Parental DNA Research Articles

O-064 Genome-wide analysis of meiotic recombination reveals a subset of oocytes with reduced frequency of recombination and increased risk of aneuploidy.

Abstract Study question Are there differences in recombination rates between male and female embryos, and is the frequency of recombination associated with the risk of aneuploidy? Summary answer There is more recombination in female meiosis than in males. Aneuploidy is associated with fewer recombination events. Genome-wide reduction in recombination predisposes oocytes to aneuploidy. What is known already Aneuploidy is believed to be the primary explanation for embryo implantation failure and miscarriage and is, therefore, of great relevance to IVF. Despite the high frequency and clinical importance of aneuploidy, the mechanisms causing a gamete to become chromosomally abnormal are only partially understood. One factor known to influence the risk of aneuploidy is meiotic recombination. An absence of recombination or atypical positioning of chiasmata predisposes to chromosomal malsegregation. Most data on the subject of meiotic recombination and aneuploidy comes from miscarriages and aneuploid births, but this provides an incomplete view since most aneuploidies are lethal at earlier developmental stages. Study design, size, duration ∼300,000 polymorphisms scattered across the genome were genotyped in DNA from 125 couples and 543 blastocysts that they produced. The inheritance of individual alleles was tracked from parents to embryos, allowing the parental origin of each chromosome to be determined. This strategy also permitted the detection of abnormal numbers of chromosomes, allowed deduction of which meiotic division aneuploidy had arisen in, and revealed the sites of meiotic recombination on each chromosome in the embryos analysed. Participants/materials, setting, methods Embryo analysis involved trophectoderm biopsy, followed by multiple displacement amplification. Parental DNA and amplified embryo samples were then tested using a microarray. 22,133 individual recombination events on 14,561 chromatids were evaluated. The relationship between aneuploidy and recombination frequency was considered. The average distance between recombination events was calculated by dividing the size of each autosome (in Mega bases – Mb) by the number of detected recombination events. An unpaired t-test was conducted for statistical analysis. Main results and the role of chance A quarter of all embryos were found to carry one or more chromosome abnormalities (altogether, 221 chromosome abnormalities were detected). Of these, 82% had a maternal (oocyte) origin, while 18% were paternal. Chromosomes 16 and 22 were the most frequently affected by aneuploidy, together accounting for 33% of all maternal chromosome abnormalities. Analysis of recombination events in maternally and paternally inherited chromosomes confirmed that female meiosis is associated with a significantly higher frequency of recombination (41.4 ±7.7 recombination events compared with 24.4±4 in chromosomes of paternal origin). Chromosomes 21 and 22 showed the lowest number of recombination events, consistent with their small size (average of 0.6 and 0.7 recombination sites per chromosome, respectively). As expected, a complete failure of recombination occurred more frequently for chromosomes with lower numbers of recombination events. Interestingly, embryos with aneuploidies of maternal origin showed evidence that the oocytes that produced them had lower recombination rates in general (not restricted to the affected chromosomes) than those producing euploid embryos (P = 0.01). Across the genome, average distances between recombination events were ∼10% lower for euploid embryos compared with aneuploid embryos (72.4 +/-14 Mb versus 81+-18 Mb) (P = 0.0004). Limitations, reasons for caution It is possible that the pattern and frequency of recombination sites that exist in sperm and oocytes might show some differences from the blastocysts tested during this study, considering that more than half of all oocytes either fail to fertilise or produce embryos that arrest before the blastocyst stage. Wider implications of the findings As expected, a higher aneuploidy rate and more recombination events were seen in female meiosis compared with male meiosis. Interestingly, aneuploidy was associated with lower levels of recombination across the whole genome, not only for the affected chromosomes, suggesting that unusually low recombination activity predisposes some oocytes to chromosome malsegregation. Trial registration number not applicable

O-304 Whole-exome sequencing in women with reproductive failure is a potentially useful diagnostic test in clinical practice

