Rapid mapping of chromosomal breakpoints: from blood to BAC in 20 days

Structural chromosome aberrations and associated segmental or chromosomal aneusomies are major causes of reproductive failure in humans. Despite the fact that carriers of reciprocal balanced translocation often have no other clinical symptoms or disease, impaired chromosome homologue pairing in meiosis and karyokinesis errors lead to over-representation of translocations carriers in the infertile population and in recurrent pregnancy loss patients. At present, clinicians have no means to select healthy germ cells or balanced zygotes in vivo, but in vitro fertilization (IVF) followed by preimplantation genetic diagnosis (PGD) offers translocation carriers a chance to select balanced or normal embryos for transfer. Although a combination of telomeric and centromeric probes can differentiate embryos that are unbalanced from normal or unbalanced ones, a seemingly random position of breakpoints in these IVF-patients poses a serious obstacle to differentiating between normal and balanced embryos, which for most translocation couples, is desirable. Using a carrier with reciprocal translocation t(4;13) as an example, we describe our state-of-the-art approach to the preparation of patient-specific DNA probes that span or 'extent' the breakpoints. With the techniques and resources described here, most breakpoints can be accurately mapped in a matter of days using carrier lymphocytes, and a few extra days are allowed for PGD-probe optimization. The optimized probes will then be suitable for interphase cell analysis, a prerequisite for PGD since blastomeres are biopsied from normally growing day 3 – embryos regardless of their position in the mitotic cell cycle. Furthermore, routine application of these rapid methods should make PGD even more affordable for translocation carriers enrolled in IVF programs

Extensible Languages for Flexible and Principled Domain Abstraction

Die meisten Programmiersprachen werden als Universalsprachen entworfen. Unabhängig von der zu entwickelnden Anwendung, stellen sie die gleichen Sprachfeatures und Sprachkonstrukte zur Verfügung. Solch universelle Sprachfeatures ignorieren jedoch die spezifischen Anforderungen, die viele Softwareprojekte mit sich bringen. Als Gegenkraft zu Universalsprachen fördern domänenspezifische Programmiersprachen, modellgetriebene Softwareentwicklung und sprachorientierte Programmierung die Verwendung von Domänenabstraktion, welche den Einsatz von domänenspezifischen Sprachfeatures und Sprachkonstrukten ermöglicht. Insbesondere erlaubt Domänenabstraktion Programmieren auf dem selben Abstraktionsniveau zu programmieren wie zu denken und vermeidet dadurch die Notwendigkeit Domänenkonzepte mit universalsprachlichen Features zu kodieren. Leider ermöglichen aktuelle Ansätze zur Domänenabstraktion nicht die Entfaltung ihres ganzen Potentials. Einerseits mangelt es den Ansätzen für interne domänenspezifische Sprachen an Flexibilität bezüglich der Syntax, statischer Analysen, und Werkzeugunterstützung, was das tatsächlich erreichte Abstraktionsniveau beschränkt. Andererseits mangelt es den Ansätzen für externe domänenspezifische Sprachen an wichtigen Prinzipien, wie beispielsweise modularem Schließen oder Komposition von Domänenabstraktionen, was die Anwendbarkeit dieser Ansätze in der Entwicklung größerer Softwaresysteme einschränkt. Wir verfolgen in der vorliegenden Doktorarbeit einen neuartigen Ansatz, welcher die Vorteile von internen und externen domänenspezifischen Sprachen vereint um flexible und prinzipientreue Domänenabstraktion zu unterstützen. Wir schlagen bibliotheksbasierte erweiterbare Programmiersprachen als Grundlage für Domänenabstraktion vor. In einer erweiterbaren Sprache kann Domänenabstraktion durch die Erweiterung der Sprache mit domänenspezifischer Syntax, statischer Analyse, und Werkzeugunterstützung erreicht werden . Dies ermöglicht Domänenabstraktionen die selbe Flexibilität wie externe domänenspezifische Sprachen. Um die Einhaltung üblicher Prinzipien zu gewährleisten, organisieren wir Spracherweiterungen als Bibliotheken und verwenden einfache Import-Anweisungen zur Aktivierung von Erweiterungen. Dies erlaubt modulares Schließen (durch die Inspektion der Import-Anweisungen), unterstützt die Komposition von Domänenabstraktionen (durch das Importieren mehrerer Erweiterungen), und ermöglicht die uniforme Selbstanwendbarkeit von Spracherweiterungen in der Entwicklung zukünftiger Erweiterungen (durch das Importieren von Erweiterungen in einer Erweiterungsdefinition). Die Organisation von Erweiterungen in Form von Bibliotheken ermöglicht Domänenabstraktionen die selbe Prinzipientreue wie interne domänenspezifische Sprachen. Wir haben die bibliotheksbasierte erweiterbare Programmiersprache SugarJ entworfen und implementiert. SugarJ Bibliotheken können Erweiterungen der Syntax, der statischen Analyse, und der Werkzeugunterstützung von SugarJ deklarieren. Eine syntaktische Erweiterung besteht dabei aus einer erweiterten Syntax und einer Transformation der erweiterten Syntax in die Basissyntax von SugarJ. Eine Erweiterung der Analyse testet Teile des abstrakten Syntaxbaums der aktuellen Datei und produziert eine Liste von Fehlern. Eine Erweiterung der Werkzeugunterstützung deklariert Dienste wie Syntaxfärbung oder Codevervollständigung für bestimmte Sprachkonstrukte. SugarJ Erweiterungen sind vollkommen selbstanwendbar: Eine erweiterte Syntax kann in eine Erweiterungsdefinition transformiert werden, eine erweiterte Analyse kann Erweiterungsdefinitionen testen, und eine erweiterte Werkzeugunterstützung kann Entwicklern beim Definieren von Erweiterungen assistieren. Um eine Quelldatei mit Erweiterungen zu verarbeiten, inspizieren der SugarJ Compiler und die SugarJ IDE die importierten Bibliotheken um die aktiven Erweiterungen zu bestimmen. Der Compiler und die IDE adaptieren den Parser, den Codegenerator, die Analyseroutine und die Werkzeugunterstützung der Quelldatei entsprechend der aktiven Erweiterungen. Wir beschreiben in der vorliegenden Doktorarbeit nicht nur das Design und die Implementierung von SugarJ, sondern berichten darüber hinaus über Erweiterungen unseres ursprünglich Designs. Insbesondere haben wir eine Generalisierung des SugarJ Compilers entworfen und implementiert, die neben Java alternative Basissprachen unterstützt. Wir haben diese Generalisierung verwendet um die bibliotheksbasierten erweiterbaren Programmiersprachen SugarHaskell, SugarProlog, und SugarFomega zu entwickeln. Weiterhin haben wir SugarJ ergänzt um polymorphe Domänenabstraktion und Kommunikationsintegrität zu unterstützen. Polymorphe Domänenabstraktion ermöglicht Programmierern mehrere Transformationen für die selbe domänenspezifische Syntax bereitzustellen. Dies erhöht die Flexibilität von SugarJ und unterstützt bekannte Szenarien aus der modellgetriebenen Entwicklung. Kommunikationsintegrität spezifiziert, dass die Komponenten eines Softwaresystems nur über explizite Kanäle kommunizieren dürfen. Im Kontext von Codegenerierung stellt dies eine interessante Eigenschaft dar, welche die Generierung von impliziten Modulabhängigkeiten untersagt. Wir haben Kommunikationsintegrität als weiteres Prinzip zu SugarJ hinzugefügt. Basierend auf SugarJ und zahlreicher Fallstudien argumentieren wir, dass flexible und prinzipientreue Domänenabstraktion ein skalierbares Programmiermodell für die Entwicklung komplexer Softwaresysteme darstellt

