Direct fluorescent labelling of clones by DOP PCR

<p>Abstract</p> <p>Background</p> <p>Array Comparative Genomic Hybridisation (array CGH) is a powerful technique for the analysis of constitutional chromosomal anomalies. Chromosomal duplications or deletions detected by array CGH need subsequently to be validated by other methods. One method of validation is Fluorescence <it>in situ </it>Hybridisation (FISH). Traditionally, fluorophores or hapten labelling is performed by nick translation or random prime labelling of purified Bacterial Artificial Chromosome (BAC) products. However, since the array targets have been generated from Degenerate Oligonucleotide Primed (DOP) amplified BAC clones, we aimed to use these DOP amplified BAC clones as the basis of an automated FISH labelling protocol. Unfortunately, labelling of DOP amplified BAC clones by traditional labelling methods resulted in high levels of background.</p> <p>Results</p> <p>We designed an improved labelling method, by means of degenerate oligonucleotides that resulted in optimal FISH probes with low background.</p> <p>Conclusion</p> <p>We generated an improved labelling method for FISH which enables the rapid generation of FISH probes without the need for isolating BAC DNA. We labelled about 900 clones with this method with a success rate of 97%.</p

Single-cell copy number variation detection

Detection of chromosomal aberrations from a single cell by array comparative genomic hybridization (single-cell array CGH), instead of from a population of cells, is an emerging technique. However, such detection is challenging because of the genome artifacts and the DNA amplification process inherent to the single cell approach. Current normalization algorithms result in inaccurate aberration detection for single-cell data. We propose a normalization method based on channel, genome composition and recurrent genome artifact corrections. We demonstrate that the proposed channel clone normalization significantly improves the copy number variation detection in both simulated and real single-cell array CGH data

Somatic Genomic Variations in Early Human Prenatal Development

Only 25 to 30% of conceptions result in a live birth. There is mounting evidence that the cause for this low fecundity is an extremely high incidence of chromosomal rearrangements occurring in the cleavage stage embryo. In this review, we gather all recent evidence for an extraordinary degree of mosaicisms in early embryogenesis. The presence of the rearrangements seen in the cleavage stage embryos can explain the origins of the placental mosaicisms seen during chorion villi sampling as well as the chromosomal anomalies seen in early miscarriages. Whereas these rearrangements often lead to implantation failure and early miscarriages, natural selection of the fittest cells in the embryo is the likely mechanism leading to healthy fetuses

Skeletal muscle repair by adult human mesenchymal stem cells from synovial membrane

We have demonstrated previously that adult human synovial membrane-derived mesenchymal stem cells (hSM-MSCs) have myogenic potential in vitro (De Bari, C., F. Dell'Accio, P. Tylzanowski, and F.P. Luyten. 2001. Arthritis Rheum. 44:1928–1942). In the present study, we have characterized their myogenic differentiation in a nude mouse model of skeletal muscle regeneration and provide proof of principle of their potential use for muscle repair in the mdx mouse model of Duchenne muscular dystrophy. When implanted into regenerating nude mouse muscle, hSM-MSCs contributed to myofibers and to long term persisting functional satellite cells. No nuclear fusion hybrids were observed between donor human cells and host mouse muscle cells. Myogenic differentiation proceeded through a molecular cascade resembling embryonic muscle development. Differentiation was sensitive to environmental cues, since hSM-MSCs injected into the bloodstream engrafted in several tissues, but acquired the muscle phenotype only within skeletal muscle. When administered into dystrophic muscles of immunosuppressed mdx mice, hSM-MSCs restored sarcolemmal expression of dystrophin, reduced central nucleation, and rescued the expression of mouse mechano growth factor

