K. Kiiski

Mutation Update: The Spectra of Nebulin Variants and Associated Myopathies

A mutation update on the nebulin gene (NEB) is necessary because of recent developments in analysis methodology, the identification of increasing numbers and novel types of variants, and a widening in the spectrum of clinical and histological phenotypes associated with this gigantic, 183 exons containing gene. Recessive pathogenic variants in NEB are the major cause of nemaline myopathy (NM), one of the most common congenital myopathies. Moreover, pathogenic NEB variants have been identified in core‐rod myopathy and in distal myopathies. In this update, we present the disease‐causing variants in NEB in 159 families, 143 families with NM, and 16 families with NM‐related myopathies. Eighty‐eight families are presented here for the first time. We summarize 86 previously published and 126 unpublished variants identified in NEB. Furthermore, we have analyzed the NEB variants deposited in the Exome Variant Server (http://evs.gs.washington.edu/EVS/), identifying that pathogenic variants are a minor fraction of all coding variants (∼7%). This indicates that nebulin tolerates substantial changes in its amino acid sequence, providing an explanation as to why variants in such a large gene result in relatively rare disorders. Lastly, we discuss the difficulties of drawing reliable genotype–phenotype correlations in NEB‐associated disease.

Targeted array comparative genomic hybridization – A new diagnostic tool for the detection of large copy number variations in nemaline myopathy-causing genes

Nemaline myopathy (NM) constitutes a heterogeneous group of congenital myopathies. Mutations in the nebulin gene (NEB) are the main cause of recessively inherited NM. NEB is one of the most largest genes in human. To date, 68 NEB mutations, mainly small deletions or point mutations have been published. The only large mutation characterized is the 2.5 kb deletion of exon 55 in the Ashkenazi Jewish population. To investigate any copy number variations in this enormous gene, we designed a novel custom comparative genomic hybridization microarray, NM-CGH, targeted towards the seven known genes causative for NM. During the validation of the NM-CGH array we identified two novel deletions in two different families. The first is the largest deletion characterized in NEB to date, (∼53 kb) encompassing 24 exons. The second deletion (1 kb) covers two exons. In both families, the copy number change was the second mutation to be characterized and shown to have been inherited from one of the healthy carrier parents. In addition to these novel mutations, copy number variation was identified in four samples in three families in the triplicate region of NEB. We conclude that this method appears promising for the detection of copy number variations in NEB.

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.

Bilateral foot-drop as predominant symptom in nebulin (NEB) gene related “core-rod” congenital myopathy

BACKGROUND Congenital myopathies (CM) are a group of rare inherited muscle disorders characterized by particular histopathological alterations on muscle biopsy. Core-rod myopathy is a CM presenting with cores and rods as distinctive muscle morphological features. METHODS/RESULTS We describe 3 young patients presenting congenital core-rod myopathy with bilateral foot-drop associated with autosomal recessive nebulin gene (NEB) mutations detected by exome sequencing. CONCLUSIONS This report illustrates that core-rod congenital myopathy with foot-drop is frequently associated with NEB gene mutations and should be considered in the differential diagnosis of early onset distal myopathies.

A Large Deletion Affecting TPM3, Causing Severe Nemaline Myopathy

Background and Objectives: Nemaline myopathy may be caused by pathogenic variants in the TPM3 gene and is then called NEM1. All previously identified disease-causing variants are point mutations including missense, nonsense and splice-site variants. The aim of the study was to identify the disease-causing gene in this patient and verify the NM diagnosis. Methods: Mutation analysis methods include our self-designed nemaline myopathy array, The Nemaline Myopathy Comparative Genomic Hybridisation Array (NM-CGH array), whole-genome array-CGH, dHPLC, Sanger sequencing and whole-exome sequencing. The diagnostic muscle biopsy was investigated further by routine histopathological methods. Results: We present here the first large (17–21 kb) aberration in the α-tropomyosinslow gene (TPM3), identified using the NM-CGH array. This homozygous deletion removes the exons 1a and 2b as well as the promoter of the TPM3 isoform encoding Tpm3.12st. The severe phenotype included paucity of movement, proximal and axial weakness and feeding difficulties requiring nasogastric tube feeding. The infant died at the age of 17.5 months. Muscle biopsy showed variation in fibre size and rods in a population of hypotrophic muscle fibres expressing slow myosin, often with internal nuclei, and abnormal immunolabelling revealing many hybrid fibres. Conclusions: This is the only copy number variation we have identified in any NM gene other than nebulin (NEB), suggesting that large deletions or duplications in these genes are very rare, yet possible, causes of NM.

