Salla Välipakka, MSc
Ma co Sa a ese, PhD
M idul Joha i, MSc
Lydia Saga h, MSc
Meha ji A umilli, MSc
Ki si Kiiski, PhD
Ame s Sáenz, PhD
Adol o Lopez de Munain,
MD, PhD
Ana-Ma ia Cobo, PhD
Ka a ina Pelin, PhD
Bja ne Udd, MD, PhD
Pe e Hackman, PhD
Co espondence o
S. Välipakka:
salla. alipakka@helsinki. i
Copy numbe a ia ion analysis inc eases
he diagnos ic yield in muscle diseases
ABSTRACT
Objec i e: Copy numbe a ian s (CNVs) we e analyzed om nex -gene a ion sequencing da a,
wi h he aim o imp o ing diagnos ic yield in skele al muscle diso de cases.
Me hods: Fou publicly a ailable bioin o ma ic analy ic ools we e used o analyze CNVs om
sequencing da a om pa ien s wi h muscle diseases. The pa ien s we e p e iously analyzed wi h
a a ge ed gene panel o single nucleo ide a ian s and small inse ions and dele ions, wi hou
achie ing inal diagnosis. Va ian s de ec ed by mul iple CNV analysis ools we e e i ied wi h
ei he a ay compa a i e genomic hyb idiza ion o PCR. The clinical signi icance o he e i ied
CNVs was in e p e ed, conside ing p e iously iden i ied a ian s, seg ega ion s udies, and clini-
cal in o ma ion o he pa ien cases.
Resul s: Combining analysis o all di e en mu a ion ypes enabled in eg a ion o esul s and iden-
i ied he inal cause o he disease in 9 myopa hy cases. Complex e ec s like compound he e o-
zygosi y o di e en mu a ion ypes and compound disease a ising om a ian s o di e en
genes we e un a eled. We iden i ied he i s la ge in agenic dele ion o he i in (TTN)gene
implica ed in he pa hogenesis o a se e e o m o myopa hy. Ou wo k also e ealed a “double-
ouble”e ec in a pa ien ca ying a single he e ozygous inse ion/dele ion mu a ion in he TTN
gene and a Becke muscula dys ophy causing dele ion in he dys ophin gene.
Conclusions: Causa i e CNVs we e iden i ied p o ing ha analysis o CNVs is essen ial o
inc easing he diagnos ic yield in muscle diseases. Complex se e e muscula dys ophy pheno-
ypes can be he esul o di e en mu a ion ypes bu also o he compound e ec o 2 di e en
gene ic diseases. Neu ol Gene 2017;3:e204; doi: 10.1212/NXG.0000000000000204
GLOSSARY
aCGH 5a ay compa a i e genomic hyb idiza ion; BMD 5Becke muscula dys ophy; CK 5c ea ine kinase; CNV 5copy
numbe a ian ; indels 5inse ions and dele ions; LGMD 5limb-gi dle muscula dys ophy; NGS 5nex -gene a ion sequenc-
ing; SNV 5single nucleo ide a ian .
Nex -gene a ion sequencing (NGS) me hods ha e become he mos common me hod o he
gene ic diagnosis o gene ically he e ogeneous diso de s.
1,2
We ha e p e iously de eloped a a -
ge ed NGS gene panel, MyoCap.
1
An upda ed e sion used he e includes p obes o he exons o
nea ly 300 myopa hy genes and candida e genes. Simila pla o ms a e cu en ly in use in many
labo a o ies.
2
The epo ed diagnos ic success a es a e signi ican ly highe han hose ob ained
by adi ional gene-by-gene sequencing.
2
Howe e , o e 50% pa ien s emain undiagnosed
when only concen a ing on single nucleo ide a ian s (SNVs) and small inse ions and dele ions
(indels).
2
Copy numbe a ian s (CNVs) a e de ined as genomic dele ions o duplica ions g ea e han
1 kb in size.
