Gene (v.498, #1)

Editorial Board (iv-v).

Fibroblast Growth Factors (FGFs) regulate prenatal and postnatal bone formation through activation of FGF receptors (FGFR) and downstream signaling events. During the last decade, major advances have been made in our understanding of the mechanisms by which FGF/FGFR signaling controls osteoprogenitor cell replication and osteoblast differentiation and function. The analysis of the phenotype induced by FGF invalidation and mutations in FGFR allowed to delineate key FGF signaling pathways that regulate osteoblastogenesis. Molecular genomic studies led to identify target genes that are controlled by FGF/FGFR signaling and govern osteoblasts. The analysis of intracellular signaling pathways showed the importance of functional crosstalks between FGF signaling and other pathways in the regulation of bone formation. These recent progresses in the mechanisms underlying FGF/FGFR signaling may provide a molecular basis for developing therapeutic strategies in human skeletal dysplasias.► Recent progresses on the role of FGF/FGFR signaling in bone formation are reviewed. ► Key FGF signaling pathways that regulate osteoblastogenesis have been identified. ► Osteoblasts are governed by genes regulated by FGF/FGFR signaling. ► Crosstalks between FGF signaling and other pathways functionally control osteoblasts. ► This knowledge will help in developing therapeutic strategies in skeletal dysplasias.
Keywords: FGF; Signaling; Osteoblast; Bone formation;

Global DNA promoter methylation in frontal cortex of alcoholics and controls by A.M. Manzardo; R.S. Henkhaus; M.G. Butler (5-12).
To determine if ethanol consumption and alcoholism cause global DNA methylation disturbances, we examined alcoholics and controls using methylation specific microarrays to detect all annotated gene and non-coding microRNA promoters and their CpG islands. DNA was isolated and immunoprecipitated from the frontal cortex of 10 alcoholics and 10 age and gender-matched controls then labeled prior to co-hybridization. A modified Kolmogorov–Smirnov test was used to predict differentially enriched regions (peaks) from log-ratio estimates of amplified vs input DNA. More than 180,000 targets were identified for each subject which correlated with > 30,000 distinct, integrated peaks or high probability methylation loci. Peaks were mapped to regions near 17,810 separate annotated genes per subject representing hypothetical methylation targets. No global methylation differences were observed between the two subject groups with 80% genetic overlap, but extreme methylation was observed in both groups at specific loci corresponding with known methylated genes (e.g., H19) and potentially other genes of unknown methylation status. Methylation density patterns targeting CpG islands visually correlated with recognized chromosome banding. Our study provides insight into global epigenetic regulation in the human brain in relationship to controls and potentially novel targets for hypothesis generation and follow-up studies of alcoholism.► High probability for methylation loci for > 180,000 targets per subject. ► Methylation peaks mapped to 17,810 hypothetical gene targets per subject. ► Methylation density patterns targeting CpG islands correlate with chromosome banding. ► Extreme methylation at loci corresponding with mammalian genes known to be methylated. ► 80% overlap of hypothetical gene targets between alcoholic and control samples.
Keywords: Alcoholism; Epigenetics; Brain; Frontal cortex; DNA methylation; Global methylation;

Low serum PON1 activity: An independent risk factor for coronary artery disease in North–West Indian type 2 diabetics by Nidhi Gupta; K.B.K. Binu; Surjit Singh; Nagarjuna V. Maturu; Yash P. Sharma; Anil Bhansali; Kiran Dip Gill (13-19).
The paraoxonase (PON1) gene polymorphisms are known to affect the PON1 activity and coronary artery disease (CAD) risk. Studies done so far have given conflicting results. In the present study, we determined the role of PON1 genetic variants and PON1 activity in the development of CAD in North–West Indian Punjabis, a distinct ethnic group, having high incidence of both CAD and type 2 diabetes. 300 angiographically proven CAD with type 2 diabetics and 250 type 2 diabetics with no clinically evident CAD were enrolled. Serum PON1 activity and genotyping of coding (Q192R, L55M) and promoter (− 909G/C, − 162A/G, − 108C/T) region polymorphisms were carried out and haplotypes were determined using PHASE software.The serum PON1 activity was significantly lower in CAD with type 2 diabetics as compared to diabetics alone (51.0 vs. 114.2 nmol/min/ml). In logistic regression model after adjusting for confounding variables, lower PON1 activity was found to be significantly associated with CAD risk in type 2 diabetics with OR being 16.8 (95% CI: 10.2–27.7). The lower serum PON1 activity, irrespective of genotypes and haplotypes is a risk factor for development of CAD in North–West Indian Punjabis with type 2 diabetics.► PON1 activity significantly lowers in CADT2DM patients as compared to T2DM. ► Lower PON1 activity significantly associated with CAD risk in T2DM with high OR. ► Irrespective of PON1 polymorphisms lower activity is a risk factor for CAD in T2DM.
Keywords: Paraoxonase1; Polymorphisms; Haplotypes; Coronary artery disease; Type 2 diabetes;

