Mutations in C1orf194, encoding a calcium regulator, cause dominant Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease is a hereditary motor and sensory neuropathy exhibiting great clinical and genetic heterogeneity. Here, the identification of two heterozygous missense mutations in the C1orf194 gene at 1p21.2-p13.2 with Charcot-Marie-Tooth disease are reported. Specifically, the p.I122N mutation was the cause of an intermediate form of Charcot-Marie-Tooth disease, and the p.K28I missense mutation predominately led to the demyelinating form. Functional studies demonstrated that the p.K28I variant significantly reduced expression of the protein, but the p.I122N variant increased. In addition, the p.I122N mutant protein exhibited the aggregation in neuroblastoma cell lines and the patient's peroneal nerve. Either gain-of-function or partial loss-of-function mutations to C1ORF194 can specify different causal mechanisms responsible for Charcot-Marie-Tooth disease with a wide range of clinical severity. Moreover, a knock-in mouse model confirmed that the C1orf194 missense mutation p.I121N led to impairments in motor and neuromuscular functions, and aberrant myelination and axonal phenotypes. The loss of normal C1ORF194 protein altered intracellular Ca2+ homeostasis and upregulated Ca2+ handling regulatory proteins. These findings describe a novel protein with vital functions in peripheral nervous systems and broaden the causes of Charcot-Marie-Tooth disease, which open new avenues for the diagnosis and treatment of related neuropathies.

Mutation in SSUH2 Causes Autosomal-Dominant Dentin Dysplasia Type I.

Dentin dysplasia type I (DDI) is an autosomal-dominant genetic disorder resulting from dentin defects. The molecular basis of DDI remains unclear. DDI exhibits unique characteristics with phenotypes featuring obliteration of pulp chambers and diminutive root, thus providing a useful model for understanding the genetics of tooth formation. Using a large Chinese family with 14 DDI patients, we mapped the gene locus responsible for DDI to 3p26.1-3p24.3 and further identified a missense mutation, c.353C>A (p.P118Q) in the SSUH2 gene on 3p26.1, which co-segregated with DDI. We showed that SSUH2 (p.P118Q) perturbed the structure and significantly reduced levels of mutant (MT) protein and mRNA compared with wild-type SSUH2. Furthermore, MT P141Q knock-in mice (+/- and -/-) had a unique partial obliteration of the pulp cavity and upregulation or downregulation of six major genes involved in odontogenesis: Dspp, Dmp1, Runx2, Pax9, Bmp2, and Dlx2. The phenotype of missing teeth was determined in zebrafish with morpholino gene knockdowns and rescued by injection of normal human mRNA. Taken together, our observations demonstrate that SSUH2 disrupts dental formation and that this novel gene, together with other odontogenesis genes, is involved in tooth development.

A novel, complex RUNX2 gene mutation causes cleidocranial dysplasia.

BACKGROUND: Haploinsufficiency of the runt-related transcription factor 2 (RUNX2) gene is known to cause cleidocranial dysplasia (CCD). Here, we investigated a complex, heterozygous RUNX2 gene mutation in a Chinese family with CCD and the pathogenesis associated with the variations. METHODS: Genomic DNA extracted from peripheral venous blood was taken from the proband, her parents and 3 siblings, and 150 normal controls. Analysis of their respective RUNX2 gene sequences was performed by PCR amplification and Sanger sequencing. Pathogenesis associated with RUNX2 mutations was investigated by performing bioinformatics, real-time PCR, western blot analysis, and subcellular localization studies. RESULTS: We identified 2 complex heterozygous mutations involving a c.398-399 insACAGCAGCAGCAGCA insertion and a c.411-412 insG frameshift mutation in exon 3 of the RUNX2 gene. The frameshift mutation changed the structure of the RUNX2 protein while did not affect its expression at the mRNA level. Transfection of HEK293T cells with a plasmid expressing the RUNX2 variant decreased the molecular weight of the variant RUNX2 protein, compared with that of the wild-type protein. Subcellular localization assays showed both nuclear and cytoplasmic localization for the mutant protein, while the wild-type protein localized to the nucleus. CONCLUSIONS: Our findings demonstrated that the novel c.398-399insACAGCAGCAGCAGCA mutation occurred alongside the c.411-412insG frameshift mutation, which resulted in RUNX2 truncation. RUNX2 haploinsufficiency was associated with CCD pathogenesis. These results extend the known mutational spectrum of the RUNX2 gene and suggest a functional role of the novel mutation in CCD pathogenesis.

A splicing mutation in VPS4B causes dentin dysplasia I.

