Research progress of myosin heavy chain genes in human genetic diseases.

Myosins constitute a large superfamily proteins, which convert chemical energy, through ATP hydrolysis, to mechanical force for diverse cellular movements, such as cell migration and muscle contraction. The class Ⅱ myosin forms the filaments in muscle and non-muscle cells as a hexameric protein complex, consisting of two myosin heavy chain (MyHC) subunits and two pairs of non-identical light chain subunits. There are several MyHC isoforms encoded by different genes of the MYH family in humans. At present, distinct mutations in different genes of the MYH family are associated with various human genetic diseases. Mutations in MYH2 are associated with skeletal myopathies, characterized by ophthalmoplegia. Mutations in MYH3 and MYH8 are associated with distal arthrogryposis syndromes. Mutations in MYH7 are associated with not only skeletal muscle diseases, such as Laing distal myopathy and myosin storage myopathy, but also hypertrophic cardiomyopathy. Mutations in MYH9 are associated with the so-called MYH9-related disease, characterized by giant platelets, thrombocytopenia and granulocyte inclusions. In this review, we briefly discuss the expression patterns of the MYH gene family and summarize the research progress in correlating the abnormalities of MYH gene family with various human genetic diseases.

The functional mechanisms and clinical application of read-through drugs.

According to previous reports, nearly one in 10 genetic diseases are caused by nonsense mutations around the world. Nonsense mutations lead to premature transcription terminations in cells, which in turn generate non-functional, truncated proteins. In recent years, read-through drugs are playing increasing prominent roles in the researches related to genetic diseases caused by nonsense mutations. However, due to the fact that the mechanisms lying behind translation termination still remain to be elucidated, the mechanistic research and clinical application of read-through drugs are facing new challenges. This review mainly discusses about the pathogenesis of genetic diseases caused by nonsense mutations, and then introduces the current clinical application of read-through drugs. Finally, we display some problems that remain to be solved and propose some possible coping strategies.

A nonsense mutation in DHTKD1 causes Charcot-Marie-Tooth disease type 2 in a large Chinese pedigree.

Charcot-Marie-Tooth (CMT) disease represents a clinically and genetically heterogeneous group of inherited neuropathies. Here, we report a five-generation family of eight affected individuals with CMT disease type 2, CMT2. Genome-wide linkage analysis showed that the disease phenotype is closely linked to chromosomal region 10p13-14, which spans 5.41 Mb between D10S585 and D10S1477. DNA-sequencing analysis revealed a nonsense mutation, c.1455T>G (p.Tyr485(∗)), in exon 8 of dehydrogenase E1 and transketolase domain-containing 1 (DHTKD1) in all eight affected individuals, but not in other unaffected individuals in this family or in 250 unrelated normal persons. DHTKD1 mRNA expression levels in peripheral blood of affected persons were observed to be half of those in unaffected individuals. In vitro studies have shown that, compared to wild-type mRNA and DHTKD1, mutant mRNA and truncated DHTKD1 are significantly decreased by rapid mRNA decay in transfected cells. Inhibition of nonsense-mediated mRNA decay by UPF1 silencing effectively rescued the decreased levels of mutant mRNA and protein. More importantly, DHTKD1 silencing was found to lead to impaired energy production, evidenced by decreased ATP, total NAD(+) and NADH, and NADH levels. In conclusion, our data demonstrate that the heterozygous nonsense mutation in DHTKD1 is one of CMT2-causative genetic alterations, implicating an important role for DHTKD1 in mitochondrial energy production and neurological development.

[Advances in genetic studies of Charcot-Marie-Tooth disease type 4 (CMT4)].

