Precision medicine in Thailand

Extraordinary advances in high throughput next generation sequencing (NGS) technology and bioinformatics are the main thrust that transforms the current state of healthcare into the era of precision medicine where clinical practice takes individual variability into account. Here, we summarize the current status of the infrastructure we have and the adoption of precision medicine in Thailand in four spheres: rare diseases, oncology, pharmacogenomics, and noncommunicable diseases. Moreover, we provide our perspectives to the future of precision medicine in Thailand, especially the manpower and ethical, legal, and social issues. We believe that with decreasing costs of NGS, increasing ability to interpret the genomic data, a greater number of actionable and available treatments, implementation of precision medicine at the public health level is not a matter of if but when.     

Newborn screening research sponsored by the NIH: From diagnostic paradigms to precision therapeutics

Newborn screening (NBS) is a successful public health initiative that effectively identifies pre-symptomatic neonates so that treatment can be initiated before the onset of irreversible morbidity and mortality. Legislation passed in 2008 has supported a system of state screening programs, educational resources, and an evidence-based review process to add conditions to a recommended universal newborn screening panel (RUSP). The Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), NIH, has promoted NBS research to advance legislative goals by supporting research that will uncover fundamental mechanisms of disease, develop treatments for NBS disorders, and promote pilot studies to test implementation of new conditions. NICHD's partnerships with other federal agencies have contributed to activities that support nominations of new conditions to the RUSP. The NIH's Newborn Sequencing In Genomic Medicine and Public Health (NSIGHT) initiative funded research projects that considered how genomic sequencing could be integrated into NBS and its ethical ramifications. Recently, the workshop, “Gene Targeted Therapies: Early Diagnosis and Equitable Delivery,” has explored the possibility of expanding NBS to include genetic diagnosis and precision, gene-based therapies. Although hurdles remain to realize such a vision, broad engagement of multiple stakeholders is essential to advance genomic medicine within NBS.     

Ethical considerations of population screening for late‐onset genetic disease

Population‐based genetic screening has been a mainstay of public health in the United States for many years. The goal of genetic screening is to identify individuals at increased risk for treatable diseases. The evolution of genetic testing to include multi‐disease panels allows for new screening applications which challenge the traditional model of clinical genetics care by the identification of late‐onset disorders in an asymptomatic fetus, child, or adult. We present two unique examples of individuals referred to a biochemical genetics clinic due to the detection of late‐onset Pompe disease by population‐based screening modalities. We review early experiences in counseling and management of pre‐symptomatic individuals and highlight some of the primary ethical factors warranting consideration as we enter the era of genomic medicine.     

目标序列捕获测序技术及其在疾病相关基因研究中的作用

目标序列捕获测序是指将感兴趣的基因组区域定制成特异性探针与目标基因组DNA在序列捕获芯片(或溶液)进行杂交,将目标基因组区域的DNA片段进行富集后再利用新一代测序技术进行测序,以获得目标基因组序列的研究策略.该项技术具有高度灵活性、特异性及覆盖率,操作便捷等特点,该文就目标序列捕获测序技术的原理及其在疾病相关基因研究中的应用作一简要综述.

Abstract：

Target sequence capture sequencing refers to a sequencing technology that uses the interested genomic region as specific probes, which are attached to chips or beads. The probes then hybridize with free target genomic DNA on sequence capture chip ( or in solution ) to enrich target genomic DNA. The enriched target genomic DNA fragments can then be amplified and studied using the next-generation sequencing technologies. The technology has a high degree of flexibility, specificity and coverage,and easy operation characteristics. The goal of this review is to outline the principle of target sequencing technology and its implication in disease-related gene research.     

目标序列捕获测序技术及其在疾病相关基因研究中的作用

目标序列捕获测序是指将感兴趣的基因组区域定制成特异性探针与目标基因组DNA在序列捕获芯片(或溶液)进行杂交,将目标基因组区域的DNA片段进行富集后再利用新一代测序技术进行测序,以获得目标基因组序列的研究策略.该项技术具有高度灵活性、特异性及覆盖率,操作便捷等特点,该文就目标序列捕获测序技术的原理及其在疾病相关基因研究中的应用作一简要综述.     

