Heterozygous De Novo Truncating Mutation of Nucleolin in an ASD Individual Disrupts Its Nucleolar Localization

Nucleolin (NCL/C23; OMIM: 164035) is a major nucleolar protein that plays a critical role in multiple processes, including ribosome assembly and maturation, chromatin decondensation, and pre-rRNA transcription. Due to its diverse functions, nucleolin has frequently been implicated in pathological processes, including cancer and viral infection. We recently identified a de novo frameshifting indel mutation of NCL, p.Gly664Glufs*70, through whole-exome sequencing of autism spectrum disorder trios. Through the transfection of constructs encoding either a wild-type human nucleolin or a mutant nucleolin with the same C-terminal sequence predicted for the autism proband, and by using co-localization with the nucleophosmin (NPM; B23) protein, we have shown that the nucleolin mutation leads to mislocalization of the NCL protein from the nucleolus to the nucleoplasm. Moreover, a construct with a nonsense mutation at the same residue, p.Gly664*, shows a very similar effect on the location of the NCL protein, thus confirming the presence of a predicted nucleolar location signal in this region of the NCL protein. Real-time fluorescence recovery experiments show significant changes in the kinetics and mobility of mutant NCL protein in the nucleoplasm of HEK293Tcells. Several other studies also report de novo NCL mutations in ASD or neurodevelopmental disorders. The altered mislocalization and dynamics of mutant NCL (p.G664Glufs*70/p.G664*) may have relevance to the etiopathlogy of NCL-related ASD and other neurodevelopmental phenotypes.

Meiotic Cas9 expression mediates gene conversion in the male and female mouse germline

Highly efficient gene conversion systems have the potential to facilitate the study of complex genetic traits using laboratory mice and, if implemented as a “gene drive,” to limit loss of biodiversity and disease transmission caused by wild rodent populations. We previously showed that such a system of gene conversion from heterozygous to homozygous after a sequence targeted CRISPR/Cas9 double-strand DNA break (DSB) is feasible in the female mouse germline. In the male germline, however, all DSBs were instead repaired by end joining (EJ) mechanisms to form an “insertion/deletion” (indel) mutation. These observations suggested that timing Cas9 expression to coincide with meiosis I is critical to favor conditions when homologous chromosomes are aligned and interchromosomal homology-directed repair (HDR) mechanisms predominate. Here, using a Cas9 knock-in allele at the Spo11 locus, we show that meiotic expression of Cas9 does indeed mediate gene conversion in the male as well as in the female germline. However, the low frequency of both HDR and indel mutation in both male and female germlines suggests that Cas9 may be expressed from the Spo11 locus at levels too low for efficient DSB formation. We suggest that more robust Cas9 expression initiated during early meiosis I may improve the efficiency of gene conversion and further increase the rate of “super-mendelian” inheritance from both male and female mice.

Identification of an SRY-Negative 46,XX Infertility Male with a Heterozygous Deletion Downstream of SOX3 Gene

Background: A male individual with a non-chimeric karyotype of 46,XX is very rare. We explored the genetic aetiology of an infertility male with 46,XX and SRY negative.Methods: The peripheral blood sample was collected from the patient and subjected to a range of genetic testing, including conventional chromosomal karyotyping, short tandem repeat (STR) analysis for chromosome 13, 18, 21, X, Y contained SRY gene, azoospermia factor (AZF) deletion analysis including SRY gene, fluorescence in situ hybridization (FISH) with specific probes for CSP X/CSP Y/SRY, chromosomal microarray analysis (CMA) for genomic copy number variations (CNVs), and whole-genome analysis(WGA) for SNV&InDel variants, and the X chromosome inactivation (XCI) analysis for AR gene.Results: The patient was found to have a 46,XX karyotype. Neither AZFa+b+c nor SRY band was detected in the electrophoresis result. FISH results of both interphase cells with CSPX/CSPY probe and metaphase cells with CSPX/CSPY/SRY probe showed two green fluorescence signals at the centromeres of X chromosomes, but no Y chromosome and SRY fluorescence signal. QF-PCR results showed that the patient had only the AMELX fluorescence peak of the X chromosome but no AMELY and SRY fluorescence peak. All results of the Karyotype, FISH, and STR did not suggest limited Y chimerism. CMA showed he had a heterozygous deletion of about 867 kb in Xq27.1 (hg19: chrX: 138,612,879-139,480,163 bp), located at 104 kb downstream of SOX3 gene, including F9, CXorf66, MCF2 and ATP11C; Meanwhile, whole-genome sequencing also found no SNV&InDel mutation associated with abnormal sex development. 75% X chromosome inactivation was detected.Conclusions: Although the pathogenicity of 46,XX male patients with SRY negative remains unclear, SOX3 expression of the acquired function may be associated with partial testis differentiation. Therefore, copy number variation of SOX3 gene and regulatory region should be performed routinely for these patients.

