DNA microextraction from dried blood spots on filter paper blotters: potential applications to newborn screening

Summary Microextraction of DNA from dried blood specimens would ease specimen transport to centralized laboratory facilities for recombinant DNA diagnosis in the same manner as use of dried blood spots allowed the broad application of screening tests to newborn populations. A method is described which reproducibly yields 0.5g DNA from the dried equivalent of 50l whole blood. Though DNA yields decreased with storage of dried specimens at room temperature, good-quality DNA was still obtained. Sufficient DNA was routinely obtained for Southern blot analysis using repetitive and unique sequences. This microextraction procedure will allow immediate application of molecular genetic technology to direct newborn screening follow-up of disorders amenable to DNA diagnosis, such as sickle cell anemia, and may eventually permit primary DNA screening for specific mutations.     

Molecular genetic diagnosis of sickle cell disease using dried blood specimens on blotters used for newborn screening

Summary The protein-based technologies used to screen newborns for sickle cell disease require confirmation with a liquid blood specimen. We have developed a strategy for rapid and specific genotypic diagnosis using DNA extracted from a dried blood spot on the filter paper blotter used to screen newborns. DNA could be microextracted from a specimen as small as a 1/8 inch diameter punched disc representing the dried equivalent of approximately 3 μl of whole blood. We utilized the DNA from a 1/4 inch diameter specimen (12 μl equivalent) for polymerase chain reaction amplification of the β-globin region spanning the sickle cell mutation with detection by allele-specific oligonucleotide probes. Molecular confirmation of genotype from the original blotter would reduce the personnel costs associated with obtaining follow-up liquid blood specimens and would provide information to the family in a more timely and less equivocal manner.     

Hi-Plex targeted sequencing is effective using DNA derived from archival dried blood spots

Many genetic epidemiology resources have collected dried blood spots (predominantly as Guthrie Cards) as an economical and efficient means of archiving sources of DNA, conferring great value to genetic screening methods that are compatible with this medium. We applied Hi-Plex to screen the breast cancer predisposition gene PALB2 in 93 Guthrie Card-derived DNA specimens previously characterized for PALB2 genetic variants via DNA derived from lymphoblastoid cell lines, whole blood, and buffy coat. Of the 93 archival Guthrie Card-derived DNAs, 92 (99%) were processed successfully and sequenced using approximately half of a MiSeq run. From these 92 DNAs, all 59 known variants were detected and no false-positive variant calls were yielded. Fully 98.13% of amplicons (5417/5520) were represented within 15-fold of the median coverage (2786 reads), and 99.98% of amplicons (5519/5520) were represented at a depth of 10 read-pairs or greater. With Hi-Plex, we show for the first time that a High-Plex amplicon-based massively parallel sequencing (MPS) system can be applied effectively to DNA prepared from dried blood spot archival specimens and, as such, can dramatically increase the scopes of both method and resource.     

DNA elution and amplification by polymerase chain reaction from dried blood spots.

A quick, sensitive and easily automatizable method for PCR amplification of genomic DNA eluted from dried blood spots is described. DNA is eluted from a 3-mm spot routinely used for neonatal screening of inherited diseases either by boiling or by sonication. A preliminary and brief spot-autoclaving step is mandatory to ensure optimal and reproducible PCR amplifications. Only 1% of the eluted DNA is required for PCR analysis allowing the execution of multiple genetic tests on the same blood spot. The method has been successfully applied to the detection of a known phenylketonuria-causing mutation and will facilitate the analysis of the genetic repository provided by Guthrie's cards stored in neonatal screening laboratories.     