The Charcot-Marie-Tooth disease (CMT) is one of the most common human inherited peripheral neuropathies. The most common pattern of inheritance is autosomal dominant, with less often occurrence autosomal recessive and X-linked dominant/recessive inheritance. CMT is generally divided into three forms: demyelinating forms (CMT1), axonal forms (CMT2) and intermediate forms (DI-CMT). The autosomal recessive form (AR-CMT1 or CMT4) is accompanied by progressive distal muscle weakness and atrophy of the limbs, pes cavus and claw-like hands. In addition, CMT4 is also characterized by early onset, rapid progression, and varying degrees of sensory loss and spinal deformities (e.g. scoliosis). Recently, 11 subtypes of CMT4 have been identified. Some of these subtypes were clear in pathogenic mechanisms, some had founder mutation, but some still had limited clinical description and mutation analysis. In this review, we summarize the latest research progresses of CMT4, including genotypes and phenotypes, pathogenic mechanisms and mouse models.

Dopamine receptor 3 might be an essential molecule in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity.

BACKGROUND: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces Parkinson's disease (PD)-like neurodegeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) via its oxidized product, 1-methyl-4-phenylpyridinium (MPP+), which is transported by the dopamine (DA) transporter into DA nerve terminals. DA receptor subtype 3 (D3 receptor) participates in neurotransmitter transport, gene regulation in the DA system, physiological accommodation via G protein-coupled superfamily receptors and other physiological processes in the nervous system. This study investigated the possible correlation between D3 receptors and MPTP-induced neurotoxicity. A series of behavioral experiments and histological analyses were conducted in D3 receptor-deficient mice, using an MPTP-induced model of PD. RESULTS: After the fourth MPTP injection, wild-type animals that received 15 mg/kg per day displayed significant neurotoxin-related bradykinesia. D3 receptor-deficient mice displayed attenuated MPTP-induced locomotor activity changes. Consistent with the behavioral observations, further neurohistological assessment showed that MPTP-induced neuronal damage in the SNpc was reduced in D3 receptor-deficient mice. CONCLUSIONS: Our study indicates that the D3 receptor might be an essential molecule in MPTP-induced PD and provides a new molecular mechanism for MPTP neurotoxicity.

Multiple synostoses syndrome is due to a missense mutation in exon 2 of FGF9 gene.

Fibroblast growth factors (FGFs) play diverse roles in several developmental processes. Mutations leading to deregulated FGF signaling can cause human skeletal dysplasias and cancer.(1,2) Here we report a missense mutation (Ser99Asp) in exon 2 of FGF9 in 12 patients with multiple synostoses syndrome (SYNS) in a large Chinese family. In vitro studies demonstrate that FGF9(S99N) is expressed and secreted as efficiently as wild-type FGF9 in transfected cells. However, FGF9(S99N) induces compromised chondrocyte proliferation and differentiation, which is accompanied by enhanced osteogenic differentiation and matrix mineralization of bone marrow-derived mesenchymal stem cells (BMSCs). Biochemical analysis reveals that S99N mutation in FGF9 leads to significantly impaired FGF signaling, as evidenced by diminished activity of Erk1/2 pathway and decreased beta-catenin and c-Myc expression when compared with wild-type FGF9. Importantly, the binding of FGF9(S99N) to its receptor is severely impaired although the dimerization ability of mutant FGF9 itself or with wild-type FGF9 is not detectably affected, providing a basis for the defective FGFR signaling. Collectively, our data demonstrate a previously uncharacterized mutation in FGF9 as one of the causes of SYNS, implicating an important role of FGF9 in normal joint development.

[Nonsense-mediated mRNA decay and human monogenic disease].

Nonsense-mediated mRNA decay (NMD) is a widespread quality control mechanism in eukaryotic cells. It can recognize and degrade aberrant transcripts harbouring a premature translational termination codon (PTC), and thereby prevent the production of C-terminally truncated proteins which might be deleterious. Approximately, 30% of human genetic diseases are caused by transcripts containing PTCs. These transcripts are potential targets of NMD. As for monogenic diseases, NMD has effects on the phenotype or mode of inheritance. Here, we explain the mechanism of this surveillance pathway, and take several neuromuscular disorders as examples to discuss its influence for human monogenic diseases. The deeper understanding for NMD will shed light on the nosogenesis and therapies of monogenic diseases.

CMT2Q-causing mutation in the Dhtkd1 gene lead to sensory defects, mitochondrial accumulation and altered metabolism in a knock-in mouse model.