BACKGROUND: Dentin dysplasia I (DDI) is a genetically heterogeneous autosomal-dominant disorder characterised by rootless teeth with abnormal pulpal morphology, the aetiology of which presents as genetically heterogeneous. METHODS AND RESULTS: Using a cohort of a large Chinese family with 10 patients with DDI, we mapped to a 9.63 Mb candidate region for DDI on chromosome 18q21.2-q21.33. We then identified a mutation IVS7+46C>G which resulted in a novel donor splice site in intron 7 of the VPS4B gene with co-segregation of all 10 affected individuals in this family. The aberrant transcripts encompassing a new insert of 45 bp in size were detected in gingival cells from affected individuals. Protein structure prediction showed that a 15-amino acid insertion altered the ATP-binding cassette of VPS4B. The mutation resulted in significantly reduced expression of mRNA and protein and altered subcellular localisation of VPS4B, indicating a loss of function of VPS4B. Using human gingival fibroblasts, the VPS4B gene was found to act as an upstream transducer linked to Wnt/ß-catenin signalling and regulating odontogenesis. Furthermore, knockdown of vps4b in zebrafish recapitulated the reduction of tooth size and absence of teeth similar to the tooth phenotype exhibited in DDI index cases, and the zebrafish mutant phenotype could be partially rescued by wild-type human VPS4B mRNA. We also observed that vps4b depletion in the zebrafish negatively regulates the expression of some major genes involved in odontogenesis. CONCLUSIONS: This study identifies VPS4B as a disease-causing gene for DDI, which is one of the important contributors to tooth formation, through the Wnt/ß-catenin signalling pathway.

Two novel copy number variations involving the α-globin gene cluster on chromosome 16 cause thalassemia in two Chinese families.

Copy number variations (CNVs) can cause many genetic disorders and the structure analysis of unknown CNVs is important for clinical diagnosis. The human α-globin gene cluster lies close to the telomere of the short arm on chromosome 16. Copy number variations of this region produce excessive or insufficient α-globin chains which imbalances the ß-globin chains, resulting in thalassemia. However, these CNVs usually cannot be precisely defined by traditional methods. Here, we designed a technique strategy and applied it to identify two CNVs involving the α-globin gene cluster causing thalassemia in two Chinese families. A novel 282 kb duplication (αααα(282)) was identified in family A and a novel 235 kb deletion (--(235)) in family B. Proband A is a coinheritance of ß(CD41-42) and αααα(282) and showed severe ß-thalassemia intermedia phenotype. Proband B is a compound heterozygote of --(235)/α(CS)α genotype and was diagnosed with hemoglobin H disease. The clinical phenotypic features of the CNVs carriers were described, together with a complete picture of molecular structure of these rearrangements. Two CNVs are novel rearrangements in α-globin clusters and the αααα(282) is the first to identify the exact insert position of a duplication region from the telomere on chromosome 16. In a conclusion, successful identification and characterization of these two novel CNVs not only demonstrates the precision and effectiveness of our strategy in analyzing the structure of unknown CNVs, but also extended the spectrum of thalassemia and provide new examples for studying genomic recombination.

A Double Heterozygous Mutation of TNNI3 Causes Hypertrophic Cardiomyopathy in a Han Chinese Family.

OBJECTIVES: To investigate the variations in the TNNI3 gene in a Chinese Han family affected by hypertrophic cardiomyopathy (HCM) and the potential molecular mechanism linking these mutations with disease. METHODS: Peripheral venous blood was acquired from family members, and TNNI3 mutations were identified by DNA sequencing. The pathophysiology of TNNI3 mutations was investigated using bioinformatics, subcellular localization determination and Western blotting. RESULTS: Sanger sequencing revealed that the proband possessed 2 heterozygous mutations, c.235C>T and c.470C>T, located at exons 4 and 6 of the TNNI3 gene. The proband (II-2) and her brother (II-1), who had been previously diagnosed with HCM, harbored both mutations whereas their healthy parents harbored only 1. Alignment of the TNNI3 amino acid sequence indicated that the two Pro residues were highly conserved across species. Subcellular localization showed that both wild-type (WT) and mutant TNNI3 proteins were localized at the cell nucleus. Western blot analysis of expression in human embryonic kidney 293T cells showed that the intracellular levels of the mutant proteins were significantly decreased compared to WT TNNI3 (p < 0.01). CONCLUSIONS: Our findings showed that a double heterozygous mutation in the TNNI3 gene is involved in the pathogenesis of HCM via haploinsufficiency. These results will inspire further studies to investigating the link between the TNNI3 gene and HCM.

[Screening of CTSC gene mutations in a Chinese pedigree affected with Papillon-Lefevre syndrome].

