Identification of deletion and duplication genotypes of the PMP22 gene using PCR-RFLP, competitive multiplex PCR, and multiplex ligation-dependent probe amplification: a comparison

We evaluated the efficacy of PCR-RFLP, competitive multiplex PCR, and a commercially available system of multiplex ligation-dependent probe amplification (MLPA) for the determination of deletion and duplication genotypes of the PMP22 gene. We compared the methods for efficiency, sensitivity, and specificity. We determined the gene dosage of the PMP22 gene via PCR-RFLP, competitive multiplex PCR, and MLPA. To demonstrate the sensitivity and accuracy of these three methods, a total of 185 samples from 42 patients with hereditary neuropathy with liability to pressure palsies (HNPP), 57 patients with Charcot-Marie-Tooth disease type 1A (CMT1A), and 86 unaffected individuals, were analyzed. Molecular diagnosis by PCR-RFLP was performed on all 185 samples; 24 HNPP deletions and 33 CMT1A duplications were identified. In contrast, 25 HNPP deletions and 38 CMT1A duplications were identified correctly using competitive multiplex PCR and MLPA. Six samples were incorrectly identified by PCR-RFLP (one HNPP deletion and five CMT1A duplications). Competitive multiplex PCR and MLPA demonstrated reliability and relative speed compared to PCR-RFLP; they were superior to PCR-RFLP for gene dosage quantification. Multiplex PCR and MLPA should be the methods of choice for detection of deletion and duplication genotypes in molecular genetic diagnoses.     

Capillary electrophoresis for the detection of PMP22 gene duplication: study in Mexican patients

Charcot-Marie-Tooth (CMT) disease is the most common inherited disorder of the human peripheral nerve, with an estimated overall prevalence of 17-40/10 000 [1]. The typical phenotype presents peroneal muscular atrophy and pes cavus [2]. CMT is usually divided into two large types, about two-thirds of the patients have CMT type 1 (CMT1), that affects the layer of myelin (demyelination). In type 2 (CMT2) the nerve fibers are affected (axonal). CMT diseases have autosomal dominant, autosomal recessive, and X-linked inheritance [1]. The most frequent subtype is 1A (CMT1A) with autosomal dominant transmission, secondary in most cases to a tandem duplication of a 1.5 Mb DNA fragment on chromosome 17p11.2-p12 [4-7]. In this region, the codification of the peripheral myelin protein 22 (PMP22) takes place. The severity of the disease varies among patients, even within the same family, from almost no symptoms to severe foot-drop and sensory loss. The PMP22 gene has four exons and is regulated by two promoters located toward the extreme 5'. The origin of the duplication that causes the disease is an uneven exchange of the chromatids during the meiosis. This unequal recombination occurs between two regions that limit the PMP22 gene, described as REP places of 24 kb, proximal and distal [3, 4].     

Mutation analysis of PAH gene and characterization of a recurrent deletion mutation in Korean patients with phenylketonuria

Phenylketonuria (PKU; MIM 261600) is an autosomal recessive metabolic disorder caused by a deficiency of phenylalanine hydroxylase (PAH; EC 1.14.16.1). Point mutations in the PAH gene are known to cause PKU in various ethnic groups, and large deletions or duplications account for up to 3% of the PAH mutations in some ethnic groups. However, a previous study could not identify approximately 14% of the mutant alleles by sequence analysis in Korean patients with PKU, which suggests that large deletions or duplication might be frequent causes of PKU in Koreans. To test this hypothesis, we performed multiplex ligation-dependent probe amplification (MLPA) for the identification of uncharacterized mutant alleles after PAH sequence analysis of 33 unrelated Korean patients with PKU. Bi-directional sequencing of the PAH exons and flanking intronic regions revealed 27 different mutations, including four novel mutations (two missense and two deletion mutations), comprising 57/66 (86%) mutant alleles. MLPA identified a large deletion that encompassed exons 5 and 6 in four patients, another large deletion that extended from exon 4 to exon 7 in one patient, and a duplication of exon 4 in one patient. Chromosomal walking characterized the deletion breakpoint of the most common large deletion that involved exons 5 and 6 (c.456_706+138del). The present study shows that the allelic frequency of exon deletion or duplication is 9% (6/66) in Korean PKU patients, which suggests that these mutations may be frequent causes of PKU in Korean subjects.     

