Genomics in Arrhythmogenic Cardiomyopathy: exploring the complexity

Head Off ice: Un ivers i ty of Padua

Depar tmen t of Card iac , Thorac ic and Vascu lar Sc iences and Pub l ic Hea l th Ph .D . COURSE: Trans la t iona l Spec ia l is t ic Med ic ine “G . B . Morgagn i”

CURRICULUM: Card io thorac ic and Vascu lar Sc iences SERIES: XXXI

Genom ics in Arrhythmogen ic Card iomyopathy:

exp lor ing the comp lex ity

Coord inator: Prof . Anna l isa Ange l in i Superv isor: Prof . Ka l l iop i P i l ichou

Ph .D . student: Rudy Ce legh in

i

Index

Abs trac t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

In troduc t ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Ep idem io logy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Pa tho log ica l f ind ings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

C l in ica l f ind ings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

D iagnos t ic cr i ter ia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

L im i ta t ions of curren t d iagnos t ic cr i ter ia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

C l in ica l tes ts enab l ing AC d iagnos is : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Lef t Dom inan t AC d iagnos is . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Mo lecu lar gene t ics of AC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

1 .10 .1 Desmosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

1 .10 .2 Non desmosoma l genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Pa thogenes is . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Conven t iona l Screen ing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Nex t Genera t ion Sequenc ing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Nex t Genera t ion Sequenc ing in Card iomyopa th ies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Gene t ic Tes t ing in AC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

L im i ta t ions of gene t ic tes t ing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

A im of the s tudy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Ma ter ia l and Me thods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

i i

Cohor t and C l in ica l Exam ina t ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Nuc le ic ac id and pro te ins ex trac t ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

DNA ex trac t ion from b lood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

DNA ex trac t ion from frozen t issue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

DNA quan t if ica t ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

3 .5 .1 Spec tropho tome tr ic me thod . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

3 .5 .2 Qub i t F luorome ter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

3 .5 .3 0 .8% Agarose ge l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Po lymerase cha in reac t ion (PCR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

3 .6 .1 S tandard PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

3 .6 .2 GC r ich PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

3 .6 .3 PCR qua l i ty on 2% agarose ge l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Pur if ica t ion of PCR produc ts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

D irec t sequenc ing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Nex t Genera t ion Sequenc ing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

3 .9 .1 L ibrary prepara t ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

3 .9 .2 L ibrary Qua l i ty Check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

3 .9 .3 Sequenc ing by Syn thes is . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

3 .9 .4 Da ta Ana lys is . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Var ian ts C lass if ica t ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Mu l t ip lex l iga t ion-dependen t probe amp l if ica t ion (MLPA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Da ta ana lys is too l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Resu l ts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Cohor t Ana lys is . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

i i i

Var ian ts ana lys is . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Var ian ts d is tr ibu t ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

Var ian ts c lass if ica t ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

Recurren t var ian ts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

Copy number var ian ts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

CDH2 screen ing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

FLNC screen ing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

Comprehens ive y ie ld of gene t ic screen ing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Geno type-Pheno type corre la t ion ana lys is . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

TTN var ian ts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

Rare var ian ts in d isease_unre la ted genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

WES screen ing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

4 .13 .1 Ga lec t in 3 (LGALS3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

D iscuss ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

B ib l iog raphy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

Append ix A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

iv

v

Abs tract

Background

Arrhy thmogen ic card iomyopa thy (AC) is a rare hear t musc le d isease charac ter ized by f ibrofa t ty myocard ia l rep lacemen t , prom inen t impa irmen t of ven tr icu lar sys to l ic func t ion and arrhy thm ias . AC pheno typ ic spec trum was revea led w ider than prev ious ly though t thanks to geno type–pheno type corre la t ion . Comb in ing mu l t ip le sources of c l in ica l informa t ion , such as gene t ic , e lec trocard iog raph ic , arrhy thm ic , morphofunc t iona l , and h is topa tho log ic f ind ings resu l ted the bes t approach to un tang le the comp lex i ty of th is d isease .

Aim

The a im of our s tudy was to assess AC gene t ic he terogene i ty , app ly ing appropr ia te gene t ic screen ing and perform ing a mu l t iparame tr ic geno type-pheno type corre la t ion tak ing in to accoun t AC var ian ts c lass if ica t ion .

Mater ia ls and Methods

A to ta l of 224 consecu t ive pa t ien ts , w i th a c l in ica l (n .192) or pos t mor tem (n .32) d iagnos is of AC , underwen t gene t ic screen ing by us ing a 174 card iac-re la ted genes pane l (Trus igh t Card io , I l lum ina) . The preva lence of Copy Number Var ia t ions (CNVs) and new ly AC-assoc ia ted genes such as FLNC and CDH2, were inves t iga ted . De ta i led c l in ica l da ta were ob ta ined on 12 - lead e le t trocard iog raphy , echocard iog raphy , and card iac magne t ic resonance w i th the purpose of perform ing a mu l t iparame tr ic geno type-pheno type corre la t ion. TTN var ian ts were eva lua ted separa te ly , due the magn i tude of the gene , as we l l as rare var ian ts in AC-unre la ted genes . F inal ly , WES was car r ied ou t on 10 AC geno type negat ive pa t ien ts in order to iden t ify new cand ida te genes invo lved in the d isease pa thogenes is .

Resu lts

We iden t if ied 95 d ifferen t rare gene t ic var ian ts in 97 (43%) of the 224 index cases . Of them , 79 var ian ts

were found in 5 ma jor desmosoma l genes (83%) , whereas 16 in AC-re la ted non desmosoma l genes

(17%) . Amer ican Co l lege of Med ica l Gene t ics (ACMG)-based var ian t c lass if ica t ion made ev iden t tha t

ha lf of desmosoma l var ian ts (39 /79, 49%) were c lass if ied as p a thogen ic / l ike ly pa thogen ic and were

predom inan t ly rad ica l (32 /39 , 78%) . Comprehens ive sequenc ing , inc lud ing new ly AC assoc ia ted genes

(FLNC and CDH2) and CNVs ana lys is , led to the iden t if ica t ion of the gene t ic cause in 10 more pa t ien ts

vi

increasing the overall yield of genetic screening from 43% to 48%. No pathogenic variants were identified in 117 (52%) patients.

Overall we re-evaluated based on the current International Task Force Criteria (ITC) the clinical phenotype of 188 out of 224 AC (84%) index cases. Of these, 94 index cases received a definite AC diagnosis at the outpatient clinic, 16 were heart-transplanted (HTx) patients and 32 sudden death (SD) victims. The remaining 18 were borderline and 28 were possibly affected by AC. 78 of the 142 (55%) definite index cases carried at least a rare variant in AC related genes whereas, only 13 of the 46 (28%) borderline/possible index cases were genotype positive.

Genotype analysis focusing on ventricular involvement highlighted that patients with Left Dominant variant (LDAC) were significantly less positive for desmosomal variants (11/42, 26%) compared to the

“classic” AC cases (75/182, 41%)(p-value 0.0065). More in deep, 19 of the 42 (45%) LDAC patients were SD victims, of whom only 4 cases (21%) were genotype positive for desmosomal rare variants.

