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Identification of a Dutch founder mutation in MUSK causing fetal akinesia deformation sequence.

Tan-Sindhunata MB, Mathijssen IB, Smit M, Baas F, de Vries JI, van der Voorn JP, Kluijt I, Hagen MA, Blom EW, Sistermans E, Meijers-Heijboer H, Waisfisz Q, Weiss MM, Groffen AJ - Eur. J. Hum. Genet. (2014)

Bottom Line: The carrier frequency in the genetic isolate was 8%, exclusively found in heterozygous carriers.Consistent with the established role of MUSK as a tyrosine kinase that orchestrates neuromuscular synaptogenesis, the fetal myopathy was accompanied by impaired acetylcholine receptor clustering and reduced tyrosine kinase activity at motor nerve endings.Taken together, the results strongly support a causal role of this founder mutation in MUSK, further expanding the gene set associated with FADS and offering new opportunities for prenatal genetic testing.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands.

ABSTRACT
Fetal akinesia deformation sequence (FADS) refers to a clinically and genetically heterogeneous group of disorders with congenital malformations related to impaired fetal movement. FADS can result from mutations in CHRNG, CHRNA1, CHRND, DOK7 and RAPSN; however, these genes only account for a minority of cases. Here we identify MUSK as a novel cause of lethal FADS. Fourteen affected fetuses from a Dutch genetic isolate were traced back to common ancestors 11 generations ago. Homozygosity mapping in two fetuses revealed MUSK as a candidate gene. All tested cases carried an identical homozygous variant c.1724T>C; p.(Ile575Thr) in the intracellular domain of MUSK. The carrier frequency in the genetic isolate was 8%, exclusively found in heterozygous carriers. Consistent with the established role of MUSK as a tyrosine kinase that orchestrates neuromuscular synaptogenesis, the fetal myopathy was accompanied by impaired acetylcholine receptor clustering and reduced tyrosine kinase activity at motor nerve endings. A functional assay in myocytes derived from human fetuses confirmed that the variant blocks MUSK-dependent motor endplate formation. Taken together, the results strongly support a causal role of this founder mutation in MUSK, further expanding the gene set associated with FADS and offering new opportunities for prenatal genetic testing.

No MeSH data available.


Related in: MedlinePlus

Wild-type but not mutant MUSK augments AChR clustering in MUSKI575T myocytes. (a) Primary myocytes derived from an affected fetus homozygous for MUSKI575T were plated on Matrigel-coated coverslips and transduced with lentiviral vectors encoding either MUSKWT or MUSKI575T. As a control, non-transduced MUSKI575T cells were used. Myocytes were treated with neuronal Agrin(3,4,8) and stained for nuclei (DAPI, displayed in green) and AChR clusters (Rhodamine-bungarotoxin, red). Representative examples from each experimental group are shown. (b) Mean±SEM values for the AChR cluster density, quantitated from 35 images from three coverslips of each group. ***P<0.001 (Student's t-test). (c) Immunoblot analysis of Flag-tagged MUSKWT and MUSKI575T constructs expressed in HEK293 cell lysates, suggesting similar yield and molecular mass.
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fig4: Wild-type but not mutant MUSK augments AChR clustering in MUSKI575T myocytes. (a) Primary myocytes derived from an affected fetus homozygous for MUSKI575T were plated on Matrigel-coated coverslips and transduced with lentiviral vectors encoding either MUSKWT or MUSKI575T. As a control, non-transduced MUSKI575T cells were used. Myocytes were treated with neuronal Agrin(3,4,8) and stained for nuclei (DAPI, displayed in green) and AChR clusters (Rhodamine-bungarotoxin, red). Representative examples from each experimental group are shown. (b) Mean±SEM values for the AChR cluster density, quantitated from 35 images from three coverslips of each group. ***P<0.001 (Student's t-test). (c) Immunoblot analysis of Flag-tagged MUSKWT and MUSKI575T constructs expressed in HEK293 cell lysates, suggesting similar yield and molecular mass.

Mentions: To corroborate the above findings in fetal muscle tissue, we next investigated the AChR-clustering activity of mutant and wild-type MUSK in a direct comparison. Wild-type and mutant MUSK were expressed in HEK293 cells with similar molecular mass (Figure 4c), migrating in SDS-PAGE as a 110-kDa protein consistent with previous studies.16, 17 In myocytes derived from a fetus homozygous for the c.1724T>C variant, overexpression of wild-type MUSK significantly increased the density of agrin-induced AChR clusters (P<0.001, n=35), whereas mutant MUSKI575T did not (P=0.65, n=35; see Figure 4a and b). Taken together, our observations demonstrate that the c.1724T>C; p.(Ile575Thr) variant strongly affects MUSK autoactivation, tyrosine phosphorylation and AChR clustering in both fetal tissue and cultured myocytes.


