Low Mosaicism Research Articles

Tin Microcrystals Synthesized by Thermal Chemical Vapor Deposition in a Hydrogen-Reducing Atmosphere

We demonstrate a synthetic route to high-quality Sn microcrystals using thermal chemical vapor deposition (CVD) in a hydrogen-reducing atmosphere. During thermal CVD with SnO2 powder, a mixture of Ar and H2 (4%) gases was used as a carrier gas to maintain a hydrogen reduction environment. Hydrogen maintained a reduction environment throughout the thermal CVD process. Samples were prepared at temperatures of 800, 900, and 1000 °C. The structural properties of the Sn microcrystals were determined using synchrotron X-ray diffraction (XRD), transmission electron microscopy, and micro-Raman spectroscopy. In particular, the atomic position order in both the out-of-plane and in-plane directions was determined through high-resolution XRD measurement in a four-circle geometry. We found that at temperatures greater than 800 °C the supersaturation of Sn and O vapors induces the formation of microcrystals by self-assembly process without additional metallic seeds. As determined by the energy dispersive X-ray analysis, oxygen atoms and/or vapor were simultaneously reduced by hydrogen gas, resulting in the formation of Sn crystals rather than Sn oxide crystals. We also found that the Sn microcrystals were mostly strain-free single-crystalline and of high quality with extremely low mosaicity. The order of the atomic positions in both the out-of-plane and in-plane directions was comparable to that of single-crystalline sapphire. Key words: tin, single crystal, thermal CVD, hydrogen reduction, x-ray diffraction

Recurrence of Trisomic Pregnancies in Four Families: A Cytogenetic and Molecular Study

AbstractThe risk for recurrence of non disjunction trisomy 21 is conventionally considered to be less than 1%. Within a span of 3 years, we observed recurrence of non disjunction trisomies in four families. The objective of the present study was to determine low level mosaicism in either of the couple and to identify the parental origin of additional chromosome 21/18. The four couples who had recurrent trisomic conceptions were investigated for the underlying mosaicism by analysis of 100 cells from peripheral blood of the couple and the parental origin of supernumerary chromosome 21/18 were identified using microsatellite markers. Low level mosaicisms in peripheral lymphocytes of couple were ruled out for all four families. Microsatellite markers have shown maternal origin of chromosomal nondisjunction for all the families and defective first meiotic division as the most common mechanism for nondisjunction. This observation raises the need for discussing the option of invasive testing while counseling the couple with an affected child with non-disjunction trisomy as the risk for recurrence of trisomies in subsequent pregnancy might not be as low as 1%.

Consequences of Mississippi River diversions on nutrient dynamics of coastal wetland soils and estuarine sediments: A review

Coastal Louisiana is dominated by the Mississippi River Deltaic Plain, which is composed of a series of overlapping and truncated sub-delta lobes that formed an extensive coastal wetland – open water shallow bay and low relief upland mosaic across the entire coastal zone (∼25,000 km2). These coastal wetlands have been eroding at an alarming rate during the past century, coincident with major modifications to the deltaic landscape. One of these former modifications included extensive leveeing of the Mississippi River, essentially isolating the river from the coastal basins. One restoration technique involves re-connecting the previously isolated coastal basins to the river via diversions, which would once again allow the influx of freshwater, sediments and nutrients into the coastal basins. We review issues on potential impacts of nutrient loading from the river as a consequence of river diversions, focused on nitrogen (N) and phosphorus (P) dynamics including denitrification related to carbon dynamics. We also examined how water residence times in these shallow systems, compared to deeper, often seasonally stratified, coastal systems, are more likely to lead to greater N removal potential in the context of a large scale river diversion helping to reduce N transport to the coastal ocean.

A novel handling-free method of mounting single protein crystals for synchrotron structure analyses at room temperature.

