In zebrafish, fast muscle-specific myosin heavy chain genes have their unique expression patterns in a well-defined and restricted region of the skeletal muscle. However, the transcriptional regulatory mechanisms involved have remained unclear. Here, we examined the regulation of spatio-temporal expression patterns of myhz1 (myhz1.1, myhz1.2 and myhz1.3) and myhz2 during their development by using transient gene and stable transgenic techniques. Embryos microinjected with different length 5'-flanking sequences of myhz1 conjugated with the enhanced green fluorescent protein (EGFP) gene showed EGFP expression in the anterior and medial subsections of somites, but not in the tail somite region. In contrast, embryos microinjected with different length 5'-flanking sequences of myhz2 showed EGFP expression exclusively at the posterior tail somite domain. Promoter deletion analyses demonstrated that reduced EGFP fluorescence typically is correlated with smaller 5'-flanking sequences. The immunohistochemical observation revealed that zebrafish larvae provided with the transient gene and those from stable transgenic lines consistently expressed EGFP in the fast muscle fibers. r-VISTA plot identified one common conserved region of about 140°bp among myhz1.1, myhz1.2 and myhz1.3. Deletion of this conserved region from the 5'-flanking sequence of each myhz1 markedly reduced EGFP expression in its unique spatial somite region. Deletion mutation analysis demonstrated that myhz2 expression in the tail somite region might be mediated by Tbx (family of transcription factors having a common DNA-binding sequence known as T-box) binding elements. In summary, 5'-flanking sequences of myhz1 and myhz2 regulate their unique expression patterns in a well-defined and restricted somite region of the skeletal muscle in zebrafish.
Mitochondria are cellular machines essential for energy production. The biogenesis of mitochondria is a highly complex and it depends on the coordination of the nuclear and mitochondrial genome. Mitochondrial DNA (mtDNA) mutations and deletions are suspected to be associated with carcinogenesis. The most described mtDNA deletion in various human cancers is called the 4977-bp common deletion (mDNA4977) and it has been explored since two decades. In spite of that, its implication in carcinogenesis still unknown and its predictive and prognostic impact remains controversial. This review article provides an overview of some of the cellular and molecular mechanisms underlying mDNA4977 formation and a detailed summary about mDNA4977 reported in various types of cancers. The current knowledges of mDNA4977 as a prognostic and predictive marker are also discussed.
Genetic diagnosis of spinal muscular atrophy (SMA) is complicated by the presence of SMN2 gene as majority of SMA patients show absence or deletion of SMN1 gene. PCR may amplify both the genes non selectively in presence of high amount of DNA. We evaluated whether allele-specific PCR for diagnostic screening of SMA is reliable in the presence of high amount of genomic DNA, which is commonly used when performing diagnostic screening using restriction enzymes.
Breast cancer is the most diagnosed malignancy and the second leading cause of cancer mortality in females. Previous association studies have identified variants on 2q35 associated with the risk of breast cancer. To identify functional susceptibility loci for breast cancer, we interrogated the 2q35 gene desert for chromatin architecture and functional variation correlated with gene expression. We report a novel intergenic breast cancer risk locus containing an enhancer copy number variation (enCNV; deletion) located approximately 400Kb upstream to IGFBP5, which overlaps an intergenic ERα-bound enhancer that loops to the IGFBP5 promoter. The enCNV is correlated with modified ERα binding and monoallelic-repression of IGFBP5 following oestrogen treatment. We investigated the association of enCNV genotype with breast cancer in 1,182 cases and 1,362 controls, and replicate our findings in an independent set of 62,533 cases and 60,966 controls from 41 case control studies and 11 GWAS. We report a dose-dependent inverse association of 2q35 enCNV genotype (percopy OR = 0.68 95%CI 0.55-0.83, P = 0.0002; replication OR = 0.77 95% CI 0.73-0.82, P = 2.1 × 10-19) and identify 13 additional linked variants (r2 > 0.8) in the 20Kb linkage block containing the enCNV (P = 3.2 × 10-15 - 5.6 × 10-17). These associations were independent of previously reported 2q35 variants, rs13387042/rs4442975 and rs16857609, and were stronger for ER-positive than ER-negative disease. Together, these results suggest that 2q35 breast cancer risk loci may be mediating their effect through IGFBP5.