Abstract Study question Could whole-exome sequencing (WES) be useful in clinical practice for primary infertile female involving oocyte or embryo defects and unexplained recurrent miscarriage (RM) patients? Summary answer Identifying key genes involved in oocyte and embryo development helps explain approximately 17% cases of reproductive failure. WES results will help optimize clinical therapeutic treatment. What is known already Dozens of genes have been reported to be the genetic causes of human infertility. Variants in these genes have specific effects on certain processes of human early reproduction and result in a spectrum of phenotypes, such as oocyte maturation arrest, fertilization defects, cleavage failure, early embryonic lethality, and recurrent miscarriage. Study design, size, duration This retrospective cohort study was conducted from July 2020 to August 2022. A total of 129 affected females were enrolled. Most of them were primary infertile women who suffered repeated failure of in vitro fertilization (≥2 cycles) due to abnormal development of oocytes or embryos (PI group, n = 125). Our cohort also comprised a small number of unexplained RM subjects without offspring (RM group, n = 4). All patients and their spouses had normal karyotype. Participants/materials, setting, methods The peripheral blood samples from all the participants and all of their available family members were obtained for DNA extraction. Genomic DNA samples from the probands were subjected to WES to identify candidate variants. Subsequently, the variants were validated by Sanger sequencing or qPCR. Familial co-segregation analyses were then carried out within the family members. The relationship between phenotype and genotype by clinical tests and the clinical records of the patients was studied. Main results and the role of chance Firstly, variants that are potentially relevant to the phenotypes of female infertility were identified in 17.05% of cases (n = 22), including TUBB8 in 14 cases, PATL2 in 2 cases, ZP2, ZP3, PANX1, TLE6, NLRP2 and NLRP7 in 1 case for each. A breakdown by phenotype revealed that we identified variants in 16.00% (n = 20) of PI group and 50% (n = 2) of RM group. Secondly, 16 TUBB8 variants were identified in 14 cases, of which 8 were novel, 3 were previously reported, and 5 were novel amino acid change occurring at the same position as another previously reported change. We found 3 TUBB8 variants (p.C211R, p.T232I, p.A342T) in 3 embryonic arrest families and 1 TUBB8 mutation (p.A102V) in a RM family, which were maternally inherited. Thirdly, 75 patients in PI group without phenotypic variant underwent ≥1 cycles of ovulation and/or frozen embryo transfer after WES, and 49.33% had achieved clinical pregnancy (n = 37). Limitations, reasons for caution The genomic DNA of parents were not available in all these 22 families, so we cannot determine whether the variant is de novo or inherited in some cases. Additionally, further functional studies should be performed to prove that these novel mutations affect protein function. Wider implications of the findings The present study expands the kinds of variants and phenotypic spectrum of gene mutations with regard to female reproductive failure. Genetic tests for causative genes involved in oocyte and embryo defects were recommended for primary infertile patients who suffered from these conditions and for unexplained RM patients. Trial registration number not applicable

O-209 The incidence of different ploidy alterations in abnormally fertilized oocytes (AFO)-derived embryos

Abstract Study question Which is the incidence of de novo ploidy alterations in different abnormally fertilized embryos categories analysed with SNP-array? Summary answer Based on the number of pronuclei (PN) identified, 0PN embryos were mostly diploid, while 1PN and >2PN-derived embryos showed increased rates of ploidy abnormalities. What is known already Recent development of SNP-array and haplotyping by sequencing approaches are contributing to expanding preimplantation genetic testing (PGT) clinical utility including ploidy level evaluation. When applied to euploid embryos derived from AFO, the identification of abnormal ploidy constitution (i.e., haploidy, triploidy, or tetraploidy) could rescue viable diploid embryos otherwise considered non-transferable in IVF. However, no definitive genetic evidence has been obtained showing the incidence of different ploidy abnormalities in each AFO-derived blastocyst category. Here we present 4 years of experience in the clinical application of a validated SNP-array based protocol and custom-made algorithm to detect ploidy defects in AFO-derived embryos. Study design, size, duration Prospective observational study evaluating the incidence of altered ploidy configurations in AFO-derived blastocyst. After PN check, AFO samples were divided as follow: absence of observed pronuclei (0PN), monopronuclear (1PN), more than two pronuclei observed (2.1PN, with one smaller additional PN and 3PN). Genetic classification combining PGT-A and ploidy analysis was performed at Igenomix Italy laboratory between May 2019 and January 2023 on 133 AFO-derived embryos (44 0PN, 59 1PN, 30 >2PN). Participants/materials, setting, methods Multi-centre study involving 293 consenting patients of advance maternal age (mean=38.6±3.9) undergoing PGT-A on MDA-WGA using Ion ReproSeq kit and IonTorrent S5 (ThermoFisher). SNP-array-based ploidy test was performed using HumanKaryomap-12 kit and NextSeq550 (Illumina). Proprietary algorithm was based on genome-wide BAF obtained: expected BAFs were 1, 0.5 or 0 for diploids,1 or 0 for haploid and 1, 0.66, 0.33 or 0 for triploids, as determined by the frequency of each allele for 300000 SNPs loci. Main results and the role of chance Preclinical validation of SNP-array-based ploidy test included 26 samples with known ploidy status and karyotype: 7 triploid re-biopsies, 7 haploid left-overs and 12 cell lines. Moreover, inter-platform comparison of 72 diploids, 9 triploids, 8 haploids embryos analysed with an alternative targeted-NGS approach, validated by Igenomix, showed 100% (95% CI= 95.94%-100%) concordance. In this study, a total of 318 AFO-derived embryos were collected for PGT-A analysis for different indications. Of these, 58.2% (n = 185/318; 95%CI=52.54-63.66) resulted as aneuploid. The remaining 133 euploid AFO-derived blastocyst where subjected to SNP-array based ploidy assessment. 0PN-derived blastocysts (n = 44/133) were diploid in 93.2% (n = 41/44; 95%CI=81.34-98.57) of cases; only 4.5% (n = 2/44; 95%CI=0.56-15.47) were haploid and 2.3% (n = 1/44; 95%CI=0.06-12.02) were polyploid. 1PN-derived blastocysts showed all possible ploidy configurations in these proportions: 59.3% (n = 35/59; 95%CI=45.74-71.93) haploid, 35.6% (n = 21/59; 95%CI=23.55-49.13) diploid and 5.1% (n = 3/59; 95%CI=1.06-14.15) polyploid. Finally, in the group of AFO where more than 2PN were observed (2.1PN or 3PN), the additional PN resulted in 66.7% (n = 20/30; 95%CI=47.19-82.71) of polyploid configurations, the remaining 33.3% (n = 10/30; 95%CI=17.29-52.81) were diploid. No haploid results were obtained from this category. Chi-square test showed significant overall correlation between PN category and ploidy status configurations (p < 0.05). Collectively, ploidy evaluation of 133 AFO-derived embryos, allowed the clinical use of 72 additional euploid/diploid embryos otherwise not considered for transfer. Limitations, reasons for caution Ploidy assessment protocol was not tested to distinguish triploids from tetraploids. Parental origin of each chromosomal set could not be determined without analysing parental DNA. The way of performing PN check could have affected AFO classification. In future studies we will focus on evaluating clinical outcomes following euploid/diploid embryo transfer. Wider implications of the findings An altered number of pronuclei is predictive of the correspondent altered ploidy status, while 0PN category doesn’t represent a clear indication for ploidy evaluation. Nevertheless, a significant proportion of euploid/diploid embryos can be rescued from all types of AFO, potentially increasing the overall chance to achieve a live birth. Trial registration number n/a