Angiotensin converting enzyme gene polymorphism in Korean patients with primary knee osteoarthritis

Angiotensin converting enzyme (ACE) plays an important role in the physiology of vasculature, blood pressure and inflammation. ACE gene, known to have insertion/deletion (I/D) polymorphism, has been widely investigated in its relation with cardiovascular and neurodegenerative diseases and longevity. ACE gene polymorphism in an inflammation associated osteoarthritis (OA) patients is not known. Here we have investigated ACE gene polymorphism in 142 Korean primary knee OA patients and 135 healthy volunteers to establish any clinical correlates between ACE polymorphism and knee osteoarthritis. Clinical parameters such as disease onset age, Kellgren-Lawrence grade and Lequesne's functional index provided additional analysis of the relationship of ACE polymorphism and clinical features of OA. Early onset OA showed significantly higher allele frequency and carriage rate of I than late onset OA. Radiographically severe and functionally poor OA showed higher carriage rate of I allele than radiographically mild and functionally good OA, respectively. This study first reports ACE gene polymorphism to be a risk factor for early onset, severe form primary knee OA.     

Distal hereditary motor neuropathy in Korean patients with a small heat shock protein 27 mutation

Distal hereditary motor neuropathy (dHMN) is a heterogeneous disorder characterized by degeneration of motor nerves in the absence of sensory abnormalities. Recently, mutations in the small heat shock protein 27 (HSP27) gene were found to cause dHMN type II or Charcot-Marie-Tooth disease type 2F (CMT2F). The authors studied 151 Korean axonal CMT or dHMN families, and found a large Korean dHMN type II family with the Ser135Phe mutation in HSP27. This mutation was inherited in an autosomal dominant manner, and was well associated with familial members with the dHMN phenotype. This mutation site is located in the alpha-crystallin domain and is highly conserved between different species. The frequency of this HSP27 mutation in Koreans was 0.6%. Magnetic resonance imaging analysis revealed that fatty infiltrations tended to progressively extend distal to proximal muscles in lower extremities. In addition, fatty infiltrations in thigh muscles progressed to affect posterior and anterior compartments but to lesser extents in medial compartment, which differs from CMT1A patients presenting with severe involvements of posterior and medial compartments but less involvement of anterior compartment. The authors describe the clinical and neuroimaging findings of the first Korean dHMN patients with the HSP27 Ser135Phe mutation. To our knowledge, this is the first report of the neuroimaging findings of dHMN type II.     

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.     

基元通量模式预测酵母生长现象

本文通过EFM预测了基因突变后的酵母细胞生长现象, 模拟预测结果和实验结果吻合很好; 与FBA方法得到的模拟结果相比较, EFM方法能更好地把基因突变和其表型(生长)联系起来.     

Lamin A/C mutations associated with familial and sporadic cases of dilated cardiomyopathy in Koreans

Dilated cardiomyopathy (DCM) is characterized by cardiac dilation and systolic dysfunction. So far sixteen genes have been shown to cause autosomal dominant familial dilated cardiomyopathy (FDC). We identified a large Korean family from the Jeju island showing a clear Mendelian inheritance of FDC. A genomewide linkage scan at 9 cM marker density identified a peak multipoint LOD score of 2.82 at D1S195. Haplotyping of the region with 15 additional markers defined a candidate interval that included a known candidate gene encoding the lamin A/C (LMNA). Sequencing of the LMNA exons revealed one missense mutation at C568T (Arg190Trp) in the alpha-helical rod domain of the LMNA gene co-segregating with FDC with conduction-system disease. The same mutation was found in patients of another Korean family with FDC without conduction-system disease. Upon screening 14 sporadic DCM cases, we found three LMNA mutations including a case having a previously described (Glu161Lys) mutation and two having novel mutations (Glu53Val and Glu186Lys). Our results suggest that variable genotypes of laminopathy are implicated in not only familial but also considerable proportion of sporadic DCM.     

