The good, the bad, and the ugly: Evolutionary and pathological aspects of gene dosage alterations

Diploid organisms contain a maternal and a paternal genome complement that is thought to provide robustness and allow developmental progression despite genetic perturbations that occur in heterozygosity. However, changes affecting gene dosage from the chromosome down to the individual gene level possess a significant pathological potential and can lead to developmental disorders (DDs). This indicates that expression from a balanced gene complement is highly relevant for proper cellular and organismal function in eukaryotes. Paradoxically, gene and whole chromosome duplications are a principal driver of evolution, while heteromorphic sex chromosomes (XY and ZW) are naturally occurring aneuploidies important for sex determination. Here, we provide an overview of the biology of gene dosage at the crossroads between evolutionary benefit and pathogenicity during disease. We describe the buffering mechanisms and cellular responses to alterations, which could provide a common ground for the understanding of DDs caused by copy number alterations.

Introgressions of Vitis rotundifolia Michx. to obtain grapevine genotypes with complex resistance to biotic and abiotic stresses

Vitis rotundifolia Michx. is one of the species of the family Vitaceae, with resistance to both biotic and abiotic stresses. The present study reports new scientific knowledge about the inheritance of resistance to downy mildew, powdery mildew and frost by V. vinifera varieties from V. rotundifolia. Recombinant lines of three hybrid populations from the crossing of the maternal genotype ♀M. 31-77-10 with V. rotundifolia hybrids were used as the object of the study. As a result of laboratory screening, more than 40 % of recombinants of the ♀M. 31-77-10× ×[DRX-M5-734+DRX-M5-753+DRX-M5-790] population showed a high degree of frost resistance (–24 °C), while 6 % of transgressive recombinants were characterized by a very high degree of resistance (–27 °С). The maternal genotype ♀M. 31-77-10 does not carry alleles of resistance to powdery mildew at the Run1 locus and in the field suffers from powdery mildew much more than the paternal genotypes. The prevalence of powdery mildew on vegetative organs in the three recombinant populations over the years varies on average between 3.2–17.1, 0.3–17.7 and 0.6–5.2 %, respectively. As a result, almost all recombinant genotypes that received a resistant allele from the paternal genome are highly resistant to powdery mildew.

Single Nucleotide Substitutions Effectively Block Cas9 and Allow for Scarless Genome Editing in Caenorhabditis elegans

In Caenorhabditis elegans, germline injection of Cas9 complexes is reliably used to achieve genome editing through homology-directed repair of Cas9-generated DNA breaks. To prevent Cas9 from targeting repaired DNA, additional blocking mutations are often incorporated into homologous repair templates. Cas9 can be blocked either by mutating the PAM sequence that is essential for Cas9 activity or by mutating the guide sequence that targets Cas9 to a specific genomic location. However, it is unclear how many nucleotides within the guide sequence should be mutated, since Cas9 can recognize “off-target” sequences that are imperfectly paired to its guide. In this study, we examined whether single-nucleotide substitutions within the guide sequence are sufficient to block Cas9 and allow for efficient genome editing. We show that a single mismatch within the guide sequence effectively blocks Cas9 and allows for recovery of edited animals. Surprisingly, we found that a low rate of edited animals can be recovered without introducing any blocking mutations, suggesting a temporal block to Cas9 activity in C. elegans. Furthermore, we show that the maternal genome of hermaphrodite animals is preferentially edited over the paternal genome. We demonstrate that maternally provided haplotypes can be selected using balancer chromosomes and propose a method of mutant isolation that greatly reduces screening efforts post-injection. Collectively, our findings expand the repertoire of genome editing strategies in C. elegans and demonstrate that extraneous blocking mutations are not required to recover edited animals when the desired mutation is located within the guide sequence.

Genomic evidence of paternal genome elimination in globular springtails

Paternal genome elimination (PGE) - a type of reproduction in which males inherit but fail to pass on the genome of their father - evolved independently in six to eight arthropod clades. Thousands of species, including several important for agriculture, reproduce via this mode of reproduction. While some of the clades are well established PGE systems, the evidence in globular springtails (Symphypleona) remains elusive, even though they represent the oldest and most species rich clade putatively reproducing via PGE. We sequenced genomic DNA from whole bodies of Allacma fusca males with sufficiently high fractions (31 - 38%) of sperm to conclusively confirm that all the sperm carry one parental haplotype only. Although it is suggestive that the single haplotype present in sperm is maternally inherited, definitive genetic proof of the parent of origin is still needed. The genomic approach we developed allows for detection of genotypic differences between germline and soma in all species with sufficiently high fraction of germline in their bodies. This opens new opportunities for scans for reproductive modes in small animals.

