Copy number variation analysis increases the diagnostic yield in muscle diseases

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. REFERENCES 1. E ila A, A umilli M, Udd B, Hackman P. Ta ge ed nex - gene a ion sequencing assay o de ec ion o mu a ions in p ima y myopa hies. Neu omuscul Diso d 2016;26:7–15. 2. Nig o V, Sa a ese M. Nex -gene a ion sequencing ap- p oaches o he diagnosis o skele al muscle diso de s. Cu Opin Neu ol 2016;29:621–627. 3. Pi ooznia M, Goes FS, Zandi PP. Whole-genome CNV analysis: ad ances in compu a ional app oaches. F on Gene 2015;6:138. 4. Tan R, Wang Y, Kleins ein SE, e al. An e alua ion o copy numbe a ia ion de ec ion ools om whole-exome sequencing da a. Hum Mu a 2014;35:899–907. 5. Piluso G, Dionisi M, Del Vecchio Blanco F, e al. Mo o chip: a compa a i e genomic hyb idiza ion mic oa ay o copy-numbe mu a ions in 245 neu omuscula diso de s. Clin Chem 2011;57:1584–1596. 6. K umm N, Sudman PH, Ko A, e al. Copy numbe a ia ion de ec ion and geno yping om exome sequence da a. Genome Res 2012;22:1525–1532. 7. F ome M, Mo an JL, Chambe K, e al. Disco e y and s a is ical geno yping o copy-numbe a ia ion om whole-exome sequencing dep h. Am J Hum Gene 2012;91:597–607. 8. Plagnol V, Cu is J, Eps ein M, e al. A obus model o ead coun da a in exome sequencing expe imen s and implica ions o copy numbe a ian calling. Bioin o - ma ics 2012;28:2747–2754. 9. Jiang Y, Old idge DA, Diskin SJ, Zhang NR. CODEX: a no maliza ion and copy numbe a ia ion de ec ion me hod o whole exome sequencing. Nucleic Acids Res 2015;43:e39. 10. Hackman P, Ma chand S, Sa pa an a J, e al. T unca ing mu a ions in C- e minal i in may cause mo e se e e ibial muscula dys ophy (TMD). Neu omuscul Diso d 2008; 18:922–928. 11. Bladen CL, Salgado D, Monges S, e al. The TREAT- NMD DMD global da abase: analysis o mo e han 7,000 duchenne muscula dys ophy mu a ions. Hum Mu a 2015;36:395–402. 12. Posey JE, Ha el T, Liu P, e al. Resolu ion o disease pheno ypes esul ing om mul ilocus genomic a ia ion. N Engl J Med 2017;376:21–31. Neu ology: Gene ics 5