Mutations in DPAGT1 Cause a Limb-Girdle Congenital Myasthenic Syndrome with Tubular Aggregates

Congenital myasthenic syndromes are a heterogeneous group of inherited disorders that arise from impaired signal transmission at the neuromuscular synapse. They are characterized by fatigable muscle weakness. We performed whole-exome sequencing to determine the underlying defect in a group of individuals with an inherited limb-girdle pattern of myasthenic weakness. We identify DPAGT1 as a gene in which mutations cause a congenital myasthenic syndrome. We describe seven different mutations found in five individuals with DPAGT1 mutations. The affected individuals share a number of common clinical features, including involvement of proximal limb muscles, response to treatment with cholinesterase inhibitors and 3,4-diaminopyridine, and the presence of tubular aggregates in muscle biopsies. Analyses of motor endplates from two of the individuals demonstrate a severe reduction of endplate acetylcholine receptors. DPAGT1 is an essential enzyme catalyzing the first committed step of N-linked protein glycosylation. Our findings underscore the importance of N-linked protein glycosylation for proper functioning of the neuromuscular junction. Using the DPAGT1-specific inhibitor tunicamycin, we show that DPAGT1 is required for efficient glycosylation of acetylcholine-receptor subunits and for efficient export of acetylcholine receptors to the cell surface. We suggest that the primary pathogenic mechanism of DPAGT1 mutations is reduced levels of acetylcholine receptors at the endplate region. These individuals share clinical features similar to those of congenital myasthenic syndrome due to GFPT1 mutations, and their disorder might be part of a larger subgroup comprising the congenital myasthenic syndromes that result from defects in the N-linked glycosylation pathway and that manifest through impaired neuromuscular transmission. Congenital myasthenic syndromes are a heterogeneous group of inherited disorders that arise from impaired signal transmission at the neuromuscular synapse. They are characterized by fatigable muscle weakness. We performed whole-exome sequencing to determine the underlying defect in a group of individuals with an inherited limb-girdle pattern of myasthenic weakness. We identify DPAGT1 as a gene in which mutations cause a congenital myasthenic syndrome. We describe seven different mutations found in five individuals with DPAGT1 mutations. The affected individuals share a number of common clinical features, including involvement of proximal limb muscles, response to treatment with cholinesterase inhibitors and 3,4-diaminopyridine, and the presence of tubular aggregates in muscle biopsies. Analyses of motor endplates from two of the individuals demonstrate a severe reduction of endplate acetylcholine receptors. DPAGT1 is an essential enzyme catalyzing the first committed step of N-linked protein glycosylation. Our findings underscore the importance of N-linked protein glycosylation for proper functioning of the neuromuscular junction. Using the DPAGT1-specific inhibitor tunicamycin, we show that DPAGT1 is required for efficient glycosylation of acetylcholine-receptor subunits and for efficient export of acetylcholine receptors to the cell surface. We suggest that the primary pathogenic mechanism of DPAGT1 mutations is reduced levels of acetylcholine receptors at the endplate region. These individuals share clinical features similar to those of congenital myasthenic syndrome due to GFPT1 mutations, and their disorder might be part of a larger subgroup comprising the congenital myasthenic syndromes that result from defects in the N-linked glycosylation pathway and that manifest through impaired neuromuscular transmission. Congenital myasthenic syndromes (CMSs) are inherited disorders of neuromuscular transmission.1Chaouch A. Beeson D. Hantaï D. Lochmüller H. 186th ENMC International Workshop: Congenital myasthenic syndromes 24-26 June 2011, Naarden, The Netherlands.Neuromuscul. Disord. 2012; 22: 566-576Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 2Engel A.G. Current status of the congenital myasthenic syndromes.Neuromuscul. Disord. 