Low Mosaicism Research Articles

Search for Hidden Germline Mutations in Bilateral Retinoblastoma

Retinoblastoma (RB) is a cancer in the retina that develops in children usually under the age of five and can cause blindness, loss of the affected eye, or death by metastasis, if not diagnosed and treated early. It arises as a result of bi‐allelic inactivation of the RB1 gene in a retinal cell that prevents the production of the pRB protein, causing uncontrollable cell proliferation in the retina. The Genetic Diagnostic Laboratory in the Perelman School of Medicine at the University of Pennsylvania has used Sanger sequencing to identify disease‐causing mutations in the RB1 gene in thousands of individuals diagnosed with bilateral retinoblastoma. Current testing focuses on coding regions and splice site consensus sequences, and various mutations have been found in 97% of individuals tested. This study aimed to identify pathogenic exonic or deep intronic mutations in the germline of six individuals which belong to the 3% of mutation‐negative cases. For three of these individuals, RNA isolated from the lymphocytes of the whole blood was converted into cDNA, RB1 transcript amplified with gene specific primers and sequenced by Sanger sequencing. The cDNA of RB1 had indicated one sample had a heterozygous deletion of exon 10, but the cause of the exonic deletion was unknown. To investigate this, several primer pairs were designed to amplify and sequence regions flanking the deleted region in the genomic DNA, and an inversion between exons 10 and 11 was suspected. For the other two patients, who are sisters, an exonic deletion of exon 2 was found but proven to be benign. For the other three individuals, previous Sanger sequencing of exons had not indicated the presence of any pathogenic mutations. An AmpliSeq panel of three hundred and four primer pairs was designed to amplify and sequence both coding and non‐coding regions spanning the RB1 gene. Amplicons were prepared using genomic DNA isolated from these three individuals and next generation sequencing on the Ion Torrent PGM platform was performed. Upon analysis, three pathogenic mutations present at low allele frequencies were identified in each individual. These were low frequency mosaic mutations that may not be detected through Sanger sequencing. Therefore, the AmpliSeq design panel can provide information about intronic mutations and low frequency mosaic mutations, and it can serve as an assay complimentary to Sanger sequencing when investigating missing mutations in RB1 gene.Support or Funding InformationJennifer Richards‐Yutz, Tappan Ganguly, Eric Toorens, Jennifer Rosado, Richard Grant, Kimberly Moran, Arupa Ganguly. Genetic Diagnostic Laboratory at the University of Pennsylvania, Philadelphia, PAThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ultrahigh vacuum dc magnetron sputter-deposition of epitaxial Pd(111)/Al2O3(0001) thin films.

Pd(111) thin films, ∼245 nm thick, are deposited on Al2O3(0001) substrates at ≈0.5Tm, where Tm is the Pd melting point, by ultrahigh vacuum dc magnetron sputtering of Pd target in pure Ar discharges. Auger electron spectra and low-energy electron diffraction patterns acquired in situ from the as-deposited samples reveal that the surfaces are compositionally pure 111-oriented Pd. Double-axis x-ray diffraction (XRD) ω-2θ scans show only the set of Pd 111 peaks from the film. In triple-axis high-resolution XRD, the full width at half maximum intensity Γω of the Pd 111 ω-rocking curve is 630 arc sec. XRD 111 pole figure obtained from the sample revealed six peaks 60°-apart at a tilt angles corresponding to Pd 111 reflections. XRD ϕ scans show six 60°-rotated 111 peaks of Pd at the same ϕ angles for 11[Formula: see text]3 of Al2O3 based on which the epitaxial crystallographic relationships between the film and the substrate are determined as [Formula: see text]ǁ[Formula: see text] with two in-plane orientations of [Formula: see text]ǁ[Formula: see text] and [Formula: see text]ǁ[Formula: see text]. Using triple axis symmetric and asymmetric reciprocal space maps, interplanar spacings of out-of-plane (111) and in-plane (11[Formula: see text]) are found to be 0.2242 ± 0.0003 and 0.1591 ± 0.0003 nm, respectively. These values are 0.18% lower than 0.2246 nm for (111) and the same, within the measurement uncertainties, as 0.1588 nm for (11[Formula: see text]) calculated from the bulk Pd lattice parameter, suggesting a small out-of-plane compressive strain and an in-plane tensile strain related to the thermal strain upon cooling the sample from the deposition temperature to room temperature. High-resolution cross-sectional transmission electron microscopy coupled with energy dispersive x-ray spectra obtained from the Pd(111)/Al2O3(0001) samples indicate that the Pd-Al2O3 interfaces are essentially atomically abrupt and dislocation-free. These results demonstrate the growth of epitaxial Pd thin films with (111) out-of-plane orientation with low mosaicity on Al2O3(0001).