Sequencing of high-complexity DNA pools for identification of nucleotide and structural variants in regions associated with complex traits

We have used targeted genomic sequencing of high-complexity DNA pools based on long-range PCR and deep DNA sequencing by the SOLiD technology. The method was used for sequencing of 286 kb from four chromosomal regions with quantitative trait loci (QTL) influencing blood plasma lipid and uric acid levels in DNA pools of 500 individuals from each of five European populations. The method shows very good precision in estimating allele frequencies as compared with individual genotyping of SNPs (r(2) = 0.95, P < 10(-16)). Validation shows that the method is able to identify novel SNPs and estimate their frequency in high-complexity DNA pools. In our five populations, 17% of all SNPs and 61% of structural variants are not available in the public databases. A large fraction of the novel variants show a limited geographic distribution, with 62% of the novel SNPs and 59% of novel structural variants being detected in only one of the populations. The large number of population-specific novel SNPs underscores the need for comprehensive sequencing of local populations in order to identify the causal variants of human traits.     

The Japanese Society of Pathology Practical Guidelines on the handling of pathological tissue samples for cancer genomic medicine

Clinical cancer genomic testing based on next-generation sequencing can help select genotype-matched therapy and provide diagnostic and prognostic information. Pathological tissue from malignant tumors obtained during routine practice are frequently used for genomic testing. This article is aimed to standardize the proper handling of pathological specimens in practice for genomic medicine based on the findings established in “Guidelines on the handling of pathological tissue samples for genomic medicine (in Japanese)” published by The Japanese Society of Pathology (JSP) in 2018. The two-part practical guidelines are based on empirical data analyses; Part 1 describes the standard preanalytic operating procedures for tissue collection, processing, and storage of formalin-fixed paraffin-embedded (FFPE) samples, while Part 2 describes the assessment and selection of FFPE samples appropriate for genomic testing, typically conducted by a pathologist. The guidelines recommend that FFPE sample blocks be used within 3 years from preparation, and the tumor content should be ≥30% (minimum 20%). The empirical data were obtained from clinical studies performed by the JSP in collaboration with leading Japanese cancer genome research projects. The Japanese Ministry of Health, Labour, and Welfare (MHLW) recommended to comply with the JSP practical guidelines in implementing cancer genomic testing under the national health insurance system in over 200 MHLW-designated core and cooperative cancer genome medicine hospitals in Japan.     

Efficient Depletion of Host DNA Contamination in Malaria Clinical Sequencing

The cost of whole-genome sequencing (WGS) is decreasing rapidly as next-generation sequencing technology continues to advance, and the prospect of making WGS available for public health applications is becoming a reality. So far, a number of studies have demonstrated the use of WGS as an epidemiological tool for typing and controlling outbreaks of microbial pathogens. Success of these applications is hugely dependent on efficient generation of clean genetic material that is free from host DNA contamination for rapid preparation of sequencing libraries. The presence of large amounts of host DNA severely affects the efficiency of characterizing pathogens using WGS and is therefore a serious impediment to clinical and epidemiological sequencing for health care and public health applications. We have developed a simple enzymatic treatment method that takes advantage of the methylation of human DNA to selectively deplete host contamination from clinical samples prior to sequencing. Using malaria clinical samples with over 80% human host DNA contamination, we show that the enzymatic treatment enriches Plasmodium falciparum DNA up to ∼9-fold and generates high-quality, nonbiased sequence reads covering >98% of 86,158 catalogued typeable single-nucleotide polymorphism loci.     