2012; 22: 99-111Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar They are a heterogeneous group of disorders in which the safety margin for neuromuscular transmission is compromised as a result of mutations in a series of different genes encoding proteins at the neuromuscular synapse. These disorders are characterized by fatigable muscle weakness, and the most commonly affected muscles are ocular, bulbar, and limb muscles. The age of onset is variable, although most cases present with the disorder in infancy or early childhood. To date, mutations in 15 different genes have been shown to lead to impaired neuromuscular transmission, although some are limited to single case reports.1Chaouch A. Beeson D. Hantaï D. Lochmüller H. 186th ENMC International Workshop: Congenital myasthenic syndromes 24-26 June 2011, Naarden, The Netherlands.Neuromuscul. Disord. 2012; 22: 566-576Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 2Engel A.G. Current status of the congenital myasthenic syndromes.Neuromuscul. Disord. 2012; 22: 99-111Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar Whereas most CMS-associated genes have a defined function at the neuromuscular junction (NMJ), the recently described GFPT1 encodes glutamine-fructose-6-phosphate transaminase 1, which is ubiquitously expressed and is involved in the synthesis of UDP-N-acetylglucosamine, a saccharide that serves as a building block for protein and lipid glycosylation. Although the exact role of GFPT1 in NMJ function is unknown, it is possible that when mutated, it impairs glycosylation and, consequently, the function of one or more component proteins of the NMJ.3Senderek J. Müller J.S. Dusl M. Strom T.M. Guergueltcheva V. Diepolder I. Laval S.H. Maxwell S. Cossins J. Krause S. et al.Hexosamine biosynthetic pathway mutations cause neuromuscular transmission defect.Am. J. Hum. Genet. 2011; 88: 162-172Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar There remain a number of CMS subtypes for which the underlying mutations have not been identified. Individuals with a predominant limb-girdle pattern of muscle weakness have been found to have mutations in DOK74Beeson D. Higuchi O. Palace J. Cossins J. Spearman H. Maxwell S. Newsom-Davis J. Burke G. Fawcett P. Motomura M. et al.Dok-7 mutations underlie a neuromuscular junction synaptopathy.Science. 2006; 313: 1975-1978Crossref PubMed Scopus (226) Google Scholar (MIM 610285) and GFPT1 (MIM 138292).3Senderek J. Müller J.S. Dusl M. Strom T.M. Guergueltcheva V. Diepolder I. Laval S.H. Maxwell S. Cossins J. Krause S. et al.Hexosamine biosynthetic pathway mutations cause neuromuscular transmission defect.Am. J. Hum. Genet. 2011; 88: 162-172Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar Although these cases share several phenotypic features, muscle biopsy has shown that the majority of individuals with GFPT1 mutations have tubular aggregates, which are not seen in muscle biopsies from individuals with DOK7-associated CMS.5Palace J. Lashley D. Newsom-Davis J. Cossins J. Maxwell S. Kennett R. Jayawant S. Yamanashi Y. Beeson D. Clinical features of the DOK7 neuromuscular junction synaptopathy.Brain. 2007; 130: 1507-1515Crossref PubMed Scopus (101) Google Scholar However, tubular aggregates have been identified in muscle biopsies of additional cases who do not have GFPT1 mutations. Here, we performed whole-exome capture and high-throughput sequencing to identify a second CMS-associated mutation that underlies a limb-girdle-type congenital myasthenia with tubular aggregates in muscle biopsy. Ethical approval for studies on CMSs was obtained from Oxfordshire Research Ethics Committees B (04.OXB.017) and C (09/H0606/74). Initially, we studied two unrelated individuals (cases 1 and 2) with tubular aggregates in muscle biopsies and without GFPT1 mutations. We performed whole-exome capture from 3 μg of genomic DNA by using Agilent SureSelect Human All Exon Kit v.2 according to the manufacturer's protocol. We sequenced the captured libraries by using 51 bp paired-end reads on Illumina HiSeq or Genome Analyzer IIx platforms. We mapped sequence data to human genome build hg19 by using