Molecular profiles and long-term outcomes of Thai children with hepatic glycogen storage disease in Thailand

BACKGROUND Thus far,genetic analysis of patients clinically diagnosed with glycogen storage diseases(GSDs)in Thailand has not been reported.AIM To evaluate the clinical and biochemical profiles,molecular analysis and long-term outcomes of Thai children diagnosed with hepatic GSD.METHODS Children aged<18 years diagnosed with hepatic GSD and followed up at King Chulalongkorn Memorial Hospital were recruited.Whole-exome sequencing(WES)was performed to identify the causative gene variants.Medical records were assessed.RESULTS All eight children with histopathologically confirmed diagnosis were classified by WES into subtypes Ia(n=1),III(n=3),VI(n=3),and IX(n=1).A total number of 10 variants were identified including G6PC(n=1),AGL(n=4),PYGL(n=5),and PHKA2(n=1).AGL had two novel variants.The clinical manifestations were hepatomegaly(n=8),doll-like facies(n=3),wasting(n=2),and stunting(n=5).All patients showed hypoglycemia,transaminitis,and dyslipidemia.The mainstay of treatment was cornstarch supplementation and high-protein and low-lactosefructose diet.After a median follow-up time of 9.59 years,height turned to normal for age in 3/5 patients and none had malnutrition.Liver enzymes,blood sugar,and lipid profiles improved in all.CONCLUSION Hepatomegaly,transaminitis,and hypoglycemia are the hallmarks of GSD confirmed by liver histopathology.Molecular analysis can confirm the diagnosis or classify the subtype that might benefit from personalized treatment,prognosis,and long-term care.

Hybrid Chiller Plant Optimization Strategies for Hotel Building on Nile River of Egypt

Hybrid chiller plants (HCPs) using multiple chillers and different energy sources are highly recommended in several energy applications in non-residential buildings such as hospitals and hotels. Time of use and cooling load profiles are significant factors that should be carefully considered either in chiller plant design or in chiller sequencing operation. This article aims to present an operation planning of HCP which consists of both electric and non-electric chillers. Four operational strategies are proposed and solved to compare their coefficients of performance and economics of running costs. A typical hotel building located on the Nile river in Egypt is selected to perform the current thermal and economic case study. The total cooling load profile of this hotel building is 4000 refrigeration tonnage (TR), which is simulated to optimize chiller sequence of operation and to select optimal design conditions of both numbers for electric and non-electric chillers used in HCP. The results of this comparative study for running cost are defined using various design configurations with different several chiller sequences available for each configuration. Then, the results of COPs, and operational running cost and initial cost are presented in this article also. The comparison aims to find the optimal design and operational sequencing for HCPs on thermal basis and economic analysis which were attached in this article. Recommendations and suggestions for future work are attached at the end of this article.

Identification and Characterization of Drought Stress-Responsive Novel microRNAs in Dongxiang Wild Rice

MicroRNAs （miRNAs） are non-coding small RNAs, which play important regulatory roles in response to biotic and abiotic stresses. Dongxiang wild rice （Oryza rufipogon, DXWR） can survive in extreme drought environment, but its molecular mechanism of drought resistance is still largely unknown. To further explore miRNA regulatory mechanisms involved in drought resistance, we identified 138 novel miRNAs in DXWR using small RNA sequencing and bioinformatics approaches, and found that the expression levels of 67 novel miRNAs were significantly affected by drought stress. In total, 200 candidate target genes were predicted and annotated for the drought stress-responsive novel miRNAs. Gene Ontology （GO） analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathways suggested that most of the target genes were related to metabolism. Stem-loop quantitative real-time PCR （qRT-PCR） results exhibited high concordance with sequencing data, which confirmed that miRNA expression patterns based on small RNA sequencing in the present study were reliable. Meanwhile, qRT-PCR validated the inverse expression patterns between several miRNAs and their target genes. These results will enhance our understanding of miRNA regulatory mechanisms in response to drought stress in DXWR, and can serve as an important reference for the protection and utilization of this valuable genetic resource.