Based on previous transcriptome studies from our laboratory we identified rare variants in LGALS3.

Specifically, sequencing of 10 index cases through WES and 140 by direct sequencing, led to the identification of 5 LGALS3 rare nucleotide variants in 7 probands (4%, 5 males, mean age 39±11 years).

Of note, two missense variants occurred in the protein carbohydrate recognition domain (CRD) conferring the loss of its binding site.

Conclusions

Comprehensive genetic analysis revealed a genetic cause in nearly half (48%) of AC patients, of which only half could be classified as P/LP. A proper phenotypic characterization increased variant finding likelihood in definite AC patients (55%). Nevertheless, half of AC patients still missed a genetic cause.

Specifically, genetic testing achieved to identify a causative variant in only ~25% of LDAC cases. Finally

a new candidate gene was identified in 4% of AC cases, supporting the fact that other genetic factors

might be involved in disease pathogenesis. Most of identified genetic variants were variants of unknown

significance (VUS), highlighting that cascade genetic screening remains mandatory to understand their

significance in disease pathogenesis.

1

Introduct ion Background

Arrhy thmogen ic card iomyopa thy (OMIM #107970 ; ORPHA247) is a rare d isease of the hear t musc le charac ter ized pa tho log ica l ly by f ibrofa t ty myocard ia l rep lacemen t and c l in ica l ly by prom inen t ven tr icu lar arrhy thm ias and impa irmen t of ven tr icu lar sys to l ic func t ion [1-4]. The or ig ina l descr ip t ion of th is d isorder , a t tr ibu ted to Marcus e t a l . [5] in 1982 a l though the c l in ica l phys io logy of th is cond i t ion was descr ibed few years ear l ier in French [6], charac ter ized 24 ind iv idua ls w i th ex tens ive subs t i tu t ion of the r igh t ven tr ic le (RV) myocard ium w i th fa t ty and f ibrous t issue . Subsequen t ly , the d isease ro le in caus ing sudden dea th in a th le tes was descr ibed in a prospec t ive c l in ica l-pa tho log ic s tudy w i th de ta i led morpho log ic and h is to log ic fea tures as we l l as the preva lence of sudden card iac dea th (SCD) in the young popu la t ion of the Vene to reg ion , Nor th-eas tern of I ta ly [1].

A t f irs t , myocy te loss w i th subsequen t f ibrofa t ty rep lacemen t was though t to be the resu l t of a congen i ta l defec t of myocard ia l deve lopmen t con tr ibu t ing to the ear ly des igna t ion of dysp las ia . Thanks to the progress of the gene t ic and pheno typ ic charac ter iza t ion the term dysp las ia was soon rep laced by the des igna t ion of card iomyopa thy , wh ich refers to a gene t ica l ly de term ined hear t musc le d isorder , and th is d isease was named as arrhy thmogen ic r igh t ven tr icu lar card iomyopa thy (ARVC) [1 , 2] .

A l though the or ig ina l d isease pheno type ha l lmark was a predom inan t RV invo lvemen t , w i th m inor and la te lef t ven tr ic le (LV) a l tera t ion , now we know tha t the d isease spec trum is w ider than prev ious ly though t . “C lass ic” ARVC is charac ter ized by RV preponderance throughou t the d isease course ; the

“b iven tr icu lar” pa t tern is def ined by para l le l invo lvemen t of bo th ven tr ic les and the “ lef t dom inan t” form LDAC is purpor ted to m irror the “c lass ic” pa t tern , w i th the LV cons is ten t ly more severe ly affec ted than the RV [7 ] . These f ind ings have led over the pas t few years to the use of the broader term

“arrhy thmogen ic card iomyopa thy” (AC) , wh ich encompasses a l l the pheno typ ic express ions .

2

Ep idem io logy

The es t ima ted preva lence of AC in the genera l popu la t ion ranges from 1 :2000 to 1 :5000 [3 , 4 , 8 ] . In the

pas t was cons idered an endem ic d isease in Nor th Eas t I ta ly (“Vene t ian d isease”) , where a sys tema t ic

inves t iga t ion of the causes of SCD in the young have iden t if ied affec ted AC ind iv idua ls more common ly

than in o ther coun tr ies , is now we l l recogn ized in human popu la t ions of d i fferen t e thn ic i ty . AC affec ts

more frequen t ly ma les than fema les (up to 3 :1) , and th is has been ascr ibed to the d irec t inf luence of sex

hormones on the pheno typ ic or to the sex-re la ted d ifferences in the amoun t and in tens i ty of exerc ise [9-

11] . I t becomes c l in ica l ly over t mos t of ten in the second-four th decade of l ife [3 , 4 , 8 ] . More rare ly ,

symp toms and s igns can appear before puber ty or in the e lder ly . However , occas iona l ly the f irs t c l in ica l

man ifes ta t ions ar ise even in pa t ien ts >70 , bu t the d iagnos is is of ten m issed because c l in ic ians do no t take

i t in to cons idera t ion th is morb id en t i ty in th is o lder age-group [3] .

3

Patho log ica l f ind ings

The ha l lmark les ion of AC is rep lacemen t of the ven tr icu lar myocard ium by f ibrofa t ty t issue [1 , 2 , 5] . Th is cond i t ion shou ld no t be confounded w i th Uh l d isease , a congen i ta l hear t defec t in wh ich the RV myocard ium fa i ls to deve lop dur ing embryon ic l ife w i th the ep icard ium app l ied d irec t ly to endocard ium in the absence of in terposed fa t [12] . In AC myocard ia l a trophy is a gene t ica l ly de term ined process tha t occurs progress ive ly w i th t ime , s tar ts from the ep icard ium and ex tends toward the endocard ium to become transmura l , resu l t ing in to prog ress ive wa l l th inn ing . As consequence , the pa thognomon ic gross fea tures of AC cons is t of RV aneurysms , whe ther s ing le or mu l t ip le , loca ted in the so-ca l led “ tr iang le of dysp las ia” ( i .e . inf low , apex and ou tf low trac t) [13] (F igure 1) .

Figure 1: Pathologicfeatures of arrhythmogenic cardiomyopathy. Classicalright ventricular(RV) variant: A, Grosstransverse section of the heart that shows anterior and posterior RV wall thinning because of myocardial atrophy and a subtricupid aneurysm. Fullthickness histology ofthe posterior(B) and anterior(C) RVfree wallthat showsfibrofattytissuereplacement.

