analysis-skeletal-muscle-defects-larval-zebrafish-birefringence-touch

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

Boston Children's Hospital, Harvard Medical School

Congenital muscular dystrophy (CMD) is a clinically and genetically heterogeneous group of inherited muscle disorders. In patients, muscle weakness is usually present at or shortly after birth and is progressive in nature. Merosin deficient congenital muscular dystrophy (MDC1A) is a form of CMD caused by a defect in the laminin-α2 gene (LAMA2). Laminin-α2 is an extracellular matrix protein that interacts with the dystrophin-dystroglycan (DGC) complex in membranes providing stability to muscle fibers. In an N-ethyl-N-nitrosourea mutagenesis screen to develop zebrafish models of neuromuscular diseases, we identified a mutant fish that exhibits severe muscular dystrophy early in development. Genetic mapping identified a splice site mutation in the lama2 gene. This splice site is highly conserved in humans and this mutation results in mis-splicing of RNA and a loss of protein function. Homozygous lama2 mutant zebrafish, designated lama2(cl501/cl501), exhibited reduced motor function and progressive degeneration of skeletal muscles and died at 8-15 days post fertilization. The skeletal muscles exhibited damaged myosepta and detachment of myofibers in the affected fish. Laminin-α2 deficiency also resulted in growth defects in the brain and eye of the mutant fish. This laminin-α2 deficient mutant fish represents a novel disease model to develop therapies for modulating splicing defects in congenital muscular dystrophies and to restore the muscle function in human patients with CMD.

A splice site mutation in laminin-α2 results in a severe muscular dystrophy and growth abnormalities in zebrafish.

&#x6574;&#x4E2A; exome &#x6D4B;&#x5E8F; &#xFF08;WES&#xFF09;&#xFF0C;&#x5176;&#x4E2D;&#x5206;&#x6790;&#x6700;&#x6CE8;&#x660E;&#x5728;&#x4EBA;&#x7C7B;&#x57FA;&#x56E0;&#x7EC4;&#x4E2D;&#x57FA;&#x56E0;&#x7684;&#x7F16;&#x7801;&#x5E8F;&#x5217;&#xFF0C;&#x662F;&#x4E00;&#x79CD;&#x7406;&#x60F3;&#x7684;&#x5B66;&#x4E60;&#x5B8C;&#x5168;&#x6E17;&#x900F;&#x7684;&#x5E38;&#x67D3;&#x8272;&#x4F53;&#x9690;&#x6027;&#x75BE;&#x75C5;&#xFF0C;&#x53CA;&#x5373;&#x4F7F;&#x51CF;&#x5C11;&#x751F;&#x5B58;&#x53D7;&#x9650;&#x5236;&#x53D7;&#x5F71;&#x54CD;&#x4E2A;&#x4EBA;&#x7684;&#x6CE8;&#x518C;&#xFF0C;&#x4E00;&#x76F4;&#x975E;&#x5E38;&#x5F3A;&#x5927;&#x4E2D;&#x627E;&#x51FA;&#x5BFC;&#x81F4;&#x75BE;&#x75C5;&#x7684;&#x57FA;&#x56E0;&#x7A81;&#x53D8;&#x3002;&#x5728;&#x6B64;&#x7814;&#x7A76;&#x4E2D;