Evolution and diversity of secretome genes in the apicomplexan parasite Theileria annulata

<b>BACKGROUND</b>: Little is known about how apicomplexan parasites have evolved to infect different host species and cell types. Theileria annulata and Theileria parva invade and transform bovine leukocytes but each species favours a different host cell lineage. Parasite-encoded proteins secreted from the intracellular macroschizont stage within the leukocyte represent a critical interface between host and pathogen systems. Genome sequencing has revealed that several Theileria-specific gene families encoding secreted proteins are positively selected at the inter-species level, indicating diversification between the species. We extend this analysis to the intra-species level, focusing on allelic diversity of two major secretome families. These families represent a well-characterised group of genes implicated in control of the host cell phenotype and a gene family of unknown function. To gain further insight into their evolution and function, this study investigates whether representative genes of these two families are diversifying or constrained within the T. annulata population. <b>RESULTS</b>: Strong evidence is provided that the sub-telomerically encoded SVSP family and the host-nucleus targeted TashAT family have evolved under contrasting pressures within natural T. annulata populations. SVSP genes were found to possess atypical codon usage and be evolving neutrally, with high levels of nucleotide substitutions and multiple indels. No evidence of geographical sub-structuring of allelic sequences was found. In contrast, TashAT family genes, implicated in control of host cell gene expression, are strongly conserved at the protein level and geographically sub-structured allelic sequences were identified among Tunisian and Turkish isolates. Although different copy numbers of DNA binding motifs were identified in alleles of TashAT proteins, motif periodicity was strongly maintained, implying conserved functional activity of these sites. <b>CONCLUSIONS</b>: This analysis provides evidence that two distinct secretome genes families have evolved under contrasting selective pressures. The data supports current hypotheses regarding the biological role of TashAT family proteins in the management of host cell phenotype that may have evolved to allow adaptation of T. annulata to a specific host cell lineage. We provide new evidence of extensive allelic diversity in representative members of the enigmatic SVSP gene family, which supports a putative role for the encoded products in subversion of the host immune response