The allele frequencies for the apolipoprotein B (apo B) 5'-Ins/Del and 3'-VNTR polymorphisms varied significantly (p < 0.01) among Singaporeans of Chinese, Malay and Indian descent. We calculated the unbiased expected heterozygosities for the 5'-Ins/Del polymorphism as 0.3357, 0.1984 and 0.2418, and for the 3'-VNTR as 0.5980, 0.5260 and 0.6749, respectively, in the Chinese, Malays and Indians. Compared to heterozygosities reported for other populations, the Singaporeans differed from most Caucasians in having significantly lower values but were closely related to other non-Caucasians. Thirteen alleles, with a bimodal distribution, were observed at the 3'-VNTR polymorphic locus; the alleles occurring most frequently among the Chinese and Malays were of 35 or 53 repeats, and among the Indians, of 37 or 47 repeats. The Del allele was associated with elevated serum cholesterol (p = 0.023), LDL-cholesterol (LDL-C) (p = 0.001) in the Chinese, and apo B (p = 0.007) in the Indians. Likewise, the larger 3'-VNTR alleles (> 41 repeats) were associated with raised cholesterol (p = 0.018), LDL-C (p = 0.025), and triglyceride (p = 0.001) in the Chinese. The two polymorphisms were not in significant linkage disequilibrium (D = -0.0029, p = 0.494) in the three ethnic groups.
A truncating mutation (1100delC) in the cell cycle checkpoint kinase-2 gene, CHEK2, has been identified as a risk factor for familial and sporadic breast cancer in some Northern and Western European populations. However, the prevalence of CHEK2*1100delC in breast cancer appears to be population dependent. We analysed the prevalence of CHEK2*1100delC in 668 breast cancer cases, of which 542 were invasive breast cancers, from a hospital-based cohort of breast cancer patients from Kuala Lumpur, Malaysia. The variant was not found in any patients in this cohort, suggesting that CHEK2*1100delC is rare in our population, and unlikely to contribute significantly to risk to breast cancer among the Malay, Chinese and Indian ethnic groups in Malaysia. This suggests that screening for this allele should not be routinely conducted in Malaysia.
The major outer membrane protein (OmpH) of 4 local Malaysian strains of Pasteurella multocida serotype B:2 were characterized in comparison to ATCC strains. Three major peptide bands of MW 26, 32 and 37 kDa were characterized using SDSPAGE. Two of these fragments, the 32 kDa and 37 kDa were observed to be more reactive with a mouse polyclonal antiserum in all of the local isolates as well as the ATCC strains in a Western blot. However, the 32 kDa fragment was found to cross react with other Gram negative bacteria. Therefore, the 37 kDa OmpH was selected as vaccine candidate. The 37 kDa ompH gene of the isolated strain 1710 was cloned into an Escherichia coli expression vector to produce large amounts of recombinant OmpH (rOmpH). The 37 kDa ompH gene of strain 1710 was sequenced. In comparison to a reference strain X-73 of the ompH of P. multocida, 39bp was found deleted in the 37 kDa ompH gene. However, the deletion did not shift the reading frame or change the amino acid sequence. The rOmpH was used in a mice protection study. Mice immunized and challenged intraperitoneally resulted 100% protection against P. multocida whilst mice immunized subcutaneously and challenged intraperitoneally only resulted 80% protection. The rOmpH is therefore a suitable candidate for vaccination field studies. The same rOmpH was also used to develop a potential diagnostic kit in an ELISA format.
Malaysia first reported H5N1 poultry case in 2004 and subsequently outbreak in poultry population in 2007. Here, a recombinant gene encoding of peptide epitopes, consisting fragments of HA1, HA2 and a polybasic cleavage site of H5N1 strain Malaysia, was amplified and cloned into pET-47b(+) bacterial expression vector. DNA sequencing and alignment analysis confirmed that the gene had no alteration and in-frame to the vector. Then, His-tagged truncated HA protein was expressed in Escherichia coli BL21 (DE3) under 1 mM IPTG induction. The protein expression was optimized under a time-course induction study and further purified using Ni-NTA agarose under reducing condition. Migration size of protein was detected at 15 kDa by Western blot using anti-His tag monoclonal antibody and demonstrated no discrepancy compared to its calculated molecular weight.
