Hmga2 deficiency is associated with allometric growth retardation, infertility, and behavioral abnormalities in mice

The high mobility group AT-hook 2 (HMGA2) protein works as an architectural regulator by binding AT-rich DNA sequences to induce conformational changes affecting transcription. Genomic deletions disrupting HMGA2 coding sequences and flanking non-coding sequences cause dwarfism in mice and rabbits. Here, CRISPR/Cas9 was used in mice to generate the Hmga2 null allele that specifically disrupts only the coding sequence. The loss of one or both alleles of Hmga2 resulted in reduced body size of 20% and 60%, respectively, compared to wild-type littermates as well as an allometric reduction in skull length in Hmga2-/- mice. Both male and female Hmga2-/- mice are infertile, whereas Hmga2+/- mice are fertile. Examination of reproductive tissues of Hmga2-/- males revealed a significantly reduced size of testis, epididymis, and seminal vesicle compared to controls, and 70% of knock-out males showed externalized penis, but no cryptorchidism was observed. Sperm analyses revealed severe oligospermia in mutant males and slightly decreased sperm viability, increased DNA damage but normal sperm chromatin compaction. Testis histology surprisingly revealed a normal seminiferous epithelium, despite the significant reduction in testis size. In addition, Hmga2-/- mice showed a significantly reduced exploratory behavior. In summary, the phenotypic effects in mouse using targeted mutagenesis confirmed that Hmga2 is affecting prenatal and postnatal growth regulation, male reproductive tissue development, and presents the first indication that Hmga2 function is required for normal mouse behavior. No specific effect, despite an allometric reduction, on craniofacial development was noted in contrast to previous reports of an altered craniofacial development in mice and rabbits carrying deletions of both coding and non-coding sequences at the 5’part of Hmga2.

Defining Genomic Probability of Progression to Identify Low-Risk Smoldering Multiple Myeloma

On an average, 1% of monoclonal gammopathy of undermined significance (MGUS) and 10% of smoldering Multiple Myeloma (SMM) progress to symptomatic MM every year within the first five years of diagnosis. The probability of progression significantly decreases for SMM patients after first 5 years. However, a distinct subset of SMM patients progress within 2 years and are re-classified as high-risk patients based on risk markers such as 20/2/20 or certain genomic features. Although recent studies have evaluated the high-risk genomic features for SMM but genomic background of SMM patients who do not progress to MM after long-term follow-up (>= 5 years) has not been described. Here, we evaluated transcriptomic and genomic changes enriched in non-progressor (NP) (no progression after 5 years of follow-up) precursor conditions (N=31) with those progressed within short period of time (N=71) and compared them with changes observed in newly diagnosed MM (N=192). Additionally, using transcriptome, epigenome and whole genome profiling we also studied additional unique samples from 18 patients at their precursor stage as well as when progressed to MM. Overall, we have observed significantly lower mutational load for NP SMM from progressor SMM (median SNV 4900 vs. 7881 p < 3e-04) with high sensitivity (0.83) and specificity (0.65) to separate NP from progressors. We have further developed a deep learning model by using more than 4500 genome wide features using ten-fold cross validation. This model indicated that not only the load but also the patterns of mutations (type, location, frequency) are different between two conditions. We also found that NP samples have significantly lower heterogeneity (p < 0.05). However, progressed samples showed similar mutational load and heterogeneity at precursor stage and MM. Among CNA differences, absence of gain or deletion of chr8 (not involving MYC region) were strong predictor of NP (OR=7.2 95% CI 2.2-24). Focal genomic loss was also significantly lower for NP (p=0.004) which was also reflected by low genome scar score (GSS) (p=0.07). Structural variant and copy number signature analysis also showed that NPs were showing significantly low exposure to non-clustered variable size genomic deletions. We observed similar frequency of primary translocations [t(11;14), t(4;14), and t(14;16)] in both progressor and NP samples as well as newly diagnosed MM. MYC translocation with any partner was not observed in NP samples, whereas 37% of progressor samples had a MYC translocations (OR=12.8). Adding all these differences including chr8 CNAs, MYC translocations, mutation burden, GSS, focal deletions, all driver mutations as well as primary translocations into recursive partitioning model to predict non-progressor SMM, we have identified a simple genomic model only involving chr8 CN changes and overall mutational burden to achieve a high sensitivity (0.82) and specificity (74%). Our transcriptomic analysis measured the distance between progressor and NP SMM as well as MM and found that NP SMM has greater difference with MM which is closer to progressor SMM. We quantified transcriptomic heterogeneity by using molecular degree of perturbation. This analysis showed that consistent with DNA changes, DNA repair pathway and MYC target genes are expressed similarly in NP SMM as in normal plasma cells compared to progressor SMM. Epigenomic analysis yielded 75 SEs regions differentially utilized between precursor and symptomatic MM stage using paired samples. The targeted genes included BMP6, PRDM1, STAT1, SERTAD2 and RAB21 and possibly regulating genes related to oncogenic KRAS activities. In conclusion, we define genomic characterization of non-progressor SMM and our results now provide the basis to develop molecular definition of SMM as well as risk driving features. Disclosures Munshi: Janssen: Consultancy; Pfizer: Consultancy; Legend: Consultancy; Novartis: Consultancy; Adaptive Biotechnology: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Takeda: Consultancy; Abbvie: Consultancy; Karyopharm: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Bristol-Myers Squibb: Consultancy.

