J. Laitila

EXPRESSION OF MULTIPLE NEBULIN ISOFORMS IN HUMAN SKELETAL MUSCLE AND BRAIN

Nebulin is a large actin‐binding protein of the skeletal muscle sarcomere. Multiple isoforms of nebulin are produced from the 183‐exon–containing nebulin gene (NEB). Mutations in NEB cause nemaline myopathy, distal myopathy, and core‐rod myopathy.

A recurrent copy number variation of the NEB triplicate region: only revealed by the targeted nemaline myopathy CGH array.

Recently, new large variants have been identified in the nebulin gene (NEB) causing nemaline myopathy (NM). NM constitutes a heterogeneous group of disorders among the congenital myopathies, and disease-causing variants in NEB are a main cause of the recessively inherited form of NM. NEB consists of 183 exons and it includes homologous sequences such as a 32-kb triplicate region (TRI), where eight exons are repeated three times (exons 82–89, 90–97, 98–105). In human, the normal copy number of NEB TRI is six (three copies in each allele). Recently, we described a custom NM-CGH microarray designed to detect copy number variations (CNVs) in the known NM genes. The array has now been updated to include all the currently known 10 NM genes. The NM-CGH array is superior in detecting CNVs, especially of the NEB TRI, that is not included in the exome capture kits. To date, we have studied 266 samples from 196 NM families using the NM-CGH microarray, and identified a novel recurrent NEB TRI variation in 13% (26/196) of the families and in 10% of the controls (6/60). An analysis of the breakpoints revealed adjacent repeat elements, which are known to predispose for rearrangements such as CNVs. The control CNV samples deviate only one copy from the normal six copies, whereas the NM samples include CNVs of up to four additional copies. Based on this study, NEB seems to tolerate deviations of one TRI copy, whereas addition of two or more copies might be pathogenic.

Two alternatively-spliced human nebulin isoforms with either exon 143 or exon 144 and their developmental regulation

Nebulin is a very large protein required for assembly of the contractile machinery in muscle. Mutations in the nebulin gene NEB are a common cause of nemaline myopathy. Nebulin mRNA is alternatively-spliced so that each mRNA contains either exon 143 or exon 144. We have produced monoclonal antibodies specific for the regions of nebulin encoded by these two exons, enabling analysis of expression of isoforms at the protein level for the first time. All antibodies recognized a protein of the expected size (600–900 kD) and stained cross-striations of sarcomeres in muscle sections. Expression of exon 143 is developmentally-regulated since newly-formed myotubes in cell culture expressed nebulin with exon 144 only; this was confirmed at the mRNA level by qPCR. In fetal muscle, nebulin with exon 143 was expressed in some myotubes by 12-weeks of gestation and strongly-expressed in most myotubes by 17-weeks. In mature human muscle, the exon 144 antibody stained all fibres, but the exon 143 antibody staining varied from very strong in some fibres to almost-undetectable in other fibres. The results show that nebulin containing exon 144 is the default isoform early in myogenesis, while regulated expression of nebulin containing exon 143 occurs at later stages of muscle development.

A nebulin super-repeat panel reveals stronger actin binding towards the ends of the super-repeat region: Nebulin binds actin stronger at the ends of the super-repeat region

Introduction: Nebulin is a giant actin‐binding protein in the thin filament of the skeletal muscle sarcomere. Studies of nebulin interactions are limited by the size, complexity, and poor solubility of the protein. We divided the nebulin super‐repeat region into a super‐repeat panel, and studied nebulin/actin interactions. Methods: Actin binding was studied using a co‐sedimentation assay with filamentous actin and 26 different nebulin super‐repeats. Results: The panel revealed notable differences in actin binding between the super‐repeats. Both ends of the super‐repeat region bound actin significantly more strongly, whereas the central part of the protein bound actin weakly. Thus, the binding between nebulin and actin formed a location‐dependent pattern of strong vs. weak binding. Discussion: The nebulin super‐repeat panel allowed us to study the actin binding of each super‐repeat individually. The panel will be a powerful tool in elucidating nebulin function in health and disease. Muscle Nerve 59:116–121, 2019

G.P.274

Mutation analysis of the known nemaline myopathy (NM)-causing genes in a Finnish patient with an unusual form of NM, with legs clearly stronger than arms, did not reveal the cause of the disease. A heterozygous TPM3 splice site mutation, delTAGG, in intron 1 was identified, inherited from the healthy father. The mutation was predicted to be recessive, but no TPM3 mutation was found on the maternal allele. Large copy number variations affecting the known nine NM genes were excluded by NM-CGH array analysis. Whole-exome sequencing revealed a heterozygous RYR1 missense mutation, p.Leu2031Phe, predicted to be probably damaging by PolyPhen-2, but the healthy father also carried this mutation. No other putative pathogenic mutation in RYR1 was found in the patient, and in myoblasts of the patient, epigenetic silencing of the maternal allele was ruled out. Exome sequencing did, however, disclose two putative pathogenic mutations in the cold shock domain protein A encoding gene, CSDA . The CSDA gene has 11 exons and encodes three different protein isoforms: 303 aa, 372 aa and 200 aa. CSDA is highly expressed in skeletal muscle and heart. Our patient was compound heterozygous for two missense mutations, p.Ser34Arg and p.Arg129Trp, in CSDA, each inherited from one of the healthy parents. The mutations are not listed in the EVS database (http://evs.gs.washington.edu/EVS/). Ser34 and Arg129 are highly conserved between species, and reside in the DNA-binding domain (cold shock domain) of CSDA. Furthermore, Ser34 is phosphorylated by ERK2 and GSK3 β , and the phosphorylation is thought to be important for the formation of nuclear CSDA complexes and binding of CSDA to single-stranded DNA. Western blot analysis of cultured myotubes from the patient confirms CSDA expression on the protein level. Further analyses are ongoing in order to quantify the expression levels and determine the intracellular location of CSDA in myoblasts from the patient and healthy controls.