Thereisthinning andresidual myocardium confinedtothe endocardialtrabeculae (trichrome stain).(Modifiedfrom Corrado et al, 2017)

4

AC pheno typ ic spec trum has been revea led w ider than prev ious ly though t thanks to geno type–pheno type corre la t ion . Pa tho log ica l fea tures can vary from a g ross ly norma l hear ts a t one end to hear ts w i th mass ive b iven tr icu lar d isease invo lvemen t . LV invo lvemen t has been observed in up to 76% of the AC hear ts s tud ied a t pos t-mor tem , usua l ly l im i ted to the subep icard ium or m idmura l layers of the free wa l l [14] . Hear ts w i th end-s tage d isease lead ing to hear t fa i lure usua l ly show huge b iven tr icu lar chamber d i la ta t ion w ith mu l t ip le free-wa l l aneurysms [2 , 14] . Moreover recen t ly , an iso la ted , non ischem ic LV f ibrofa t ty scar , as seen e i ther a t pos tmor tem exam ina t ion or by pos t-con tras t card iac magne t ic resonance (CMR) sequences , has been repor ted to be a no t uncommon myocard ia l subs tra te of l ife- threa ten ing ven tr icu lar arrhy thm ias and SCD in young peop le and a th le tes [15 , 16] .

H is to log ica l exam ina t ion revea ls is lands of surv iv ing myocy tes , in terspersed w i th f ibrous and fa t ty t issue [1 , 2 ]. Replacemen t -type f ibros is and myocy te degenera t ive changes shou ld a lways be iden t if ied because fa t ty inf i l tra t ion of the RV is no t a suff ic ien t morpho log ic ha l lmark of AC [17-19]. Of no te in tramyocard ia l fa t is presen t f is io log ica l ly in the RV an tero la tera l and ap ica l reg ion and increases w i th age and body s ize . Myocy te degenera t ion and dea th are of ten assoc ia ted w i th inf lamma tory inf i l tra tes repor ted in up to 75% o f hear ts a t au topsy . Ra ther than be ing a con t inuous , ongo ing process , d isease progress ion may occur through per iod ic acu te burs ts of an o therw ise s tab le d isease m im ick ing myocard i t is or myocard ia l infarc t ion w i th norma l coronary ar ter ies . The de tec t ion of v ira l genomes led to cons idera t ion of an infec t ive v ira l cause , bu t i t is mos t l ike ly tha t e i ther v iruses are innocen t bys tanders or myocard ia l ce l l degenera t ion may serve as a m i l ieu favour ing v ira l a t tach .

C l in ica l f ind ings

The pheno typ ic express ion of AC var ies cons iderab ly , rang ing from the c l in ica l prof i les of asymp toma t ic

fam i ly members w i th concea led s truc tura l abnorma l i t ies and no arrhy thm ias to symp toma t ic pa t ien ts

exper ienc ing arrhy thm ic card iac arres t or undergo ing card iac transp lan ta t ion because of refrac tory hear t

fa i lure [3 , 14 , 20-23]. The mos t common c l in ica l presen ta t ion cons is ts of ven tr icu lar arrhy thm ias and

re la ted symp toms /even ts , wh ich inc lude pa lp i ta t ions , syncopa l ep isodes (mos t ly occurr ing dur ing

phys ica l exercise) , and card iac arres t . Less -common presen ta t ions are RV or b iven tr icu lar d i la ta t ion ,

w i th or w i thou t hear t fa i lure symp toms , m im ick ing d i la ted card iomyopa thy (DCM) .

5

However, often non-specific clinical features comprise myocarditis or fake chest pain, dynamic ST-T wave changes on basal 12- lead elettrocardiogram (ECG) and myocardial enzymes release with normal coronary arteries.

Sudden cardiac death may occur unexpectedly as first symptom in previously asymptomatic individuals, mostly young people and competitive athletes, with a previously undiagnosed (and unsuspected) AC [1, 24-26].

The prognosis of AC is related either to ventricular electric instability, which may lead to arrhythmic SCD, or progression of ventricular muscle disease resulting in RV or biventricular systolic dysfunction.

The natural history of the disease can be divided into four phases, based on clinical and pathological observations [3, 27, 28]:

 A “concealed” phase typical of very early AC. Disease is frequently characterized by the absence of clinical symptoms, but subtle structural changes and minor ventricular arrhythmia may be recognized. Even if subjects are mainly asymptomatic, however they may be at risk of SCD, especially during physical exercise [24].

 In the “overt electrical disorder” phase, patients manifest symptomatic ventricular arrhythmias, palpitations, syncopes and pre-syncopes. This phase is characterized by the presence of functional and morphological abnormalities of the RV and/or Left ventricle (LV), usually detectable by cardiac imaging techniques.

 The “right ventricular dysfunction” phase is characterized by further extension of disease involving the RV myocardium, leadings to impaired contractility and isolated right heart failure.

 The most advanced phase is characterized by LV involvement leading to biventricular (BV) heart

failure, which is difficult to distinguish from DCM. However, LV involvement may be detected

also in earlier phases of the disease [7].

6

D iagnos t ic cr iter ia

The bes t s tra tegy to reach the c l in ica l d iagnos is cons is ts in comb in ing mu l t ip le sources of c l in ica l informa t ion , such as gene t ic , e lec trocard iog raph ic , arrhy thm ic , morphofunc t iona l , and h is topa tho log ic f ind ings . AC cr i ter ia were f irs t proposed by an In terna t iona l Task Force (ITC) in 1994 [29] to fac i l i ta te d isease d iagnos is . The cr i ter ia were in i t ia l ly des igned to prov ide adequa te spec if ic i ty for the r igh t- dom inan t var ian t of AC among index cases w i th over t c l in ica l man ifes ta t ions . Based on the ir d iagnos t ic sens i t iv i ty and /or spec if ic i ty , cr i ter ia were c lass if ied as ma jor and m inor , and the d iagnos is of AC was reached in the presence of 2 ma jor cr i ter ia , 1 ma jor p lus 2 m inor , or 4 m inor cr i ter ia from d ifferen t ca tegor ies . La ter , these cr i ter ia were mod if ied in order to increase sens i t iv i ty in de tec t ing m i lder forms of AC [30] . The mod if ied cr i ter ia fac i l i ta ted the recogn i t ion of pa t ien ts’ re la t ives affec ted based on d ifferen t c l in ica l fea tures compr is ing s truc tura l , h is to log ica l and arrhy thm ic abnorma l i t ies a long w i th a de ta i led fam i l ia l h is tory of d isease and SCD (Tab le 1) These d iagnos t ic cr i ter ia in adu l ts were demons tra ted va l id a lso in the ped ia tr ic age , excep t ion made for the presence of inver ted T wave on the r igh t precord ia l leads in ch i ldren  <  12 years of age . To da te , the AC d iagnos t ic cr i ter ia inc lude the presence of a pa thogene t ic var ian t in AC re la ted genes as a ma jor cr i ter ion to es tab l ish the d iagnos is , d ifferen t from a l l o ther forms of card iomyopa thy .

L im itat ions o f current d iagnos t ic cr iter ia

S ince bo th the 1994 and 2010 gu ide l ines were deve loped to d iagnose the or ig ina l r igh t-dom inan t d isease

pheno type they d id no t inc lude spec if ic cr i ter ia for d iagnos ing LV invo lvemen t and the more recen t ly

recogn ized lef t-s ided pheno typ ic var ian ts [7 , 31 ] . Moreover , pecu l iar i t ies of d iagnos is in the paed ia tr ic

popu la t ion , wh ich represen ts approx ima te ly one-s ix th of the overa l l ARVC-popu la t ion , were no t

addressed .