A commentary on identification of the rare compound heterozygous variants in the NEB gene in a Korean family with intellectual disability, epilepsy and early-childhood-onset generalized muscle weakness.

A commentary on identification of the rare compound heterozygous variants in the NEB gene in a Korean family with intellectual disability, epilepsy and early-childhood-onset generalized muscle weakness

G.P.270

Using our self-designed NM-CGH microarray, which we have developed to identify large copy number variations in the currently known nine nemaline myopathy (NM) genes, we identified the first large (>20kb) aberration in the alpha-tropomyosin gene ( TPM3 ). This homozygous mutation deletes the promoter as well as the muscle- specific exons 1 and 2 of TPM3 . The promoter and exon 1 of the non-muscle isoform seem to be intact. The deletion also spans the micro-RNA-encoding MIR190B gene and the last two exons of the C1orf189 gene upstream of TPM3 . Our attempts to sequence the promoter and exon 1 of TPM3 have constantly failed, supporting the array result, showing an approximately 6kb deletion including the musclespecific exons 1 and 2 as well as regions upstream from the gene. Before analyzing the patient sample using our array, we had analyzed other NM genes including nebulin ( NEB ), in which we identified a heterozygous nonsense mutation in exon 42. No second mutation, however, was identified in NEB and this nonsense mutation likely has a modifying impact on the phenotype of the patient. We also performed whole-exome sequencing, but no other pathogenic mutations were revealed. The patient was born to consanguineous parents. He deceased at the age of approximately 1.5years. His biopsy confirmed the diagnosis of NM. Unfortunately, to date, no further clinical details are available. We have screened 250 samples from 185 NM and related myopathy families using the NM-CGH-array and identified nine large aberrations in NEB in nine families, but we have not detected such mutations in other NM genes except the one discussed here. Therefore large aberrations in known NM genes other than NEB are likely a very rare cause of NM. The NM-CGH microarray method is currently available for mutation analysis in our laboratory.

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.

C.P.13 The clinical spectrum of entities caused by mutations in the nebulin gene

Abstract Nebulin is one of the main components of the thin filament of the muscle sarcomere and the gene encoding nebulin ( NEB ) is, with its 183 exons, one of the biggest genes in the human genome. Mutations in NEB are the most common cause of autosomal recessive nemaline myopathy (NM), which is diagnosed on the basis of muscle weakness and protein aggregates called nemaline bodies in the muscle fibres of the patients. In 2007 we published NEB mutations in four Finnish distal myopathy families with no readily detectable nemaline bodies in their biopsies and in 2011 we described two NM patients with distal muscle weakness. In addition, we have identified NEB mutations in two core-rod myopathy patients; the first one was described in 2009. Usually patients with NEB mutation have type 1 predominance, but we have encountered two patients with type 2 predominance. To date, we have identified more than 140 mutations in NEB in some 100 families. There are no clear mutational hotspots, and mutations are rarely recurrent among the usually compound heterozygous patients. The first large deletion in NEB was published by Anderson et al. in 2004 and to date we have identified three additional large deletions in NM families using a self-designed mutational microarray. Mutations in NEB can cause a variety of different phenotypes with differences in distribution of muscle weakness, severity and histological findings. Our novel combination of methods facilitates mutation identification in the enormous NEB gene, permitting more distinct genotype-phenotype correlations and possibly the discovery of further entities caused by mutation of NEB .