Fractal genome sequences by Guenter Albrecht-Buehler (20-27).
The existence of fractal sets of DNA sequences have long been suspected on the basis of statistical analyses of genome data. In this article we identify for the first time explicitly the GA-sequences as a class of fractal genomic sequences that are easy to recognize and to extract, and are scattered densely throughout the chromosomes of a large number of genomes from different species and kingdoms including the human genome.Their existence and their fractality may have significant consequences for our understanding of the origin and evolution of genomes. Furthermore, as universal and natural markers they may be used to chart and explore the non-coding regions.► First explicit identification of a class of fractal genomic sequences (FGS). ► FGS are found in a large number of different species and kingdoms. ► FGS are easy to recognize and to extract from the genomes. ► FGS are useful as universal natural markers of the non-coding regions of genomes. ► The existence of FGS's casts a novel light on the origin of genomes.
Keywords: GA-sequences; Fractals; Heat shock; Genome navigation; Evolution;

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC: 1.1.1.34), an enzyme catalyzing the first committed step in the mevalonic acid (MVA) pathway for the biosynthesis of isoprenoids, has been reported to be involved in the fruit size determination through the regulation of early cell division. In litchi, the cell number achieved by this early cell division determines the final fruit size, but whether HMGR plays any role in this process was unknown. In this study, we set out to address this question with gene cloning and expression analysis in fruits of different pheno- or genotypes. We found that the litchi genome includes two HMGR homologues, denoted as LcHMG1 and LcHMG2. Despite 70% sequence identity at the amino acid level, they exhibited distinct expression patterns during litchi fruit development. LcHMG1 expression was highest in the early stage of fruit development, correlated with the high level of cell division. Absolute levels of LcHMG1 expression varied among fruits of different pheno- or genotypes, with expression in large-fruited types reaching higher levels for longer duration compared to that in small-fruited types. The expression patterns for LcHMG1 strongly suggest that this gene is involved in early cell division and fruit size determination in litchi. In contrast, LcHMG2 was most highly expressed in the late stage of fruit development, in association with biosynthesis of isoprenoid compounds required for later cell enlargement. These findings provided new insights on the function of HMGR genes during fruit development.► Two HMGR genes were isolated from a fruit crop litchi. ► We examined the relationship between cell division activity and HMGR gene expression. ► LcHMG1 accumulated to a higher level in large-fruited pheno- or genotypes. ► LcHMG1 was highly correlated with the early cell division activity. ► LcHMG2 is related to the cell enlargement in late fruit development.
Keywords: HMGR genes; Litchi; Fruit size; Cell division activity; Gene expression;

Microarray identification of conserved microRNAs in Pinellia pedatisecta by Bo Wang; Miao Dong; Wenduo Chen; Xuefeng Liu; Ruijuan Feng; Tao Xu (36-40).
Large numbers of microRNAs (miRNAs) reportedly play important roles in plant development. However, none has been reported in Pinellia pedatisecta, an important aroid medicinal plant that possesses the only pedate leaf blades and the largest tubers and inflorescences among all Pinellia species. To detect the miRNAs from P. pedatisecta, an in situ synthesized custom miRNA microarray was employed, following the verification for the presence of the miRNAs through reverse transcription polymerase chain reaction (RT-PCR) and the quantitative RT-PCR (qRT-PCR). A total of 99 miRNAs belonging to 22 miRNA families were identified. The RT-PCR was applied to 14 miRNAs detected to validate the microarray results. The qRT-PCR that targeted seven miRNAs showed different expression levels of miRNAs in different tissues. The current research is the first to report on the miRNAs in P. pedatisecta and will enable further investigation of their roles in P. pedatisecta development.► We first detected numerous miRNAs in Pinellia pedatisecta employing microarray. ► We validated the results using RT-PCR and qRT-PCR. ► The miRNAs existed in different tissues with different expression levels.
Keywords: MicroRNA; Microarray; Pinellia pedatisecta;