265 Awardee Talk: Maternal and Paternal Contributions to Fertility

Using over 56,000 pregnancy diagnostic records in beef cattle, a meta-analysis was conducted to objectively determine that 28.4 % of embryos will not develop past day 7 of gestation with most embryonic losses occurring before day 4. Furthermore, by day 30 of gestation, 47.9 % of cows submitted to a single insemination at day 0 will not be pregnant and pregnancy loss between days 32 and 100 was 5.8%. Reproductive success, however, is highly variable and influenced by maternal and paternal factors. Maternal characteristics, including subspecies, parity and reproductive tract size, intersect with breeding management decisions regarding estrus expression and detection to influence conception and pregnancy rates. The maternal endocrine environment, from estradiol associated with estrus around fertilization to elevated prostaglandins during the embryonic to fetal transition period, play critical roles in pregnancy success. Despite the numerous maternal factors involved with pregnancy establishment, sire contribution to reproductive failure should not be overlooked. Sires are reported to influence pregnancy loss during the second month of gestation in both beef and dairy herds. These differences in sire field fertility could not be explained by differences in semen characteristics nor differences in sire conception rate (SCR) score. To understand parental contribution to pregnancy development during the second month of gestation we developed parthenogenetic (PA) embryos (embryos lacking paternal genome). We observed that PA conceptus had well developed trophectoderm tissue at day 31 of gestation but no site of embryo attachment to the endometrium. Moreover, conceptus secretion such as pregnancy-associated glycoproteins and interferon-stimulated genes were not found in maternal circulation. These results suggest that paternal genetics is required for post-elongated embryo attachment to endometrium. Further exploring the maternal vs paternal contribution to pregnancy development can help elucidate the mechanism that drives reproductive failure in cattle.

Single Nucleotide Substitutions Effectively Block Cas9 and Allow for Scarless Genome Editing in Caenorhabditis elegans

In Caenorhabditis elegans, germline injection of Cas9 complexes is reliably used to achieve genome editing through homology-directed repair of Cas9-generated DNA breaks. To prevent Cas9 from targeting repaired DNA, additional blocking mutations are often incorporated into homologous repair templates. Cas9 can be blocked either by mutating the PAM sequence that is essential for Cas9 activity or by mutating the guide sequence that targets Cas9 to a specific genomic location. However, it is unclear how many nucleotides within the guide sequence should be mutated, since Cas9 can recognize off-target sequences that are imperfectly paired to its guide. In this study, we examined whether single-nucleotide substitutions within the guide sequence are sufficient to block Cas9 and allow for efficient genome editing. We show that a single mismatch within the guide sequence effectively blocks Cas9 and allows for recovery of edited animals. Surprisingly, we found that a low rate of edited animals can be recovered without introducing any blocking mutations, suggesting a temporal block to Cas9 activity in C. elegans. Furthermore, we show that the maternal genome of hermaphrodite animals is preferentially edited over the paternal genome. We demonstrate that maternally provided haplotypes can be selected using balancer chromosomes and propose a method of mutant isolation that greatly reduces screening efforts post-injection. Collectively, our findings expand the repertoire of genome editing strategies in C. elegans and demonstrate that extraneous blocking mutations are not required to recover edited animals when the desired mutation is located within the guide sequence.

Impact of Paternal Genome with a High DNA Fragmentation Index (>60%) on Early Embryonic Development

: Objectives: The objective of this study is to propose thresholds of the sperm DNA fragmentation rate (IFA≤30% IFA31%-60% IFA>60%), in order to assess the clinical effects of the paternal genome on intra cytoplasmic sperm injection parameters, in particular the effect of the latter on early embryonic development. Materials and Methods: The procedure is a retrospective study, which involved 101 patients enrolled in an ICSI program with their partners. The index of spermatic DNA fragmentation rate was measured using the Sperm Chromatin Dispersion assay. Results: There is a negative correlation between high levels of the spermatic DNA fragmentation index and spermiological characteristics: Concentration P=0.002 and mobility P=0.0001. For ICSI results, there are different observations on the existence of a correlation between the spermatic DNA fragmentation index and fertility rate. On the other hand, the rate of sperm DNA fragmentation does not seem to influence early embryonic development, and even couples whose partners have a high fragmentation index manage to obtain the best quality embryos (P=0.002). We observe a decrease in the rate of implantation with an increase in the rate of alteration of the sperm genome, but this remains insignificant P > 0.05. Conclusion: ICSI remains the only alternative for men with a high rate of sperm DNA fragmentation. Moreover, the operator seems to influence the results more than is suggested. This does not exclude the paternal effect which may influence the quality of the concepltus later on. Keywords: DNA Fragmentation Index, ICSI, Fertilization Rate, Embryos Quality.