The human genome contains many submicroscopic copy number variations which includes deletions, duplications and insertions. Although conventional karyotyping remains an important diagnostic tool in evaluating a dysmorphic patient with mental retardation, molecular diagnostic technology such as array comparative genomic hybridization (aCGH) has proven to be sensitive and reliable in detecting these submicroscopic anomalies. A 3 month-old infant with dysmorphic facies, microcephaly and global developmental delay was referred for genetic evaluation. Preliminary karyotyping which was confounded by the quality of metaphase spread was normal; however, aCGH detected a 30.6Mb deletion from 5p15.33-p13.3. This case illustrates the usefulness of aCGH as an adjunctive investigative tool for detecting chromosomal imbalances.
Haemoglobin (Hb) Adana (HBA2:c.179>A) interacts with deletional and nondeletional α-thalassaemia mutations to produce HbH disorders with varying clinical manifestations from asymptomatic to severe anaemia with significant hepatosplenomegaly. Hb Adana carriers are generally asymptomatic and haemoglobin subtyping is unable to detect this highly unstable α-haemoglobin variant. This study identified 13 patients with compound heterozygosity for Hb Adana with either the 3.7 kb gene deletion (-α(3.7)), Hb Constant Spring (HbCS) (HBA2:c.427T>C) or Hb Paksé (HBA2:429A>T). Multiplex Amplification Refractory Mutation System was used for the detection of five deletional and six nondeletional α-thalassaemia mutations. Duplex-PCR was used to confirm Hb Paksé and HbCS. Results showed 84.6% of the Hb Adana patients were Malays. Using DNA studies, compound heterozygosity for Hb Adana and HbCS (α(codon 59)α/α(CS)α) was confirmed in 11 patients. A novel point in this investigation was that DNA studies confirmed Hb Paksé for the first time in a Malaysian patient (α(codon 59)α/α(Paksé)α) after nine years of being misdiagnosis with Hb Adana and HbCS (α(codon 59)α/α(CS)α). Thus, the reliance on haematology studies and Hb subtyping to detect Hb variants is inadequate in countries where thalassaemia is prevalent and caused by a wide spectrum of mutations.
Dystonia is a heterogenous group of movement disorders whose clinical spectrum is very wide. At least 13 different genes and gene loci have been reported. While a 3-bp deletion in the DYT1 gene is the most frequent cause of early limb-onset, generalized dystonia, it has also been found in non-generalized forms of sporadic dystonia. An 18-bp deletion in the DYT1 gene has also been reported.
BACKGROUND: The most common autosomal form of Chronic Granulomatous Disease, p47-phox deficient CGD, generally features a GT (deltaGT) deletion in the GTGT sequence at the start of exon 2 on the NCF-1 gene. This consistency is due to the coexistence of and the recombination between 2 homologous pseudogenes (psi s) and NCF-1. The GTGT: deltaGT ratio mirrors the NCF-I: NCF-1 psi ratio and is 2:4 in normal individuals.
OBJECTIVE: To determine the molecular basis of the Autosomal-CGD in a family with 2 children, a male and female, affected by the disease. The female patient suffered recurrent infection, retinitis pigmentosa and discoid lupus.
METHODS: Chemiluminescence (CL) was used to study the respiratory burst, while genetic analysis was done by RT-PCR, PCR, deltaGT and the 20bp gene scans.
RESULTS: The CL response of the patient was profoundly low. The patient's p47-phox band was absent in the RT-PCR for NADPH-oxidase component mRNAs. The deltaGT scan showed that the patient's GTGT: deltaGT ratio was 0:6, the parents' and the younger brother's was 1:5 and the younger sister's was 2:4. Examination of other NCF-1/ NCF-1 psi s differences showed that the father had a compound deltaGT allele ie. deltaGT-20bp, inherited by the patient, and that both parents had compound GTGT alleles with a single 30bp segment in intron 1.
CONCLUSIONS: The patient was a classic, homozygous deltaGT p47-phox deficient CGD with one allele harbouring a compound deltaGT-20bp gene. The deltaGT and 20bp gene scans offer a relatively simple and efficient means of defining a p47-phox deficient CGD patient.
