Diagnosis of a rare double heterozygous Hb D Punjab/Hb Q India hemoglobinopathy using Sebia capillary zone electrophoresis.

In India, hemoglobinopathies constitute a major genetic disorder and hemoglobin variants such as Hb S, Hb D Punjab, and Hb E are the most common ones. Other variants include Hb Q India, Hb Lepore, Hb J Meerut, Hb D Iran, etc. These variants show heterozygous state along with beta thalassemia. However, compound heterozygosities among these variants are very rare. Ethylenediaminetetraacetic acid whole blood sample received for routine thalassemia screening was subjected to alkaline electrophoresis using automated capillary zone electrophoresis. Suspecting the presence of rare variants, further analysis was carried out using Bio-Rad D10 and Tosoh G8 high-performance liquid chromatography (HPLC) systems. Capillary zone electrophoretograms showed the presence of peaks in zone Hb A, Hb D, a fused peak in Hb A2, and a small peak in Z1 zone. Bio-Rad and Tosoh chromatograms also indicated the presence of four peaks which are identified as Hb A, Hb D Punjab, Hb Q India, and hybrid of Hb D Punjab/Hb Q India. A peak in Hb D zone of capillary was due to co-migration of Hb D Punjab and Hb Q India variants. Small peak in Z1 zone indicated the presence of alpha chain variant Hb Q India. The findings were further confirmed by HPLC results and molecular genetic studies. The present study reports for the 1st time a rare hemoglobinopathy of double heterozygosity for Hb D Punjab, Hb Q India on Capillarys 2 Flex Piercing analyzer and is forth reported case for this rare hemoglobinopathy.

Absence of altered expression of optineurin in primary open angle glaucoma patients.

PURPOSE: To investigate the expression level of the optineurin gene (OPTN) in the blood of primary open angle glaucoma (POAG) patients to determine if altered expression is playing a role in primary open angle glaucoma systemically. METHODS: Patients (n=47) were eligible for inclusion if they met standard clinical criteria for POAG, including age greater than 40 years, intraocular pressure ≥21 mmHg in at least one eye before treatment, normal-appearing anterior chamber angles bilaterally on gonioscopy, and optic nerve injury characteristic of POAG. Control subjects (n=27) were recruited who were free from glaucoma by examination. DNA from patient was sequenced to look for possible mutations in the coding region of OPTN or its promoter. RNA was extracted from leukocytes of patients and controls and converted to cDNA by reverse transcriptase enzyme, and quantitative PCR was used to assess expression levels of OPTN and the ß-globulin gene. The ratio of OPTN expression to ß-globulin gene expression for POAG patients was compared to that of controls and to clinical characteristics of POAG patients. RESULTS: No mutation(s) were detected in any of the patients after sequencing the full OPTN gene and its promoter region. Mean OPTN (p≤0.35), and ß-globulin (p≤0.48) gene expression values were statistically similar in POAG patients and controls. OPTN/ß-globulin (p≤0.83) ratios were also indistinguishable between POAG patients and controls. OPTN/ß-globulin ratios were not significantly associated with age, sex, or ethnicity of patients within the POAG group. Similarly, OPTN/ß-globulin ratios were not significantly affected by ethnicity or clinical parameters related to POAG severity including maximum intraocular pressure, vertical cup-to-disk ratio, static perimetry mean deviation, or static perimetry pattern standard deviation. CONCLUSIONS: OPTN expression is not altered in the blood of POAG patients, suggesting that OPTN expression is not changed systemically and implying that other mechanisms are involved in POAG pathogenesis.

Unaltered myocilin expression in the blood of primary open angle glaucoma patients.

