Limb girdle muscle dystrophy 2L (ANO5opathy) in Norway. Marijke Van Ghelue

Transcription

1 Limb girdle muscle dystrophy 2L (ANO5opathy) in Norway Marijke Van Ghelue

2 What is Limb-Girdle Muscular Dystrophy? Group of disorders affecting voluntary muscles, o mainly around the hips and shoulders the pelvic and shoulder girdles, also known as the limb girdles. Proximal muscles are most affected in LGMD. The distal muscles are affected late in LGMD, if at all. Over time (usually many years), the person with LGMD loses muscle bulk and strength. Eventually, he/she may need a wheelchair. LGMD can begin in childhood, adolescence, young adulthood or adulthood Both genders are affected

3 Inheritance Disorder Gene Product Chromosome Location Autosomal Dominant (8) LGMD1A LGMD1B LGMD1C LGMD1D LGMD1E LGMD1F LGMD1G LGMD1H Myotilin Lamin A/C Caveolin-3 DNAJB6 Desmin TNPO3?? 5q31 1q21 3p25 7q36 2q35 7q32 4q21 3p23 Autosomal Recessive (19) LGMD2A LGMD2B LGMD2C LGMD2D LGMD2E LGMD2F LGMD2G LGMD2H LGMD2I LGMD2J LGMD2K LGMD2L LGMD2M LGMD2N LGMD2O LGMD2P LGMD2Q LGMD2R LGMD2S Calpain-3 Dysferlin Gamma-sarcoglycan Alpha-sarcoglycan Beta-sarcoglycan Delta-sarcoglycan Telethonin TRIM32 FKRP Titin POMT1 ANO5 Fukutin POMT2 POMGnT1 DAG1 Plectin Desmin TRAPPC11 15q15 2p q12 17q21 4q12 5q33 17q12 9q33 19q13.3 2q24 9q34 11p14 9q31 14q24 1p32 3p21 8q24 2q35 4q35

4 Mercuri and Muntoni 2013 The Lancet 381, (9869):

5 History of LGMD2L/ANO5opathy 14 French Canadian patients from eight different families Quadriceps myopathy that evolved into an LGMD All demonstrate quadriceps femoris asymmetrical atrophy CK values were variable from normal to 6000 U/l. Clinical evaluations and MRI studies demonstrate a variable intrafamilial and interfamilial phenotype. Muscle pathology showed no sign of active inflammation increased endomysial connective tissue associated with basal lamina duplication collagen disorganization Bolduc et al., 2010 The American Journal of Human Genetics 86,

6 Bolduc et al., 2010 The American Journal of Human Genetics 86,

7 Bolduc et al., 2010 The American Journal of Human Genetics 86, Atrophy of the biceps in patient IX-11. Atrophy of the medial part of the quadriceps in patient IX-15

8 Homozygosity analysis via AutoSNPa22 identified a 4.7 Mb region of homozygosity on chromosome 11p14.3-p15 contained 11 annotated genes: 1. NAV2 (MIM ) 2. DBX1 3. HTATIP2 (MIM605628) 4. PRMT3 (MIM ) 5. SLC6A5 (MIM ) 6. NELL1 (MIM ) 7. ANO5/TMEM16E 8. FANCF (MIM603467) 9. GAS2 (MIM ) 10.SLC17A6 (MIM ) 11.SVIP.

9 Miyoshi distal myopathy Weakness that begins in the lower extremities, especially in the calf muscles. It can progress to other muscles as well. Symptoms usually begin between 15 and 30 years of age. The genetic defects that cause Miyoshi myopathy are in the gene for the dysferlin protein Defects in the dysferlin gene also can cause limb-girdle muscular dystrophy 2B, which results in muscle weakness in and around the hips and shoulders.

10 (B) In the MMD3 Dutch family IV, the mutation identified is c.191 dupa in exon 5 inherited as homozygous. The same mutation is present as heterozygous in the LGMD2L Family XXIX.

