> Experimental oncology    2022

Rapid Low-cost Detection of Type 2calr Mutation by Allele-specific rt-pcr for Diagnosis of Myeloproliferative Neoplasms

Dybkov MV, Zavelevich MP, Gluzman DF, Telegeev GD.

DOI：10.32471/exp-oncology.2312-8852.vol-44-no-1.17329

Abstract
Background: Approximately 15% to 24% of essential thrombocythemia (ET) and 25-35% of primary myelofibrosis cases carry a mutation in the calreticulin (CALR) gene. Sanger sequencing, qPCR, high resolution melt or targeted next generation sequencing usually used to detect these mutations are expensive and require costly equipment. Nevertheless, type 1 CALR mutations are detectable by using polymerase chain reaction (PCR) and agarose gel electrophoresis.

Aim: To offer the use of the allele-specific reverse transcription (RT) PCR for rapid low-cost detection of the type 2 mutation in the CALR gene.

Materials and methods: Allele-specific primers designed for detecting type 2 mutation (5-bp insertion; c.1154_1155 ins TTGTC) of the CALR gene were used for allele-specific RT-PCR analysis of cDNA of the patient with JAK2-, MPL-negative ET, whose mutation in CALR gene has been identified by Sanger sequencing. RT-PCR samples were analyzed by agarose gel electrophoresis.

Results: The type 2 mutation (K385fs*47 ins5) in CALR gene was detected by Sanger sequencing in JAK2- and MPL-negative ET patient. The cDNA obtained was then re-analyzed by using allele-specific RT-PCR with newly designed primers. Normal and type 2 mutation alleles of the CALR gene were detected by gel electrophoresis. The results of allele-specific RT-PCR were consistent with the data of Sanger sequencing.

Conclusion: Allele-specific RT-PCR analysis may be used for the fast low-cost detection of the major type 2 mutation (ins 5) of the CALR gene in patients with MPNs.

> Molecular Medicine Reports    2021

Comparative analysis of the autism-related variants between different autistic children in a family pedigree

Luxi Shen, Panyuan Li, Tianjin Zheng, Meichen Luo, Shao Zhang, Yuting Huang, Yongwu Hu, HongZhi Li

DOI：10.3892/mmr.2021.12336

Abstract
The present study aimed to provide evidence for the genetic heterogeneity of familial autism spectrum disorder (aSd), which might help to improve our understanding of the complex polygenic basis of this disease. Whole-exome sequencing (WeS) was performed on two autistic children in a family pedigree, and reasonable conditions were set for prelim-inarily screening variant annotations. Sanger sequencing was used to verify the preliminarily screened variants and to determine the possible sources. in addition, autism-related genes were screened according to autism databases, and their variants were compared between two autistic children. The results showed that there were 21 genes respectively for autistic children IV2 and IV4, preliminarily screened from all variants based on the harmfulness (high) and quality (high or medium) of the variants, as well as the association between mutant genes and autism in human gene mutation database. Furthermore, candidate autism‑related genes were screened according to the evidence score of >4 in the autism KnowledgeBase (AutismKB) database or ≥3 in the AutDB database. A total of 11 and 10 candidate autism‑related genes were identified in the autistic children IV2 and IV4, respectively. Candidate genes with an evidence score of >16 in autismKB were credible autism‑related genes, which included LAMC3, JMJD1C and CACNA1H in child IV2, as well as SCN1A, SETD5,CHD7 and KCNMA1 in child IV4. Other than the c.G1499A mutation of SCN1A, which is known to be associated with Dravet syndrome, the specific missense variant loci of other six highly credible putative autism‑related genes were reported for the first time, to the best of the authors' knowledge, in the present study. These credible autism-related variants were inherited not only from immediate family members but also from extended family members. in summary, the present study established a reasonable and feasible method for screening credible autism‑related genes from WeS results, which by be worth extending into clinical practice. The different credible autism-related genes between the two autistic children indicated a complex polygenic architecture of aSd, which may assist in the early diagnosis of this disease.