Heat-shock-mediated elimination of the nptII marker gene in transgenic apple (Malus  ×  domestica Borkh.) by Katja Herzog; Henryk Flachowsky; Holger B. Deising; Magda-Viola Hanke (41-49).
Production of marker-free genetically modified (GM) plants is one of the major challenges of molecular fruit breeding. Employing clean vector technologies, allowing the removal of undesired DNA sequences from GM plants, this goal can be achieved. The present study describes the establishment of a clean vector system in apple Malus  ×  domestica Borkh., which is based on the use of the neomycin phosphotransferase II gene (nptII) as selectable marker gene and kanamycin/paramomycin as selective agent. The nptII gene can be removed after selection of GM shoots via site-specific excision mediated by heat-shock-inducible expression of the budding yeast FLP recombinase driven by the soybean Gmhsp17.5-E promoter. We created a monitoring vector containing the nptII and the flp gene as a box flanked by two direct repeats of the flp recognition target (FRT) sites. The FRT-flanked box separates the gusA reporter gene from the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter. Consequently, GUS expression does only occur after elimination of the FRT-flanked box. Transformation experiments using the monitoring vector resulted in a total of nine transgenic lines. These lines were investigated for transgenicity by PCR, RT-PCR and Southern hybridization. Among different temperature regimes tested, exposure to 42 °C for 3.5 to 4 h led to efficient induction of FLP-mediated recombination and removal of the nptII marker gene. A second round of shoot regeneration from leaf explants led to GM apple plants completely free of the nptII gene.► We produced transgenic apple clones containing the nptII, flp and the gusA genes. ► nptII and flp genes are flanked by two FRT recognition sites. ► Both genes were excised by heat-shock induced expression of the FLP recombinase. ► GusA expression was used to monitor where excision was taking place. ► The system was successful used to produce marker-free apple plants.
Keywords: Transformation; Marker-free; Site-specific recombination; Fruit tree;

Xenobiotic transport proteins are involved in cellular defence against accumulation of xenobiotics participating in multixenobiotic resistance (MXR). In order to study the transcriptional regulation of MXR genes in fish exposed to common chemical pollutants we selected the thicklip grey mullet (Chelon labrosus), since mugilids are widespread in highly degraded estuarine environments where they have to survive through development and adulthood. Partial sequences belonging to genes coding for members of 3 different families of ATP binding cassette (ABC) transporter proteins (ABCB1; ABCB11; ABCC2; ABCC3; ABCG2) and a vault protein (major vault protein, MVP) were amplified and sequenced from mullet liver. Their liver and brain transcription levels were examined in juvenile mullets under exposure to perfluorooctane sulfonate (PFOS) and to fresh (F) and weathered (WF) Prestige-like heavy fuel oil for 2 and 16 days. In liver, PFOS significantly up-regulated transcription of abcb1, abcb11 and abcg2 while in brain only abcb11 was up-regulated. Both fuel treatments significantly down-regulated abcb11 in liver at day 2 while abcc2 was only down-regulated by WF. mvp was significantly up-regulated by F and down-regulated by WF at day 2 in the liver. At day 16 only a significant up-regulation of abcb1 in the F group was recorded. Brain abcc3 and abcg2 were down-regulated by both fuels at day 2, while abcb1 and abcc2 were only down-regulated by F exposure. After 16 days of exposure only abcb11 and abcg2 were regulated. In conclusion, exposure to organic xenobiotics significantly alters transcription levels of genes participating in xenobiotic efflux, especially after short periods of exposure. Efflux transporter gene transcription profiling could thus constitute a promising tool to assess exposure to common pollutants.► Gene sequences of 6 xenobiotic efflux proteins were cloned in mullet Chelon labrosus. ► Efflux protein transcription activity was analysed after exposure to PFOS and fuel oil. ► Xenobiotic efflux proteins are transcribed in liver and brain of juvenile mullets. ► Exposure to organic xenobiotics regulates transcription of efflux genes in juveniles.
Keywords: Multixenobiotic resistance; ATP binding cassette transporters; Major vault protein; Liver; Brain; Chelon labrosus;