We have developed a handling-free mounting method for X-ray crystallography of protein crystals at room temperatures-the glass capillary method. In this method, crystals were nucleated and grown on the capillary walls, and then, growth solutions were gently removed. The procedures for collecting data on the crystals were conducted by simply setting the capillary on the goniometer of a synchrotron beamline without touching the crystals. Crystal quality was characterized using mosaicity, resolution at I/σ(I) = 2, I/σ(I) at resolution = 2.0 Å, Rmerge, and completeness. Wilson plots were also used to characterize the quality of crystals. In particular, all samples showed very low mosaicity; the handling-free method successfully retained their low mosaicity and effectively maintained the crystal quality.

Why Day 3 biopsy mosaicism is blamed for the lack of success of PGD-A?

Introduction Preimplantation Genetic Diagnosis- Aneuploidy Screening (PGD-A) is a technique commonly used in conjunction with IVF treatments. PGD-A is intended to identify those embryos with greater likelihood to implant. It is offered to patients at increased risk of producing chromosomally abnormal embryos (increased maternal age, Recurrent miscarriage, IVF failure and severe male factor). Technical related Error rates of PGD-A have fallen from 7% when using FISH to less than 1% when using modern techniques such as Next Generation Sequencing (NGS). Another source of error that is related to the physiology of the embryos is mosaicism described as the presence of two or more cells lines within an embryo. The advantage of NGS made the detection of mosaicism easier due to the i) higher sensitivity in aneuploidy detection and ii) the reliability of the technique making non-mosaicism related error minimal. The objective of our study is to assess the mosaicism rate and the misdiagnosis rate due to euploid/aneuploid mosaic embryos on day 3 biopsy. Material and Methods Embryos from patients undergoing PGD-A were biopsied on day 3 and sent to our laboratory for Aneuploidy Screening by NGS. Blind Reanalysis ofembryonic cells from embryos diagnosed as aneuploid but with good morphology and development was performed. Rebiopsy was carried out either on day 4, day 5 or day 6. Both, original analysis and reanalysis were performed by NGS (MySeq, Illumina platform). Result(s) PGD-A was performed in our laboratory on 29309 embryos at cleavage stage from 2016 to date. Rebiopsy for 173 embryos, originally diagnosed as aneuploidy, were blindly reanalyzed. There was 118 out of 173 showed aneuploidy (91.3%) while 15 out of 173 were found euploids (8.7%). The aneuploid embryos were divided into 3 categories: first where 118 embryos out of 173 (68.2%) showed the same initial result; second, 37 of the 173 (21.4%) showed at least one aneuploidy that was present in the initial result and third 3 out 173 (1.7%) showed a different an inverted figure of deletion/duplication for the same chromosome. Conclusions This study shows a very high confirmation rate (more than 90%) for day 3 biopsies. Our misdiagnosis rate after day 3 biopsy is 8.7%, due to mosaicism. Our data support the usefulness of day 3 biopsy PGD-A by NGS in places where day 5 biopsy is not possible. The mosaic rate on a relatively high number of embryos observed in this study is in line with other similar reported data. Our reanalysis was based on rebiopsies on embryos showing a good division after the first biopsy which might explain the low mosaic rate. Despite the high accuracy in day 3 biopsy diagnosis, implantation/pregnancy rate is still in the range of 50-60% implicating the necessity of finding more embryonic viability markers allowing a better outcome after IVF/PGD-A.

Cell-free DNA analysis in renal cell carcinoma: Comparison with tumor sequencing and correlation with response to therapy.

