<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N backbone resonance assignment of the lamin C-terminal region specific to prelamin A

Lamins are the main components of the nucleoskeleton. They form a protein meshwork that underlies the inner nuclear membrane. Mutations in the LMNA gene coding for A-type lamins (lamins A and C) cause a large panel of human diseases, referred to as laminopathies. These diseases include muscular dystrophies, lipodystrophies and premature aging diseases. Lamin A exhibits a C-terminal region that is different from lamin C and is post-translationally modified. It is produced as prelamin A and it is then farnesylated, cleaved, carboxymethylated and cleaved again in order to become mature lamin A. In patients with the severe Hutchinson–Gilford progeria syndrome, a specific single point mutation in LMNA leads to an aberrant splicing of the LMNA gene preventing the post-translational processing of prelamin A. This leads to the accumulation of a permanently farnesylated lamin A mutant lacking 50 amino acids named progerin. We here report the NMR ¹H, ¹⁵N, ¹³CO, ¹³Cα and ¹³Cβ chemical shift assignment of the C-terminal region that is specific to prelamin A, from amino acid 567 to amino acid 664. We also report the NMR ¹H, ¹⁵N, ¹³CO, ¹³Cα and ¹³Cβ chemical shift assignment of the C-terminal region of the progerin variant, from amino acid 567 to amino acid 614. Analysis of these chemical shift data confirms that both prelamin A and progerin C-terminal domains are largely disordered and identifies a common partially populated α-helix from amino acid 576 to amino acid 585. This helix is well conserved from fishes to mammals.     

Novel 1q22-q23.1 duplication in a patient with lambdoid and metopic craniosynostosis,muscular hypotonia,and psychomotor retardation

Craniosynostosis (CS) refers to the group of craniofacial malformations characterized by the premature closure of one or more cranial sutures. The disorder is clinically and genetically heterogeneous and occurs usually as an isolated trait, but can also be syndromic. In 30–60% of patients, CS is caused by known genetic factors; however, in the rest of the cases, causative molecular lesions remain unknown. In this paper, we report on a sporadic male patient affected by complex CS (metopic and unilateral lambdoid synostosis), muscular hypotonia, psychomotor retardation, and facial dysmorphism. Since a subset of CS results from submicroscopic chromosomal aberrations, we performed array comparative genomic hybridization (array CGH) in order to identify possibly causative copy-number variation. Array CGH followed by breakpoint sequencing revealed a previously unreported de novo 1.26 Mb duplication at chromosome 1q22-q23.1 that encompassed two genes involved in osteoblast differentiation: BGLAP, encoding osteocalcin (OCN), and LMNA, encoding lamin A/C. OCN is a major component of bone extracellular matrix and a marker of osteogenesis, whereas mutations in LMNA cause several genetic disorders called laminopathies, including mandibuloacral dysostosis (MAD) that manifests with low bone mass, severe bone deformities, and delayed closure of the cranial sutures. Since LMNA and BGLAP overexpression promote osteoblast differentiation and calcification, phenotype of our patient may result from misexpression of the genes. Based on our findings, we hypothesize that both LMNA and BGLAP may be implicated in the pathogenesis of CS in humans. However, further studies are needed to establish the exact pathomechanism underlying development of this defect.     

Modifier locus of the skeletal muscle involvement in Emery–Dreifuss muscular dystrophy

Autosomal dominant Emery–Dreifuss muscular dystrophy is caused by mutations in LMNA gene encoding lamins A and C. The disease is characterized by early onset joint contractures during childhood associated with humero-peroneal muscular wasting and weakness, and by the development of a cardiac disease in adulthood. Important intra-familial variability characterized by a wide range of age at onset of myopathic symptoms (AOMS) has been recurrently reported, suggesting the contribution of a modifier gene. Our objective was to identify a modifier locus of AOMS in relation with the LMNA mutation. To map the modifier locus, we genotyped 291 microsatellite markers in 59 individuals of a large French family, where 19 patients carrying the same LMNA mutation, exhibited wide range of AOMS. We performed Bayesian Markov Chain Monte Carlo-based joint segregation and linkage methods implemented in the Loki^© software, and detected a strong linkage signal on chromosome 2 between markers D2S143 and D2S2244 (211 cM) with a Bayes factor of 28.7 (empirical p value = 0.0032). The linked region harbours two main candidate genes, DES and MYL1 encoding desmin and light chain of myosin. Importantly, the impact of the genotype on the phenotype for this locus showed an overdominant effect with AOMS 2 years earlier for the homozygotes of the rare allele and 37 years earlier for the heterozygotes than the homozygotes for the common allele. These results provide important highlights for the natural history and for the physiopathology of Emery–Dreifuss muscular dystrophy.     