Next generation sequencing to detect low grade mosaicism and its effect on the live birth rate

Introduction: Chromosomal aneuploidy is highly prevalent in human embryos. It is a reason for the relatively low success rates of IVF cycles. PGS by aCGH has been used to identify euploid embryos in order to improve the clinical outcome of IVF treatments. However a percentage of morphologically normal euploid blastocysts fail to implant or result in a pregnancy loss. The chromosomal mosaicism has been considered as a feasible explanation for some failures after the transfer of euploid embryos. The incorporation of NGS to PGS has improved the mosaicism detection compared with aCGH. The aim of this work is to assess if PGS by NGS is able to improve the live birth rate compared with aCGH.

Embryos classified as low-grade mosaic (

Mosaic preimplantation embryos contain two or more cell lines with different chromosome content, the consequence of errors in chromosome segregation occurring during mitotic divisions. Recently the NGS was validated and implemented for PGS. There are several different platforms, with different resolutions, that can detect mosaicism to a different extent. Veriseq NGS (Illumina) that was used in our study, can detect mosaicism when aneuploidy is present in 20%-80% of TE cells biopsied from a blastocyst. The purpose of the present study was to retrospectively analyze the pregnancy outcome of replaced low rate mosaic embryos (20%-50% abnormal cells in the trophoctoderm biopsy) as diagnosed with the use of hr-NGS. The molecular analyses were provided by Nexgenomics, LLC, Pasadena, CA. The platform used was the Veryseq NGS (Illumina). Nexgenomics position is to use an artificial cutoff of 50% to classify all the mosaicism events as normal or abnormal. Mosaicism <20% is considered not differentiable from technical noise. Low rate mosaic embryos were transferred when euploid embryos were not available. This study included only patients that received a single embryo transfer of low rate mosaic embryos and for which the delivery of a newborn could be confirmed. The patients generating the control group (only single euploid embryo transferred) were matched with the patients of the study group for age, embryo quality, use of an egg donor and/or gestational carrier and period of IVF treatment. All pregnancies that went to term were confirmed, by means of chorionic villus sampling or amniocentesis, to have a normal Karyotype. The results are summarized in Table 1. A total of 33 low mosaic embryos were transferred and they resulted in an overall live birth rate of 51.5%, similar to the live birth rate obtained from the transfer of euploid embryos (48.5%). No birth defects were reported.Table 1Comparison of the pregnancy outcomes of low rate mosaic and control embryos according to the mosaicism type and the chromosomes involved.Type of mosaicismSegmental*Single-Double1-12**Single-Double13-22***X-YComplexTotalControlsPAge38.8 ± 6.440.8 ± 6.936.3 ± 5.0-34.639.1 ± 6.439.3 ± 6.3NSN of transfer cycles13127013366NSEmbryos transferred13127013366NSEmbryos Implanted875-02039NSImplantation rate (IR)61.5%58.3%71.4%-060.6%59.1%NSAbortions111--37NSAbortion rate (AR)12.5%14.3%20%--15%17.9%NSDeliveries764-01732NSLive birth rate (LBR)53.8%50.0%57.1%-051.5%48.5%NS*Segmental: regarding a piece of chromosome**Single-Double 1-12: regarding one or two entire chromosomes from number 1 to number 12***Single-Double 13-22: regarding one or two entire chromosomes from number 13 to number 12-22 Open table in a new tab *Segmental: regarding a piece of chromosome **Single-Double 1-12: regarding one or two entire chromosomes from number 1 to number 12 ***Single-Double 13-22: regarding one or two entire chromosomes from number 13 to number 12-22 Our results indicate that low rate mosaic embryos have the same IR and LBR when compared to euploid embryos. Due to the low number of embryos in our study, further studies are required to verify this observation, but for clinical purposes, these embryos could be considered euploid. As far as we know this is the second study that provides data about the LBR of the mosaic embryos (Greco et al., 2015). A recent study (Munnè et al., 2017) concluded that, overall, mosaic embryos implant less and miscarry more than euploid embryos. However, embryos with >40% abnormal cells and complex mosaic embryos had the lower ongoing implantation rate. We transferred only one complex mosaic embryo and low rate mosaic embryos, which can explain the difference in our findings.