PURPOSE: To investigate the expression of the myocilin gene (MYOC) in the blood of primary open angle glaucoma (POAG) patients to determine if altered systemic expression is playing a role. METHODS: Patients (n=47) were eligible for inclusion if they met standard clinical criteria for POAG. Control subjects (n=27) were recruited who were free from glaucoma by examination. RNA was extracted from leukocytes of patients and controls and converted to cDNA by reverse transcriptase enzyme, and quantitative PCR was used to assess expression levels of MYOC and the house keeping gene ß-globulin (HBB). The ratio of MYOC expression to HBB expression for POAG patients was compared to that of controls and to clinical characteristics of POAG patients. RESULTS: Mean gene expression values were statistically similar in POAG patients and controls for both MYOC (p≤0.55) and HBB (p≤0.48). MYOC/HBB ratios were also statistically indistinguishable between POAG patients and controls (p≤0.90). MYOC/HBB ratios were not significantly associated with age, sex, or ethnicity of patients within the POAG group. Similarly, MYOC/HBB ratios were not significantly associated with clinical parameters related to POAG severity, including maximum intraocular pressure, vertical cup-to-disk ratio, static perimetry mean deviation, or static perimetry pattern standard deviation. CONCLUSIONS: MYOC expression is not altered in the blood of POAG patients, unlike MYOC expression in trabecular meshwork (TM) cultures. These results suggests that MYOC expression is not altered systemically but rather that MYOC expression may contribute to POAG pathogenesis in specific tissues such as TM.

Identification of tristetraprolin as a factor that modulates the stability of the TAFI transcript through binding to the 3'-untranslated region.

BACKGROUND: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a basic carboxypeptidase zymogen encoded by the human gene CPB2. TAFI constitutes a molecular link between coagulation and fibrinolysis, and between coagulation and inflammation. The 3'-untranslated region (UTR) of the human CPB2 mRNA plays a key role in regulating CPB2 mRNA abundance, but the exact mechanisms that mediate this regulation are largely unexplored. OBJECTIVES: To pinpoint cis-acting elements in the CPB2 3'-UTR that act as stability determinants and to identify protein factors binding to these sites. METHODS: We constructed a series of plasmids encoding mRNAs containing rabbit ß-globin sequences (as a reporter) fused to sequences of the CPB2 3'-UTR (encompassing 5' and internal deletions). These plasmids were transfected into HepG2 (human hepatoma) cells and the stability of the fusion transcripts measured. We performed a series of gel mobility shift analyses using RNA probes encompassing putative (in)stability elements. RESULTS: We identified one element conferring stability and three elements conferring instability. Supershift assays identified the protein bound to the site between the second and third polyadenylation sites as tristetraprolin (TTP). Mutation of the TTP site abolished TTP binding in gel mobility shift assays and also stabilized ß-globin/CPB2 fusion transcripts. TTP knockdown stabilized the fusion transcript containing the TTP site, but not a fusion transcript in which this site was mutated. CONCLUSIONS: Our findings are indicative of a role for TTP in constitutive, and perhaps regulated, control of CPB2 mRNA stability and hence abundance.

Prototypic chromatin insulator cHS4 protects retroviral transgene from silencing in Schistosoma mansoni.

Vesicular stomatitis virus glycoprotein (VSVG) pseudotyped murine leukemia virus (MLV) virions can transduce schistosomes, leading to chromosomal integration of reporter transgenes. To develop VSVG-MLV for functional genomics in schistosomes, the influence of the chicken ß-globin cHS4 element, a prototypic chromatin insulator, on transgene expression was examined. Plasmid pLNHX encoding the MLV 5'- and 3'-Long Terminal Repeats flanking the neomycin phosphotransferase gene (neo) was modified to include, within the U3 region of the 3'-LTR, active components of cHS4 insulator, the 250 bp core fused to the 400 bp 3'-region. Cultured larvae of Schistosoma mansoni were transduced with virions from producer cells transfected with control or cHS4-bearing plasmids. Schistosomules transduced with cHS4 virions expressed 2-20 times higher levels of neo than controls, while carrying comparable numbers of integrated proviral transgenes. The findings not only demonstrated that cHS4 was active in schistosomes but also they represent the first report of activity of cHS4 in any Lophotrochozoan species, which has significant implications for evolutionary conservation of heterochromatin regulation. The findings advance prospects for transgenesis in functional genomics of the schistosome genome to discover intervention targets because they provide the means to enhance and extend transgene activity including for vector based RNA interference.

