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Biochemical Genetics (v.51, #3-4)

MicroRNAs as Markers for Neurally Committed CD133+/CD34+ Stem Cells Derived from Human Umbilical Cord Blood by Maryam Hafizi; Amir Atashi; Behnaz Bakhshandeh; Mahboubeh Kabiri; Samad Nadri; Reza Haji Hosseini; Masoud Soleimani (pp. 175-188).

Neural differentiation of the CD133+/CD34+ subpopulation of human umbilical cord blood stem cells was investigated, and neuro-miR (mir-9 and mir-124) expression was examined. An efficient induction protocol for neural differentiation of hematopoietic stem cells together with the exclusion of retinoic acid in this process was also studied. Transcription of some neural markers such as microtubule-associated protein-2, beta-tubulin III, and neuron-specific enolase was evaluated by real-time PCR, immunocytochemistry, and western blotting. Increased expression of neural indicators in the treated cells confirmed the appropriate neural differentiation, which supported the high efficiency of our defined neuronal induction protocol. Verified high expression of neuro-miRNAs along with neuronal specific proteins not only strengthens the regulatory role of miRNAs in determining stem cell fate but also introduces these miRNAs as novel indicators of neural differentiation. These data highlight the prominent therapeutic potential of hematopoietic stem cells for use in cell therapy of neurodegenerative diseases.

Keywords: Hematopoietic stem cells; MicroRNAs; Neural differentiation; CD133+/CD34+

Improved Detection of Deletions and Duplications in the DMD Gene Using the Multiplex Ligation-Dependent Probe Amplification (MLPA) Method by Ivona Sansović; Ingeborg Barišić; Katja Dumić (pp. 189-201).

The multiplex ligation-dependent probe amplification (MLPA) assay is the most powerful tool in screening for deletions and duplications in the dystrophin gene in patients with Duchenne and Becker muscular dystrophy (DMD/BMD). The efficacy of the assay was validated by testing 20 unrelated male patients with DMD/BMD who had already been screened by multiplex PCR (mPCR). We detected two duplications that had been missed by mPCR. In one DMD patient showing an ambiguous MLPA result, a novel mutation (c.3808_3809insG) was identified. MLPA improved the mutation detection rate of mPCR by 15 %. The results of our study (1) confirmed MLPA to be the method of choice for detecting DMD gene rearrangements in DMD/BMD patients, (2) showed that ambiguous MLPA amplification products should be verified by other methods, and (3) indicated that the MLPA method could be used in screening even for small mutations located in the probe-binding regions.

Keywords: MLPA; Multiplex PCR; DMD; Muscular dystrophy

Expression of Activin Receptor-like Kinase 7 in Adipose Tissues by Masaru Murakami; Mitsuyuki Shirai; Ryo Ooishi; Asako Tsuburaya; Kumiko Asai; Osamu Hashimoto; Kenji Ogawa; Yoshii Nishino; Masayuki Funaba (pp. 202-210).

The tissue distribution of activin receptor-like kinase 7 (Alk7) expression, the signaling ability of Alk7 variants, and Alk7 expression in response to β₃-adrenergic receptor activation were examined. Expression levels of Alk7 varied greatly among tissues but were highest in white adipose tissue and brown adipose tissue. In addition to full-length Alk7 (Alk7-v1), Alk7-v3, an Alk7 variant, was expressed in adipose tissues, brain, and ovary. Nodal transmits signals via Alk7 in cooperation with its coreceptor, Cripto. Evaluation of the ability of Alk7 variants to confer Nodal signaling using luciferase-based reporter assays showed that Alk7-v3 does not transmit Nodal-Cripto-mediated signals. Expression of Alk7 was down-regulated in brown but not in white adipose tissue treated with CL316,243, a β₃-adrenergic receptor agonist. These results suggest involvement of Alk7 in modulation of metabolism in the adipose tissues in response to β₃-adrenergic receptor activation.

Keywords: Alk7; Tgf-β family; Variants; Adipose tissue

Genetic Variability of PRNP in Chinese Indigenous Goats by Rongyan Zhou; Xianglong Li; Jianzhong Xi; Lanhui Li; Zhenhong Zhang; Zhujun Zhao (pp. 211-222).

Polymorphism of the prion protein gene (PRNP) is usually associated with scrapie susceptibility or resistance. To determine the variability of PRNP in Chinese indigenous goat breeds, we isolated genomic DNA from goat blood and amplified and sequenced the coding region of the gene. We identified 10 polymorphic sites that gave rise to 28 haplotypes. Clear frequency differences were found between northern and southern breeds and confirmed by genetic distance analysis, except for the Tangshan dairy goat. Phylogeographic analysis supported the idea that northern and southern breeds might be considered separate clusters, except for the Tangshan dairy goat. The finding of significant differences in allele distribution in northern and southern goats, especially if involved in modulating resistance/susceptibility, needs to be carefully considered for the feasibility of selection plans for resistance to scrapie.