High levels of Paleolithic Y-chromosome lineages characterize Serbia by Maria Regueiro; Luis Rivera; Tatjana Damnjanovic; Ljiljana Lukovic; Jelena Milasin; Rene J. Herrera (59-67).
Whether present-day European genetic variation and its distribution patterns can be attributed primarily to the initial peopling of Europe by anatomically modern humans during the Paleolithic, or to latter Near Eastern Neolithic input is still the subject of debate. Southeastern Europe has been a crossroads for several cultures since Paleolithic times and the Balkans, specifically, would have been part of the route used by Neolithic farmers to enter Europe. Given its geographic location in the heart of the Balkan Peninsula at the intersection of Central and Southeastern Europe, Serbia represents a key geographical location that may provide insight to elucidate the interactions between indigenous Paleolithic people and agricultural colonists from the Fertile Crescent. In this study, we examine, for the first time, the Y-chromosome constitution of the general Serbian population. A total of 103 individuals were sampled and their DNA analyzed for 104 Y-chromosome bi-allelic markers and 17 associated STR loci. Our results indicate that approximately 58% of Serbian Y-chromosomes (I1-M253, I2a-P37.2 and R1a1a-M198) belong to lineages believed to be pre-Neolithic. On the other hand, the signature of putative Near Eastern Neolithic lineages, including E1b1b1a1-M78, G2a-P15, J1-M267, J2-M172 and R1b1a2-M269 accounts for 39% of the Y-chromosome. Haplogroup frequency distributions in Western and Eastern Europe reveal a spotted landscape of paleolithic Y chromosomes, undermining continental-wide generalizations. Furthermore, an examination of the distribution of Y-chromosome filiations in Europe indicates extreme levels of Paleolithic lineages in a region encompassing Serbia, Bosnia-Herzegovina and Croatia, possibly the result of Neolithic migrations encroaching on Paleolithic populations against the Adriatic Sea.► 103 individuals from Serbia were typed for 104 Y-SNPs for the first time. ► Samples within E-M96, I-M258, J-M172 and R-M173 were typed for 17 Y-STR loci. ► The majority of Serbian Y-chromosomes (58%) belong to pre-Neolithic lineages. ► Signature of Near Eastern Neolithic lineages accounts for 39% of the Y-chromosome.
Keywords: Y-chromosome; Neolithic transition; Agricultural revolution; Serbia; Y-STRs; Y-SNPs;

Clinical and biological significance of Cdk10 in hepatocellular carcinoma by Xiang-yu Zhong; Xiao-xue Xu; Jian-hua Yu; Gui-xing Jiang; Yang Yu; Sheng Tai; Zhi-dong Wang; Yun-fu Cui (68-74).
Cyclin-dependent kinase 10 (Cdk10) is a Cdc2-related kinase and plays an essential role in the progression from the G2 to M phase of the cell cycle. However, relative little is known about its expression pattern, clinical relevance, and biological function in hepatocellular carcinoma (HCC). In the present study, we investigated the mRNA and protein expression levels of Cdk10 in 127 pairs of HCC samples and adjacent nontumorous liver tissues and evaluated its clinical significance. Additionally, we assessed the effects of restoration of Cdk10 on cell proliferation and drug sensitivity in HCC cells. We showed that the Cdk10 mRNA and protein expression was markedly decreased in HCC samples compared to adjacent nontumorous liver tissues. Quantitative real-time polymerase chain reaction and immunohistochemical studies revealed that reduced Cdk10 expression was significantly associated with alpha-fetoprotein levels, tumor size, and tumor stage. Ectopic expression of Cdk10 reduced HCC cell proliferation, blocked the cell cycle at the G0-G1 phase, as well as inhibited cell migration and anchorage-independent growth. Additionally, Cdk10 overexpression enhanced the chemosensitivity of HCC cells to cisplatin and epidoxorubicin, two chemotherapeutic agents commonly used in HCC. These data collectively demonstrate that reduced Cdk10 expression is closely linked to HCC development and progression. Restoration of its expression may have therapeutic benefits in treating this malignancy.► HCC tissues show downregulation of Cdk10. ► Cdk10 reduction correlates with AFP levels, tumor size, and tumor stage in HCC. ► Cdk10 restoration inhibits HCC cell growth and migration. ► Cdk10 overexpression confers chemosensitivity to HCC cells.
Keywords: HCC; Cdk10; clinical significance; proliferation; drug resistance;

nMETR: Technique for facile recovery of hypomethylation genomic tags by Konstantin Baskaev; Andrew Garazha; Nurshat Gaifullin; Maria V. Suntsova; Anastassia A. Zabolotneva; Anton A. Buzdin (75-80).
Genome-wide methylation studies frequently lack adequate controls to estimate proportions of background reads in the resulting datasets. To generate appropriate control pools, we developed technique termed nMETR (non-methylated tag recovery) based on digestion of genomic DNA with methylation-sensitive restriction enzyme, ligation of adapter oligonucleotide and PCR amplification of non-methylated sites associated with genomic repetitive elements. The protocol takes only two working days to generate amplicons for deep sequencing. We applied nMETR for human DNA using BspFNI enzyme and retrotransposon Alu-specific primers. 454-sequencing enabled identification of 1113 nMETR tag sites, of them ~ 65% were parts of CpG islands. Representation of reads inversely correlated with methylation levels, thus confirming nMETR fidelity. We created software that eliminates background reads and enables to map and annotate individual tags on human genome. nMETR tags may serve as the controls for large-scale epigenetic studies and for identifying unmethylated transposable elements located close to genomic CpG islands.► We developed new experimental technique termed nMETR (non-methylated tag recovery). ► nMETR may be used to identify hypomethylated DNA tags. ► nMETR is based on digestion of DNA with methylation-sensitive restriction enzyme. ► 2nd step includes ligation of adapter oligonucleotide and further PCR amplification. ► Complete protocol takes only 48 hours to generate amplicons for deep sequencing.
Keywords: Epigenetics; Methylation; Genome wide analysis; Genomic repeats; Next generation sequencing; Alu retrotransposon;