A melting curve analysis--based PCR assay for one-step genotyping of ß-thalassemia mutations a multicenter validation.

The increasing number of disease-causing mutations demands a simple, direct, and cost-effective diagnostic genotyping technique capable of detecting multiple mutations. This study validated the efficacy of a novel melting curve analysis-based genotyping assay (MeltPro HBB assay) for 24 ß-thalassemia mutations in the Chinese population. The diagnostic potential of this assay was evaluated in 1022 pretyped genomic DNA samples, including 909 clinical cases of ß-thalassemia minor or major, using a double-blind analysis in a multicenter validation study. Reproducibility of the assay was 100%, and the limit of detection was 10 pg per reaction. All 24 ß-thalassemia mutations were accurately genotyped, and ß-thalassemia genotypes were correctly determined in all 1022 samples, yielding overall sensitivity and specificity of 100%. The concordance rate was 99.4% between this assay and the reference method. It was concluded that the MeltPro HBB assay is useful for reliable genotyping of multiple ß-thalassemia mutations in clinical settings and may have potential as a versatile method for rapid genotyping of known mutations because of its high throughput, accuracy, ease of use, and low cost.

Down-regulation of OPA1 in patients with primary open angle glaucoma.

PURPOSE: Heterozygous optic atrophy type1 (OPA1) mutations are responsible for dominant optic atrophy, and the down regulation of OPA1 expression in patients with Leber hereditary optic neuropathy may imply that Opa1 protein levels in mitochondria play a role in other spontaneous optic neuropathies as well. Mitochondrial and metabolic abnormalities may put the optic nerve at risk in primary open angle glaucoma (POAG), and this preliminary study was designed to investigate whether altered OPA1 expression might be present in the progressive optic neuropathy of POAG. METHODS: Patients were eligible for inclusion if they met standard clinical criteria for POAG, including age greater than 40 years, intraocular pressure ≥ 21 mmHg in at least one eye before treatment, normal-appearing anterior chamber angles bilaterally on gonioscopy, and optic nerve injury characteristic of POAG. RNA was extracted from leukocytes and converted to cDNA by reverse transcriptase enzyme, and real time PCR was used to assess expression levels of OPA1 and the ß-globulin (HBB) housekeeping gene. The ratio of OPA1 expression to HBB expression (OPA1/HBB) for POAG patients was compared to that of controls and to clinical characteristics of POAG patients. RESULTS: Forty-three POAG patients and 27 controls were completely phenotyped with a full ophthalmologic examination and static perimetry. Mean age (POAG 67.9 years; controls 61.8 years) and sex (POAG 26 males/17 females; controls 11/16) were similar for the two groups. Mean OPA1/HBB of POAG patients (1.16, SD 0.26) was 18% lower than controls (1.41, SD 0.50), and this difference was statistically significant (p≤0.021). OPA1 expression differed between the groups (p≤0.037), but HBB expression did not differ (p≤0.24). OPA1/HBB was not correlated with any clinical feature of POAG patients. CONCLUSIONS: Transcriptional analysis of peripheral blood leucocytes is a limited model system for studying the consequences of mitochondrial abnormalities in the optic nerve. Nevertheless, OPA1 is known to affect mitochondrial stability and has now been implicated in several spontaneous optic neuropathies. Decreased OPA1 expression in POAG patients is another indication that mitochondrial function, and possibly mitochondrially-induced apoptosis, may play a role in the development of POAG.

Mitochondrial C150T polymorphism increases the risk of cervical cancer and HPV infection.