The origin of sickle cell alleles in Israel

Summary Molecular genetic studies were undertaken to determine the source of chromosomes carrying the sickle cell allele in Israeli patients. Analysis of restriction fragment length polymorphism (RFLP) patterns (haplotypes) along the -globin gene cluster was performed on 31 sickle chromosomes obtained from 10 unrelated families living in Israel. One is a Caucasian Jewish family, recently found to be carrying the sickle allele, and the other 9 are Arab families of various communities. The Jewish family, previously noted not to carry African red blood cell markers, was discovered to have the most common African haplotype of the -globin gene cluster, Benin. Similarly, 8 of the Arab families were also found to carry the Benin haplotype, whereas the ninth has the CAR (Central African Republic or Bantu) haplotype. The results suggest that sickle alleles in Israel originated in Africa, probably in two different regions, and migrated north into Arab and Jewish populations.     

Improved screening test for abnormal hemoglobins from dried blood samples

Summary A method is described wherin blood samples taken from adults or newborns and dried on filter paper can be used for hemoglobin analysis within 2 years after sampling. The samples are eluted in 8 M urea in the presence of 5% 2-mercaptoethanol and 2% of the neutral detergent Nonidet P-40. Then the individual , , , and chains are separated by means of electrofocusing in 8 M urea-PAA gels. Up to 96 samples can be applied to a gel using multiple syringes. Several hundred samples can be analyzed daily by one person. This method may be especially useful for preventive programs against sickle cell anemia as well as for human mutation monitoring systems.Abbreviations used in this paper PAA polyacrylamide - PAG polyacrylamide gel - TCA trichloroacetic acid - PKU phenylketonuria     

Herbarium tale: the utility of dry specimens for DNA barcoding Juncaceae

Phylogenetic and barcoding studies usually use fresh plant tissues as sources of DNA and have successfully amplified DNA for various loci. The use of dried samples, however, is often necessary due to the frequent inaccessibility of fresh rare plants or their parts for genetic analyses or barcoding. The difficulty in obtaining amplifiable DNA is a major restriction of the use of herbarium specimens for DNA analyses. Recent study has highlighted the crucial issues for comparing herbarium and fresh plants for barcoding. We analysed the performance of samples of the family Juncaceae from various herbarium specimens of different ages with fresh plant material in PCRs and the sequences of seven loci (rbcL, rpoC1, trnL-F intergenic spacer, trnL intron, and psbA-trnH from chloroplast DNA; atp1 from mitochondrial DNA; and ITS1-5.8S-ITS2 from nuclear DNA) using a combination of 28 primers. The herbarium specimens amplified well and may thus be successfully applied for both phylogenetic analyses and barcoding for the Juncaceae family. Amplifying DNA was more difficult from dried herbarium specimens than fresh samples but could be successful in most cases when appropriate internal primers were designed or methods were optimised. Using the set of universal primers recommended by the Consortium for the Barcode of Life and designing specific primers for a particular group of interest were both useful. Specimen age and amplicon length had limited detrimental effects on amplification success for most of the Juncaceae loci tested.     

From a dry bone to a genetic portrait: a case study of sickle cell anemia

The potential and reliability of DNA analysis for the identification of human remains are demonstrated by the study of a recent bone sample, which represented a documented case of sickle cell anemia. beta-globin gene sequences obtained from the specimen revealed homozygosity for the sickle cell mutation, proving the authenticity of the retrieved residual DNA. Further investigation of mitochondrial and Y chromosome DNA polymorphic markers indicated that this sample came from a male of maternal West African (possibly Yoruban) and paternal Bantu lineages. The medical record, which became available after the DNA analyses had been completed, revealed that it belonged to a Jamaican black male. These findings are consistent with this individual being a descendent of Africans brought to Jamaica during the trans-Atlantic slave trade. This study exemplifies how a "reverse population genetics" approach can be applied to reconstruct a genetic profile from a bone specimen of an unknown individual.     

Detection of IL28B SNP DNA from buccal epithelial cells, small amounts of serum, and dried blood spots

Background & Aims

Point mutations in the coding region of the interleukin 28 gene (rs12979860) have recently been identified for predicting the outcome of treatment of hepatitis C virus infection. This polymorphism detection was based on whole blood DNA extraction. Alternatively, DNA for genetic diagnosis has been derived from buccal epithelial cells (BEC), dried blood spots (DBS), and genomic DNA from serum. The aim of the study was to investigate the reliability and accuracy of alternative routes of testing for single nucleotide polymorphism allele rs12979860CC.

