Implications of genetic testing and informed consent before and after genetic testing in individuals with cancer

Recent advancements in next generation sequencing have allowed for genetic information become more readily available in the clinical setting for those affected by cancer and by treating clinicians.Given the lack of access to geneticists,medical oncologists and other treating physicians have begun ordering and interpreting genetic tests for individuals with cancer through the process of"mainstreaming".While this process has allowed for quicker access to genetic tests,the process of"mainstreaming"has also brought several challenges including the dissemination of variants of unknown significance results,ordering of appropriate tests,and accurate interpretation of genetic results with appropriate followup testing and interventions.In this editorial,we seek to explore the process of informed consent of individuals before obtaining genetic testing and offer potential solutions to optimize the informed consent process including categorization of results as well as a layered consent model.

Current scenario of the genetic testing for rare neurological disorders exploiting next generation sequencing

Next generation sequencing is currently a cornerstone of genetic testing in routine diagnostics,allowing for the detection of sequence variants with so far unprecedented large scale,mainly in genetically heterogenous diseases,such as neurological disorders.It is a fast-moving field,where new wet enrichment protocols and bioinformatics tools are constantly being developed to overcome initial limitations.Despite the as yet undiscussed advantages,however,there are still some challenges in data analysis and the interpretation of variants.In this review,we address the current state of next generation sequencing diagnostic testing for inherited human disorders,particularly giving an overview of the available high-throughput sequencing approaches;including targeted,whole-exome and whole-genome sequencing;and discussing the main critical aspects of the bioinformatic process,from raw data analysis to molecular diagnosis.

Comparison of next generation sequencing-based and methylated DNA immunoprecipitation-based approaches for fetal aneuploidy non-invasive prenatal testing

Over the past few years, many researchers have attempted to develop non-invasive prenatal testing methods in order to investigate the genetic status of the fetus. The aim is to avoid invasive procedures such as chorionic villus and amniotic fluid sampling, which result in a significant risk for pregnancy loss. The discovery of cell free fetal DNA circulating in the maternal blood has great potential for the development of non-invasive prenatal testing(NIPT) methodologies. Such strategies have been successfully applied for the determination of the fetal rhesus status and inherited monogenic disease but the field of fetal aneuploidy investigation seems to be more challenging. The main reason for this is that the maternal cell free DNA in the mother's plasma is far more abundant, and because it is identical to half of the corresponding fetal DNA. Approaches developed are mainly based on next generation sequencing(NGS) technologies and epigenetic genetic modifications, such as fetal-maternal DNA differential methylation. At present, genetic services for non-invasive fetal aneuploidy detection are offered using NGS-based approaches but, for reasons that are presented herein, they still serve as screening tests which are not readily accessed by the majority of couples. Here we discuss the limitations of both strategies for NIPT and the future potential of the methods developed.

Endoscopic ultrasound-guided fine-needle aspiration pancreatic adenocarcinoma samples yield adequate DNA for next-generation sequencing:A cohort analysis

BACKGROUND Genetic tests are increasingly performed for the management of unresectable pancreatic cancer.For genotyping aimed samples current guidelines recommend using core specimens,although based on moderate quality evidence.However,in clinical practice among the endoscopic ultrasound(EUS) guided tissue acquisition methods,fine needle aspiration(FNA) is the most widely performed.AIM To assess the adequacy for next generation sequencing(NGS) of the DNA yielded from EUS-FNA pancreatic adenocarcinoma(PDAC) samples.METHODS Between November 2018 and December 2021,105 patients with PDAC confirmed by EUS-FNA were included in the study at our tertiary gastroenterology center.Either 22 gauge(G) or 19G FNA needles were used.One pass was dedicated to DNA extraction.DNA concentration and purity(A260/280,A260/230) were assessed by spectrophotometry.We assessed the differences in DNA parameters according to needle size and tumor characteristics(size,location) and the adequacy of the extracted DNA for NGS(defined as A260/280 ≥ 1.7,and DNA yield:≥ 10 ng for amplicon based NGS,≥ 50 ng for whole exome sequencing [WES],≥ 100 ng for whole genome sequencing [WGS]) by analysis of variance and ttest respectively.Moreover,we compared DNA purity parameters across the different DNA yield categories.RESULTS Our cohort included 49% male patients,aged 67.02 ± 8.38 years.The 22G needle was used in 71%of the cases.The DNA parameters across our samples varied as follows:DNA yield:1289 ng(inter quartile range:534.75-3101),A260/280 = 1.85(1.79-1.86),A260/230 = 2.2(1.72-2.36).DNA yield was > 10 ng in all samples and > 100 ng in 93% of them(one sample < 50 ng).There were no significant differences in the concentration and A260/280 between samples by needle size.Needle size was the only independent predictor of A260/230 which was higher in the 22G samples(P =0.038).NGS adequacy rate was 90% for 19G samples regardless of NGS type,and for 22G samples it reached 89% for WGS adequacy and 91% for WES and amplicon based NGS.Samples with DNA yield > 100 ng had significantly higher A260/280(1.89 ± 0.32 vs 1.34 ± 0.42,P = 0.013).Tumor characteristics were not corelated with the DNA parameters.CONCLUSION EUS-FNA PDAC samples yield DNA adequate for subsequent NGS.DNA amount was similar between 22G and 19G FNA needles.DNA purity parameters may vary indirectly with needle size.