Novoalign software (Novocraft Technologies). The duplicate reads generated as a result of PCR amplification were filtered out, and only reads that mapped uniquely to the genome were used for further analysis. Aligned sequence data was visualized with GBrowse6Stein L.D. Mungall C. Shu S. Caudy M. Mangone M. Day A. Nickerson E. Stajich J.E. Harris T.W. Arva A. Lewis S. The generic genome browser: A building block for a model organism system database.Genome Res. 2002; 12: 1599-1610Crossref PubMed Scopus (908) Google Scholar and the UCSC genome browser.7Kent W.J. Sugnet C.W. Furey T.S. Roskin K.M. Pringle T.H. Zahler A.M. Haussler D. The human genome browser at UCSC.Genome Res. 2002; 12: 996-1006Crossref PubMed Scopus (6711) Google Scholar Variants were called with either Samtools8Li H. Handsaker B. Wysoker A. Fennell T. Ruan J. Homer N. Marth G. Abecasis G. Durbin R. 1000 Genome Project Data Processing SubgroupThe Sequence Alignment/Map format and SAMtools.Bioinformatics. 2009; 25: 2078-2079Crossref PubMed Scopus (31476) Google Scholar or Platypus9Rimmer, A., Mathieson, I., McVean, G., and Lunter, G. (2012). Platypus program: Integrated Variant Caller. The Wellcome Trust Centre for Human Genetics, University of Oxford (http://www.well.ox.ac.uk/platypus).Google Scholar programs. Variants were filtered out if they were present in dbSNP13210Sherry S.T. Ward M.H. Kholodov M. Baker J. Phan L. Smigielski E.M. Sirotkin K. dbSNP: The NCBI database of genetic variation.Nucleic Acids Res. 2001; 29: 308-311Crossref PubMed Scopus (4830) Google Scholar (unless they were annotated as medically associated SNPs). This filtering narrowed the list of variants to 1,574 and 1,287 variants per exome for cases 1 and 2, respectively (see Table S1, available online). Functional annotation of the variants with ANNOVAR software11Wang K. Li M. Hakonarson H. ANNOVAR: Functional annotation of genetic variants from high-throughput sequencing data.Nucleic Acids Res. 2010; 38: e164Crossref PubMed Scopus (7856) Google Scholar allowed us to separate nonsynonymous substitutions, splicing mutations, and mutations in 3′ UTRs or 5′ UTRs, further limiting the number of interesting variants to 377 and 300 per exome for cases 1 and 2, respectively. CMSs are commonly inherited in an autosomal-recessive manner. Thus, we focused on the genes that had either homozygous variants or contained two or more heterozygous variants. Among these, 34 genes had potential mutations in both analyzed individuals. Further filtering of these variants with our in-house database of 14 exomes from cases with unrelated disorders allowed us to eliminate all but one gene—DPAGT1 (RefSeq NM_001382.3), which encodes dolichyl-phosphate (UDP-N-acetylglucosamine) N-acetylglucosaminephosphotransferase 1. Case 1 had two heterozygous nonsynonymous mutations, c.324G>C (p.Met108Ile) and c.349G>A (p.Val117Ile), whereas case 2 had a heterozygous frameshift single-nucleotide duplication, c.699dup (p.Thr234Hisfs∗116), along with the nonsynonymous mutation c.349G>A (p.Val117Ile). We confirmed the presence of all four mutations in the genome of analyzed individuals by using Sanger sequencing. To determine whether DPAGT1 mutations are present in other CMS cases, we performed Sanger sequencing of DPAGT1 on a cohort of 31 unrelated cases of suspected CMS with varying phenotypic features, and we identified two more individuals who had two heterozygous nonsynonymous mutations in this gene. Case 3 had a combination of mutations c.358C>A (p.Leu120Met) and c.791T>G (p.Val264Gly), whereas case 4 had mutations c.478G>A (p.Gly160Ser) and c.574G>A (p.Gly192Ser) (Table 1). All four individuals with DPAGT1 mutations were genetically screened for possible mutations in GFPT1 or in other known CMS-associated genes, and all proved to be negative.Table 1Clinical Details of Cases with DPAGT1 MutationsCase 1Case 2Case 3Case 4Case 5GendermalefemalemalefemalefemaleCurrent age43 years57 years25 years58 years6 yearsAge when assessed43 years53 years17 years48 years6 yearsMutationsc.324G>C (p.Met108Ile) and c.349G>A (p.Val117Ile)c.349G>A (p.Val117Ile) and c.699dup (p.Thr234Hisfs∗116)c.358C>A (p.Leu120Met) and c.791T>G (p.Val264Gly)c.478G>A (p.Gly160Ser) and c.574G>A (p.Gly192Ser)c.358C>A (p.Leu120Met) and c.791T>G (p.Val264Gly)Progressionstable but with long-term fluctuationsslowly progressivesome improvement during teenage yearsslowly progressivestable in infancy and small improvement in childhoodAge of onset2.5 years7 years0.5 years2 yearsin first at and is to with and and motor and on response to The are not and Research in a The are not and Research To whether DPAGT1 mutations with we performed Sanger sequencing of DPAGT1 in all available the mutations a pattern and with the 1). case 1, from was available for analysis. However, these two mutations were 51 of in our sequencing we that sequencing contained only one or the other of these mutations. reads contained both or of the mutations, that these two mutations were present on two different and were most inherited from two different case 2, genomic DNA was available from four of the were heterozygous for the frameshift one was heterozygous for and one had case was heterozygous for one of the mutations. case 3 had one affected and one The affected had the DPAGT1 mutations as case whereas the only one of the mutations. case were not available for analysis. Thus, the of the DPAGT1 mutations is with the that the mutations underlie the CMS of the DPAGT1 variants described were present in S.T. Ward M.H. Kholodov M. Baker J. Phan L. Smigielski E.M. Sirotkin K. dbSNP: The NCBI database of genetic variation.Nucleic Acids Res. 2001; 29: 308-311Crossref PubMed Scopus (4830) Google Scholar or the 1000 Project of human genome from 2010; PubMed Scopus Google Scholar we the Variant Variant Project Scholar and found that this database one of our mutations, but the of this mutation is 2 in in the or in in the All identified individuals are of and the described in the Variant are for our of the mutation that the mutation is present in the in a heterozygous and is with the of the mutation Thus, it is that of the described DPAGT1 variants are common they are to cause The mutation c.699dup a in the in the of DPAGT1 and a the is that protein be from this The is the of in might be that the of this be to of protein was in with a the DPAGT1 c.699dup All in DPAGT1 present in of the protein 2) and might the function of the although and in C. and To further whether the mutations are to the and function of the we used S. L. A. P. A and for 2010; PubMed Scopus Google Scholar which that all were to be (Table all identified individuals had at one mutation in 3 of that the part of the protein that this encodes might be for a function to neuromuscular transmission. All five cases with mutations in DPAGT1 show a CMS All had features that a disorder of neuromuscular transmission on features were a on 3 and and on (Table 1). They share a number of clinical features that might be used for this of They have involvement of ocular, and muscle be affected in other of the most affected muscles in cases with DPAGT1 mutations are proximal limb muscles, although some muscle be affected (Table 1). is seen for V. Müller J.S. Dusl M. J. A. C. Maxwell S. J. A. et myasthenic syndrome with tubular aggregates by GFPT1 2011; Google Scholar the age of onset to be for other it in at or early To date, all analyzed cases have had tubular aggregates present in their muscle biopsies (Table 1, of all cases a response to and two from 3,4-diaminopyridine, which acetylcholine from the Thus, individuals with mutations in DPAGT1 have clinical features show to V. Müller J.S. Dusl M. J. A. C. Maxwell S. J. A. et myasthenic syndrome with tubular aggregates by GFPT1 2011; Google Scholar that from individuals with other of DPAGT1 encodes dolichyl-phosphate (UDP-N-acetylglucosamine) N-acetylglucosaminephosphotransferase 1. This enzyme is essential for N-linked protein and of 2009; PubMed Scopus Google Scholar N-linked protein glycosylation in the and with the of the on the lipid the is from the lipid the of DPAGT1 is a protein that the first committed step of the of from to is in all and has that is to the and of a encoding Full Text PDF PubMed Google Scholar with out the essential role of this Marth A in the gene in PubMed Scopus Google Scholar To date, two of mutations in DPAGT1 describe a severe congenital disorder of glycosylation J.S. D. D. R. of a congenital disorder of 22: PubMed Scopus Google Scholar, J. S. C. S. G. P. L. R. T. Congenital disorder of glycosylation the clinical and of a Genet. 