Germline de novo mutations in families with Mendelian cancer syndromes caused by defects in DNA repair

DNA repair defects underlie many cancer syndromes. We tested whether de novo germline mutations (DNMs) are increased in families with germline defects in polymerase proofreading or base excision repair. A parent with a single germline POLE or POLD1 mutation, or biallelic MUTYH mutations, had 3-4 fold increased DNMs over sex-matched controls. POLE had the largest effect. The DNMs carried mutational signatures of the appropriate DNA repair deficiency. No DNM increase occurred in offspring of MUTYH heterozygous parents. Parental DNA repair defects caused about 20–150 DNMs per child, additional to the ~60 found in controls, but almost all extra DNMs occurred in non-coding regions. No increase in post-zygotic mutations was detected, excepting a child with bi-allelic MUTYH mutations who was excluded from the main analysis; she had received chemotherapy and may have undergone oligoclonal haematopoiesis. Inherited DNA repair defects associated with base pair-level mutations increase DNMs, but phenotypic consequences appear unlikely.

The disease-causing mutation p.F907I reveals a novel pathogenic mechanism for POLγ-related diseases

Mutations in the catalytic domain of mitochondrial DNA polymerase γ (POLγ) cause a broad spectrum of clinical conditions. POLγ mutations impair mitochondrial DNA replication, thereby causing deletions and/or depletion of mitochondrial DNA, which in turn impair biogenesis of the oxidative phosphorylation system. We here identify a patient with a homozygous p.F907I mutation in POLγ, manifesting a severe clinical phenotype with developmental arrest and rapid loss of skills from 18 months of age. Magnetic resonance imaging of the brain revealed extensive white matter abnormalities, Southern blot of muscle mtDNA demonstrated depletion of mtDNA and the patient deceased at 23 months of age. Interestingly, the p.F907I mutation does not affect POLγ activity on single-stranded DNA or its proofreading activity. Instead, the mutation affects unwinding of parental double-stranded DNA at the replication fork, impairing the ability of the POLγ to support leading-strand DNA synthesis with the TWINKLE helicase. Our results thus reveal a novel pathogenic mechanism for POLγ-related diseases.

Protocol of the Double-Click Seq Method to Evaluate the Deposition Bias of Chromatin Proteins.

Symmetrical deposition of parental and newly synthesized chromatin proteins over both sister chromatids is important for the maintenance of epigenetic integrity. However, the mechanisms to maintain equal distribution of parental and newly synthesized chromatid proteins over sister chromatids remains largely unknown. Here, we describe the protocol for the recently developed double-click seq method that enables mapping of asymmetry in the deposition of parental and newly synthesized chromatin proteins on both sister chromatids in DNA replication. The method involved metabolic labeling of new chromatin proteins with l-Azidohomoalanine (AHA) and newly synthesized DNA with Ethynyl-2'-deoxyuridine (EdU) followed by two subsequent click reactions for biotinylation and subsequently by corresponding separation steps. This enables isolation of parental DNA that was bound to nucleosomes containing new chromatin proteins. Sequencing of these DNA samples and mapping around origins of replication in the cellular DNA enables estimation of the asymmetry in deposition of chromatin proteins over the leading and lagging strand in DNA replication. Altogether, this method contributes to the toolbox to understand histone deposition in DNA replication. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Metabolic labeling with AHA and EdU and isolation of nuclei Basic Protocol 2: First click reaction, MNase digestion and streptavidin enrichment of labeled nucleosomes Basic Protocol 3: Second click reaction, Replication-Enriched Nucleosome Sequencing (RENS) Protocol.