Quantitative NanoLC/NSI+-HRMS Method for 1,3-Butadiene Induced bis-N7-guanine DNA-DNA Cross-Links in Urine

1,3-Butadiene (BD) is a common environmental and industrial chemical widely used in plastic and rubber manufacturing and also present in cigarette smoke and automobile exhaust. BD is classified as a known human carcinogen based on evidence of carcinogenicity in laboratory animals treated with BD by inhalation and epidemiological studies revealing an increased risk of leukemia and lymphohematopoietic cancers in workers occupationally exposed to BD. Upon exposure via inhalation, BD is bioactivated to several toxic epoxides including 3,4-epoxy-1-butene (EB), 3,4-epoxy-1,2-butanediol (EBD), and 1,2,3,4-diepoxybutane (DEB); these are conjugated with glutathione and excreted as 2-(N-acetyl-L-cystein-S-yl)-1-hydroxybut-3-ene/1-(N-acetyl-L-cystein-S-yl)-2-hydroxybut-3-ene (MHBMA), 4-(N-acetyl-L-cystein-S-yl)-1,2-dihydroxybutane (DHBMA), and 1,4-bis-(N-acetyl-L-cystein-S-yl)butane-2,3-diol (bis-BDMA). Exposure to DEB generates monoalkylated DNA adducts, DNA-DNA crosslinks, and DNA-protein crosslinks, which can cause base substitutions, genomic rearrangements, and large genomic deletions. In this study, we developed a quantitative nanoLC/NSI+-HRMS methodology for 1,4-bis-(gua-7-yl)-2,3-butanediol (bis-N7G-BD) adducts in urine (LOD: 0.1 fmol/mL urine, LOQ: 1.0 fmol/mL urine). This novel method was used to quantify bis-N7G-BD in urine of mice treated with 590 ± 150 ppm BD for 2 weeks (6 h/day, 5 days/week). Bis-N7G-BD was detected in urine of male and female BD-exposed mice (574.6 ± 206.0 and 571.1 ± 163.4 pg/mg of creatinine, respectively). In addition, major urinary metabolites of BD, bis-BDMA, MHBMA and DHBMA, were measured in the same samples. Urinary bis-N7G-BD adduct levels correlated with DEB-derived metabolite bis-BDMA (r = 0.80, Pearson correlation), but not with the EB-derived DNA adducts (EB-GII) or EB-derived metabolites MHBMA and DHBMA (r = 0.24, r = 0.14, r = 0.18, respectively, Pearson correlations). Urinary bis-N7G-BD could be employed as a novel non-invasive biomarker of exposure to BD and bioactivation to its most mutagenic metabolite, DEB. This method will be useful for future studies of 1,3-butadiene exposure and metabolism.