> Asian Journal of Andrology    2021

A new TEX11 mutation causes azoospermia and testicular meiotic arrest

Xiao-Chen Yu, Meng-Jing Li, Fei-Fei Cai, Si-Jie Yang, Hong-Bin Liu, Hao-Bo Zhang

DOI：10.4103/aja.aja_8_21

Abstract
There are many unknown genetic factors that lead to infertility in nonobstructive azoospermia men. Here, we performed whole-exome sequencing in blood samples obtained from 40 azoospermia patients with meiotic arrest and found a novel c.151_154del(p.D51fs) frame-shift mutation in exon 3 of the testis expressed 11 (TEX11) gene in one patient. Sanger sequencing analysis of the patient and 288 fertile men was performed to validate the mutation. Immunohistochemical analysis showed TEX11 expression in late-pachytene spermatocytes and in round spermatids in fertile human testes. In contrast, testes of the patient with TEX11 mutation underwent meiotic arrest and lacked TEX11 expression. Western blotting of human embryonic kidney (HEK293) cells transfected with a vector for the p.D51fs TEX11 variant detected no TEX11 expression. In conclusion, we identified a novel frame-shift mutation in the TEX11 gene in an azoospermia patient, emphasizing that this gene should be included in genetic screening panels for the clinical evaluation of azoospermia patients.

> Cell Research    2019

Endogenous reverse transcriptase and RNase H-mediated antiviral mechanism in embryonic stem cells

Junyu Wu, Chunyan Wu, Fan Xing, Liu Cao, Weijie Zeng, Liping Guo, Ping Li, Yongheng Zhong, Hualian Jiang, Manhui Luo, Guang Shi, Lang Bu, Yanxi Ji, Panpan Hou, Hong Peng, Junjiu Huang, Chunmei Li, Deyin Guocorresponding author

DOI：10.1038/s41422-021-00524-7

Abstract
Nucleic acid-based systems play important roles in antiviral defense, including CRISPR/Cas that adopts RNA-guided DNA cleavage to prevent DNA phage infection and RNA interference (RNAi) that employs RNA-guided RNA cleavage to defend against RNA virus infection. Here, we report a novel type of nucleic acid-based antiviral system that exists in mouse embryonic stem cells (mESCs), which suppresses RNA virus infection by DNA-mediated RNA cleavage. We found that the viral RNA of encephalomyocarditis virus can be reverse transcribed into complementary DNA (vcDNA) by the reverse transcriptase (RTase) encoded by endogenous retrovirus-like elements in mESCs. The vcDNA is negative-sense single-stranded and forms DNA/RNA hybrid with viral RNA. The viral RNA in the heteroduplex is subsequently destroyed by cellular RNase H1, leading to robust suppression of viral growth. Furthermore, either inhibition of the RTase activity or depletion of endogenous RNase H1 results in the promotion of virus proliferation. Altogether, our results provide intriguing insights into the antiviral mechanism of mESCs and the antiviral function of endogenized retroviruses and cellular RNase H. Such a natural nucleic acid-based antiviral mechanism in mESCs is referred to as ERASE (endogenous RTase/RNase H-mediated antiviral system), which is an addition to the previously known nucleic acid-based antiviral mechanisms including CRISPR/Cas in bacteria and RNAi in plants and invertebrates.

> Cancer Letters    2020

PTEN deficiency facilitates the therapeutic vulnerability to proteasome inhibitor bortezomib in gallbladder cancer

Tian-YiJiang, Xiao-FanFeng, ZhengFang, Xiao-WenCui, Yun-KaiLin, Yu-FeiPan, ChunYang, Zhi-WenDing, Yong-JieZhang, Ye-XiongTan, Hong-YangWang, Li-WeiDonga

