Mitochondrial tRNA(Leu(UUR)) mutation m.3302A > G presenting as childhood-onset severe myopathy: threshold determination through segregation study

Mitochondrial tRNA(Leu(UUR)) mutation m.3302A > G is associated with respiratory chain complex I deficiency and has been described as a rare cause of mostly adult-onset slowly progressive myopathy. Five families with 11 patients have been described so far; 5 of them died young due to cardiorespiratory failure. Here, we report on a segregation study in a family with an index patient who already presented at the age of 18 months with proximal muscular hypotonia, abnormal fatigability, and lactic acidosis. This early-onset myopathy was rapidly progressive. At 8 years, the patient is wheel-chair bound, requires nocturnal assisted ventilation, and suffers from recurrent respiratory infections. Severe complex I deficiency and nearly homoplasmy for m.3302A > G were found in muscle. We collected blood, hair, buccal swabs and muscle biopsies from asymptomatic adults in this pedigree and determined heteroplasmy levels in these tissues as well as OXPHOS activities in muscle. All participating asymptomatic adults had normal OXPHOS activities. In contrast to earlier reports, we found surprisingly little variation of heteroplasmy levels in different tissues of the same individual. Up to 45% mutation load in muscle and up to 38% mutation load in other tissues were found in non-affected adults. The phenotypic spectrum of tRNA(Leu(UUR)) m.3302A > G mutation seems to be wider than previously described. A threshold of more than 45% heteroplasmy in muscle seems to be necessary to alter complex I activity leading to clinical manifestation. The presented data may be helpful for prognostic considerations and counseling in affected families.

Ultrastructural changes in diaphragm neuromuscular junctions in a severe mouse model for Spinal Muscular Atrophy and their prevention by bifunctional U7 snRNA correcting SMN2 splicing

In Spinal Muscular Atrophy (SMA), the SMN1 gene is deleted or inactivated. Because of a splicing problem, the second copy gene, SMN2, generates insufficient amounts of functional SMN protein, leading to the death of spinal cord motoneurons. For a "severe" mouse SMA model (Smn -/-, hSMN2 +/+; with affected pups dying at 5-7 days), which most closely mimicks the genetic set-up in human SMA patients, we characterise SMA-related ultrastructural changes in neuromuscular junctions (NMJs) of two striated muscles with discrete functions. In the diaphragm, but not the soleus muscle of 4-days old SMA mice, mitochondria on both sides of the NMJs degenerate, and perisynaptic Schwann cells as well as endoneurial fibroblasts show striking changes in morphology. Importantly, NMJs of SMA mice in which a modified U7 snRNA corrects SMN2 splicing and delays or prevents SMA symptoms are normal. This ultrastructural study reveals novel features of NMJ alterations - in particular the involvement of perisynaptic Schwann cells - that may be relevant for human SMA pathogenesis.

The Trypanosoma brucei MitoCarta and its regulation and splicing pattern during development

It has long been known that trypanosomes regulate mitochondrial biogenesis during the life cycle of the parasite; however, the mitochondrial protein inventory (MitoCarta) and its regulation remain unknown. We present a novel computational method for genome-wide prediction of mitochondrial proteins using a support vector machine-based classifier with approximately 90% prediction accuracy. Using this method, we predicted the mitochondrial localization of 468 proteins with high confidence and have experimentally verified the localization of a subset of these proteins. We then applied a recently developed parallel sequencing technology to determine the expression profiles and the splicing patterns of a total of 1065 predicted MitoCarta transcripts during the development of the parasite, and showed that 435 of the transcripts significantly changed their expressions while 630 remain unchanged in any of the three life stages analyzed. Furthermore, we identified 298 alternatively splicing events, a small subset of which could lead to dual localization of the corresponding proteins.

Mitochondrial tRNA import and its consequences for mitochondrial translation

The mitochondrial genomes of most eukaryotes lack a variable number of tRNA genes. This lack is compensated for by import of a small fraction of the corresponding cytosolic tRNAs. There are two broad mechanisms for the import of tRNAs into mitochondria. In the first one, the tRNA is coimported together with a mitochondrial precursor protein along the protein import pathway. It applies to the yeast tRNA(Lys) and has been elucidated in great detail. In the second more vaguely defined mechanism, which is mainly found in plants and protozoa, tRNAs are directly imported independent of cytosolic factors. However, results in plants indicate that direct import of tRNAs may nevertheless require some components of the protein import machinery. All imported tRNAs in all systems are of the eukaryotic type but need to be functionally integrated into the mitochondrial translation system of bacterial descent. For some tRNAs, this is not trivial and requires unique evolutionary adaptations.