Bursts of recent transposable element activity and signatures of element removal in the monarch genome, Danaus plexippus

BackgroundLepidoptera (butterflies and moths) are an important model system in ecology and evolution. A high-quality chromosomal genome assembly of the monarch butterfly (Danaus plexippus), famous for its North American migration, is available but lacks an in-depth transposable element (TE) annotation. This provides an opportunity to explore host-TE interactions, and the impact TEs have in shaping the monarch genome.Results6.47% of the monarch genome is comprised of TEs, a reduction of 6.59% compared to the original TE annotation performed on the draft genome assembly. TE content is low compared to two closely related species, Danaus chrysippus (26.70%) and Danaus melanippus (11.87%). The biggest contributors to genome size in the monarch are LINEs and Penelope-like elements, and 37.7% of TE content is contributed by five newly identified TE families (two LINE, two Penelope-like, and one SINE). Some young DNA TE families show similar activity profiles to these LINEs, with their success putatively due to horizontal transposon transfer from species sharing the same environment. There are several recent peaks of TE activity in the monarch, with little evidence for peaks of activity more anciently. LINE fragments demonstrate signatures of genomic deletions as reported by studies on Heliconius butterflies, indicating a high rate of TE turnover. Given previous associations in other species, we investigated the association of TEs with wing colouration and immune genes. We find a single unclassified element 7kb upstream of the myosin gene locus, associated with wing colouration, and 49 immune genes with TEs within 5kb upstream of the transcription start site, presenting the potential for the involvement of TEs in regulatory functions.ConclusionsWe provide an in-depth TE annotation and analysis of TE diversity and evolution for the monarch genome. We identify highly successful novel DNA TE families, mirroring the activity profile of the most successful LINEs. We also find evidence of ongoing TE expansion and removal in the monarch, highlighting the dynamic nature of repeat content in genomes over time. Further in-depth comparative studies across closely related species will be beneficial to our understanding of the evolutionary dynamics of TEs and the processes leading to their contrasting distributions.

Co-variation of viral recombination with single nucleotide variants during virus evolution revealed by CoVaMa

Adaptation of viruses to their environments occurs through the acquisition of both novel Single-Nucleotide Variants (SNV) and recombination events including insertions, deletions, and duplications. The co-occurrence of SNVs in individual viral genomes during their evolution has been well-described. However, unlike covariation of SNVs, studying the correlation between recombination events with each other or with SNVs has been hampered by their inherent genetic complexity and a lack of bioinformatic tools. Here, we expanded our previously reported CoVaMa pipeline (v0.1) to measure linkage disequilibrium between recombination events and SNVs within both short-read and long-read sequencing datasets. We demonstrate this approach using long-read nanopore sequencing data acquired from Flock House virus (FHV) serially passaged in vitro. We found SNVs that were either correlated or anti-correlated with large genomic deletions generated by nonhomologous recombination that give rise to Defective-RNAs. We also analyzed NGS data from longitudinal HIV samples derived from a patient undergoing antiretroviral therapy who proceeded to virological failure. We found correlations between insertions in the p6Gag and mutations in Gag cleavage sites. This report confirms previous findings and provides insights on novel associations between SNVs and specific recombination events within the viral genome and their role in viral evolution.

