Dilated cardiomyopathy and limb-girdle muscular dystrophy-dystroglycanopathy due to novel pathogenic variants in the DPM3 gene.

Deficiency of Dolichol-P-mannose synthase subunit 3 (DPM3) affects the N-glycosylation and O-mannosylation pathways that are respectively involved in congenital disorders of glycosylation (CDG) and alpha-dystroglycanopathies. Herein, we describe novel pathogenic variants in the DPM3 gene in two unrelated male patients. They developed dilated cardiomyopathy in their late teens, limb-girdle muscular dystrophy - one patient in childhood and the other in adulthood. In both patients, next generation sequencing found in the DPM3 gene a heterozygous deletion and a heterozygous pathogenic missense mutation in exon 2 (c.41T>C, p.Leu14Pro). Electrophoresis of serum transferrin found an abnormal N-glycosylation profile suggestive of CDG type 1 (decreased tetrasialotransferrin, increased disialo- and asialotransferrin). Only two cases of DPM3 gene mutations with limb-girdle muscular dystrophy-dystroglycanopathy have been reported previously. The present study highlights several aspects related to DPM3 gene mutations such as mild to moderately severe limb-girdle muscular dystrophy, dilated cardiomyopathy, and abnormal N-glycosylation profile suggestive of CDG type 1.

Significant response to immune therapies in a case of subacute necrotizing myopathy and FKRP mutations.

Necrotizing myopathies can be encountered in various conditions as acquired myopathies (toxic or autoimmune) or muscular dystrophies. We report a twenty-year-old Caucasian woman who presented with clinical findings suggestive of an inflammatory myopathy: subacute onset of lower limb muscle weakness, myalgia, weight loss and absence of family history. The serum creatine kinase level was elevated at 4738 IU/L (normal range, 25-175 IU/L). Muscle biopsy was consistent with necrotizing myopathy. The patient showed significant clinical improvement following corticosteroid, azathioprine and intravenous immunoglobulin treatments. Biological tests revealed no specific autoantibodies associated with necrotizing autoimmune myopathies. Immunohistochemical staining for sarcolemmal proteins in muscle biopsy samples finally led to a diagnosis of limb-girdle muscular dystrophy 2I (fukutin-related protein gene mutations). The response to immune therapies suggested a possible inflammatory component associated with the muscular dystrophy and highlighted the potential benefit of corticosteroid treatment in patients with LGMD2I and subacute onset.

Three novel CYP11B1 mutations in congenital adrenal hyperplasia due to steroid 11Beta-hydroxylase deficiency in a moroccan population.

BACKGROUND/AIMS: Steroid 11beta-hydroxylase deficiency (11OHD), the second cause of congenital adrenal hyperplasia (CAH), accounts only for 5% of all CAH. To date, only 51 different mutations have been reported with poor clinical and biological data. Most of them could be considered as private mutations except one, p.R448H, identified especially in Moroccan Jews but also in Caucasian patients. As two other CYP11B1 mutations have a high incidence in Tunisian patients, we report from another Maghreb population the clinical, follow-up and molecular genetics of 5 Moroccan patients with classical 11OHD. METHODS: Patients belonging to 3 families were recruited on clinical data. The diagnosis was confirmed by 11-deoxycortisol determination. Sequencing of the CYP11B1 gene and molecular modeling were performed. RESULTS: Clinical, hormonal and follow-up data were consistent with a severe form of 11OHD. Gender reassignment and evolution of hypertension were discussed. Three novel mutations, p.Ala259Asp, p.Gly446Val and IVS5+2T>G were identified. As each patient was homozygous for one mutation, we could deduce from their phenotype and our modeling studies that the p.Gly446Val mutation was more severe than p.Ala259Asp. CONCLUSION: This study shows a good correlation between phenotype and genotype. Each CYP11B1 mutation is new and private, contrasting with the high incidence of two Tunisian mutations.

Phenotype-genotype correlations of 13 rare CYP21A2 mutations detected in 46 patients affected with 21-hydroxylase deficiency and in one carrier.

CONTEXT: Steroid 21-hydroxylase deficiency is the most common enzymatic defect causing congenital adrenal hyperplasia with genotype/phenotype relationships for common mutations. Novel mutations of the CYP21A2 gene must be well studied to propose right genetic counseling for patients. OBJECTIVE: Thirteen CYP21 mutations have been studied. A detailed description of phenotype was performed for all mutations (p.I77T, p.L167P, p.I230T, p.R233K, p.G291S, p.G292D, p.E320K, p.R341P, p.R354H, p.R369W, p.R408C, p.G424S, and p.R426H). In vitro and in silico studies were performed only for those not previously described (p.L167P, p.I230T, p.R233K, p.G292D, p.E320K, and p.R369W). RESULTS: Regarding phenotype, patients with 10 of these mutations had a classical form. A patient with isolated p.I230T presented with nonclassical form and a patient with the association p.I230T + p.V281L in cis presented with a more severe phenotype. The p.R233K mutation was detected in a carrier partner. A patient with p.R369W presented with an intermediate form. Functional studies showed that all mutations except p.I230T and p.R369W decreased enzyme activity more than p.P30L: severity of p.R369W was intermediate between p.P30L and p.V281L, and finally p.I230T was less severe than p.V281L. Mutation analysis in a three-dimensional model structure of the CYP21 protein explained the observed in vitro effects, severe mutations being implicated in important functional domains of the protein. CONCLUSION: According to phenotype and functional studies, 11 of the mutations described, except the isolated p.R369W and p.I230T, may be responsible for a severe phenotype underlying the necessity to manage children having them. The p.I230T is a nonclassical mutation, and for the p.R369W, we need more cases to precise its severity.