22q11.2 deletion syndrome

22q11.2 deletion syndrome (22q11.2DS) is the most common chromosomal microdeletion disorder, estimated to result mainly from de novo non-homologous meiotic recombination events occurring in approximately 1 in every 1,000 fetuses. The first description in the English language of the constellation of findings now known to be due to this chromosomal difference was made in the 1960s in children with DiGeorge syndrome, who presented with the clinical triad of immunodeficiency, hypoparathyroidism and congenital heart disease. The syndrome is now known to have a heterogeneous presentation that includes multiple additional congenital anomalies and later-onset conditions, such as palatal, gastrointestinal and renal abnormalities, autoimmune disease, variable cognitive delays, behavioural phenotypes and psychiatric illness - all far extending the original description of DiGeorge syndrome. Management requires a multidisciplinary approach involving paediatrics, general medicine, surgery, psychiatry, psychology, interventional therapies (physical, occupational, speech, language and behavioural) and genetic counselling. Although common, lack of recognition of the condition and/or lack of familiarity with genetic testing methods, together with the wide variability of clinical presentation, delays diagnosis. Early diagnosis, preferably prenatally or neonatally, could improve outcomes, thus stressing the importance of universal screening. Equally important, 22q11.2DS has become a model for understanding rare and frequent congenital anomalies, medical conditions, psychiatric and developmental disorders, and may provide a platform to better understand these disorders while affording opportunities for translational strategies across the lifespan for both patients with 22q11.2DS and those with these associated features in the general population

NIPTmer : rapid k-mer-based software package for detection of fetal aneuploidies

Non-invasive prenatal testing (NIPT) is a recent and rapidly evolving method for detecting genetic lesions, such as aneuploidies, of a fetus. However, there is a need for faster and cheaper laboratory and analysis methods to make NIPT more widely accessible. We have developed a novel software package for detection of fetal aneuploidies from next-generation low-coverage whole genome sequencing data. Our tool - NIPTmer - is based on counting pre-defined per-chromosome sets of unique k-mers from raw sequencing data, and applying linear regression model on the counts. Additionally, the filtering process used for k-mer list creation allows one to take into account the genetic variance in a specific sample, thus reducing the source of uncertainty. The processing time of one sample is less than 10 CPU-minutes on a high-end workstation. NIPTmer was validated on a cohort of 583 NIPT samples and it correctly predicted 37 non-mosaic fetal aneuploidies. NIPTmer has the potential to reduce significantly the time and complexity of NIPT post-sequencing analysis compared to mapping-based methods. For non-commercial users the software package is freely available at http://bioinfo.ut.ee/NIPTMer/.Peer reviewe

Genome-wide haplotyping embryos developing from 0PN and 1PN zygotes increases transferrable embryos in PGT-M.

STUDY QUESTION Can genome-wide haplotyping increase success following preimplantation genetic testing for a monogenic disorder (PGT-M) by including zygotes with absence of pronuclei (0PN) or the presence of only one pronucleus (1PN)? SUMMARY ANSWER Genome-wide haplotyping 0PNs and 1PNs increases the number of PGT-M cycles reaching embryo transfer (ET) by 81% and the live-birth rate by 75%. WHAT IS KNOWN ALREADY Although a significant subset of 0PN and 1PN zygotes can develop into balanced, diploid and developmentally competent embryos, they are usually discarded because parental diploidy detection is not part of the routine work-up of PGT-M. STUDY DESIGN, SIZE, DURATION This prospective cohort study evaluated the pronuclear number in 2229 zygotes from 2337 injected metaphase II (MII) oocytes in 268 cycles. PGT-M for 0PN and 1PN embryos developing into Day 5/6 blastocysts with adequate quality for vitrification was performed in 42 of the 268 cycles (15.7%). In these 42 cycles, we genome-wide haplotyped 216 good quality embryos corresponding to 49 0PNs, 15 1PNs and 152 2PNs. The reported outcomes include parental contribution to embryonic ploidy, embryonic aneuploidy, genetic diagnosis for the monogenic disorder, cycles reaching ETs, pregnancy and live birth rates (LBR) for unaffected offspring. PARTICIPANTS/MATERIALS, SETTING, METHODS Blastomere DNA was whole-genome amplified and hybridized on the Illumina Human CytoSNP12V2.1.1 BeadChip arrays. Subsequently, genome-wide haplotyping and copy-number profiling was applied to investigate the embryonic genome architecture. Bi-parental, unaffected embryos were transferred regardless of their initial zygotic PN score. MAIN RESULTS AND THE ROLE OF CHANCE A staggering 75.51% of 0PN and 42.86% of 1PN blastocysts are diploid bi-parental allowing accurate genetic diagnosis for the monogenic disorder. In total, 31% (13/42) of the PGT-M cycles reached ET or could repeat ET with an unaffected 0PN or 1PN embryo. The LBR per initiated cycle increased from 9.52 to 16.67%. LIMITATIONS, REASONS FOR CAUTION The clinical efficacy of the routine inclusion of 0PN and 1PN zygotes in PGT-M cycles should be confirmed in larger cohorts from multicenter studies. WIDER IMPLICATIONS OF THE FINDINGS Genome-wide haplotyping allows the inclusion of 0PN and 1PN embryos and subsequently increases the cycles reaching ET following PGT-M and potentially PGT for aneuploidy (PGT-A) and chromosomal structural rearrangements (PGT-SR). Establishing measures of clinical efficacy could lead to an update of the ESHRE guidelines which advise against the use of these zygotes. STUDY FUNDING/COMPETING INTEREST(S) SymBioSys (PFV/10/016 and C1/018 to J.R.V. and T.V.), the Horizon 2020 WIDENLIFE: 692065 to J.R.V., T.V., E.D., A.D. and M.Z.E. M.Z.E., T.V. and J.R.V. co-invented haplarithmisis (‘Haplotyping and copy-number typing using polymorphic variant allelic frequencies’), which has been licensed to Agilent Technologies. H.M. is fully supported by the (FWO) (ZKD1543-ASP/16). The authors have no competing interests to declare