Keywords: PRNP ; Goat; Variability; Genetic distance; Allele distribution

Genetic Diversity of Winter Wheat (Triticum aestivum L.) Revealed by SSR Markers by Funda Senturk Akfirat; Ahu Altinkut Uncuoglu (pp. 223-229).

Genetic Polymorphisms and the Risk of Myocardial Infarction in Patients Under 45 Years of Age by Agata Sakowicz; Wojciech Fendler; Malgorzata Lelonek; Bartosz Sakowicz; Tadeusz Pietrucha (pp. 230-242).

This study investigates the potential role of 17 chosen polymorphisms in 15 candidate genes and the risk of myocardial infarction in patients under 45 years of age. The study consists of 271 patients with myocardial infarction and 141 controls. The analysis of genetic polymorphisms was performed using the PCR–RFLP method. Of the chosen polymorphisms, two (Leu125Val PECAM1 and A1/A2 FVII) are related to myocardial infarction and two (C677T MTHFR and 5A/6A MMP3) to advanced stenosis in arterial vessels (> 75%). We also found that the frequency of some combinations among the analyzed genes and environmental factors varied between the patient and control groups.

Keywords: Myocardial infarction; Coronary artery disease; Atherothrombosis; Polymorphism

Molecular Variability among Isolates of Fusarium oxysporum Associated with Root Rot Disease of Agave tequilana by Karla L. Vega-Ramos; J. Xavier Uvalle-Bueno; Juan F. Gómez-Leyva (pp. 243-255).

In this study, 115 isolates of Fusarium oxysporum from roots of Agave tequilana Weber cv azul plants and soil in commercial plantations in western Mexico were characterized using morphological and molecular methods. Genetic analyses of monosporic isolates included restriction enzyme analysis of rDNA (ARDRA) using HaeIII and HinfI, and genetic diversity was determined using Box-PCR molecular markers. Box-PCR analysis generated 14 groups. The groups correlated highly with the geographic location of the isolate and sample type. These results demonstrate the usefulness of ARDRA and Box-PCR techniques in the molecular characterization of the Fusarium genus for the discrimination of pathogenic isolates.

Keywords: Fusarium ; Molecular markers; Vascular wilt; ARDRA; Box-PCR

Correlation of Peroxisome Proliferator-Activated Receptor (PPAR-γ) mRNA Expression with Pro12Ala Polymorphism in Obesity by Rym Berhouma; Soumaya Kouidhi; Myriam Ammar; Hafawa Abid; Hajer Ennafaa; Amel Benammar-Elgaaied (pp. 256-263).

Our study aimed to analyze whether the expression of PPARγ mRNA in subcutaneous adipocyte tissue correlates with Pro12Ala PPARγ2 polymorphism in the obesity context. We found that mRNA expression of PPARγ in subcutaneous adipose tissue was greater in obese subjects (P < 0.05) than in the nonobese control group. Concurrently, genotyping of the Pro12Ala polymorphism showed that obese subjects possess a significantly higher frequency of the Pro/Pro genotype than nonobese controls (90.5 vs 79.5%; P = 0.03), suggesting that this genotype is involved in an increased risk of obesity in the Tunisian population. Taken together, our results demonstrate that the Pro12 allele is accompanied by an overexpression of PPARγ mRNA in subcutaneous adipocyte tissue, suggesting that the PPARγ Pro12Ala variant may contribute to the observed variability in PPARγ mRNA expression and consequently in body mass index and insulin sensitivity in the general population.

Keywords: Obesity; PPARγ expression; Pro12Ala polymorphism; Adipocytes

Contribution of Genetic Variation rs266882 to Prostate-Specific Antigen Levels in Healthy Controls with Serum PSA Below 2.0 ng/ml by Jaeman Song; Heeyoon Park; Gilho Lee (pp. 264-274).

We evaluated the impact of genetic variation in the prostate-specific antigen (PSA) gene (rs266882) on serum PSA levels in healthy men as well as risk factors for benign prostate hypertrophy (BPH) and prostate cancer. The study population comprised 91 men with PSA levels below 2.0 ng/ml as healthy controls, 78 men with PSA 2–10 ng/ml as a BPH group, and 128 prostate cancer patients, all in Korea. DNA was amplified by polymerase chain reaction and the product was sequenced. We found that PSA levels were associated with a G/A polymorphism only in healthy controls. The transition, however, was not associated with PSA levels of BPH and cancer patients, nor was it a risk factor. In conclusion, this genetic factor is important for determining serum PSA levels in the naive group, whereas the disruption of prostatic architecture in BPH or prostate cancer may be a major determining factor for PSA levels.

Keywords: Serum prostate-specific antigen; RS266882; Prostate cancer; Genotyping

Genetic Diversity of Tomistoma schlegelii Inferred from mtDNA Markers by Taranjeet Kaur; Jeffrine Rovie Ryan Japning; Mohamad Shahbudin Sabki; Irvan Sidik; Lee Kim Chong; Alan H. K. Ong (pp. 275-295).

