutation 2014

Titin/connectin – ruler of the sarcomeredomain layout of cardiac titin isoforms & known interactions

• Telethonin / T-cap (MLP, Myostatin, Calsarcins)

• sAnkyrin (Telethonin, RyR)

• α-Actinin(MLP. Actin)

• Obscurin (sAnk, Novex3)

• Molecular Ruler

I-bandZ-disk A-band M-band

• αB-crystallin • DRAL/FHL-2

(MM-CK, AK, PFK, β-Catenin, AR, ...)

• MARP (Ankrd2, CARP, DARP),• Myopalladin• Calpain-1, calpain-3• Smyd2/HSP90• Protein kinases A, G

• I-band= Spring, Molecular Ruler

• Myosin• MyBP-C• MyBP-H

• Molecular Ruler

• Myomesin,M-Protein

• DRAL/FHL-2 (MM-CK, AK, PFK, β-Catenin, AR, ...)

• p94/calpain-3• Nbr1 (p62, MuRF)

• Obscurin• MURFs• Myospryn

I15

NH2

I1 N2-B I20

I28 I29

I58-I61

I68 - I79

I55

I58

I59

N2-A PEVK

COOH

(cardiac N2B)

(cardiac N2A + N2B)

M1is2 M10

TK

Ig-like

FN3-like

Insertion Sequence

Kinase

MW > 3000 kDa Gene > 300 kb

117

Z-repeatsZ1

21

364 exons 169 Ig domains 132 fibronectin domains

Titin: a highly modular giant protein

multifunctional

modularBigdynamicelastic

Too big to fail…?

Titin mutations: Frequent cause of myopathies

Stop-gained and missense mutations

• LGMD2J, TMD (rec/dom, (Ig169/M10)

• Other distal myopathies

• Salih Myopathy (rec., C-term truncating)

• Hereditary Myopathy with Early Respiratory Failure, HMERF (dom, Fn119)

• Autosomal Recessive Multi-minicore Disease with Heart Disease (AR MmD-HD) (rec. missense/truncating, compound-heterozygous)

• Centronuclear Myopathy, CNM with/without cardiomyopathy (rec. truncating)

• Left ventricular compaction, LVNC (dom?, Ig2/Z2)

• Dilated cardiomyopathy, DCM (rec/dom? Truncating, mostly A-band) Peripartum cardiomyopathy

• Hypertrophic cardiomyopathy, HCM (rec/dom? Missense, mostly A-band)

Disease entities linked to titin

Titin truncating mutations Frequent in dilated cardiomyopathies

Titin variants in 1000 genomes (all)

Status May 2016

1000 genomes….

A glimpse on human evolution

goodbad…ugly…

How can we discriminate them?

0

2

5

7

9

0 1993 3985 5978 7970 9963 11956 13948 15941 17933 19926 21919 23911 25904

Z I I A M

N2B

PEVK

Residues (in 1000)

Incidence

Kinase

HCM EOM HMERF

Titin missense variants cause a wide variety of diseases

TMD/LGMD2J

Chauveau et al., HMG 2014

A recurrent, recessive TK mutation leading to myopathy: W34072R

Compound heterozygous, truncation after Z-disk (maternal), missense in kinase (paternal). No FH Cardiomyopathy, transplantation at age 3, skeletal myopathy.

New case (A. Ferreiro, Paris): Compound heterozygous, truncation in skeletal I-band (paternal), W34072R missense in kinase (maternal). No FH No cardiomyopathy, only skeletal myopathy.

W34072R is a recurrent, recessive pathogenic mutation in TK, a constitutively expressed domain.

With Ana Ferreiro, Carsten Bönnemann

A recessive TK mutation leading to myopathy: W34072R

Soluble, monomeric, no aggregation

Chauveau et al., HMG 2014

Computational models of TTN Fn3 domains are too inaccurate for mutation impact analysis

(iTasser)

Experimental structures and homology-based structural models required for accurate impact prediction

SG Tm: 54°C

WT Tm: 64°C

A63 WT (1.65Å)A63 SG (1.43Å)

A63 WT A63 SG

Ser Gly

Temperature (°C)

Rel

ativ

e Fl

uore

scen

ce

D zone C zoneI / A

1 2 3 4 5 6 1 2 3 4 5 6 7 8 9 10 11A63

serine side chain forms H-bond with valine.

S14141G variant destabilises A63 domain

Common Titin missense variants are not destabilising

D zone C zoneI / A

1 2 3 4 5 6 1 2 3 4 5 6 7 8 9 10 11A81 A110

RC:62°C

WT:62°C

WT:62°C

IV:63°C

A81 A110

Temperature (°C)

Rel

ativ

e Fl

uore

scen

ce

Temperature (°C)

TITINdb, a webtool to assess titinopathy-linked missense variants

Laddach, A., Gautel, M., and Fraternali, F., TITINdb-a computational tool to assess titin's role as a disease gene. Bioinformatics, 2017. 33(21): p. 3482-3485.