655 Background: Massively parallel sequencing (MPS) of circulating-free DNA (cfDNA) is seeing increasing use in multiple cancer types. There is little data on its use in metastatic renal cell carcinoma (mRCC) as a tool for prognostication and disease monitoring. Methods: cfDNA was extracted from 63 blood samples of 40 metastatic RCC patients (pts). Serial samples were obtained in 12 of 40 (30%) pts (median = 1, range = 1-7). cfDNA was used for targeted MPS using a custom bait-set of 27 genes commonly mutated in RCC. Variants observed in at least 3 reads, in both read directions, and at an allele frequency (AF) of ≥0.5% for single nucleotide variants (SNV), or in 2 reads and AF of ≥0.2% for small indels, were candidate variants validated by Sanger sequencing or amplicon MPS (aMPS). All mutations identified in cfDNA were also assessed in matched patient WBC DNA using aMPS and Sanger sequencing. Tumor specimens from 23 pts were also sequenced in parallel using our institutional OncoPanel assay that assesses 275-447 cancer-associated genes and results were compared with those seen in the cfDNA. Results: Thirty-one of 38 (82%) candidate variants were validated in 17 of 40 pts. Ten of those (32%) from 10 pts were also detected in WBC DNA, 3 of which were germline and 7 were at low mosaic frequency and likely reflected clonal hematopoiesis (CH). The remaining 21 variants validated in cfDNA were in TP53 (6), PBRM1 (3), SETD2 (3), VHL (2), ATM (2), NF2 (2), PTEN (1), PIK3CA (1), and MTOR (1). Two of 17 (12%) pts without tumor mutation analysis had 4 validated variants seen in cfDNA only. 10 of 23 (43%) pts with tumor mutation analysis had one or more variants seen in both tumor DNA and cfDNA. Three of the 23 had mutations seen only in cfDNA. Pts with any mutation in cfDNA (n = 14) had a significantly shorter overall survival in comparison to those without a finding (p < 0.001). Among 12 pts with serial samples, 5 had cfDNA variants identified. Response to therapy correlated with variant prevalence in all 5, including 2 with significant partial responses. Conclusions: This study suggests that paired tumor–cfDNA analysis has value in the assessment of response to therapy in RCC. Further analysis is proceeding.

Rapid and high-efficient generation of mutant mice using freeze-thawed embryos of the C57BL/6J strain

BackgroundThe CRISPR/Cas9 technique has undergone many modifications to decrease the effort and shorten the time needed for efficient production of mutant mice. The use of fresh embryos consumes time and effort during oocytes preparation and fertilization before every experiment, and freeze-thawed embryos overcome this limitation. However, cryopreservation of 1-cell embryos is challenging. New methodWe introduce a protocol that combines a modified method for cryopreserving 1-cell C57BL/6J embryos with optimized electroporation conditions that were used to deliver CRISPR reagents into embryos, 1 h after thawing. ResultsFreeze-thawed 1-cell embryos showed similar survival rates and surprisingly high developmental rates compared to fresh embryos. Using our protocol, we generated several lines of mutant mice: knockout mice via non-homologous end joining (NHEJ) and knock-in mice via homology-directed repair (HDR) with high-efficient mutation rates (100%, 75% respectively) and a low mosaic rate within 4 weeks. Comparison with existing method (s)Our protocol associates the use of freeze-thawed embryos from an inbred strain and electroporation, and can be performed by laboratory personnel with basic training in embryo manipulation to generate mutant mice within short time periods. ConclusionWe developed a simple, economic, and robust protocol facilitating the generation of genetically modified mice, bypassing the need of backcrossing, with a high efficiency and a low mosaic rate. It makes the preparation of mouse models of human diseases a simple task with unprecedented ease, pace, and efficiency.

Interceptación y escorrentía del bosque altoandino en la reserva forestal protectora “El Malmo”