During a survey of control region (D-loop) sequence variances in 142 cervical cancer (CC) patients and 136 controls, all Chinese women, including both HPV-positive (human papillomavirus) and HPV-negative subjects, we determined that the C150T polymorphism increased the CC risk in a case-control study (OR=3.0, 95% CI=1.8-5.0, P<0.05). HPV-positive individuals were more likely to carry the C150T polymorphism than HPV-negative controls (OR=5.8, 95% CI=2.6-13.2, P=2.3×10(-5)). HPV-positive CC patients were more likely to carry the C150T polymorphism than HPV-negative controls (OR=4.9, 95% CI=2.6-9.3, P=9.9×10(-7)). In all subjects, an increased risk of HPV infection was also associated with the C150T polymorphism (OR=4.5, 95% CI=2.5-8.1, P=6.6×10(-7)). However, no significant difference in the frequency of other alleles was found at the variable sites in D146, D152, D310 and D514. These results indicated that the C150T polymorphism increased the risk of HPV infection and CC progression. Additionally, we assessed the association of mtDNA copy number with CC risk or the C150T polymorphism in 45 CC patients and 43 controls. There was no significant association of mtDNA copy number with CC risk or the C150T polymorphism. To the best of our knowledge, this is the first report to suggest that mtDNA C150T polymorphism was positively associated with HPV infection and subsequent CC risk among Chinese women.

Importance of housekeeping gene selection for accurate reverse transcription-quantitative polymerase chain reaction in a wound healing model.

Studies in the field of wound healing have utilized a variety of different housekeeping genes for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. However, nearly all of these studies assume that the selected normalization gene is stably expressed throughout the course of the repair process. The purpose of our current investigation was to identify the most stable housekeeping genes for studying gene expression in mouse wound healing using RT-qPCR. To identify which housekeeping genes are optimal for studying gene expression in wound healing, we examined all articles published in Wound Repair and Regeneration that cited RT-qPCR during the period of January/February 2008 until July/August 2009. We determined that ACTß, GAPDH, 18S, and ß2M were the most frequently used housekeeping genes in human, mouse, and pig studies. We also investigated nine commonly used housekeeping genes that are not generally used in wound healing models: GUS, TBP, RPLP2, ATP5B, SDHA, UBC, CANX, CYC1, and YWHAZ. We observed that wounded and unwounded tissues have contrasting housekeeping gene expression stability. The results demonstrate that commonly used housekeeping genes must be validated as accurate normalizing genes for each individual experimental condition.

Host beta-globin gene fragments in crevicular fluid as a biomarker in periodontal health and disease.

BACKGROUND AND OBJECTIVE: Leukocytes and epithelium are the first line of defense in preventing bacterial invasion into periodontium. Some of these cells die in gingival crevicular fluid, whereupon their DNA is spilled out. The present study was designed to investigate the profile of host beta-globin gene fragments in the gingival crevicular fluid of various periodontal conditions. MATERIAL AND METHODS: Gingival crevicular fluid from 40 teeth with chronic periodontitis, 30 with gingivitis and 22 that were clinically healthy were centrifuged (3,000 g, 10 min). The supernatant (cell-free gingival crevicular fluid) was centrifuged again (13,000 g, 10 min), resulting in the pellet and the supernatant as debris and debris-free fractions, respectively. Specific primers for amplifying 110 bp, 536 bp and 2 kb amplicons of human beta-globin gene were used to investigate host DNA by quantitative and qualitative polymerase chain reaction. RESULTS: The periodontitis group showed the largest amount of host beta-globin gene fragments, while the healthy group had the lowest. In the debris and debris-free fractions, the 536 bp and 2 kb amplicons were more often detected in the periodontitis group than in the other groups. Interestingly, the presence of 2 kb amplicon in the debris fraction could be used to discriminate periodontitis from gingivitis and healthy groups because we found it in 85% of periodontitis samples but only in 13% of gingivitis samples, and it was absent in the healthy group. CONCLUSION: This study shows the different DNA profiles of cell-free gingival crevicular fluid in periodontal health and disease. It suggests that the quantity and quality of host DNA are dependent on the disease conditions. Therefore, the beta-globin gene fragments in cell-free gingival crevicular fluid may be a potential biomarker of periodontal disease progression.

Cell-free DNA in the plasma of patients with systemic sclerosis.