&#xFF0C;&#x6211;&#x4EEC;&#x4E0E;&#x8840;&#x7F18;&#x7684;&#x5BB6;&#x8C31;&#xFF0C;&#x751A;&#x81F3;&#x5355;&#x4E2A;&#x53D7;&#x5F71;&#x54CD;&#x7684;&#x4E2A;&#x4F53;&#x662F;&#x53EF;&#x7528;&#xFF0C;&#x4EE5;&#x786E;&#x5B9A;&#x8D1F;&#x8D23;&#x4E25;&#x91CD;&#x5F71;&#x54CD;&#x53D1;&#x5C55;&#x7684;&#x5927;&#x8111;&#x3001; &#x773C;&#x775B;&#x548C;&#x808C;&#x8089;&#x57FA;&#x56E0;&#x5F02;&#x6784;&#x7684;&#x5E38;&#x67D3;&#x8272;&#x4F53;&#x9690;&#x6027;&#x969C;&#x788D;&#x7EFC;&#x5408;&#x5F81;&#x6C83;&#x514B;&#x534E;&#x5B9D; (WWS) &#x57FA;&#x56E0;&#x7A81;&#x53D8;&#x662F;&#x5185;&#x5BB9;&#x4E30;&#x5BCC;&#x7684; homozygosity &#x5206;&#x6790;&#x76F8;&#x7ED3;&#x5408;&#x996E;&#x6C34;&#x548C;&#x73AF;&#x5883;&#x536B;&#x751F;&#x3002;&#x5DF2;&#x77E5; 7 &#x4E2A;&#x57FA;&#x56E0;&#x7684;&#x7A81;&#x53D8;&#x5BFC;&#x81F4; WWS &#x548C;&#x89E3;&#x91CA; 50%-60%&#x7684;&#x60C5;&#x51B5;&#x4E0B;&#xFF0C;&#x4F46;&#x591A;&#x4E2A;&#x989D;&#x5916;&#x57FA;&#x56E0;&#x9884;&#x8BA1;&#x4F1A;&#x7A81;&#x53D8;&#x56E0;&#x4E3A;&#x4E0D;&#x660E;&#x539F;&#x56E0;&#x7684;&#x60C5;&#x51B5;&#x4E0B;&#x663E;&#x793A;&#x4E0D;&#x540C;&#x7684;&#x57FA;&#x56E0;&#x4F4D;&#x70B9;&#x7684;&#x6697;&#x793A;&#x6027;&#x8054;&#x7CFB;&#x3002;&#x6211;&#x4EEC;&#x8FD1;&#x4EB2; WWS &#x53D7;&#x707E;&#x5BB6;&#x5EAD;&#x4E2D;&#x4F7F;&#x7528;&#x996E;&#x6C34;&#x548C;&#x73AF;&#x5883;&#x536B;&#x751F;&#xFF0C;&#x5728; GTDC2 &#xFF08;&#x4E5F;&#x79F0;&#x4E3A; AGO61&#xFF09; &#x4E2D;&#x627E;&#x5230;&#x591A;&#x4E2A;&#x6709;&#x5BB3;&#x7A81;&#x53D8;&#x3002;GTDC2 &#x7684;&#x9884;&#x6D4B;&#x4F5C;&#x7528;&#xFF0C;&#x5982;&#x65E0;&#x7684;&#x7CD6;&#x662F;&#x7B26;&#x5408;&#x5176;&#x4ED6;&#x5DF2;&#x77E5;&#x7684; WWS &#x7684;&#x7A81;&#x53D8;&#x548C;&#x53C2;&#x4E0E;&#x7684;&#x8DE8;&#x819C;&#x53D7;&#x4F53;&#x808C;&#x7CD6;&#x57FA;&#x5316;&#x7684;&#x57FA;&#x56E0;&#x7684;&#x529F;&#x80FD;&#x3002;&#x56E0;&#x6B64;&#xFF0C;&#x5728;&#x5F00;&#x53D1;&#x8FC7;&#x7A0B;&#x4E2D;&#x63A2;&#x7D22;&#x7684; GTDC2 &#x673A;&#x80FD;&#x4E27;&#x5931;&#x7684;&#x4F5C;&#x7528;&#xFF0C;&#x6211;&#x4EEC;&#x4F7F;&#x7528; morpholino &#x4ECB;&#x5BFC;&#x7EC4;&#x5408;&#x5F0F;&#x7684;&#x5176;&#x6591;&#x9A6C;&#x9C7C; ortholog&#xFF0C;gtdc2&#x3002;&#x6211;&#x4EEC;&#x53D1;&#x73B0;&#xFF0C;gtdc2 &#x7EC4;&#x5408;&#x5F0F;&#x5728;&#x6591;&#x9A6C;&#x9C7C;&#x4E2D;&#x7684;&#x590D;&#x5236;&#x6240;&#x6709; WWS &#x529F;&#x80FD; &#xFF08;&#x8111;&#x79EF;&#x6C34;&#x3001; &#x773C;&#x90E8;&#x7F3A;&#x9677;&#x4EE5;&#x53CA;&#x808C;&#x8425;&#x517B;&#x4E0D;&#x826F;&#x75C7;&#xFF09;&#xFF0C;&#x5F3A;&#x70C8;&#x5EFA;&#x8BAE; GTDC2 &#x57FA;&#x56E0;&#x7A81;&#x53D8;&#x5BFC;&#x81F4; WWS&#x3002;