Attitudes towards Genetic Information Delivered by High-Throughput Sequencing among Molecular Geneticists,Genetic Counselors,Medical Advisors and Students in France

High-throughput sequencing technologies performed in the clinical setting have the potential to reveal diverse genetic information. Whether it is initially targeted or unsolicited, strictly medical or not, or even information on a carrier status as part of preconception screening, access to genetic information needs to be managed. The aim of the current study was to gather potential attitudes of various stakeholders towards the sharing of genetic information from next-generation sequencing, and more specifically towards incidental findings, predictive findings, non-medical information and carrier status. Answers from a total number of 1631 individuals belonging to four different groups (45 molecular geneticists, 65 genetic counselors, 56 medical advisors to the state insurance plan, and 1465 university students) were collected through online questionnaires. Overall, the study reflects preferences towards the return of health risks related to serious diseases when effective treatment is available and information on reproductive risks. The importance of the perceived medical utility, both for disease prevention and treatment, was the main distinguishing feature. Attitudes from genetic health professionals were found more reluctant to receive a wide range of information. Hands-on experience with the practice of genetic testing is likely to influence perception of the utility of the genetic information that should be delivered. At the same time, perceptions of preconception genetic carrier screening brought out less differences between participants. Better understanding of the underlying interest in genomic information and thorough education on its value and usage are key elements to the adoption of future guidelines and policy that respect bioethical principles.     

Scaffolding a Caenorhabditis nematode genome with RNA-seq

Efficient sequencing of animal and plant genomes by next-generation technology should allow many neglected organisms of biological and medical importance to be better understood. As a test case, we have assembled a draft genome of Caenorhabditis sp. 3 PS1010 through a combination of direct sequencing and scaffolding with RNA-seq. We first sequenced genomic DNA and mixed-stage cDNA using paired 75-nt reads from an Illumina GAII. A set of 230 million genomic reads yielded an 80-Mb assembly, with a supercontig N50 of 5.0 kb, covering 90% of 429 kb from previously published genomic contigs. Mixed-stage poly(A)(+) cDNA gave 47.3 million mappable 75-mers (including 5.1 million spliced reads), which separately assembled into 17.8 Mb of cDNA, with an N50 of 1.06 kb. By further scaffolding our genomic supercontigs with cDNA, we increased their N50 to 9.4 kb, nearly double the average gene size in C. elegans. We predicted 22,851 protein-coding genes, and detected expression in 78% of them. Multigenome alignment and data filtering identified 2672 DNA elements conserved between PS1010 and C. elegans that are likely to encode regulatory sequences or previously unknown ncRNAs. Genomic and cDNA sequencing followed by joint assembly is a rapid and useful strategy for biological analysis.     

Next-generation sequencing technologies and their application to the study and control of bacterial infections

BackgroundWith the efficiency and the decreasing cost of next-generation sequencing, the technology is being rapidly introduced into clinical and public health laboratory practice.AimsThe historical background and principles of first-, second- and third-generation sequencing are described, as are the characteristics of the most commonly used sequencing instruments.SourcesPeer-reviewed literature, white papers and meeting reports.Content and implicationsNext-generation sequencing is a technology that could potentially replace many traditional microbiological workflows, providing clinicians and public health specialists with more actionable information than hitherto achievable. Examples of the clinical and public health uses of the technology are provided. The challenge of comparability of different sequencing platforms is discussed. Finally, the future directions of the technology integrating it with laboratory management and public health surveillance systems, and moving it towards performing sequencing directly from the clinical specimen (metagenomics), could lead to yet another fundamental transformation of clinical diagnostics and public health surveillance.     

Next-generation carrier screening

PurposeCarrier screening for recessive Mendelian disorders traditionally employs focused genotyping to interrogate limited sets of mutations most prevalent in specific ethnic groups. We sought to develop a next-generation DNA sequencing–based workflow to enable analysis of a more comprehensive set of disease-causing mutations.MethodsWe utilized molecular inversion probes to capture the protein-coding regions of 15 genes from genomic DNA isolated from whole blood and sequenced those regions using the Illumina HiSeq 2000 (Illumina, San Diego, CA). To assess the quality of the resulting data, we measured both the fraction of the targeted region yielding high-quality genotype calls, and the sensitivity and specificity of those calls by comparison with conventional Sanger sequencing across hundreds of samples. Finally, to improve the overall accuracy for detecting insertions and deletions, we introduce a novel assembly-based approach that substantially increases sensitivity without reducing specificity.ResultsWe generated high-quality sequence for at least 99.8% of targeted base pairs in samples derived from blood and achieved high concordance with Sanger sequencing (sensitivity >99.9%, specificity >99.999%). Our novel algorithm is capable of detecting insertions and deletions inaccessible by current methods.ConclusionOur next-generation DNA sequencing–based approach yields the accuracy and completeness necessary for a carrier screening test.Genet Med16 2, 132–140.     