Recombination analysis of autosomal short tandem repeats in Chinese Han families

Recombination fractions between forensic STRs can be extrapolated from the International HapMap Project, but the concordance between recombination fractions predicated from genetic maps and derived from observation of STR transmissions in families is still ambiguous for autosomal STRs because of limited family studies. Therefore, the main goal of this study is to compare recombination fractions estimated by pedigree analysis with those derived from HapMap phase SNP data. Genotypes of nine autosomal STR pairs (TPOX‐D2S1772, D5S818‐CSF1PO, D7S3048‐D7S820, D8S1132‐D8S1179, TH01‐D11S2368, vWA‐D12S391, D13S325‐D13S317, D18S51‐D18S1364, and D21S11‐PentaD) from 207 two‐generation families with two to five children (the number of families with five, four, three, and two children was 2, 3, 20, and 182, respectively) were used to analyze the recombination. The linkage analysis showed that significant linkage was observed at six STR pairs (D5S818‐CSF1PO, D8S1132‐D8S1179, TH01‐D11S2368, vWA‐D12S391, D13S325‐D13S317, and D18S51‐D18S1364) with genetic distances <36.22 cM in HapMap. Their recombination fractions calculated from family data were very close to those derived from HapMap. However, three STR pairs of TPOX‐D2S1772, D7S3048‐D7S820, and D21S11‐PentaD showed no significant linkage with genetic distances from 43.38 to 91.49 cM. Our results indicate that recombination fractions extrapolated from HapMap can provide a substitute if empirical data are unavailable for the linkage STR pair with a genetic distance spanned <36.22 cM.     

Acoustic,viscometric and optical properties of binary mixtures of tetrahydrofuran with 1-propanol and 2-propanol

The values estimated from various mixing rules for the ultrasonic velocity, viscosity and refractive index have been compared with the respective values measured earlier at 293, 303, and 313?K over the entire mole fraction range of two binary mixtures of tetrahydrofuran (THF) with 1-propanol (1-p) and 2-propanol (2-p). There is an excellent agreement between the experimental values of ultrasonic velocity and of refractive index with the respective values obtained from the mixing rules. The mixing rules for viscosity provide values agreeing broadly with those obtained from experimental measurements. The relative merits and interrelations of these mixing rules are discussed.     

Advances in Large Gap Peripheral Nerve Injury Repair and Regeneration with Bridging Nerve Guidance Conduits

Peripheral nerve injury is a common complication of accidents and diseases. The traditional autologous nerve graft approach remains the gold standard for the treatment of nerve injuries. While sources of autologous nerve grafts are very limited and difficult to obtain. Nerve guidance conduits are widely used in the treatment of peripheral nerve injuries as an alternative to nerve autografts and allografts. However, the development of nerve conduits does not meet the needs of large gap peripheral nerve injury. Functional nerve conduits can provide a good microenvironment for axon elongation and myelin regeneration. Herein, the manufacturing methods and different design types of functional bridging nerve conduits for nerve conduits combined with electrical or magnetic stimulation and loaded with Schwann cells, etc., are summarized. It summarizes the literature and finds that the technical solutions of functional nerve conduits with electrical stimulation, magnetic stimulation and nerve conduits combined with Schwann cells can be used as effective strategies for bridging large gap nerve injury and provide an effective way for the study of large gap nerve injury repair. In addition, functional nerve conduits provide a new way to construct delivery systems for drugs and growth factors in vivo.     