The aim of the present study was to evaluate the concentration of cell-free DNA (cf-DNA) in the plasma of patients with systemic sclerosis (SSc) and to examine the correlation of cf-DNA with clinical variables of the disease. The study population consisted of 122 SSc patients and 16 healthy controls. Epidemiological and clinical data were collected by direct assessment. The beta-globin gene was used to determine the total amount of DNA in the plasma by real-time quantitative PCR analysis. cf-DNA was found in all patients (mean concentration 1,420.7 copies/ml) and controls (mean concentration 1,462.5), with no significant difference. In SSc patients, no correlation was found between cf-DNA and the type of organ involvement, but patients with active disease presented significantly higher cf-DNA concentrations than those with inactive disease (p < 0.05). Our data suggest that cf-DNA could provide a useful biomarker for the assessment of disease activity in SSc patients.

Sickle cell anemia in northern Israel: screening and prevention.

BACKGROUND: Sickle cell anemia is a hemolytic anemia caused by a single mutation in position 6 of the beta globin molecule. About 80 patients with SCA in northern Israel are currently receiving treatment. OBJECTIVES: To assess a screening program in northern Israel aimed at detecting couples at risk for having offspring with SCA. METHODS: Since 1987, screening for beta thalassemia in pregnant women in northern Israel has been conducted, and from 1999 all the samples were also tested for hemoglobin S, Hgb C, Hgb D, Hgb O Arab and others. RESULTS: During the 20 year period 1987-2006 a total of 69,340 women were screened; 114 couples who carried Hgb S were detected and 187 prenatal diagnoses were performed in couples at risk for having an offspring with Hgb S. The mean gestational age was 13 +/- 4 weeks. Fifty-four of those diagnoses revealed affected fetuses and in 4 cases the couple declined to perform therapeutic abortion. CONCLUSIONS: The economic burden to the health services for treating SCA patients is about U.S.$ 7000 per year, and the institution of prevention programs has proven cost-effective in populations with a high frequency of carriers. Since our program is aimed to also detect beta thalassemia, a disease that is more frequent in this area (> 2.5%), the added cost for the prevention of SCA is less significant despite the low incidence of the S gene in our population, namely < 1%.

Genotyping of mutation in the beta-globin gene using DNA microarrays.

Genotyping using DNA microarrays is a cost-efficient method compared with real-time PCR and DNA sequencing. Here, a DNA microarray-based method using allele-specific oligo hybridization is demonstrated. This method relies on immobilization of probes that are specific for wild-type sequences or the mutated sequences, respectively. The method makes use of agarose film-coated glass slides, unmodified DNA probes, target preparation using T7 in vitro transcription, labeling of target using biotin labels, and detection using alkaline phosphatase precipitation reaction. Visualization is performed using a desktop computer scanner. Because the biotin/strepavidin chemistry is utilized, the method described here is compatible with many different detection methods. The demonstrated colorimetric detection of mutations has an expected error frequency of about 5 x 10(-7) per mutation or better. Given this low error frequency, an array diagnosing 100 different mutations would misclassify about 1 patient in 100,000.

Highly specific and sensitive electrochemical genotyping via gap ligation reaction and surface hybridization detection.

This paper developed a novel electrochemical genotyping strategy based on gap ligation reaction with surface hybridization detection. This strategy utilized homogeneous enzymatic reactions to generate molecular beacon-structured allele-specific products that could be cooperatively annealed to capture probes stably immobilized on the surface via disulfide anchors, thus allowing ultrasensitive surface hybridization detection of the allele-specific products through redox tags in close proximity to the electrode. Such a unique biphasic architecture provided a universal methodology for incorporating enzymatic discrimination reactions in electrochemical genotyping with desirable reproducibility, high efficiency and no interferences from interficial steric hindrance. The developed technique was demonstrated to show intrinsic high sensitivity for direct genomic analysis, and excellent specificity with discriminativity of single nucleotide variations.

Evaluation of beta globin mRNA as an early marker of haemoglobin response to epoetin treatment.