2012; PubMed Scopus Google Scholar cases with this disorder have been had mutation along with an splicing mutation and only of the DPAGT1 J.S. D. D. R. of a congenital disorder of 22: PubMed Scopus Google Scholar The other two cases were from a and had homozygous mutation J. S. C. S. G. P. L. R. T. Congenital disorder of glycosylation the clinical and of a Genet. 2012; PubMed Scopus Google Scholar The of the was reduced to of that of the All cases had severe clinical All had and and one had The with homozygous mutations the first of whereas the other case of All are to have had a severe that neuromuscular transmission might have been The of the individuals with CMSs described in this were limited to neuromuscular and the of were not in of these of we are not in a to the in these CMS cases are different from those of the individuals with the is that in the cases that we the DPAGT1 mutations are to DPAGT1 A of protein might be to function in other at the is of that all of our cases have a in and the of the protein by this have a role for function at the of disorders of glycosylation are with a glycosylation E. and of congenital disorders of 2007; PubMed Scopus Google Scholar is an protein that has two to two the most common of is However, when glycosylation is are and The of the of these of is used as a for different of congenital disorders of glycosylation. of the for the glycosylation levels is by the of by E. and of congenital disorders of 2007; PubMed Scopus Google Scholar, I. S. M. R. M. of of seven 2006; Google Scholar this was performed on the from two of the DPAGT1 CMS cases (cases 3 and both levels of Case 3 had and case had levels are an presence of and These demonstrate that these two CMS cases have a defect in and they that the described variants in DPAGT1 are The described for the glycosylation might be for of individuals with similar However, it is whether the glycosylation be to the in glycosylation in CMS The clinical features of case 1 have been described Fawcett Beeson D. C. D. and associated with impaired neuromuscular transmission in a group of with 2006; PubMed Scopus Google Scholar A muscle biopsy has been characterized as possible and of the of the with that the number of per NMJ was in this per NMJ in the affected per NMJ in This was by a reduction of the of and Fawcett Beeson D. C. D. and associated with impaired neuromuscular transmission in a group of with 2006; PubMed Scopus Google Scholar studies of the that the of in the was five that in the the of the at NMJ was in the affected and in the in separate in These features are seen in acetylcholine in which they to impaired neuromuscular R. C. C. S. M. A. Beeson D. and levels in syndrome due to 2001; PubMed Google Scholar, K. M. L. et myasthenic syndromes due to mutations in the acetylcholine and of Genet. PubMed Scopus Google Scholar, to the safety for neuromuscular 2011; PubMed Scopus Google Scholar, C. P. Fawcett M. L. J. et is reduced at neuromuscular of with both inherited and acetylcholine PubMed Scopus Google Scholar The primary defect of reduced the of the to the A of the of endplate is of This be to the for in the muscle by both the number and of which are in the and the amplification by the of the of the of neuromuscular transmission and it is PubMed Scopus Google Scholar with the from the muscle biopsy from case 1, a muscle biopsy from case 2 a reduction in endplate in the for and in Thus, both analyzed biopsies show a reduction of endplate with the of endplate all the affected individuals to and two individuals from both of these are for A.G. Current status of the congenital myasthenic syndromes.Neuromuscul. Disord. 