个体基因组时代即将到来

buddy2008

转帖自丁香园  heyyou翻译
Published at www.nejm.org March 10, 2010 (10.1056/NEJMe1001090)

Individual Genomes on the Horizon
Richard P. Lifton, M.D., Ph.D.

Physicians have long recognized that pinpointing specific causes of disease in individual patients enables therapies that are the most likely to confer benefit with the fewest adverse effects. We also recognize the potential for disease prevention through identification of specific risk factors and mitigation of their effects. For a century, we have known that many of these risk factors are genetic. In the past 20 years, the genomic revolution has translated this knowledge into a new understanding of disease: mutations that cause more than 2000 mendelian diseases have been identified, which has led to the rewriting of textbooks of pathophysiology of every organ system and the identification of rational targets for therapeutic intervention. Genes also play a major role in risk for virtually every common disease, affording the possibility of identifying persons who have a specific inherited predisposition.

The field has been driven by saltatory leaps in technology. The development of complete genetic maps of the human genome fueled the mapping and identification of genes underlying mendelian traits in nuclear families. Subsequently, the ability to inexpensively genotype hundreds of thousands of common sequence variants across the genome enabled the discovery of common variants contributing to common diseases in large cohorts of case patients and controls.

Building on the complete sequence of the human genome, spectacular reductions in the cost of DNA sequencing now point to a coming era of genomics based on identification of rare variants that confer disease risk in individual patients. When the sequencing of the first human genome was initiated, the cost to produce 1 million bases of sequence was $100,000. The development of new technologies that permit simultaneous sequencing of hundreds of millions of DNA templates has recently driven the cost to sequence 1 million bases to under $1.

This advance creates myriad opportunities for the use of DNA sequencing in gene discovery. For example, the discovery of the comprehensive set of somatic mutations in cancer1 and suspected de novo mutations underlying diseases ranging from congenital malformations to autism become tractable goals. Similarly, common variants have explained only a small fraction of the inherited risk for most common diseases, findings that suggest a role for rare variants with relatively large effect,2,3 which can be discovered by sequencing large cohorts. Finally, thousands of known and suspected mendelian traits that have thus far eluded understanding will most likely be solvable with the use of high-throughput sequencing.

Genome sequencing will also have a role in translating these discoveries into clinical diagnosis. Traditionally, the genetic diagnosis of a mendelian disorder relied on the establishment of a clinical diagnosis followed by the sequencing of previously implicated genes. Practical limitations of this approach include frequent diagnostic uncertainties, which thwart efforts to define a short list of genes for sequencing. Similar limitations arise for diseases in which mutations in many genes can cause the same disease. Sequencing these genes one by one is cumbersome and limits the number that can be efficiently examined. Supplanting this approach with routine sequencing of all the genes is consequently attractive and, more importantly, scalable. Although daunting challenges, such as distinguishing clinically significant mutations from nonconsequential variation, remain, the cost to sequence all the genes in the genome with the use of new technology is already approaching the fee charged to sequence single genes in some diagnostic laboratories.

In this issue of the Journal, Lupski and colleagues report on their study that shows the power of this new technology.4 They used whole-genome sequencing to make a specific diagnosis in a family in which four siblings were affected by Charcot–Marie–Tooth disease, a peripheral polyneuropathy. Mutations in 31 known genes and additional unidentified loci can produce Charcot–Marie–Tooth disease. The investigators produced nearly 90 billion base pairs of genomic sequence in one affected subject (sufficient to ensure that both alleles at nearly every base pair have been sampled repeatedly) and identified variations from the reference sequence. As expected, they found a large number of common and novel variants. When they examined genes known to be mutated in patients with Charcot–Marie–Tooth disease, they found two compelling mutations in SH3TC2 (the SH3 domain and tetratricopeptide repeats 2 gene), which causes autosomal recessive Charcot–Marie–Tooth disease. They also found complete cosegregation of these mutations with disease status in the family, providing convincing evidence that these SH3TC2 mutations are the cause of Charcot–Marie–Tooth disease in this family.