Expression and purification of an anti-Foot-and-mouth disease virus single chain variable antibody fragment in tobacco plants.

Low-cost recombinant antibodies could provide a new strategy to control Foot-and-mouth disease virus (FMDV) outbreaks by passive immunization of susceptible animals. In this study, a single chain variable antibody fragment (scFv) recognizing FMDV coat protein VP1 was expressed in transgenic tobacco plants. To enhance the accumulation of scFv protein, the codon-usage of a murine hybridoma-derived scFv gene was adjusted to mimic highly expressed tobacco genes and fused to an elastin-like polypeptide (ELP) tag. This scFv-ELP fusion accumulated up to 0.8% of total soluble leaf protein in transgenic tobacco. To recover scFv-ELP protein from the leaf extract, a simple and scalable purification strategy was established. Purified scFv-ELP fusion was cleaved to separate the scFv portion. Finally, it was shown that the purified scFv proteins retained their capacity to bind the FMDV in the absence or presence of ELP fusion.

p.H62L, a rare mutation of the CYP21 gene identified in two forms of 21-hydroxylase deficiency.

CONTEXT: Steroid 21-hydroxylase deficiency is the most common enzymatic defect causing congenital adrenal hyperplasia with good genotype/phenotype relationships for common mutations. To determine the severity of rare mutations is essential for genetic counseling and better understanding of the structure-function of the cytochrome P450c21. OBJECTIVE: The p.H62L mutation was the most frequent of 60 new mutations detected in 2900 steroid 21-hydroxylase deficiency patients, either isolated or associated on the same allele with a mild mutation (p.P453S, p.P30L, or partial promoter). Because phenotypes seemed to differ between patients with isolated or associated p.H62L, a detailed phenotype description and functional studies were performed. RESULTS: Regarding phenotype, patients with isolated p.H62L had a nonclassical form, whereas patients with the association p.H62L + mild mutation had a simple virilizing form. Functional studies showed that p.H62L reduced the conversion of the two substrates, progesterone and 17-hydroxyprogesterone, in the same way as the mild p.P453S; the association p.H62L + p.P453S decreased enzymatic activity more strongly while conserving residual activity at a level intermediate between p.P453S and p.I172N. This suggested that p.H62L was a mild mutation, whereas a synergistic effect occurred when it was associated. Analysis of p.H62L in a three-dimensional model structure of the CYP21 protein explained the observed in vitro effects, the H62 being located in a domain implied in membrane anchoring. CONCLUSION: According to phenotype and functional studies, p.H62L is a mild mutation, responsible for a more severe phenotype when associated with another mild mutation. These data are important for patient management and genetic counseling.

Post-transcriptional and developmental regulation of a CMS-associated mitochondrial gene region by a nuclear restorer gene.

Transcripts of the mitochondrial gene region orf224/atp6, which is associated with the Polima or pol cytoplasmic male sterility (CMS) of Brassica napus, differ among fertile, sterile and nuclear-restored plants. We show here that the effects of the restorer gene Rfp on orf224/atp6 transcripts varies among different floral organs. Relative to monocistronic atp6 transcripts, levels of the dicistronic transcripts spanning orf224 and atp6 are dramatically reduced in petals, stamens and carpels, but not sepals, of restored flowers. In pol CMS plants, the relative levels of different orf224/atp6 transcripts are similar among the floral organs. Analysis of guanylyltransferase-labeled mtRNA indicates that only the dicistronic 2.2 and 1.9 kb orf224/atp6 transcripts carry an initiator 5' terminus; hence the 1.4 and 1.3 kb transcripts of restored plants, as well as the 1.1 kb atp6 transcript common to all genotypes, are generated by RNA processing and not de novo initiation. Although steady-state levels of dicistronic transcripts in flower buds are lower in restored than in sterile plants, run-on transcription experiments show that these transcripts are synthesized at the same rate in both types of flowers. These findings imply that the restorer gene acts by conditioning the removal of sequences from the 5' end of dicistronic transcripts in a developmentally regulated manner. Run-on transcription experiments indicate that the single 1.1 kb atp6 transcript of nap cytoplasm is also generated by removal of sequences from the 5' end of a precursor. We suggest that specific endonucleolytic cleavage of a precursor RNA, followed by non-specific 3' to 5' exonuclease action, may represent a common mechanism for tailoring transcripts in plant mitochondria.

An open reading frame for a protein involved in cytochrome c biogenesis is split into two parts in Brassica mitochondria.