DOI：10.1016/j.canlet.2020.11.016

Abstract
Gallbladder cancer (GBC) is an aggressive malignancy of biliary tract with poor prognosis. Although several studies have shown the frequency of relevant genetic alterations, there are few genetic models or translational studies that really benefit for GBC treatment in the era of precision medicine. By targeted sequencing and immunohistochemistry staining, we identified that phosphate and tension homology deleted on chromosome ten (PTEN) was frequently altered in GBC specimens, and loss of PTEN expression was independently correlated with poor survival outcomes. Further drug screening assays revealed proteasome inhibitor bortezomib as a promising agent for GBC treatment, and knockdown of PTEN increased bortezomib efficacy both in vivo and in vitro. Therapeutic evaluation of patient derived xenografts (PDXs) strongly supported the utilization of bortezomib in PTEN deficient GBC. Mechanically, functional PTEN inhibited ARE-dependent transcriptional activity, the same machinery regulating the transcription of proteasome subunits, thus PTEN suppressed proteasome activity and bortezomib sensitivity. Through siRNA screening, we identified the ARE-related transcriptional suppressor BACH1 involved in PTEN-mediated proteasome inhibition and regulated by PTEN-AKT1 axis. In summary, our study indicates that proteasome activity represents a prime therapeutic target in PTEN-deficient GBC tumors, which is worthy of further clinical validation.

> Science Translational Medicine    2020

PTEN status determines chemosensitivity to proteasome inhibition in cholangiocarcinoma

Tian-Yi Jiang, Yu-Fei Pan, Zheng-Hua Wan, Yun-Kai Lin, Bin Zhu, Zhen-gang Yuan, Yun-Han Ma, Yuan-Yuan Shi, Tian-Mei Zeng, Li-Wei Dong,  Ye-Xiong Tan, Hong-Yang Wang

DOI：10.1126/scitranslmed.aay0152

Abstract
Patient-derived xenografts (PDXs) and PDX-derived cells (PDCs) are useful in preclinical research. We performed a drug screening assay using PDCs and identified proteasome inhibitors as promising drugs for cholangiocarcinoma (CCA) treatment. Furthermore, we determined that phosphate and tensin homology deleted on chromosome ten (PTEN) deficiency promotes protein synthesis and proteasome subunit expression and proteolytic activity, creating a dependency on the proteasome for cancer cell growth and survival. Thus, targeting the proteasome machinery with the inhibitor bortezomib inhibited the proliferation and survival of CCA cells lacking functional PTEN. Therapeutic evaluation of PDXs, autochthonous mouse models, and patients confirmed this dependency on the proteasome. Mechanistically, we found that PTEN promoted the nuclear translocation of FOXO1, resulting in the increased expression of BACH1 and MAFF. BACH1 and MAFF are transcriptional regulators that recognize the antioxidant response element, which is present in genes encoding proteasome subunits. PTEN induced the accumulation and nuclear translocation of these proteins, which directly repressed the transcription of genes encoding proteasome subunits. We revealed that the PTEN-proteasome axis is a potential target for therapy in PTEN-deficient CCA and other PTEN-deficient cancers.

> Molecular Genetics & Genomic Medicine    2019

Identification of gene variants in 130 Han Chinese patients with hypospadias by targeted next‐generation sequencing

Wanyu Zhang, Jinxiu Shi, Chenhui Zhang, Xincheng Jiang, Junqi Wang, Wei Wang, Defen Wang, Jihong Ni, Lifen Chen, Wenli Lu, Yuan Xiao, Weijing Ye, Zhiya Dong

DOI：10.1002/mgg3.827

Abstract
Background: Hypospadias is a common congenital malformation of male external genitalia, which mainly manifests as an abnormal urethral opening on the ventral side of the penis. The etiology and clinical phenotype of hypospadias is highly heterogeneous, and its clinical diagnosis is challenging. Currently, over 70% of patients have an unknown etiology. Here, we performed a targeted analysis of gene mutations in 130 patients with hypospadias of unknown etiology to find the precise genetic cause.

Methods: We developed a targeted next‐generation sequencing (NGS) panel, encompassing the exon coding regions of 105 genes involved in external genitalia and urogenital tract development and performed sequencing analysis on 130 children with hypospadias of unknown etiology.