Hypoxia-inducible factors (HIFs) are transcription factors that respond to changes in oxygen tension in the cellular environment. In this study, we identified full-length cDNAs of HIF-1α, HIF-2α and HIF-3α in an endangered hypoxia-sensitive fish species, Chinese sucker. The HIF-1α/2α/3α cDNAs are 3890, 3230 and 3374 bp in length, encoding 780, 782 and 632 amino acid residues, respectively. The real-time PCR results suggested that HIF-1α and HIF-3α mRNAs were highly expressed in liver and gonads, followed by spleen and muscle. Moreover, HIF-1α and HIF-3α transcription factors revealed similar developmental expression patterns, with the lowest expression at 48 h post-fertilization, and reaching the highest expression level at 360 h post-fertilization. Short-term hypoxia exposure (2.2, 2.8 and 3.2 mg/L dissolved oxygen for 24 h) increased mRNA levels of HIF-1α and HIF-3α. HIF-2α mRNA showed similar expression patterns with that of HIF-1α and HIF-3α, however, its expression was extremely low in the spatio-temporal expression patterns and hypoxia treatment. This is the first report describing the potential to identify hypoxia-sensitive/tolerant fishes according to the number of the serine residues of fish oxygen-dependent degradation (ODD) domain. It was suggested that Cyprinomorpha fishes, with less than 40 serine residues in fish ODD domain were hypoxia-sensitive fishes and more than 40 serine residues in this domain were hypoxia-tolerant fishes.► Hypoxia-inducible factor alpha genes-HIF-1α/2α/3α of Chinese sucker were identified. ► Chinese sucker HIF-αs were widely expressed in various tissues. ► Chinese sucker HIF-αs showed highest expression at 360 hpf. ► Short-term hypoxia exposure increased mRNA levels of HIF-αs.
Keywords: Chinese sucker; Hypoxia-inducible factor alpha; Gene expression;

Aberrant expression of FcγRIIIA (CD16) contributes to the development of atherosclerosis by Ye Huang; Huijun Yin; Jingshang Wang; Qian Liu; Caifeng Wu; Keji Chen (91-95).
Previous studies have documented that Fc receptor III A of immunoglobulin G (FcγRIIIA, also named CD16) is involved in the development of coronary heart disease (CHD). However, the mechanism responsible for FcγRIIIA's in contribution to CHD development remains largely unclear. Herein, we investigated the possible role of FcγRIIIA in the development of atherosclerosis. Our results showed that the elevated level of FcγRIIIA on monocytes closely correlated to the adhesive efficiency of human umbilical vein endothelial cells (HUVECs) in vitro. Importantly, we also observed increased population of CD16+ monocytes and elevated CD16 level on monocytes in ApoE−/− mice with characterized atherosclerosis after feeding with high-fat diet for 10 weeks. The enhancement of CD16 on monocytes closely correlated to increased content of MMP-9 in aorta and increased inflammatory cytokines in sera. In addition, similar to simvastatin, recombinant human M-CSF represented a robust inhibitory influence on plaque instability and inflammation. Taken together, these data established that FcγRIIIA (CD16)-mediated signaling orchestrated by interaction between monocytes and HUVECs, coupled with inflammatory cytokine stimulation and MMP activation, as a fundamental pathway linked to the development of atherosclerotic formation. Inhibition of FcγRIIIA or its signaling thus might represent a promising approach for the prevention and treatment of CHD.► FcγRIIIA deregulation on monocytes correlates to the adhesion with HUVECs. ► Increased CD16 on monocytes in ApoE−/− mice with atherosclerosis. ► FcγRIIIA-mediated signaling is coupled with inflammation and MMP activation.
Keywords: CHD; FcγRIIIA; Destabilization; Adhesion; Monocytes; Inflammation;