Methods

Blood, plasma, and sera samples from 200 patients were extracted (400 µL). Buccal smears were tested using an FTA card. To simulate postal delay, we tested the influence of storage at ambient temperature on the different sources of DNA at five time points (baseline, 48 h, 6 days, 9 days, and 12 days)

Results

There was 100% concordance between blood, plasma, sera, and BEC, validating the use of DNA extracted from BEC collected on cytology brushes for genetic testing. Genetic variations in HPTR1 gene were detected using smear technique in blood smear (3620 copies) as well as in buccal smears (5870 copies). These results are similar to those for whole blood diluted at 1/10. A minimum of 0.04 µL, 4 µL, and 40 µL was necessary to obtain exploitable results respectively for whole blood, sera, and plasma. No significant variation between each time point was observed for the different sources of DNA. IL28B SNPs analysis at these different time points showed the same results using the four sources of DNA.

Conclusion

We demonstrated that genomic DNA extraction from buccal cells, small amounts of serum, and dried blood spots is an alternative to DNA extracted from peripheral blood cells and is helpful in retrospective and prospective studies for multiple genetic markers, specifically in hard-to-reach individuals.     

DNA extraction and PCR amplification method suitable for fresh, herbarium-stored, lichenized, and other fungi

This paper presents a DNA extraction method suitable for fresh, herbarium-stored, lichenized and other fungal specimens. The method is fast and reliable, comparatively inexpensive and is suitable for obtaining PCR amplification quality DNA from large numbers of samples in a short time. The method has been tested with over 300 samples ofAscochyta, Phyllosticta, Ramalina, Parmelia andPhysconia. Amplifiable fungal DNA was extracted from pure cultures, leaf lesions, whole thalli and dissected only-fungal sections of lichenized fungi. In addition, the method has proved suitable for use with herbarium specimens of both lichenized and non-lichenized fungi, stored as dried pure cultures or dried infected plant material.     

Normal Muscle Oxygen Consumption and Fatigability in Sickle Cell Patients Despite Reduced Microvascular Oxygenation and Hemorheological Abnormalities

Background/Aim

Although it has been hypothesized that muscle metabolism and fatigability could be impaired in sickle cell patients, no study has addressed this issue.

Methods

We compared muscle metabolism and function (muscle microvascular oxygenation, microvascular blood flow, muscle oxygen consumption and muscle microvascular oxygenation variability, which reflects vasomotion activity, maximal muscle force and local muscle fatigability) and the hemorheological profile at rest between 16 healthy subjects (AA), 20 sickle cell-hemoglobin C disease (SC) patients and 16 sickle cell anemia (SS) patients.

Results

Muscle microvascular oxygenation was reduced in SS patients compared to the SC and AA groups and this reduction was not related to hemorhelogical abnormalities. No difference was observed between the three groups for oxygen consumption and vasomotion activity. Muscle microvascular blood flow was higher in SS patients compared to the AA group, and tended to be higher compared to the SC group. Multivariate analysis revealed that muscle oxygen consumption was independently associated with muscle microvascular blood flow in the two sickle cell groups (SC and SS). Finally, despite reduced muscle force in sickle cell patients, their local muscle fatigability was similar to that of the healthy subjects.

Conclusions

Sickle cell patients have normal resting muscle oxygen consumption and fatigability despite hemorheological alterations and, for SS patients only, reduced muscle microvascular oxygenation and increased microvascular blood flow. Two alternative mechanisms can be proposed for SS patients: 1) the increased muscle microvascular blood flow is a way to compensate for the lower muscle microvascular oxygenation to maintain muscle oxygen consumption to normal values or 2) the reduced microvascular oxygenation coupled with a normal resting muscle oxygen consumption could indicate that there is slight hypoxia within the muscle which is not sufficient to limit mitochondrial respiration but increases muscle microvascular blood flow.     