Inter‐group variability in seed dispersal by white‐handed gibbons in mosaic forest

AbstractSeed dispersers, like white‐handed gibbons (Hylobates lar), can display wide inter‐group variability in response to distribution and abundance of resources in their habitat. In different home ranges, they can modify their movement patterns along with the shape and scale of seed shadow produced. However, the effect of inter‐group variability on the destination of dispersed seeds is still poorly explained. In this study, we evaluate how seed dispersal patterns of this arboreal territorial frugivore varies between two neighboring groups, one inhabiting high quality evergreen forest and one inhabiting low quality mosaic forest. We predicted a difference in seed dispersal distance between the two groups (longer in the poor quality forest). We hypothesized that this difference would be explained by differences in home range size, daily path length, and ranging tortuosity. After 6 months of data collection, the evergreen group had a smaller home range (12.4 ha) than the mosaic group (20.9 ha), significantly longer daily path lengths (1507 m vs. 1114 m respectively) and greater tortuosity (39.1 vs. 16.1 respectively). Using gut passage times and displacement rates, we estimated the median seed dispersal distance as 163 m for the evergreen group (high quality forest) and of 116 m for the mosaic group (low quality forest). This contradiction with our initial prediction can be explained in term of social context, resource distribution, and habitat quality. Our results indicate that gibbons are dispersers of seeds between habitats and that dispersal distances provided by gibbons are influenced by a range of factors, including habitat and social context.

Formation of crystalline silicon-germanium thin films on silicon substrates by solid phase crystallization

We have researched on the formation of crystalline silicon-germanium (c-SiGe) films as a new material for bottom cells of silicon-based multi-junction solar cells. We conducted solid phase crystallization (SPC) from amorphous SiGe (a-SiGe) precursors with 80–70% Ge fraction deposited on n-type [100] single crystalline silicon (c-Si) substrates. Preferential crystal growth following the orientation of c-Si substrates is successfully done by the SPC at temperatures from 500 to 950°C, although X-ray diffraction indicates that the c-SiGe films have the mosaic structures consisting of crystalline domains with several tens of nanometers. Lower SPC temperature is more appropriate to obtain better crystallinity SiGe with larger domain size and lower mosaicity as long as the crystallization occurs. The inter-diffusion between the a-SiGe precursors and Si substrates occurs at relatively high SPC temperatures (≥850°C), and the Ge fraction in the c-SiGe films becomes lower than that in the a-SiGe precursors.

The extent of chromosomal mosaicism influences the clinical outcome of in vitro fertilization treatments