Los bosques tropicales son importantes a nivel mundial por su biodiversidad y los servicios ecosistémicos que proveen, son claves en el ciclo global del agua. Los cambios y presiones de origen antrópico que afectan a los bosques tropicales inciden en el papel fundamental de estos en la provisión de agua. En este trabajo se evalúa la relación de las coberturas vegetales presentes en el Bosque altoandino de la Reserva Forestal Protectora “El Malmo” con la calidad y cantidad de interceptación y escorrentía; la zona de vida analizada comprende cuatro tipos de coberturas: bosque denso altoandino, vegetación secundaria baja, plantación de latifoliadas y mosaico de pastos con espacios naturales. Se instalaron ocho montajes (dos por cobertura) compuestos cada uno de una parcela de escorrentía y un medidor de precipitación bajo el dosel; el levantamiento de datos se efectúo cada ocho días durante 24 semanas. Los resultados indican que la interceptación de la precipitación no difiere en cada cubierta, mientras que si se ve afectada la escorrentía superficial y su calidad respecto a sedimentos, cambios que obedecen fuertemente a las diferencias en las condiciones físicas del suelo. La cobertura que permite las mejores dimensiones de calidad y cantidad de agua es el bosque denso altoandino. La influencia de la intervención antrópica en el área y la presencia de invasoras afectan negativamente estas variables. Este trabajo aporta conocimiento sobre el comportamiento hidrológico de la Reserva, para su uso en la gestión forestal. Así mismo, genera información, hasta el momento inexistente, sobre la relación interceptación/escorrentía en bosques de la región Cundiboyacense, convirtiéndose en punto inicial de comparación para nuevas investigaciones en ecosistemas altoandinos.

Multiplex Ligation-Dependent Probe Amplification (MLPA) for Prenatal Diagnosis of Common Aneuploidies.

Multiplex Ligation-dependent Probe Amplification (MLPA) is a method to determine the copy number of up to 60 genomic DNA sequences in a single multiplex PCR based reaction.MLPA probes consist of two oligonucleotides that can hybridize next to each other on a certain DNA sequence of interest, where they are ligated. All ligated probes are subsequently amplified by PCR using a single set of primers. Each amplified MLPA probe has a unique length and can be visualized and quantified by capillary electrophoresis. As the primers are almost 100% consumed in the PCR reaction, the quantity of each PCR amplicon is proportional to the number of copies of each probe target sequence in the DNA sample. A trisomy 21 can therefore be detected by an approximately 50% increased signal of each chromosome 21 specific probe relative to reference samples.MLPA with the P095 Aneuploidy probemix for chromosomes 13, 18, 21, X and Y has been used as a rapid detection method on large numbers of samples from uncultured amniotic fluid or from chorionic villi. As compared to FISH and karyotyping, MLPA is more rapid, has a higher throughput, and is less expensive. MLPA however cannot detect low grade mosaicism, female triploidies, and copy number neutral chromosome abnormalities such as inversions and translocations.

Low temperature (Ts/Tm < 0.1) epitaxial growth of HfN/MgO(001) via reactive HiPIMS with metal-ion synchronized substrate bias

Low-temperature epitaxial growth of refractory transition-metal nitride thin films by means of physical vapor deposition has been a recurring theme in advanced thin-film technology for several years. In the present study, 150-nm-thick epitaxial HfN layers are grown on MgO(001) by reactive high-power impulse magnetron sputtering (HiPIMS) with no external substrate heating. Maximum film-growth temperatures Ts due to plasma heating range from 70 to 150 °C, corresponding to Ts/Tm = 0.10–0.12 (in which Tm is the HfN melting point in K). During HiPIMS, gas and sputtered metal-ion fluxes incident at the growing film surface are separated in time due to strong gas rarefaction and the transition to a metal-ion-dominated plasma. In the present experiments, a negative bias of 100 V is applied to the substrate, either continuously during the entire deposition or synchronized with the metal-rich portion of the ion flux. Two different sputtering-gas mixtures, Ar/N2 and Kr/N2, are employed in order to probe effects associated with the noble-gas mass and ionization potential. The combination of x-ray diffraction, high-resolution reciprocal-lattice maps, and high-resolution cross-sectional transmission electron microscopy analyses establishes that all HfN films have a cube-on-cube orientational relationship with the substrate, i.e., [001]HfN||[001]MgO and (100)HfN||(100)MgO. Layers grown with a continuous substrate bias, in either Ar/N2 or Kr/N2, exhibit a relatively high mosaicity and a high concentration of trapped inert gas. In distinct contrast, layers grown in Kr/N2 with the substrate bias synchronized to the metal-ion-rich portion of HiPIMS pulses have much lower mosaicity, no measurable inert-gas incorporation, and a hardness of 25.7 GPa, in good agreement with the results for epitaxial HfN(001) layers grown at Ts = 650 °C (Ts/Tm = 0.26). The room-temperature film resistivity is 70 μΩ cm, which is 3.2–10 times lower than reported values for polycrystalline-HfN layers grown at Ts = 400 °C.