7

Table 1: Comparison of original and revised Task Force criteria for diagnosis of AC [29, 30]

8

9

C l in ica l tes ts enab l ing AC d iagnos is :

Twe lve- lead ECG is a va luab le d iagnos t ic tes t in AC because i t records repo lar iza t ion and depo lar iza t ion abnorma l i t ies in the ma jor i ty of probands w i th a def in i t ive d iagnos is of AC . Nega t ive T waves in the an ter ior precord ia l leads (V1 through V4) are the mos t common f ind ing . Depo lar iza t ion abnorma l i t ies inc lude incomp le te (rare ly comp le te) r igh t bund le branch b lock (RBBB) , pro longa t ion of r igh t precord ia l QRS dura t ion w i th a de layed S-wave ups troke ( term ina l ac t iva t ion de lay >55 ms) , QRS fragmen ta t ion , and pos t-exc i ta t ion eps i lon waves ( ie , sma l l amp l i tude po ten t ia ls occurr ing a t the end of QRS comp lex /beg inn ing of the ST segmen t) [32 , 33] .

S igna l-averaged e lec trocard iogram (SAECG) a l lows the reg is tra t ion of low amp l i tude po ten t ia ls w i th in the end of the QRS comp lex ( la te po ten t ia l) tha t are no t w ide enough to be ev iden t on the 12- lead ECG . Ho l ter ECG mon i tor ing is used to reg is ter the e lec tr ic ac t iv i ty of the card iovascu lar sys tem for a per iod of norma l ly 24 h , w i th par t icu lar a t ten t ion to the d iurna l rhy thm f luc tua t ions .

Echocard iography (Echo) represen ts the f irs t- l ine imag ing approach for eva lua t ing pa t ien ts w i th suspec ted AC or for screen ing of fam i ly members , a l low ing ser ia l exam ina t ions w i th the a im to assess the d isease onse t and prog ress ion .

CMR imag ing is ano ther non- invas ive t issue charac ter iza t ion techn ique tha t a l lows the de tec t ion of morpho log ica l and s truc tura l abnorma l i t ies of the ven tr icu lar wa l l l ike m icro aneurysms , ca lcu la t ion of RV and LV vo lumes and e jec t ion frac t ion [34-36] .

Endomyocard ia l b iopsy (EMB) is used to de tec t myocy tes in d iverse s tages of ce l l dea th and of f ibrofa t ty rep lacemen t (ma jor cr i ter ion) . However , a nega t ive b iopsy is no t enough to exc lude AC because of the segmen ta l na ture of the d isease , espec ia l ly dur ing ear ly s tages [37] . The sens i t iv i ty of EMB for AC is low if the myocard ia l samp les are taken from the sep tum , wh ich is a reg ion uncommon ly invo lved by the d isease . EMB canno t be rou t ine ly recommended for d iagnos is of AC and shou ld be reserved for se lec ted pa t ien ts par t icu lar ly probands w i th a sporad ic form of AC , in whom the f ina l d iagnos is depends on h is to log ic exc lus ion of phenocop ies such as DCM , myocard i t is , or sarco idos is .

Three-d imens iona l e lec troana tom ic vo l tage mapp ing by CARTO sys tem (B iosense , D iamond Bar , CA)

may be of s ign if ican t added va lue for the d iagnos is of AC because i t has the po ten t ia l to iden t ify and

quan t ify RV reg ions of scar w i th low-amp l i tude e lec tr ic s igna ls , wh ich typ ica l ly show frac t iona t ion ,

doub le po ten t ia ls , or conduc t ion de lay [38 , 39] . However , i t is no t recommended as a rou t ine d iagnos t ic

too l because i t is invas ive , expens ive , and h igh ly opera tor dependen t .

10

Le ft Dom inant AC d iagnos is

Wh i le the rev ised cr i ter ia eas i ly ca tch the c lass ica l RV var ian t , there is lack of spec if ic d iagnos t ic gu ide l ines for non-c lass ica l d isease pa t terns , thus exp la in ing the under-recogn i t ion of the LV one . ECG abnorma l i t ies such as la tera l or infero la tera l T-wave invers ion ( leads V5 , V6 , LI , and aVL ) , low vo l tage QRS comp lex on per iphera l leads and RBBB /po lymorph ic ven tr icu lar arrhy thm ias sugges t a lef t-s ided invo lvemen t [7 , 31 , 40] . Gado l in ium -enhanced CMR a far more-sens i t ive ind ica tor of even ear ly or m inor lef t-s ided d isease , and is frequen t ly de tec ted in a wa l l segmen t w i thou t a concom i tan t morphofunc t iona l abnorma l i ty , thus preced ing the onse t of LV dysfunc t ion or d i la ta t ion [7 , 41 , 42].

Typ ica l ly , LV la te gado l in ium enhancemen t invo lves the infero la tera l and infero-sep ta l reg ions , and affec ts the subep icard ia l or m idwa l l layers .

Therapy

The mos t impor tan t ob jec t ive of c l in ica l trea tmen t of AC pa t ien ts is p reven t ion of SCD . Curren t

therapeu t ic op t ions inc lude l ifes ty le changes , β-b lockers , an t iarrhy thm ic drugs (AADs) , ca the ter

ab la t ion , ICD , and hear t transp lan ta t ion (HTx) . Recen t ly , a task force of exper ts from bo th Europe and

the Un i ted S ta tes produced a consensus documen t for trea tmen t of AC [43] . Phys ica l exerc ise is one of

the mos t impor tan t r isk fac tors , wh ich promo tes the pheno typ ic express ion of the d isease and tr iggers

l ife -threa ten ing ven tr icu lar arrhy thm ias in AC [11]. Accord ing ly , curren t gu ide l ines recommended the

exc lus ion of compe t i t ive or endurance spor t ac t iv i ty of affec ted ind iv idua ls bu t a lso desmosome gene

var ian ts carr iers .

11

Mo lecu lar genet ics o f AC

In 1982 , Marcus e t a l . advoca ted the inher i ted na ture of AC , w i th the descr ip t ion of two affec ted adu l t cases in the same fam i ly . AC is curren t ly cons idered an au tosoma l dom inan t d isease w i th reduced pene trance and var iab le c l in ica l express ion [8 ] a l though , recurren t descr ip t ion of compound / d igen ic he terozygo tes and homozygo tes pa t ien ts have been assoc ia ted w i th severe forms of the d isease [9 , 44]

w i th or w i thou t cu taneous abnorma l i t ies [45-51] .