Genetic and Epigenetic Impact of Chronic Inflammation on Colon Mucosa Cells

Chronic inflammation increases cancer risk, and cancer development is characterized by stepwise accumulation of genetic and epigenetic alterations. During chronic inflammation, infectious agents and intrinsic mediators of inflammatory responses can induce genetic and epigenetic changes. This study tried to evaluate both the genetic and epigenetic influence of chronic inflammation on colon mucosa cells. Repetitive dextran sulfate sodium (DSS) treatment induced chronic colitis model. Whole-exome sequencing (WES) (200× coverage) was performed to detect somatic variations in colon mucosa cells. With the use of whole-genome bisulfite sequencing (BS) at 34-fold coverage (17-fold per strand), the methylome of both the colitis and control tissue was comparatively analyzed. Bioinformatics assay showed that there was no significant single-nucleotide polymorphism/insertion or deletion (SNP/InDel) mutation accumulation in colitis tissue, while it accumulated in aged mice. Forty-eight genes with SNP/InDel mutation were overlapped in the three colitis tissues, two (Wnt3a and Lama2) of which are in the cancer development-related signaling pathway. Differentially methylated region (DMR) assay showed that many genes in the colitis tissue are enriched in the cancer development-related signaling pathway, such as PI3K–AKT, Ras, Wnt, TGF-beta, and MAPK signaling pathway. Together, these data suggested that even though chronic inflammation did not obviously increase genetic mutation accumulation, it could both genetically and epigenetically alter some genes related to cancer development.

Distinct mutational profile and immune microenvironment in microsatellite-unstable and POLE-mutated tumors

IntroductionMismatch repair (MMR)-deficient and DNA polymerase epsilon (POLE)-mutated tumors exhibit a high tumor mutation burden (TMB) and have been proven to be associated with good responses to immune checkpoint inhibitor treatments. However, the relationship between mutational characteristics of MMR-deficient and POLE-mutated tumors and the spatial architecture of tumor-infiltrating lymphocytes (TILs) has not been fully evaluated.MethodsWe retrieved microsatellite instability-high (MSI-high, N=20) and POLE-mutated (N=47) cases from the clinical next-generation sequencing cohort at Asan Medical Center. Whole-slide immunostaining for CD3, CD4, CD8, FoxP3 and PD-1 were performed with tissue samples of colorectal and gastric cancer (N=24) and the tumor-positive TIL cell densities were correlated with the tumor’s mutational features. The findings were compared with the results of similar analyses in The Cancer Genome Atlas-Colorectal Adenocarcinoma (TCGA-COADREAD) cohort (N=592).ResultsThe MSI-high group showed significantly higher overall TMBs with a number of insertion/deletion (indel) mutations relative to the POLE-mutated group (median TMB; 83.6 vs 12.5/Mb). Oncogenic/likely-oncogenic POLE mutations were identified with ultrahypermutations (≥100 mutations/Mb) (2/47, 4.3%). Concurrent POLE mutations of unknown significance and MSI-high cases were identified in eight cases (8/67, 11%), and two of these colorectal cancers had multiple POLE mutations, showing an ultramutated phenotype (378.1 and 484.4/Mb) and low indel mutation burdens with complete loss of MSH-6 or PMS-2, which was similar to the mutational profile of the POLE-inactivated tumors. Intratumoral CD3-positive, CD4-positive, CD8-positive, FoxP3-positive and PD-1-positive TIL cell densities were more strongly correlated with the indel mutation burden than with the total TMB (correlation coefficient, 0.61–0.73 vs 0.23–0.38). In addition, PI3K/AKT/mTOR pathway mutations were commonly found in MSI-high tumors (75%) but not in POLE-mutated tumors.ConclusionsIndel mutation burden rather than total TMB could serve as a predictor of high TILs in both MSI-high and POLE-mutated tumors. Multiple uncharacterized/non-pathogenic POLE mutations occurring via MMR deficiency within MSI-high tumors may have combined pathogenic roles. A mutated PI3K/AKT/mTOR pathway may be a biomarker that can be used to stratify patients with advanced MSI-high tumors for immune therapy.

