Compound heterozygous c.598_612del and c.1746-20C > G CAPN3 genotype cause autosomal recessive limb-girdle muscular dystrophy-1: a case report

Background Autosomal recessive limb–girdle muscular dystrophy-1 (LGMDR1), also known as calpainopathy, is a genetically heterogeneous disorder characterised by progression of muscle weakness. Homozygous or compound heterozygous variants in the CAPN3 gene are known genetic causes of this condition. The aim of this study was to confirm the molecular consequences of the CAPN3 variant NG_008660.1(NM_000070.3):c.1746-20C > G of an individual with suspected LGMDR1 by extensive complementary DNA (cDNA) analysis. Case presentation In the present study, we report on a male with proximal muscular weakness in his lower limbs. Compound heterozygous NM_000070.3:c.598_612del and NG_008660.1(NM_000070.3):c.1746-20C > G genotype was detected on the CAPN3 gene by targeted next-generation sequencing (NGS). To confirm the pathogenicity of the variant c.1746-20C > G, we conducted genetic analysis based on Sanger sequencing of the proband’s cDNA sample. The results revealed that this splicing variant disrupts the original 3′ splice site on intron 13, thus leading to the skipping of the DNA fragment involving exon 14 and possibly exon 15. However, the lack of exon 15 in the CAPN3 isoforms present in a blood sample was explained by cell-specific alternative splicing rather than an aberrant splicing mechanism. In silico the c.1746-20C > G splicing variant consequently resulted in frameshift and formation of a premature termination codon (NP_000061.1:p.(Glu582Aspfs*62)). Conclusions Based on the results of our study and the literature we reviewed, both c.598_612del and c.1746-20C > G variants are pathogenic and together cause LGMDR1. Therefore, extensive mRNA and/or cDNA analysis of splicing variants is critical to understand the pathogenesis of the disease.

New Insectotoxin from Tibellus Oblongus Spider Venom Presents Novel Adaptation of ICK Fold

The Tibellus oblongus spider is an active predator that does not spin webs and remains poorly investigated in terms of venom composition. Here, we present a new toxin, named Tbo-IT2, predicted by cDNA analysis of venom glands transcriptome. The presence of Tbo-IT2 in the venom was confirmed by proteomic analyses using the LC-MS and MS/MS techniques. The distinctive features of Tbo-IT2 are the low similarity of primary structure with known animal toxins and the unusual motif of 10 cysteine residues distribution. Recombinant Tbo-IT2 (rTbo-IT2), produced in E. coli using the thioredoxin fusion protein strategy, was structurally and functionally studied. rTbo-IT2 showed insecticidal activity on larvae of the housefly Musca domestica (LD100 200 μg/g) and no activity on the panel of expressed neuronal receptors and ion channels. The spatial structure of the peptide was determined in a water solution by NMR spectroscopy. The Tbo-IT2 structure is a new example of evolutionary adaptation of a well-known inhibitor cystine knot (ICK) fold to 5 disulfide bonds configuration, which determines additional conformational stability and gives opportunities for insectotoxicity and probably some other interesting features.

Effects of RTTN Gene Mutations and the Need for Complementary cDNA Analysis for Some Transcripts

Introduction: Prenatal WES analysis is currently required in prenatal diagnosis in case of multiple congenital anomalies and in families where some genetic diseases are reported. However, with development of prenatal WES, practitioners are sometimes facing a lot of challenge regarding interpretation of the genetic results. Method: Prenatal WES analysis. Result: Detection of two different RTTN gene transcripts in fetal WES. One of the transcripts showed RTTN homozygous gene mutation while the other transcript was normal. Discussion: This result emphasizes difficulty of genetic counselling in case of absence of prenatal radiological findings or late findings. Conservative fetal follow up was advised because of absence of any positive radiological finding. Conclusion: This article presents the multidisciplinary approach in prenatal and postnatal counselling for cases with quiry fetal genetic findings and illustrates the urgent need for development of transcriptome analysis for the fetus with WES findings of uncertain significance.

Effects of RTTN Gene Mutations and the Need for Complementary cDNA Analysis for Some Transcripts

Introduction: Prenatal WES analysis is currently required in prenatal diagnosis in case of multiple congenital anomalies and in families where some genetic diseases are reported. However, with development of prenatal WES, practitioners are sometimes facing a lot of challenge regarding interpretation of the genetic results. Method: Prenatal WES analysis. Result: Detection of two different RTTN gene transcripts in fetal WES. One of the transcripts showed RTTN homozygous gene mutation while the other transcript was normal. Discussion: This result emphasizes difficulty of genetic counselling in case of absence of prenatal radiological findings or late findings. Conservative fetal follow up was advised because of absence of any positive radiological finding. Conclusion: This article presents the multidisciplinary approach in prenatal and postnatal counselling for cases with quiry fetal genetic findings and illustrates the urgent need for development of transcriptome analysis for the fetus with WES findings of uncertain significance.

Controlling the hypermutation: Exploitation of the MutS protein in Pseudomonas aeruginosa

Pseudomonas aeruginosa infections commonly develop in individuals with cystic fibrosis (CF), and its adaptation in such an unfavourable condition is always found to be related to hypermutation. In fact, most of the hypermutation is due to the defects in mutS gene which involves in the mismatch repair mechanism, causing the acceleration of mutation rate and adaptive evolution. In order to rheostatically express the MutS protein and achieve “hypomutation” (in which the rate of mutation is lower than that of wild type strain), an exogenous mutS gene with rhamnose-inducible promoter was cloned into MPAO1 mutS::Tn mutant strain. Present findings demonstrate that this system is tightly-controlled and stable, with less rifampicin-resistant mutant frequency and more fluorescence intensity from a GFP-tagged MutS expressing cells were observed when the concentration of the inducer increases. Interestingly, the results from Western blot analysis show that less MutS protein is required to suppress hypermutation in the wild type strain, as compared to our construct that behaves similar to the wild type but obviously needs more MutS expression to achieve such state. This indicates that the exogenous MutS might be lacking of other important protein to work efficiently in mismatch recognition. Therefore, based on our cDNA analysis, we found that fdxA gene next to the mutS gene is in the same operon, which could suggest that they might be functionally related in the DNA repair machinery.

Novel HADHB mutations in a patient with mitochondrial trifunctional protein deficiency

We encountered a patient with mitochondrial trifunctional protein deficiency in whom the corresponding mutations were not identified by a DNA panel for newborn screening for targeted diseases. After diagnosis confirmation by an enzyme assay and immunoblotting using the autopsied liver, the re-evaluation of the panel data indicated a heterozygous deletion of exons 6–9 that was later confirmed at the genomic level. cDNA analysis also identified exonization of the 5′ region of intron 9 caused by a deep intronic mutation, c.811 + 82A>G.