Compound heterozygous mutations of TYMP as underlying causes of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE).

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), an autosomal recessive multiorgan disease, frequently associated with mutations in the thymidine phosphorylase (TYMP) gene. TYMP encodes thymidine phosphorylase (TP), which has an essential role in the nucleotide salvage pathway for mitochondrial DNA (mtDNA) replication. This study reports an MNGIE patient with novel compound heterozygous missense mutations (Thr151Pro and Leu270Pro) in TYMP. Each mutation was inherited from one parent. Neither mutation was found in the controls and the mutation sites were well conserved between different species. Neither large deletion nor causative point mutations were found in the mtDNA. The patient presented with MNGIE symptoms, including gastrointestinal discomfort, external ophthalmoplegia, pigmentary retinopathy and demyelinating type diffuse sensory motor polyneuropathy. The patient demonstrated an early-onset but mild phenotype, with 9.6% TP activity; therefore, patients with these compound heterozygous mutations may exhibit a mild phenotype with a variable onset age according to TP activity level.

Inheritance of Charcot-Marie-Tooth disease 1A with rare nonrecurrent genomic rearrangement.

Rare copy number variations by the nonrecurrent rearrangements involving PMP22 have been recently suggested to be associated with CMT1A peripheral neuropathy. As a mechanism of the nonrecurrent rearrangement, replication-based fork stalling template switching (FoSTeS) by microhomology-mediated break-induced replication (MMBIR) has been proposed. We found three Korean CMT1A families with putative nonrecurrent duplication. The duplications were identified by microsatellite typing and applying a CGH microarray. The breakpoint sequences in two families suggested an Alu-Alu-mediated rearrangement with the FoSTeS by the MMBIR, and a two-step rearrangement of the replication-based FoSTeS/MMBIR and meiosis-based recombination. The two-step mechanism has still not been reported. Segregation analysis of 17p12 microsatellite markers and breakpoint junction analysis suggested that the nonrecurrent rearrangements are stably inherited without alteration of junction sequence; however, they may allow some alteration of the genomic contents in duplication across generations by recombination event. It might be the first study on the pedigree analysis of the large CMT1A families with nonrecurrent rearrangements. It seems that the exact mechanism of the nonrecurrent rearrangements in the CMT1A may have a far more complex process than has been expected.

Clinicopathologic efficacy of copper bromide plus/yellow laser (578 nm with 511 nm) for treatment of melasma in Asian patients.

BACKGROUND: Melasma is a common pigmentary disorder in Asians. Although the pathogenesis of melasma is not yet fully understood, there are several hypotheses supporting angiogenetic factors related to some types of melasma. OBJECTIVE: To test the efficacy of copper bromide laser in the treatment of Korean women with melasma. MATERIALS AND METHODS: Clinical parameters included physician and patient assessment and Melasma Area and Severity Index score. The intensity of pigmentation and erythema was measured using a chromometer. To evaluate histopathologic changes, punch biopsies from melasma were obtained from four patients. Immunohistochemical staining for Melan-A, endothelin 1, CD34, and vascular endothelial growth factor (VEGF) antigen of the melasma lesions was observed. RESULTS: Mean MASI score decreased dramatically after treatment. Patients exhibited telangiectatic erythema within the melasma lesion. The values of L(*) reflecting intensity of pigmentation increased, and the values of a(*) as the measurement of redness decreased after the treatments. Expression of Melan-A, CD34, endothelin-1, and VEGF decreased after treatment. CONCLUSION: The potential application of an antiangiogenetic laser for the treatment of melasma specially accompanied by pronounced telangiectasia in Asian skin is a possible treatment option.

Mutational analysis of whole mitochondrial DNA in patients with MELAS and MERRF diseases.

Mitochondrial diseases are clinically and genetically heterogeneous disorders, which make the exact diagnosis and classification difficult. The purpose of this study was to identify pathogenic mtDNA mutations in 61 Korean unrelated families (or isolated patients) with MELAS or MERRF. In particular, the mtDNA sequences were completely determined for 49 patients. From the mutational analysis of mtDNA obtained from blood, 5 confirmed pathogenic mutations were identified in 17 families, and 4 unreported pathogenically suspected mutations were identified in 4 families. The m.3243A>G in the tRNA(Leu(UUR))was predominantly observed in 10 MELAS families, and followed by m.8344A>G in the tRNA(Lys) of 4 MERRF families. Most pathogenic mutations showed heteroplasmy, and the rates were considerably different within the familial members. Patients with a higher rate of mutations showed a tendency of having more severe clinical phenotypes, but not in all cases. This study will be helpful for the molecular diagnosis of mitochondrial diseases, as well as establishment of mtDNA database in Koreans.