First evidence of maternally inherited mosaicism in TGFBR1 and subtle primary myocardial changes in Loeys-Dietz syndrome: a case report

BackgroundLoeys-Dietz syndrome (LDS) is a rare multisystemic disorder characterized by vascular and skeletal abnormalities, with considerable intra- and interfamilial variability.Case presentationWe report the case of an 8-year-old male with clinical features of two distinct genetic disorders, namely LDS, manifesting in the first months by progressive aortic root dilatation, arterial tortuosity, bifid uvula, and inguinal hernias and oculocutaneous albinism (OCA) manifesting by white hair and skin that does not tan, nystagmus, reduced iris pigment with iris translucency, and reduced retinal pigment). We identified previously reported, homozygous mutations of TYR, c.1A > G (p.Met1Val) and heterozygous, missense mutation of TGFBR1, c.1460G > A (p.Arg487Gln). Family history revealed that his mother underwent multiple surgical repairs for recurrent hemorrhage originating from the buccal artery. Molecular studies confirmed a maternally inherited low grade TGFBR1 mutation somatic mosaicism (18% in peripheral blood leukocytes, 18% in buccal cells and 10% in hair root cells). Maternal cardiac investigations revealed peculiar cardiovascular features: mild tortuosity at the aortic arch, dilatation of the proximal abdominal aorta, multiple deep left ventricular myocardial crypts, and dysplastic mitral valve. TGFBR2 germline mosaicism has been described in three fathers of children carrying TGFBR2 mutations but, to the best of our knowledge, no case of maternally inherited TGFBR1 mutation mosaicism has been reported so far.ConclusionsThis case report suggests that individuals with somatic mosaicism might be at risk for mild and unusual forms of LDS but germline mosaicism can lead to full blown picture of the disease in offspring.

Embryos classified as low-grade mosaic (

The Preimplantation Genetic Diagnosis International Society (PGDIS) recommend the use of the high resolution next-generation sequencing (hr-NGS) that can detect mosaicism in excess of 20%. Therefore, embryos with less than 20% aneuploidy in the TE sample are considered as euploid and routinely transferred. Those over 80% as aneuploid and subsequently discarded. The 20-80% range as mosaic and transferred with caution, only in absence of euploid embryos. These cutoffs are currently debated. We recently reported the birth of 17 healthy babies from the transfer of 33 low grade (<50%) mosaic embryos with an overall live birth rate (LBR) and abortion rate (AR) similar to the LBR and AR obtained with euploid embryos (Rubino et al., 2018). The current study reports a grater number of cases. Retrospective analysis of 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. 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. NGS was performed by Nexgenomics, LLC, Pasadena by means of platform 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. The patients of the study group were matched 1:2 with the patients of the control group (only single euploid embryo transferred) 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 to have a normal karyotype. The results are summarized in Table 1. No birth defects were reported. We reported the birth of 33 babies with confirmed normal karyotype after the transfer of 55 low grade mosaic embryos. LBR and AR were similar to the ones obtained with the transfer of euploid embryos suggesting that low grade mosaic embryos (<50%) can have the same potential to give healthy newborn as euploid embryos and the PGDIS guidelines needs to be reconsidered. As far as we know this is the second study that provides data about the LBR of mosaic embryos (Greco et al., 2015). No deliveries were obtained from the transfer of complex mosaic embryos.Table 1Comparison of the pregnancy outcomes of low rate mosaic and control embryosType of mosaicismSegmental*Single-Double 1-12**Single-Double 13-22**X-YComplexTotalControlsAge38.8±6.440.8±6.936.3±5.049.136.2±4.039.1±6.439.3±6.3N of transfer cycles2215121555110Embryos transferred2215121555110Embryos Implanted16109113773Implantation rate (IR)72.3%66.7%75.0%-067.3%66.4%Abortions11101410Abortion rate (AR)6.3%10.0%11.1%--10.8%13.7%Deliveries1598103363Live birth rate(LBR)68.2%60.0%66.7%--60.0%57.3%*Segmental: regarding a piece of chromosome;**Single-double 1-12: regarding one of two entire chromosomes from number 1 to number 12;***Single-double 13-22: regarding one or two entire chromosomes from number 13 to number 22. Open table in a new tab