Analysis of the NF1 gene by temperature gradient gel electrophoresis reveals a high incidence of mutations in exon 4b

A total of 196 unrelated patients with neurofibromatosis type 1 (NF1) was screened for mutations in exons 4a-c of the NF1 gene by temperature gradient gel electrophoresis (TGGE) of polymerase chain reaction (PCR)-amplified genomic DNA fragments using intron-based primers. DNA samples with abnormal TGGE band patterns were subjected to sequence analysis. Sequence alterations were identified in ten patients (5.1%): 496delGT (1), 499delTGTT (4), T528A = D176E (2), T539A = L180X (1), 540insA (1), C574T = R192X (1). Thus, a total of six different mutations was identified in exon 4b but none in exons 4a and 4c. Only the missense mutation D176E, which we assume to be a nonpathogenic polymorphism, and the 4-base pair (bp) deletion 499delTGTT have been described before. The reason for the high incidence of mutations in exon 4b is obviously a tetranucleotide tandem repeat comprising nucleotides 495-502 (TGTTTGTT) that may give rise to slipped mispairing and subsequent deletion of one repeat unit during replication. Additionally, the recurrent 4 bp deletion was found as a second hit in a malignant schwannoma of a further NF1 patient, suggesting that microlesions may be as frequent among somatic as among germline mutations. This is the first report of a systematic study of NF1 exons 4a-c in a large group of NF1 patients.     

Electrochemical Interpretation of Propagation of the Change in the Membrane Potential Using the Goldman‐Hodgkin‐Katz Equation

Nerve conduction has been frequently explained by the Hodgkin‐Huxley equation based on the flow of K⁺ and Na⁺ across the cell membrane. By considering the relation between the membrane potential and the membrane current based on the Goldman‐Hodgkin‐Katz equation, it becomes clear that the conventional analysis using the voltage‐clamp method is not correct and that the hyperpolarization condition is artificially made. Taking into account the channel functions and the electronic properties, we suggested a new propagation mechanism. When the nerve cell is excited by an external stimulus, the ligand‐gated channels at the synapse serve as an electric power source to propagate the change in the membrane potential to the synapse terminal along the axon and the voltage‐gated channels at the axon locally assist the directional propagation along the axon.     

Ribosomal protein mutations in Korean patients with Diamond-Blackfan anemia

Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by hypoproliferative anemia, associated physical malformations and a predisposition to cancer. DBA has been associated with mutations and deletions in the large and small ribosomal protein genes, and genetic aberrations have been detected in ∼50–60% of patients. In this study, nine Korean DBA patients were screened for mutations in eight known DBA genes (RPS19, RPS24, RPS17, RPS10, RPS26, RPL35A, RPL5 and RPL11) using the direct sequencing method. Mutations in RPS19, RPS26 and RPS17 were detected in four, two and one patient, respectively. Among the mutations detected in RPS19, two mutations were novel (c.26T>A, c.357-2A>G). For the mutation-negative cases, array-CGH analysis was performed to identify copy-number variations, and no deletions involving the known DBA gene regions were identified. The relative mRNA expression of RPS19 estimated using real-time quantitative PCR analysis revealed two- to fourfold reductions in RPS19 mRNA expression in three patients with RPS19 mutations, and p53 protein expression analysis by immunohistochemistry showed variable but significant nuclear staining in the DBA patients. In conclusion, heterozygous mutations in the known DBA genes RPS19, RPS26 and RPS17 were detected in seven out of nine Korean DBA patients. Among these patients, RPS19 was the most frequently mutated gene. In addition, decreased RPS19 mRNA expression and p53 overexpression were observed in the Korean DBA patients, which supports the hypothesis that haploinsufficiency and p53 hyperactivation represent a central pathway underlying the pathogenesis of DBA.     

Electrical conducting bis(oxalato)platinate complex with direct connection of CuII ions