A G613A missense in the Hutchinson’s progeria lamin A/C gene causes a lone,autosomal dominant atrioventricular block

Background

LMNA/C mutations have been linked to the premature aging syndrome Hutchinson’s progeria, dilated cardiomyopathy 1A, skeletal myopathies (such as the autosomal dominant variant of Emery-Dreifuss muscular dystrophy and limb-girdle muscular dystrophy), Charcot-Marie-Tooth disorder type 2B1, mandibuloacral dysplasia, autosomal dominant partial lipodystrophy, and axonal neuropathy. Atrioventricular block (AVB) can be associated with several cardiac disorders and it can also be a highly heritable, primitive disease.One of the most common pathologies associated with AVB is dilated cardiomyopathy (DCM), which is characterized by cardiac dilatation and reduced systolic function. In this case, onset has been correlated with several mutations in genes essential for the proper maturation of cardiomyocytes, such as the gene for lamin A/C. However, no clear genotype–phenotype relationship has been reported to date between LMNA/C mutations and cardiomyopathies.

Results

DNA and medical histories were collected from (n?=?11) members of different generations of one family, the proband of which was implanted with a pacemaker for lone, type II AVB. Exome sequencing analysis was performed on three relatives with AVB, and the mutations therein identified validated in a further three AVB-affected family members.In the initial three AVB family members, we identified 10 shared nonsynonymous single-nucleotide variations with a rare or unreported allele frequency in the 1000 Genomes Project database. Follow-up genetic screening in the additional three affected relatives disclosed a correlation between the lone AVB phenotype and the single-nucleotide polymorphism rs56816490, which generates an E317K change in lamin A/C. Although this mutation has already been described by others in a DCM-affected proband with familiarity for AVB and sudden death, the absence of DCM in our large, AVB-affected family is indicative of genotype–phenotype correlation between rs56816490 and a familial, autosomal dominant form of lone AVB.

Conclusions

Screening for G613A in LMNA/C in patients with lone AVB and their relatives might prevent sudden death in families affected by AVB but without familiarity for DCM. Lone AVB is an age-related disease caused by mutations in LMN A/C gene rather than a complication of DCM.

     

Myofibrillar myopathy in the genomic context

Myofibrillar myopathy (MFM) is a group of inherited muscular disorders characterized by myofibril dissolution and abnormal accumulation of degradation products. The diagnosis of muscular disorders based on clinical presentation is difficult due to phenotypic heterogeneity and overlapping symptoms. In addition, precise diagnosis does not always explain the disease etiopathology or the highly variable clinical course even among patients diagnosed with the same type of myopathy. The advent of high-throughput next-generation sequencing (NGS) has provided a successful and cost-effective strategy for identification of novel causative genes in myopathies, including MFM. So far, pathogenic mutations associated with MFM phenotype, including atypical MFM-like cases, have been identified in 17 genes: DES, CRYAB, MYOT, ZASP, FLNC, BAG3, FHL1, TTN, DNAJB6, PLEC, LMNA, ACTA1, HSPB8, KY, PYROXD1, and SQSTM + TIA1 (digenic). Most of these genes are also associated with other forms of muscle diseases. In addition, in many MFM patients, numerous genomic variants in muscle-related genes have been identified. The various myopathies and muscular dystrophies seem to form a single disease continuum; therefore, gene identification in one disease impacts the genetic etiology of the others. In this review, we describe the heterogeneity of the MFM genetic background focusing on the role of rare variants, the importance of whole genome sequencing in the identification of novel disease-associated mutations, and the emerging concept of variant load as the basis of the phenotypic heterogeneity.     