-3

-2.25

-1.5

-0.75

0

-40 -30 -20 -10 0 10

Titinopathy-linked missense variants destabilise domains

mCSM

ΔTm

In early-onset myopathies and TMD, TTN mutations strongly destabilise the domain by more than 15 degrees.

> Established semi-automated pipeline for mutation assessment

TMD, Bel Mutation, impact questioned

Neutral, R24947C

Neutral, I27775V

unfolded

Myopathies caused by misfolded toxic proteins

Desminopathies desmin Crystallinopathies αB-crystallin Myofibrillar myopathies BAG3, filamin-C centronuclear myopathies titin Hypertrophic cardiomyopathy many; titin? Dilated cardiomyopathy many; titin?

Neurodegeneration by misfolded toxic proteins

Tipping, K.W., P. van Oosten-Hawle, E.W. Hewitt, and S.E. Radford Trends Biochem Sci, 2015. 40: 719-27.

Conclusions

• TTN is the major scaffold of the sarcomere and a major gene for hereditary myopathies

• Recessive missense variants can become dominant in compound heterozygous settings when expressed with recessive truncating variants

• Discriminate bad from neutral and good?

• Truncating as well as destabilising missense variants frequent in “normal” population

• Accurate assessment of missense variants requires a rational assessment strategy including structural and functional data.

• Destabilising mutations can be recessive or dominant

• TITINdb designed to help with initial assessment of TTN missense variants

King’s College London Alexander AlexandrovichBirgit BrandmeierAtsushi FukuzawaAndrea GhisleniMark HoltKatharina JennichesAy Lin KhoEva MasierioRoksana NikoopourJing QiMartin ReesLuke SmithJessica Stuart

Anna LaddachFranca Fraternali

Mark Pfuhl

Stefano PernigoRoberto Steiner

Sergi Garcia-Manyes

King’s BHF Centre for Research Excellence

Göttingen UniversityJing QiKaomei Guan

Max-Planck-Institute DortmundStefan Raunser

Max-Perutz-Labs ViennaKristina Djinovic-Carugo

EMBL HamburgMatthias Wilmanns

Oxford UniversityKatja GehmlichHugh Watkins

University CologneMarcus Krüger

UKE HamburgLucie Carrier

UCL/Bart’s Luis LopesPetros SyrrisPerry Elliot

UCL ICH/GOSH Francesco Muntoni

Evelina London Children’s Hospital/ Tom Cullup, Heinz JungbluthGSTT Gerald Carr-White

West of Scotland Regional Genetics Glasgow Cheryl Longman

Université Paris Diderot-CNRS Claire ChauveauVirginie CarmignacAna Ferreiro

NIH Bethesda Carsten Bönnemann

University Helsinki Peter HackmanAnna ViholaBjarne Udd

Cologne University Hospital Sebahattin Cirhak

Göttingen University Hospital Elke HobbiebrunkenGabriele DekomienEkkehard Wilichowski

Radboud University Medical Centre Nicol VoermansNijmegen

Hôpital des Enfants Joël FlussGenève

Titin mutations in myopathies: open questions

• Why do truncating mutations mostly appear to be silent?

• Do missense mutations lead to phenotypic penetrance of truncating mutations, or vice versa?

• Do recessive missense mutations cause similar phenotypes in compound heterozygosity or homozygosity?

• What is the cellular fate of truncated titin? • What is the cellular fate of missense titin?

• Is perturbed protein homeostasis a common denominator?

• How does perturbed proteostasis impact on myocyte function?

Sarcomeric M-band links to protein quality control Disease implications

Fukuzawa et al., JCS 2008

Titin

Obsl1

Obscurin

Titin

Myomesin

Nbr1

Cullin-7

MURFs myospryn

SQSTM1

COP9

Calpain-3

TK mutations in HCM, specific myopathies

LGMD2A

LGMD2J, Salih myopathy

• Titin, a molecular ruler – blueprint to determine the length of myosin filaments,

the length of the I-band, and the thickness of the Z-disk

• Titin, a molecular spring – maintains myosin filaments centrally in the sarcomere – Ensures balanced forces between both halves of

sarcomere

• Titin, a “signal transducer” – Titin organises numerous cellular communication

pathways

Titin: a multitasking giant The biggest protein in the human body

myomesin

α-actinin

myosin filaments

M ZZ I IAA

NH2 COOHTitin

actin filaments

NH2

titin

COOH

The sarcomere and regulators of sarcomeric turnover

} }}MyBP-C

ZASP/cypher

calsarcin/myozenin/FATZNbr1/SQSTM1

Obscurin/Obsl1 *

myotilin

MURF1/2/3

calpain-3

MLP

BAG3

γ-filaminCapZ

Fbxl22

tropomodulin myopalladin

MARP

FHL2

telethonin

NH2Nebulin(nebulette)

COOH