The giant nebulin protein is a fundamental structural component of the thin filaments of the striated muscle sarcomere. Nebulin binds to actin and the size of nebulin correlates with actin filament length, suggesting that nebulin may determine the length of the thin filaments during myofibrillogenesis. We have previously described the genomic organization of the 3 0 end of the nebulin gene (NEB), and identified 18 different NEB mutations in patients with autosomal recessive nemaline myopathy. Here we present the genomic organization of the entire nebulin gene, and the identification of numerous alternatively spliced mRNAs. The gene comprises 183 exons spanning 249 kb of the genomic sequence. The translation initiation codon is in exon 3, and the stop codon and the 3 0 UTR are in exon 183. There are four regions with alternatively spliced exons, that is, exons 63 -66, 82-105, 143 -144 and 166 -177, giving rise to a number of different transcripts. The alternatively spliced exons 143 -144 give rise to two different transcripts varying between muscle types and between muscles of different developmental stages. The alternatively spliced exons 166 -177 express at least 20 different transcripts in adult human tibialis anterior muscle alone. Preliminary results show several transcripts in both of the two remaining alternatively spliced regions. Extensive alternative splicing of NEB may explain why nemaline myopathy patients with homozygous truncating mutations show expression of the carboxy-terminus of the nebulin protein contrary to expectations . The use of alternative transcripts might also explain why severe phenotypes are rare among patients with two truncating mutations.
BackgroundMüllerian aplasia (MA) is a congenital disorder of the female reproductive tract with absence of uterus and vagina with paramount impact on a woman’s life. Despite intense research, no major genes have been found to explain the complex genetic etiology.Methods and ResultsWe have used several genetic methods to study 112 patients with MA. aCGH identified CNVs in 8/50 patients (16%), including 16p11.2 and 17q12 deletions previously associated with MA. Subsequently, another four patients were shown to carry the ~0.53 Mb deletion in 16p11.2. More importantly, sequencing of TBX6, residing within 16p11.2, revealed two patients carrying a splice site mutation. Two previously reported TBX6 variants in exon 4 and 6 were shown to have a significantly higher frequency in patients (8% and 5%, respectively) than in controls (2% each). We also sequenced LHX1 and found three apparently pathogenic missense variants in 5/112 patients. Altogether, we identified either CNVs or variations in TBX6 or LHX1 in 30/112 (26.8%) MA patients. CNVs were found in 12/112 (10.7%), patients, novel variants in TBX6 or LHX1 in 7/112 (6.3%), and rare variants in TBX6 in 15/112 (13.4%) patients. Furthermore, four of our patients (4/112, 3.6%) were shown to carry variants in both TBX6 and LHX1 or a CNV in combination with TBX6 variants lending support to the complex genetic etiology of MA.ConclusionsWe have identified TBX6 as a new gene associated with MA. Our results also support the relevance of LHX1 and CNVs in the development of this congenital malformation.
Nemaline myopathy (NM) is a clinically and genetically heterogeneous disorder of skeletal muscle caused by mutations in at least five different genes encoding thin filament proteins of the striated muscle sarcomere. We have previously described 18 different mutations in the last 42 exons of the nebulin gene (NEB) in 18 families with NM. Here we report 45 novel NEB mutations detected by denaturing high-performance liquid chromatography (dHPLC) and sequence analysis of all 183 NEB exons in NM patients from 44 families. Altogether we have identified, including the deletion of exon 55 identified in the Ashkenazi Jewish population, 64 different mutations in NEB segregating with autosomal recessive NM in 55 families. The majority (55%) of the mutations in NEB are frameshift or nonsense mutations predicted to cause premature truncation of nebulin. Point mutations (25%) or deletions (3%) affecting conserved splice signals are predicted in the majority of cases to cause in-frame exon skipping, possibly leading to impaired nebulin-tropomyosin interaction along the thin filament. Patients in 18 families had one of nine missense mutations (14%) affecting conserved amino acids at or in the vicinity of actin or tropomyosin binding sites. In addition, we found the exon 55 deletion in four families. The majority of the patients (in 49/55 families) were shown to be compound heterozygous for two different mutations. The mutations were found in both constitutively and alternatively expressed exons throughout the NEB gene, and there were no obvious mutational hotspots. Patients with more severe clinical pictures tended to have mutations predicted to be more disruptive than patients with milder forms.
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