The major histocompatibility complex of reindeer

The major histocompatibility complex (MHC) is a system of closely linked genes showing an extremely high degree of polymorphism. These genes are major elements in the government of specific immune reactions. Consequently they may represent a genetic marker system well suited to investigate variability in selective pressure from disease agents on different populations. On this background we have started investigation of the MHC complex in reindeer (Rangifer tarandus L). The MHC complex consist of polymorphic regions as well as regions conserved during evolution which should allow the use of cross-species reagents. We have shown that human MHC gene probes hybridize with genomic DNA from reindeer, and thus can be used as a tool in reindeer MHC research. By RFLP (restriction fragment length polymorphism) analysis using these probes we have also been able to show polymorphism in MHC related genes from reindeer

A GWAS SNP for Schizophrenia Is Linked to the Internal MIR137 Promoter and Supports Differential Allele-Specific Expression

Single nucleotide polymorphisms (SNPs) within the MIR137 gene locus have been shown to confer risk for schizophrenia through genome-wide association studies (GWAS). The expression levels of microRNA-137 (miR-137) and its validated gene targets have also been shown to be disrupted in several neuropsychiatric conditions, including schizophrenia. Regulation of miR-137 expression is thus imperative for normal neuronal functioning. We previously characterised an internal promoter domain within the MIR137 gene that contained a variable number tandem repeat (VNTR) polymorphism and could alter the in vitro levels of miR-137 in a stimulus-induced and allele-specific manner. We now demonstrate that haplotype tagging-SNP analysis linked the rs1625579 GWAS SNP for schizophrenia to this internal MIR137 promoter through a proxy SNP rs2660304 located at this domain. We postulated that the rs2660304 promoter SNP may act as predisposing factor for schizophrenia through altering the levels of miR-137 expression in a genotype-dependent manner. Reporter gene analysis of the internal MIR137 promoter containing the common VNTR variant demonstrated genotype-dependent differences in promoter activity with respect to rs2660304. In line with previous reports, the major allele of the rs2660304 proxy SNP, which has previously been linked with schizophrenia risk through genetic association, resulted in downregulation of reporter gene expression in a tissue culture model. The genetic influence of the rs2660304 proxy SNP on the transcriptional activity of the internal MIR137 promoter, and thus the levels of miR-137 expression, therefore offers a distinct regulatory mechanism to explain the functional significance of the rs1625579 GWAS SNP for schizophrenia risk

Reduced Polymorphism in Domains Involved in Protein-Protein Interactions

Genome sequencing of various individuals or isolates of the same species allows studying the polymorphism level of specific proteins and protein domains. Here we ask whether domains that are known to be involved in mediating protein-protein interactions show lower polymorphism than other domains. To this end we take advantage of a recent genome sequence dataset of 39 Saccahromyces cerevisiae strains and the experimentally determined protein interaction network of the laboratory strain. We analyze the polymorphism in domain residues involved in interactions at various levels of resolution, depending on their likelihood to be interaction mediators. We find that domains involved in interactions are less polymorphic than other domains. Furthermore, as the likelihood of a residue to be involved in interaction increases, its polymorphism decreases. Our results suggest that purifying selection operates on domains capable of mediating protein interactions to maintain their function

A Study Of Human T-Cell Lines Generated From Multiple Sclerosis Patients And Controls By Stimulation With Peptides Of Myelin Basic Protein

We generated T-cell lines from the peripheral blood of controls and of patients with multiple sclerosis (MS) by stimulation with overlapping synthetic peptides representing the entire sequences of all four isoforms of human myelin basic protein (MBP). The T-cell lines reacted to a wide range of epitopes in the major isoforms of MBP and to epitopes that were present only in the minor isoforms. Many MS patients and controls had T-cells responding to one or more cryptic MBP epitopes, as indicated by the generation of a peptide-specific T-cell line(s) by stimulation with synthetic peptides but not by stimulation with whole MBP. About one-third of the peptide-generated lines were cytotoxic. Although we have shown that this technique of peptide stimulation is effective in generating human antiviral cytotoxic CD8+ T-cell lines, all the cytotoxic MBP-specific lines generated by this method were predominantly CD4+. Our study did not reveal any significant differences, between MS patients and controls, in reactivity to epitopes within any of the isoforms of MBP

Interleukin-1 Receptor Antagonist (IL-1RN) Gene Variable Number Tandem Repeats (VNTR) Polymorphism Association in men Infertility in Erbil City /Kurdistan Iraq

