Diagnosis of Fanconi Anemia: Mutation Analysis by Multiplex Ligation-Dependent Probe Amplification and PCR-Based Sanger Sequencing

Fanconi anemia (FA) is a rare inherited disease characterized by developmental defects, short stature, bone marrow failure, and a high risk of malignancies. FA is heterogeneous: 15 genetic subtypes have been distinguished so far. A clinical diagnosis of FA needs to be confirmed by testing cells for sensitivity to cross-linking agents in a chromosomal breakage test. As a second step, DNA testing can be employed to elucidate the genetic subtype of the patient and to identify the familial mutations. This knowledge allows preimplantation genetic diagnosis (PGD) and enables prenatal DNA testing in future pregnancies. Although simultaneous testing of all FA genes by next generation sequencing will be possible in the near future, this technique will not be available immediately for all laboratories. In addition, in populations with strong founder mutations, a limited test using Sanger sequencing and MLPA will be a cost-effective alternative. We describe a strategy and optimized conditions for the screening of FANCA, FANCB, FANCC, FANCE, FANCF, and FANCG and present the results obtained in a cohort of 54 patients referred to our diagnostic service since 2008. In addition, the follow up with respect to genetic counseling and carrier screening in the families is discussed

Reducing Discomfort While Measuring Crown-Heel Length in Neonates

To assess the degree of discomfort caused by length measurement in neonates, performed with one or both lower limbs extended, on the first and second day after birth, with either one or both lower limbs extended. METHODS: Healthy full-term neonates were systematically sampled during the months of February and March 2004. Crown-heel length was measured, using a 1-mm precision neonatometer, at approximately 8 h and 32 h after birth, with one and both lower limbs extended. The Neonatal Facial Coding System was used to assess discomfort during measurements. Data were analysed by parametric and non-parametric tests as appropriate. RESULTS: Whatever the measurement technique, discomfort scores are significantly higher during the length measurement than at baseline. Whenever length measurements are performed, discomfort scores are significantly higher when extending both lower limbs rather than one lower limb (p < 0.006). The measured length is greater with one lower limb extended; however, the difference decreases over time, being 0.19 cm (95% CI 0.1-0.3; p < 0.001) at approximately 32 h of age. No significant differences in length were found between measurements at approximately 8 or 32 h, regardless of the technique used. The best correlation between length measurements with one or both lower limbs extended was observed at approximately 32 h after birth (r = 0.98). CONCLUSION: Measuring crown-heel length is a distressful procedure for the neonate. Measurements with one lower limb extended result in less discomfort than when both lower limbs are extended, without decreasing the accuracy

A molecular genetic toolbox for Yarrowia lipolytica

Background: Yarrowia lipolytica is an ascomycete yeast used in biotechnological research for its abilities to secrete high concentrations of proteins and accumulate lipids. Genetic tools have been made in a variety of backgrounds with varying similarity to a comprehensively sequenced strain. Results: We have developed a set of genetic and molecular tools in order to expand capabilities of Y. lipolytica for both biological research and industrial bioengineering applications. In this work, we generated a set of isogenic auxotrophic strains with decreased non-homologous end joining for targeted DNA incorporation. Genome sequencing, assembly, and annotation of this genetic background uncovers previously unidentified genes in Y. lipolytica. To complement these strains, we constructed plasmids with Y. lipolytica-optimized superfolder GFP for targeted overexpression and fluorescent tagging. We used these tools to build the "Yarrowia lipolytica Cell Atlas," a collection of strains with endogenous fluorescently tagged organelles in the same genetic background, in order to define organelle morphology in live cells. Conclusions: These molecular and isogenetic tools are useful for live assessment of organelle-specific protein expression, and for localization of lipid biosynthetic enzymes or other proteins in Y. lipolytica. This work provides the Yarrowia community with tools for cell biology and metabolism research in Y. lipolytica for further development of biofuels and natural products

The T7-Primer Is a Source of Experimental Bias and Introduces Variability between Microarray Platforms

Eberwine(-like) amplification of mRNA adds distinct 6–10 bp nucleotide stretches to the 5′ end of amplified RNA transcripts. Analysis of over six thousand microarrays reveals that probes containing motifs complementary to these stretches are associated with aberrantly high signals up to a hundred fold the signal observed in unaffected probes. This is not observed when total RNA is used as target source. Different T7 primer sequences are used in different laboratories and platforms and consequently different T7 primer bias is observed in different datasets. This will hamper efforts to compare data sets across platforms

Leucine Biosynthesis Is Involved in Regulating High Lipid Accumulation in Yarrowia lipolytica

The yeast Yarrowia lipolytica is a potent accumulator of lipids, and lipogenesis in this organism can be influenced by a variety of factors, such as genetics and environmental conditions. Using a multifactorial study, we elucidated the effects of both genetic and environmental factors on regulation of lipogenesis in Y. lipolytica and identified how two opposite regulatory states both result in lipid accumulation. This study involved comparison of a strain overexpressing diacylglycerol acyltransferase (DGA1) with a control strain grown under either nitrogen or carbon limitation conditions. A strong correlation was observed between the responses on the transcript and protein levels. Combination of DGA1 overexpression with nitrogen limitation resulted in a high level of lipid accumulation accompanied by downregulation of several amino acid biosynthetic pathways, including that of leucine in particular, and these changes were further correlated with a decrease in metabolic fluxes. This downregulation was supported by the measured decrease in the level of 2-isopropylmalate, an intermediate of leucine biosynthesis. Combining the multi-omics data with putative transcription factor binding motifs uncovered a contradictory role for TORC1 in controlling lipid accumulation, likely mediated through 2-isopropylmalate and a Leu3-like transcription factor