Reactions of K1.62[Pt(ox)2].2H2O and [Cu(bpy)(H2O)3](NO3)2 yielded partially oxidized one-dimensional (1D) bis(oxalato)platinates of [Cu(bpy)(H2O)n]6[Pt(ox)2]7.7H2O (n = 2, 3, or 4) (1) and [Cu(bpy)(H2O)n]8[Pt(ox)2]10.8H2O (n = 3 or 4) (2). The average oxidation numbers of the platinum ions in 1 and 2 are +2.29 and +2.40, respectively. Complexes 1 and 2 crystallize in the triclinic P and monoclinic C2/c space groups, respectively, and the [Pt(ox)2]n- anions are stacked along the crystallographic b axis with 7-fold periodicity for 1 and 10-fold periodicity for 2. In 1, an oxalato ligand in the platinum chain directly coordinates to a paramagnetic [Cu(bpy)(H2O)3]2+ ion, whereas no such direct coordination was observed for 2. The electrical conductivity of 2 at room temperature along the platinum chain is approximately 3 orders of magnitude smaller (sigma||= 1.3 x 10(-3) S cm(-1)) than that of 1 (sigma|| = 0.9-0.5 S cm(-1)), and the activation energies of 1 and 2 are 29 and 67 meV, respectively. The longest inter-platinum distances in 1 and 2 are 2.762 and 3.0082 A, respectively, and this is responsible for the lower electrical conductivity of 2. An X-ray oscillation photograph taken along the b axis of 1 reveals the 7-fold periodicity in the 1D chain, consistent with the period of the Peierls distortion estimated from the degree of partial oxidation. The semiconducting state of 1 can therefore be regarded as a commensurate Peierls state. The magnetoresistance of 1 at ambient pressure indicates no interaction between conduction electrons in the platinum chain and local spins of the paramagnetic CuII ions. Application of hydrostatic pressures of up to 3 GPa enhances electrical conduction, as is often seen as the usual pressure effect on the electrical conductivity, which is due to enhanced orbital (Pt-5dz2) overlap by pressure application.     

Sensory properties of oxide films with high concentrations of conduction electrons

The dependence of a sensor’s response to hydrogen on the temperature and hydrogen pressure in an indium oxide nanostructured film is measured. A theory of sensor’s response to reducing gases in nanostructured semiconducting oxides with high concentrations of electrons in the conduction band is developed (using the example of In₂O₃). It is shown that the capture of conduction electrons by adsorbed oxygen redistributes the electrons in nanoparticles and reduces the surface electron density and the conductivity of a system; the conductivity is proportional to the electron density in nanoparticle contacts, i.e., to the surface electron density. It is found that atomic oxygen ions react with reducing gases (H₂, CO) during adsorption of the latter: electrons are released and enter the volumes of nanoparticles; the conductivity of the system grows, creating the sensory effect. Using a model developed earlier to describe the distribution of conduction electrons in a semiconductor nanoparticle, a kinetic scheme corresponding to the above scenario is built and corresponding equations are solved. As a result, a theoretical dependence of a sensor’s sensitivity to temperature is found that describes the experimental data well.

     

C_4H_4Y(Y=O,S,Se)与BX_3(X=H,F,Cl)作用的计算化学研究

采用量子化学的密度泛函B3LYP和二阶微扰MP2(full)方法对C4H4Y(Y=O,S,Se)与BX3(X=H,F,Cl)形成的电子授受型复合物进行了研究,所得18个复合物的构型包括BX3位于C=C双键上方的π-p作用型和B与O,S,Se直接作用的n-p作用型.体系C4H4Y-BH3以n-p作用型较为稳定,体系C4H4Y-BF3,C4H4Y-BCl3的π-p和n-p作用型复合物稳定性相当.对各复合物的几何构型、振动频率和自然键轨道分析表明,复合物的形成过程中均存在几何构型的改变、电荷的转移和振动频率的变化,它们的变化规律与复合物稳定性的变化规律基本一致,即按H,F,Cl的顺序依次降低.     