Recent advances of single-cell RNA sequencing technology in mesenchymal stem cell research

Mesenchymal stem cells(MSCs)are multipotent stromal cells with great potential for clinical applications.However,little is known about their cell heterogeneity at a single-cell resolution,which severely impedes the development of MSC therapy.In this review,we focus on advances in the identification of novel surface markers and functional subpopulations of MSCs made by single-cell RNA sequencing and discuss their participation in the pathophysiology of stem cells and related diseases.The challenges and future directions of single-cell RNA sequencing in MSCs are also addressed in this review.

Clinical and genetic characteristics of retinoblastoma patients in a single center with four novel RB1 variants

AIM:To assess the clinical and genetic characteristics of children diagnosed with retinoblastoma(RB)at Gazi University Faculty of Medicine’s Department of Pediatric Oncology.METHODS:All cases diagnosed with RB and received treatment and follow-up in the Ophthalmology and Pediatric Oncology Department,October 2016 to May 2021 were evaluated retrospectively.The RB1 gene was analyzed by next-generation sequencing(NGS)technique in DNAs obtained from peripheral blood samples of the patients.RESULTS:This study included 53 cases with 67 RBaffected eyes during the study period.The mean age was 24.6(median:18.5,range:3–151)mo.There were 15(22.3%)Group D eyes and 39(58.2%)Group E eyes.The RB1 gene was sequenced by the NGS method in 19 patients.Heterozygous RB1:NM_000321.3:c.54_76del(p.Glu19AlafsTer4)variant was detected in a 15-month-old female with bilateral RB.Heterozygous RB1:NM_000321.3:c.1814+3A>T variant was detected in a 5.5-month-old male with bilateral RB.The intronic RB1:NM_000321.3:c.1332+4A>G variant was detected in patient 14,a 13-month-old male with unilateral RB.The RB1:NM_000321.3:c.575_576del(p.Lys192SerfsTer10)variant was found in an 18-month-old female with an allele frequency of 37%.These variants have not been reported in the literature and mutation databases.CONCLUSION:Four novel variants are described and one of them is found in two different patients.This data is crucial for assessing prognosis.It serves as a guide for estimating the long-term risk of secondary malignancy as well as the short-term risk of developing additional malignancies in the same eye and the other eye.

三株新型鸭源微RNA病毒分离毒株的全基因组序列分析

分离新型鸭源微RNA病毒进行全基因组测序并进行遗传进化分析。对本实验室2021年不同来源的种鸭和肉鸭病料进行PCR检测,初步确定存在一种未知分类的新型微RNA病毒感染。取病死鸭病料组织处理后接种SPF鸡胚分离病毒,设计引物对分离到的病毒进行PCR检测,通过重叠PCR方法进行全基因组扩增测序。将分离病毒各蛋白氨基酸序列两两比对,同时选取GenBank数据库中微RNA病毒代表毒株序列绘制系统进化树,并对主要蛋白P1、2C、3D序列比对分析。结果显示:共分离到三株微RNA病毒,分别命名为21101株、21016株和21075株(GenBank登录号:OQ927377~OQ927379)。基因组长度分别为7445、7445和7447 bp,均包含一个编码2141个氨基酸的开放阅读框(ORF),可划分为P1、P2、P3三个部分,符合微RNA病毒序列特征。基于全基因组序列遗传进化分析发现,三株分离病毒与本实验室前期分离的Duck/FC22/China/2017(GenBank登录号:MN102111)毒株及上海兽医研究所分离的Duck/AH15/CHN/2015(GenBank登录号:MT681985)位于同一分支,与鸭甲型肝炎病毒(Duck hepatitis A virus,DHAV)遗传距离最近。分离的三株鸭源微RNA病毒进行全基因组测序及遗传进化分析发现,与目前已知的两株微RNA毒株为同一类新型鸭源微RNA病毒。