3
CNVs cause mic odele ion and mic oduplica ion synd omes, and hey ha e also
F om he Folkhälsan Ins i u e o Gene ics (S.V., M.S., M.J., L.S., M.A., K.K., K.P., B.U., P.H.), Medicum, Facul y o Biological and En i on-
men al Sciences (K.P.), Uni e si y o Helsinki, Finland; Neu omuscula Resea ch Cen e (B.U.), Tampe e Uni e si y and Uni e si y Hospi al,
Finland; Depa men o Neu ology (B.U.), Vaasa Cen al Hospi al, Finland; Biodonos ia Heal h Resea ch Ins i u e (A.S., A.L.D.M), Neu osciences
A ea, CIBERNED, Uni e si y o he Basque Coun y, San Sebas ián, Spain; and Cen e de Ré é ence Maladies Neu omusculai es (GNMH)
(A.-M.C.), Hôpi al Ma in APHP, Hendaye, F ance.
Funding in o ma ion and disclosu es a e p o ided a he end o he a icle. Go o Neu ology.o g/ng o ull disclosu e o ms. The A icle P ocessing
Cha ge was unded by he au ho s.
This is an open access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion-NonComme cial-NoDe i a i es License 4.0 (CC
BY-NC-ND), which pe mi s downloading and sha ing he wo k p o ided i is p ope ly ci ed. The wo k canno be changed in any way o used
comme cially wi hou pe mission om he jou nal.
Neu ology.o g/ng Copy igh © 2017 The Au ho (s). Published by Wol e s Kluwe Heal h, Inc. on behal o he Ame ican Academy o Neu ology. 1
been associa ed wi h se e al complex dis-
eases.
3,4
Gene ally, s udies aiming o he iden-
i ica ion o causa i e disease a ian s in
skele al muscle diso de s ha e no sys ema i-
cally used CNV sc eening. Mul iplex liga ion-
dependen p obe ampli ica ion has a lowe
h oughpu when he amoun o in es iga ed
genes inc eases.
5
A ay compa a i e genomic
hyb idiza ion (aCGH) has long been consid-
e ed he only eliable and obus pla o m o
CNV disco e y.
4
Howe e , in NGS s udies,
he diagnos ic e alua ion may end wi h a dis-
co e y o a single pa hogenic o likely pa ho-
genic mu a ion be o e he u iliza ion o
complemen a y me hods, which may lead o
an unde es ima ion o CNV con ibu ion o
diseases. Recen ly, se e al CNV analysis ools
o NGS da a ha e been de eloped and a e in
use o ou ine diagnosis.
3,4
He e, we desc ibe
he de ec ion o CNVs om NGS da a wi h
a combina ion o al eady a ailable bioin o -
ma ic ools.
METHODS S anda d p o ocol app o als, egis a ions,
and pa ien consen s. DNA samples o muscle disease pa ien s
and heal hy amily membe s we e ob ained om clinicians in di -
e en coun ies. The s udy was app o ed by he Coo dina ing
E hics Commi ee o he Hospi al Dis ic o Helsinki and Uusi-
maa. The samples we e ob ained acco ding o he Helsinki decla-
a ion. W i en in o med consen was ob ained om all pa ien s.
CNV assessmen om NGS da a. CNVs we e analyzed in
smalle ba ches om NGS da a alignmen iles (.bam) ob ained
by analyzing DNA om 791 myopa hy pa ien s wi h MyoCap.
1
We used 4 CNV analysis p og ams: Copy Numbe In e ence
F om Exome Reads (CoNIFER) 0.2.2,
6
eXome-Hidden Ma -
ko Model (XHMM) 1.1,
7
ExomeDep h 1.1.10,
8
and COpy
numbe De ec ion by EXome sequencing (CODEX) 1.4.0,
9
wi h he ecommended de aul se ings o each p og am.
A minimum o 1-bp o e lap was used o de e mine whe he calls
in e sec ing be ween di e en p og ams o igina ed om he same
CNV. In his a icle, we p io i ize he speci ic cases wi h a e y
high clinical in e es , ocusing on 7 a ian s de ec ed by mul iple
p og ams and e i ied by using independen ools.