Recently, we have demonstrated that mosaic embryos hold the potential to implant and result in the birth of healthy babies1. The aim of this study was to determine whether the extent of chromosomal mosaicism can influence the development potential of mosaic embryos. The transfer of mosaic embryos at different aneuploidy percentage was offered to 77 women for whom IVF had resulted in no euploid embryos between May 2013-March 2016. The clinical outcome obtained after transfer of mosaic embryos with low (<50%) and high (≥50%) aneuploidy percentage, was compared with those resulting from a control group of 251 euploid blastocysts. We aimed to assess if there was any statistically significant difference in the development potential between these groups. All embryos were cultured to blastocyst stage; trophectoderm biopsy was performed on Day-5 of development or Day6/7 for slow growing embryos. Comprehensive chromosome screening PGS was performed using either NGS or array-CGH methodologies. Transfers of mosaic embryos with a high percentage of chromosomally abnormal cells (≥50%) resulted in a live birth rate of 15.1% and involved a miscarriage rate of 9%. In contrast, mosaic embryos with a lower aneuploidy percentage (<50%) resulted in a live birth rate of 42.2%, with a miscarriage occurring in 7.0% of the transfers. All pregnancies that went to term were confirmed, through sampling of the chorionic villi and/or amniotic liquid, to have a normal karyotype. Mosaic embryos with high aneuploidy percentage (≥50%) showed significantly lower implantation rate (24.4% vs 54.6%; P=0.001) and live birth rate (15.1% vs 46.6%; P=0.001) than euploid blastocysts. In contrast, embryos with lower aneuploidy percentage (<50%) have similar clinical outcomes of those euploid. The biochemical pregnancy rate and miscarriage rate were not significantly different between the two groups (Table 1). The results of this study further confirm that mosaic embryos can develop into healthy euploid newborns. We demonstrated that the extent of mosaicism affects the IVF success rate. Priority for transfer should be given to mosaic embryos with low mosaicism levels.Table 1Comparison of clinical outcomes in euploid embryos vs mosaic embryo different mosaicism levelEuploidMosaic with <50%pMosaic with >50%pNo. of embryos transferred2514533No. of transfer2504433No. of positive beta hCG160 (64%)26 (59%)0.411 (33.3%)0.001No. of Biochemical pregnancies24 (9.6%)5 (11.36%)0.83 (9.1%)0.8No. of embryos implanted137 (54.6%)22 (48.9%)0.58 (24.4%)0.01No. of Early abortion20 (8.0%)3 (6.8%)0.83 (9.1%)0.9No. of Ongoing clinical pregnancies per ET117 (46.6%)19 (42.2 %)0.75 (15.1%)0.001No. of Pregnancies went to term116 (46.4%)18 (41%)0.45 (15.1%)0.001No. of Babies Born117 (46.6%)19 (42.2 %)0.75 (15.1%)0.001 Open table in a new tab

A Systematic Investigation of PIK3CA Mutations in Isolated Macrodactyly: Indication for Accurate Classification, Diagnosis and Potential Novel Therapeutics

Grant received from: Beijing Talent Funding There is no financial information to disclose. Macrodactyly, a rare congenital digital anomaly, has been challenging the hand surgery society. The recent discovery of somatic PIK3CA mutations in a few isolated macrodactyly patients shed light on the fundamental understanding in pathogenesis of this disabling deformity. We set up to systematically test the association between PIK3CA mutations with isolated macrodactyly in order to establish a molecular pathophysiology-based diagnosis, classification and possibly a novel therapeutic strategy. Overgrowth tissues including skin, nerve, adipose tissues from clinically diagnosed isolated macrodactyly patients (N = 12) were removed during routine surgical debulking or correction procedures and preserved as formalin-fixed-paraffin-embedded (FFPE) blocks. DNA recovered from these FFPE samples were tested for PIK3CA mutation status using a modified and targeted Sanger DNA sequencing method with greatly improved sensitivity for detecting low level somatic mosaic mutations. PIK3CA mutation level, tissue(s) of occurring, type of mutations and their genomic locations were analyzed. PIK3CA mutations were detected from affected tissues in 9 out of the 12 patients studied, with mutation level ranging from 8 to 27%. The mutations detected include p.His1047Arg (N = 4), p.His1047Leu (N = 2), p.545Glu > Lys (N = 2) and p.542Glu > Lys (N = 1). These are codons in the PIK3CA gene that are frequently mutated in cancers. In terms of the tissue sources in which a mutation was found, adipose tissue has the highest mutation detection rate (100%), followed by nerve (83%) and skin (71%). No mutations found in bone tissues from two patients with PIK3CA mutations detected in other tissues of the same patients. Patients and/or tissues negative for PIK3CA mutations are subjected to downstream analysis using high throughput next generation DNA sequencing. (Figs. 40-1, 40-2) •A high proportion (75%) of isolated macrodactyly patients harbors activating PIK3CA mutations that lead to digital overgrowth as a result of abnormally activated Akt-PI3K signaling.•Adipose and nerve tissues provide the highest PIK3CA mutation detection yield among all the sources of affected tissues.•A modified Sanger DNA sequencing method is a cost-effective way to exam PIK3CA mutations in isolated macrodactyly patients clinically.•Patients negative for PIK3CA mutations may harbor other PIK3CA mutations beyond the detection of our assay or due to different molecular mechanism.•To our knowledge, our study is the largest study linking PIK3CA mutations to isolated macrodactyly.Figure 40-2PIK3CA mutations in isolated macrodactyly patients. PIK3CA gene structure with critical domains, targeted regions, and mutations identified in isolated macrodactyly patients are summarized. Red arrows indicate three targeted regions (p.418-435, p.527-555, and p.1009-1058) by Sanger DNA sequencing. Mutations previously reported and numbers of patients are depicted above the gene structure and those found in our patients are shown under. Cancer hotspot mutations are highlighted in red.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Thermoelectric La-doped SrTiO3 epitaxial layers with single-crystal quality: from nano to micrometers