Exome &#x6D4B;&#x5E8F;&#x548C;&#x6591;&#x9A6C;&#x9C7C;&#x7684;&#x529F;&#x80FD;&#x6027;&#x9A8C;&#x8BC1;&#x6807;&#x8BC6; GTDC2 &#x7A81;&#x53D8;&#x4E3A;&#x6C83;&#x514B;&#x6C83;&#x4F2F;&#x683C;&#x7EFC;&#x5408;&#x5F81;&#x7684;&#x4E00;&#x4E2A;&#x539F;&#x56E0;&#x3002;

Whole-exome sequencing (WES), which analyzes the coding sequence of most annotated genes in the human genome, is an ideal approach to studying fully penetrant autosomal-recessive diseases, and it has been very powerful in identifying disease-causing mutations even when enrollment of affected individuals is limited by reduced survival. In this study, we combined WES with homozygosity analysis of consanguineous pedigrees, which are informative even when a single affected individual is available, to identify genetic mutations responsible for Walker-Warburg syndrome (WWS), a genetically heterogeneous autosomal-recessive disorder that severely affects the development of the brain, eyes, and muscle. Mutations in seven genes are known to cause WWS and explain 50%-60% of cases, but multiple additional genes are expected to be mutated because unexplained cases show suggestive linkage to diverse loci. Using WES in consanguineous WWS-affected families, we found multiple deleterious mutations in GTDC2 (also known as AGO61). GTDC2's predicted role as an uncharacterized glycosyltransferase is consistent with the function of other genes that are known to be mutated in WWS and that are involved in the glycosylation of the transmembrane receptor dystroglycan. Therefore, to explore the role of GTDC2 loss of function during development, we used morpholino-mediated knockdown of its zebrafish ortholog, gtdc2. We found that gtdc2 knockdown in zebrafish replicates all WWS features (hydrocephalus, ocular defects, and muscular dystrophy), strongly suggesting that GTDC2 mutations cause WWS.

