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AUTHORS: Wayne W. Grody, MD, PhD, FCAP, FACMG,a Barry H. Thompson, MD, MS, FAAP, FACMG,b and Louanne abstract c Hudgins, MD, FAAP, FACMG As medical has progressed from a descriptive entity to one aDivisions of and Molecular , focused on the functional relationship between and clinical dis- Departments of Pathology and Laboratory , Pediatrics, and Genetics, UCLA School of Medicine, Los Angeles, orders, emphasis has been placed on genomics. Genomics, a subelement California; bAmerican College of Medical Genomics and Genetics, of genetics, is the study of the genome, the sum total of all the genes of Bethesda, ; and cDivision of Medical Genetics, an . The , which is contained in the 23 pairs of Department of Pediatrics, Stanford University School of Medicine/Lucile Packard Children’s Hospital, Stanford, California nuclear and in the mitochondrial DNA of each , com- prises .6 billion of . There are some 23 000 KEY WORDS , , genomics, next-generation -coding genes, a surprisingly small fraction of the total genetic sequencing, primary care, whole-, whole- material, with the remainder composed of noncoding DNA, regulatory genome sequencing sequences, and . The Human , launched in ABBREVIATION 1990, produced a draft of the genome in 2001 and then a finished DTC—direct-to-consumer sequence in 2003, on the 50th anniversary of the initial publication of www.pediatrics.org/cgi/doi/10.1542/peds.2013-1032E Watson and Crick’s paper on the double-helical structure of DNA. Since doi:10.1542/peds.2013-1032E then, this mass of genetic information has been translated at an ever- Accepted for publication Aug 28, 2013 increasing pace into useable knowledge applicable to clinical medi- Address correspondence to Louanne Hudgins, MD, FAAP, FACMG, cine. The recent advent of massively parallel DNA sequencing (also Division of Medical Genetics, Department of Pediatrics, Stanford known as shotgun, high-throughput, and next-generation sequencing) University School of Medicine/Lucile Packard Children’s Hospital, 300 Pasteur Dr, H315, Stanford, CA 94305-5208. E-mail: has brought whole-genome analysis into the clinic for the first time, [email protected] and most of the current applications are directed at children with PEDIATRICS (ISSN Numbers: Print, 0031-4005; Online, 1098-4275). congenital conditions that are undiagnosable by using standard ge- Copyright © 2013 by the American Academy of Pediatrics netic tests for single- disorders. Thus, pediatricians must be- FINANCIAL DISCLOSURE: The authors have indicated they have come familiar with this technology, what it can and cannot offer, no financial relationships relevant to this article to disclose. and its technical and ethical challenges. Here, we address the con- FUNDING: This effort was supported by grant UC7MC21713 from cepts of human genomic analysis and its clinical applicability for the Health Resources and Services Administration’s Maternal primary care providers. Pediatrics 2013;132:S211–S215 and Child Health Bureau. The Genetics in Primary Care Institute is a cooperative agreement between the American Academy of Pediatrics and the Maternal and Child Health Bureau. POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose.

PEDIATRICS Volume 132, Supplement 3, December 2013 S211 Downloaded from www.aappublications.org/news by guest on October 2, 2021 INTRODUCTION been enhanced by the development single genes, as we do in the current paper. Semantics aside, the ultimate The term “genetic testing” can encom- of molecular techniques, particularly fl goal of the field of genomics is to pass the detection and characteriza- uorescence in situ hybridization: the identify how in multiple tion of any analyte that is determined useoflabeledDNAprobesthathybridize genes interact with each other and the or predicted by one’s inheritance. In the to intact chromosomes to reveal sub- environment, a goal that may not be broadest sense of the term, genetic microscopic deletions or duplications achieved for several years. testing includes, for example, mea- of a particular region of the genome. surements of cholesterol levels or Even more recently, chromosomal plasma metabolites such as amino microarrays, consisting of as many as 1 TECHNICAL ASPECTS OF DNA acids and microscopic examination of million DNA probes bound to a solid SEQUENCING support, have been used to interrogate a peripheral blood smear for sickle- For most of the .25-year history of shaped red cells or spherocytes. regions of the entire genome. This method is capable of detecting dele- clinical , and for These tests reflect the effects or signs the entire duration of the Human Ge- of genetic hyperlipidemias, errors of tions and duplications of chromosomal regions at much finer resolution than nome Project, DNA sequencing was metabolism, and hemolytic performed on semiautomated capil- afforded by standard . In fact, anemias, respectively. Such biochemical lary instruments by the power and diagnostic yield of genetic testing has been available for using the biochemical method known chromosomalmicroarraysaresomuch many decades. However, the most fun- as dideoxy-chain-termination, or Sanger greater that the American College of damental form of genetic testing clearly sequencing, after its inventor. This highly Medical Genetics and Genomics has is the direct analysis of the genetic accurate, although relatively slow, recommended that this test replace material itself (chromosomes, genes, method can provide the DNA sequence of karyotype analysis as the standard or the entire genome), which falls un- a targeted and limited region of 150 to first-tier test in the diagnostic evalua- der the domains of cytogenetics and 200 nucleotides in a single run of 1 to 2 tion of patients with congenital mal- . Biochemical ge- days. This length of DNA represents formations, nonspecific dysmorphic netics, cytogenetics, and molecular only a small fraction of the total length features, developmental delay/intellectual genetics are the 3 laboratory sub- of most genes, let alone the entire ge- disability, and autism.1 specialties recognized by the American nome, which explains why sequencing Board of Medical Genetics. Finally, a new technology called next- the first human genome took 13 years Theadventofclinicalcytogeneticsdates generation DNA sequencing has and cost approximately $3 billion. Ob- to 1959, with the discovery that children placed within