High doses of CRISPR/Cas9 ribonucleoprotein efficiently induce gene knockout with low mosaicism in the hydrozoan Clytia hemisphaerica through microhomology-mediated deletion

Targeted mutagenesis using CRISPR/Cas9 technology has been shown to be a powerful approach to examine gene function in diverse metazoan species. One common drawback is that mixed genotypes, and thus variable phenotypes, arise in the F0 generation because incorrect DNA repair produces different mutations amongst cells of the developing embryo. We report here an effective method for gene knockout (KO) in the hydrozoan Clytia hemisphaerica, by injection into the egg of Cas9/sgRNA ribonucleoprotein complex (RNP). Expected phenotypes were observed in the F0 generation when targeting endogenous GFP genes, which abolished fluorescence in embryos, or CheRfx123 (that codes for a conserved master transcriptional regulator for ciliogenesis) which caused sperm motility defects. When high concentrations of Cas9 RNP were used, the mutations in target genes at F0 polyp or jellyfish stages were not random but consisted predominantly of one or two specific deletions between pairs of short microhomologies flanking the cleavage site. Such microhomology-mediated (MM) deletion is most likely caused by microhomology-mediated end-joining (MMEJ), which may be favoured in early stage embryos. This finding makes it very easy to isolate uniform, largely non-mosaic mutants with predictable genotypes in the F0 generation in Clytia, allowing rapid and reliable phenotype assessment.

Mosaic secret-fragment-visible data hiding for secure image transmission based on two-step energy matching

Recently, a new information hiding technology was proposed to hide a secret image into secret-fragment-visible mosaic image for secure image transmission. However, the previous method pays less attention to the block matching in the transform, and may obtain a low quality mosaic transform in some cases. In this paper, we propose to improve the secret-fragment-visible data hiding based the two-step energy function, which returns a weighted value of standard deviation and total variation of block. We match all the blocks according to the energy function and take the zero-mean normalized cross correlation (ZNCC) as the similarity measurement to optimize the matching. The interpolation optimization algorithm is used to stimulate the total ZNCC changes along with the parameter selection, and helps to solve the optimization quickly. Since more block's statistical properties are employed, the image blocks can be matched accurately, and a low distortion is introduced in the transform. Experimental results show that the proposed method can obtain a better visual quality of secret image after extraction and also requires less bits to encode the residuals.

A mosaic form of microphthalmia with linear skin defects

BackgroundMicrophthalmia with linear skin defects (MLS) syndrome is a rare neurodevelopmental X-dominant disorder. It presents in females as it is normally lethal in males. Three causative genes for MLS syndrome (OMIM 309801) have been identified all taking part in mitochondrial respiratory chain and oxidative phosphorylation. In our case, we describe a newborn with mosaic deletion encompassing HCCS gene resulting in unilateral microphthalmia and facial skin lesions.Case presentationA girl was born with caesarean section at 40 weeks of gestation. Clinical findings revealed anophthalmia of the left eye. The left eyelids were intact, the orbit was empty and the right eye was normal, without any abnormalities. She had typical linear skin defects on the left cheek, one on the left side of the neck, and two on the 3th and 4th fingers of the left hand. The other clinical findings and the neurological exam were normal. US of the brain and EEG were normal. Molecular karyotyping using BlueGnome CytoChip Oligo 4× 180K array was performed detecting an approximately 18% mosaic 3.3 Mb deletion (arr[GRCh37] Xp22.31p22.2(8,622,553_11,887,361)× 1[0.18]). FISH using RPCI11-768H20 BAC clone on cultivated interphase and metaphase lymphocytes was used to confirm the array results. The observed deletion was present in 29% of cells (46,XX,ish del(p22.2p22.31)(RPCI11-768H20)[60/205]).ConclusionsIn this report we present a female proband with MLS syndrome. To our knowledge, there have been only few other cases of mosaic MLS syndrome described in the literature. Our case shows that low grade mosaicism does not preclude full clinical presentation and further supports the critical role of the X inactivation pattern in the development of the clinical findings.