Minimal-Length Interoperability Test Sequences Generation via Genetic Algorithm

A novel interoperability test sequences optimization scheme is proposed in which the genetic algorithm （GA） is used to obtain the minimal-length interoperability test sequences. During our work, the basic interoperability test sequences are generated based on the minimal-complete-coverage criterion, which removes the redundancy from conformance test sequences. Then interoperability sequences minimization problem can be considered as an instance of the set covering problem, and the GA is applied to remove redundancy in interoperability transitions. The results show that compared to conventional algorithm, the proposed algorithm is more practical to avoid the state space explosion problem, for it can reduce the length of the test sequences and maintain the same transition coverage.

Class hierarchical test case generation algorithm based on expanded EMDPN model

A new model of event and message driven Petri network(EMDPN) based on the characteristic of class interaction for messages passing between two objects was extended. Using EMDPN interaction graph, a class hierarchical test-case generation algorithm with cooperated paths (copaths) was proposed, which can be used to solve the problems resulting from the class inheritance mechanism encountered in object-oriented software testing such as oracle, message transfer errors, and unreachable statement. Finally, the testing sufficiency was analyzed with the ordered sequence testing criterion(OSC). The results indicate that the test cases stemmed from newly proposed automatic algorithm of copaths generation satisfies synchronization message sequences testing criteria, therefore the proposed new algorithm of copaths generation has a good coverage rate.

Monogenic diabetes in children:An underdiagnosed and poorly managed clinical dilemma

Monogenic diabetes,constituting 1%-2%of global diabetes cases,arises from single gene defects with distinctive inheritance patterns.Despite over 50 associated genetic disorders,accurate diagnoses and management of monogenic diabetes remain inadequate,underscoring insufficient clinician awareness.The disease spectrum encompasses maturity-onset diabetes of the young(MODY),characterized by distinct genetic mutations affecting insulin secretion,and neonatal diabetes mellitus(NDM)-a heterogeneous group of severe hyperglycemic disorders in infants.Mitochondrial diabetes,autoimmune monogenic diabetes,genetic insulin resistance and lipodystrophy syndromes further diversify the monogenic diabetes landscape.A tailored approach based on phenotypic and biochemical factors to identify candidates for genetic screening is recommended for suspected cases of MODY.NDM diagnosis warrants immediate molecular genetic testing for infants under six months.Identifying these genetic defects presents a unique opportunity for precision medicine.Ongoing research aimed to develop cost-effective genetic testing methods and gene-based therapy can facilitate appropriate identification and optimize clinical outcomes.Identification and study of new genes offer a valuable opportunity to gain deeper insights into pancreatic cell biology and the pathogenic mechanisms underlying common forms of diabetes.The clinical review published in the recent issue of World Journal of Diabetes is such an attempt to fill-in our knowledge gap about this enigmatic disease.

New era of cystic fibrosis:Full mutational analysis and personalized therapy

Despite its apparently simple genetics,cystic fibrosis(CF) is a rather complex genetic disease.A lot of variability in the steps of the path from the cystic fibrosis transmembrane conductance regulator(CFTR) gene to the clinical manifestations originates an uncertain genotype- phenotype relationship.A major determinant of this uncertainty is the incomplete knowledge of the CFTR mutated genotypes,due to the high number of CFTR mutations and to the higher number of their combinations in trans and in cis.Also the very limited knowledge of functional effects of CFTR mutated alleles severely impairs our diagnostic and prognostic ability.The final phenotypic modulation exerted by CFTR modifier genes and interactome further complicates the framework.The next generation sequencing approach is a rapid,lowcost and high-throughput tool that allows a near complete structural characterization of CFTR mutated genotypes,as well as of genotypes of several other genes cooperating to the final CF clinical manifestations.This powerful method perfectly complements the new personalized therapeutic approach for CF.Drugs active on specific CFTR mutational classes are already available for CF patients or are in phase 3 trials.A complete genetic characterization has been becoming crucial for a correct personalized therapy.However,the need of a functional classification of each CFTR mutation potently arises.Future big efforts towards an ever more detailed knowledge of both structural and functional CFTR defects,coupled to parallel personalized therapeutic interventions decisive for CF cure can be foreseen.

Generation method of minimal-complete-coverage interoperability test sequence based on digraph

Even if two implementations of a protocol pass the conformance testing,it cannot guarantee that they can interoperate properly;so direct testing of interoperation is considered indispensable.During the interoperability testing,a minimal number of test sequences are expected to check as many as possible implementation errors.By using minimal-complete-coverage criterion,the test sequence generation based on digraph can produce more effective test sequences.