The recogn i t ion of d isease fam i l ia l na ture [52] led to the iden t if ica t ion of the f irs t d isease-caus ing gene mu ta t ion in a recess ive syndrom ic form of AC known as Naxos d isease [53 , 54] . Naxos d isease was f irs t descr ibed in 1986 as a fam i l ia l , au tosoma l recess ive d isease charac ter ized by ha ir and sk in abnorma l i t ies (woo l ly ha ir and pa lmop lan tar kera toderma) exh ib i t ing an AC- l ike card iomyopa thy [55] . The typ ica l ha ir pheno type is presen t a t b ir th a long w i th the ery thema on the pa lms of the hands wh ich gaffers w i th in the f irs t years of l ife , wh i le the card iac pheno type appears on ly dur ing ado lescence or ear ly adu l thood . In the or ig ina l descr ip t ion , 9 affec ted sub jec ts from 4 Greek fam i l ies of the Naxos is land were inves t iga ted by l inkage ana lys is lead ing to the iden t if ica t ion of a gene t ic locus on the long arm of chromosome 17 (17q21 .2) [56] . Subsequen t d irec t sequenc ing of the P lakog lob in ( JUP) gene in a l l affec ted sub jec ts iden t if ied a 2bp-de le t ion lead ing to a frame sh if t w i th consequen t in troduc t ion of a prema ture s top codon af ter 11 am ino ac id [53] .

L inkage ana lys is were a lso used to iden t ify the f irs t d isease caus ing mu ta t ion in a dom inan t AC form [54] . S ix affec ted sub jec ts from an I ta l ian fam i ly of the Vene to Reg ion carr ied a he terozygous m issense mu ta t ion c .897C>G th is t ime in the Desmop lak in ( DSP) gene , loca ted on the shor t arm of chromosome 6 (6p24 .3) . Ear l ier , a homozygous DSP de le t ion was descr ibed for ano ther syndrom ic form , known as Carva ja l syndrome [57] . Carva ja l syndrome was f irs t descr ibed in 1998 as a fam i l ia l , au tosoma l recess ive d isorder charac ter ized by ep idermo ly t ic pa lmop lan tar kera toderma , woo l ly ha ir , and hear t anoma l ies . In the or ig ina l descr ip t ion , 18 sub jec ts from 3 Ecuador fam i l ies , were inves t iga ted by l inkage ana lys is and subsequen t d irec t sequenc ing lead ing to the iden t if ica t ion of a homozygous s ing le-nuc leo t ide de le t ion c .7901de lG , wh ich crea tes a prema ture s top codon depr iv ing DSP of i ts C-doma in [58 , 59].

DSP and JUP are bo th componen ts of the card iac desmosome , therefore cand ida te gene approaches revea led the gene t ic he terogene i ty of AC w i th the iden t if ica t ion of 15 gene t ic loc i (

Tab le 2) .

12

Nowadays AC is defined as a disease of the desmosomes since causative variants are found in genes coding for desmosomal proteins: JUP, DSP, plakophilin-2 (PKP2), desmoglein-2 (DSG2), and democollin-2 (DSC2) [54, 60-64]. Isolated reports, accounting for less than 1-3% of cases, are caused by variants in nondesmosomal genes, such as transforming growth factor-β-3 (TGFβ3), cardiac ryanodine receptor (RYR2), transmembrane protein 43 (TMEM43), lamin A/C (LMNA), desmin (DES), catenin alpha 3 (CTNNA3), titin (TTN), phospholamban (PLN), filamin C (FLNC) and N-cadherin (CDH2) [65-74].

Table 2: AC genetic loci. Abbreviations AD: autosomal dominant; AR: autosomal recessive.

MIM entry Locus Gene ID Gene name Mode of

transmission Reference

#611528 17q21.2 JUP Plakoglobin AD [60]

#601214 AR [53]

#607450 6p24.3 DSP Desmoplakin AD [54]

#605676 AR [57]

#609040 12p11.21 PKP2 Plakophilin-2 AD/AR [61]

#610193 18q12.11 DSG2 Desmoglein-2 AD/AR [62]

#610476 18q12.1 DSC2 Desmocollin-2 AD/AR [63]

Non-desmosomal genes

#600996 1q43 RYR2 Cardiac Ryanodine Receptor 2 AD [65]

#107970 14q24.3 TGF-β3 Transforming growth factor-beta 3 AD [66]

#604400 3p25.1 TMEM43 Transmembrane Protein 43 AD [67]

2q35 DES Desmin AD [68]

6q22.31 PLN Phospholamban AD [71]

2q31.2 TTN Titin AD [69]

1q22 LMNA Lamin A/C AD [70]

#615616 10q21.3 CTNNA3 alpha-1-catenin AD [72]

7q32.1 FLNC Filamin C AD [73]

18q12.1 CDH2 Cadherin 2 AD [74]

Independent studies identified large deletions in PKP2 gene, suggesting a pathogenic role of copy

number variants (CNVs) in the disease due to haploinsufficiency [75-78]. First, Cox et al.[75] described

3 large PKP2 deletions in a 149 Dutch AC cases, reporting as prevalent as ≈2%. The authors highlighted

that the CNVs analysis should be extended not only to PKP2 but also in other desmosomal genes. Since

then, single case carrying CNVs in PKP2 have been reported [76-78] but only recently a systematic

comprehensive CNVs screening of was performed by our group. Pilichou et al., [79] reported for the first

time CNVs in desmosomal cadherin-related genes (DSG2, DSC2) and estimated the prevalence of PKP2-

13

related CNVs to 5.6% (9 of the 160) in genotype-negative proband. Although CNVs may confer

haploinsufficiency, the reduced disease penetrance in family carriers (≈32%) was similar to the one

observed in other point variants suggesting that other factors has been involved in the development and

progression of the disease.

14

1.10.1 Desmosomes

Desmosome junctional complexes are particularly abundant in tissues subjected to mechanical stress like the epithelium and the myocardium, where these molecules mediate mechanical anchorage of cardiomyocytes by connecting the cytoskeleton to the cell membranes of adjacent cells (Figure 2). As well as may play a role in cell-cell communication, tissue differentiation, and apoptosis [80].

Figure 2: Schematic representation of desmosome modified from [79]

These electron-dense symmetrical structures that appear as dense membrane-associated plaques divided

by a central mid-line of intercellular space are called desmoglea and intracellular plaque. The

intracellular plaque is commonly described as composed of two areas: the outer dense plaque, and a

dense inner plaque [81]. The desmosomal structure comprises transmembrane adhesive glycoproteins

(components of the cadherin superfamily) and cytoplasmic proteins (components of the plakin and

armadillo families). The outer dense plaque is where the cytoplasmic domains of the cadherins (DSG2,

DSC2) attach to plakins (DSP) via armadillo proteins (PKP2 and JUP). The inner dense plaque is located

where plakins attach to the intermediate filaments of the cell.

15

Desmoplakin_DSP (6p24.3) belongs to the plakin protein family which includes a large number of proteins such as plectin, envoplakin and periplakin, all mediating the link between different junctional complexes and the cytoskeleton. DSP is composed by an N-terminal domain required for both the localization to the desmosome and interaction with plakophilin and cadherin family proteins, a central coiled-coil rod domain involved in the protein dimerization and a C-terminal domain that interacts directly with the intermediate filaments. It is expressed in all tissues containing desmosomes [82] and an alternative splicing of the DSP precursor messenger RNA (mRNA) produces two isoforms, differing in the length of the central -helical domains: DSP I, predominantly expressed in heart, comprise 2871 amino acids whereas DSP II only 2271 amino acids.