Novel mutation in the FOXC2 gene in three generations of a family with lymphoedema–distichiasis syndrome by Edyta Sutkowska; Justyna Gil; Agnieszka Stembalska; Aneta Hill-Bator; Andrzej Szuba (96-99).
Lymphoedema–distichiasis syndrome (LDS, OMIM #153400) is a genetic disorder with an autosomal dominant pattern of inheritance caused by mutations in the FOXC2 gene. Affected individuals typically present with lower extremity lymphoedema and distichiasis. The most common types of mutations in FOXC2 gene include small deletions and insertions, but duplications, duplications-insertions, missense and nonsense mutations were also found.Herein, we describe three generations of a family diagnosed with LDS caused by a new mutation in the FOXC2 gene. This mutation is a frameshift due to a deletion of two nucleotides (CC) in C repeats between C586 and C591. This mutation leads to protein truncation as a result of an earlier insertion of a stop codon.To the best of our knowledge, this is the first description of this mutation in the literature and could be coupled with an atypical lymphoscintigram.► We describe a new mutation in the FOXC2 gene in LDS family members. ► Lymphoscintigram shows atypical for LDS invisible proximal lymphnodes. ► This new mutation could be coupled with an atypical patient's lymphoscintigram.
Keywords: FOXC2 gene; Mutation; Lymphoedema; Distichiasis; Heterogeneity;

Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by mutations in low-density lipoprotein receptor (LDLR), apolipoprotein B-100 (APOB), and proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. This study investigated FH patients carrying common mutations in Taiwan and compared them to FH southeastern Asians. Causal FH mutations were identified by exon-by-exon sequencing with/without multiplex ligation-dependent probe amplification among 208 Taiwanese with clinically diagnosed FH. Haplotype analyses among probands and family members were undertaken using TaqMan® Assays. Totally, LDLR mutations were found in 118 probands, consisting of 61 different loci, and APOB 10579C>T mutations in 12 probands. Three mutations (64delG, 1661C>T, and 2099A>G) were novel. LDLR 986G>A (13.1%), 1747C>T (10.8%), and APOB 10579C>T (9.2%) were common mutations with no differences in phenotypes. LDLR 1747C>T associated with one haplotype (CAAGCCCCATGG/(dTA)n-112nt); LDLR 986G>A with two. APOB 10579C>T associated with the same LDLR binding-domain pattern in Taiwanese and southeastern Asians. We concluded that LDLR 986G>A, 1747C>T and APOB 10579C>T are common mutations, with combined frequency of approximately 33%. The presence of different haplotypes associated with FH common mutations in Taiwan indicates multiple historical migrations, probable multiple recurrent origins from southern China, and haplotype homologies reflect the presence of common ancestors in southern China.► Three common mutations of familial hypercholesterolemia are present in Taiwan. ► We detect the presence of different haplotype patterns for each FH mutations. ► The causes of FH mutations in Taiwan are from migrations and recurrent origins.
Keywords: Chinese; Familial hypercholesterolemia; Haplotype; Mutation; Taiwanese;

The dual behavior of heat shock protein 70 and asymmetric dimethylarginine in relation to serum CRP levels in type 2 diabetes by Manouchehr Nakhjavani; Afsaneh Morteza; Firuzeh Asgarani; Omid Khalilzadeh; Zaniar Ghazizadeh; Seyede Zahra Bathaie; Alireza Esteghamati (107-111).
Experimental evidence suggests that heat shock proteins (HSP) and asymmetric dimethylarginine (ADMA) are induced in the state of chronic inflammation and stress conditions. They are both inhibitors of nitric oxide synthase (NOS). The aim of this study was to evaluate the correlation between ADMA and HSP70, in patients with type 2 diabetes with respect to serum levels of C reactive protein (CRP).We quantified serum HSP70, ADMA and CRP in 80 newly-diagnosed patients with type 2 diabetes plus 80 age-, sex and BMI-matched healthy controls. The patients and controls were also stratified into groups of high and low CRP levels (cut-point: 2.5 mg/ml).Patients with type 2 diabetes had significantly higher serum HSP70 (0.52 [0.51–0.66] vs. 0.27 [0.26–0.36], p < 0.001), ADMA (0.86 [0.81–0.92] vs. 0.72 [0.71–0.85], p < 0.05) and CRP (2.9 [1.7–3.4] vs. 1.6[1.2–2.3], p < 0.05) compared with healthy controls. Serum HSP70 and ADMA levels were significantly correlated in patients with high CRP levels (r = 0.89, p < 0.01), whereas there were no correlation in patients with low CRP (r = − 0.37, p = 0.07) and controls. This correlation was significant (r = 0.77, p < 0.001) in patients with high CRP and also in patients with low CRP levels (r = − 0.51, p < 0.05), after multiple adjustments for LDL and HDL levels.We showed that, in a state of high inflammation; serum levels of ADMA parallel the HSP70 levels. However in low inflammation, they are negatively correlated. The duality in HSP70 and ADMA correlation may be related to the duality of NOS function in low and high CRP levels.► We studied the correlation of HSP70and ADMA in diabetic patients and controls. ► We examine if the correlation changes in the high and low inflammation. ► HSP70 and ADMA were positively correlated in patients with high CRP. ► HSP70 and ADMA were negatively correlated in patients with low CRP. ► HSP70 and ADMA were not correlated in controls.
Keywords: HSP70; Heat-shock proteins; N,N-dimethylarginine (asymmetric dimethylarginine); C-reactive protein; Type 2 diabetes mellitus;