A simple DNA diagnostic method for human genetic disorders

Summary A fast, reliable, and simple technique for detecting point mutations in unfractionated human DNA has been developed. Oligonucleotide probes complementary to either sickle- or normal -globin DNA are labeled by primer extension and hybridized to DNA applied to nitrocellulose paper in a dot-blot format. A short hybridization time (about 1 h) and low probe concentration (about 1 nM) yield low background and high specificity. Double-blind trials show 100% agreement with restriction fragment length polymorphism (RFLP) analysis of DNA from normal, sickle, and heterozygous subjects.     

Hemoglobin abnormalities

Summary In order to evaluate the polymorphism of hemoglobin in a population of Equatorial Africa, we undertook a prospective study of 146 births at a rural maternity hospital close to Brazzaville (P.R. Congo). This showed among the mothers 31 (22%) carriers of the sickle cell trait (AS), six with mutation, and two with -thalassemia triat. Among the children, 27 (18.5%) had sickle cell trait and one had sickle cell homozygosity. The frequency of the HbF Sardinia trait was 7.5%. This and other studies suggested a dilution gradient from Europe to Africa. Hemoglobin Bart's could be visually detected in 23.3% of the new-born babies. We attempted to distinguish between those infants with a high level of Hb Bart's (Bart's ++ group: 13.7%) and a group with a detectable Hb Bart's level that in our experimental conditions is between 1 and 2% (Bart's + group: 9.6%). Mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) were 88.6 ±5.7 fl and 29.0±2.1 pg in the Bart's ++ group; 94.5±10.9 fl and 30.6±4.1 pg in the Bart's + group; whereas they were 101.0±8.7 fl and 33.9±2.5 pg in the control group. Since iron dificiencies are very rare in new-borns and selecting according to published data on black people as homozygous -thalassemia of the type I (-/-), individuals of the Bart's ++ group whose MCV was below 95 fl and MCV below 30 pg, the gene frequency is estimated to be 34% and that of heterozygotes (-/) 45%. These high frequencies were confirmed in AS mothers: 45% showed a significant decrease of the S fraction.     

A rapid method for detection of Y-chromosomal DNA from dried blood specimens by the polymerase chain reaction

Summary The alphoid satellite family is the only repetitive DNA family showing chromosome specificity. We have developed a simple, rapid, and reliable test for sex diagnosis based on detection of these sequences in undigested genomic DNA using the polymerase chain reaction. In our test, dried blood specimens were the source of DNA. When female DNA was used as a template for the reaction, only the expected 130-bp X-chromosome-specific fragment was detected, while with male DNA both the expected 170-bp Y-chromosome-specific and X-chromosome-specific fragments were detected. The Y-chromosome-specific fragment was further characterized by restriction enzyme analysis. The Y fragment was detectable when DNA obtained from an equivalent of 10 l of spotted blood was used in the reaction, whereas detection of the X fragment was possible with DNA from an equivalent of 5 l of blood. Our test may find various applications in newborn screening and in forensic science.     

Candidate Sequence Variants and Fetal Hemoglobin in Children with Sickle Cell Disease Treated with Hydroxyurea

Background

Fetal hemoglobin level is a heritable complex trait that strongly correlates swith the clinical severity of sickle cell disease. Only few genetic loci have been identified as robustly associated with fetal hemoglobin in patients with sickle cell disease, primarily adults. The sole approved pharmacologic therapy for this disease is hydroxyurea, with effects largely attributable to induction of fetal hemoglobin.

Methodology/Principal Findings

In a multi-site observational analysis of children with sickle cell disease, candidate single nucleotide polymorphisms associated with baseline fetal hemoglobin levels in adult sickle cell disease were examined in children at baseline and induced by hydroxyurea therapy. For baseline levels, single marker analysis demonstrated significant association with BCL11A and the beta and epsilon globin loci (HBB and HBE, respectively), with an additive attributable variance from these loci of 23%. Among a subset of children on hydroxyurea, baseline fetal hemoglobin levels explained 33% of the variance in induced levels. The variant in HBE accounted for an additional 13% of the variance in induced levels, while variants in the HBB and BCL11A loci did not contribute beyond baseline levels.