11 LGMD2L Allelic with Gnathodiaphyseal dysplasia : Dominant Distal myopathy (MMD3): Recessive Genotype-Phenotype correlations Variable phenotypes, mild & severe, with most mutations

12 ANO5 gene Exons: 22 Coding exons: 22 Transcript length: 6,651 bps Translation length: 913 amino acids

13 Anoctamin 5 protein Member of family of proteins with 8 transmembrane domains Anoctamins 1, 2 &3 are calcium-activated chloride channels (CaCC) ANO3 mutations cause: Dominant Craniocervical Dystonia (DYT23) Integral membrane glycoprotein Intracellular Functions: Myogenesis; Osteogenesis Mutations may cause: Defective membrane repair

14 Anoctamin protein family

15 Clinical Variability Intrafamilial: Prominent Variant syndromes Onset Distal myopathy Normal strength High CK Age: Mean = 33 years; Range 11 to 51 years Weakness: Quadriceps, Asymmetric Muscle Atrophy: Quadriceps; Biceps brachii; Lower leg Asymmetric Weakness Legs > Arms Scapular: Biceps Pelvic: Psoas; Quadriceps Posterior leg: See MMD3 Distal arms: Normal strength Face: Few patients Progression: Slow

16 Anette Skogstad, Birgitte Hestholm (2010) # investigated Exon 5 Exon 8 Exon hetr.c.191dupa, p.asn64lysfsx15 hetr.c.692g>t, p.gly231val hetr.c.191dupa, p.asn64lysfsx15 hetr.c.191dupa, p.asn64lysfsx15 hetr.c.2272c>t, p.arg758cys

17 2013 # investigated Exon 2 Exon 5 Exon 8 Exon 9 Exon 14 Exon 20 Exon c.191dupa, c.41-1g>a total 12 p.asn64lysfs*15 c.242a>g, p.asp81gly c.191dupa, p.asn64lysfs*15 c.191dupa, p.asn64lysfs*15 c.191dupa, p.asn64lysfs*15 c.191dupa, p.asn64lysfs*15 c.692g>t, p.gly231val c.692g>t, p.gly231val c.775a>t, p.lys259* c.775a>t, p.lys259* c.775a>t, p.lys259* c.1405t>c, p.trp469arg c.2272c>t, p.arg758cys c.2585_2586deltg, p.val862glyfs*16

18 Exon deletion exon ?? c.41-1g>a c.148c>t (p.arg50*) c.172c>t (p.arg58trp) c.191dupa (p.asn64lysfsx15) c.220c>t (p.arg74*) c.242a>g (p.asp81gly) c.294g>a (p.ala98ala) c.295-1g>a c.323t>g (p.leu108arg) c.352del c.397a>t G (p.ile133phe) c.400c>t (p.his 134Tyr) c.692g>t (p.gly231val) c.762+5g>t c.775a>t (p.lys259*) c.892g>a (p.glu298lys) c.1025g>a (p.cys341tyr) c.1066t>c (p.cys356arg) c.1066t>g (p.cys356gly) c.1261c>t (p.gln421*) c.1295c>g (p.ala432glyfsx49) c.1391delcinsa c.1405t>c T (p.trp469arg) c g> c.1520del A c.1609t>c T (p.ser537pro) c.1627dup c.1643c>t A (p.thr548ile) c.1733t>c (p.phe578ser) c a>g c.1965g>c (p.trp655cys) c.2018a>g (p.tyr673cys) c.2101a>g (p.asn701asp) c.2141c>g (p.thr714ser) c g> c.2272c>t A (p.arg758cys) c.2311_2312delc A c.2433t>a (p.tyr811*) c.2516t>g (p.met839arg)

19 Green label: dominant mutations causing gnathodiaphyseal dysplasia. Blue label: previously reported recessive mutations. Red label: mutations reported by Penttilä S et al., 2012 Encased blue labels: mutations also found in our study. Encased red labels: new mutations found in our study p.trp469arg c.2585_2586delgt p.val865glyfs16* p.lys259* Penttilä S et al., 2012 Neurology. 78(12):

