Mutations in KLHL40 are a frequent cause of severe autosomal-recessive nemaline myopathy.

Nemaline myopathy (NEM) is a common congenital myopathy. At the very severe end of the NEM clinical spectrum are genetically unresolved cases of autosomal-recessive fetal akinesia sequence. We studied a multinational cohort of 143 severe-NEM-affected families lacking genetic diagnosis. We performed whole-exome sequencing of six families and targeted gene sequencing of additional families. We identified 19 mutations in KLHL40 (kelch-like family member 40) in 28 apparently unrelated NEM kindreds of various ethnicities. Accounting for up to 28% of the tested individuals in the Japanese cohort, KLHL40 mutations were found to be the most common cause of this severe form of NEM. Clinical features of affected individuals were severe and distinctive and included fetal akinesia or hypokinesia and contractures, fractures, respiratory failure, and swallowing difficulties at birth. Molecular modeling suggested that the missense substitutions would destabilize the protein. Protein studies showed that KLHL40 is a striated-muscle-specific protein that is absent in KLHL40-associated NEM skeletal muscle. In zebrafish, klhl40a and klhl40b expression is largely confined to the myotome and skeletal muscle, and knockdown of these isoforms results in disruption of muscle structure and loss of movement. We identified KLHL40 mutations as a frequent cause of severe autosomal-recessive NEM and showed that it plays a key role in muscle development and function. Screening of KLHL40 should be a priority in individuals who are affected by autosomal-recessive NEM and who present with prenatal symptoms and/or contractures and in all Japanese individuals with severe NEM.

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.

The exon 55 deletion in the nebulin gene--one single founder mutation with world-wide occurrence.

In 2004, Anderson et al. reported a homozygous 2502 bp deletion including exon 55 of the nebulin gene in five Ashkenazi Jewish probands with nemaline myopathy. We determined the occurrence of this deletion in a world-wide series of 355 nemaline myopathy probands with no previously known mutation in other genes and found the mutation in 14 probands, two of whom represented families previously ascertained by Anderson et al. Two of the families were not of known Ashkenazi Jewish descent but they had the haplotype known to segregate with this mutation. In all but two of eight homozygous patients, the clinical picture was more severe than in typical nemaline myopathy.

[Clinical and pathologic analysis of an autosomal recessive kindred with nemaline myopathy].

OBJECTIVE: To investigate the clinical, pathological and ultrastructural features of nemaline myopathy (NM). METHODS: The clinical manifestations of four patients in a rare autosomal recessive kindred with nemaline myopathy were analyzed retrospectively. Biopsied specimens of left gastrocnemius from the proband were detected and observed through light microscope using enzymatic histochemical methods for histopathological changes and through electron microscope for ultrastructural features. RESULTS: Four affected siblings in this kindred had an onset at birth or fetal stage, among whom two case were respiratory independent with delayed attainment of motor milestones and general muscle atrophy complying with typical form of NM, whereas the other two did not achieve adequate spontaneous movement or breathing, and died at neonatal period according with severe form of NM. Other clinical characteristics of elongated face, tent-shaped mouth and high-arched palate were also found. The proband had normal serum muscle enzymes and the karyotypic analysis showed a normal G band. Brain magnetic resonance image (MRI) indicated no abnormality. Electromyogram (EMG) showed typical muscle-derived damages of biceps, triceps, brachioradial muscle, vastus medialis muscle, anterior tibial muscle and gastrocnemius with normal motor conduction velocity of bilateral tibial nerves and common peroneal nerves. Myofibrillar atrophy was found through light microscopy with fiber type 1 predominance shown by ATP enzyme staining, yet without indication of lipid or glycogen deposition by ORO or PAS staining. Modified gomori trichrome (MGT) treatment resulted in dark-red staining of nemaline bodies in myofibril cytoplasm. And it was also observed as purple-red staining followed by hematoxylin-eosin (HE) treatment. Electron microscopic observation by lead-uranium double staining showed widened interstitial myofibrils, focal myofilament disorganization, partial myofilament atrophy, focal dissolution or necrosis, partial myofibrils nucleus pyknosis, numerous subsarcolemmal and perinuclear rod-like structures, partial nemaline bodies connected with Z discs, and mitochondria vacuolation or disappearance. CONCLUSION: NM is among congenital myopathies characterized by marked clinical and pathological heterogeneity. The diagnosis of NM should be based upon typical clinical and histopathological features.

Nemaline myopathy caused by absence of alpha-skeletal muscle actin.

OBJECTIVE: To investigate seven congenital myopathy patients from six families: one French Gypsy, one Spanish Gypsy, four British Pakistanis, and one British Indian. Three patients required mechanical ventilation from birth, five died before 22 months, one is ventilator-dependent, but one, at 30 months, is sitting with minimal support. All parents were unaffected. METHODS: The alpha-skeletal muscle actin gene (ACTA1) was sequenced. Available muscle biopsies were investigated by standard histological and electron microscopic techniques. The expression of various proteins was determined by immunohistochemistry, western blotting, or both. RESULTS: Three homozygous ACTA1 null mutations were identified: p.Arg41X in the French patient, p.Tyr364fsX in the Spanish patient, and p.Asp181fsX10 in all five British patients. An absence of alpha-skeletal muscle actin protein but presence of alpha-cardiac actin was shown in all muscle biopsies examined, with more alpha-cardiac actin in the biopsy from the child with the greatest muscle function. Muscle biopsies from all patients exhibited nemaline bodies whereas three also contained zebra bodies. INTERPRETATION: The seven patients have recessive nemaline myopathy caused by absence of alpha-skeletal muscle actin. The level of retention of alpha-cardiac actin, the skeletal muscle fetal actin isoform, may determine alpha-skeletal muscle actin disease severity. This has implications for possible future therapy.