Key words: Chronic Granulomatous Disease, Primary Immunodeficiency, NCF-1, p47-phox, NADPH-oxidas
The authors suggest a simplification for the current molecular genetic testing of spinal muscular atrophy (SMA). Deletion analysis of SMN1 exon 7 alone may be necessary and sufficient for the diagnosis of SMA. It is based on sole contribution of survival motor neuron 1 (SMN1) exon 7 to SMA pathogenesis.
Currently, the effects of chronic, continuous low dose environmental irradiation on the mitochondrial genome of resident small mammals are unknown. Using the bank vole (Myodes glareolus) as a model system, we tested the hypothesis that approximately 50 generations of exposure to the Chernobyl environment has significantly altered genetic diversity of the mitochondrial genome. Using deep sequencing, we compared mitochondrial genomes from 131 individuals from reference sites with radioactive contamination comparable to that present in northern Ukraine before the 26 April 1986 meltdown, to populations where substantial fallout was deposited following the nuclear accident. Population genetic variables revealed significant differences among populations from contaminated and uncontaminated localities. Therefore, we rejected the null hypothesis of no significant genetic effect from 50 generations of exposure to the environment created by the Chernobyl meltdown. Samples from contaminated localities exhibited significantly higher numbers of haplotypes and polymorphic loci, elevated genetic diversity, and a significantly higher average number of substitutions per site across mitochondrial gene regions. Observed genetic variation was dominated by synonymous mutations, which may indicate a history of purify selection against nonsynonymous or insertion/deletion mutations. These significant differences were not attributable to sample size artifacts. The observed increase in mitochondrial genomic diversity in voles from radioactive sites is consistent with the possibility that chronic, continuous irradiation resulting from the Chernobyl disaster has produced an accelerated mutation rate in this species over the last 25 years. Our results, being the first to demonstrate this phenomenon in a wild mammalian species, are important for understanding genetic consequences of exposure to low-dose radiation sources.
LMP-1, an Epstein-Barr viral (EBV) latency protein, is considered a viral oncogene because of its ability to transform rodent fibroblasts in vivo and render them tumorigenic in nude mice. In human B cells, EBV LMP-1 induces DNA synthesis and abrogates apoptosis. LMP-1 is expressed in EBV-transformed lymphoblastoid cell lines, nasopharyngeal carcinoma (NPC), a subset of Hodgkin's disease (HD), and in EBV-associated lymphoproliferative disorders (EBV-LPDs). Recently, focused deletions near the 3' end of the LMP-1 gene (del-LMP-1, amino acids 346-355), in a region functionally related to the half-life to the LMP-1 protein, have been reported frequently in human immunodeficiency virus (HIV)-associated HD (100%) and EBV+ Malaysian and Danish peripheral T-cell lymphomas (100%, 61% respectively), but less frequently in cases of HD not associated with HIV (28%, 33%) and infectious mononucleosis (33%). To further investigate the potential relationship of del-LMP-1 to EBV-LPDs associated with immunosuppression or immunodeficiency, we studied 39 EBV-associated lymphoproliferations (10 benign, 29 malignant) from four distinct clinical settings: posttransplant (4 malignant, 1 reactive); HIV+ (18 malignant, 2 reactive); nonimmunodeficiency malignant lymphoma (ML) (7 cases); and sporadic EBV infection with lymphoid hyperplasia (7 cases). The presence of EBV within lymphoid cells was confirmed by EBV EBER1 RNA in situ hybridization or by polymerase chain reaction (PCR) analysis. EBV strain type and LMP-1 deletion status were determined by PCR. EBV strain types segregated into two distinct distributions: HIV+ (9 A; 11 B) and non-HIV (19 A, 0 B), consistent with previous reports. Overall, del-LMP-1 were found in 1 of 5 (20%) Burkitt lymphomas (BL); 17 of 24 (71%) aggressive non-Hodgkin's lymphoma (agg-NHL), and 2 of 10 (20%) reactive lymphoid proliferations. Of the agg-NHLs, del-LMP-1 were present in 4 of 4 PT-ML (100%); 10 of 15 HIV+ ML (67%); and 3 of 5 nonimmunodeficiency malignant lymphoma (ML, 60%). A total of 2 of 7 (28%) sporadic EBV-associated lymphoid hyperplasias contained a del-LMP-1. All del-LMP-1 were identical by DNA sequence analysis. No correlation was identified between the presence of del-LMP-1 and the EBV strain type observed. The high incidence of del-LMP-1 observed in agg-NHLs (71%), in contrast to the relatively low incidence observed in reactive lymphoid proliferations (28%), suggests that the deleted form may be preferentially selected in lymphomatous processes. All posttransplant agg-NHLs contained a del-LMP-1, and a similar frequency of del-LMP-1 was observed in both HIV-associated ML (66%) and nonimmunodeficiency ML (60%), suggesting that impairment of immune function alone is not a requirement for the expansion of malignant cells infected by EBV stains containing the deleted LMP-1 gene.