Conclusions/Significance

These findings clarify the overlap between baseline and hydroxyurea-induced fetal hemoglobin levels in pediatric disease. Studies assessing influences of specific sequence variants in these and other genetic loci in larger populations and in unusual hydroxyurea responders are needed to further understand the maintenance and therapeutic induction of fetal hemoglobin in pediatric sickle cell disease.     

Phase 1 Study of the E-Selectin Inhibitor GMI 1070 in Patients with Sickle Cell Anemia

Background

Sickle cell anemia is an inherited disorder of hemoglobin that leads to a variety of acute and chronic complications. Abnormal cellular adhesion, mediated in part by selectins, has been implicated in the pathophysiology of the vaso-occlusion seen in sickle cell anemia, and selectin inhibition was able to restore blood flow in a mouse model of sickle cell disease.

Methods

We performed a Phase 1 study of the selectin inhibitor GMI 1070 in patients with sickle cell anemia. Fifteen patients who were clinically stable received GMI 1070 in two infusions.

Results

The drug was well tolerated without significant adverse events. There was a modest increase in total peripheral white blood cell count without clinical symptoms. Plasma concentrations were well-described by a two-compartment model with an elimination T_1/2 of 7.7 hours and CLr of 19.6 mL/hour/kg. Computer-assisted intravital microscopy showed transient increases in red blood cell velocity in 3 of the 4 patients studied.

Conclusions

GMI 1070 was safe in stable patients with sickle cell anemia, and there was suggestion of increased blood flow in a subset of patients. At some time points between 4 and 48 hours after treatment with GMI 1070, there were significant decreases in biomarkers of endothelial activation (sE-selectin, sP-selectin, sICAM), leukocyte activation (MAC-1, LFA-1, PM aggregates) and the coagulation cascade (tissue factor, thrombin-antithrombin complexes). Development of GMI 1070 for the treatment of acute vaso-occlusive crisis is ongoing.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00911495","term_id":"NCT00911495"}}NCT00911495     

用干血纸片扩增人雄激素受体基因

雄激素不敏感综合征(AIS)为一类主要与雄激素受体(AR)基因缺陷密切相关的X-连锁隐性遗传病.为进一步阐明AIS的发病机制,建立了用干血纸片直接PCR扩增或将干血纸片中的血细胞洗脱裂解后进行PCR扩增雄激素受体(AR)基因的方法,结合已建立的SSCP分析及DNA直接测序等方法,可对AR基因进行突变分析.干血纸片取样及保存容易,便于邮寄,适用于外地,特别是边远地区患者的取样.该法不仅为AIS患者的AR基因突变分析和家系调查提供简便易行的方法,也适用于PCR基础上的其他各种基因的突变分析.     

The role of fibrocytes in sickle cell lung disease

Background

Interstitial lung disease is a frequent complication in sickle cell disease and is characterized by vascular remodeling and interstitial fibrosis. Bone marrow-derived fibrocytes have been shown to contribute to the pathogenesis of other interstitial lung diseases. The goal of this study was to define the contribution of fibrocytes to the pathogenesis of sickle cell lung disease.

Methodology/Principal Findings

Fibrocytes were quantified and characterized in subjects with sickle cell disease or healthy controls, and in a model of sickle cell disease, the NY1DD mouse. The role of the chemokine ligand CXCL12 in trafficking of fibrocytes and phenotype of lung disease was examined in the animal model. We found elevated concentration of activated fibrocytes in the peripheral blood of subjects with sickle cell disease, which increased further during vaso-occlusive crises. There was a similar elevations in the numbers and activation phenotype of fibrocytes in the bone marrow, blood, and lungs of the NY1DD mouse, both at baseline and under conditions of hypoxia/re-oxygenation. In both subjects with sickle cell disease and the mouse model, fibrocytes expressed a hierarchy of chemokine receptors, with CXCR4 expressed on most fibrocytes, and CCR2 and CCR7 expressed on a smaller subset of cells. Depletion of the CXCR4 ligand, CXCL12, in the mouse model resulted in a marked reduction of fibrocyte trafficking into the lungs, reduced lung collagen content and improved lung compliance and histology.