The business of genomic testing: a survey of early adopters

PurposeThe practice of “genomic” (or “personalized”) medicine requires the availability of appropriate diagnostic testing. Our study objective was to identify the reasons for health systems to bring next-generation sequencing into their clinical laboratories and to understand the process by which such decisions were made. Such information may be of value to other health systems seeking to provide next-generation sequencing testing to their patient populations.MethodsA standardized open-ended interview was conducted with the laboratory medical directors and/or department of pathology chairs of 13 different academic institutions in 10 different states.ResultsGenomic testing for cancer dominated the institutional decision making, with three primary reasons: more effective delivery of cancer care, the perceived need for institutional leadership in the field of genomics, and the premise that genomics will eventually be cost-effective. Barriers to implementation included implementation cost; the time and effort needed to maintain this newer testing; challenges in interpreting genetic variants; establishing the bioinformatics infrastructure; and curating data from medical, ethical, and legal standpoints. Ultimate success depended on alignment with institutional strengths and priorities and working closely with institutional clinical programs.ConclusionThese early adopters uniformly viewed genomic analysis as an imperative for developing their expertise in the implementation and practice of genomic medicine.Genet Med16 12, 954–961.     

Motivators for participation in a whole-genome sequencing study: implications for translational genomics research

The promise of personalized medicine depends on the ability to integrate genetic sequencing information into disease risk assessment for individuals. As genomic sequencing technology enters the realm of clinical care, its scale necessitates answers to key social and behavioral research questions about the complexities of understanding, communicating, and ultimately using sequence information to improve health. Our study captured the motivations and expectations of research participants who consented to participate in a research protocol, ClinSeq, which offers to return a subset of the data generated through high-throughput sequencing. We present findings from an exploratory study of 322 participants, most of whom identified themselves as white, non-Hispanic, and coming from higher socio-economic groups. Participants aged 45-65 years answered open-ended questions about the reasons they consented to ClinSeq and about what they anticipated would come of genomic sequencing. Two main reasons for participating were as follows: a conviction to altruism in promoting research, and a desire to learn more about genetic factors that contribute to one's own health risk. Overall, participants expected genomic research to help improve understanding of disease causes and treatments. Our findings offer a first glimpse into the motivations and expectations of individuals seeking their own genomic information, and provide initial insights into the value these early adopters of technology place on information generated by high-throughput sequencing studies.     

Maternal cell-free DNA–based screening for fetal microdeletion and the importance of careful diagnostic follow-up

BackgroundNoninvasive prenatal screening (NIPS) by next-generation sequencing of cell-free DNA (cfDNA) in maternal plasma is used to screen for common aneuploidies such as trisomy 21 in high risk pregnancies. NIPS can identify fetal genomic microdeletions; however, sensitivity and specificity have not been systematically evaluated. Commercial companies have begun to offer expanded panels including screening for common microdeletion syndromes such as 22q11.2 deletion (DiGeorge syndrome) without reporting the genomic coordinates or whether the deletion is maternal or fetal. Here we describe a phenotypically normal mother and fetus who tested positive for atypical 22q deletion via maternal plasma cfDNA testing.MethodsWe performed cfDNA sequencing on saved maternal plasma obtained at 11 weeks of gestation from a phenotypically normal woman with a singleton pregnancy whose earlier screening at a commercial laboratory was reported to be positive for a 22q11.2 microdeletion. Fluorescence in situ hybridization and chromosomal microarray diagnostic genetic tests were done postnatally.ConclusionNIPS detected a 22q microdeletion that, upon diagnostic workup, did not include the DiGeorge critical region. Diagnostic prenatal or postnatal testing with chromosomal microarray and appropriate parental studies to determine precise genomic coordinates and inheritance should follow a positive microdeletion NIPS result.     