CBL Syndrome: At the Intersection of Inborn Error of Immunity and Cancer Predisposition

CBL syndrome is a form of RASopathies due to heterozygous germline mutations of the Casitas B-lineage lymphoma (CBL) gene. CBL encodes the cbl family of proteins which function as E3 ubiquitin ligases that negatively regulate intracellular signaling by various receptor tyrosine kinases including RAS-MAPK pathway. Individuals with CBL syndrome have variable clinical presentation but phenotypically resembles Noonan syndrome with predisposition to JMML. Patients affected by RASopathies are at risk to develop autoimmune disorders given the role of Ras-MAPK signaling pathway in peripheral tolerance. Prior mice studies suggested the role of cbl protein as negative regulator of TCR-mediated signaling. We describe a case of CBL syndrome presenting with multiple autoimmune conditions and reviewed reported cases in literature.A 14-year old male presented with pancytopenia, autoimmune thyroiditis and organomegaly. His past medical history was remarkable for ADHD, speech delay, short stature, idiopathic hepatosplenomegaly and history of acute disseminated encephalomyelitis at age 5. His bone marrow evaluation was normal. A somatic NGS panel identified VUS in CBL (c.1193A>G, p.H398R) with VAF of 53%. Autoimmune work up showed elevated ANA, DNA antibody, RNP antibody titer and mildly positive anti-C3 antibody. Immunophenotyping revealed normal Ig profile, NK cytopenia, T cell lymphopenia, decreased naïve T cells and elevated CD8+ effector memory T cell activation. ALPS genetic panel revealed paternally inherited VUS in CTLA4 which was later determined to be benign. Since his cytopenias did not respond to IVIG, he was treated with corticosteroid with improvement in counts although he remained thrombocytopenic. A skin biopsy was performed and sequencing of patient’s fibroblast DNA demonstrated a germline origin of CBL variant. Parental DNA sequencing confirmed a de novo event. Literature review revealed 28 germline CBL mutations (Figure 1), most of which were missense mutations (78%). At least nine of these mutations (32%) have been associated with immune dysregulation including autoimmune cytopenias, aHUS, RA, autoimmune thyroiditis, vasculitis and HLH. JMML has been reported in more than half of these germline mutations. [Display omitted] In conclusion, immune dysregulation is a common feature of CBL syndrome in addition to JMML. CBL syndrome should be considered in the differential diagnosis of individuals with ALPS-like phenotypes.

RAD51 bypasses the CMG helicase to promote replication fork reversal.

Replication fork reversal safeguards genome integrity as a replication stress response. DNA translocases and the RAD51 recombinase catalyze reversal. However, it remains unknown why RAD51 is required and what happens to the replication machinery during reversal. We find that RAD51 uses its strand exchange activity to circumvent the replicative helicase, which remains bound to the stalled fork. RAD51 is not required for fork reversal if the helicase is unloaded. Thus, we propose that RAD51 creates a parental DNA duplex behind the helicase that is used as a substrate by the DNA translocases for branch migration to create a reversed fork structure. Our data explain how fork reversal happens while maintaining the helicase in a position poised to restart DNA synthesis and complete genome duplication.

Effects of Thinking Maps in the Development of Higher Order Thinking Skills among Elementary Science Students

The study involved using thinking maps to develop higher order thinking skills (HOTS) of fifth-grade science students in Starline Elementary School. It utilized the quasi-experimental design to assess the effects of Thinking Maps on the development of higher order thinking skills (HOTS) through a pretest and a posttest. The descriptive- correlational design was used to describe the students’ profiles, examine their perceptions of the effect of using Thinking Maps on learning science, and investigate the relationship between profile variables and achievement in science. This study was conducted from April through May during the school year 2021-2022. Purposive sampling was used to determine the number of respondents. Students’ achievement in science was determined from the Individual Student Performance on class tests for CKLA Science based on Parent Vue (Synergy) and DnA Illuminate District Assessment. Results showed a significant correlation of the students’ profile variables such as sex, age, and economic status to their achievement in science using Thinking Maps. Results also indicated a significant difference between the respondents’ pretest and posttest scores after using Thinking Maps. There was a significant difference in terms of developing higher order thinking skills, organizing, providing evidence, reasoning, and clarity. As to their perceptions about Thinking Maps, the students generally strongly agree that using them has improved their essay-writing skills and their interest in learning science. However, there was no significant relationship between the academic achievement of the respondents and their perception of Thinking Maps.