The genetic diversity of the endangered crocodile Tomistoma schlegelii was characterized using the protein coding ND 6-tRNA^glu-cyt b and the cytochrome b-control region (cyt b-CR) markers. Concatenate data revealed six haplotypes with an overall haplotype diversity of 0.769 ± 0.039; nucleotide diversity was 0.00535 ± 0.00172. A nearest-neighbor analysis showed that all individuals clustered with four geographic regions (Sumatra, Peninsular Malaysia, Sarawak, and East Kalimantan) and were genetically differentiated. With the exception of the individuals from haplotype H2, which occurred in both Peninsular Malaysia and Sarawak, all other haplotypes were geographically distinct. The H4 lineage, which was found to be the most divergent, clustered exclusively in the basal clade in all phylogenetic trees, and the haplotype network was unconnected at the 95% reconnection limit, suggesting further investigation to establish its possible status as a distinct evolutionary significant unit or a cryptic species.

Keywords: Tomistoma schlegelii ; mtDNA; Genetic diversity; Population differentiation; Divergent lineage

Molecular Cloning and Evolutionary Analysis of the HOG-Signaling Pathway Genes from Saccharomyces cerevisiae Rice Wine Isolates by Yudong Li; Wei Chen; Yugang Shi; Xinle Liang (pp. 296-305).

The high osmolarity glycerol (HOG) signaling pathway is crucial for yeast to cope with high osmolarity. Here, we showed that Saccharomyces cerevisiae rice wine isolates exhibited higher tolerance to osmotic stress, which was associated with the evolution of HOG pathway genes. Phylogenetic analysis of HOG genes revealed that Chinese rice wine strains were closely related to sake strains, indicating a common origin of rice wine strains. The DNA sequence diversity analysis showed that higher levels of polymorphism tended to accumulate on the osmosensor genes (MSB2 and SLN1), suggesting that most changes in a signaling transduction pathway were concentrated in the receptors. Moreover, the rapid evolution of osmosensors (Sln1/Msb2) and transcription factor (Msn4) might experience positive selection. Our results imply that the evolution of HOG pathway genes in S. cerevisiae rice wine strains is associated with their adaptation to high osmotic environments.

Keywords: HOG pathway; Evolution; Osmotic stress; Rice wine; Yeast

Molecular Characterization of Primary Gene Pool of Chickpea Based on ISSR Markers by Pooja Choudhary; Suruchi M. Khanna; Pradeep K. Jain; Chellapilla Bharadwaj; Jitendra Kumar; Pramesh C. Lakhera; Ramamurthy Srinivasan (pp. 306-322).

Genetic diversity and relationships within and among members of the primary gene pool of chickpea, including 38 accessions of Cicer arietinum, six of C. reticulatum,, and four of C. echinospermum, were investigated using 31 ISSR markers. The study revealed moderate diversity, detecting 141 fragments, of which 79 (56%) were polymorphic. Averages were 0.125 for polymorphic information content, 0.350 for marker index, and 0.715 for resolving power. The UPGMA dendrogram and the principal coordinate analysis revealed a clear differentiation between wild and cultivated accessions. The clustering pattern did not strictly follow the grouping of accessions by geographic origin but was in good agreement with the pedigree data and the seed type. The study demonstrates that ISSRs provide promising marker tools in revealing genetic diversity and relationships in chickpea and can contribute to efficient identification, conservation, and utilization of germplasm for plant improvement through conventional as well as molecular breeding approaches.

Keywords: Chickpea; Genetic diversity; Molecular markers; Principal coordinate analysis

Contribution of TGFβ1 Codon 10 Polymorphism to High Myopia in an Ethnic Kashmiri Population from India by Shabhat Rasool; Ishfaq Ahmed; Rubiya Dar; Sheikh Gazalla Ayub; Sabia Rashid; Tariq Jan; Tahir Ahmed; Niyaz A. Naikoo; Khurshid I. Andrabi (pp. 323-333).

This study looks at novel variants of the TGFβ1 gene and their potential association with high myopia in an ethnic population from Kashmir, India. Allele frequencies of 247 Kashmiri subjects (from India) with high myopia and 176 ethnically matched healthy controls were tested for Hardy–Weinberg disequilibrium. The genotype and allele frequencies were evaluated using chi-square or Fisher’s exact tests. One of the three SNPs in codon 10 showed a significant difference between patients and control subjects (rs1982073: p genotype = 0.003, p allele = 0.001). There were no statistically significant differences between patients and control subjects for the other two SNPs, rs1800471 at codon 25 and a novel variant at codon 52. SNP rs1982073, substituting proline with leucine, appeared to be significantly associated with high myopia (p < 0.05). In silico predictions show that substitutions are likely to have an impact on the structure and functional properties of the protein, making it imperative to understand their functional consequences in relation to high myopia.

Keywords: Myopia; Ethnic; Polymorphism; CSGE; TGFβ1; Novel

Molecular Phylogeny of Mullets (Teleosti: Mugilidae) in Iran Based on Mitochondrial DNA by Mahboobeh Nematzadeh; Sohrab Rezvani Gillkolaei; Mohammad Kazem Khalesi; Faramarz Laloei (pp. 334-340).

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Biochemical Genetics (v.51, #3-4)

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Biochemical Genetics (v.51, #3-4)