Identification and association of novel lncRNA <Emphasis Type="BoldItalic">pouMU1</Emphasis> gene mutations with chicken performance traits

The biological functions of long noncoding RNAs (lncRNAs), which play an important role in regulating development and gene expression, may be affected by variations in lncRNA gene loci or associated genomic sequences. However, the functions of many lncRNAs remain unknown. To analyse correlations between mutations in pouMU1 with chicken growth and carcass traits, 860 chickens from a Gushi \(\times \) Anka F2 resource population and 96 Lushi, Xichuan, Changshun and recessive white chickens were used to evaluate the genetic effect of the pouMU1 gene. We performed quantitative real-time polymerase chain reaction (qRT-PCR) to analyse the relative expression levels of pouMU1 in nine different tissues and stages of development. pouMU1 expression was highest in pectoralis and leg muscles, whereas no expression was observed in the heart, liver and abdominal fat. Using direct sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods, two novel sequence mutations (g.1198A>G and g.1238-1239del/insGA) were detected in the pouMU1 gene. SPSS software was used for statistical analysis in association studies. Based on the association data, the presence of both variants was significantly associated with leg muscle fibre width and leg muscle fibre roundness (\(P < 0.05\)) and highly associated with leg muscle fibre girth and body weight at 0 week of age (\(P < 0.01\)). These data suggest that pouMU1 might participate in regulating chicken muscle development and growth, and the findings offer new insight into the functions of sequence mutations in lncRNAs.     

Association of polymorphic markers of chemokine genes,their receptors,and <Emphasis Type="Italic">CD14</Emphasis> gene with coronary atherosclerosis

Atherosclerosis represents an inflammatory response to the disturbance of the endothelial layer in the arterial bloodstream. In the present study, an analysis of associations of polymorphic markers for the genes controlling synthesis of proteins involved in atherosclerosis pathogenesis in coronary atherosclerosis (CA) patients (217 subjects) and in a control group (250 subjects) was conducted. The following genes were examined: rs991804 (CCL2 gene), rs1126579 (CXCR2 gene), rs4074 (CXCL1 gene), rs4073 (CXCL8 gene), rs333 (CCR5 gene), rs2471859 (CXCR4 gene), rs1801157 (CXCL12 gene), and rs2569190 (CD14 gene). Using the Monte Carlo and Markov chain (APSampler) method, allele/genotype combinations associated with both low and high CA risk were revealed. The most important findings included the following: CXCR4*T/T + CCL2*C + CCR5*I/I (P_perm = 1 × 10^–6, OR = 0.44, 95% CI 0.3–0.63), CXCR2*C + CD14*C + CXCL12*G + CCL2*C + CCR5*D (P_perm = 4 × 10^–6, OR = 5.78, 95% CI 2.34–14.28), CD14*C + CCL2*C/C + CCR5*D (P_perm = 6.3 × 10^–6, OR = 5.81, 95% CI 2.17–15.56), CXCL8*A + CXCR2*C + CD14*T + CXCR4*C (P_perm = 0.01, OR = 3.21, 95% CI 1.63–6.31).     

Phenotype–Genotype Analysis of Chinese Patients with Early-Onset LMNA-Related Muscular Dystrophy

This study aimed to analyze the correlation between the phenotype and genotype of Chinese patients with early-onset lamin A (LMNA)-related muscular dystrophy (MD). The clinical and myopathological data of 21 Chinese pediatric patients with early-onset LMNA-related MD were collected and analyzed. LMNA gene mutation analysis was performed by direct sequencing of genomic DNA. Sublocalization of wild-type and mutant proteins were observed by immunofluorescence using cultured fibroblasts and human embryonic kidney 293 (HEK 293) cell. Seven patients were diagnosed with Emery-Dreifuss muscular dystrophy (EDMD) and 14 were diagnosed with LMNA-associated congenital muscular dystrophy (L-CMD). Four biopsy specimens from the L-CMD cases exhibited inflammatory changes. Abnormal nuclear morphology was observed with both transmission electron microscopy and lamin A/C staining. We identified 10 novel and nine known LMNA gene mutations in the 21 patients. Some mutations (c.91G>A, c.94_96delAAG, c.116A>G, c.745C>T, c.746G>A, and c.1580G>C) were well correlated with EDMD or L-CMD. LMNA-related MD has a common symptom triad of muscle weakness, joint contractures, and cardiac involvement, but the severity of symptoms and disease progression differ greatly. Inflammatory change in biopsied muscle is a characteristic of early-stage L-CMD. Phenotype–genotype analysis determines that some mutations are well correlated with LMNA-related MD.     