基于事件消息驱动的Petri网的类族测试用例生成技术的研究

在类族交互测试的正交阵列测试系统(OATS)生成测试用例的方法中,存在类的状态盲目组合而导致测试用例"爆炸"以及标准正交阵列不易扩充的问题。提出了基于EMDPN的类的状态组合的标记关联递推算法,通过类族之间的层次级别与状态级别的优选组合产生交互路径,在证明生成交互路径数定理的基础上,得到了基于EMDPN模型的扩展的正交阵列测试系统(EOATS)的类族交互测试用例的生成方法,优化OATS。在讨论类族交互测试的覆盖率问题时,给出了类族交互测试的同步消息序列测试标准(SMSC),阐明了新的EOATS方法具有良好的覆盖性。

基于UML状态图的类测试技术

针对基于状态的类测试技术缺陷检测率较低的问题,提出一种使用等价类划分和边界值分析等功能性测试方法构建UML状态图的方法,描述基于W方法的测试序列生成策略,使用Mujava变异工具对方法的有效性进行检测。实验结果表明,该测试策略具有较高的缺陷检测率。

在带OCL约束的状态图下测试线索的自动生成

提出一种带OCL约束的状态图测试线索的自动生成方法。该方法自动解析类图和带OCL约束的状态图的XML模型文件,获取相应的状态节点邻接表,再结合状态对—事件约束集合生成带OCL约束的测试线索。实例研究表明,通过OCL约束冲突判断可避免不可行测试线索的生成,减少测试用例数目,达到降低测试成本的目的。实验结果证明了该方法的可行性和有效性。

基于有向同步交互图的类族测试用例生成算法

根据类对象之间的消息通信产生的同步消息、消息传递以及消息等待的交互特征,设计了一种有向同步交互图(DSIG)模型,提出了类继承的同步交互路径(SIAPaths)测试用例生成方法,给出了自动生成同步交互路径的算法。同时,提出了同步交互序列测试充分性准则,并从基本交互路径序列和基本交互消息序列的测试覆盖率两方面阐述了测试的可行性。

基于顺序图的软件可靠性测试用例生成方法

将面向对象技术特别是UML中的建模要素应用到软件可靠性测试过程是一个值得研究的问题。对基于UML的操作剖面开发以及基于UML顺序图的测试用例生成方法进行了研究。重点讨论了测试用例生成方法的步骤、具体的建模活动以及自动生成测试用例的“基于模型的代码设计”方法,给出了相应的算法。最后对所讨论方法的有效性进行了分析,从理论上说明了方法的可行性。

基于JPF的类数据流测试生成技术

通过设置陷阱性质,用时序逻辑公式表示数据流测试的覆盖准则,将测试生成问题简化成模型检测中寻找反例的问题,自动生成满足数据流覆盖准则的类方法测试调用序列,提出了一种适用于类方法调用序列自动生成的搜索算法,并在程序模型检测器JPF上实现。算法分析和实验结果表明,该算法能生成高效的方法调用序列并明显减少测试生成代价。

一种基于UML的多态性测试线索生成方法

首先分析了多态性的成因及形式化表示;然后借用控制流图的思想分析协作图,将类图中的信息结合到协作图中,对传统的函数间受限控制流图IRCFG进行多态性扩展并带上类图的基本信息;最后分析了测试覆盖准则并给出测试线索的生成方法。

类状态测试模型

针对类状态测试可能遗漏数据错误问题,利用数据流分析技术对生成的测试序列进行分析,找出数据流异常进而对其改进,能较好地解决数据错误问题。为解决在状态测试执行时需要插装代码来跟踪状态转换以便进行可行性检测分析,以及需要通过改写被测类代码来解决类封装性限制进行类状态验证,导致测试脚本臃肿、强聚合和被测类代码版本不一致问题,提出了一种基于AOP类状态测试执行方法,它通过分离关注点、突破类封装性限制,能有效地解决这些问题。基于上述技术和方法,提出一种类状态测试模型,实例分析和实验结果表明,该模型能较好地实现类状态测试。

Investigation on the genetic-inconsistent paternity cases using the MiSeq FGx system

Mutations might challenge the paternity index calculation in forensic identification.While many studies have focussed on the autosomal short tandem repeats(A-STR),the mutation status of sex chromosomes and single nucleotide polymorphism(SNP)remain blank.Next generation sequencing(NGS),known as high throughput and large sequence polymorphism,is a promising tool for forensic genetics.To describe the mutation landscapes in the paternity cases with genetic inconsistencies,a total of 63 parentage confirmed paternity cases contained at least one mismatched locus have been collected.The mutations were subsequently evaluated using Verogen’s MPSForenSeqTM DNASignature Kit and a microsatellite instability(MSI)detection kit.The result showed 98.41%(62/63)of the cases had no additional autosomal mutations even when the number of A-STRs increased to 27.As for the sex chromosomes,about 11.11%(7/63)of the cases exhibited either X-STR or Y-STR mutations.D2S1338,FGAand Penta Ewere the most frequent altered STRs,which suggested they might be the mutation hotspots.In addition,a male with sex chromosome abnormality was observed accidently,whose genotype might be 47,XXY,rather than MSI.Nearly 56.90%of the STR loci possessed isoalleles,which might result in higher STR polymorphisms.No Mendelian incompatibility was detected among the SNP markers,which indicated that SNP was a more reliable genetic marker in the genetic-inconsistent paternity cases.