An optimized protocol for microarray validation by quantitative PCR using amplified amino allyl labeled RNA

<p>Abstract</p> <p>Background</p> <p>Validation of microarrays data by quantitative real-time PCR (qPCR) is often limited by the low amount of available RNA. This raised the possibility to perform validation experiments on the amplified amino allyl labeled RNA (AA-aRNA) leftover from microarrays. To test this possibility, we used an ongoing study of our laboratory aiming at identifying new biomarkers of graft rejection by the transcriptomic analysis of blood cells from brain-dead organ donors.</p> <p>Results</p> <p>qPCR for ACTB performed on AA-aRNA from 15 donors provided Cq values 8 cycles higher than when original RNA was used (P < 0.001), suggesting a strong inhibition of qPCR performed on AA-aRNA. When expression levels of 5 other genes were measured in AA-aRNA generated from a universal reference RNA, qPCR sensitivity and efficiency were decreased. This prevented the quantification of one low-abundant gene, which was readily quantified in un-amplified and un-labeled RNA. To overcome this limitation, we modified the reverse transcription (RT) protocol that generates cDNA from AA-aRNA as follows: addition of a denaturation step and 2-min incubation at room temperature to improve random primers annealing, a transcription initiation step to improve RT, and a final treatment with RNase H to degrade remaining RNA. Tested on universal reference AA-aRNA, these modifications provided a gain of 3.4 Cq (average from 5 genes, P < 0.001) and an increase of qPCR efficiency (from -1.96 to -2.88; P = 0.02). They also allowed for the detection of a low-abundant gene that was previously undetectable. Tested on AA-aRNA from 15 brain-dead organ donors, RT optimization provided a gain of 2.7 cycles (average from 7 genes, P = 0.004). Finally, qPCR results significantly correlated with microarrays.</p> <p>Conclusion</p> <p>We present here an optimized RT protocol for validation of microarrays by qPCR from AA-aRNA. This is particularly valuable in experiments where limited amount of RNA is available.</p

Use of Non-Amplified RNA Samples for Microarray Analysis of Gene Expression

Demand for high quality gene expression data has driven the development of revolutionary microarray technologies. The quality of the data is affected by the performance of the microarray platform as well as how the nucleic acid targets are prepared. The most common method for target nucleic acid preparation includes in vitro transcription amplification of the sample RNA. Although this method requires a small amount of starting material and is reported to have high reproducibility, there are also technical disadvantages such as amplification bias and the long, laborious protocol. Using RNA derived from human brain, breast and colon, we demonstrate that a non-amplification method, which was previously shown to be inferior, could be transformed to a highly quantitative method with a dynamic range of five orders of magnitude. Furthermore, the correlation coefficient calculated by comparing microarray assays using non-amplified samples with qRT-PCR assays was approximately 0.9, a value much higher than when samples were prepared using amplification methods. Our results were also compared with data from various microarray platforms studied in the MicroArray Quality Control (MAQC) project. In combination with micro-columnar 3D-Gene™ microarray, this non-amplification method is applicable to a variety of genetic analyses, including biomarker screening and diagnostic tests for cancer

BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons

<p>Abstract</p> <p>Background</p> <p>Visualisation of genome comparisons is invaluable for helping to determine genotypic differences between closely related prokaryotes. New visualisation and abstraction methods are required in order to improve the validation, interpretation and communication of genome sequence information; especially with the increasing amount of data arising from next-generation sequencing projects. Visualising a prokaryote genome as a circular image has become a powerful means of displaying informative comparisons of one genome to a number of others. Several programs, imaging libraries and internet resources already exist for this purpose, however, most are either limited in the number of comparisons they can show, are unable to adequately utilise draft genome sequence data, or require a knowledge of command-line scripting for implementation. Currently, there is no freely available desktop application that enables users to rapidly visualise comparisons between hundreds of draft or complete genomes in a single image.</p> <p>Results</p> <p>BLAST Ring Image Generator (BRIG) can generate images that show multiple prokaryote genome comparisons, without an arbitrary limit on the number of genomes compared. The output image shows similarity between a central reference sequence and other sequences as a set of concentric rings, where BLAST matches are coloured on a sliding scale indicating a defined percentage identity. Images can also include draft genome assembly information to show read coverage, assembly breakpoints and collapsed repeats. In addition, BRIG supports the mapping of unassembled sequencing reads against one or more central reference sequences. Many types of custom data and annotations can be shown using BRIG, making it a versatile approach for visualising a range of genomic comparison data. BRIG is readily accessible to any user, as it assumes no specialist computational knowledge and will perform all required file parsing and BLAST comparisons automatically.</p> <p>Conclusions</p> <p>There is a clear need for a user-friendly program that can produce genome comparisons for a large number of prokaryote genomes with an emphasis on rapidly utilising unfinished or unassembled genome data. Here we present BRIG, a cross-platform application that enables the interactive generation of comparative genomic images via a simple graphical-user interface. BRIG is freely available for all operating systems at <url>http://sourceforge.net/projects/brig/</url>.</p