The sequence production for this project cost less than $50,000. More traditional approaches could have obtained the same answers; nonetheless, the study provides a striking proof of principle. Moreover, there is every reason to believe that the cost of sequencing will continue to plummet. Owing to innovation and intense competition, the cost of sequence production 2 years from now will almost certainly be at most one tenth of the current cost of using current technologies. Moreover, there are widespread efforts to advance new technologies to achieve further drastic drops in cost.5

In addition, large cost reductions can be achieved by shrinking the target for sequencing. Protein-encoding exons of the roughly 23,000 genes in humans constitute approximately 1% of the genome but harbor about 90% of all mutations with large effects. Efficient methods for whole-exome sequencing (that is, sequencing of all the exons in a genome) have recently been reported,6,7 and their usefulness for both clinical diagnosis7 and disease-gene identification8 has been shown. Current costs for whole-exome sequencing are only about $4,000, and as long as expense remains a factor, a 90 to 95% reduction of that cost will be significant. Notably, this approach could have led to the same conclusion far less expensively in the current study.

It is increasingly clear that the cost is fast approaching a threshold at which DNA sequencing will become a routine part of the diagnostic armamentarium. This raises many critical questions. Who will benefit from comprehensive sequencing? When in a person's life should sequencing be done? How should we deal with the many variants of uncertain clinical significance? How should we interpret changes found outside of genes? How should we effectively communicate the results to patients in ways that will improve health without inducing neurosis? These questions have far-reaching implications for the education of health care professionals and patients as well as for health and social policy. Lupski and colleagues provide a glimpse of the future for which we need to prepare.

Disclosure forms provided by the author are available with the full text of this article at NEJM.org.

Source Information

From the Departments of Genetics and Internal Medicine, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT.

This article (10.1056/NEJMe1001090) was published on March 10, 2010, at NEJM.org.

References

Parsons DW, Jones S, Zhang X, et al. An integrated genomic analysis of human glioblastoma multiforme. Science 2008;321:1807-1812. [Free Full Text]

Cohen JC, Kiss RS, Pertsemlidis A, Marcel YL, McPherson R, Hobbs HH. Multiple rare alleles contribute to low plasma levels of HDL cholesterol. Science 2004;305:869-872. [Free Full Text]

Ji W, Foo J-N, O'Roak BJ, et al. Rare independent mutations in renal salt handling genes contribute to blood pressure variation. Nat Genet 2008;40:592-599. [CrossRef][Web of Science][Medline]

Lupski JR, Reid JG, Gonzaga-Jauregui C, et al. Whole-genome sequencing in a patient with Charcot–Marie–Tooth neuropathy. N Engl J Med 2010. DOI: 10.1056/NEJMoa0908094.
Drmanac R, Sparks AB, Callow MJ, et al. Human genome sequencing using unchained base reads on self-assembling DNA nanoarrays. Science 2010;327:78-81. [Free Full Text]

Ng SB, Turner EH, Robertson PD, et al. Targeted capture and massively parallel sequencing of 12 human exomes. Nature 2009;461:272-276. [CrossRef][Web of Science][Medline]

Choi M, Scholl UI, Ji W, et al. Genetic diagnosis by whole exome capture and massively parallel DNA sequencing. Proc Natl Acad Sci U S A 2009;106:19096-19101. [Free Full Text]

Ng SB, Buckingham KJ, Lee C, et al. Exome sequencing identifies the cause of a mendelian disorder. Nat Genet 2010;42:30-35. [CrossRef][Web of Science][Medline]

个体基因组时代即将到来

Physicians have long recognized that pinpointing specific causes of disease in individual patients enables therapies that are the most likely to confer benefit with the fewest adverse effects. We also recognize the potential for disease prevention through identification of specific risk factors and mitigation of their effects. For a century, we have known that many of these risk factors are genetic. In the past 20 years, the genomic revolution has translated this knowledge into a new understanding of disease: mutations that cause more than 2000 mendelian diseases have been identified, which has led to the rewriting of textbooks of pathophysiology of every organ system and the identification of rational targets for therapeutic intervention. Genes also play a major role in risk for virtually every common disease, affording the possibility of identifying persons who have a specific inherited predisposition.
医生在很久以前就认识到，如果能够精确定位病人的特异性病因，就能够以最小的副作用将病人治愈。我们也认识到，通过确定特定的危险因素并对它们的作用进行控制就能够预防疾病。一个世纪以来，人们认识到，许多危险因素是有遗传性的。在过去的20年里，基因组***将这种知识转变为我们对疾病的新认识：引起2000种孟德尔疾病的突变得以确认，这使得每个器官系统的病理生理学教科书需要重写，并且为治疗干预提供了合适的靶点。实际上基因在每种常见疾病风险也起到重要的作用，从而使我们有可能确定那些具有特异遗传易感性的个体。

The field has been driven by saltatory leaps in technology. The development of complete genetic maps of the human genome fueled the mapping and identification of genes underlying mendelian traits in nuclear families. Subsequently, the ability to inexpensively genotype hundreds of thousands of common sequence variants across the genome enabled the discovery of common variants contributing to common diseases in large cohorts of case patients and controls.