<Emphasis Type="Italic">Sfi</Emphasis>NX: a method for assembly of protein coding sequences with high success rates

Objective

Concatenation of two NdeI–XhoI gene fragments via an oligonucleotide linker on a plasmid vector with an SfiI site was performed to evaluate success rates in construction of polycistronic genes expressible in Escherichia coli.

Results

A series of plasmids with an SfiI site between the selection marker and the replication origin were constructed. The three wheat eEF1B subunit genes inserted between the NdeI and XhoI sites of pET-22b were transferred to the SfiI-containing plasmid with a spectinomycin-resistance gene. Then, the marker gene in the resultant plasmids was substituted with the ampicillin-resistance gene. These plasmids were used for concatenation of two different genes via a linker oligonucleotide containing a ribosome-binding site. During these operations, 42 clones were picked up out of which 41 had the intended product plasmid.

Conclusion

This method, named as the SfiNX method, is useful for trial-and-error based testing of different combinations of fusion and co-expression partners for optimization of recombinant protein production.

     

Mapping disease‐related missense mutations in the immunoglobulin‐like fold domain of lamin A/C reveals novel genotype–phenotype associations for laminopathies

Mutations in A‐type nuclear lamins cause laminopathies. However, genotype–phenotype correlations using the 340 missense mutations within the LMNA gene are unclear: partially due to the limited availability of three‐dimensional structure. The immunoglobulin (Ig)‐like fold domain has been solved, and using bioinformatics tools (including Polyphen‐2, Fold X, Parameter OPtimized Surfaces, and PocketPicker) we characterized 56 missense mutations for position, surface exposure, change in charge and effect on Ig‐like fold stability. We find that 21 of the 27 mutations associated with a skeletal muscle phenotype are distributed throughout the Ig‐like fold, are nonsurface exposed and predicted to disrupt overall stability of the Ig‐like fold domain. Intriguingly, the remaining 6 mutations clustered, had higher surface exposure, and did not affect stability. The majority of 9 lipodystrophy or 10 premature aging syndrome mutations also did not disrupt Ig‐like fold domain stability and were surface exposed and clustered in distinct regions that overlap predicted binding pockets. Although buried, the 10 cardiac mutations had no other consistent properties. Finally, most lipodystrophy and premature aging mutations resulted in a ‐1 net charge change, whereas skeletal muscle mutations caused no consistent net charge changes. Since premature aging, lipodystrophy and the subset of 6 skeletal muscle mutations cluster tightly in distinct, charged regions, they likely affect lamin A/C –protein/DNA/RNA interactions: providing a consistent genotype–phenotype relationship for mutations in this domain. Thus, this subgroup of skeletal muscle laminopathies that we term the ‘Skeletal muscle cluster’, may have a distinct pathological mechanism. These novel associations refine the ability to predict clinical features caused by certain LMNA missense mutations. Proteins 2014; 82:904–915. © 2013 Wiley Periodicals, Inc.     

Dissociation of Systemic Glucose Homeostasis from Triacylglyceride Accumulation by Reduced <Emphasis Type="Italic">Acsl6</Emphasis> Expression in Skeletal Muscle

Long chain acyl-CoA synthetase (ACSL) is an enzyme that activates fatty acids before they are further metabolized. ACSL6 is the one of main ACSL isoforms exclusively expressed in skeletal muscle, but the consequences of the suppression of this gene in systemic glucose homeostasis has yet to be reported. Hence, we investigated the roles of ACSL6 gene in glucose tolerance and TAG distribution in physiological conditions. Eight-week-old male C57BL/6J mice were administered with control or Acsl6 siRNAs and then fed with either AIN-93 control diet or high fat diet. At seven days after the first siRNA injection, oral glucose tolerance tests and TAG quantification were performed. In vivo administration of Acsl6 siRNA decreased Acsl6 expression only in skeletal muscle under AIN-93 or a high fat diet. However Acsl6 siRNA injection to animals increased TAG accumulation in the liver without the change of Acsl6 expression. Atelocollagen mediated Acsl6 suppression enhanced whole-body glucose tolerance coinciding with decreased TAG accumulation in skeletal muscle of mice fed an AIN-93 diet. However, the improved glucose tolerance by Acsl6 reduction was ablated by high fat diet. Moreover reduced Acsl6 did not alter the phosphorylation of insulin signaling proteins in skeletal muscle. These results suggest that Acsl6 reduction in skeletal muscle enhances glucose homeostasis and dissociates the insulin responses from TAG accumulation in skeletal muscle.     