CNV alida ion. PCR was pe o med o con i m CNVs in pa-
ien s I, IIIa, IIIb, IV, V, VI, and VII. P ime s we e designed
using P ime 3 4.0.0 (p ime 3.u .ee) ( able e-1, h p://links.
lww.com/NXG/A12), and PCR was pe o med wi h D eamTaq
DNA Polyme ase (The mo Fishe Scien i ic, Wal ham, MA)
( igu e e-1). A cus om aCGH (manusc ip in p epa a ion),
in es iga ing 187 o he genes included in MyoCap, was used o
con i m CNV de ec ed in pa ien IIa. Seg ega ion s udy in he
amily o pa ien IIa was pe o med by PCR ( able e-1).
RESULTS Table 1 shows he clinical ea u es o pa-
ien s in his s udy, gene ic indings, amoun o p o-
g ams ha de ec ed he CNV, and he CNV
e i ica ion me hod.
Pa ien I was iden i ied o ha e a he e ozygous
FINmaj mu a ion, an 11-bp inse ion/dele ion in
he i in (TTN) gene.
10
This a ian causes dominan
ibial muscula dys ophy, cha ac e ized by a la e age
a onse , no mal o sligh ly ele a ed c ea ine kinase
(CK) le els, and a mild dis al pheno ype. Howe e ,
his pa ien has p oximal weakness and a e y high
Table 1 Pa ien and a ian ea u es
ID Sex Onse Fi s symp om Muscle weakness CK
P e ious gene ic
es s Gene ic indings
De ec ed by n
p og ams/ e i ied wi h
IM Ea ly adul Weakness P oximal LL 9x CAPN3
a
;FKRP
a
;
LMNA
a
T;RIM32
a
TTN: c.107780_107790
delinsTGAAAGAAAAA1;DMD:
c.(643811_6439-1) _
(c.821711_c.8218-1)del
3/PCR
IIa; IIb M In ancy Weakness, exe cise
in ole ance
P oximal and dis al LL 11x CAPN3
a
;DYSF
a
TTN: c.107889delA:p.
(Lys35963Asn s*)1c.
(785511_7856-1)_
(970311_9704-1)del
4/aCGH
IIIa; IIIb F In ancy Weakness, walking
di icul ies
P oximal LL, Gowe s1.10x CAPN3
a
;ANO5
a
;WES SGCD: c.(?_-1) _(50511_506-1)
del hom
4/PCR
IV M In ancy Walking di icul ies P oximal .dis al;
Gowe s1
20x DMD
b
CAPN3: c.(309 11_310-1) _
(*1_?) hom
2/PCR
VM Ea ly adul Walking di icul ies P oximal LL 5–6x None DMD: c.(643811_6439-1) _
(c.821711_c.8218-1)del
3/PCR
VI M Ea ly adul Walking di icul ies Muscle a ophy; cal
hype ophy
10x None DMD: c.(643811_6439-1)_
(c.709811_c.7099-1)del
2/PCR
VII M In ancy Walking di icul ies P oximal LL, Gowe s1.10x None DMD: c.(592211_5923-1) _
(629011_6291-1)del
2/PCR
Abb e ia ions: aCGH 5a ay compa a i e genomic hyb idiza ion; CK 5c ea ine kinase; hom 5homozygous; LL 5lowe limbs; NA 5no a ailable.
Gene ic indings a e desc ibed using he e e ence ansc ip s: NM_001267550.1 o TTN, NM_004006.1 o DMD, NM_000231.2 o SGCD, and
NM_000070.2 o CAPN3.
a
Sange sequencing.
b
Mul iplex liga ion-dependen p obe ampli ica ion.
2Neu ology: Gene ics
CK le el. We excluded he p esence o o he dele e-
ious a ian s in TTN. Su p isingly, he pa ien was
ound o ha e a p e iously epo ed dys ophin
(DMD) dele ion (exons 45–55) known o cause
Becke muscula dys ophy (BMD).
11
Pa ien IIa has a p e iously epo ed Ibe ian
ameshi mu a ion, p.(Lys35963Asn s*), in he las
exon o TTN usually de e mining 1 componen o
he ecessi e dis al i inopa hy pheno ype.