技术的突飞猛进带动了这个领域的发展。人类基因组遗传图谱的完成推动了核心家系中孟德尔性状相关基因的作图与确认。随后，基因组中对成百上千常见结构变异基因分型费用不断降低，从而使人们能够在大规模病例和对照人群中发现造成常见疾病的常见变异

Building on the complete sequence of the human genome, spectacular reductions in the cost of DNA sequencing now point to a coming era of genomics based on identification of rare variants that confer disease risk in individual patients. When the sequencing of the first human genome was initiated, the cost to produce 1 million bases of sequence was $100,000. The development of new technologies that permit simultaneous sequencing of hundreds of millions of DNA templates has recently driven the cost to sequence 1 million bases to under $1.
基于人类基因组全测序的完成，当前DNA测序费用的急剧降低，基于确定导致病人疾病风险升高的罕见变异的基于基因组时代即将到来。当人类基因组测序刚刚开始时，对一百万个碱基测序的费用为十万美元。现在的新技术可以同时上百万DNA模板同时测序，并使一百万个碱基测序的费用降低到一美元以下。

This advance creates myriad opportunities for the use of DNA sequencing in gene discovery. For example, the discovery of the comprehensive set of somatic mutations in cancer1 and suspected de novo mutations underlying diseases ranging from congenital malformations to autism become tractable goals. Similarly, common variants have explained only a small fraction of the inherited risk for most common diseases, findings that suggest a role for rare variants with relatively large effect,2,3 which can be discovered by sequencing large cohorts. Finally, thousands of known and suspected mendelian traits that have thus far eluded understanding will most likely be solvable with the use of high-throughput sequencing.
这些进展为DNA测序技术在寻找基因的领域提供了很大的机会。例如，在肿瘤中发现一系列体细胞突变，从先天畸形到自闭症等疾病中发现可疑新生突变。常见变异仅能解释多数常见疾病遗传风险的一小部分，类似的，研究表明一些效应相对较大的罕见变异也在遗传易感性中起一定作用，而这些罕见变异能够通过对大规模人群进行测序来发现。

Genome sequencing will also have a role in translating these discoveries into clinical diagnosis. Traditionally, the genetic diagnosis of a mendelian disorder relied on the establishment of a clinical diagnosis followed by the sequencing of previously implicated genes. Practical limitations of this approach include frequent diagnostic uncertainties, which thwart efforts to define a short list of genes for sequencing. Similar limitations arise for diseases in which mutations in many genes can cause the same disease. Sequencing these genes one by one is cumbersome and limits the number that can be efficiently examined. Supplanting this approach with routine sequencing of all the genes is consequently attractive and, more importantly, scalable. Although daunting challenges, such as distinguishing clinically significant mutations from nonconsequential variation, remain, the cost to sequence all the genes in the genome with the use of new technology is already approaching the fee charged to sequence single genes in some diagnostic laboratories.
全基因组测序同样能够帮助我们将这些发现转变为临床诊断方法。通常上，孟德尔疾病的遗传诊断主要依靠临床诊断以及随后对疾病已知相关基因的测序。这种方法在实践中有很多局限性，包括诊断往往有不确定性，从而要对许多基因进行检验。当某种疾病可能由于许多基因中的突变造成时也会出现这种限制。逐个对这些基因进行测序效率很低并且限制了可以检测的基因数目。因此，用对所有基因进行常规测序的方法代替当前方法很有诱惑力，并且是可以实现的。