High-quality thermoelectric La0.2Sr0.8TiO3 (LSTO) films, with thicknesses ranging from 20 nm to 0.7 μm, have been epitaxially grown on SrTiO3(001) substrates by enhanced solid-source oxide molecular-beam epitaxy. All films are atomically flat (with rms roughness < 0.2 nm), with low mosaicity (<0.1°), and present very low electrical resistivity (<5 × 10−4 Ω cm at room temperature), one order of magnitude lower than standard commercial Nb-doped SrTiO3 single-crystalline substrate. The conservation of transport properties within this thickness range has been confirmed by thermoelectric measurements where Seebeck coefficients of approximately –60 μV/K have been recorded for all films. These LSTO films can be integrated on Si for non-volatile memory structures or opto-microelectronic devices, functioning as transparent conductors or thermoelectric elements.

Structural and electrical properties of K3Li2Nb5O15 thin film grown by pulsed laser deposition

Potassium lithium niobate K3Li2Nb5O15 (KLN) with tetragonal tungsten bronze-type structure (TTB) thin film was successfully deposited on MgO substrate by pulsed laser ablation method. The substrate was coated by a perovskite conductive layer of La0.5Sr0.5CoO3. X-ray diffraction revealed (001) orientation with low mosaicity. The oriented film had smooth and droplets-free surface as evidenced by reflection high electron energy diffraction and atomic force microscopy. Using Pt top electrode, dielectric measurements indicated stable dielectric permittivity of about 400 up to 105Hz. A dielectric relaxation was also observed. Moreover, the capacitance voltage measurements of the obtained (001)KLN film show ferroelectric behavior. Our results provide useful insights into the growth of tetragonal tungsten and other complex materials. These results will be useful for the growth and integration of epitaxial TTB thin films on perovskite and Si substrates.

CRISPR/Cas9-mediated somatic and germline gene correction to restore hemostasis in hemophilia B mice.

Hemophilia B (HB) is an X-linked disorder caused by defects of F9 encoded coagulation factor IX, which is an ideal model for gene therapy. Most existing HB gene therapies are based on viral mediated gene supplementation, which could increase immunoreaction. In this study, CRISPR/Cas9 system was used for gene correction in an F9 mutant HB mouse model in both adult mice (in vivo) and in germline cells (ex vivo). In vivo, naked Cas9-sgRNA plasmid and donor DNA were delivered to HB mice livers to recover the mutation via hydrodynamic tail vein (HTV) injection. 62.5% of the HTV-treated mice showed a detectable gene correction (>1%) in the F9 alleles of hepatocytes, which was sufficient to remit the coagulation deficiency. Ex vivo, three different forms of Cas9 were microinjected into germline cells of HB mice to investigate their efficiency and safety in gene correction. Cas9 protein showed higher gene recovery rates, less embryo toxicity, and lower mosaic repair percentage, making it more suitable for germline gene therapy. Our study strongly supports that CRISPR/Cas9-mediated genome editing is feasible in gene therapy of genetic disorders.