A plant mitochondrial gene with sequence similarity to ccl1, a bacterial gene involved in cytochrome c biogenesis, occurs as a single open reading frame in wheat, Oenothera, carrot and Marchantia mtDNAs. In Brassica napus "Polima" or Brassica campestris mtDNA, however, this open reading frame is split into two segments that are located 60 kb apart on the master-circle form of the genome. Although transcripts of the split ORF are edited in a manner similar to that of a functional gene, transcripts that span both portions of the ORF are not evident in gel-blot hybridization experiments. Low-abundance transcripts that span both portions of the split ORF can be detected by RT-PCR, but these contain an additional 54-bp sequence, inserted between the two segments, that is unrelated to the corresponding sequences of other plants. Since this additional sequence introduces an in-frame stop codon, no transcripts have been found that could be translated to yield a protein product of a size similar to that present in other plant species. An antiserum directed against the product of the corresponding wheat gene detects polypeptides of similar size in wheat and Brassica mitochondria. This antiserum, however, immunoprecipitates a labelled polypeptide from the products of wheat, but not Brassica, in organello protein synthesis. Gel-blot analysis of total Brassica DNA indicates that sequences capable of hybridizing with the ORF are present in the nuclear genome. We suggest that the functional form of the Brassica gene may reside in the nucleus.

Nuclear genes associated with a single Brassica CMS restorer locus influence transcripts of three different mitochondrial gene regions.

Previous studies have shown that the mitochondrial orf224/atp6 gene region is correlated with the Polima (pol) cytoplasmic male sterility (CMS) of Brassica napus. We now extend this correlation by showing that the effects of nuclear fertility restoration on orf224/atp6 transcripts cosegregate with the pol restorer gene Rfp1 in genetic crosses. We also show, however, that the recessive rfp1 allele, or a very tightly linked gene, acts as a dominant gene, designated Mmt (modifier of mitochondrial transcripts), in controlling the presence of additional smaller transcripts of the nad4 gene and a gene possibly involved in cytochrome c biogenesis. A common sequence, TTGTGG, maps immediately downstream of the 5' termini of both of the transcripts specific to plants with the Mmt gene and may serve as a recognition motif in generation of these transcripts. A similar sequence, TTGTTG, that may be recognized by the product of the alternate allele (or haplotype), Rfp1, is found within orf224 just downstream of the major 5' transcript terminus specific to fertility restored plants. Our results suggest that Rfp1/ Mmt is a novel nuclear genetic locus that affects the expression of multiple mitochondrial gene regions, with different alleles or haplotypes exerting specific effects on different mitochondrial genes.

Characterization of transcription initiation sites on the soybean mitochondrial genome allows identification of a transcription-associated sequence motif.

Transcription initiation sites on the soybean mitochondrial genome have been characterized by sequence analysis of in vitro-capped soybean mtRNAs and corresponding mtDNA regions. The most abundant, discrete soybean mtRNA species labeled by guanylyltransferase and [alpha-32P]GTP are shown to correspond to the major transcript of the atp9 gene and to a group of small RNAs consisting of a discrete 80 nucleotide (nt) species plus heterogeneous species ranging in size from 133 to 148 nt. The 133-148 nt RNAs represent a set of transcripts with a common 5' terminus and ragged 3' ends, while the 80 nt RNA corresponds to positions 53-133 of the 133 nt species. The major, discrete in vitro-capped RNA species thus correspond to primary transcripts originating at three sites located in two regions of the soybean mitochondrial genome. The sequences extending from 13 nucleotides upstream to 8 nucleotides downstream of the initiation sites for the atp9 and 133-148 nt transcripts are identical at 18 of 21 positions. Sequences closely resembling this motif are located at some other 5' transcript termini of dicot plant mitochondria. Less closely related sequences are found at transcription initiation sites of wheat and maize mitochondria.

A preliminary analysis of Fatty Acid synthesis in pea roots.

Subcellular fractions from pea (Pisum sativum L.) roots have been prepared by differential centrifugation techniques. Greater than 50% of the recovered plastids can be isolated by centrifugation at 500g for 5 minutes. Plastids of this fraction are largely free from mitochondrial and microsomal contamination as judged by marker enzyme analysis. De novo fatty acid biosynthesis in pea roots occurs in the plastids. Isolated pea root plastids are capable of fatty acid synthesis from acetate at rates up to 4.3 nanomoles per hour per milligram protein. ATP, bicarbonate, and either Mg(2+) or Mn(2+) are all absolutely required for activity. Coenzyme A at 0.5 millimolar improved activity by 60%. Reduced nucleotides were not essential but activity was greatest in the presence of 0.5 millimolar of both NADH and NADPH. The addition of 0.5 millimolar glycerol-3-phosphate increased activity by 25%. The in vitro and in vivo products of fatty acid synthesis from acetate were primarily palmitate, stearate, and oleate, the proportions of which were dependent on experimental treatments. Fatty acids synthesized by pea root plastids were recovered in primarily phosphatidic acid and diacylglycerol or as water soluble derivatives and the free acids. Lesser amounts were found in phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and monogalactosyldiacylglycerol.