Exome sequencing and functional validation in zebrafish identify GTDC2 mutations as a cause of Walker-Warburg syndrome.

Le s&eacute;quen&ccedil;age de l&#39;exome-ensemble (WES), qui analyse la s&eacute;quence codante de g&egrave;nes plus annot&eacute;s dans le g&eacute;nome humain, est une approche id&eacute;ale pour &eacute;tudier enti&egrave;rement par ressuage maladies autosomique r&eacute;cessive, et il a &eacute;t&eacute; tr&egrave;s puissant dans l&#39;identification des mutations pathog&egrave;nes, m&ecirc;me lorsque l&#39;inscription des personnes touch&eacute;es est limit&eacute;e par la r&eacute;duction de la survie. Dans cette &eacute;tude, nous avons combin&eacute; WES avec analyse d&#39;homozygotie des pedigrees consanguins, qui ont un caract&egrave;re informatif m&ecirc;me si une seule personne touch&eacute;e est disponible, pour identifier les mutations g&eacute;n&eacute;tiques responsables de syndrome de Walker-Warburg (WWS), une affection autosomique r&eacute;cessive g&eacute;n&eacute;tiquement h&eacute;t&eacute;rog&egrave;ne qui affecte gravement le d&eacute;veloppement du cerveau, des yeux et le muscle. Mutations dans les sept g&egrave;nes sont connues pour causer WWS et expliquer les 50-60 % des cas, mais plusieurs autres g&egrave;nes sont cens&eacute;s &ecirc;tre mut&eacute; car cas inexpliqu&eacute;s montrent le lien suggestif avec locus divers. &Agrave; l&#39;aide de WES en familles consanguines WWS touch&eacute;s, nous avons trouv&eacute; plusieurs mutations d&eacute;l&eacute;t&egrave;res dans GTDC2 (&eacute;galement connu comme AGO61). GTDC2 pr&eacute;dit r&ocirc;le puisqu&#39;une glycosyltransf&eacute;rase ind&eacute;termin&eacute; est compatible avec la fonction d&#39;autres g&egrave;nes qui sont connus pour &ecirc;tre mut&eacute; &agrave; WWS et qui sont impliqu&eacute;s dans la glycosylation de la dystroglycane r&eacute;cepteurs transmembranaires. Par cons&eacute;quent, afin d&#39;&eacute;tudier le r&ocirc;le de GTDC2 perte de fonction au cours du d&eacute;veloppement, nous avons utilis&eacute; morpholino-mediated knockdown de ses orthologues de poisson-z&egrave;bre, gtdc2. Nous avons trouv&eacute; que gtdc2 pr&eacute;cipitation chez le poisson z&egrave;bre fournit toutes les caract&eacute;ristiques WWS (hydroc&eacute;phalie, anomalies oculaires et la dystrophie musculaire), ce qui sugg&egrave;re fortement que les mutations de GTDC2 causent WWS.

S&eacute;quen&ccedil;age de l&#39;exome et validation fonctionnelle chez le poisson z&egrave;bre identifient les mutations de le GTDC2 comme une cause du syndrome de Walker-Warburg.

Ganz-Exome-Sequenzierung (WES), die die kodierende Sequenz am meisten kommentierte Gene im menschlichen Genom analysiert, ist ein idealer Ansatz, um vollst&auml;ndig Eindringpr&uuml;fung autosomal-rezessive Krankheiten, und es ist sehr leistungsf&auml;hig in identifizierende krankheitsverursachende Mutationen gewesen, auch wenn die Registrierung des betroffenen durch reduzierte &uuml;berleben begrenzt ist. In dieser Studie haben wir WES kombiniert, mit Syndroms Analyse der blutsverwandten Stammb&auml;ume, die informativ sind, selbst wenn eine betroffene Einzelperson ist verf&uuml;gbar, verantwortlich f&uuml;r Walker-Warburg-Syndrom (WWS), eine genetisch heterogene Erkrankung autosomal-rezessive genetische Mutationen identifiziert, die die Entwicklung des Gehirns, der Augen und der Muskel stark beeinflusst. Mutationen in Genen sieben sind bekannterma&szlig;en verursachen WWS und 50-60 % der F&auml;lle zu erkl&auml;ren, aber mehrere zus&auml;tzliche Gene sollen ge&auml;ndert werden, da ungekl&auml;rte F&auml;lle suggestiven Verbindung zu verschiedenen Loci zeigen. Mit WES in konsanguinen WWS-betroffenen Familien, fanden wir mehrere sch&auml;dliche Mutationen in GTDC2 (auch bekannt als AGO61). GTDC2 des vorhergesagt Rolle, wie ein uncharakterisiertes Glycosyltransferase steht im Einklang mit der Funktion anderer Gene, die bekannt, um in WWS mutiert sein und die Glykosylierung von Transmembran Rezeptor-Dystroglycan beteiligt sind. Daher verwendet, um die Rolle der GTDC2 Verlust der Funktion w&auml;hrend der Entwicklung zu untersuchen, wir Morpholino-vermittelte Niederschlag von seiner Zebrafisch-Ortholog, gtdc2. Wir fanden, dass gtdc2 Niederschlag im Zebrafisch repliziert alle WWS-Features (Hydrozephalus, Augendefekte und Muskeldystrophie), stark darauf hindeutet, dass GTDC2 Mutationen WWS f&uuml;hren.