Conclusions

These data support the notion that activated fibrocytes play a significant role in the pathogenesis of sickle cell lung disease.     

Proteomic analysis of ERK1/2-mediated human sickle red blood cell membrane protein phosphorylation

Background

In sickle cell disease (SCD), the mitogen-activated protein kinase (MAPK) ERK1/2 is constitutively active and can be inducible by agonist-stimulation only in sickle but not in normal human red blood cells (RBCs). ERK1/2 is involved in activation of ICAM-4-mediated sickle RBC adhesion to the endothelium. However, other effects of the ERK1/2 activation in sickle RBCs leading to the complex SCD pathophysiology, such as alteration of RBC hemorheology are unknown.

Results

To further characterize global ERK1/2-induced changes in membrane protein phosphorylation within human RBCs, a label-free quantitative phosphoproteomic analysis was applied to sickle and normal RBC membrane ghosts pre-treated with U0126, a specific inhibitor of MEK1/2, the upstream kinase of ERK1/2, in the presence or absence of recombinant active ERK2. Across eight unique treatment groups, 375 phosphopeptides from 155 phosphoproteins were quantified with an average technical coefficient of variation in peak intensity of 19.8%. Sickle RBC treatment with U0126 decreased thirty-six phosphopeptides from twenty-one phosphoproteins involved in regulation of not only RBC shape, flexibility, cell morphology maintenance and adhesion, but also glucose and glutamate transport, cAMP production, degradation of misfolded proteins and receptor ubiquitination. Glycophorin A was the most affected protein in sickle RBCs by this ERK1/2 pathway, which contained 12 unique phosphorylated peptides, suggesting that in addition to its effect on sickle RBC adhesion, increased glycophorin A phosphorylation via the ERK1/2 pathway may also affect glycophorin A interactions with band 3, which could result in decreases in both anion transport by band 3 and band 3 trafficking. The abundance of twelve of the thirty-six phosphopeptides were subsequently increased in normal RBCs co-incubated with recombinant ERK2 and therefore represent specific MEK1/2 phospho-inhibitory targets mediated via ERK2.

Conclusions

These findings expand upon the current model for the involvement of ERK1/2 signaling in RBCs. These findings also identify additional protein targets of this pathway other than the RBC adhesion molecule ICAM-4 and enhance the understanding of the mechanism of small molecule inhibitors of MEK/1/2/ERK1/2, which could be effective in ameliorating RBC hemorheology and adhesion, the hallmarks of SCD.     

Genetic Defects as Tumor Markers

Carcinogenesis is long-term multistep accumulation of defects of genes responsible for cell division, DNA repair, and apoptosis. The functions of these genes are known both for norm and for pathologies caused by their damage and resulting in asocial cell behavior. Owing to the recent progress in studying the mechanisms of carcinogenesis, some genetic defects may be considered from the applied point of view (as tumor markers rather than as pathogenetic factors) and employed in diagnostics. Thus detection of mutant alleles in biological fluids (e.g., beyond the tumor) suggests higher risk of carcinogenesis. Genetic defects are a new class of tumor markers and have a substantial diagnostic potential. In contrast to known protein markers (-fetoprotein, etc.) used in clinical practice, DNA markers are oncospecific (as these are in direct cause-and-effect relationships with carcinogenesis) and universal (as there is not a single tumor cell without a genetic defect). Analysis of DNA markers may be employed not only in diagnostics or tumor growth monitoring (assessment of treatment efficiency, early detection of recurrence or metastasis), but also (prospectively) in screening (tumor detection at the presymptomatic stage, identification of high-risk groups). Theoretical grounds, prospects, problems, and methods of this new field are considered.