Identifying parental and cell-division origins of aneuploidy in the human blastocyst

Preimplantation genetic testing commonly employs simplistic copy-number analyses to screen for aneuploidy in blastocyst trophectoderm biopsies. Interpreting intermediate copy number alone as evidence of mosaicism has led to suboptimal estimation of its prevalence. Because mosaicism originates from mitotic nondisjunction, utilizing SNP microarray technology to identify the cell-division origins of aneuploidy might provide a more accurate estimation of its prevalence. The present study develops and validates a method of determining the cell-division origin of aneuploidy in the human blastocyst by using both genotyping and copy-number data in parallel. The concordance of predicted origins with expected results was demonstrated in a series of truth models (99%-100%). This included determination of X chromosome origins from a subset of normal male embryos, determination of the origins of translocation chromosome-related imbalances via embryos from couples with structural rearrangements, and prediction of either mitotic or meiotic origins via multiple rebiopsies of embryos with aneuploidy. In a cohort of blastocysts with parental DNA (n= 2,277), 71% were euploid, 27% were meiotic aneuploid, and 2% were mitotic aneuploid, indicating a low frequency of bona fide mosaicism in the human blastocyst (mean maternal age: 34.4). Chromosome-specific trisomies in the blastocyst were also consistent with observations previously established in products of conception. The ability to accurately identify mitotic-origin aneuploidy in the blastocyst could benefit and better inform individuals whose IVF cycle results in all aneuploid embryos. Clinical trials with this methodology might also help provide a definitive answer regarding the reproductive potential of bona fide mosaic embryos.

DNA methylation remodeling in F1 hybrids.

F1 hybrids derived from a cross between two inbred parental lines often display widespread changes in DNA methylation patterns relative to their parents. To which extent these changes drive non-additive gene expression levels and phenotypic heterosis in F1 individuals is not fully resolved. Current mechanistic models propose that DNA methylation remodeling in hybrids is the result of epigenetic interactions between parental alleles via small interfering RNA (sRNA). These models have strong empirical support but are limited to genomic regions where the two parental lines differ in DNA methylation status. However, most remodeling events occur in parental regions with similar methylation patterns, and seem to be strongly conditioned by distally acting factors, even in isogenic hybrid systems. The molecular basis of these distal interactions is currently unknown, and will likely emerge as an active area of research in the future. Despite these gaps in our molecular understanding, parental DNA methylation states are statistically associated with heterosis, independent of genetic information, and may serve as biomarkers in crop breeding.

Improved clinical utility of preimplantation genetic testing through the integration of ploidy and common pathogenic microdeletions analyses.

Can chromosomal abnormalities beyond copy-number aneuploidies (i.e. ploidy level and microdeletions (MDs)) be detected using a preimplantation genetic testing (PGT) platform? The proposed integrated approach accurately assesses ploidy level and the most common pathogenic microdeletions causative of genomic disorders, expanding the clinical utility of PGT. Standard methodologies employed in preimplantation genetic testing for aneuploidy (PGT-A) identify chromosomal aneuploidies but cannot determine ploidy level nor the presence of recurrent pathogenic MDs responsible for genomic disorders. Transferring embryos carrying these abnormalities can result in miscarriage, molar pregnancy, and intellectual disabilities and developmental delay in offspring. The development of a testing strategy that integrates their assessment can resolve current limitations and add valuable information regarding the genetic constitution of embryos, which is not evaluated in PGT providing new level of clinical utility and valuable knowledge for further understanding of the genomic causes of implantation failure and early pregnancy loss. To the best of our knowledge, MDs have never been studied in preimplantation human embryos up to date. This is a retrospective cohort analysis including blastocyst biopsies collected between February 2018 and November 2021 at multiple collaborating IVF clinics from prospective parents of European ancestry below the age of 45, using autologous gametes and undergoing ICSI for all oocytes. Ploidy level determination was validated using 164 embryonic samples of known ploidy status (147 diploids, 9 triploids, and 8 haploids). Detection of nine common MD syndromes (-4p=Wolf-Hirschhorn, -8q=Langer-Giedion, -1p=1p36 deletion, -22q=DiGeorge, -5p=Cri-du-Chat, -15q=Prader-Willi/Angelman, -11q=Jacobsen, -17p=Smith-Magenis) was developed and tested using 28 positive controls and 97 negative controls. Later, the methodology was blindly applied in the analysis of: (i) 100 two pronuclei (2PN)-derived blastocysts that were previously defined as uniformly euploid by standard PGT-A; (ii) 99 euploid embryos whose transfer resulted in pregnancy loss. The methodology is based on targeted next-generation sequencing of selected polymorphisms across the genome and enriched within critical regions of included MD syndromes. Sequencing data (i.e. allelic frequencies) were analyzed by a probabilistic model which estimated the likelihood of ploidy level and MD presence, accounting for both sequencing noise and population genetics patterns (i.e. linkage disequilibrium, LD, correlations) observed in 2504 whole-genome sequencing data from the 1000 Genome Project database. Analysis of phased parental haplotypes obtained by single-nucleotide polymorphism (SNP)-array genotyping was performed to confirm the presence of MD. In the analytical validation phase, this strategy showed extremely high accuracy both in ploidy classification (100%, CI: 98.1-100%) and in the identification of six out of eight MDs (99.2%, CI: 98.5-99.8%). To improve MD detection based on loss of heterozygosity (LOH), common haploblocks were analyzed based on haplotype frequency and LOH occurrence in a reference population, thus developing two further mathematical models. As a result, chr1p36 and chr4p16.3 regions were excluded from MD identification due to their poor reliability, whilst a clinical workflow which incorporated parental DNA information was developed to enhance the identification of MDs. During the clinical application phase, one case of triploidy was detected among 2PN-derived blastocysts (i) and one pathogenic MD (-22q11.21) was retrospectively identified among the biopsy specimens of transferred embryos that resulted in miscarriage (ii). For the latter case, family-based analysis revealed the same MD in different sibling embryos (n = 2/5) from non-carrier parents, suggesting the presence of germline mosaicism in the female partner. When embryos are selected for transfer based on their genetic constitution, this strategy can identify embryos with ploidy abnormalities and/or MDs beyond aneuploidies, with an estimated incidence of 1.5% (n = 3/202, 95% CI: 0.5-4.5%) among euploid embryos. Epidemiological studies will be required to accurately assess the incidence of ploidy alterations and MDs in preimplantation embryos and particularly in euploid miscarriages. Despite the high accuracy of the assay developed, the use of parental DNA to support diagnostic calling can further increase the precision of the assay. This novel assay significantly expands the clinical utility of PGT-A by integrating the most common pathogenic MDs (both de novo and inherited ones) responsible for genomic disorders, which are usually evaluated at a later stage through invasive prenatal testing. From a basic research standpoint, this approach will help to elucidate fundamental biological and clinical questions related to the genetics of implantation failure and pregnancy loss of otherwise euploid embryos. No external funding was used for this study. S.C., M.F., F.C., P.Z., I.P., L.G., C.P., M.P., D.B., J.J.-A., D.B.-J., J.M.-V., and C.R. are employees of Igenomix and C.S. is the head of the scientific board of Igenomix. A.C. and L.P. are employees of JUNO GENETICS. Igenomix and JUNO GENETICS are companies providing reproductive genetic services. N/A.