Identification of a Dutch founder mutation in MUSK causing fetal akinesia deformation sequence.

Tan-Sindhunata MB, Mathijssen IB, Smit M, Baas F, de Vries JI, van der Voorn JP, Kluijt I, Hagen MA, Blom EW, Sistermans E, Meijers-Heijboer H, Waisfisz Q, Weiss MM, Groffen AJ - Eur. J. Hum. Genet. (2014)

Wild-type but not mutant MUSK augments AChR clustering in MUSKI575T myocytes. (a) Primary myocytes derived from an affected fetus homozygous for MUSKI575T were plated on Matrigel-coated coverslips and transduced with lentiviral vectors encoding either MUSKWT or MUSKI575T. As a control, non-transduced MUSKI575T cells were used. Myocytes were treated with neuronal Agrin(3,4,8) and stained for nuclei (DAPI, displayed in green) and AChR clusters (Rhodamine-bungarotoxin, red). Representative examples from each experimental group are shown. (b) Mean±SEM values for the AChR cluster density, quantitated from 35 images from three coverslips of each group. ***P<0.001 (Student's t-test). (c) Immunoblot analysis of Flag-tagged MUSKWT and MUSKI575T constructs expressed in HEK293 cell lysates, suggesting similar yield and molecular mass.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4538208&req=5

fig4: Wild-type but not mutant MUSK augments AChR clustering in MUSKI575T myocytes. (a) Primary myocytes derived from an affected fetus homozygous for MUSKI575T were plated on Matrigel-coated coverslips and transduced with lentiviral vectors encoding either MUSKWT or MUSKI575T. As a control, non-transduced MUSKI575T cells were used. Myocytes were treated with neuronal Agrin(3,4,8) and stained for nuclei (DAPI, displayed in green) and AChR clusters (Rhodamine-bungarotoxin, red). Representative examples from each experimental group are shown. (b) Mean±SEM values for the AChR cluster density, quantitated from 35 images from three coverslips of each group. ***P<0.001 (Student's t-test). (c) Immunoblot analysis of Flag-tagged MUSKWT and MUSKI575T constructs expressed in HEK293 cell lysates, suggesting similar yield and molecular mass.
Mentions: To corroborate the above findings in fetal muscle tissue, we next investigated the AChR-clustering activity of mutant and wild-type MUSK in a direct comparison. Wild-type and mutant MUSK were expressed in HEK293 cells with similar molecular mass (Figure 4c), migrating in SDS-PAGE as a 110-kDa protein consistent with previous studies.16, 17 In myocytes derived from a fetus homozygous for the c.1724T>C variant, overexpression of wild-type MUSK significantly increased the density of agrin-induced AChR clusters (P<0.001, n=35), whereas mutant MUSKI575T did not (P=0.65, n=35; see Figure 4a and b). Taken together, our observations demonstrate that the c.1724T>C; p.(Ile575Thr) variant strongly affects MUSK autoactivation, tyrosine phosphorylation and AChR clustering in both fetal tissue and cultured myocytes.

Bottom Line: The carrier frequency in the genetic isolate was 8%, exclusively found in heterozygous carriers.Consistent with the established role of MUSK as a tyrosine kinase that orchestrates neuromuscular synaptogenesis, the fetal myopathy was accompanied by impaired acetylcholine receptor clustering and reduced tyrosine kinase activity at motor nerve endings.Taken together, the results strongly support a causal role of this founder mutation in MUSK, further expanding the gene set associated with FADS and offering new opportunities for prenatal genetic testing.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands.

ABSTRACT
Fetal akinesia deformation sequence (FADS) refers to a clinically and genetically heterogeneous group of disorders with congenital malformations related to impaired fetal movement. FADS can result from mutations in CHRNG, CHRNA1, CHRND, DOK7 and RAPSN; however, these genes only account for a minority of cases. Here we identify MUSK as a novel cause of lethal FADS. Fourteen affected fetuses from a Dutch genetic isolate were traced back to common ancestors 11 generations ago. Homozygosity mapping in two fetuses revealed MUSK as a candidate gene. All tested cases carried an identical homozygous variant c.1724T>C; p.(Ile575Thr) in the intracellular domain of MUSK. The carrier frequency in the genetic isolate was 8%, exclusively found in heterozygous carriers. Consistent with the established role of MUSK as a tyrosine kinase that orchestrates neuromuscular synaptogenesis, the fetal myopathy was accompanied by impaired acetylcholine receptor clustering and reduced tyrosine kinase activity at motor nerve endings. A functional assay in myocytes derived from human fetuses confirmed that the variant blocks MUSK-dependent motor endplate formation. Taken together, the results strongly support a causal role of this founder mutation in MUSK, further expanding the gene set associated with FADS and offering new opportunities for prenatal genetic testing.

No MeSH data available.


Related in: MedlinePlus