Exome-Sequenzierung und funktionelle Validierung im Zebrafisch identifizieren GTDC2 Mutationen als Ursache von Walker-Warburg-Syndrom.

Intero-exome sequenziamento (WES), che analizza la sequenza codifica di pi&ugrave; con annotazioni geni nel genoma umano, &egrave; un approccio ideale per studiare malattie autosomica recessiva completamente penetranti, ed &egrave; stato molto potente nell&#39;identificare mutazioni che causano malattia anche quando la registrazione delle persone affette &egrave; limitata dalla ridotta sopravvivenza. In questo studio, abbiamo combinato WES con analisi di omozigosi di consanguinei pedigree, che sono di carattere informativo anche quando un singolo individuo interessato &egrave; disponibile, per identificare le mutazioni genetiche responsabili della sindrome di Walker-Warburg (WWS), una malattia autosomica recessiva geneticamente eterogenea che colpisce gravemente lo sviluppo del cervello, occhi e muscolare. Mutazioni nei sette geni sono noti per causare WWS e spiegare il 50-60% dei casi, ma pi&ugrave; geni supplementari dovrebbero essere mutati perch&eacute; inspiegabili casi mostrano suggestivo collegamento a diversi loci. Utilizzando WES in famiglie consanguinee WWS-colpiti, abbiamo trovato pi&ugrave; deleterie mutazioni in GTDC2 (noto anche come AGO61). GTDC2 previsto ruolo come un glycosyltransferase atipici &egrave; coerente con la funzione di altri geni che sono noti per essere mutato in WWS e che sono coinvolti nella glicosilazione del distroglicano recettore transmembrana. Pertanto, per esplorare il ruolo di GTDC2 perdita di funzione durante lo sviluppo, abbiamo usato atterramento oligonucleotides-mediata del suo ortholog zebrafish, gtdc2. Abbiamo trovato che gtdc2 atterramento in zebrafish replica tutte le caratteristiche WWS (idrocefalo, difetti oculari e distrofia muscolare), fortemente suggerendo che le GTDC2 mutazioni causano WWS.

Sequenziamento Exome e validazione funzionale in zebrafish identificare mutazioni GTDC2 come causa della sindrome di Walker-Warburg.