The first pathogenic nucleotide variants described in DSP families with autosomal dominant striated palmoplantar keratoderma, were a heterozygous nonsense p.Gln331Ter and a splicing site c.939+1G>A variants [83] but cardiac alterations were not studied. Since then, more than 100 DSP pathogenic variants have been detected in 5 to 16% of AC cases [62, 75, 84].

Plakophilin-2_PKP2 (12p11.21) the predominant protein isotype expressed in heart, belongs to the

armadillo family of proteins and interacts directly with both, desmosomal cadherins and DSP [85]. Two

alternatively spliced mRNA transcripts give origin to two protein isoforms: transcript 2b (881 amino

acid) and transcript 2a (837amino acid) [86]. PKP2 comprise an amino-terminal head domain and nine

armadillo repeat motifs and it’s essential for heart morphogenesis and proper localization of DSP in mice

[87]. After the link to disease pathogenesis through a haploinsufficiency mechanism which bring to

defect cell-cell contact [61], more than 120 PKP2 pathogenic variants have been reported to AC

accounting for approximately 10 to 45% of reported cases [61, 62, 75, 84]. Most variants show a

dominant inheritance with reduced penetrance, but recessive and compound heterozygous variants have

also been identified in several patients. Moreover, large deletions involving PKP2 have also been

described in subset of affected individuals [75-79].

16

Desmoglein-2_DSG2 (18q12.1) is a desmosomal cadherin belonging to the cadherin superfamily, mediating calcium dependent cell-cell adhesion. Desmosomes bears four isoforms of desmoglein (DSG 1 to 4), showing tissue specific expression patterns [88]. DSG2 is expressed in all tissues bearing desmosomes but seems to be the only isoform expressed in cardiac tissue [89, 90]. DSG2 has an intracellular anchoring domain interacting with DSP, a transmembrane domain, four extracellular cadherin domains each containing a calcium binding site that stabilizes the structure and function of cadherins, a small signal and a preprotein domain. Since 2006 when DSG2 was linked for the first time to AC through the detection of 9 missense variants in 8 Italian families affected by AC affecting highly conserved amino acids, more than 50 DSG2 variants have been found in 2 to 20% of AC patients [62, 75, 84]. Of note in 2006 the first compound heterozygote AC patients was reported.

Desmocollin-2_DSC2 (18q12.1), is another glycoprotein, member of the cadherin superfamily mediating calcium dependent cell-cell adhesion. Three desmocollin isoforms (DSC 1, 2 and 3) are currently known, of which DSC2 is the only isoform present in cardiac tissue even though ubiquitously expressed [88]. DSC2 is composed of a signal domain, a preprotein domain followed by four highly conserved extracellular subdomains and an extracellular anchor domain at the N-terminus. In 2006 Syrris and colleagues described the presence of heterozygous DSC2 frameshift variants in 4 AC affected probands without variants in other desmosomal genes [63]. Since them, less than 50 DSC2 nucleotide variants have been reported, accounting for 1 to 3% of AC cases [63, 75, 84, 91, 92].

Plakoglobin_JUP (17q21), is another armadillo protein of desmosomes. It is present both, in adherens

junctions and desmosomes, where it binds to the cytoplasmic domain of cadherin acting as a linker

molecule between the inner and outer parts of the desmosomal plaque. JUP comprise an N-terminal

domain, a central domain containing highly conserved armadillo repeats and a C-terminal domain. JUP

has only recently been linked to the dominant AC form, after the description of an in frame insertion of

a serine residue p.Ser39_Lys40insSer in the N-terminus of the protein in a small German family [60]. To

date, less than 20 JUP variants have been reported in approximately 1% of AC cases [60, 84] [75].

17

1.10.2 Non desmosomal genes

Isolated reports, accounts for less than 1-3%, associated variants in non-desmosomal genes with a clinical phenotype resembling AC

Transmembrane protein 43_TMEM43 (3p25.1) acts as a nuclear membrane organizer. TMEM43 variants were first identified in Emery–Dreifuss muscular dystrophy patients. In contrast to classic AC, TMEM43 variants carriers exhibit right ventricular aneurysms and ventricular arrhythmias before birth [67], the phenotypic penetrance of TMEM43 variants is 100% and frequency is less than 1%. Recent studies reported the founder effect of TMEM43 variants, probably caused by migration from continental Europe to Canada [93].

Desmin_DES (2q35) is a type of intermediate filament in myocytes, often related to myofibrillar myopathies. DES variants carriers often have both myopathy and cardiomyopathy [68]. The first DES variants p.Arg454Trp, affecting the localization of DSP and PKP2 at the intercalated disk, was identified in a family showing an early onset of conduction system disorder without variants in other desmosomal genes [94]. The frequency of DES is about 1–2%.

Titin_TTN (2q31.2) is a sarcomeric protein, the largest protein in mammals, which contributes to force transmission at the Z line and resting tension in the I band region. TTN variants were reported in other cardiomyopathies such as HypertrophicCardiomyopathy (HCM) and DCM. In AC, TTN variant carriers display various phenotypes, including biventricular dysfunction and conduction block. Eight unique missense TTN variants were identified in 7 families with AC-like phenotype and without desmosomal genes variants, including a prominent p. Thr2896Ile variant which showed complete cosegregation in one large family [69]. The role of TTN variants for AC is still a matter of study.

Lamin A/C_LMNA (1q22) is located in the inner nuclear membrane and is fundamental for cell

stabilization. LMNA variants are reported in various systemic diseases, including Emery–Dreifuss

muscular dystrophy, and in dilated cardiomyopathy patients with sinus bradycardia and conduction

disturbance. In 2012, four LMNA variants were reported in AC-like patients with conduction disturbances

[70] but variants frequency has not been yet determined as LMNA variants were often found in AC_DCM

patients [95].

18

Phospholamban_PLN (6q22.31) is a protein necessary for calcium handling in cardiac contractions and PLN was known as a causative gene for DCM. The only reported PLN variant so far, p.Arg14del, was described in a Dutch patients with AC [71] which seems to be a founder one.

Alpha-T-catenin_CTNNA3 (10q21) is a cadherin which binds to plakophilins in cell adherens junctions between cardiomyocytes. Only two CTNNA3 AC-related variants have been reported to date leading to a lack of interaction with beta-catenin [72].

Sodium voltage-gated channel alpha subunit 5_SCN5A (3p22.2) is a subunit of the cardiac sodium channel, and its mutations have been linked to various cardiac diseases, most ‘sine materia’, including long QT syndrome type 3, Brugada syndrome with non-ischaemic ST-segment elevation, progressive cardiac atrioventricular conduction disease, and DCM. In 2008, an SCN5A loss-of function variant was reported in a 58-year-old patient with AC and frequent non-sustained ventricular tachycardia in the absence of ST-segment elevation [96]. Recently, a large multicentre collaborative study reported that

~2% of AC probands have a pathogenic SCN5A mutations, expanding the spectrum of phenotypes associated with variants in this gene [97].