Cytogenetic Characterization and Metabolomic Differences of Full-Sib Progenies of Saccharum spp.

Sugarcane smut is a worldwide fungal disease. Disease resistance breeding is the most economical and effective measure to prevent and control sugarcane smut. The cytogenetic characteristics and metabolomic differences of sugarcane F1s are closely related to disease resistance. Zhongzhe 1 and G160 sugarcane from the same parents (ROC25 and Yunzhe89-7) were used; the plants were grown in accordance with the barrel method. When the seedlings had 4-5 leaves, genomic in situ hybridization (GISH) was performed; digoxigenin (DIG)-labeled female parental (ROC25)DNA and biotin-labeled male parental (Yunzhe89-7) DNA were used as probes, and the karyotypes of two hybrids were analyzed. The new sugarcane smut-resistant variety (Zhongzhe 1) and the susceptible variety (G160) derived from the same parent were analyzed via gas chromatography-mass spectrometry technology (GC-MS) to compare the metabolomic differences between them. GISH analysis revealed that the chromosome ploidy number of Zhongzhe 1 sugarcane and G160 sugarcane were 114 and 110, respectively. However, the two contain different numbers of chromosomes from the female (ROC25) and male (Yunzhe89-7) parents. Moreover, 258 significantly changed metabolites were identified in smut-resistant Zhongzhe 1, as compared with the smut-susceptible G160 sugarcane: 56 flavonoids, 52 phenolic acids, 30 lipids, 26 organic acids, 26 amino acids and derivatives, 19 nucleotides and derivatives, 5 alkaloids, 9 terpenoids, and 35 others. Multivariate statistical analysis revealed a distinct difference in metabolic pathways between Zhongzhe 1 sugarcane and G160, and both of these varieties had unique functional metabolites. Differences in chromosome composition may constitute the genetic basis for the difference in resistance to smut disease between Zhongzhe 1 sugarcane and G160 sugarcane, and a high accumulation of flavonoids, lipids, terpenoids and tannins may constitute the basis of resistance to smut disease for the Zhongzhe 1 variety.

Single molecule studies reveal that MutS's binding characteristics are mismatch type and context dependent and correlate with mismatch repair efficiency in vivo.