10
Com-
pa ed wi h he o he ca ie s o his a ian , his
pa ien has a mo e se e e disease p og ession wi h
p oximal weakness, loss o ambula ion be o e he
age o 40 yea s, and ma ked hype CKemia. The
pa ien had no u he causa i e SNVs o indels in
he TTN gene. We iden i ied a la ge dele ion in TTN
(exons 34–41; igu e 1A) in ans wi h he Ibe ian
ameshi a ian in he p oband IIa as well as in
pa ien IIb, a simila ly a ec ed b o he . Thei heal hy
ela i es a e he e ozygous o only one o he a o e-
men ioned TTN mu a ions demons a ing he eces-
si e e ec o he de ec ed dele ion. The se e e dis al
and p oximal i inopa hies we e hus caused by he
compound he e ozygosi y o he Ibe ian ameshi
and he dele ion ( igu e 2).
Figu e 1 Visualiza ions o copy numbe a ian s o pa ien s IIa and IIIa
CoNIFER isualiza ions o a he e ozygous dele ion in he TTN in pa ien IIa (A) and a homozygous dele ion in he SGCD in pa ien IIIa (B) accompanied wi h
co esponding egions isualized wi h In eg a i e Genomics Viewe (IGV). In CoNIFER isualiza ions, he ed line co esponds o he ead dep hs o sample
wi h dele ion and black lines co espond o con ol samples. In he IGV isualiza ion, he i s ow shows he sample wi h dele ion and he second ow shows
a con ol sample wi hou dele ion.
Neu ology: Gene ics 3
Pa ien s IIIa and IIIb, wi h a se e e limb-gi dle
muscula dys ophy (LGMD) pheno ype, a e he
daugh e s o i s -cousin pa en s. Sange sequencing
o candida e genes (CAPN3 and ANO5), MyoCap,
and whole-exome sequencing had been pe o med
wi hou iden i ying he causa i e a ian . All he
CNV de ec ion p og ams iden i ied a homozygous
dele ion in he SGCD gene (exons 1–5) ( igu e 1B).
Pa ien IV is he child o consanguineous pa en s,
and he has been su e ing om lowe limb muscula
weakness in he lowe limbs since he age o 7. Dele-
ions and duplica ions in he DMD gene had been
excluded. Immunochemis y showed no mal s aining
o dys ophin as well as o he sa coglycans. A homo-
zygous dele ion in CAPN3 (exons 2–8) was de ec ed.
In 2 males wi h an LGMD-like pheno ype (pa-
ien s V and VI), we ound p e iously epo ed
DMD dele ions explaining he obse ed p oximal mus-
cula weakness. Bo h pa ien s ha e in- ame dele ions
(exons 45–55 and exons 45–48) causing BMD.
11
Pa ien VII wi h a Duchenne pheno ype was also
included in ou sc eening. As expec ed, a p e iously
epo ed ou -o - ame DMD dele ion (exons 42–43)
was iden i ied.
11
DISCUSSION Genomes a e usually analyzed o
SNVs and indels, bu s udies o CNVs a e o en
unde ep esen ed. CNVs in muscle diseases ha e
p e iously been s udied wi h complemen a y me h-
ods like a ge ed aCGH, which s ill emains he gold
s anda d echnique o CNV de ec ion.
4,5
He e, we
show ha combining analysis o all di e en mu a-
ion ypes enables in eg a ion o esul s and iden i-
ies he inal cause o he disease in se e al cases.
Complex e ec s like compound he e ozygosi y in
pa ien s IIa and IIb and compound gene ic disease
a ising om a ian s o di e en genes in pa ien I
can be un a eled. I has been sugges ed ha he
numbe o pa ien s wi h a combina ion o 2 o mo e
gene ic diseases is p obably unde es ima ed.
12
The
pheno ypic complexi y in hese pa ien s may e o-
neously be in e p e ed as a new gene ic disease wi h
uniden i ied gene ic de ec o as a pheno ypic expan-
sion o a single disease.
12
The p oximal pheno ype
seen in pa ien I is mainly due o BMD, as he
ypical an e io lowe -leg muscle lesions o he
FINmaj TTN mu a ion may de elop only a e age
60. Howe e , he iden i ica ion o mul ilocus
genomic a ian sisc ucial o ap ope gene ic
counseling in he amily.