&#x4EBA;&#x9593;&#x306E;&#x30B2;&#x30CE;&#x30E0;&#x306E;&#x6700;&#x3082;&#x6CE8;&#x91C8;&#x3055;&#x308C;&#x305F;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x30B3;&#x30FC;&#x30C9;&#x914D;&#x5217;&#x3092;&#x5206;&#x6790;&#x3057;&#x3001;&#x5168;&#x4F53; - &#x30A8;&#x30AD;&#x30BD;&#x30FC;&#x30E0; &#xFF08;&#x30A6;&#x30A7;&#x30B9;&#xFF09; &#x306F;&#x3067;&#x3042;&#x308A;&#x3001;&#x7406;&#x60F3;&#x7684;&#x306A;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x306F;&#x5B8C;&#x5168;&#x306B;&#x6D78;&#x900F;&#x306E;&#x5E38;&#x67D3;&#x8272;&#x4F53;&#x52A3;&#x6027;&#x75BE;&#x60A3;&#x306E;&#x52C9;&#x5F37;&#x306B;&#x3082;&#x5F71;&#x97FF;&#x3092;&#x53D7;&#x3051;&#x308B;&#x500B;&#x4EBA;&#x306E;&#x767B;&#x9332;&#x306F;&#x3001;&#x524A;&#x6E1B;&#x306E;&#x751F;&#x5B58;&#x306B;&#x3088;&#x3063;&#x3066;&#x5236;&#x9650;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x5834;&#x5408;&#x3092;&#x8B58;&#x5225;&#x3059;&#x308B;&#x75C5;&#x6C17;&#x3092;&#x5F15;&#x304D;&#x8D77;&#x3053;&#x3059;&#x7A81;&#x7136;&#x5909;&#x7570;&#x3067;&#x975E;&#x5E38;&#x306B;&#x5F37;&#x529B;&#x306A;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x672C;&#x7814;&#x7A76;&#x3067;&#x306F;&#x3001;&#x540C;&#x80DE;&#x306E;&#x5206;&#x6790;&#x3082;&#x5F71;&#x97FF;&#x3092;&#x53D7;&#x3051;&#x308B;&#x500B;&#x4EBA;&#x30A6;&#x30A9;&#x30FC;&#x30AB;&#x30FC;&#x3001;&#x30A6;&#x30A9;&#x30FC;&#x30D0;&#x30FC;&#x30B0;&#x75C7;&#x5019;&#x7FA4; (WWS&#xFF09; &#x6DF1;&#x523B;&#x306A;&#x8133;&#x3001;&#x76EE;&#x3068;&#x7B4B;&#x8089;&#x306E;&#x958B;&#x767A;&#x306B;&#x5F71;&#x97FF;&#x3092;&#x4E0E;&#x3048;&#x308B;&#x907A;&#x4F1D;&#x7684;&#x7570;&#x7A2E;&#x5E38;&#x67D3;&#x8272;&#x4F53;&#x52A3;&#x6027;&#x75BE;&#x60A3;&#x306E;&#x8CAC;&#x4EFB;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x7A81;&#x7136;&#x5909;&#x7570;&#x3092;&#x8B58;&#x5225;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x5229;&#x7528;&#x53EF;&#x80FD;&#x306A;&#x5834;&#x5408;&#x306F;&#x6709;&#x76CA;&#x3067;&#x3059;&#x8FD1;&#x89AA;&#x306E;&#x5BB6;&#x7CFB;&#x306E;&#x30A6;&#x30A7;&#x30B9;&#x3092;&#x7D44;&#x307F;&#x5408;&#x308F;&#x305B;&#x307E;&#x3059;&#x3002;WWS &#x306E;&#x539F;&#x56E0;&#x306E;&#x5834;&#x5408;&#x3001;50-60% &#x3092;&#x8AAC;&#x660E;&#x3059;&#x308B; 7 &#x3064;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x7A81;&#x7136;&#x5909;&#x7570;&#x304C;&#x77E5;&#x3089;&#x308C;&#x3066;&#x3044;&#x308B;&#x304C;&#x3001;&#x8907;&#x6570;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x591A;&#x69D8;&#x306A;&#x907A;&#x4F1D;&#x5B50;&#x5EA7;&#x3078;&#x306E;&#x30EA;&#x30F3;&#x30B1;&#x30FC;&#x30B8;&#x3092;&#x6311;&#x767A;&#x7684;&#x306A;&#x539F;&#x56E0;&#x4E0D;&#x660E;&#x306E;&#x5834;&#x5408;&#x3092;&#x793A;&#x3059;&#x305F;&#x3081;&#x306B;&#x5909;&#x7570;&#x3059;&#x308B;&#x3068;&#x4E88;&#x60F3;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x30A6;&#x30A7;&#x30B9;&#x8FD1;&#x89AA;&#x5BB6;&#x65CF; WWS &#x5F71;&#x97FF;&#x3092;&#x53D7;&#x3051;&#x308B;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3001;&#x306B;&#x304A;&#x3044;&#x3066; &#xFF08;&#x3082; AGO61 &#x3068;&#x3057;&#x3066;&#x77E5;&#x3089;&#x308C;&#x3066;&#x3044;&#x308B;&#xFF09; GTDC2 &#x3067;&#x8907;&#x6570;&#x6709;&#x5BB3;&#x306A;&#x7A81;&#x7136;&#x5909;&#x7570;&#x3092;&#x898B;&#x3064;&#x3051;&#x305F;&#x3002;GTDC2 &#x306E;&#x5F79;&#x5272;&#x3001;&#x672A;&#x540C;&#x5B9A;&#x306E;&#x7CD6;&#x8EE2;&#x79FB;&#x9175;&#x7D20;&#x3001;WWS &#x306B;&#x5909;&#x7570;&#x3059;&#x308B;&#x77E5;&#x3089;&#x308C;&#x3066;&#x3044;&#x308B;&#x3057;&#x3001;&#x819C;&#x8CAB;&#x901A;&#x578B;&#x53D7;&#x5BB9;&#x4F53;&#x30B8;&#x30B9;&#x30C8;&#x30ED;&#x30B0;&#x30EA;&#x30AB;&#x30F3;&#x7CD6;&#x9396;&#x306E;&#x95A2;&#x4E0E;&#x3057;&#x3066;&#x3044;&#x308B;&#x4ED6;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x6A5F;&#x80FD;&#x3068;&#x4E00;&#x8CAB;&#x6027;&#x306E;&#x3042;&#x308B;&#x4E88;&#x6E2C;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;&#x958B;&#x767A;&#x4E2D;&#x306E; GTDC2 &#x640D;&#x5931;&#x95A2;&#x6570;&#x306E;&#x5F79;&#x5272;&#x3092;&#x63A2;&#x7D22;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#x305D;&#x306E;&#x30BC;&#x30D6;&#x30E9;&#x30D5;&#x30A3;&#x30C3;&#x30B7;&#x30E5; &#x30AA;&#x30EB;&#x30BD;&#x30ED;&#x30B0;&#x3001;gtdc2 &#x306E; morpholino &#x3092;&#x4ECB;&#x3057;&#x305F;&#x30CE;&#x30C3;&#x30AF;&#x30C0;&#x30A6;&#x30F3;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x6211;&#x3005; &#x306F; gtdc2 &#x304C;&#x898B;&#x3064;&#x304B;&#x308A;&#x307E;&#x3057;&#x305F;&#x30CE;&#x30C3;&#x30AF;&#x30C0;&#x30A6;&#x30F3; &#x30BC;&#x30D6;&#x30E9;&#x30D5;&#x30A3;&#x30C3;&#x30B7;&#x30E5;&#x306E;&#x8907;&#x88FD;&#x3059;&#x3079;&#x3066; WWS &#x6A5F;&#x80FD; &#xFF08;&#x6C34;&#x982D;&#x75C7;&#x3001;&#x773C;&#x306E;&#x6B20;&#x9665;&#x3001;&#x7B4B;&#x30B8;&#x30B9;&#x30C8;&#x30ED;&#x30D5;&#x30A3;&#x30FC;) &#x5F37;&#x304F; WWS GTDC2 &#x7A81;&#x7136;&#x5909;&#x7570;&#x3092;&#x5F15;&#x304D;&#x8D77;&#x3053;&#x3059;&#x3053;&#x3068;&#x3092;&#x793A;&#x5506;&#x3057;&#x307E;&#x3059;&#x3002;