30. Clinical utility of NGS in the understanding of crosstalk between nuclear cancer genes and mitochondrial genome in tumorigenesis

Reprogramming of energy metabolism is one of the hallmarks of cancers. Dynamic bioenergetics changes can be evidenced by the detection of deleterious mutations in nuclear or mitochondrial genes related to energy metabolism and cancer during tumorigenesis. In order to identify these molecular changes, we developed a comprehensive next-generation sequencing (NGS) approach by target capture-sequencing of 183 cancer-related genes with an average read depth of approximately 1000X. The deep coverage facilitates simultaneous detection of exonic copy number variants (CNVs) and single nucleotide variants (SNVs) at low somatic mosaicism in one comprehensive assessment. Additionally, the circular mitochondrial genome is analyzed as a single amplicon using one pair of back to back primers for the long-range PCR-based enrichment coupled with massively parallel sequencing at an average coverage of ∼20,000X. Thirty Trio (tumor, adjacent tissue, and blood) of colon cancers (20 carcinomas and 10 polyps) were studied. Variants detected are classified according to ACMG guidelines. Out of 30 Trio samples, germline variants were found in 11 patients, 4 of which have pathogenic mutations. Somatic nuclear driver mutations are present in all tumors at >15%. As for mtDNA variants, 1 out of 30 carries deleterious germline mtDNA mutations and 5 harbor somatic pathogenic mtDNA variants. Upon completion of data analysis of 30 Trio samples, it demonstrates energy reprogramming is the pathway intimately correlated with tumorigenesis even though every tumor develops its unique mechanism leading to tumorigenesis.

Efficient Multiplex Genome Editing Induces Precise, and Self-Ligated Type Mutations in Tomato Plants.

Several expression systems for multiple guide RNA (gRNAs) have been developed in the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) system to induce multiple-gene modifications in plants. Here, we evaluated mutation efficiencies in the tomato genome using multiplex CRISPR/Cas9 vectors consisting of various Cas9 expression promoters with multiple gRNA expression combinations. In transgenic tomato calli induced with these vectors, mutation patterns varied depending on the promoters used to express Cas9. By using the tomato ELONGATION FACTOR-1α (SlEF1α) promoter to drive Cas9, occurrence of various types of mutations with high efficiency was detected in the tomato genome. Furthermore, sequence analysis showed that the majority of mutations using the multiplex system with the SlEF1α promoter corresponded to specific mutation pattern of deletions produced by self-ligation at two target sites of CRISPR/Cas9 with low mosaic mutations. These results suggest that optimizing the Cas9 expression promoter used in CRISPR/Cas9-mediated mutation improves multiplex genome editing, and could be used effectively to disrupt functional domains precisely in the tomato genome.

Somatic copy number gains of α-synuclein (SNCA) in Parkinson's disease and multiple system atrophy brains.