Maple syrup urine disease (MSUD) is an autosomal recessive metabolic disorder that is caused by mutations in the subunits of the branched-chain α-ketoacid dehydrogenase (BCKD) complex. BCKD is a mitochondrial complex encoded by four nuclear genes (BCKDHA, BCKDHB, DBT, and DLD) and is involved in the metabolism of branched-chain amino acids (BCAAs). In this study, we investigated the DNA sequences of BCKDHA, BCKDHB and DBT genes for mutations in a Chinese newborn with the classic form of MSUD and predicted the associated conformational changes using molecular modeling. We identified two previously unreported mutations in the BCKDHB gene, R170H (c.509 G > A) in exon 5 and Q346R (c.1037 A > G) in exon 9. In silico analysis of the two novel missense mutations revealed that the mutation R170H-β alters the spatial orientation with both Y195-β′ and S206-α, which results in unstable β–β′ assembly and an unstable K+ ion binding loop of the α subunit, respectively; The Q346R mutation is predicted to disrupt the spatial conformation between Q346-β and I357-β′, which reduces the affinity of the β–β′ subunits. These results indicate that R170-β and Q346-β are crucial for the activity of the E1 component. These two novel mutations, R170H and Q346R result in the patient's clinical manifestation of the classic form of MSUD.► We identified two novel mutations R170H and Q346R in the BCKDHB gene. ► In silico analysis revealed that two mutations destabilize β–β′ subunit assembly. ► It provided a simple and rapid method to predict the severity of MSUD.
Keywords: Maple syrup urine disease; Branched-chain a-ketoacid dehydrogenase complex; BCKDHB gene; Mutation; in silico analysis;

Association between TPO gene polymorphisms and Anti-TPO level in Tehranian population: TLGS by Bita Faam; Maryam Sadat Daneshpour; Fereidoun Azizi; Marzieh Salehi; Mehdi Hedayati (116-119).
Thyroid peroxidase (TPO) gene variations are one cause of thyroid autoimmune diseases. The aim of this study was to examine the association between the T1936C, T2229C and A2257C polymorphisms of the TPO gene and Anti-TPO level.In this case–control study, 188 individuals (86 males and 102 females), aged 20–80 years, were randomly selected from among the Tehran Lipid and Glucose Study (TLGS) population. A2257C and T2229C SNPs were detected with RFLP by use of BsrI and Eco57I as the restriction enzymes respectively, while the T1936C SNP was determined with ARMS-PCR.In the presence of the C allele of T1936C, Anti-TPO level was significantly increased (CC: 238 ± 43.3, CT: 47.7 ± 15.9, TT: 74.1 ± 11.3 IU/L p = 0.002); however, this association was attenuated after adjustment for sex and age (p = 0.059). No significant difference, before and after adjustment, was found in Anti-TPO level in the presence of T2229C SNP (CC: 129.1 ± 24.5, CT: 43.5 ± 12.6, TT: 126.5 ± 13.8 IU/L p = 0.196). The association between A2257C and Anti-TPO level was only significant after adjustment for potential confounders (p = 0.007). The association between ATC and CTT haplotypes and Anti-TPO level was significant (p = 0.023, 0.021 respectively), the association between CTT and Anti-TPO concentration was also significant after adjustment for sex (p = 0.014).The results of the present study confirmed the association between TPO gene polymorphisms and Anti-TPO level in the Tehranian population.► We assess the association between TPO SNPs and Anti-TPO levels among Tehranians. ► Polymorphisms were examined with RFLP, ARMS PCR methods. ► Genetic variations of TPO gene were associated with Anti-TPO levels. ► Potential confounders as sex and age had an influence on the mentioned association.
Keywords: Thyroid peroxidase gene; Polymorphism; Anti-TPO; TLGS;