OBJECTIVE: To analyze the clinical phenotype of a Chinese pedigree affected with Papillon-Lefevre syndrome(PLS) and detect mutation of CTSC gene. METHODS: Clinical phenotypes were noted, and oral examination for the proband was carried out for the clinical diagnosis of PLS. PCR and Sanger sequencing were used to identify potential mutation of the CTSC gene. Functional effect of the mutation was predicted with SIFT and PolyPhen-2. Swiss-Port was used to predict the tertiary structure of wild type and mutant proteins. The mRNA and protein expression were analyzed by real-time PCR and Western blotting. RESULTS: A homozygous mutation c.901G>A (p.G301S) in exon 7 of CTSC gene was identified in the patient. Both parents of the patient had carried a heterozygous c.901G>A mutation. The mutation was located in the conserved region of CTSC enzyme and was predicted to be damaging by changing the structure of the protein, which could affect the activity of Cathepsin C. However, no significant difference was found in the expression of p.G301S variant at the mRNA and protein levels compared with that of the wild type CTSC gene. CONCLUSION: The c.901G>A mutation of the CTSC gene was first reported in China, which has expanded its mutation spectrum.

Novel loss-of-function PRRT2 mutation causes paroxysmal kinesigenic dyskinesia in a Han Chinese family.

BACKGROUND: Mutations in proline-rich transmembrane protein 2 (PRRT2) are a cause of paroxysmal kinesigenic dyskinesia (PKD). In this study, we investigated the PRRT2 gene mutation in a Chinese Han family with PKD and study the pathogenesis of the mutation with PRRT2 gene. METHODS: Peripheral venous blood was taken from the family members. Sanger sequencing was used for novel mutation sequencing. For the pathogenesis with the novel mutation was analyzed by bioinformatics, real-time PCR, subcellular localization and Western blot. RESULTS: The Sanger sequencing showed a novel mutation, c.186-187delGC, a deletion mutation, in exon 2 of the PRRT2 gene, the frameshift mutation generated a truncated protein that was stably expressed in transfected Human embryonic kidney (HEK) 293 cells. A subcellular localization assay in COS-7 cells with GFP-tagged protein showed nuclear localization for the mutant protein while the wild-type protein was localized in membranes. Co-transfection of HEK293 cells with wild-type and mutant expression plasmids cells did not influence mRNA or protein expression from the wild-type plasmid. CONCLUSIONS: Our findings demonstrated that the c.186-187delGC mutation resulted in a truncated protein from the PRRT2 gene to involve in PKD pathogenesis with haploinsufficiency. The results extend the mutation spectrum of the PRRT2 gene and provide a new example for studying the pathogenesis of the mutated PRRT2 gene.

The Effects of Habitual Foot Strike Patterns on the Morphology and Mechanical Function of the Medial Gastrocnemius-Achilles Tendon Unit.

As a crucial and vulnerable component of the lower extremities, the medial gastrocnemius-Achilles tendon unit (gMTU) plays a significant role in sport performance and injury prevention during long-distance running. However, how habitual foot strike patterns influence the morphology of the gMTU remains unclear. Therefore, this study aimed to explore the effects of two main foot strike patterns on the morphological and mechanical characteristics of the gMTU. Long-distance male runners with habitual forefoot (FFS group, n = 10) and rearfoot strike patterns (RFS group, n = 10) and male non-runners (NR group, n = 10) were recruited. A Terason uSmart 3300 ultrasonography system was used to image the medial gastrocnemius (MG) and Achilles tendon, Image J software to analyze the morphology, and a dynamometer to determine plantar flexion torque during maximal voluntary isometric contractions. The participants first performed a 5-minute warm up; then, the morphological measurements of MG and AT were recorded in a static condition; finally, the MVICs test was conducted to investigate the mechanical function of the gMTU. One-way ANOVA and nonparametric tests were used for data analysis. The significance level was set at a p value of <0.05. The muscle fascicle length (FL) (FFS: 67.3 ± 12.7, RFS: 62.5 ± 7.6, NRs: 55.9 ± 2.0, η2 = 0.187), normalized FL (FFS: 0.36 ± 0.48, RFS: 0.18 ± 0.03, NRs: 0.16 ± 0.01, η2 = 0.237), and pennation angle (PA) (FFS: 16.2 ± 1.9, RFS: 18.9 ± 2.8, NRs: 19.3 ± 2.4, η2 = 0.280) significantly differed between the groups. Specifically, the FL and normalized FL were longer in the FFS group than in the NR group (p < 0.05), while the PA was smaller in the FFS group than in the NR group (p < 0.05). Conclusion: Long-term running with a forefoot strike pattern could significantly affect the FL and PA of the MG. A forefoot strike pattern could lead to a longer FL and a smaller PA, indicating an FFS pattern could protect the MG from strain under repetitive high loads.