Pipeline for generating stable large genomic deletions in zebrafish, from small domains to whole gene excisions

Here we describe a short feasibility study and methodological framework for the production of stable, CRISPR/Cas9-based, large genomic deletions in zebrafish, ranging from several base pairs (bp) to hundreds of kilobases (kb). Using a cocktail of four sgRNAs targeting a single genomic region mixed with a marker-sgRNA against the pigmentation gene tyrosinase (tyr), we demonstrate that one can easily and accurately excise genomic regions such as promoters, protein domains, specific exons or whole genes. We exemplify this technique with a complex gene family, neurexins, composed of three duplicated genes with multiple promoters and intricate splicing processes leading to thousands of isoforms. We precisely deleted small regions such as their transmembrane domains (150 bp deletion in average) to their entire genomic locus (300 kb deletion for nrxn1a for instance). We find that both the concentration and ratio of Cas9/sgRNAs are critical for the successful generation of these large deletions and, interestingly, that in our study their transmission frequency does not seem to decrease with increasing distance between sgRNA target sites. Considering the growing reports and debate about genetically compensated small indel mutants, the use of large-deletion approaches is likely to be widely adopted in studies of gene function. This strategy will also be key to the study of non-coding genomic regions. Note that we are also describing here a custom method to produce the sgRNAs, which proved to be faster and more robust than the ones traditionally used in the community to date.

Resurrecting essential amino acid biosynthesis in a mammalian cell

Major genomic deletions in independent eukaryotic lineages have led to repeated ancestral loss of biosynthesis pathways for nine of the twenty canonical amino acids1. While the evolutionary forces driving these polyphyletic deletion events are not well understood, the consequence is that extant metazoans are unable to produce nine essential amino acids (EAAs). Previous studies have highlighted that EAA biosynthesis tends to be more energetically costly2,3, raising the possibility that these pathways were lost from organisms with access to abundant EAAs in the environment4,5. It is unclear whether present-day metazoans can reaccept these pathways to resurrect biosynthetic capabilities that were lost long ago or whether evolution has rendered EAA pathways incompatible with metazoan metabolism. Here, we report progress on a large-scale synthetic genomics effort to reestablish EAA biosynthetic functionality in a mammalian cell. We designed codon-optimized biosynthesis pathways based on genes mined from Escherichia coli. These pathways were de novo synthesized in 3 kilobase chunks, assembled in yeasto and genomically integrated into a Chinese Hamster Ovary (CHO) cell line. One synthetic pathway produced valine at a sufficient level for cell viability and proliferation, and thus represents a successful example of metazoan EAA biosynthesis restoration. This prototrophic CHO line grows in valine-free medium, and metabolomics using labeled precursors verified de novo biosynthesis of valine. RNA-seq profiling of the valine prototrophic CHO line showed that the synthetic pathway minimally disrupted the cellular transcriptome. Furthermore, valine prototrophic cells exhibited transcriptional signatures associated with rescue from nutritional starvation. This work demonstrates that mammalian metabolism is amenable to restoration of ancient core pathways, thus paving a path for genome-scale efforts to synthetically restore metabolic functions to the metazoan lineage.

Rapid generation of maternal mutants via oocyte transgenic expression of CRISPR-Cas9 and sgRNAs in zebrafish

Maternal products are exclusive factors to drive oogenesis and early embryonic development. As disrupting maternal gene functions is either time-consuming or technically challenging, early developmental programs regulated by maternal factors remain mostly elusive. We provide a transgenic approach to inactivate maternal genes in zebrafish primary oocytes. By introducing three tandem single guide RNA (sgRNA) expression cassettes and a green fluorescent protein (GFP) reporter into Tg(zpc:zcas9) embryos, we efficiently obtained maternal nanog and ctnnb2 mutants among GFP-positive F1 offspring. Notably, most of these maternal mutants displayed either sgRNA site–spanning genomic deletions or unintended large deletions extending distantly from the sgRNA targets, suggesting a prominent deletion-prone tendency of genome editing in the oocyte. Thus, our method allows maternal gene knockout in the absence of viable and fertile homozygous mutant adults. This approach is particularly time-saving and can be applied for functional screening of maternal factors and generating genomic deletions in zebrafish.