20 Clinical findings Pasient Debut Pareser/atrofier Respirasjon Hjerteaff. amb Myal gier Pas.1 68 år Pas.2 57 år Pas.3 70 år Pas.4 45 år Pas.5 70 år Pas.6 43 år Pas.7 26 år Pas.8 50 år Pas.9 30 år 50 Nedsatt kraft hoftefleksjon, kneekstensjon, knefleksjon, plantarfleksjon i ankelledd. Paresene mest uttalte i bakre leggmuskler. Lett atrofi i bakre leggmuskler bilat. Ca. 50 Atrofier quadriceps hø.>>ve. og gastrocnemius bilateralt. Nedsatt kraft bakre leggmuskler, Hyperekstensjon kneledd bilateralt. Trendelenburg positiv ve. sida. Gowers positiv. 67 Lett atrofi frem og bakre lårmuskler Redusert kraft hofteekstensjon og knefleksjon bilateralt. 16 Lett atrofi lårmuskler ve. sida. Lett redusert kraft hoftefleksjon og knefleksjon ve. sida Andre sympt. Normal Nei Ja Nei Trettbarhet i muskulaturen Normal Nei Ja Nei Nei Moderat restriktiv ventilasjonsforstyrrelse, relatert til muskelsykd.? Atrieflimmer/ pacemaker, relatert til muskelsykdom? Ja Sviing i muskulaturen Normal Nei Ja Ja Stivhet og smerter i muskulaturen, særlig ve. bein. 43 Lett atrofi gastrocnemius medialt bilateralt. Normal Nei Ja Lette myalgier ved aktivitet 17 Lett atrofi periscapulært, lett atrofi pectoralis hø. sida, lette pareser i skulderbelte. Pareser for hoftefleksjon, kneekstensjon og bakre leggmuskler, atrofi quadriceps, hypertrofi leggmuskler ve.>hø.trendelenburg positiv, Gowers positiv Normal Nei Ja Nei Rabdomyolyse i 17 års alderen??????? 24 Lett atrofi vastus lateralis hø. sida, lett nedsatt kraft hoftefleksjon og fingerabduksjon. Normal Nei Ja Diffus Nei 20 Hypertrofi leggmuskler bilateralt, hø. > ve. Normal Nei Ja Ja Nedsatt utholdenhet, slitenhet Nei

21 Clinical findings Pasient CK EMG MRI Pas.1 68 år Pas.2 57 år Pas.3 70 år Pas.4 45 år Pas.5 70 år Pas.6 43 år Pas.7 26 år Pas.8 50 år Pas.9 30 år 2160 nevrogene forandringer, ikke sikkert myopatisk myopatisk Atrofi og fettinfiltrasjon biceps fem., semimembranosus, semitendinosus, quadriceps hø.>ve.. Lett ødem sartorius ve>hø.. Uttalt ødem og fettinfiltrasjon soelus og gastrocnemius lette nevrogene forandringer Fettinfiltrasjon biceps femoris, semimembranosus, adductor magnus Ødem semimembranosus, semitendinosus og gluteus maximus. MR av leggene normalt Uttalt myopatisk Fettinfiltrasjon og atrofi gastocnemius medialt >> lateralt på begge sider. Ødem i soleus, gastrocnemius lateralt hø. sida. Fettinfiltrasjon/atrofi gluteus maximus ve.>hø., semimembranosus, semitendinosus, U/l myopatisk beceps femoris. MR overarm: normalt MR bekken og lår/legg: fettinfiltrasjon/atrofi/lett ødem biceps og semitendinosus. Semimembranosus lett ødem hø. sida maks myopatisk Fettinfiltrasjon gastrocnemius bilateralt, ødem soleus og laterale gastrocnemius. Uttalt fettinfiltrasjon quadriceps og hamstrings bilateralt, litt ødem vastus lateralis Ingen Ingen opplysninger hos oss i Ingen opplysninger hos oss i journalen. opplysninger journalen. hos oss i journalen myopatisk Fettinfiltrasjon/atrofi tensor fasciae latae, semimembranosus, semitendinosus, biceps femoris, mediale gastrocnemius og mediale soleus. Lett atrofi vastus intermedius/medialis, adductor magnus. Lett ødem mediale soleus bil Meget lett myopatisk MR bekken/lår/legger: ødem og noe fettinfiltrasjon i bakre leggmuskler