Rapid and reliable genotyping of polymorphic loci modifying correct splicing of CFTR pre-mRNA using mass spectrometry

We describe a fast and unambiguous method for haplotyping the (TG)mTn repeat in IVS8 and determining three other single nucleotide polymorphisms (SNPs) in exons 10, 14a and 24 in the cystic fibrosis transmembrane conductance regulator (CFTR) gene affecting correct splicing of the CFTR pre-mRNA using primer extension and mass spectrometry. The diagnostic products are generated by primer extension (PEX) reactions, which require a single detection primer complementary to a region downstream of a target strand's variable site. On addition of a polymerase and an appropriate mixture of dNTP's and 2', 3'-dideoxynucleotide triphosphates (ddNTP's), the primer is extended through the mutation region until the first ddNTP is incorporated and the mass of the extension products determines the composition of the variable site. Analysis of patient DNA assigned the correct and unambiguous haplotype for the (TG)mTn repeat in intron 8 of the CFTR gene. Additional crucial SNPs influencing correct splicing in exon 10, 14 and 24 can easily be detected by biplexing the assay to genotype allelic variants important for correct splicing of the CFTR pre-mRNA. Different PEX reactions with subsequent mass spectrometry generate sufficient data, to enable unambiguous and easy haplotyping of the (TG)mTn repeat in the CFTR gene. The method can be easily extended to the inclusion of additional SNPs of interest by biplexing some of the PEX reactions. All experimental steps required for PEX are amenable to the high degree of automation desirable for a high-throughput diagnostic setting, facilitating the work of clinicians involved in the diagnosis of non-classic cystic fibrosis.

Point mutation tRNA(Ser(UCN)) in a child with hearing loss and myoclonus epilepsy

We report on a family with a 12-year-old boy who suffered from a maternally inherited syndrome characterized by a combination of sensorineural hearing loss, myoclonus epilepsy, ataxia, severe psychomotor retardation, short stature, and diabetes mellitus. First, he showed a muscular hypotonia with hearing loss; later, he developed a myoclonus epilepsy, growth failure, and severe psychomotor retardation. At the age of 10 years, he developed diabetes mellitus. After initiation of combined ubiquinone and vitamin C treatment, we observed a progression in psychomotor development. Lactate and pyruvate levels in blood and cerebrospinal fluid were normal. No ragged red fibers or ultrastructural abnormalities were seen in a skeletal muscle biopsy. Biochemical assays of respiratory chain complex activities revealed decreased activity of complexes I and IV. By sequence analysis of mitochondrial DNA encoding transfer ribonucleic acids (RNAs), a homoplasmic T to C substitution at nucleotide position 7512 was found affecting a highly conserved base pair in the tRNA(ser(UCN)) acceptor stem. Asymptomatic family members of the maternal line were heteroplasmic for the mutation in blood samples. Analysis of mitochondrial DNA in patients with hearing loss and myoclonus epilepsy is recommended, even in the absence of laboratory findings. Therapeutically, ubiquinone and antioxidants can be beneficial.

Age and gender-dependent alternative splicing of P/Q-type calcium channel EF-hand

Ca(v)2.1 Ca(2+) channels (P/Q-type), which participate in various key roles in the CNS by mediating calcium influx, are extensively spliced. One of its alternatively-spliced exons is 37, which forms part of the EF hand. The expression of exon 37a (EFa form), but not exon 37b (EFb form), confers the channel an activity-dependent enhancement of channel opening known as Ca(2+)-dependent facilitation (CDF). In this study, we analyzed the trend of EF hand splice variant distributions in mouse, rat and human brain tissues. We observed a developmental switch in rodents, as well as an age and gender bias in human brain tissues, suggestive of a possible role of these EF hand splice variants in neurophysiological specialization. A parallel study performed on rodent brains showed that the data drawn from human and rodent tissues may not necessarily correlate in the process of aging.