Association analysis of alcohol metabolizing enzymes <Emphasis Type="Italic">ADH1B,ADH7, CYP2E1</Emphasis> gene polymorphism with risk for coronary atherosclerosis

The allele and genotype distribution of two alcohol dehydrogenase genes ADH1B (exon 3 polymorphism A/G (47His)), ADH7 (intron 5 polymorphism G/C) and cytochrome P450 2E1 gene (CYP2E1; 5′-flanking region G/C and intron 6 T/A polymorphisms) were examined in Russian (Tomsk, n = 125) healthy population and in coronary atherosclerosis patients (CA, n = 92). The genotype frequencies followed the Hardy-Weinberg equilibrium and the alleles were in linkage equilibrium or gametic equilibrium in the control sample. Only two CYP2E1 gene polymorphisms were in linkage disequilibrium. The frequencies of the derived alleles at ADH1B ^* G (+MslI) allele, CYP2E1 ^* C2 (+PstI) allele and CYP2E1 ^* C (-DraI) allele were 8.48 ± 1.86, 1.20 ± 0.69, and 10.00 ± 1.90%, respectively. The ADH7 gene polymorphism showed a high level of heterozygosity; the frequency of the ADH7 ^* C (-StyI) allele was 44.58 ± 3.21%. A significantly higher frequency of CYP2E1 PstI C2 allele has been revealed in the CA group (P = 0.043; OR = 4.23; 95% CI 1.03–20.01). The tendency to significant effect of A1A2 genotype in ADH1B MslI polymorphism was observed for systolic blood pressure in the control group (P = 0.068). The statistically significant two-way interaction effects of ADH7 StyI and CYP2E1 DraI on diastolic blood pressure (P = 0.029) and on the serum high density lipoprotein level (P = 0.042) were also revealed. Association of A1A2 genotype in ADH1B MslI polymorphism with reduced amount in a serum of a very low density lipoprotein level (P = 0.045) have also been shown. This may result from multifunctional activity of alcohol metabolizing enzymes and their involvement in many metabolic and free radical reactions in the body.     

Adaptive mutation related to cellulose producibility in <Emphasis Type="Italic">Komagataeibacter medellinensis</Emphasis> (<Emphasis Type="Italic">Gluconacetobacter xylinus</Emphasis>) NBRC 3288

Gluconacetobacter xylinus (formerly Acetobacter xylinum and presently Komagataeibacter medellinensis) is known to produce cellulose as a stable pellicle. However, it is also well known to lose this ability very easily. We investigated the on and off mechanisms of cellulose producibility in two independent cellulose-producing strains, R1 and R2. Both these strains were isolated through a repetitive static culture of a non-cellulose-producing K. medellinensis NBRC 3288 parental strain. Two cellulose synthase operons, types I and II, of this strain are truncated by the frameshift mutation in the bcsBI gene and transposon insertion in the bcsCII gene, respectively. The draft genome sequencing of R1 and R2 strains revealed that in both strains the bcsBI gene was restored by deletion of a nucleotide in its C-rich region. This result suggests that the mutations in the bcsBI gene are responsible for the on and off mechanism of cellulose producibility. When we looked at the genomic DNA sequences of other Komagataeibacter species, several non-cellulose-producing strains were found to contain similar defects in the type I and/or type II cellulose synthase operons. Furthermore, the phylogenetic relationship among cellulose synthase genes conserved in other bacterial species was analyzed. We observed that the cellulose genes in the Komagataeibacter shared sequence similarities with the γ-proteobacterial species but not with the α-proteobacteria and that the type I and type II operons could be diverged from a same ancestor in Komagataeibacter.     