Results: In total, 25 patients with hypospadias (19.2%) were found to have 20 mutations among the nine genes involved in external genitalia and urogenital tract development, including 16 reported and four novel mutation sites. Twenty‐two patients (16.9%) had diagnostic variants. Multiple genetic mutations were identified in three of the 25 patients. Hypospadias combined with micropenis was the most common phenotype (68%) in 25 patients.

Conclusions: Higher frequency mutations were identified in SRD5A2 (52%) and AR (24%) in our patient cohort. Middle or posterior hypospadias with micropenis may be significant indicators of genetic variations. Polygenic inheritance may be a rare genetic cause of hypospadia

> Genetic Testing and Molecular Biomarkers    2019

Design of a Targeted Sequencing Assay to Detect Rare Mutations in Circulating Tumor DNA

Jianxia Chen, Jun Chen, Fusheng He, Yiqiong Huang, Shan Lu, Haibo Fan, Mingbang Wang, Ruihuan Xu

DOI：10.1089/gtmb.2018.0173

Abstract
Background: Qualitative and quantitative detection of circulating tumor DNA (ctDNA) is a liquid biopsy technology used for early cancer diagnosis. However, the plasma ctDNA content is extremely low, so it is difficult to detect somatic mutations of tumors using conventional sequencing methods. Target region sequencing (TRS) technology, through enrichment of the target genomic region followed by next generation sequencing, overcomes this challenge and has been widely used in ctDNA sequencing.

Methods: We designed a ctDNA sequencing panel to capture 128 tumor genes, and tested the performance of the panel by running TRS for ctDNA of a clear cell renal cell carcinoma (ccRCC) patient and 12 breast cancer patients.

Results: TRS using the new ctDNA panel at more than 500 × coverage depth achieved almost the same accuracy as traditional whole-exome sequencing (WES). PBRM1 p.L641V was detected in the plasma sample of the ccRCC patient with an allele frequency of 0.2%. The ctDNA of 12 breast cancer patients was sequenced at a depth of 500-fold, achieving 99.89% coverage; 34 genes were detected with mutations, including the drug target genes BRCA2, PTEN, TP53, APC, KDR, and NOTCH2.

Conclusions: This TRS new ctDNA panel can be used to detect mutations in cell-free DNA from multiple types of cancer.

> Human Genome Variation    2019

Targeted next-generation sequencing identified a known EMD mutation in a Chinese patient with Emery-Dreifuss muscular dystrophy

Xiafei Dai, Chenqing Zheng, Xuepin Chen, Yibin Tang, Hongmei Zhang, Chao Yan, Huihui Ma, Xiaoping Li

DOI：10.1038/s41439-019-0072-8

Abstract
Emery-Dreifuss muscular dystrophy (EDMD) is a rare X-linked recessive disease characterized by the clinical triad of early childhood joint contractures, progressive weakness in muscles and cardiac involvement and can result in sudden death. Targeted next-generation sequencing was performed for a Chinese patient with EDMD and the previously reported mutation [NM_000117.2: c.251_255del (p.Leu84Profs*7)] in exon 3 of the emerin gene (EMD) was identified.

> International Journal of Biological Sciences    2019

Evaluation of the effects of sequence length and microsatellite instability on single-guide RNA activity and specificity

Changzhi Zhao , Yunlong Wang , Xiongwei Nie , Xiaosong Han , Hailong Liu , Guanglei Li , Gaojuan Yang, Jinxue Ruan , Yunlong Ma , Xinyun Li, Huijun Cheng, Shuhong Zhao, Yaping Fang, Shengsong Xie