The α-synuclein protein, encoded by SNCA, has a key role in the pathogenesis of Parkinson's disease and other synucleinopathies. Although usually sporadic, Parkinson's disease can result from inherited copy number variants in SNCA and other genes. We have hypothesized a role of somatic SNCA mutations, leading to mosaicism, in sporadic synucleinopathies. The evidence for mosaicism in healthy and diseased brain is increasing rapidly, with somatic copy number gains of APP reported in Alzheimer's brain. Here we demonstrate somatic SNCA copy number gains in synucleinopathies (Parkinson's disease and multiple system atrophy), focusing on substantia nigra. We selected sporadic cases with relatively young onset or short disease duration, and first excluded high level copy number variant mosaicism by DNA analysis using digital PCR for SNCA, and/or customized array comparative genomic hybridization. To detect low level SNCA copy number variant mosaicism, we used fluorescent in situ hybridization with oligonucleotide custom-designed probes for SNCA, validated on brain and fibroblasts with known copy number variants. We determined SNCA copy number in nigral dopaminergic neurons and other cells in frozen nigra sections from 40 cases with Parkinson's disease and five with multiple system atrophy, and 25 controls, in a blinded fashion. Parkinson's disease cases were significantly more likely than controls to have any SNCA gains in dopaminergic neurons (P = 0.0036), and overall (P = 0.0052). The average proportion of dopaminergic neurons with gains in each nigra was significantly higher in Parkinson's disease than controls (0.78% versus 0.45%; P = 0.017). There was a negative correlation between the proportion of dopaminergic neurons with gains and onset age in Parkinson's disease (P = 0.013), but not with disease duration, or age of death in cases or controls. Cases with tremor at onset were less likely to have gains (P = 0.035). All multiple system atrophy cases had gains, and the highest levels in dopaminergic neurons were in two of these cases (2.76%, 2.48%). We performed selective validation with different probes after dye swapping. All three control probes used showed minimal or no gains (≤0.1% in dopaminergic neurons). We also found occasional SNCA gains in frontal neurons of cases with Parkinson's disease, and the putamen of one multiple system atrophy case. We present evidence of somatic SNCA gains in brain, more commonly in nigral dopaminergic neurons of Parkinson's disease than controls, negatively correlated with onset age, and possibly commonest in some multiple system atrophy cases. Somatic SNCA gains may be a risk factor for sporadic synucleinopathies, or a result of the disease process.10.1093/brain/awy157_video1awy157media15813519976001.

Unusual association of a unique CAG interruption in 5' of DM1 CTG repeats with intergenerational contractions and low somatic mosaicism.

Myotonic dystrophy type 1 (DM1) is a dominant multisystemic disorder associated with high variability of symptoms and anticipation. DM1 is caused by an unstable CTG repeat expansion that usually increases in successive generations and tissues. DM1 family pedigrees have shown that ∼90% and 10% of transmissions result in expansions and contractions of the CTG repeat, respectively. To date, the mechanisms of CTG repeat contraction remain poorly documented in DM1. In this report, we identified two new DM1 families with apparent contractions and no worsening of DM1 symptoms in two and three successive maternal transmissions. A new and unique CAG interruption was found in 5' of the CTG expansion in one family, whereas multiple 5' CCG interruptions were detected in the second family. We showed that these interruptions are associated with maternal intergenerational contractions and low somatic mosaicism in blood. By specific triplet-prime PCR, we observed that CTG repeat changes (contractions/expansions) occur preferentially in 3' of the interruptions for both families.

CGH Array and Karyotype as Complementary Tools in Prenatal Diagnosis: Prenatal Diagnosis of a 4q Derivative Chromosome from Maternal 4q;11q Translocation

ABSTRACTBackground: There is controversy whether chromosomal microarray (CMA) can replace karyotyping in prenatal diagnosis. Chromosomal microarray may detect more clinically significant chromosomal imbalances than karyotyping in a shorter time but does not detect inversions, triploidies or low mosaicisms. Case report: Amniocentesis was performed in the late second trimester based on ultrasound abnormalities. A CMA, obtained at 10 days, demonstrated a terminal deletion in 4q34.3-q35.2 and a duplication in 11q21-q25. The karyotype results, obtained 1 week later, showed a derivative chromosome 4 inherited from a maternal balanced 4;11 translocation. Conclusion: CMA and karyotype were complementary in this case, together permitting a more accurate diagnosis and genetic counseling than if only one method was used.