Molecular cloning and expression of the IL-10 gene from guinea pigs by Vijaya R. Dirisala; Amminikutty Jeevan; Gregory Bix; Teizo Yoshimura; David N. McMurray (120-127).
The Guinea pig (Cavia porcellus) is one of the most relevant small animals for modeling human tuberculosis (TB) in terms of susceptibility to low dose aerosol infection, the organization of granulomas, extrapulmonary dissemination and vaccine-induced protection. It is also considered to be a gold standard for a number of other infectious and non-infectious diseases; however, this animal model has a major disadvantage due to the lack of readily available immunological reagents. In the present study, we successfully cloned a cDNA for the critical Th2 cytokine, interleukin-10 (IL-10), from inbred Strain 2 guinea pigs using the DNA sequence information provided by the genome project. The complete open reading frame (ORF) consists of 537 base pairs which encodes a protein of 179 amino acids. This cDNA sequence exhibited 87% homology with human IL-10. Surprisingly, it showed only 84% homology with the previously published IL-10 sequence from the C4-deficient (C4D) guinea pig, leading us to clone IL-10 cDNA from the Hartley strain of guinea pig. The IL-10 gene from the Hartley strain showed 100% homology with the IL-10 sequence of Strain 2 guinea pigs. In order to validate the only published IL-10 sequence existing in Genbank reported from C4D guinea pigs, genomic DNA was isolated from tissues of C4D guinea pigs. Amplification with various sets of primers showed that the IL-10 sequence reported from C4D guinea pigs contained numerous errors. Hence the IL-10 sequence that is being reported by us replaces the earlier sequence making our IL-10 sequence to be the first one accurate from guinea pig. Recombinant guinea pig IL-10 proteins were subsequently expressed in both prokaryotic and eukaryotic cells, purified and were confirmed by N-terminal sequencing. Polyclonal anti-IL-10 antibodies were generated in rabbits using the recombinant IL-10 protein expressed in this study. Taken together, our results indicate that the DNA sequence information provided by the genome project is useful to directly clone much needed cDNAs necessary to study TB in the guinea pig. The newly cloned guinea pig IL-10 cDNA and recombinant proteins will serve as valuable resources for immunological studies in the guinea pig model of TB and other diseases.► Cloned IL-10 cDNA from strain 2 and Hartley strain of guinea pigs. ► Validated IL-10 sequence from C4D and C2D guinea pigs. ► Expressed guinea pig IL-10 in both prokaryotic and eukaryotic expression systems. ► Rabbit polyclonal antiserum reacted strongly and specifically with rgp-IL-10.
Keywords: IL-10 cDNA; Guinea pig; Recombinant protein;

Small supernumerary marker chromosomes (sSMCs) are a heterogeneous group with regards to their clinical effects as well as their chromosomal origin and their shape. The sSMCs are associated with mental retardation and dysmorphic features. Multiple sSMCs are rarely reported. We report four sSMCs in a case of dysmorphic features and intellectual disabilities. Among the four sSMCs, one sSMC confirmed to be chromosome 5 derived sSMC using fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY). The sSMCs were de novo originated as parental chromosomal analysis revealed normal karyotypes. The sSMC derived from chromosome 5 might be associated with mental retardation and dysmorphic features in the present case. However the remaining three sSMCs might have originated from repetitive sequences of chromosomes.► Four denovo marker chromosomes detected. ► Identified as chromosome 5. ► Associated with disease.
Keywords: Small supernumerary marker chromosome; Multiple sSMCs; Chromosome aberrations; Genotype–phenotype; Spectral karyotype;

A novel mutation in the ABCD1 gene of a Korean boy diagnosed with X-linked adrenoleukodystrophy by Jeong A. Park; Kyung Ran Jun; Sung-Hee Han; Gu-Hwan Kim; Han-Wook Yoo; Yun Jung Hur (131-133).
X-linked adrenoleukodystrophy (ALD; MIM #300100) is a neurodegenerative disorder caused by mutations in the ABCD1 adrenoleukodystrophy protein gene. The ABCD1 gene mutations have been reported by laboratories in China and Japan, but not in Korea. This case report describes a Korean boy diagnosed with X-ALD. Direct sequencing for the ABCD1 gene in this boy and his mother detected Tyr620His missense mutation, caused by cDNA nucleotide change 1858 T > C in exon 8 (c.1858T > C). This missense variant was novel and predicted to be possibly damaging by the PolyPhen and SIFT prediction software. Moreover, this is the first report in Korean.► We diagnose a childhood cALD with a novel Tyr620His missense mutation. ► Tyr620His missense mutation was caused by the cDNA nucleotide transition 1858 T > C. ► This mutation was located on exon 8, containing the ATP-binding domain. ► Our patient showed very rapidly progressing neurodevelopmental regression.
Keywords: Adrenoleukodystrophy; Missense mutation;