Filamin C_FLNC (7q32.1) is a muscle specific filamin which directly interacts with the dystrophin associated glycoproteins and the integrin complexes that link the subsarcolemmal actin cytoskeleton to the extracellular matrix. At intercalated discs, FLNC is located in the fascia adherens where myofibre ends reach the sarcolemma, adjacent to the position of desmosomal junctions. Its role in the attachment of the sarcomere’s Z-disc to the sarcolemma and to the intercalated discs allows cell to-cell mechanical force transduction. Truncating FLNC variants in 28 families have been linked to particular overlapping phenotypes of dilated and left-dominant arrhythmogenic cardiomyopathies complicated by frequent premature sudden death, a dominant mode of transmission, and an unusual penetrance of higher than 97% in carriers older than 40 years of age [73].

Cadherin 2_CDH2 (18q12.1) also known as N-cadherin, is a protein that mediates calcium ion- dependent adhesion in the brain and skeletal and cardiac muscle. Recently, two different studies identified two variants in this gene linked to an AC phenotype [74, 98].

Variants in the regulatory region of transforming growth factor beta-3_TGFB3 (14q24.3) gene have

also been reported, but their pathogenicity is still a matter of debate [66].

19

F ina l ly , the ryanod ine recep tor 2 _RYR2 (1q43) gene encodes a pro te in of the sarcop lasm ic re t icu lum , necessary for card iac con trac t ion by con tro l l ing the ca lc ium ions . RYR2 var ian ts are nowadays assoc ia ted w i th ca techo lam inerg ic po lymorph ic ven tr icu lar tachycard ia (CPVT) ra ther than w i th AC , as or ig ina l ly be l ieved [65] .

Pathogenes is

Severa l theor ies have been pos tu la ted to exp la in the pa thogenes is of AC : dyson togen ic , degenera t ive , inf lamma tory , transd ifferen t ia t iona l .

The dyson togen ic theory [99 ] cons ider AC a m i lder form of Uh l’s anoma ly [12] wh ich is a congen i ta l hear t defec t charac ter ized by hypop las ia of the RV myocard ium a t b ir th however , myocy te loss has been demons tra ted to occur prog ress ive ly s tar t ing from ch i ldhood [100] . The degenera t ive theory wh ich was pos tu la ted in 1996 , cons idered AC as a consequence of myocy te dea th , e i ther by necros is or apop tos is , due to inher i ted u l tra-s truc tura l defec ts [101 , 102] . Fur ther , recen t s tud ies have demons tra ted tha t the key in i t ia t ing phenomenon in the cascade of even ts tha t lead to f ibrofa t ty rep lacemen t of the norma l myocard ium is myocy te necros is [103 , 104] .

In the inf lamma tory theory , the d isease is cons idered the consequence of preced ing myocard i t is , s ince myocard ia l inf lamma t ion is a common fea ture in hear ts w i th AC [105 , 106] . The v ira l myocard i t is may over lap an a lready affec ted hear t acce lera t ing the d isease prog ress ion , ra ther than be ing a pr imary fac tor in the e t io logy of the d isease [107] .

The transd ifferen t ia t ion theory sugges ts tha t card iac myocy tes undergo a me tamorphos is lead ing to became ad ipoc i tes [108] . Th is theory seems deba tab le due to the l im i ted ded ifferen t ia t ion capab i l i t ies of adu l t card iomyocy tes , indeed recen t s tud ies suppor ted the idea tha t

ad ipocy tes in AC der ive from progen i tor ce l ls of the second hear t f ie ld , wh ich g ive r ise to the bu lbus

cord is and the pu lmonary infund ibu lum [109 ] .

20

Transgenic animal models, that mimic the human AC phenotype (mice and zebrafish) and induced pluripotent stem cells (iPSCs) from affected patients, are useful tools to investigate mechanical and/or functional disruption of cell junctions by aberrant desmosomal proteins leading to cardiomyocyte death and subsequent repair with fibrous and fatty tissue

Reduced cell-cell adhesion has been demonstrated by using monolayers of neonatal rat ventricular myocytes in which PKP2 was silenced [110]. However, when expressing mutant forms of either PKP2 or JUP, cells exhibited preserved intercellular adhesion and abnormal signalling in response to mechanical stress, advocating questions on a primary role of cell-cell adhesion in AC pathogenesis [111].

Parallel , Asimaki and colleagues demonstrated that a reduced junctional signal for JUP seems to be a hallmark of the disease in myocardial samples from AC patients, suggesting its possible role in intracellular signalling rather than adhesion [112], as propose by other groups [113, 114].

Marian group for the first time demonstrate in a Dsp-deficient mouse model that an abnormal distribution of intercalated disc protein leads to suppression of the canonical WNT/β-catenin/Tcf/Lef pathway a known regulator of adipogenesis, fibrogenesis and apoptosis [113]. Moreover, knocking down DSP in HL-1 cells causes the translocation of JUP into the nucleus, where it interferes with β-catenin/TCF transcriptional activity, leading to an adipogenic switch. Thereafter, the same group demonstrated that most of the adipocytes in AC originate from cardiac progenitors cells of the embryonic second heart field [109]. Furthermore, in mice overexpressing cardiac truncated JUP, suppression of the canonical WNT signaling led to adipogenesis in c-kit + cardiac progenitor cells [115].

A lower expression protein kinase C α, a signalling molecule which localises to the intercalated disc, has

been reported analysing molecular remodelling of the intercalated disc [116] .As a result, neurofibromin-

2 (or Merlin) becomes activated and in turn phosphorylates and inactivates the effector of the Hippo

pathway, Yes -associated p rotein, YAP [117]. YAP is then free to bind to β-catenin and plakoglobin and

therefore drives adipogenesis in myocardial tissue.The Hippo pathway, among other functions, regulates

cell differentiation and homeostasis [118]. Interestingly, the activation of the Hippo pathway inhibits the

canonical WNT pathway, reinforcing the hypothesis that the two pathways might be implicated in the

pathogenesis of AC [119].

21

Further, among the others, Kim and colleagues successfully reprogrammed patient-derived dermal fibroblasts into iPSCs generating human cardiomyocytes for in vivo modelling. iPSCs-derived cardiomyocytes from AC patients with PKP2 mutations, demonstrated that the abnormal JUP nuclear translocation and decreased β-catenin activity is insufficient to reproduce the pathologic phenotype [120].

Remarkably, transgenic experimental animal models and iPSC-derived cardiomyocytes demonstrated only abnormal “lipogenesis”, but not adipocyte formation/transdifferentiation. Thus, cells other than cardiomyocytes must be involved in the abnormal adipogenesis and fibrosis which is an essential feature of AC phenotype [121]. A role of cardiac mesenchymal stromal cells as a source of adipocytes in AC has been recently suggested [122].

In most AC cases a reduction of connexin-43 expression at the intercellular junction has been highlighted, underlying that compromised mechanical coupling might also account for abnormal electrical coupling through gap-junction remodelling. Desmosomes, gap junctions and sodium channels are functionally coupled and modification of one constituent can affect the function and integrity of the others [4].