ACMG statement on noninvasive prenatal screening for fetal aneuploidy

Noninvasive assessment of the fetal genome is now possible using next-generation sequencing technologies. The isolation of fetal DNA fragments from maternal circulation in sufficient quantity and sizes, together with proprietary bioinformatics tools, now allows patients the option of noninvasive fetal aneuploidy screening. However, obstetric care providers must become familiar with the advantages and disadvantages of the utilization of this approach as analysis of cell-free fetal DNA moves into clinical practice. Once informed, clinicians can provide efficient pretest and posttest counseling with the goal of avoiding patient harm. It is in the public’s best interest that test results contain key elements and that laboratories adhere to established quality control and proficiency testing standards. The analysis of cell-free fetal DNA in maternal circulation for fetal aneuploidy screening is likely the first of major steps toward the eventual application of whole fetal genome/whole fetal exome sequencing.     

Whole cancer genome sequencing by next-generation methods

Traditional approaches to sequence analysis are widely used to guide therapy for patients with lung and colorectal cancer and for patients with melanoma, sarcomas (eg, gastrointestinal stromal tumor), and subtypes of leukemia and lymphoma. The next-generation sequencing (NGS) approach holds a number of potential advantages over traditional methods, including the ability to fully sequence large numbers of genes (hundreds to thousands) in a single test and simultaneously detect deletions, insertions, copy number alterations, translocations, and exome-wide base substitutions (including known "hot-spot mutations") in all known cancer-related genes. Adoption of clinical NGS testing will place significant demands on laboratory infrastructure and will require extensive computational expertise and a deep knowledge of cancer medicine and biology to generate truly useful "clinically actionable" reports. It is anticipated that continuing advances in NGS technology will lower the overall cost, speed the turnaround time, increase the breadth of genome sequencing, detect epigenetic markers and other important genomic parameters, and become applicable to smaller and smaller specimens, including circulating tumor cells and circulating free DNA in plasma.     

Pyrosequencing-based SNP allele frequency estimation in DNA pools

Association screening involving numerous genetic markers is facilitated by the analysis of pooled DNA samples rather than individual samples. Several genotyping methods have shown high accuracy and precision of allele frequency estimation in pools. Here, we expand the validation of SNP allele frequency estimation in DNA pools using Pyrosequencing by analyzing 186 pools for three SNPs representing complex sequencing cases. The correlation coefficient between estimated and true allele frequencies ranged between 0.979 and 0.996 and tended to increase with pool size, whereas the difference between estimated and true allele frequencies was 2.37+/-0.11%, in post-PCR pools. The precision was 1.73%. Pool size had no significant effect on accuracy and precision. A comparison between post-PCR and pre-PCR pools showed that for pre-PCR pooling efforts to accurately quantify the genomic DNA samples to be pooled and subsequently amplified are critical. To conclude, Pyrosequencing can be used for allele frequency estimation in DNA pools of SNPs with complex sequencing scenarios with accuracy and precision values in ranges comparable with those of other SNP typing techniques. Considering the ease of use, short run and analysis times, and little instrument maintenance requirements, Pyrosequencing may even be a preferred option.     

Understanding the potential of state-based public health genomics programs to mitigate disparities in access to clinical genetic services

PurposeState health agencies (SHAs) have developed public health genomics (PHG) programs that play an instrumental role in advancing precision public health, but there is limited research on their approaches. This study examines how PHG programs attempt to mitigate or forestall health disparities and inequities in the utilization of genomic medicine.MethodsWe compared PHG programs in three states: Connecticut, Michigan, and Utah. We analyzed 85 in-depth interviews with SHA internal and external collaborators and program documents. We employed a qualitative coding process to capture themes relating to health disparities and inequities.ResultsEach SHA implemented population-level approaches to identify individuals who carry genetic variants that increase risk of hereditary cancers. However, each SHA developed a unique strategy—which we label public health action repertoires—to reach specific subgroups who faced barriers in accessing genetic services. These strategies varied across states given demographics of the state population, state-level partnerships, and availability of healthcare services.ConclusionOur findings illustrate the imperative of tailoring PHG programs to local demographic characteristics and existing community resources. Furthermore, our study highlights how integrating genomics into precision public health will require multilevel, multisector collaboration to optimize efficacy and equity.