Microarray-Based Differential Expression Monitoring of 79 Novel Genes in Human Fetal Tissues

ESTs fragments which represents corresponding novel genes were obtained by sequencing and bioinformatics analysis of human fet al kidney cDNA library. Microarray was prepared by using these novel EST fragmen ts by automatic spotting. Expression patters of 79 ESTs of novel genes from huma n fetal kidney were analyzed in fetal brain and fetal heart tissues of 20\|week\ | and 26\|week\|age fetus by performing of cDNA chip hybridization. This provide s clues for studying exact functions of the novel genes. 8 genes were obtained w hich were expressed differentially in the fetal brain and heart of 20\|week\| an d 26\|week\|age respectively. Then differentially expressed genes were identifie d by Northern analysis. The more exact function of the novel genes is under stud y.

Clinical applications of high-throughput genetic diagnosis in inherited retinal dystrophies: Present challenges and future directions

The advent of next generation sequencing(NGS) tech-niques has greatly simplified the molecular diagnosis and gene identification in very rare and highly heterogeneous Mendelian disorders. Over the last two years, these approaches, especially whole exome sequencing(WES), alone or combined with homozygosity mapping and linkage analysis, have proved to be successful in the identification of more than 25 new causative retinal dystrophy genes. NGS-approaches have also identified a wealth of new mutations in previously reported genes and have provided more comprehensive information concerning the landscape of genotype-phenotype correlations and the genetic complexity/diversity of human control populations. Although whole genome sequencing is far more informative than WES, the functional meaning of the genetic variants identified by the latter can be more easily interpreted, and final diagnosis of inherited retinal dystrophies is extremely successful, reaching 80%, particularly for recessive cases. Even considering the present limitations of WES, the reductions in costs and time, the continual technical improvements, the implementation of refined bioinformatic tools and the unbiased comprehensive genetic information it provides, make WES a very promising diagnostic tool for routine clinical and genetic diagnosis in the future.

(Z)-ETHYL 3-BROMO-4,4,4-TRIFLUORO-2-BUTENOATE AS A NOVEL CF_3-CONTAINING BUILDING BLOCK: Its Preparation and Pd-Catalysed Reaction with Alkynes and Organozinc Reagents

A novel CF_3-containing building block, (Z)-ethyl 3-bromo-4,4,4-trifluoro-2-butenoate, was easily prepared from CF_3CBr_3, the former reacted with alkynes Or organozinc reagents in the presence of Pd complexes to afford useful CF_3-containing intermediates in good yield.

NOVEL INTEGRATED DESIGN STRATEGIES FOR NET-ZERO-ENERGY SOLAR BUILDINGS(NZESBS)IN NANJING,CHINA

The connotations and denotations of the term net-zero-energy solar buildings(NZESBs)have been in constant flux because of continuous developments in solar heating technology,solar photovoltaic(PV)technology,building energy-storage technology,regional energy-storage technology,and energy-management systems.This paper focuses on innovative strategies for implementing NZESBs in Nanjing,China.These strategies include integrated architectural design,including passive solar design(respecting climatic characteristics and conducting integrated planning based on the environment,building orientation,distance between buildings,build-ing shape,ratio of window area to wall area,and building envelope)and active solar design(integration of the solar-energy-collecting end of the system–collectors and PV panels–with the building surface–roof,wall surfaces,balconies,and sun-shading devices–and the integration of solar-energy transfer and storage equip-ment with the building).Some Nanjing-specific recommendations and findings on NZESBs are proposed.The results illustrate that NZESBs can be realized in Nanjing if solar energy technologies are appropriately integrated with the character-istics of Nanjing’s geography,climate and buildings.