Germline mosaicism is a pitfall in PGD for X-linked disorders. Single sperm typing detects very low frequency paternal gonadal mosaicism in a case of recurrent chondrodysplasia punctata misattributed to a maternal origin

This manuscript presents a molecularly demonstrated gonadal mosaicism from paternal origin for X-linked dominant chondrodysplasia punctata by single sperm typing. A couple who had experienced two medical terminations of pregnancy of female fetuses was referred to our pre-implantation genetic diagnosis (PGD) centre with the diagnosis of maternally derived gonadal mosaicism. Indeed, genetic analyses of different DNA samples - including semen - from the healthy parents failed to detect the variant found in the fetuses. Six embryos, all male, were obtained during the PGD cycle. The causative variant was not detected in any embryo, whereas five embryos had inherited the 'at-risk' maternal haplotype. The assumption of a maternal gonadal mosaicism was still possible, but this finding allowed us to consider the possibility of a paternal rather than maternal gonadal mosaicism. It prompted us to perform extensive single sperm analyses, demonstrating a low-frequency paternal germline mosaicism, which led to completely different haplotype phasing and PGD counselling. In conclusion, this case further exemplifies that germline mosaicism is a pitfall in PGD where diagnosis largely relies on linkage analysis and suggests that tracing the parental inheritance through polar body analysis and/or single sperm typing experiments is of major importance for adequate genetic counselling and accurate PGD. © 2016 John Wiley & Sons, Ltd.

Strained monolayer germanene with 1 × 1 lattice on Sb(111)

Monolayer germanene, the germanium analog of graphene, has been successfully synthesized on Sb(111) surface via molecular beam epitaxy. Scanning tunneling microscopy revealed a dendrite structure at low Ge coverage and mosaic patterns at high coverage, both with local 1 × 1 lattice. First-principle calculations confirmed the 1 × 1 low-buckled structure of germanene. The dendrite and mosaic patterns stem from strain modulation induced by large lattice mismatch between germanene and Sb substrate. This work provide a new system to explore the physical properties and applications of germanene.

Nanometer-Thick Gold on Silicon as a Proxy for Single-Crystal Gold for the Electrodeposition of Epitaxial Cuprous Oxide Thin Films.

Single-crystal Au is an excellent substrate for electrochemical epitaxial growth due to its chemical inertness, but the high cost of bulk Au single crystals prohibits their use in practical applications. Here, we show that ultrathin epitaxial films of Au electrodeposited onto Si(111), Si(100), and Si(110) wafers can serve as an inexpensive proxy for bulk single-crystal Au for the deposition of epitaxial films of cuprous oxide (Cu2O). The Au films range in thickness from 7.7 nm for a film deposited for 5 min to 28.3 nm for a film deposited for 30 min. The film thicknesses are measured by low-angle X-ray reflectivity and X-ray Laue oscillations. High-resolution TEM shows that there is not an interfacial SiOx layer between the Si and Au. The Au films deposited on the Si(111) substrates are smoother and have lower mosaic spread than those deposited onto Si(100) and Si(110). The mosaic spread of the Au(111) layer on Si(111) is only 0.15° for a 28.3 nm thick film. Au films deposited onto degenerate Si(111) exhibit ohmic behavior, whereas Au films deposited onto n-type Si(111) with a resistivity of 1.15 Ω·cm are rectifying with a barrier height of 0.85 eV. The Au and the Cu2O follow the out-of-plane and in-plane orientations of the Si substrates, as determined by X-ray pole figures. The Au and Cu2O films deposited on Si(100) and Si(110) are both twinned. The films grown on Si(100) have twins with a [221] orientation, and the films grown on Si(110) have twins with a [411] orientation. An interface model is proposed for all Si orientations, in which the -24.9% mismatch for the Au/Si system is reduced to only +0.13% by a coincident site lattice in which 4 unit meshes of Au coincide with 3 unit meshes of Si. Although this study only considers the deposition of epitaxial Cu2O films on electrodeposited Au/Si, the thin Au films should serve as high-quality substrates for the deposition of a wide variety of epitaxial materials.

Efficient biallelic mutation in porcine parthenotes using a CRISPR-Cas9 system

The parthenotes represent ideal models mimicking the embryonic development and characterizing the function of maternal genomes as well as an alternative source of pluripotent cell lines. Besides, parthenogenetically activated (PA) embryos serve as a rapid assay system to maximize the efficiency of generating genetically modified pig CRISPR/Cas9 system, an efficient and multiplex gene editing tool, has been utilized to modify the genome of porcine parthenotes. However, lower biallelic mutation rate and high mosaicism frequency were observed. Here, we aimed to enhance the biallelic mutation rate with reduced mosaicism by optimization of the concentration and injection time of the Cas9/sgRNA mixture in porcine parthenotes. The results showed that the efficient biallelic mutation (93%) and low mosaicism (33%) could be achieved in porcine parthenotes by cytoplasmic injection of Cas9 mRNA/sgRNA (125/12.5 ng/μl) after 8 h of parthenogenetical activation. Thus, our study provides an effective strategy for increasing the biallelic mutation rate and population homogeneity of genetically modified parthenotes, which will strengthen the role of parthenotes in uncovering early embryonic development and assessing the mutation efficiency due to the simplicity and adaptability of CRISPR/Cas9.