Single-cell paired-end genome sequencing reveals structural variation per cell cycle

The nature and pace of genome mutation is largely unknown. Because standard methods sequence DNA from populations of cells, the genetic composition of individual cells is lost, de novo mutations in cells are concealed within the bulk signal and per cell cycle mutation rates and mechanisms remain elusive. Although single-cell genome analyses could resolve these problems, such analyses are error-prone because of whole-genome amplification (WGA) artefacts and are limited in the types of DNA mutation that can be discerned. We developed methods for paired-end sequence analysis of single-cell WGA products that enable (i) detecting multiple classes of DNA mutation, (ii) distinguishing DNA copy number changes from allelic WGA-amplification artefacts by the discovery of matching aberrantly mapping read pairs among the surfeit of paired-end WGA and mapping artefacts and (iii) delineating the break points and architecture of structural variants. By applying the methods, we capture DNA copy number changes acquired over one cell cycle in breast cancer cells and in blastomeres derived from a human zygote after in vitro fertilization. Furthermore, we were able to discover and fine-map a heritable inter-chromosomal rearrangement t(1;16)(p36;p12) by sequencing a single blastomere. The methods will expedite applications in basic genome research and provide a stepping stone to novel approaches for clinical genetic diagnosis

Pathogenesis of vestibular schwannoma in ring chromosome 22

<p>Abstract</p> <p>Background</p> <p>Ring chromosome 22 is a rare human constitutional cytogenetic abnormality. Clinical features of neurofibromatosis type 1 and 2 as well as different tumour types have been reported in patients with ring chromosome 22. The pathogenesis of these tumours is not always clear yet.</p> <p>Methods</p> <p>We report on a female patient with a ring chromosome 22 presenting with severe mental retardation, autistic behaviour, café-au-lait macules and facial dysmorphism. Peripheral blood lymphocytes were karyotyped and array CGH was performed on extracted DNA. At the age of 20 years she was diagnosed with a unilateral vestibular schwannoma. Tumour cells were analyzed by karyotyping, array CGH and <it>NF2 </it>mutation analysis.</p> <p>Results</p> <p>Karyotype on peripheral blood lymphocytes revealed a ring chromosome 22 in all analyzed cells. A 1 Mb array CGH experiment on peripheral blood DNA showed a deletion of 5 terminal clones on the long arm of chromosome 22. Genetic analysis of vestibular schwannoma tissue revealed loss of the ring chromosome 22 and a somatic second hit in the <it>NF2 </it>gene on the remaining chromosome 22.</p> <p>Conclusion</p> <p>We conclude that tumours can arise by the combination of loss of the ring chromosome and a pathogenic <it>NF2 </it>mutation on the remaining chromosome 22 in patients with ring chromosome 22. Our findings indicate that patients with a ring 22 should be monitored for NF2-related tumours starting in adolescence.</p