A MELAS syndrome family harboring two mutations in mitochondrial genome.

Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is a genetically heterogeneous mitochondrial disorder with variable clinical symptoms. Here, from the sequencing of the entire mitochondrial genome, we report a Korean MELAS family harboring two homoplasmic missense mutations, which were reported 9957T>C (Phe251Leu) transition mutation in the cytochrome c oxidase subunit 3 (COX3) gene and a novel 13849A>C (Asn505His) transversion mutation in the NADH dehydrogenase subunit 5 (ND5) gene. Neither of these mutations was found in 205 normal controls. Both mutations were identified from the proband and his mother, but not his father. The patients showed cataract symptom in addition to MELAS phenotype. We believe that the 9957T>C mutation is pathogenic, however, the 13849A>C mutation is of unclear significance. It is likely that the 13849A>C mutation might function as the secondary mutation which increase the expressivity of overlapping phenotypes of MELAS and cataract. This study also demonstrates the importance of full sequencing of mtDNA for the molecular genetic understanding of mitochondrial disorders.

Inhibitory effects of plant extracts on tyrosinase, L-DOPA oxidation, and melanin synthesis.

For medical, pharmacological, and cosmetic reasons, the demand for effective and safe depigmentating agents has increased. In this study, 101 plant extracts (methanol or water extracts) were screened for their inhibitory activities against tyrosinase, (L-3, 4,-dihydroxyphenylalanine) L-DOPA oxidation, and melanin biosynthesis in B16 mouse melanoma cells. Of the extracts examined, 31 showed over 50% inhibition of mushroom tyrosinase at a concentration of 666 microg/Ml, and 11 inhibited L-DOPA auto-oxidation at this concentration. In particular, extracts of Broussonetia kazinoki var. humilis (leaves and stems), Broussonetia papyrifera (leaves and bark), Cornus officinalis (fruit), Rhus javanica (gallnut), and Pinus densiflora (leaves) inhibited both tyrosinase activity and L-DOPA oxidation in a concentration-dependent manner. Seventeen plant extracts that inhibited tyrosinase were further tested for their inhibitory effects on melanogenesis. In B16 mouse melanoma cells, extracts of Acorus gramineus, Capsella bursa-pastoris, Morus bombycis, Perilla frutescens var. crispa, Quercus dentate (bark), Rhus javanica (gallnut), Schizopepon bryoniaefolius, or Sophora flavescens markedly inhibited (>50%) melanin synthesis at 50 microg/Ml. These plants represent a potential source of novel whitening agents for ultraviolet (UV)-sensitive skin.

Rapid diagnosis of CMT1A duplications and HNPP deletions by multiplex microsatellite PCR.

Charcot-Marie-Tooth (CMT) disease and hereditary neuropathy with liability to pressure palsies (HNPP) are frequent forms of genetically heterogeneous peripheral neuropathies. Reciprocal unequal crossover between flanking CMT1A-REPs on chromosome 17p11.2-p12 is a major cause of CMT type 1A (CMT1A) and HNPP. The importance of a sensitive and rapid method for identifying the CMT1A duplication and HNPP deletion is being emphasized. In the present study, we established a molecular diagnostic method for the CMT1A duplication and HNPP deletion based on hexaplex PCR of 6 microsatellite markers (D17S921, D17S9B, D17S9A, D17S918, D17S4A and D17S2230). The method is highly time-, cost- and sample-saving because the six markers are amplified by a single PCR reaction and resolved with a single capillary in 3 h. Several statistical and forensic estimates indicated that most of these markers are likely to be useful for diagnosing the peripheral neuropathies. Reproducibility, as determined by concordance between independent tests, was estimated to be 100%. The likelihood that genotypes of all six markers are homozygous in randomly selected individuals was calculated to be 1.6 x 10(-4) which indicates that the statistical error rate for this diagnosis of HNPP deletion is only 0.016%.

Peripheral effect of alpha-melanocyte-stimulating hormone on fatty acid oxidation in skeletal muscle.