DOI：10.7150/ijbs.37152

Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology is effective for genome editing and now widely used in life science research. However, the key factors determining its editing efficiency and off-target cleavage activity for single-guide RNA (sgRNA) are poorly documented. Here, we systematically evaluated the effects of sgRNA length on genome editing efficiency and specificity. Results showed that sgRNA 5′-end lengths can alter genome editing activity. Although the number of predicted off-target sites significantly increased after sgRNA length truncation, sgRNAs with different lengths were highly specific. Because only a few predicted off-targets had detectable cleavage activity as determined by Target capture sequencing (TargetSeq). Interestingly, > 20% of the predicted off-targets contained microsatellites for selected sgRNAs targeting the dystrophin gene, which can produce genomic instability and interfere with accurate assessment of off-target cleavage activity. We found that sgRNA activity and specificity can be sensitively detected by TargetSeq in combination with in silico prediction. Checking whether the on-and off-targets contain microsatellites is necessary to improve the accuracy of analyzing the efficiency of genome editing. Our research provides new features and novel strategies for the accurate assessment of CRISPR sgRNA activity and specificity.

> Oral Diseases    2019

Novel PITX2 mutations identified in Axenfeld–Rieger syndrome and the pattern of PITX2‐related tooth agenesis

Zhuangzhuang Fan, Shichen Sun, Haochen Liu, Miao Yu, Ziyuan Liu, Sing‐Wai Wong, Yang Liu, Dong Han, Hailan Feng

DOI：10.1111/odi.13196

Abstract
Objectives: To investigate the mutations in patients with Axenfeld-Rieger syndrome (ARS) and the pattern of PITX2-related tooth agenesis.

Methods: Whole-exome sequencing (WES) and copy number variation (CNV) array were used to screen the mutations in four ARS probands. After Sanger sequencing and quantitative polymerase chain reaction (qPCR) validation, secondary structure prediction and dual-luciferase assay were employed to investigate the functional impact. Eighteen PITX2-mutated patients with definite dental records were retrieved from our database and literatures, and the pattern of PITX2-related tooth agenesis was analyzed.

Results: A novel de novo segmental deletion of chromosome 4q25 (GRCh37/hg19 chr4:111, 320, 052-111, 754, 236) encompassing PITX2 and three novel PITX2 mutations c.148C > T, c.257G > A, and c.630insCG were identified. Preliminary functional studies indicated the transactivation capacity of mutant PITX2 on Distal-less homeobox 2 (DLX2) promoter was compromised. The maxillary teeth showed significantly higher rate of agenesis (57.94%) than the mandibular teeth (44.05%). The most often missing teeth were upper lateral incisors (83.33%) and upper second premolars (69.44%). Teeth with the least agenesis rate were the lower second molars (19.44%) and lower first molars (8.33%).

Conclusions: We identified a novel 4q25 microdeletion including PITX2 and three novel PITX2 mutations, and statistically analyzed the PITX2-related tooth agenesis pattern.

> Molecular Cytogenetics    2019

A novel 14q13.1-21.1 deletion identified by CNV-Seq in a patient with brain-lung-thyroid syndrome, tooth agenesis and immunodeficiency.

Xuyun Hu, Jun Liu, Ruolan Guo, Jun Guo, Zhipeng Zhao, Wei Li, Baoping Xu, Chanjuan Hao

DOI：10.1186/s13039-019-0463-z

Abstract
Background: Chromosome 14q11-q22 deletion syndrome (OMIM 613457) is a rare genomic disorder. The phenotype heterogeneity depends on the deletion size, breakpoints and genes deleted. Critical genes like FOXG1, NKX2-1, PAX9 were identified.

Case presentation: We performed whole exome sequencing (WES) and copy number variation sequencing (CNV-seq) for a patient with mild speech and motor developmental delay, short stature, recurrent pulmonary infections, tooth agenesis and triad of brain-lung-thyroid syndrome. By using CNV-seq, we identified a 3.1 Mb de novo interstitial deletion of the 14q13.2q21.1 region encompassing 17 OMIM genes including NKX2-1, PAX9 and NFKBIA. Our patient's phenotype is consistent with other published 14q13 deletion patients.

Conclusion: Our results showed the combination of WES and CNV-seq is an effective diagnostic strategy for patients with genetic or genomic disorders. After reviewing published patients, we also proposed a new critical region for 14q13 deletion syndrome with is a more benign disorder compared to 14q11-q22 deletion syndrome.