Reactions of Silicon Atoms with Methanol: A Combined Matrix‐Spectroscopic and Density Functional Theory Study

The reaction of silicon atoms with methanol ( 4 ) has been studied in an argon matrix at 10 K. In the initial step a triplet n‐adduct T‐5 between a silicon atom and 4 is formed. It cannot be detected directly as long as a low concentration of 4 is used. But T‐5 must be generated since upon simultaneous irradiation during cocondensation methylsilanone ( 15 ) is found. Without irradiation T‐5 undergoes immediately O,H insertion and methoxysilylene ( S‐7‐c ) is isolated, which establishes a photoequilibrium between the s,trans ( S‐7‐t ) and s,cis form ( S‐7‐c ). If a high concentration of 4 is applied the silylenes exist as complexes S‐17 . The next step needs photochemical activation. Products are dimethoxysilane ( 3 ) and (hydroxy)(methoxy)methylsilane ( 19 ). The picture becomes even more complicated when deuteromethanol ( [D]4 ) is treated with silicon atoms. In this case the primarily formed n‐adduct ( [D]T‐5 ) is stable under matrix conditions. As long as a low concentration is applied, the subsequent step has to be induced by long wavelength irradiation and leads – different from the undeuterated case – to O,CH₃ insertion, giving (hydroxy)methylsilylenes ( [D]S‐11‐c ) and ( [D]S‐11‐t ). If a high concentration is used O,D insertion is preferred instead and even without irradiation the first observable products are methanol‐solvated methoxysilylenes ( [D₂]S‐17‐c ) and ( [D₂]S‐17‐t ). Subsequent irradiation of complexes [D₂]S‐17 gives in accordance with the protonated series a mixture of [D₂]3 and [D₂]19 . Our goal, to find a way to dimethylsilanediol ( [D₂]2 ), was finally reached by preparing silylenes [D]S‐11 in a diluted matrix, its specific solvation with [D]4 and final irradiation of complexes [D₂]S‐18 . The structural elucidation of all new species is based on the comparison of the experimental observations with density functional theory calculations. Upon cocondensation of silicon atoms with pure methanol at 77 K dimethoxysilane ( 3 ) and 1,1,2‐trimethoxydisilane ( 25 ) are produced. The also present methoxysilanes 22‐24 have to be regarded as secondary products of 3 .     

超声刀在甲状腺手术中保护喉返神经及入喉处甲状腺组织的应用效果观察

目的探讨超声刀在甲状腺手术中对于喉返神经及入喉处甲状腺组织的保护作用。方法选取2016年1月至12月间广东省遂溪县人民医院收治的120例甲状腺疾病的患者,随机分为对照组和观察组,每组60例,对照组采用传统电刀手术,观察组采用超声刀进行手术,比较两种手术方式的效果以及对喉返神经及入喉处甲状腺组织的影响。结果相较于传统电刀手术,采用超声刀可明显缩短腺体切除时间和手术总时间,减少术中出血量,差异具有统计学意义(P0.05);两种手术方式在术后引流量和住院时间方面比较,差异无统计学意义(P0.05);观察组患者喉返神经损伤以及甲状旁腺功能低下的发生率低于对照组,但是两组间差异无统计学意义(P0.05),所有观察组患者的声音嘶哑现象均于术后3个月内恢复,而对照组有3例于3~6个月恢复,2例1年以上未好转。结论在甲状腺手术最终采用超声刀可明显缩短手术时间,减少术中出血量,利于术中对喉返神经和入喉处甲状腺组织的辨认和保护,是一种安全可行的手术方法。     

Genetic polymorphism of 13 non‐CODIS STR loci in three national populations from China

The aim of this study was to investigate a 13 non‐CODIS STR loci database using three national populations from China. A new multiplex PCR system that simultaneously amplified 13 loci in the same PCR reaction was developed. This multiplex system included the 13 STR markers (D3S2402, D3S2452, D3S1766, D3S4554, D3S2388, D3S3051, D3S3053, D4S2364, D4S2404, AC001348A, AC001348B, D17S975, and D17S1294), which were successfully analyzed by using 441 DNA samples from three national populations in China (154 Mongol, 177 Kazakh, and 110 Uigur). Allele frequencies and mutation rates of the 13 non‐CODIS STR loci were investigated. A total of 4–10 alleles at each locus were observed and altogether 84, 88, and 87 alleles for the all selected loci were found in the Mongol, Kazakh, and Uigur, respectively. Eight mutations were detected from the 13 selected loci in 9880 meioses in kinship cases. These results indicate that this multiplex system may provide significant polymorphic information for kinship testing and relationship investigations.