To study the peripheral effects of melanocortin on fuel homeostasis in skeletal muscle, we assessed palmitate oxidation and AMP kinase activity in alpha-melanocyte-stimulating hormone (alpha-MSH)-treated muscle cells. After alpha-MSH treatment, carnitine palmitoyltransferase-1 and fatty acid oxidation (FAO) increased in a dose-dependent manner. A strong melanocortin agonist, NDP-MSH, also stimulated FAO in primary culture muscle cells and C2C12 cells. However, [Glu6]alpha-MSH-ND, which has ample MC4R and MC3R agonistic activity, stimulated FAO only at high concentrations (10(-5) M). JKC-363, a selective MC4R antagonist, did not suppress alpha-MSH-induced FAO. Meanwhile, SHU9119, which has both antagonistic activity on MC3R and MC4R and agonistic activity on both MC1R and MC5R, increased the effect of alpha-MSH on FAO in both C2C12 and primary muscle cells. Small interference RNA against MC5R suppressed the alpha-MSH-induced FAO effectively. cAMP analogues mimicked the effect of alpha-MSH on FAO, and the effects of both alpha-MSH and cAMP analogue-mediated FAO were antagonized by a protein kinase A inhibitor (H89) and a cAMP antagonist ((Rp)-cAMP). Acetyl-CoA carboxylase activity was suppressed by alpha-MSH and cAMP analogues by phosphorylation through AMP-activated protein kinase activation in C2C12 cells. Taken together, these results suggest that alpha-MSH increases FAO in skeletal muscle, in which MC5R may play a major role. Furthermore, these results suggest that alpha-MSH-induced FAO involves cAMP-protein kinase A-mediated AMP-activated protein kinase activation.

Mutational analysis of PMP22, MPZ, GJB1, EGR2 and NEFL in Korean Charcot-Marie-Tooth neuropathy patients.

We examined CMT1A duplication of 17p11.2-p12, mutations of PMP22, MPZ (P0), GJB1 (Cx32), EGR2 and NEFL genes in 57 Korean families with patients diagnosed as having Charcot-Marie-Tooth (CMT) disease. The CMT1A duplication was present in 53.6% of 28 CMT type 1 patients. In the 42 CMT families without CMT1A duplication, 10 pathogenic mutations were found in 9 families. The 10 mutations were not detected in 105 healthy controls. Seven mutations (c.318delT (p.Ala106fs) in PMP22, c.352G>A (p.Asp118Asn), c.449-1G>T (3'-splice site), c.706A>G (p.Lys236Glu) in MPZ, c.407T>C (p.Val136Ala)[corrected], c.502T>C (p.Cys168Arg) in GJB1, and c.1001T>C (p.Leu334Pro) in NEFL) were determined to be novel. The mutation frequencies of PMP22 and MPZ were similar to those found in several European populations, however, it appeared that mutations in GJB1 are less frequent in East Asian CMT patients than in Eur opean patients. We described the identified mutations and phenotype-genotype correlations based on nerve conduction studies.

Identification and kinetics analysis of a novel heparin-binding site (KEDK) in human tenascin-C.

The interaction between tenascin-C (TN-C), a multi-subunit extracellular matrix protein, and heparin was examined using a surface plasmon resonance-based technique on a Biacore system. The aims of the present study were to examine the affinity of fibronectin type III repeats of TN-C fragments (TNIII) for heparin, to investigate the role of the TNIII4 domains in the binding of TN-C to heparin, and to delineate a sequence of amino acids within the TNIII4 domain, which mediates cooperative heparin binding. At a physiological salt concentration, and pH 7.4, TNIII3-5 binds to heparin with high affinity (K(D) = 30 nm). However, a major heparin-binding site in TNIII5 produces a modest affinity binding at a K(D) near 4 microm, and a second site in TNIII4 enhances the binding by several orders of magnitude, although it was far too weak to produce an observable binding of TNIII4 by itself. Moreover, mutagenesis of the KEDK sequence in the TNIII4 domain resulted in the significant reduction of heparin-binding affinity. In addition, residues in the KEDK sequences are conserved in TN-C throughout mammalian evolution. Thus the structure-based sequence alignment, mutagenesis, and sequence conservation data together reveal a KEDK sequence in TNIII4 suggestive of a minor heparin-binding site. Finally, we demonstrate that TNIII4 contains binding sites for heparin sulfate proteoglycan and enhances the heparin sulfate proteoglycan-dependent human gingival fibroblast adhesion to TNIII5, thus providing the biological significance of heparin-binding site of TNIII4. These results suggest that the heparin-binding sites may traverse TNIII4-5 and thus require KEDK in TNIII4 for optimal heparin-binding.

Production of recombinant human tenascin-C module containing a cell adhesion recognition motif of RGD.

Recombinant human tenascin peptide (hTNCIII3) that includes the Arg-Gly-Asp (RGD) cell recognition site was expressed in Escherichia coli using a prokaryotic expression system. Addition of recombinant hTNCIII3 peptide enhanced cell adhesion and survival of human chondrocytes by about 3-fold in each case.

Expression and characterization of recombinant NH2-terminal cell binding fragment of vitronectin in E. coli.

A recombinant peptide fragment of vitronectin (rVN143), that includes the Arg-Gly-Asp (RGD) cell recognition site, was expressed in Escherichia coli using a prokaryotic expression system. The addition of recombinant rVN143 peptide enhances cell adhesion and proliferation similar (approximately 70%) to those of native VN.