Mosaic ratio quantification of isochromosome 12p in Pallister-Killian syndrome using droplet digital PCR.

BackgroundPallister–Killian syndrome (PKS) is a prototypic mosaic aneuploidy syndrome caused by mosaic supernumerary marker isochromosome 12p. Cells possessing the isochromosome 12p rapidly diminish after birth in the peripheral blood, often necessitating a skin biopsy for diagnosis. Therefore, a genomic testing that is capable of detecting low percent mosaic isochromosome 12p is preferred for the diagnosis of PKS.MethodsThe utility of the droplet digital PCR system in quantifying the mosaic ratio of isochromosome 12p in PKS was evaluated.ResultsDroplet digital PCR was able to precisely quantify isochromosome 12p mosaic ratio, and copy number measured by droplet digital PCR was correlated well with that of fluorescence in situ hybridization analysis.ConclusionDroplet digital PCR should be considered as an effective tool for both clinical and research analytics to precisely quantify mosaic genomic copy number alterations or mosaic mutations.

Polyposis Caused by Low APC Mosaicism

Purpose: To present a patient with familial adenomatous polyposis (FAP) caused by a low level of somatic mosaicism. Case description: A twenty-one year old female presented with rectal bleeding and abdominal pain. She underwent a colonoscopy and esophagogastroduodenoscopy which revealed extensive polyposis. There was no family history of polyps or early onset colon cancer in her family. Methodology: Next-generation sequencing (NGS) analysis was performed using the ColoSeqTM panel on DNA extracted from both peripheral blood lymphocytes and colonic polyps. RESULTS: Molecular analysis detected the p.E1408X deleterious mutation in the APC gene in in 12 of 276 (4%) reads of the DNA in the peripheral blood leukocytes and in 30% of the DNA from colonic polyps. Conclusion: We report that low level of 4% APC mosaicism led to florid polyposis. Our report highlights the power of deep next-generation sequencing to identify mosaic mutations that are missed by traditional approaches. Though somatic APC mosaicism has previously been reported to cause polyposis syndrome in a few cases, it has been underestimated as a cause of polyposis coli. This case should reinforce the need to search for mosaicism in all patients with a personal history of polyposis and no family history.

Accurate Detection of Low Mosaic Mutations Associated with Therapy Resistance in Pediatric Acute Lymphoblastic Leukemia Using Single Molecule Tagging and Deep-Sequencing