直接测序法用于四川骨髓库汉族人群HLA-C等位基因分布的研究

目的建立HLA-C等位基因通用引物直接测序法,研究中华骨髓库四川分库(简称四川骨髓库)中川籍汉族人群HLA-C等位基因分布。方法在四川骨髓库已获HLA-A/B/DRB1高分辨分型结果的600余份川籍汉族街头无关自愿捐献者标本中,随机选择244份标本,采用PCR-SBT对HLA-C位点测序分型,通用引物测序后产生的模棱两可分型结果,分别采用加测相应外显子和组特异性测序方法确认;获得所有标本确切的高分结果后,计算HLA-C各等位基因分布频率,并与其他人群比较。结果 244份标本中直接出HLA-C位点高分辨结果的比例为27.87%(68/244),须加测外显子1、5、6和7的为61.07%(149/244),须组特异性测序的为47.54%(116/244);共检出HLA-C等位基因25个,其中等位基因频率>10%的3个:C*01∶02、C*07∶02和C*03∶04,等位基因频率>1%的12个,累计频率95.69%;四川汉族人群HLA-C等位基因的分布与中国南方人群最为接近,与美国高加索人群和黑人的差异最大。另外,发现了1例新等位基因C*06∶45,它与同源性最高的C*06∶02在第187位碱基存在1个点突变:G>T,使密码子39由GAC>TAC,导致编码的氨基酸由天门冬氨酸(Asp)>酪氨酸(Tyr);此位点在此之前尚未发现过碱基突变。结论建立了针对HLA-C基因第2—4外显子的通用引物直接测序法,并提出了模棱两可结果的解决策略,所测标本均获得确切高分结果;四川汉族人群中HLA-C等位基因呈现多样性并存在自身特点。

应用cDNA芯片分析79个新基因的人胚组织表达谱

大规模cDNA测序和生物信息学技术相结合 ,得到来自于商品化的人胚肾cDNA文库 79个代表新基因的表达序列标签 (EST) .随后 ,采用高速度机械手制备这些cDNA的基因芯片 ,用于鉴定 79个新基因的ESTs在2 0周、 2 6周两个胚胎时期 6种组织中的基因表达状况 ,以研究这些EST片段代表的新基因功能提供线索 .通过芯片杂交及结果分析 ,得到同一个组织两个不同时相 8个差异表达的基因 。

HLA新等位基因HLA-B_*67:07测序分析及确认

目的人类白细胞抗原B新等位基因核苷酸序列分析及确认。方法采用序列特异性寡核苷酸探针技术(SSO)对2015年中国造血干细胞捐献者资料库(CMDP)人库数据进行HLA常规检测,结果显示有1个磁珠反应异常,进一步选择多聚酶链反应-测序(PCR-SBT)分型发现1个与HLA-B*67相关的有1个碱基不匹配的等位基因,选择对应的组特异性引物对该等位基因进行2次分离式测序,确认突变的等位基因和突变位点。结果发现1个与HLA-B~*67:01:01序列相近的新等位基因,实验结果表明在HLA-B~*67:0:01第3外显子370位置G>A,其突变造成HLA-B~*67:01:01氨基酸序列中100位的氨基端由甘氨酸(Gly)变成丝氨酸(Ser)。结论本次实验确认了1个新的HLA等位基因,2016年3月30日被世界卫生组织HLA因子命名委员会正式命名为HLA-B~*67:07。

HLA新等位基因HLA-A*11∶230序列分析及确认

目的对人类白细胞抗原(HLA)-A*11新等位基因核苷酸序列分析及确认。方法采用多聚酶链反应-测序(PCR-SBT)在对2015年中国造血干细胞捐献者资料库(CMDP)入库数据进行HLA常规分型中,应用组序列特异性引物(GSSP)单链测序方法针对常规SBT检测中A位点无匹配结果的2例无亲缘关系陕西汉族供者样本进一步进行确证实验,并运用聚合酶链式反应-序列特异性寡核苷酸探针技术(PCR-SSO)进行对照实验。结果经确证实验发现2例无亲缘关系供者携带同一HLA-A新等位基因序列,与同源基因HLA-A*11∶01序列相比,均在第2外显子98位置发生A>G突变,该突变造成氨基酸序列9位由酪氨酸(TAC)变成半胱氨酸(TGC);而2例样本的PCR-SSO结果均显示为HLA-A的常见等位基因组合,未见异常反应格局。结论经WHO HLA因子命名委员会正式命名的新等位基因HLA-A*11:230的发现和鉴定表明,对于常规测序无匹配结果的样本应进一步深入分析,即使以常见等位基因组合为分型结果的SSO方法仍存在HLA新等位基因漏检的可能,需引起注意。