P–055 Methylation dynamics of the sperm epigenome after chemotherapy: a case study

Study question What is the evolution of the sperm epigenome after chemotherapy in a patient with testicular cancer (TC)? Summary answer These new data on epigenetic recovery profil after TC are useful tools for counseling and reassuring these patients. What is known already An important issue for young men affected TC is how TC and its treatment will affect, transiently or permanently, their future reproductive health. The consequences of cancer treatment on the sperm epigenome during the recovery periods are topical issues of ascendant significance as epigenetic modifications to the paternal genome may have deleterious effects on the offspring. Study design, size, duration Here we report the epigenomic profiling of frozen sperm from a TC patient before and after the treatment at different time points (6, 9, 12 and 24 months) by using RRBS analysis (Reduced representation bisulfite sequencing method). Participants/materials, setting, methods A testicular tumor (testicular germ cell tumor) was diagnosed in a 30 years old patient. A cryopreservation of spermatozoa was proposed before treatment.Semen samples were obtained 2 times before treatment and 4 times after treatment (6, 9, 12 and 24 months following the initiation of treatment). Main results and the role of chance Upon collection, sampling after chemotherapy ranged from 0,6 to 4,2 million per sperm straw between 6 and 24 months after the treatment, always increasing. In order to capture the direct effect of the treatment on the methylation changes, the DMR detection has been operated between pre-chemotherapy samples (pair-wise) and the time point of 6 months. Among the 179 hqDMRs, 74 are differentially methylated between the PreCT and PostCT6m samples (16 hyper- and 68 hypo-methylated) associated with 49 DMGs (15 hyper- and 34 hypo-methylated). We further sub-clustered the 74 hqDMRs between PreCT and PostCT6m into 6 patterns, 3 hyper- and 3 hypo-methylated. Briefly, patterns P1 and P4 include hqDMRs that quickly get back to their pre-treatment methylation status just after 9th months onwards. Patterns P2 and P5 include hqDMRs that slowly get back to their pre-treatment methylation status between 12 and 24 months after treatment. Patterns P3 and P6 include hqDMRs that remain hyper- or hypo-methylated even after 24 months. We have intersected the genes (DMGs) associated with the detected hqDMRs with those known to be important or expressed during embryogenesis. We thus detected that 7 hyper-methylated and 6 hypomethylated DMGs were involved (or expressed) during embryonic / fetal development. Limitations, reasons for caution This study involves a single patient. As the patient made no major changes in his personal way of life, we hypothesized that sperm parameter variations may be attributable to the BEP treatment. Wider implications of the findings: The altered methylated status of those DMGs important for early development might modify their expression pattern and thus affect their function during key stages of embryogenesis leading to potential developmental disorders. It is important to notice that among the 110 DMGs none of them correspond to known imprinted genes. Trial registration number Not applicable

Complementary role of p57kip2 immunostaining in diagnosing hydatidiform mole subtypes

Objectives Gestational trophoblastic disease comprises of a spectrum of pregnancy-related tumours which includes complete (CHM) and partial hydatidiform moles (PHM). Accurate diagnosis and subclassification of HM subtypes are crucial as prognosis differs. Histopathological examination using haemotoxylin and eosin (H&E) staining remains the basis for diagnosing HM, with only 80% accuracy. p57kip2 is a cyclin-dependent kinase inhibitor (CDKI) protein and is strongly paternally imprinted, being expressed from maternal allele. Therefore, complete mole (CHM) with only paternal genome has nearly absent expression of p57kip2 compared to partial mole (PHM) having both paternal and maternal genomes. This study is aimed to determine usefulness of p57kip2 immunohistochemistry (IHC) analysis in the diagnosis of HM subtypes. Methods A total of 82 archived paraffin embedded HM tissues with subtypes classified based on H&E staining – 39 (47.5%) CHM, 41 (50.0%) PHM and two (2.43%) unclassified molar pregnancy were retrieved. All tissue samples were subjected for p57kip2 IHC analysis and HM subtypes were then reclassified. Results A total of 66 cases (80.5%) were re-classified as CHM, 14 cases (17.1%) as PHM and two cases (2.4%) were decidual and cystic tissues. Analysis using p57kip2 immunostaining showed a diagnostic discrepancy of 33.0% from routine H&E staining and helps to improve the characterisation of the HM subtypes specifically at early gestations which have less distinctive morphologies. Conclusions IHC using p57kip2 monoclonal antibody should be considered as a routine ancillary test to H&E in improving the diagnosis of HM subtypes particularly in developing countries with limited resources.