عائلة الحريك الخلوي -1 لها أدوار متعددة في الجهاز التناسلي الذكري، ومن بينها مضادات مستقبلات الحريك الخلوي -1 (IL-1RN) الموجودة في الغدد التناسلية الذكرية، حيث تزداد نشاطها عند الاصابات بالعدوى والالتهاب. تهدف الدراسة الحالية إلى التحقق من وجود صلة محتملة لتعدد الأشكال المترادفة المترادفة (VNTR) للجين IL-1RN مع العقم عند الذكور. شملت مجموعات الدراسة المسجلين 100 من الرجال المصابين بالعقم و 100 من الرجال الأصحاء. حيث تم تحليل السوائل المنوية للمجموعات المشاركة. تم جمع عينات الدم المحيطي لتقييم أو الكشف عن تعدد الأشكال المترادفة المترادفة (VNTR) لجين مضادات مستقبلات الحريك الخلوي -1 (IL-1RN) لنمطين من الاليلاتأليل IL-1RN1 يتوافق مع 410 زوج قاعديIL-11RN2 يتوافق مع 240وج قاعدي كعلامة على العقم عند الذكور، باستخدام تقنية PCR حددت النتائج ارتفاع وتيرة التغاير الاليلي IL-1RN2   (26٪) ، واثنين من التغاير الاليلي VNTR ناقلات IL-1RN1 و IL-1RN2  (16٪) من الرجال المصابين بالعقم مع تأثيرات كبيرة على حركة الحيوانات المنوية واشكالها (P<0.000-0.002) على التوالي. هذه الدراسة التى تم تقييمها في إقليم كردستان (أربيل – العراق) والتى حددت تأثيراً كبيراً لتعدد الأشكال المترادفة المتعاقبة VNTR لجين IL-1RN في مسببات العقم عند الذكور خاصة على حركية الحيوانات المنوية واشكالها، ولا سيما ناقلات أليلية البديل IL-1RN2.The interleukin-1 family has multifaceted roles in men٫s reproductive syste. Out  of these is interleukin-1 receptor antagonist (IL-1RN) which exists in men gonads, and in case of infection and inflammatory process, its activity is increased.  The current study aims to verify a possible linkage of Variable Number Tandem Repeat (VNTR) polymorphism of the IL-1RN gene with human men infertility. The study groups enrolled included 100 infertile men and 100 fertile and healthy men. Their seminal fluids were subjected to analysis. Also peripheral blood samples were collected for the assessment or detection of polymorphic Variable Number  Tandem Repeats (VNTR) polymorphism of interleukin-1 receptor antagonist gene (IL-1RN). Two alleles, namely IL-1RN1 allele corresponding to 410bp fragment and IL-11RN2 that corresponding to 240bp fragments,  are a marker for human men infertility, detected by PCR technique. The results delineated a high frequency of IL-1RN2 allelic gene variants (26%), and two VNTR allelic gene variants carriers IL-1RN1 and IL-1RN2 (16%) among  infertile men with significant impacts on sperm motility and morphology (P< 0.000-0.002) respectively. This prospective study inKurdistan region (Erbil –Iraq)  defined a significant impact of VNTR polymorphism of IL-1RN gene in the etiology of men infertility especially on sperm motility and morphology; particularly carriers of IL-1RN2 allelic variant

Comparative Analysis of Tandem Repeats from Hundreds of Species Reveals Unique Insights into Centromere Evolution

Centromeres are essential for chromosome segregation, yet their DNA sequences evolve rapidly. In most animals and plants that have been studied, centromeres contain megabase-scale arrays of tandem repeats. Despite their importance, very little is known about the degree to which centromere tandem repeats share common properties between different species across different phyla. We used bioinformatic methods to identify high-copy tandem repeats from 282 species using publicly available genomic sequence and our own data. The assumption that the most abundant tandem repeat is the centromere DNA was true for most species whose centromeres have been previously characterized, suggesting this is a general property of genomes. Our methods are compatible with all current sequencing technologies. Long Pacific Biosciences sequence reads allowed us to find tandem repeat monomers up to 1,419 bp. High-copy centromere tandem repeats were found in almost all animal and plant genomes, but repeat monomers were highly variable in sequence composition and in length. Furthermore, phylogenetic analysis of sequence homology showed little evidence of sequence conservation beyond ~50 million years of divergence. We find that despite an overall lack of sequence conservation, centromere tandem repeats from diverse species showed similar modes of evolution, including the appearance of higher order repeat structures in which several polymorphic monomers make up a larger repeating unit. While centromere position in most eukaryotes is epigenetically determined, our results indicate that tandem repeats are highly prevalent at centromeres of both animals and plants. This suggests a functional role for such repeats, perhaps in promoting concerted evolution of centromere DNA across chromosomes