Efficient Generation of P53 Biallelic Mutations in Diannan Miniature Pigs Using RNA-Guided Base Editing

The base editing 3 (BE3) system, a single-base gene editing technology developed using CRISPR/Cas9n, has a broad range of applications for human disease model construction and gene therapy, as it is highly efficient, accurate, and non-destructive. P53 mutations are present in more than 50% of human malignancies. Due to the similarities between humans and pigs at the molecular level, pig models carrying P53 mutations can be used to research the mechanism of tumorigenesis and improve tumor diagnosis and treatment. According to pathogenic mutations of the human P53 gene at W146* and Q100*, sgRNAs were designed to target exon 4 and exon 5 of the porcine P53 gene. The target editing efficiencies of the two sgRNAs were 61.9% and 50.0%, respectively. The editing efficiency of the BE3 system was highest (about 60%) when C (or G) was at the 5th base. Puromycin screening revealed that 75.0% (21/28) and 68.7% (22/32) of cell colonies contained a P53 mutation at sgRNA-Exon5 and sgRNA-Exon4, respectively. The reconstructed embryos from sgRNA-Exon5-5# were transferred into six recipient gilts, all of which aborted. The reconstructed embryos from sgRNA-Exon4-7# were transferred into 6 recipient gilts, 3 of which became pregnant, resulting in 14 live and 3 dead piglets. Sequencing analyses of the target site confirmed 1 P53 monoallelic mutation and 16 biallelic mutations. The qPCR analysis showed that the P53 mRNA expression level was significantly decreased in different tissues of the P53 mutant piglets (p < 0.05). Additionally, confocal microscopy and western blot analysis revealed an absence of P53 expression in the P53 mutant fibroblasts, livers, and lung tissues. In conclusion, a porcine cancer model with a P53 point mutation can be obtained via the BE3 system and somatic cell nuclear transfer (SCNT).

Case Report: Characterizing the Role of the STXBP2-R190C Monoallelic Mutation Found in a Patient With Hemophagocytic Syndrome and Langerhans Cell Histiocytosis

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory disorder. HLH can be considered as a threshold disease depending on the trigger and the residual NK-cell cytotoxicity. In this study, we analyzed the molecular and functional impact of a novel monoallelic mutation found in a patient with two episodes of HLH. A 9-month-old child was diagnosed at 2 months of age with cutaneous Langerhans cell histiocytosis (LCH). After successful treatment, the patient developed an HLH episode. At 16 month of age, the patient went through an HSCT losing the engraftment 5 months later concomitant with an HLH relapse. The genetic study revealed a monoallelic mutation in the STXBP2 gene (.pArg190Cys). We transfected COS7 cells to analyze the STXBP2-R190C expression and to test the interaction with STX11. We used the RBL-2H3 cell line expressing STXBP2-WT-EGFP or R190C-EGFP for degranulation assays. Mutation STXBP2-R190C did not affect protein expression or interaction with syntaxin-11. However, we have demonstrated that STXBP2-R190C mutation diminishes degranulation in the RBL-2H3 cell line compared with the RBL-2H3 cell line transfected with STXBP2-WT or nontransfected. These results suggest that STXBP2-R190C mutation acts as a modifier of the degranulation process producing a decrease in degranulation. Therefore, under homeostatic conditions, the presence of one copy of STXBP2-R190 could generate sufficient degranulation capacity. However, it is likely that early in life when adaptive immune system functions are not sufficiently developed, an infection may not be resolved with this genetic background, leading to a hyperinflammation syndrome and eventually develop HLH. This analysis highlights the need for functional testing of new mutations to validate their role in genetic susceptibility and to establish the best possible treatment for these patients.

TSC1 loss-of-function increases risk for tauopathy by inducing tau acetylation and preventing autophagy-mediated tau clearance

Age-associated neurodegenerative disorders demonstrating tau-laden intracellular inclusions, including Alzheimer’s disease (AD), frontotemporal lobar degeneration (FTLD) and progressive supranuclear palsy (PSP), are collectively known as tauopathies. The vast majority of human tauopathies accumulate non-mutant tau rather than mutant forms of the protein, yet cell and animal models for non-mutant tauopathies are lacking. We previously linked a monoallelic mutation in the TSC1 gene to tau accumulation and FTLD. Now, we have identified new variants in TSC1 that predisposed to other tauopathies such as AD and PSP. These new TSC1 risk variants significantly decreased the half-life of TSC1/hamartin in vitro. Cellular and murine models of TSC1 haploinsufficiency (TSC1+/-) accumulated tau protein that exhibited aberrant acetylation on six lysine residues. Tau acetylation hindered its lysosomal degradation via chaperone-mediated autophagy leading to neuronal tau accumulation. Enhanced tau acetylation in TSC1+/- models was achieved through both an increase in p300 acetyltransferase activity and a decrease in SIRT1 deacetylase levels. Pharmacological modulation of either enzyme restored tau levels. Together, these studies substantiate TSC1 as a novel tauopathy risk gene and advance TSC1 haploinsufficiency as a new genetic model for tauopathy. In addition, these results promote acetylated tau as a rational target for diagnostic and therapeutic modalities in multiple tauopathies.