Moreover, cardiac sodium current was found to be reduced in experimental models of AC [104, 123- 126]. These findings suggest that life-threatening ventricular arrhythmias precede structural abnormalities due to electrical uncoupling and reduced sodium current. However, this hypothesis remains to be proven in human AC patients.

Finally, high-throughput drug screening identified SB216763, a compound able to prevent heart failure

and normalize survival in a transgenic zebrafish model of AC with cardiac specific expression of human

JUP deletion [126]. Experiments in neonatal rat ventricular myocytes expressing the same JUP mutation

and in cardiac myocytes derived from iPSCs of two PKP2 mutation carriers further supported its efficacy

in restoring the subcellular distribution of JUP, connexin-43 and SCNA5. Interestingly, SB216763 was

already known as an activator of the canonical WNT signaling pathway and these data, together with

those by Lombardi and Kim may led to the identification of a curative therapy in AC, by targeting a final

common pathway of disease pathogenesis [109, 120].

22

Convent iona l Screen ing

D irec t Sanger sequenc ing deve loped in the 70s [127] has been wor ldw ide chosen for DNA sequenc ing un t i l Nex t Genera t ion Sequenc ing (NGS) era . Desp i te var ious mod if ica t ions and improvemen ts inc lud ing i ts par t ia l au toma t ion , i t rema ins re la t ive ly t ime-consum ing and expens ive cons ider ing the g row ing need of sequenc ing larger DNA por t ions . In gene t ica l ly he terogeneous d iseases , the cos t and effor t of DNA sequenc ing is of ten cons iderab le and numerous DNA pre-ana ly t ic techn iques have been deve loped for the de tec t ion of po in t var ian ts and sma l l de le t ions . Pre-ana ly t ic techn iques , such as dena tur ing g rad ien t ge l e lec trophores is (DGGE) , s ing le-s trand conforma t ion po lymorph ism (SSCP) , chem ica l c leavage me thod (CCM) , dena tur ing h igh performance l iqu id chroma tog raphy (DHPLC) , a l l fo l lowed by d irec t sequenc ing of aberran t conformers or e lu t ion prof i les a l lowed to m in im ize sequenc ing to a subse t of abnorma l PCR produc ts . DGGE is a techn ique deve loped by F ischer and Lerman [128]

tha t can iden t ify homodup lex mo lecu les tha t d iffer by s ing le bp subs t i tu t ions us ing e lec trophoresed through a grad ien t of increas ing concen tra t ion of a dena tur ing agen t . SSCP ana lys is is based on the d ifferen t ia l e lec trophore t ic mob i l i ty of s ing le-s trand DNA mo lecu les tha t d iffer by a s ing le base [129].

Therma l ly dena tured DNA is e lec trophoresed , and those s ing le s tranded DNA fragmen ts tha t take up an a l tered conforma t ion show up as aberran t ly m ig ra t ing bands on the e lec trophore t ic ge l . In CCM PCR he terodup lexes are incuba ted w i th two m isma tch-spec if ic reagen ts : Hydroxy lam ine mod if ies unpa ired cy tos ine and po tass ium permangana te mod if ies unpa ired thym ine . He terodup lexes are then c leaved a t the s i te of the mod if ied m isma tched base and separa ted by e lec trophores is . DHPLC techn ique is based on the de tec t ion of he terodup lexes con ta in ing a var ian ts or po lymorph ism by reduced co lumn re ten t ion of he terodup lexes compared to the respec t ive homodup lexes under par t ia l ly dena tur ing cond i t ions [130]

DHPLC , under op t im ized cond i t ions , is cos teffec t ive , h igh ly accura te , rap id , and eff ic ien t for var ian ts

de tec t ion . A d isadvan tage of DHPLC is the requ iremen t and ma in tenance of a spec ia l ized and expens ive

equ ipmen t and op t im iza t ion of each reac t ion requ ired ach iev ing the h ighes t sens i t iv i ty for var ian t

de tec t ion , moreover i t can a lso be d iff icu l t to iden t ify homozygo tes un less the samp le is sp iked w i th a

known con tro l . The adven t of NGS has revo lu t ion ized sequenc ing in term of cos t , t ime and spec trum of

genes ana lysed .

23

Next Generat ion Sequenc ing

S ince the conc lus ion of the Human Genome Pro jec t in 2000 , based on Sanger techno logy , wh ich requ ired abou t 13 years [131 , 132] and 70 m i l l ion do l lars [133] , deep sequenc ing techno log ies were deve loped to sequence in shor t range of t ime who le genomes and exomes .

NGS increases throughpu t and to decreases sequenc ing cos ts a l low ing to sh if t the in teres t from the research of var ian ts in spec if ic DNA reg ions to the iden t if ica t ion of var ian ts from a genome-w ide sequenc ing da ta . Th is has led to advances in d iagnos t ics and sc ien t if ic research w i th the de tec t ion of var ian ts l inked to Mende l ian and comp lex d iseases [134-137] . NGS has been successfu l ly app l ied for ins tance to the iden t if ica t ion of gene t ic defec ts of a var ie ty of d isorders such as cancer [138 , 139] , neuro log ica l d iseases [140 , 141] , in te l lec tua l d isab i l i ty [142 , 143] , m i tochondr ia l dysfunc t ions [144 , 145] , card iovascu lar d iseases [146 , 147] . App l ica t ions of NGS has the po ten t ia l to focus on the ana lys is of en t ire genomes (Who le Genome Sequenc ing-WGS) , on the so le cod ing par t of the genome (Who le Exome Sequenc ing , WES) or on spec if ic targe t genes (Targe ted Resequenc ing , TR) . WES is the se lec t ive sequenc ing of the cod ing sequence of the human genome and less expens ive in compar ison to WGS s ince the exome represen ts on ly abou t 1% of the genome . In the c l in ica l se t t ing , WES can offer a new approach to d iagnose pa t ien ts w i th an unc lear c l in ica l d iagnos is and whom who are nega t ive from c lass ica l screen ing of d isease-re la ted genes , enab l ing the research in genes and nuc leo t ide var ian ts no t ye t assoc ia ted to the d isease .

However , the in troduc t ion of WES for c l in ica l labora tor ies rema ins cha l leng ing , espec ia l ly because of

the labor ious and accura te process of b io informa t ics ana lys is and da ta in terpre ta t ion tha t are requ ired to

iden t ify cand ida te genes and causa l var ian ts . For these reason in the c l in ica l prac t ice TR resu l ted the f irs t

screen ing s tra tegy to reach h igher coverage of exon ic reg ions of in teres t wh i le reduc ing the sequenc ing

cos t and t ime . These rap id , accura te , and re la t ive ly low cos t me thod a l lows a h igh throughpu t , geno type-

based approach to mo lecu lar d iagnos is .TR may rap id ly screen a t once large pane ls of genes assoc ia ted

w i th a par t icu lar pheno type or may prov ide d ifferen t ia l d iagnos is in d iseases tha t presen t w i th a typ ica l

man ifes ta t ions , or for wh ich no t a l l gene t ic var ian ts are known .