22 Pasient Genetikk Muskelbiopsi Pas.1 68 år Pas.2 57 år Pas.3 70 år Pas.4 45 år Pas.5 70 år Pas.6 43 år Pas.7 26 år Pas.8 50 år Pas.9 30 år Exon5: Hom.c.191dupA, p.asn64lysfs15* Exon5: Hom c.191dupa, p.asn64lysfs15* Exon5: Hom.c.242A>G, p.asp81gly Exon9: Hom. c.775a>t, p.lys259* Exon5: Het.c.191dupA, p.asn64lysfs15* Exon5: Het.c.191dupA, p.asn64lysfs15* Exon5: Het.c.191dupA, p.asn64lysfs15* Exon5: Het.c.191dupA, p.asn64lysfs15* Exon9: Hetr.c.775A>T, p.lys259* Exon8: Het.c.692G>T, p.gly231val Exon9: Het.c.775A>T, p.lys259* Exon20: Het.c.2272C>T, p.arg758cys Exon14: Het.c.1405T>C, p.trp469arg Exon22: Hetr.c.2585_2586delTG, p.val862glyfs16* Vastus lateralis: Kaliberveksling, ingen nekroser, endomyseal fibrose, grupper med atrofiske fibre, opptil 20 fibre samlet, subsarcolemmalt opphopning av NADH pos. materiale i noen type 1 fibre, targetdannelse i type 1 fibre. Type 1 dominans. Abnorme mitochondrier med parakrystallinske legemer, streaming av myofilamenter. I alt forenlig med nevrogen myopati. Vastus lateralis: Mange atrofiske fibre, antydning til type 1 atrofi. Opphopning av abnorme myofibrillære strukturer i muskelfibrene ved EM Hø. vastus: ca. 50% inskutte kjerner, ved EM dessuten spredte atrofiske fibre. Ve. vastus: lett kaliberveksling, enkelte atrofiske fibre, fokal opphopning av NADH-pos. materiale subsarcolemmalt i enkelte type I fibre. 2 blå fibre ved COX Lett kaliberveksling, noen nekrotiske fibre, atrofiske og regenerte fibre Kaliberveksling, grupper av atrofiske fibre, nekrotiske fibre, 1 inklusjonslegeme i 1 fiber Ingen opplysninger hos oss i journalen. Kaliberveksling, noen nekrotiske fibre, atrofiske fibre, dels i grupper, ca. 30% inskutte kjerner, flekkvis opphopning av amorft materiale subsarcolemmalt ved NADH Biopsi fra vastus lateralis: lett kaliberveksling, enkeltliggende atrofiske og regenererte fibre, ingen nekroser. Minimal fibrose. 5% inskutte kjerner

23 Patient nr. 3: MRI of upper and lower limbs shows fatty infiltration of m. biceps femoris, m. semitendinosus on both sides and right semimembranosus.

24 Acknowledgment Christoph M. Wahl Kjell A. Arntzen Hanne Halvorsen Monica Ingebrigtsen Anette Skogstad Birgitte Hestholm Sissel Løseth Svein Ivar Mellgren Sigurd Lindahl Christoffer Jonsrud.

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26 CaCC CaCCs play key roles epithelial secretion (Kunzelmann et al. 2007) Membrane excitability in cardiac muscle and neurons (Andre et al.2003; Guo et al. 2008) olfactory transduction (Matthews& Reisert, 2003) regulation of vascular tone (Angermann et al. 2006) photoreception (Lalonde et al. 2008) Reponsible for polyspermia blockade in Xenopus lævis (Miledi, 1982; Barish,1983) involved in control of cell volume

27 Function of ANO5 What do we know Expressed during embryogenesis In somites and in myotomal cells Mostly expressed in skeletal and cardiac muscle Expression increased in mdx mice Upregulated during myogenic differentiation of cultured cells ANO5 and dysferlin show similar distribution in C2C12 myotubes and mucle tissue Speculations Calcium activated chloride channel (CaCC)? Responsible for chloride current needed during membrane repair Important for dysferlin dependent muscle membrane repair pathway? Importance for calcium influx known to occur with membrane damage

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29 Contractures: Unusual Cardiac: Usually normal Laboratory MRI: Early atrophy of medial quadriceps, hamstrings & adductor magnus Serum CK: Normal to 35,000 EMG: Myopathic; Some with spontaneous activity MRI: Asymmetric involvement Pathology: Muscle Muscle fibers Size: Varied Necrosis & Regeneration Internal nuclei Splitting Internal architecture: Irregular; Whorled Vacuoles: 2 patients Ultrastructure: Multifocal sarcolemmal lesions

30 B. Udd, 2012 Neuromuscular disorders 22 (1): 5 12

31 Mitsuhashi and Kang Seminars in Pediatric Neurology 19, (4):

32 Anoctamins ANion selective and have eight (OCT) transmembrane segments Gene family with currently 10 members The first member of the family, called GDD1 and now known as ANO5, was found as the gene responsible for gnathodiaphyseal dysplasia (Tsutsumi et al. 2004). The gene family was then assembled from bioinformatic analyses (Katoh & Katoh, 2003; Katoh & Katoh, 2005) ANO genes code Anoctamin/TMEM16 family members which are Ca2+-activated Cl channels (CaCC) or putative CaCC

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