Chronic Eosinophilic Leukaemia Associated with JAK2 Exon 13 Insertion/Deletion Mutations

Chronic eosinophilic leukaemia, not otherwise specified (CEL, NOS), is a diagnosis of exclusion made in cases in which there is clonal eosinophilia but an absence of genetic aberrations that define other disease subtypes. There is a need for further characterization of these cases in order to inform risk stratification and management. The importance of <i>JAK2</i> mutations in myeloproliferative neoplasms (MPN) as a whole is well established, although their role specifically in eosinophilic disorders is less clear, with only a minority of cases demonstrating <i>JAK2</i> abnormalities. Here, we report 2 cases with an exon 13 insertion-deletion (indel) mutation in <i>JAK2:</i> one with CEL-NOS and the second with an unspecified eosinophilic disorder. <i>JAK2</i> indels were not detected in a screen of suspected MPN cases (<i>n</i> = 592) without eosinophilia that tested negative for common MPN driver mutations. Our findings thus provide further evidence for a specific association between this rare mutation and clonal eosinophilic disorders.

Genetic diversity of H’mong short tail dog based on sequencing of the D-loop hypervariable -1 region (HV1)

The H'mong short tail dog is breed indigenous dogs, distributed in mountainousareas of northern Vietnam. H'mong short tail dog possesses many valuable properties such as intelligence, agility, good health, good shape, human friendliness, ease of training and it can fully meet the needs of war Dogs intelligence, strength, good parenting, people friendly and more importantly, still keeping wild characteristics of hunting dogs. The total 45 samples (blood) collected from 45 individuals in two provinces of Northern Vietnam (Ha Giang and Lao Cai), were used to assess genetic diversity based on sequencing hypervariable – 1 region (HV1) in D-loop genes. In the current study showed that genetic diversity of H'mong short tail dog was high with nucleotide diversity (Pi = 0.00801), haplotype diversity (Hd = 0.96162) and average number of nucleotide differences (Kt = 5.18384). Furthermore, 25 different haplotypes were recorded and divided into four main groups: A, B, C, and E. Of which, seven new haplotypes in haplogroups A (An1 to An7) and 18 haplotypes have been published in the world. In addition, H'mong short tail dog was found rare haplogroups (B1, C2, E1 and E4). Notably, there is none individuals contain haplotype of haplogroups (D and F). H'mong short tail dog were identified 38 single nucleotide polymorphisms, including 32 nucleotide base substitution/base insertion and 6 nucleotide indel mutation. Almost mutation was transversion (31/32) and only one nucleotide transition mutations. Phylogenetic tree shown that H'mong short tail dog have close relationship with dogs origin from East Asia (China, Japan and Korea).

Exploration of Genetic Variants within the Goat A-Kinase Anchoring Protein 12 (AKAP12) Gene and Their Effects on Growth Traits

The A-kinase anchoring protein 12 gene (AKAP12) is a scaffold protein, which can target multiple signal transduction effectors, can promote mitosis and cytokinesis and plays an important role in the regulation of growth and development. In our previous study, P1–7 bp (intron 3) and P2–13 bp (3′UTR) indels within the AKAP12 gene significantly influenced AKAP12 gene expression. Therefore, this study aimed to identify the association between these two genetic variations and growth-related traits in Shaanbei white cashmere goats (SBWC) (n = 1405). Herein, we identified two non-linkage insertions/deletions (indels). Notably, we found that the P1–7 bp indel mutation was related to the height at hip cross (HHC; p < 0.05) and the P2–13 bp indel was associated with body weight, body length, chest depth, chest width, hip width, chest circumference and cannon (bone) circumference in SBWC goats (p < 0.05). Overall, the two indels’ mutations of AKAP12 affected growth traits in goats. Compared to the P1–7 bp indel, the P2–13 bp indel is more suitable for the breeding of goat growth traits.