HLA新等位基因HLA-B* 13∶42的确认及序列分析

目的认定1个人类白细胞抗原(HLA)新等位基因并分析其核苷酸序列。方法应用PCR-SBT进行HLA常规分型发现1个与HLA-B*13相关的异常等位基因,用针对于B*13的位点特异性SSSP引物测序,确认与同源性最高的HLA等位基因序列的差异。结果发现1个标本的HLA-B位点核苷酸序列与所有已知HLA-B位点等位基因核苷酸序列不一致,与同源性最高的等位基因B*13:02:01的差异是在第3外显子445位的G>T,其突变导致密码子GCC>TCC,结果造成B*13:02:01氨基酸序列中125位的Ala丙氨酸(A)变为丝氨酸(S)。结论该等位基因为HLA-B位点的1个新等位基因,该基因被世界卫生组织(WHO)HLA命名委员会命名为HLA-B*13:42。

NOTCH3基因测序发现一个新的致病突变CADASIL诊断的复杂性

目的通过一个新发现的伴皮质下梗死和白质脑病的常染色体显性遗传性脑动脉病(cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy,CADASIL)致病基因,说明基因测序对于CADASIL诊断的必要性及CADASIL诊断的复杂性。方法对1例疑似CADASIL患者的临床表现、实验室检查、影像学检查、家系及基因测序进行分析。临床上主要表现为反复缺血性卒中及进行性记忆力下降;实验室检查排除一些高凝疾病及血管炎;头部MRI提示皮质下白质广泛对侧性信号异常及多部位的新旧梗死病灶;家系分析患者家族成员存在偏头痛、反复卒中及记忆力下降;基因测序提示NOTCH3基因3号外显子存在一个基因突变(c.331G>T p.Gly111Cys),而这个突变位点截止到目前并未报道过。结果结合患者临床表现、影像学、家系及基因测序分析确诊该患者为CADASIL,通过分析CADASIL突变基因的致病机制确认该患者存在的突变基因为CADASIL一个新发现的致病突变。结论新的CADASIL致病基因突变的发现强调了基因测序及提高对突变基因致病机制认识的重要性。

中国南方汉族人群中一个常见型新等位基因HLA-C＊08：22的基因频率调查和分析

本研究调查南方汉族人群中1个常见型的HLA新等位基因C*08:22的基因频率。对163份南方汉族无血缘关系个体血样进行常规的HLA-C基因第2、第3和第4外显子测序分型;对所检出的32份带有C*08:01:01/08:22模棱两可结果的样本,进一步增加HLA-C基因的第5和第6外显子测序。在C*08:22被发现和获得WHOHLA因子命名委员会认可前,对40例被鉴定携带C*08:01:01等位基因的无血缘关系供/受者进行HLA-C基因外显子2-6再测序。所有的序列均导入Assign 3.5 SBT软件,分析等位基因型。结果表明,32份无血缘关系个体检出C*08:01:01/08:22模棱两可结果的样本中,发现3例样本鉴定为C*08:22,南方汉族无血缘关系个体中C*08:22的基因频率为0.92%。回顾性分析以往40例携带C*08:01:01等位基因的无血缘关系供/受者对发现,其中2对供/受者实际为C*08:22。结论:C*08:22在南方汉族中为常见型的HLA等位基因,当测序分型出现C*08:01:01/08:22模棱两可的结果时,不能轻易视为罕见型等位基因而错误地排除。