&#x30A8;&#x30AD;&#x30BD;&#x30FC;&#x30E0;&#x3068;&#x6A5F;&#x80FD;&#x691C;&#x8A3C;&#x30BC;&#x30D6;&#x30E9;&#x30D5;&#x30A3;&#x30C3;&#x30B7;&#x30E5;&#x306E;&#x30A6;&#x30A9;&#x30FC;&#x30AB;&#x30FC;&#x3001;&#x30A6;&#x30A9;&#x30FC;&#x30D0;&#x30FC;&#x30B0;&#x75C7;&#x5019;&#x7FA4;&#x306E;&#x539F;&#x56E0;&#x3068;&#x3057;&#x3066; GTDC2 &#x7A81;&#x7136;&#x5909;&#x7570;&#x3092;&#x8B58;&#x5225;&#x3057;&#x307E;&#x3059;&#x3002;

&#xC778;&#xAC04; &#xAC8C;&#xB188;&#xC5D0; &#xAC00;&#xC7A5; &#xC8FC;&#xC11D;&#xC774; &#xCD94;&#xAC00; &#xB41C; &#xC720;&#xC804;&#xC790;&#xC758; &#xCF54;&#xB529; &#xC21C;&#xC11C;&#xB97C; &#xBD84;&#xC11D; &#xD558; &#xC5EC; &#xC804;&#xCCB4; exome &#xC2DC;&#xD000;&#xC2F1; (WES) &#xC644;&#xC804;&#xD788; &#xCE68;&#xD22C; &#xC0C1; &#xC5FC;&#xC0C9;&#xCCB4; &#xC5F4;&#xC131; &#xC9C8;&#xD658;&#xC744; &#xACF5;&#xBD80; &#xD558; &#xACE0; &#xC774;&#xC0C1;&#xC801;&#xC778; &#xC811;&#xADFC; &#xC774;&#xBA70; &#xAC10;&#xC18C; &#xC0DD;&#xC874;&#xC5D0; &#xC758;&#xD574; &#xC601;&#xD5A5;&#xC744; &#xBC1B;&#xB294; &#xAC1C;&#xC778;&#xC758; &#xB4F1;&#xB85D;&#xC740; &#xC81C;&#xD55C; &#xD558;&#xB294; &#xACBD;&#xC6B0;&#xC5D0; &#xC9C8;&#xBCD1;&#xC744; &#xC77C;&#xC73C;&#xD0A4;&#xB294; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xC2DD;&#xBCC4;&#xC5D0; &#xB9E4;&#xC6B0; &#xAC15;&#xB825;&#xD55C; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC774; &#xC5F0;&#xAD6C;&#xC5D0;&#xC11C; &#xC6B0;&#xB9AC;&#xB294; &#xD558;&#xB098;&#xC758; &#xC601;&#xD5A5;&#xC744; &#xBC1B;&#xB294; &#xAC1C;&#xC778;&#xC740; &#xC720;&#xC804;&#xC790; &#xBCC0;&#xC774; &#xC6CC;&#xCEE4; &#xBC14;&#xB974; &#xBD80;&#xB974; &#xD06C; &#xC99D;&#xD6C4;&#xAD70; (WWS) &#xC2EC;&#xAC01; &#xD558; &#xAC8C; &#xB1CC;, &#xB208;, &#xADFC;&#xC721;&#xC758; &#xBC1C;&#xB2EC;&#xC5D0; &#xC601;&#xD5A5;&#xC744; &#xBBF8;&#xCE58;&#xB294; &#xC720;&#xC804;&#xC790; &#xC720;&#xD615;&#xC774; &#xB2E4;&#xB978; &#xC0C1; &#xC5FC;&#xC0C9;&#xCCB4; &#xC5F4;&#xC131; &#xC7A5;&#xC560;&#xC5D0; &#xB300; &#xD55C; &#xCC45;&#xC784;&#xC744; &#xC2DD;&#xBCC4; &#xD558;&#xAE30; &#xC704;&#xD574; &#xC0AC;&#xC6A9;&#xD560; &#xC218; &#xC788;&#xB294; &#xC815;&#xBCF4;&#xB97C; &#xC81C;&#xACF5; &#xD558;&#xC9C0; &#xC54A;&#xC740; consanguineous &#xD608;&#xD1B5;&#xC758; homozygosity &#xBD84;&#xC11D; &#xC6E8;&#xC2A4; &#xACB0;&#xD569;. 7 &#xAC1C;&#xC758; &#xC720;&#xC804;&#xC790;&#xC5D0; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; WWS &#xBC1C;&#xC0DD; &#xD558; &#xACE0; 50%-60%&#xC758; &#xACBD;&#xC6B0;, &#xC124;&#xBA85;&#xC5D0; &#xC54C;&#xB824;&#xC838; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xD558;&#xC9C0;&#xB9CC; &#xC5EC;&#xB7EC; &#xCD94;&#xAC00;&#xC801;&#xC778; &#xC720;&#xC804;&#xC790; &#xC6D0;&#xC778; &#xBD88;&#xBA85;&#xB41C;&#xC758; &#xACBD;&#xC6B0; &#xB2E4;&#xC591; &#xD55C; loci &#xC554;&#xC2DC; &#xB9C1;&#xD06C; &#xD45C;&#xC2DC; &#xD558;&#xAE30; &#xB54C;&#xBB38;&#xC5D0; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xB420; &#xAC83;&#xC73C;&#xB85C; &#xC608;&#xC0C1; &#xB41C;&#xB2E4;. GTDC2 (AGO61 &#xB77C;&#xACE0;&#xB3C4; &#xD568;)&#xC5D0; &#xC5EC;&#xB7EC; &#xD574;&#xB85C;&#xC6B4; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xBC1C;&#xACAC; &#xC6E8;&#xC2A4;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; consanguineous WWS &#xC601;&#xD5A5;&#xC744; &#xBC1B;&#xB294; &#xAC00;&#xC871;&#xC5D0;&#xC11C;, &#xC6B0;&#xB9AC;. Gtdc2&#xC758; &#xC0C8;&#xB86D;&#xAC70;&#xB098; glycosyltransferase &#xC77C;&#xCE58; &#xD558;&#xB294; WWS&#xC5D0;&#xC11C; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774;&#xC73C;&#xB85C; &#xC54C;&#xB824;&#xC838; &#xC788;&#xC73C;&#xBA70; &#xADF8; &#xB9C9; &#xD6A1;&#xB2E8; &#xC218;&#xC6A9; &#xCCB4; dystroglycan glycosylation&#xC5D0; &#xAD00;&#xB828; &#xB41C; &#xB2E4;&#xB978; &#xC720;&#xC804;&#xC790;&#xC758; &#xAE30;&#xB2A5;&#xC73C;&#xB85C; &#xC5ED;&#xD560;&#xC744; &#xC608;&#xCE21; &#xD588;&#xB2E4;. &#xB530;&#xB77C;&#xC11C;, &#xAC1C;&#xBC1C; &#xD558;&#xB294; &#xB3D9;&#xC548; &#xAE30;&#xB2A5;&#xC758; &#xC190;&#xC2E4; gtdc2&#xC758; &#xC5ED;&#xD560;&#xC744; &#xD0D0;&#xAD6C; &#xD558;&#xB294; zebrafish ortholog, gtdc2&#xC758; morpholino &#xC911;&#xC7AC; &#xCD5C;&#xC800;&#xC758; &#xC0AC;&#xC6A9; &#xD588;&#xC2B5;&#xB2C8;&#xB2E4;. &#xADF8; gtdc2&#xB97C; &#xCC3E;&#xC558;&#xC2B5;&#xB2C8;&#xB2E4;. &#xCD5C;&#xC800;&#xC758; zebrafish&#xC758; &#xBCF5;&#xC81C; &#xBAA8;&#xB4E0; WWS &#xAE30;&#xB2A5; (&#xC218; &#xB450; &#xC99D;, &#xB208; &#xACB0;&#xD568; &#xBC0F; &#xADFC; &#xC704;&#xCD95; &#xC99D;), GTDC2 &#xBCC0;&#xC774; &#xBC1C;&#xC0DD;&#xD560; WWS &#xAC15;&#xB825; &#xD558; &#xAC8C; &#xC81C;&#xC548; &#xD558;&#xB294; &#xBC29;&#xBC95;.