During DNA replication, DNA polymerases can incorrectly incorporate a non-complementary nucleotide against the parental DNA strand, leading to mutations and ultimately debilitating diseases if left unrepaired. All organisms possess a mismatch repair system that scans, recognizes, and repairs DNA mismatches. MutS is one of the first repair proteins to arrive at a mismatch. Upon recognition, MutS exchanges bound ADP molecules to ATP, and undergoes a conformational change to form a sliding clamp and slide away from the mismatch. MutS will then recruit other repair proteins to the site and resolve the error. Previous in vivo studies from our lab suggest that the mismatch repair efficiency in E. coli is hypervariable not only for different mismatched bases, but also for their local sequence context. Deletion studies showed that MutS is responsible for the observed hypervariability. To understand the molecular mechanism underlying sequence dependence of MutS, we employed single molecule FRET (fluorescence resonance energy transfer) to directly detect MutS binding to a mismatch, sliding clamp formation, and dissociation. The binding characteristics of E. coli MutS showed a striking dependence of the mismatch type and the overall residence time of MutS on a mismatch-containing DNA in vitro was strongly correlated with the in vivo mismatch repair efficiencies. Furthermore, the sliding clamp formation yield in the presence of ATP was correlated with in vivo mismatch repair efficiencies. These data support in vivo repair trends and help clarify mechanistic basis by which mismatches are differentially repaired. Future experiments will be conducted using additional DNA mismatches with different sequence contexts to understand the generalizability of this observation.

Development of an Amplicon Nanopore Sequencing Strategy for Detection of Mutations Conferring Intermediate Resistance to Vancomycin in Staphylococcus aureus Strains.

Staphylococcus aureus is a major cause of bacteremia and other hospital-acquired infections. The cell-wall active antibiotic vancomycin is commonly used to treat both methicillin-resistant (MRSA) and sensitive (MSSA) infections. Vancomycin intermediate S. aureus (VISA) variants can arise through de novo mutations. Here, we performed pilot experiments to develop a combined PCR/long-read sequencing-based method for detection of previously known VISA-causing mutations. Primers were designed to generate 10 amplicons covering 16 genes associated with the VISA phenotype. We sequenced amplicon pools as long reads with Oxford Nanopore adapter ligation on Flongle flow cells. We then detected mutations by mapping reads against a parental consensus or known reference sequence and comparing called variants against a database of known VISA mutations from laboratory selection. Each amplicon in the pool was sequenced to high (>1,000×) coverage, and no relationship was found between amplicon length and coverage. We also were able to detect the causative mutation (walK 646C>G) in a VISA mutant derived from the USA300 strain (N384-3 from parental strain N384). Mixing mutant (N384-3) and parental (N384) DNA at various ratios from 0 to 1 mutant suggested a mutation detection threshold of the average minor allele frequency (6.5%) at 95% confidence (two standard errors above mean mutation frequency). The study lays the groundwork for direct S. aureus antibiotic resistance genotype inference using rapid nanopore sequencing from clinical samples. IMPORTANCE Bacteremia mortality is known to increase rapidly with time after infection, making rapid diagnostics and treatment necessary. Successful treatment depends on correct administration of antibiotics based on knowledge of strain antibiotic susceptibility. Staphylococcus aureus is a major causative agent of bacteremia that is also commonly antibiotic resistant. In this work, we develop a method to accelerate detection of a complex, polygenic antibiotic resistance phenotype in S. aureus, vancomycin-intermediate resistance (VISA), through long-read genomic sequencing of amplicons representing genes most commonly mutated in VISA selection. This method both rapidly identifies VISA genotypes and incorporates the most comprehensive database of VISA genetic determinants known to date.

Gynogenetic Cirrhinus mrigala produced using irradiated sperm of Cyprinus carpio exhibit better cold tolerance

Artificial gynogenesis is an important method for accelerating the selective breeding of varieties and populations. In this study, ultraviolet-irradiated sperm of Common carp (Cyprinus carpio) was used to activate the mature eggs of mrigal carp (Cirrhinus mrigala, MC), after cold shock at 4–8 °C for 16 min to double the chromosomes, a population of gynogenetic mrigal carp (GMC) was obtained. The fertilization rate, hatching rate and survival rate of GMC exceeded 62.2%, 6.3% and 2.9%, respectively. Comparative analyses of morphological traits, DNA content, chromosome numbers and 5S rDNA sequences showed no significant differences between MC and GMC. However, microsatellite (MFW1, CCA15) analysis revealed that paternal DNA fragments were inserted and a parent DNA fragment was deleted in the GMC. The GMC population survived the natural winter for more than 50 days with a temperature below 10 °C, while all the normal MC individuals perished. In addition, lower critical thermal minimum (CTMin) and lethal temperature (TLD50) values were detected in GMC than that in MC. Our results indicated that the artificial gynogenesis method improved the cold tolerance of mrigal carp. The results of this study are of great significance for fish breeding and application.

Innate immunity mediator STING modulates nascent DNA metabolism at stalled forks in human cells.