Is Anodal Transcranial Direct Current Stimulation an Effective Ergogenic Technology in Lower Extremity Sensorimotor Control for Healthy Population? A Narrative Review.

Anodal transcranial direct current stimulation (a-tDCS) aims to hone motor skills and improve the quality of life. However, the non-repeatability of experimental results and the inconsistency of research conclusions have become a common phenomenon, which may be due to the imprecision of the experimental protocol, great variability of the participant characteristics within the group, and the irregularities of quantitative indicators. The aim of this study systematically summarised and analysed the effect of a-tDCS on lower extremity sensorimotor control under different experimental conditions. This narrative review was performed following the PRISMA guidelines until June 2022 in Web of Science, PubMed, Science Direct, Google Scholar, and Scopus. The findings of the present study demonstrated that a-tDCS can effectively improve the capabilities of lower extremity sensorimotor control, particularly in gait speed and time-on-task. Thus, a-tDCS can be used as an effective ergogenic technology to facilitate physical performance. In-depth and rigorous experimental protocol with larger sample sizes and combining brain imaging technology to explore the mechanism have a profound impact on the development of tDCS.

The effect of Tai Chi exercise on postural time-to-contact in manual fitting task among older adults.

BACKGROUND: A fall would impact elderly population's quality of life, which associate with diminished physical and psychological function, and can even be life-threatening. Tai Chi has been used to improve age-related postural instability in locomotion. However, it does not fully explain the mechanism of a lower risk of falling among the Tai Chi population compared to other healthy older adults. RESEARCH QUESTION: The maintenance of postural stability is more complicated than minimizing postural movements. Postural time to contact is an important temporal measure of postural stability under fitting tasks, which might further clarify the benefits of long term Tai Chi exercise. METHODS: Participants were required to fit a block (90 × 90 mm) through two different openings (130 × 130 mm and 100 × 100 mm) at two different distances (arm's length or 130 % of arm's length). Kistler forceplate and Vicon system were used to collect center of pressure and kinematic data, respectively. Postural time to contact was used to assess instantaneous perturbation for postural system. RESULTS: Tai Chi group exhibited significant longer postural time to contact in quiet standing and shorter postural time to contact in fitting tasks, expecting for close-small condition, compared to the brisk walking and sedentary groups (p < .05). In addition, both large and small opening condition, Tai Chi group showed a shorter postural time to contact than brisk walking and sedentary groups (p < .0001). SIGNIFICANCE: Long term Tai Chi exercise would promote the regulation of posture and decrease the postural constrain to increase the overall stability when performing fitting tasks. Therefore, Tai Chi exercise can be considered as a feasible method to enhance postural control and stability in older adult.

Tai Chi Can Improve Postural Stability as Measured by Resistance to Perturbation Related to Upper Limb Movement among Healthy Older Adults.

Purpose. The aim of the study was to examine the effects of Tai Chi (TC) training on postural control when upright standing was perturbed by upper limb movement. Methods. Three groups, TC, Brisk walk (BW), and sedentary (SE), of thirty-six participants aged from 65 to 75 years were recruited from local community centers. Participants performed static balance task (quiet standing for 30 s with eyes open and closed) and fitting task (two different reaching distances X three different opening sizes to fit objects through). During tasks, the COP data was recorded while standing on the force plate. Criteria measures calculated from COP data were the maximum displacement in anterior-posterior (AP) and medial-lateral (ML) directions, the 95% confidence ellipse area (95% area), and the mean velocity. Results. No significant effect was observed in the static balance task. For fitting tasks, the group effect was observed in all directions on COP 95% area (p < 0.05) and the TC group showed reduced area. The tests of subject contrasts showed significant trends for reaching different distances and fitting different openings conditions in all directions, the 95% area, and the mean velocity (p < 0.05). Conclusion. Compared to the other two groups, long-term TC exercise helps in reducing the effects of upper body perturbation as measured by posture sway.

Small molecule inhibitors of E. coli primase, a novel bacterial target.

Bacterial primase is essential for DNA replication in Gram-positive and Gram-negative bacteria. It is also structurally distinct from eukaryotic primases, and therefore an attractive, but under-explored, target for therapeutic intervention. We applied virtual screening to discover primase inhibitors, and subsequently several commercially available analogs of these initial hits showed potent primase inhibition and in vitro antibacterial activity. This work provides a 3D pharmacophore for primase ligands, SAR trends, and leads that can be further optimized.