An improved Diagnostic PCR Assay for identification of Cryptic Heterozygosity for CGG Triplet Repeat Alleles in the Fragile X Gene (FMR1)

<p>Abstract</p> <p>Background</p> <p>Fragile X syndrome (OMIM #300624) is the most common, recognised, heritable cause of mental retardation. Widespread testing is warranted by the relatively high frequency of the disorder, the benefits of early detection and the identification of related carriers whose offspring are at a 1 in 2 risk of inheriting the expanded pathogenic mutation. However, cost-effective screening of mentally retarded individuals has been impeded by the lack of a single, simple laboratory test. Currently, Fragile X syndrome can be excluded in males and a majority of females using a simple high-throughput PCR test. Due to the limited sensitivity of the PCR test, we find in our diagnostic service that approximately 40% of females appear homozygous and a labour intensive and expensive Southern blot test is required to distinguish these from females carrying one normal allele and an expanded allele.</p> <p>Results</p> <p>We describe an improved PCR test which displays a high level of precision allowing alleles differing by a single triplet to be resolved. Using the new assay, we detected 46/83 (53%) cryptic heterozygotes previously labelled as homozygotes. The assay also extended the range of repeats amplifiable, up to 170 CGG repeats in males and 130 CGG repeats in females. Combined with the high precision, the assay also improves discrimination of normal (CGG repeats < 45) from grey zone (45 < CGG repeats < 54) alleles and grey zone alleles from small premutations (55 < CGG repeats < 100).</p> <p>Conclusion</p> <p>Use of this PCR test provides significantly improved precision and amplification of longer alleles. The number of follow-up Southern blot tests required is reduced (up to 50%) with consequent improvement in turnaround time and cost.</p

Detection of skewed X-chromosome inactivation in Fragile X syndrome and X chromosome aneuploidy using quantitative melt analysis.

Methylation of the fragile X mental retardation 1 (FMR1) exon 1/intron 1 boundary positioned fragile X related epigenetic element 2 (FREE2), reveals skewed X-chromosome inactivation (XCI) in fragile X syndrome full mutation (FM: CGG &gt; 200) females. XCI skewing has been also linked to abnormal X-linked gene expression with the broader clinical impact for sex chromosome aneuploidies (SCAs). In this study, 10 FREE2 CpG sites were targeted using methylation specific quantitative melt analysis (MS-QMA), including 3 sites that could not be analysed with previously used EpiTYPER system. The method was applied for detection of skewed XCI in FM females and in different types of SCA. We tested venous blood and saliva DNA collected from 107 controls (CGG &lt; 40), and 148 FM and 90 SCA individuals. MS-QMA identified: (i) most SCAs if combined with a Y chromosome test; (ii) locus-specific XCI skewing towards the hypomethylated state in FM females; and (iii) skewed XCI towards the hypermethylated state in SCA with 3 or more X chromosomes, and in 5% of the 47,XXY individuals. MS-QMA output also showed significant correlation with the EpiTYPER reference method in FM males and females (P &lt; 0.0001) and SCAs (P &lt; 0.05). In conclusion, we demonstrate use of MS-QMA to quantify skewed XCI in two applications with diagnostic utility

Open Sequence Initiative: a part submission standard to complement modern DNA assembly techniques

The discipline of synthetic biology emphasizes the application of engineering principles such as standardization, abstraction, modularity, and rational design to complex biological systems. The archetypical example of such standardization is BioBrick RFC[10], introduced in 2003 by Tom Knight at MIT. BioBricks are stored on a standard plasmid, pSB1C3, which contains prefix and suffix sequences flanking the DNA sequence specifying a biological part. The prefix and suffix sequences contain two pairs of 6 base-pair (bp) restriction enzyme sites (EcoRI+XbaI and SpeI+PstI), which can be used for both part assembly and quality control. BioBricks are intended to be well- characterized biological parts, such as genes or promoters, that function in a predictable fashion and can be readily combined to make complex systems. The rules of the RFC[10] BioBrick assembly method require that none of the restriction sites used in the prefix and suffix be present in the parts themselves. This requirement can be an onerous imposition for iGEM teams developing large, novel parts, such as genes or entire operons that are obtained by amplifying DNA sequences from environmental samples or microorganisms. While iGEM teams may use methods such as site-directed mutagenesis to remove illegal restriction sites from a part's sequence, it is certainly possible that this mutation will alter the functionality of the part – a very undesirable outcome. In addition, the mutagenesis of illegal restriction sites is an unnecessary burden on teams, given the limited time and resources available to teams during each year’s iGEM competition. Efforts spent mutagenizing sites would be better spent characterizing and improving parts. This RFC proposes an alternative submission standard to eliminate these problems

Kamo ide hrvatsko maloljetničko kazneno zakonodavstvo? - 1. dio

U radu se obrađuje kazneni postupak prema maloljetnicima pred sudom za mladež u svjetlu predstojećih izmjena Zakona o sudovima za mladež. Autor, najprije, ukazuje na bitne značajke hrvatskog kaznenog postupka prema maloljetnicima, te analizira kako su u postojeće maloljetničko zakonodavstvo ugrađena temeljna procesna prava maloljetnika iz odgovarajućih međunarodnih dokumenata (tzv. Pekinška pravila, Konvencija o pravima djeteta). Usporedni pregled organizacije i nadležnosti sudova za maloljetnike u 12 europskih zemalja pokazuje da u Europi dominiraju dva modela maloljetničkog pravosuđa: sudski i zaštitni model. S obzirom na dominantnu ulogu suca za mladež tijekom čitavog kaznenog postupka prema maloljetnicima, hrvatski sud za mladež može se ubrojiti u welfare model po kojemu sudac za mladež vrši ne samo istražnu funkciju u pripremnom postupku, nego i sudsku funkciju u postupku pred vijećem za mladež. Nakon prikaza i analize odluke Europskog suda za ljudska prava u predmetu Nortier v. The Netherlands koja se može primijeniti i na naše maloljetničko pravosuđe, te prikaza rezultata ankete provedene među sucima za mladež u Hrvatskoj, autor dolazi do zaključka da objedinjenje istražne i sudske funkcije kod suca za mladež ne predstavlja kršenje prava maloljetnika na nepristran sud iz čl. 6. st. 1. Konvencije za zaštitu ljudskih prava i temeljnih sloboda. Na kraju rada autor ukazuje na eventualne posljedice do kojih bi moglo doći uslijed dosljednog odvajanja istražne i sudske funkcije u postupku pred sudom za mladež

Measurement of χ c1 and χ c2 production with s√ = 7 TeV pp collisions at ATLAS

The prompt and non-prompt production cross-sections for the χ c1 and χ c2 charmonium states are measured in pp collisions at s√ = 7 TeV with the ATLAS detector at the LHC using 4.5 fb−1 of integrated luminosity. The χ c states are reconstructed through the radiative decay χ c → J/ψγ (with J/ψ → μ + μ −) where photons are reconstructed from γ → e + e − conversions. The production rate of the χ c2 state relative to the χ c1 state is measured for prompt and non-prompt χ c as a function of J/ψ transverse momentum. The prompt χ c cross-sections are combined with existing measurements of prompt J/ψ production to derive the fraction of prompt J/ψ produced in feed-down from χ c decays. The fractions of χ c1 and χ c2 produced in b-hadron decays are also measured