Detection of 15-bp Deletion Mutation within PLAG1 Gene and Its Effects on Growth Traits in Goats

Stature and weight are important growth and development traits for animals, which also significantly affect the productivity of livestock. Polymorphic adenoma gene 1 (PLAG1) is located in the growth-related quantitative trait nucleotides (QTN), and its variation has been determined to significantly affect the body stature of bovines. This study found that novel 15-bp InDel could significantly influence important growth traits in goats. The frequencies of genotypes of the 15-bp mutation and relationship with core growth traits such as body weight, body height, height at hip cross, chest circumference, hip width and body index were explored in 1581 individuals among 4 Chinese native goat breeds. The most frequent genotypes of Shaanbei white Cashmere goat (SWCG), Inner Mongolia White Cashmere goat (IMCG) and Guanzhong Dairy goat (GZDG) were II genotypes (insertion/insertion), and the frequency of ID genotype (insertion/deletion) was found to be slightly higher than that of II genotype in Hainan Black goat (HNBG), showing that the frequency of the I allele was higher than that of the D allele. In adult goats, there were significant differences between 15-bp variation and body weight, chest circumference and body height traits in SWCG (p < 0.05). Furthermore, the locus was also found to be significantly correlated with the body index of HNBG (p = 0.044) and hip width in GZDG (p = 0.002). In regard to lambs, there were significant differences in height at the hip cross of SWCG (p = 0.036) and hip width in IMWC (p = 0.005). The corresponding results suggest that the 15-bp InDel mutation of PLAG1 is associated with the regulation of important growth characteristics of both adult and lamb of goats, which may serve as efficient molecular markers for goat breeding.

Zebrafish Cre/lox regulated UFlip alleles generated by CRISPR/Cas targeted integration provide cell-type specific conditional gene inactivation

The ability to regulate gene activity spatially and temporally is essential to investigate cell type specific gene function during development and in postembryonic processes and disease models. The Cre/lox system has been widely used for performing cell and tissue-specific conditional analysis of gene function in zebrafish, but simple and efficient methods for isolation of stable, Cre/lox regulated alleles are lacking. Here we applied our GeneWeld CRISPR/Cas9 short homology-directed targeted integration strategy to generate floxed conditional alleles that provide robust gene knockdown and strong loss of function phenotypes. A universal targeting vector, UFlip, with sites for cloning short 24-48 bp homology arms flanking a floxed mRFP gene trap plus secondary reporter cassette, was integrated into an intron in hdac1, rbbp4, and rb1. Active, gene off orientation hdac1-UFlip-Off and rb1-UFlip-Off integration alleles result in >99% reduction of gene expression in homozygotes and recapitulate known indel loss of function phenotypes. Passive, gene on orientation rbbp4-UFlip-On and rb1-UFlip-On integration alleles do not cause phenotypes in trans-heterozygous combination with an indel mutation. Cre recombinase injection leads to recombination at alternating pairs of loxP and lox2272 sites, inverting and locking the cassette into the active, gene off orientation, and the expected mutant phenotypes. In combination with our endogenous neural progenitor Cre drivers we demonstrate rbbp4-UFlip-On and rb1-UFlip-On gene inactivation phenotypes can be restricted to specific neural cell populations. Replacement of the UFlip mRFP primary reporter gene trap with a 2A-RFP in rbbp4-UFlip-Off, or 2A-KalTA4 in rb1-UFlip-Off, shows strong RFP expression in wild type or UAS:RFP injected embryos, respectively. Together these results validate a simplified approach for efficient isolation of highly mutagenic Cre/lox responsive conditional gene alleles to advance zebrafish Cre recombinase genetics.