2012; 22: 99-111Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar These are with the that DPAGT1 mutations cause a of endplate is a of subunits and The is in the to the of acetylcholine PubMed Scopus Google Scholar All four subunits of are H. N. Y. S. Y. K. of acetylcholine from and of the J. PubMed Scopus Google Scholar is required for the of the through the of and K. of the on the acetylcholine Res. Res. PubMed Scopus Google Scholar, T. K. at the the of Res. Res. PubMed Scopus Google Scholar is possible that in DPAGT1 subunits are and to be to the To whether DPAGT1 is required for we used an to levels of expressed on the cell surface. this of and subunits were either with DPAGT1 or with an The the was either to or to 1 the of was with is a inhibitor of L. The of in the of PubMed Scopus Google Scholar and has been shown to glycosylation and export to the cell R. S. J. of glycosylation with of acetylcholine subunits in muscle Full Text PDF PubMed Google Scholar We whether of DPAGT1 this of on of to the had a on export in with the of the receptors were to to the cell However, DPAGT1 were to treatment with and levels of at the cell surface. of the DPAGT1 c.699dup not levels of the that the in the presence of the The result is with the that if DPAGT1 was from the with this mutation of the cell from these that glycosylation of the most of the four was treatment with in with the However, glycosylation was when but not the DPAGT1 c.699dup was in the All other in DPAGT1 were to with not This is with in which it was shown that DPAGT1 with the protein the inhibitor and it N. in of the are essential in PubMed Scopus Google Scholar, N. has and a PubMed Scopus Google Scholar We that DPAGT1 is required for export to the cell and the are with of endplate in the affected individuals. A of the biopsy from case 1 is the presence of tubular aggregates the muscle aggregates are commonly found in individuals with GFPT1 mutations but are from the muscles of individuals with DOK7-associated these individuals have a similar limb-girdle pattern of muscle weakness. aggregates were in of the analyzed individuals with DPAGT1 mutations (Table 1). The exact and of tubular aggregates is not at They are characterized as with different and M. M. I. and of tubular aggregates of muscle 22: Google Scholar, S. aggregates in a of protein Disord. 2012; 22: Full Text Full Text PDF PubMed Scopus Google Scholar They are to arise from of aggregates are and have been in cases with or in some cases with and in most individuals with CMS by mutations in J. Müller J.S. Dusl M. Strom T.M. Guergueltcheva V. Diepolder I. Laval S.H. Maxwell S. Cossins J. Krause S. et al.Hexosamine biosynthetic pathway mutations cause neuromuscular transmission defect.Am. J. Hum. Genet. 2011; 88: 162-172Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, G. V. G. E. biopsy and cell in muscle J. PubMed Scopus Google Scholar, J. S. and tubular aggregates in 2006; PubMed Scopus Google Scholar DPAGT1 and GFPT1 are required for protein which in is essential for protein and A. B. The of 2011; PubMed Scopus Google Scholar might be that in the individuals with compromised function of DPAGT1 and and proteins are not this lead to their and in the and the of tubular we identify DPAGT1 as a gene in which mutations cause a similar to is involved in protein glycosylation. However, whereas the pathogenic mechanism of GFPT1 has not been we suggest that the primary pathogenic mechanism of DPAGT1 mutations is of export of to the cell and reduced levels of endplate The that two genes involved in the protein glycosylation pathway lead to the of CMSs that might be a series of genes in which mutations might function at the We do not the from the mutations we identified are to muscle and the NMJ in Further of DPAGT1 mutations the role of glycosylation for and function at the neuromuscular and have potential for transmission in the We the at the Wellcome Trust Centre for Human by Wellcome Trust and Research for the of the sequencing We are to and for their Platypus of We for is a of the Wellcome and is by a from the Wellcome We are for from the Research the and the We the cases with congenital myasthenic syndromes and their for in this and for their which was obtained with approval from Oxfordshire Research Ethics Committees B (04.OXB.017) and C (09/H0606/74). with and The for data are as Variant in

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