Charcot-Marie-Tooth disease (CMT) is a group of inherited neurological disorders of the peripheral nervous system. CMT is subdivided into two main types: a demyelinating form, known as CMT1, and an axonal form, known as CMT2. Nearly 30 genes have been identified as a cause of CMT2. One of these is the 'dehydrogenase E1 and transketolase domain containing 1' (DHTKD1) gene. We previously demonstrated that a nonsense mutation [c.1455 T > G (p.Y485*)] in exon 8 of DHTKD1 is one of the disease-causing mutations in CMT2Q (MIM 615025). The aim of the current study was to investigate whether human disease-causing mutations in the Dhtkd1 gene cause CMT2Q phenotypes in a mouse model in order to investigate the physiological function and pathogenic mechanisms associated with mutations in the Dhtkd1 gene in vivo. Therefore, we generated a knock-in mouse model with the Dhtkd1Y486* point mutation. We observed that the Dhtkd1 expression level in sciatic nerve of knock-in mice was significantly lower than in wild-type mice. Moreover, a histopathological phenotype was observed, reminiscent of a peripheral neuropathy, including reduced large axon diameter and abnormal myelination in peripheral nerves. The knock-in mice also displayed clear sensory defects, while no abnormalities in the motor performance were observed. In addition, accumulation of mitochondria and an elevated energy metabolic state was observed in the knock-in mice. Taken together, our study indicates that the Dhtkd1Y486* knock-in mice partially recapitulate the clinical phenotypes of CMT2Q patients and we hypothesize that there might be a compensatory effect from the elevated metabolic state in the knock-in mice that enables them to maintain their normal locomotor function.

[Cloning and structural analysis of mouse genomic nucleophosmin gene].

Nucleophosmin (NPM) is an abundant nucleolar phosphoprotein. NPM gene involved chromosomal translocations were found in the patients with anaplastic large cell lymphomas (ALCL), myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL). To generate NPM gene knockout mice and study its biological function in vivo, we screened the lambda phage genomic library derived from 129S1 mice with mouse NPM cDNA probe. A positive phage clone which contained the full-length NPM genomic DNA was obtained and the insert of 15.3 kb genomic DNA in this clone was sequenced with shotgun method. BLAST analysis showed that the sequence of insert are 99.8% identity to that of NPM gene of C57BL/6 mouse strain. Based on the sequence, bioinformatics analysis on genomic structure of NPM and the transcription factor binding sites in the NPM 5' flanking region were performed.

Two nonsense mutations cause protein C deficiency by nonsense-mediated mRNA decay.

INTRODUCTION: Protein C deficiency is a genetic disorder caused by mutations in the protein C gene (PROC). More than 10% of nonsense and frameshift mutations carrying premature termination codons have been identified in PROC, but the exact molecular mechanisms of these mutations on the pathogenesis of protein C deficiency remain unclear. OBJECTIVE: The aim of this study is to investigate whether nonsense-mediated mRNA decay (NMD) can be a mechanism accounting for protein C deficiency. METHODS: PROC of genomic DNA was amplified and sequenced. Recombinant plasmids expressing wild-type (wt) and mutant EGFP-protein C (EGFP-PC) cDNA were constructed and transiently transfected into human embryonic kidney cells using lipofectamine. Expression of mRNAs and proteins of EGFP-PC and NMD factor UPF1 were analyzed by qPCR and Western blot. RESULTS: DNA sequencing revealed a novel heterozygous nonsense mutation (p.Trp247*) in patient 1 and two compound heterozygous mutations (p.Phe181Val and p.Arg199*) in patient 2. Expression studies showed that cells transfected with the mutant plasmids expressed significantly lower levels of EGFP-PC mRNAs and proteins compared to cells transfected with the wt plasmid. A translation inhibitor cycloheximide and UPF1 small interfering RNA (UPF1 siRNA) significantly increased mRNA or protein expression of EGFP-PC in cells transfected with the mutant plasmids. CONCLUSION: Two PROC nonsense mutations (p.Trp247* and p.Arg199*) trigger NMD, resulting in protein C deficiency.