Introduction: Recent work has demonstrated that relapses in pediatric acute lymphoblastic leukemia (ALL) can arise from minor subclones present at diagnosis. Several genes have been associated with therapy resistance in these subclones, including the Ras pathway genes KRAS, NRAS, and PTPN11, the H3K36 methyltransferase NSD2 (WHSC1), and the 5'-nucleotidase NT5C2. Retrospective backtracking of these relapse-associated alterations has demonstrated that these alterations are frequently present at time of diagnosis in minor subclones, sometimes in less than a few percent of the cells. The prognostic value of subclonal alterations in these genes at time of diagnosis, however, is less well understood. Prospective screening of subclonal mutations, without prior knowledge of the mutation status, requires extra specificity and sensitivity. Accurate quantification of the subclonal burden of these mutations will provide potential for following the subclonal dynamics during early stages of treatment, and could be informative for adapting therapy. The aim of this study was to develop a targeted next generation sequencing assay to perform quantitative detection of subclonal mutations in the selected genes. We used single molecule molecular inversion probes (smMIPs), an approach that applies single molecule tagging to correct for amplification biases (Hiatt et al., Genome Research. 2013, 23: 843-854), an artifact that becomes relevant in case of low mosaic mutations.Method: We designed a pool of 77 smMIP oligonucleotides targeting the coding sequences of five genes associated with therapy resistance in BCP-ALL, including KRAS, NRAS, PTPN11, NT5C2, and WHSC1. The smMIPs tiled a total of 4124bp of genomic sequence, including hotspot regions of the genes. To demonstrate the potential of this method, we applied this newly designed smMIP panel on 22 BCP-ALL diagnosis samples to retrospective backtrack mutations in KRAS (n=11), NRAS (n=8) and PTPN11 (n=3) that were previously characterized at relapse. We used 100ng of genomic DNA per sample as input, which is the equivalent of 15,000 haploid copies. Sequencing was performed on the Illumina NextSeq platform with pair-end sequencing, data were analyzed by SeqNext v4.2.2.Result: The average read depth obtained varied per gene from 30,081x (NRAS) to 65,749x (PTPN11). Sequencing reads with the same molecular tag were clustered into one tag-defined read group, in which random errors caused by library construction and sequencing were eliminated. These so-called single molecule consensus reads (smc-reads) were comprised of, on average, 139 individual sequencing reads. Using the smMIP approach, 19 out of the 22 Ras pathway mutations identified at relapse were detectable at diagnosis, of which 10 had a low mutant allele frequency (varying from 0.52-8.31%), which is in line with our previous ultra-deep backtracking result. Taking advantage of the known position of the mutations at relapse, we established the noise level in the diagnosis samples by analyzing variant calls outside the hotspot regions. The noise level was varied between samples from 0.03% to 0.24% (average 0.06%). Based on these background settings, we subsequently searched for novel mutations and identified 1 mutation in NT5C2 (p.P534S, 0.38%), 2 hotspot mutations in WHSC1 (p.E1099K, 0.17% and 0.27%), as well as many additional subclonal mutations in KRAS, NRAS and PTPN11. The latter finding suggests the presence of multiple Ras-mutated subclones in individual cases, of which only a subset survive from chemotherapy and grow out in the relapse clone.Conclusions: Taken together, single molecule tagging based smMIP technology allows the accurate detection of low mosaic mutations. These findings illustrate the need for the current ongoing prospective mutation screens in unbiased cohorts of diagnosis samples to determine the prognostic value of subclonal mutations in these five genes on the risk of relapse. DisclosuresNo relevant conflicts of interest to declare.

In situ X-ray data collection and structure phasing of protein crystals at Structural Biology Center 19-ID.

A prototype of a 96-well plate scanner for in situ data collection has been developed at the Structural Biology Center (SBC) beamline 19-ID, located at the Advanced Photon Source, USA. The applicability of this instrument for protein crystal diffraction screening and data collection at ambient temperature has been demonstrated. Several different protein crystals, including selenium-labeled, were used for data collection and successful SAD phasing. Without the common procedure of crystal handling and subsequent cryo-cooling for data collection at T = 100 K, crystals in a crystallization buffer show remarkably low mosaicity (<0.1°) until deterioration by radiation damage occurs. Data presented here show that cryo-cooling can cause some unexpected structural changes. Based on the results of this study, the integration of the plate scanner into the 19-ID end-station with automated controls is being prepared. With improvement of hardware and software, in situ data collection will become available for the SBC user program including remote access.

Controlling the Structural Properties of Single Step, Dip Coated ZnO Seed Layers for Growing Perfectly Aligned Nanowire Arrays

Mastering the structural ordering of ZnO seed layers by sol–gel process in terms of ultrathin thickness (i.e, <10 nm), strong c-axis texture, low mosaicity, low porosity, and low roughness is a critical challenge for the formation of well-ordered ZnO nanowires in solution. The effects of the solution concentration, of the withdrawal speed, and of the annealing process on the formation mechanisms of ZnO seed layers deposited by single dip process are revealed. The size and density of primary clusters in the sol are found to govern the evolution of the film thickness and nanoparticle average diameter through the solution concentration. The Landau–Levich theory modeling the dragging process accounts for the evolution of the film thickness only before annealing and over a reduced range of withdrawal speeds. The texture mechanisms along the c-axis are driven by particle/particle interactions during annealing and explained in the light of thermodynamic considerations. They are further determined locally by elec...