Background: The cGAS/STING pathway, part of the innate immune response to foreign DNA, can be activated by cell's own DNA arising from the processing of the genome, including the degradation of nascent DNA at arrested replication forks, which can be upregulated in cancer cells. Recent evidence raises a possibility that the cGAS/STING pathway may also modulate the very processes that trigger it, e.g., DNA damage repair or processing of stalled forks. Methods: We manipulated STING levels in human cells by depleting or re-expressing it, and assessed the effects of STING on replication using microfluidics-assisted replication track analysis, or maRTA, a DNA fiber assay, as well as immuno-precipitation of nascent DNA, or iPOND. We also assessed STING subcellular distribution and its ability to activate. Results: Depletion of STING suppressed and its re-expression in STING-deficient cancer cells upregulated the degradation of nascent DNA at arrested replication forks. Replication fork arrest was accompanied by the STING pathway activation, and a STING mutant that does not activate the pathway failed to upregulate nascent DNA degradation. cGAS was required for STING's effect on degradation, but this requirement could be bypassed by treating cells with a STING agonist. Cells expressing inactive STING had a reduced level of RPA on parental and nascent DNA of arrested forks and a reduced CHK1 activation compared to cells with the wild type STING. STING also affected unperturbed fork progression in a subset of cell lines. STING fractionated to the nuclear fractions enriched for structural components of chromatin and nuclear envelope, and furthermore, it associated with the chromatin of arrested replication forks as well as post-replicative chromatin. Conclusion: Our data highlight STING as a determinant of stalled replication fork integrity, thus revealing a novel connection between the replication stress and innate immune responses.

Frequency of de novo variants and parental mosaicism in families with inactivating PTH/PTHrP signaling disorder type 2.

iPPSD2 (which includes PHP1A and PPHP/POH) is a rare inherited autosomal dominant endocrine disorder caused by inactivating GNAS pathogenic variants. A high percentage of de novo cases has been suggested. In rare cases, parental mosaicism has been described, but its real frequency is unknown. A retrospective study including a series of 95 genetically confirmed iPPSD2 probands. The frequency of de novo cases was evaluated and the distribution of the type of variants was compared according to the type of inheritance. The putative involved allele was determined by reverse transcriptase PCR (RT-PCR) or allele specific oligonucleotide RT-PCR (ASO-RT-PCR). The possibility of GNAS mosaicism was studied by next-generation sequencing (NGS) on the corresponding parental DNA. In 41 patients the variant was of de novo origin and in 24 the origin could not be established. In both cases 66.67% of variants generated a truncated or absent protein whereas the rest of the variants were missense or in-frame deletion/duplication. Parental origin was studied in 45 of those patients and determined in 35. Curiously, the percentage of de novo variants at the paternal allele was higher than when paternally inherited (31.1% vs 6.67%). NGS detected mosaicism in three independent families: one from paternal DNA (allelic ratio 10%) and two from maternal DNA (allelic ratio 10% and 2%). De novo pathogenic variants are frequent in iPPSD2 (around 45%). Parental mosaicism is infrequent (8.11%) but should be analyzed with NGS, taking into account its importance in genetic counselling.

The Roles of Nucleoid-Associated Proteins and Topoisomerases in Chromosome Structure, Strand Segregation and the Generation of Phenotypic Heterogeneity in Bacteria.

How to adapt to a changing environment is a fundamental, recurrent problem confronting cells. One solution is for cells to organise their constituents into a limited number of spatially extended, functionally relevant, macromolecular assemblies or hyperstructures, and then to segregate these hyperstructures asymmetrically into daughter cells. This asymmetric segregation becomes a particularly powerful way of generating a coherent phenotypic diversity when the segregation of certain hyperstructures is with only one of the parental DNA strands and when this pattern of segregation continues over successive generations. Candidate hyperstructures for such asymmetric segregation in prokaryotes include those containing the Nucleoid-Associated Proteins (NAPs) and the topoisomerases. Another solution to the problem of creating a coherent phenotypic diversity is by creating a growth-environment-dependent gradient of supercoiling generated along the replication origin-to-terminus axis of the bacterial chromosome. This gradient is modulated by transcription, NAPs and topoisomerases. Here, we focus primarily on two topoisomerases, TopoIV and DNA gyrase in Escherichia coli, on three of its NAPs (H-NS, HU and IHF), and on the single-stranded binding protein, SSB. We propose that the combination of supercoiling-gradient-dependent and strand-segregation-dependent topoisomerase activities result in significant differences in the supercoiling of daughter chromosomes and hence in the phenotypes of daughter cells.

Parent-of-origin detection and chromosome-scale haplotyping using long-read DNA methylation sequencing and Strand-seq

Hundreds of loci in human genomes have alleles that are methylated differentially according to their parent of origin. These imprinted loci generally show little variation across tissues, individuals, and populations. We show that such loci can be used to distinguish the maternal and paternal homologs for all human autosomes without the need for the parental DNA. We integrate methylation-detecting nanopore sequencing with the long-range phase information in Strand-seq data to determine the parent of origin of chromosome-length haplotypes for both DNA sequence and DNA methylation in five trios with diverse genetic backgrounds. The parent of origin was correctly inferred for all autosomes with an average mismatch error rate of 0.31% for SNVs and 1.89% for insertions or deletions (indels). Because our method can determine whether an inherited disease allele originated from the mother or the father, we predict that it will improve the diagnosis and management of many genetic diseases.