A case report and literature review of monoallelic mutation of GHR

Background Monoallelic mutations of GHR have been described in idiopathic short stature (ISS), although the significance of these remain unclear. We report a case of ISS with novel monoallelic S219L mutation of GHR and discuss the possible significance of monoallelic GHR mutation in ISS. Case presentation The proband, a 13.9-year-old Japanese boy, had severe short stature (−3.8 standard deviation [SD]). Serum insulin-like growth factor (IGF)-I level and growth hormone (GH) secretion was normal. His parents were nonconsanguineous and had normal stature. Genetic analyses revealed a novel monoallelic missense variation in exon 7 of GHR (S219L). The proband’s mother had the same variation. S219L might be the novel mutation judging from there being no registration of it as a single-nucleotide polymorphism (SNP) in any database, evolutional conservation of Ser219, in silico analyses, and computational molecular visualization analysis. Furthermore, a review of the literature showed that the median height of missense mutation carriers of GHR was relatively low. Conclusions We propose the possibility that monoallelic mutation of GHR increases the susceptibility to short stature.

Association between monoallelic TSHR mutations and congenital hypothyroidism: a statistical approach

Objective Biallelic TSHR mutations cause congenital hypothyroidism (CH). Serum TSH levels of monoallelic mutation carriers range from normal to mildly elevated, and thus the size of its effect remains unclear. The objectives were to examine the association between monoallelic TSHR mutations and positivity at newborn screening (NBS) for CH, and to test whether the association was modified by another genetic factor. Subjects and methods We enrolled 395 patients that had a positive result in NBS and sequenced TSHR. Monoallelic TSHR mutation carriers were further sequenced for DUOX2. Molecular functions of the mutations were verified in vitro. The frequency of the mutations in the study subjects was compared with a theoretical value in the Japanese general population. Odds ratio (OR) for NBS positivity associated with the mutation was calculated. Using Bayes’ theorem, we estimated a posterior probability of NBS positivity given the mutation. Results Twenty-six monoallelic TSHR mutation carriers were found. Four out of the 26 also had a monoallelic DUOX2 mutation (double heterozygotes). The frequencies of monoallelic TSHR mutation carriers (6.6%) and double heterozygotes (1.0%) were significantly higher than those in the general population (0.58% and 0.0087%, respectively). OR for NBS positivity of having a monoallelic TSHR mutation or being a double heterozygote was 12.0 or 117.9, respectively. Posterior probability of NBS positivity was 0.38% in monoallelic TSHR mutation carriers and 3.8% in double heterozygotes. Conclusions Monoallelic TSHR mutations are significantly associated with NBS positivity, and the association is further strengthened by the coexistence of monoallelic DUOX2 mutations.

GNRHR biallelic and digenic mutations in patients with normosmic congenital hypogonadotropic hypogonadism

Objective Normosmic congenital hypogonadotropic hypogonadism (nCHH) is a rare disorder characterised by lack of pubertal development and infertility, due to deficient production, secretion or action of gonadotropin-releasing hormone (GnRH) and, unlike Kallmann syndrome, is associated with a normal sense of smell. Mutations in the GNRHR gene cause autosomal recessive nCHH. The aim of this study was to determine the prevalence of GNRHR mutations in a group of 40 patients with nCHH. Design Cross-sectional study of 40 unrelated patients with nCHH. Methods Patients were screened for mutations in the GNRHR gene by DNA sequencing. Results GNRHR mutations were identified in five of 40 patients studied. Four patients had biallelic mutations (including a novel frameshift deletion p.Phe313Metfs*3, in two families) in agreement with autosomal recessive inheritance. One patient had a heterozygous GNRHR mutation associated with a heterozygous PROKR2 mutation, thus suggesting a possible role of synergistic heterozygosity in the pathogenesis of the disorder. Conclusions This study further expands the spectrum of known genetic defects associated with nCHH. Although GNRHR mutations are usually biallelic and inherited in an autosomal recessive manner, the presence of a monoallelic mutation in a patient should raise the possibility of a digenic/oligogenic cause of nCHH.