Exome &#xC2DC;&#xD000;&#xC2F1; &#xBC0F; &#xAE30;&#xB2A5;&#xC801; &#xC720;&#xD6A8;&#xC131; &#xAC80;&#xC0AC; zebrafish&#xC758; &#xC6CC;&#xCEE4; &#xBC14;&#xB974; &#xBD80;&#xB974; &#xD06C; &#xC99D;&#xD6C4;&#xAD70;&#xC758; &#xC6D0;&#xC778;&#xC73C;&#xB85C; GTDC2 &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xC2DD;&#xBCC4;&#xD569;&#xB2C8;&#xB2E4;.

La secuencia entera-exoma (WES), que analiza la secuencia de codificaci&oacute;n de genes m&aacute;s comentadas en el genoma humano, es un enfoque ideal para estudiar enfermedades totalmente penetrantes de recesivo de un autosoma, y ha sido muy potente en la identificaci&oacute;n de las mutaciones que causan enfermedades incluso cuando la inscripci&oacute;n de las personas afectadas se ve limitada por la supervivencia reducida. En este estudio, combinamos a WES con an&aacute;lisis de calidad de homozigoto de pedigr&iacute;es consangu&iacute;neas, que son de caracter informativo incluso cuando un individuo afectado est&aacute; disponible, para identificar mutaciones gen&eacute;ticas responsables del s&iacute;ndrome de Walker-Warburg (WWS), un desorden recesivo de un autosoma gen&eacute;tico heterog&eacute;neo que afecta gravemente el desarrollo del cerebro, ojos y m&uacute;sculo. Mutaciones en siete genes son conocidas por causar WWS y explicar el 50% - 60% de los casos, pero se espera que varios genes adicionales para ser transformado debido a casos inexplicables muestran sugerentes vinculaciones a diversos lugares geom&eacute;tricos. Usando a WES en familias consangu&iacute;neas afectadas WWS, encontramos m&uacute;ltiples mutaciones delet&eacute;reas en GTDC2 (tambi&eacute;n conocido como AGO61). GTDC2 predijo el papel como un desacostumbrado glicosiltransferasa concuerda con la funci&oacute;n de otros genes que se sabe para ser transformado en WWS y que participan en la glicosilaci&oacute;n de la dystroglycan receptor transmembrana. Por lo tanto, para explorar el papel de GTDC2 p&eacute;rdida de funci&oacute;n durante el desarrollo, utilizamos Morfolino mediada por la ca&iacute;da de su ort&oacute;logo de pez cebra, gtdc2. Encontramos que gtdc2 ca&iacute;da en el pez cebra Replica todas las caracter&iacute;sticas WWS (hidrocefalia, defectos oculares y distrofia muscular), sugiriendo fuertemente que las mutaciones de GTDC2 causar WWS.

Secuenciaci&oacute;n del exoma y validaci&oacute;n funcional en el pez cebra identifican mutaciones GTDC2 como causa del s&iacute;ndrome de Walker-Warburg.

X-linked myotubular myopathy (XLMTM) is a congenital disorder caused by mutations of the myotubularin gene, MTM1. Myotubularin belongs to a large family of conserved lipid phosphatases that include both catalytically active and inactive myotubularin-related proteins (i.e., "MTMRs"). Biochemically, catalytically inactive MTMRs have been shown to form heteroligomers with active members within the myotubularin family through protein-protein interactions. However, the pathophysiological significance of catalytically inactive MTMRs remains unknown in muscle. By in vitro as well as in vivo studies, we have identified that catalytically inactive myotubularin-related protein 12 (MTMR12) binds to myotubularin in skeletal muscle. Knockdown of the mtmr12 gene in zebrafish resulted in skeletal muscle defects and impaired motor function. Analysis of mtmr12 morphant fish showed pathological changes with central nucleation, disorganized Triads, myofiber hypotrophy and whorled membrane structures similar to those seen in X-linked myotubular myopathy. Biochemical studies showed that deficiency of MTMR12 results in reduced levels of myotubularin protein in zebrafish and mammalian C2C12 cells. Loss of myotubularin also resulted in reduction of MTMR12 protein in C2C12 cells, mice and humans. Moreover, XLMTM mutations within the myotubularin interaction domain disrupted binding to MTMR12 in cell culture. Analysis of human XLMTM patient myotubes showed that mutations that disrupt the interaction between myotubularin and MTMR12 proteins result in reduction of both myotubularin and MTMR12. These studies strongly support the concept that interactions between myotubularin and MTMR12 are required for the stability of their functional protein complex in normal skeletal muscles. This work highlights an important physiological function of catalytically inactive phosphatases in the pathophysiology of myotubular myopathy and suggests a novel therapeutic approach through identification of drugs that could stabilize the myotubularin-MTMR12 complex and hence ameliorate this disorder.

Loss of catalytically inactive lipid phosphatase myotubularin-related protein 12 impairs myotubularin stability and promotes centronuclear myopathy in zebrafish.

Nemaline myopathy (NM) is a rare congenital muscle disorder primarily affecting skeletal muscles that results in neonatal death in severe cases as a result of associated respiratory insufficiency. NM is thought to be a disease of sarcomeric thin filaments as six of eight known genes whose mutation can cause NM encode components of that structure, however, recent discoveries of mutations in non-thin filament genes has called this model in question. We performed whole-exome sequencing and have identified recessive small deletions and missense changes in the Kelch-like family member 41 gene (KLHL41) in four individuals from unrelated NM families. Sanger sequencing of 116 unrelated individuals with NM identified compound heterozygous changes in KLHL41 in a fifth family. Mutations in KLHL41 showed a clear phenotype-genotype correlation: Frameshift mutations resulted in severe phenotypes with neonatal death, whereas missense changes resulted in impaired motor function with survival into late childhood and/or early adulthood. Functional studies in zebrafish showed that loss of Klhl41 results in highly diminished motor function and myofibrillar disorganization, with nemaline body formation, the pathological hallmark of NM. These studies expand the genetic heterogeneity of NM and implicate a critical role of BTB-Kelch family members in maintenance of sarcomeric integrity in NM.

Identification of KLHL41 Mutations Implicates BTB-Kelch-Mediated Ubiquitination as an Alternate Pathway to Myofibrillar Disruption in Nemaline Myopathy.

Vandana A. Gupta

Division of Genetics and Genomics,
Manton Center for Orphan Disease Research,
Division of Genetics and Genomics, Manton Center for Orphan Disease Research

Boston Children's Hospital, Harvard Medical School

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Vandana A. Gupta

Division of Genetics and Genomics,Manton Center for Orphan Disease Research,Division of Genetics and Genomics, Manton Center for Orphan Disease Research

Boston Children's Hospital, Harvard Medical School

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Division of Genetics and Genomics,
Manton Center for Orphan Disease Research,
Division of Genetics and Genomics, Manton Center for Orphan Disease Research