In this issue of the Journal, Lupski and colleagues report on their study that shows the power of this new technology.4 They used whole-genome sequencing to make a specific diagnosis in a family in which four siblings were affected by Charcot–Marie–Tooth disease, a peripheral polyneuropathy. Mutations in 31 known genes and additional unidentified loci can produce Charcot–Marie–Tooth disease. The investigators produced nearly 90 billion base pairs of genomic sequence in one affected subject (sufficient to ensure that both alleles at nearly every base pair have been sampled repeatedly) and identified variations from the reference sequence. As expected, they found a large number of common and novel variants. When they examined genes known to be mutated in patients with Charcot–Marie–Tooth disease, they found two compelling mutations in SH3TC2 (the SH3 domain and tetratricopeptide repeats 2 gene), which causes autosomal recessive Charcot–Marie–Tooth disease. They also found complete cosegregation of these mutations with disease status in the family, providing convincing evidence that these SH3TC2 mutations are the cause of Charcot–Marie–Tooth disease in this family.
在本期杂志中，Lupski及其同事发表的研究显示了这种新方法的能力。他们用全基因组测序的方法对一个家庭进行特殊的诊断，在这个家庭中，有四个兄弟姐妹患有腓骨肌萎缩症-一种末梢神经疾病。31个已知基因中的突变和额外未确定的位点能够导致这种疾病。研究者对其中一个患者全基因组中近九百亿个碱基对进行测序（足以保证几乎每个碱基对的两个等位基因都能被重复抽样）并从参考序列中确定了相关变异。和预期一样，他们发现了大量常见变异和新变异。当在病人中检测那些已知会造成腓骨肌萎缩症的基因时，他们在SH3TC2基因中发现了两个值得关注的突变，这两个突变能够导致隐性遗传的腓骨肌萎缩症。他们同样发现，在该家系中这些突变与疾病状态完全共分离，确切表明SH3TC2基因的变异造成了该家系中的腓骨肌萎缩症。

The sequence production for this project cost less than $50,000. More traditional approaches could have obtained the same answers; nonetheless, the study provides a striking proof of principle. Moreover, there is every reason to believe that the cost of sequencing will continue to plummet. Owing to innovation and intense competition, the cost of sequence production 2 years from now will almost certainly be at most one tenth of the current cost of using current technologies. Moreover, there are widespread efforts to advance new technologies to achieve further drastic drops in cost.5
这个项目的测序成果成本少于五万美元。较为传统的方法可能会得到同样的结果；然而这项研究为这一原理提供了确凿的证据。并且我们完全有理由相信测序的费用仍会急剧下降。由于技术革新和激烈的竞争，可以肯定两年后测序费用将是当前测序技术所需费用的十分之一。另外许多努力将会促使新技术不断发展，从而使成本急剧下降。

In addition, large cost reductions can be achieved by shrinking the target for sequencing. Protein-encoding exons of the roughly 23,000 genes in humans constitute approximately 1% of the genome but harbor about 90% of all mutations with large effects. Efficient methods for whole-exome sequencing (that is, sequencing of all the exons in a genome) have recently been reported,6,7 and their usefulness for both clinical diagnosis7 and disease-gene identification8 has been shown. Current costs for whole-exome sequencing are only about $4,000, and as long as expense remains a factor, a 90 to 95% reduction of that cost will be significant. Notably, this approach could have led to the same conclusion far less expensively in the current study.
此外，通过减少测序长度也能够大幅降低费用。人类约23000个基因的蛋白编码外显子约构成全基因组的1%，但却聚集了所有具有较大遗传效应突变中的90%。有研究报道了全外显子测序的高效方法（也就是对基因组中 所有外显子进行测序），并且这种方法在临床诊断和疾病基因鉴定方面的作用已经得到了验证。当前全基因组测序的费用仅约为4000美元，并且只要仍为一个影响因素，费用可定还能够降低90-95%。显然，这种方法能够是当前研究以更为低廉的费用得到相同的结论。

It is increasingly clear that the cost is fast approaching a threshold at which DNA sequencing will become a routine part of the diagnostic armamentarium. This raises many critical questions. Who will benefit from comprehensive sequencing? When in a person's life should sequencing be done? How should we deal with the many variants of uncertain clinical significance? How should we interpret changes found outside of genes? How should we effectively communicate the results to patients in ways that will improve health without inducing neurosis? These questions have far-reaching implications for the education of health care professionals and patients as well as for health and social policy. Lupski and colleagues provide a glimpse of the future for which we need to prepare.
现在我们已经越来越清楚的认识到，费用即将不再是门槛，DNA测序将成为诊断过程中的常规组成部分。这会带来许多关键性的问题。谁将在全面测序中受益？一个人应当在何时测序？对于许多不能确定临床意义的变异我们如何处理？我们应当怎样解释基因以外的变异？为了能改善病人健康并且不带来紧张情绪，我们应该怎样有效的将结果告诉给病人？这些问题对医护人员的教育和病人都有深远的影响，对健康和社会政策也是如此。Lupski及其同事为我们提供了一个我们需要准备应对的未来场面。