No fitness cost of glyphosate resistance endowed by massive <Emphasis Type="Italic">EPSPS</Emphasis> gene amplification in <Emphasis Type="Italic">Amaranthus palmeri</Emphasis>

Amplification of the EPSPS gene has been previously identified as the glyphosate resistance mechanism in many populations of Amaranthus palmeri, a major weed pest in US agriculture. Here, we evaluate the effects of EPSPS gene amplification on both the level of glyphosate resistance and fitness cost of resistance. A. palmeri individuals resistant to glyphosate by expressing a wide range of EPSPS gene copy numbers were evaluated under competitive conditions in the presence or absence of glyphosate. Survival rates to glyphosate and fitness traits of plants under intra-specific competition were assessed. Plants with higher amplification of the EPSPS gene (53-fold) showed high levels of glyphosate resistance, whereas less amplification of the EPSPS gene (21-fold) endowed a lower level of glyphosate resistance. Without glyphosate but under competitive conditions, plants exhibiting up to 76-fold EPSPS gene amplification exhibited similar height, and biomass allocation to vegetative and reproductive organs, compared to glyphosate susceptible A. palmeri plants with no amplification of the EPSPS gene. Both the additive effects of EPSPS gene amplification on the level of glyphosate resistance and the lack of associated fitness costs are key factors contributing to EPSPS gene amplification as a widespread and important glyphosate resistance mechanism likely to become much more evident in weed plant species.     

Altered chromosomal positioning, compaction, and gene expression with a lamin A/C gene mutation

Background

Lamins A and C, encoded by the LMNA gene, are filamentous proteins that form the core scaffold of the nuclear lamina. Dominant LMNA gene mutations cause multiple human diseases including cardiac and skeletal myopathies. The nuclear lamina is thought to regulate gene expression by its direct interaction with chromatin. LMNA gene mutations may mediate disease by disrupting normal gene expression.

Methods/Findings

To investigate the hypothesis that mutant lamin A/C changes the lamina''s ability to interact with chromatin, we studied gene misexpression resulting from the cardiomyopathic LMNA E161K mutation and correlated this with changes in chromosome positioning. We identified clusters of misexpressed genes and examined the nuclear positioning of two such genomic clusters, each harboring genes relevant to striated muscle disease including LMO7 and MBNL2. Both gene clusters were found to be more centrally positioned in LMNA-mutant nuclei. Additionally, these loci were less compacted. In LMNA mutant heart and fibroblasts, we found that chromosome 13 had a disproportionately high fraction of misexpressed genes. Using three-dimensional fluorescence in situ hybridization we found that the entire territory of chromosome 13 was displaced towards the center of the nucleus in LMNA mutant fibroblasts. Additional cardiomyopathic LMNA gene mutations were also shown to have abnormal positioning of chromosome 13, although in the opposite direction.

Conclusions

These data support a model in which LMNA mutations perturb the intranuclear positioning and compaction of chromosomal domains and provide a mechanism by which gene expression may be altered.     

Cytoplasmic localization of PML particles in laminopathies

There is growing evidence that laminopathies, diseases associated with mutations in the LMNA gene, are caused by a combination of mechanical and gene regulatory distortions. Strikingly, there is a large variability in disease symptoms between individual patients carrying an identical LMNA mutation. This is why classical genetic screens for mutations appear to have limited predictive value for disease development. Recently, the widespread occurrence of repetitive nuclear ruptures has been described in fibroblast cultures from various laminopathy patients. Since this phenomenon was strongly correlated with disease severity, the identification of biomarkers that report on these rupture events could have diagnostic relevance. One such candidate marker is the PML nuclear body, a structure that is normally confined to the nuclear interior, but leaks out of the nucleus upon nuclear rupture. Here, we show that a variety of laminopathies shows the presence of these cytoplasmic PML particles (PML CPs), and that the amount of these protein aggregates increases with severity of the disease. In addition, between clinically healthy individuals, carrying LMNA mutations, significant differences can be found. Therefore, we postulate that detection of PML CPs in patient fibroblasts could become a valuable marker for diagnosis of disease development.