Approximately 60% of anaemic cancer patients respond to epoetin treatment. An early marker of response would be valuable in order to avoid ineffective treatment. We have previously shown that beta globin mRNA increases rapidly after epoetin beta treatment of healthy controls. In the present study we have evaluated whether a change of this marker during the first 2 weeks of epoetin treatment could predict later Hb response in anaemic cancer patients. Twenty cancer patients with Hb <11 g/dl received epoetin beta (NeoRecormon) 10,000 IU three times weekly during 6 weeks. Hb, reticulocytes and beta-globin mRNA were followed. The latter was measured quantitatively using PCR via the 5' nuclease assay. Eleven patients responded with a Hb increase of >1 g/dl, nine were nonresponders. All responders increased in beta-globin mRNA within 2 weeks, mean 7.7 x base-line. With a cut-off of an increase of 3 x base-line value, we obtained a specificity of 45% and a sensitivity of 91% for the prediction of a later increase of Hb >1 g/dl. With a cut-off of 4x base-line, the specificity increased to 66%, but the sensitivity decreased to 82%. Beta globin mRNA increases before Hb in all responding patients. However, some non-responding patients also show an increase, and there is a trade-off between specificity and sensitivity as the cut-off level is set at different levels. Compared to reticulocyte count, beta-globin mRNA is more reliable in the individual patient, but the clinical usefulness of the assay needs to be evaluated in further studies.

Human papilloma virus in skin, mouth and uterine cervix in female renal transplant recipients with or without a history of cutaneous squamous cell carcinoma.

Some human papillomaviruses are thought to be associated with skin cancer. In this pilot study, 21 female renal transplant carriers, 10 with a history of skin squamous cell carcinoma and 11 without, together with 9 age-matched healthy women were investigated for human papillomavirus DNA in sun-exposed (forehead) and less sun-exposed (buttock) skin, mouth and uterine cervix. Paraffin-embedded tumours from 9 of the patients with a history of squamous cell carcinoma were analysed. Healthy skin from both the healthy and the immunosuppressed individuals harboured a wide variety of papillomaviruses. In the healthy individuals, samples from less sun-exposed skin showed a lower prevalence of human papillomavirus DNA than corresponding samples from the immunosuppressed patients (4/9 and 7/9, respectively). Among the immunosuppressed patients, human papillomavirus DNA was found as frequently in buttock samples (17/21) as in forehead samples (17/20). There was no increased prevalence of human papillomavirus in the cervix or mouth samples from the immunosuppressed patients.

Single-stranded DNA-binding proteins regulate the abundance of LIM domain and LIM domain-binding proteins.

The LIM domain-binding protein Ldb1 is an essential cofactor of LIM-homeodomain (LIM-HD) and LIM-only (LMO) proteins in development. The stoichiometry of Ldb1, LIM-HD, and LMO proteins is tightly controlled in the cell and is likely a critical determinant of their biological actions. Single-stranded DNA-binding proteins (SSBPs) were recently shown to interact with Ldb1 and are also important in developmental programs. We establish here that two mammalian SSBPs, SSBP2 and SSBP3, contribute to an erythroid DNA-binding complex that contains the transcription factors Tal1 and GATA-1, the LIM domain protein Lmo2, and Ldb1 and binds a bipartite E-box-GATA DNA sequence motif. In addition, SSBP2 was found to augment transcription of the Protein 4.2 (P4.2) gene, a direct target of the E-box-GATA-binding complex, in an Ldb1-dependent manner and to increase endogenous Ldb1 and Lmo2 protein levels, E-box-GATA DNA-binding activity, and P4.2 and beta-globin expression in erythroid progenitors. Finally, SSBP2 was demonstrated to inhibit Ldb1 and Lmo2 interaction with the E3 ubiquitin ligase RLIM, prevent RLIM-mediated Ldb1 ubiquitination, and protect Ldb1 and Lmo2 from proteasomal degradation. These results define a novel biochemical function for SSBPs in regulating the abundance of LIM domain and LIM domain-binding proteins.