The LHb expression is up-regulated during puberty in female zebrafish. However, the molecular mechanism underlying how LHb expression is regulated during puberty remains largely unknown. In this study, we found that the mRNA expression levels of lhb, fshb and cyp19a1b were up-regulated along with the puberty onset in zebrafish. Among the three nuclear estrogen receptors (nERs), the esr2b is the only type whose expression is significantly up-regulated during puberty onset in the pituitary. However, in situ hybridization results revealed that lhb mRNA was colocalized with esr1 and esr2a but not esr2b. Exposure to estradiol (E2) significantly stimulates LHb expression in both wild-type and kiss1-/-;kiss2-/-;gnrh3-/- triple knockout pubertal zebrafish. Moreover, exposure of cultured pituitary cells to E2 increased the LHb expression, indicating that the estrogenic effect on LHb expression could be acted at the pituitary level. Finally, we cloned and analyzed the promoter of lhb by luciferase assay. Our results indicated that the E2 responsive regions of lhb promoter for ERα and ERβ2 are identical, suggesting that ERα and ERβ2 could bind to the same half ERE region of the promoter of lhb, exhibiting a classical ERE-dependent pathway. In summary, we demonstrate that E2 could directly act on the pituitary level to stimulate LHb transcription during puberty in zebrafish.
Haemoglobin E (Hb E) is a variant of structurally abnormal haemoglobin that can be found very commonly in the Asian countries particularly the Southeast Asian [1]. [H1] Alpha thalassaemia is a red cell disorder which is caused by deletion or mutation of one or more of the four alpha globin genes leading to absence or decrease in production of alpha globin peptides [2]. This disorder is far more common in South East Asian regions and in Malaysia itself, and the gene frequency is about 4.1% [2]. The interactions of Hb E and alpha thalassaemia are evident in Kelantan which is bordered by southern Thailand. Using capillary electrophoresis (CE), a reduction of Hb E level is noticed as compared to Hb E heterozygotes. DNA analysis should be done to determine the presence of concurrent alpha thalassaemia variant. This study was done to evaluate haematological parameters using automated blood counters, morphology of red cells, Hb separation and quantitation of Hb fractions using CE and molecular analysis for alpha thalassemia. The study also aimed to discover cut off point of Hb E level in heterozygous Hb E patients with concurrent deletional alpha thalassaemia by CE.
A combination of genetic and functional approaches has identified three independent breast cancer risk loci at 2q35. A recent fine-scale mapping analysis to refine these associations resulted in 1 (signal 1), 5 (signal 2), and 42 (signal 3) credible causal variants at these loci. We used publicly available in silico DNase I and ChIP-seq data with in vitro reporter gene and CRISPR assays to annotate signals 2 and 3. We identified putative regulatory elements that enhanced cell-type-specific transcription from the IGFBP5 promoter at both signals (30- to 40-fold increased expression by the putative regulatory element at signal 2, 2- to 3-fold by the putative regulatory element at signal 3). We further identified one of the five credible causal variants at signal 2, a 1.4 kb deletion (esv3594306), as the likely causal variant; the deletion allele of this variant was associated with an average additional increase in IGFBP5 expression of 1.3-fold (MCF-7) and 2.2-fold (T-47D). We propose a model in which the deletion allele of esv3594306 juxtaposes two transcription factor binding regions (annotated by estrogen receptor alpha ChIP-seq peaks) to generate a single extended regulatory element. This regulatory element increases cell-type-specific expression of the tumor suppressor gene IGFBP5 and, thereby, reduces risk of estrogen receptor-positive breast cancer (odds ratio = 0.77, 95% CI 0.74-0.81, p = 3.1 × 10-31).