The combina ion o 4 analysis ools aided us o
iden i y al eady known and p e iously unknown
CNVs. CNVs can explain some o he missing he -
i abili y and undiagnosed cases in skele al muscle
diso de s. An NGS-based s a egy o CNV de ec-
ion will be o g ea alue o inc easing he diagnos-
ic yield in pa ien s a ec ed by mendelian muscle
diseases. Cu en echnology does no adequa ely
cap u e epea expansion diseases such as myo onic
dys ophy o diseases ela ed o o he epe i i e ele-
men s. Long- ead sequencing echnologies may u -
he help he iden i ica ion and mapping o CNVs as
well as o epea expansions. Howe e , he clinical
in e p e a ion o CNVs emains challenging, in pa -
icula o CNVs iden i ied in genes wi hou p e i-
ously epo ed disease causing dele ions o
duplica ions. The inclusion o CNV da a in public
da abases, e.g., ExAC, could help in pa hogenici y
assessmen o CNVs.
AUTHOR CONTRIBUTIONS
Salla Välipakka: s udy concep and design, acquisi ion o da a, analysis
and in e p e a ion o da a, and d a ing he manusc ip o in ellec ual
con en . Ma co Sa a ese and M idul Joha i: s udy concep and design,
acquisi ion o da a, analysis and in e p e a ion o da a, and e ising he
manusc ip o in ellec ual con en . Lydia Saga h, Meha ji A umilli, and
Ki si Kiiski: acquisi ion o da a, analysis and in e p e a ion o da a, and
e ising he manusc ip o in ellec ual con en . Ame s Sáenz, Adol o
Lopez De Munain, and Ana-Ma ia Cobo: analysis and in e p e a ion o
da a. Ka a ina Pelin, Bja ne Udd, and Pe e Hackman: s udy concep and
design and e ising he manusc ip o in ellec ual con en .
ACKNOWLEDGMENT
The au ho s hank Me ja Soininen and Helena Luque o hei echnical
help and Sini Pen ilä, Tiina Suominen, and Sa a Leh inen o acquisi ion
o samples. They also hank all he pa ien s and amily membe s as well as
he clinicians who p o ided samples.
STUDY FUNDING
This s udy was suppo ed by he Folkhälsan Resea ch Founda ion, he
Jane and Aa os E kko Founda ion, he Academy o Finland (no. 138491,
B.U.), he Sig id Jusélius Founda ion, he Associa ion F ançaise con e les
Myopa hies, and he O ion Resea ch Founda ion s .
DISCLOSURE
S. Välipakka has ecei ed esea ch suppo om he Folkhälsan Resea ch
Founda ion. M. Sa a ese has ecei ed esea ch suppo om he Associa ion
F ançaise con e les Myopa hies and O ion Resea ch Founda ion. M. Joha i,
Figu e 2 Pedig ee o pa ien s IIa and IIb
Seg ega ion o he TTN Ibe ian ibial muscula dys ophy a ian , p.(Lys35963Asn s*), and
dele ion in he TTN (exons 34–41) in he amily o pa ien s IIa and IIb. The a ec ed b o he s
IIa and IIb (black squa es) a e compound he e ozygous o he a ian s. Thei heal hy pa en s
and siblings a e he e ozygous o only one o he a ian s. Gene ic indings a e desc ibed
using he e e ence ansc ip NM_001267550.1 o TTN.
4Neu ology: Gene ics
L. Saga h, M. A umilli, and K. Kiiski epo no disclosu es. A. Sáenz has
ecei ed esea ch suppo om Heal h Resea ch Fund (PI13-00722) o he
Spanish Minis y o Economy and Compe i i eness, he Eu opean Unión
(Eu opean Regional De elopmen Fund). A. Lopez De Munain has ecei ed
a el unding om Sano i. A. Cobo and K. Pelin epo no disclosu es. B.
Udd has se ed on he edi o ial boa d o Neu omuscula Diso de s and has
ecei ed g an s om Finska Läka esällskape (20,000 eu os 2017), he Sig id
Juselius Founda ion (gene al g an o he s udy o myopa hies), he Jane and
Aa os E kko Founda ion, and he Vasa Cen al Hospi al Resea ch Founda-
ion (g an pa ly o myopa hies). P. Hackman epo s no disclosu es. Go o
Neu ology.o g/ng o ull disclosu e o ms.
Recei ed Augus 22, 2017. Accep ed in inal o m Sep embe 28, 2017.
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