1例HLA新等位基因B*15:201*的发现及确认

目的 1个人类白细胞抗原(human leucocyte antigen,HLA)新等位基因的认定和序列分析。方法应用多聚酶链式反应—基于测序的分型技术(polymerase chain reaction-sequence based typing,PCR-SBT)对中国造血干细胞捐献者资料库河北分库捐献者进行HLA高分辨率分型,发现1个HLA-B位点的异常结果,应用序列特异性测序引物(sequence specific sequencing primer,SSSP)分离测序,确认发生突变的等位基因及发生突变的位置。结果发现1例标本的HLA-B位点分型结果不能认定为任何已知结果,利用SSSP引物进行分离测序后发现1条链的等位基因核苷酸序列与所有已知HLA-B位点等位基因核苷酸序列不一致,与同源性最高的等位基因B*15:01:01:01的差异是在第2外显子662位的A>T,其突变导致密码子CAT>CTT,结果造成B*15:01:01:01氨基酸序列中221位的组氨酸(H)变为亮氨酸(L)。结论该等位基因为HLA-B位点的1个新等位基因,被世界卫生组织(WHO)HLA系统命名委员会命名为HLA-B*15:201。

WHO HLA命名委员会命名的新等位基因HLA-A*24:327序列分析及确认

目的：发现一个人类白细胞抗原（HLA）-A新等位基因并对其核苷酸序列进行分析及确认。方法采用序列特异性寡核苷酸探针技术（SSO）对2015年中国造血干细胞捐献者资料库（CMDP）陕西分库入库数据进行 HLA常规检测，针对罕见结果进一步选择多聚酶链反应-测序（PCR-SBT）和组序列特异性引物（GSSP）进行确证试验，明确突变位点。结果 SSO分型结果显示为罕见等位基因，经PCR-SBT复核无完全匹配结果，提示疑似新等位基因，经 GSSP确证发现该基因与同源基因 HLA-B＊24∶02∶01∶01相比，在第3外显子544位置发生碱基变异由 G＞A，该突变造成氨基酸序列158位由丙氨酸（GCC）变成苏氨酸（ACC）。结论确认了一个新的 HLA-A等位基因，2015年12月31日被世界卫生组织HLA命名委员会正式命名为 HLA-A＊24∶327。

一例白血病患者及家系HLA-B基因全长序列及18个点突变分析

背景:人类白细胞抗原HLA经过长期进化形成丰富的多态性,近几年由于受检人数增加及HLA分型技术快速发展,HLA新基因不断被发现。目的:采用下一代测序技术对1例白血病患者及家系HLA-B基因的全长序列及18个点突变进行分析。方法:应用序列特异性寡核苷酸探针杂交(PCR-SSOP)及聚合酶链反应-直接测序法(PCR-SBT)发现患者的HLA-B结果异常。为了鉴定该基因,采用下一代测序技术对该基因全长进行测序,同时采集患者父亲、母亲及2位同胞姐妹的血样进行HLA基因的遗传学分析。结果与结论:应用序列特异性寡核苷酸探针杂交及聚合酶链反应-直接测序法均提示该样本HLA-B无完全匹配的基因型。应用下一代测序技术分析发现,与同源性最高的等位基因B*15:09:01相比,该基因在外显子、内含子和3′UTR共存在18个碱基突变。5个外显子碱基突变位于第3,4外显子,分别为:第486位G→C、第583位T→C、第636位T→C、第652位A→G和第756位C→T,导致5个相应密码子发生改变,其中2个碱基替换为错义突变,第171位酪氨酸(Tyr)→组氨酸(His)、第194位异亮氨酸(Ile)→缬氨酸(Val)。家系调查显示患者HLA-B新基因来源于父亲。新基因序列已递交给Genbank数据库(MG595995)。应用下一代测序技术鉴定了1个HLA-B新等位基因,该基因于2017年12月被WHOHLA因子命名委员会正式命名为HLA-B*15:435。