reach the ultimate, most viously, an effort of that magnitude with Down syndrome have 47 chromo- fundamental, and highest-resolution could never remotely be translated into somes in their cells rather than the genetic analysis currently conceiv- a clinical test. able: the precise identification and or- normal 46, the abnormality being an One reason for the slow pace of Sanger extra copy of 21 (trisomy dering of all 6 billion nucleotides in the sequencing is that it is based on 21).Sincethattime,countlessdefectsin human genome. This technology, more daughter strand synthesis of a small chromosomal number, structure, and than any other, has opened the way to stretch of DNA that is selected by using position have been observed and as- true genomic medicine. the hybridization of specific oligonucle- sociated with various congenital (and It is important to point out some se- otide primers to just that region. The neoplastic) disorders. In a sense, chro- mantic distinctions related to the primers serve as start sites for mosomal analysis (also known as kar- evolving field of genomics. Most agree DNA to make complemen- yotype analysis) can be considered the that the term “genetics” refers to the tary strands that terminate whenever first genome-wide test because it study of single genes in . Many a particular de- includes all the nuclear genetic mate- refer to “genomics” as the study of all rivative is incorporated into the elon- rial of the cell. Such analysis is limited, genes in the genome and the inter- gating strand, and the resulting however, by the resolution of the light actions among them and their envi- sequence is deduced by measuring ; any deletion, duplication, ronments, as noted in the Executive the sizes of the terminated fragments insertion, or translocation that is too Summary. However, others use the on the capillary electrophoresis in- small to be observed under standard term “genomics” to describe whole- strument. In contrast, next-generation, magnification will go undetected. More exome and whole-genome sequenc- or massively parallel, sequencing recently, the field of cytogenetics has ing aimed at identifying mutations in breaks up the whole genome into .300

S212 GRODY et al Downloaded from www.aappublications.org/news by guest on October 2, 2021 SUPPLEMENT ARTICLE million small fragments, which are disorders are believed to be due to DIAGNOSIS AND DISCOVERY then universally primed for DNA poly- mutations in the coding regions, this THROUGH NEXT-GENERATION merase copying by using 4-color fluo- approach allows laboratories to focus SEQUENCING rescently labeled nucleotides and are exclusively on those regions and elim- Whether conducted by using a whole- analyzed on instruments that take in- inate the tremendous mass of non- exome or whole-genome approach, stantaneous snapshots of the added coding DNA in the genome, which the application of next-generation se- nucleotides after each round of syn- would otherwise add greatly to the quencing to children who appear to thesis across the entire genome. Each sequencing load and produce 100 have a syndrome but are undiagnosed fragment is copied, or “covered,” be- times as much data to interpret. The has already produced some dramatic tween 10 and $100 times, and the ag- downside, however, is that - successes. Some of these are in the gregated fluorescent photos of all of capture techniques are not 100% effi- form of new gene discovery, whereas the synthesis products comprise cient: 3% to 5% of the will not be others involve detection of mutations “cluster arrays” representing 4 to 5 captured and sequenced. Therefore, in known genes that were not sus- terabytes of computer data. The align- the commonly used term “whole- pected to be associated with the par- ment software then reconstructs the exome sequencing” is actually a mis- ticular patient’s phenotype or were entire genome from these hundreds of nomer. If some of the missing exons are not practical to query by using tradi- millions of DNA fragments. Depending deemed essential for ruling out a par- tional genetic testing methods. In just on the particular instrument used, the ticular suspected diagnosis, they must method can provide the sequence of 3 be specifically targeted and sequenced the past 3 years, new causative genes for at least 150 heritable disorders to 10 gigabases of DNA in a single run,2 by using any of a variety of work- easily enough to cover the entire hap- around techniques based on poly- have been discovered by using these 3 loid or diploid genome of an individual. merase chain reaction. methods. Often this 1 test will put an end to the long and expensive di- The instruments and reagents are ex- Depending on the diagnostic question agnostic odyssey that these patients pensive, but the net cost per sample to be answered, some laboratories will (and their parents) typically endure. per run has been decreasing over the furtherrestrictthe exonsbeingqueried For that reason, we hope it will be last few years and now stands at $2500 to just a panel of genes known to be widely accepted for by in- to $6000 per individual, depending on associated with the disorder sus- surance carriers, especially when depth of coverage and other factors. pected. Examples of commonly ordered they understand that the cost, while This cost is well within the range of gene panels, consisting of 20 to 100 appreciable, is less than the aggre- currently accepted single-gene se- genes per disorder, are shown in gate cost of sequencing 2 or 3 in- quencing tests, such as those for Table 1. A key advantage to this approach dividual genes 1 at a time. BRCA1 and BRCA2. Thus, genome se- is that it eliminates the possibility of quencing is now a legitimate test to unwanted, off-target results (as dis- consider for certain clinical situations. cussed in The “Incidentalome” section), THE “INCIDENTALOME” but it has the disadvantage that most of Probably the biggest challenge in the existing gene panels yield positive EXOME SEQUENCING VERSUS performing whole-exome and whole- results in significantly less GENOME SEQUENCING genome sequencing on a clinical ba- than one-half of the tested patients with Most laboratories currently using next- the associated phenotype. sis is dealing with the huge quantity of generation sequencing are applying it unexpectedsequencevariantsthat are to just the small subset (∼1.5%) of the inevitably detected. In just the exome total human genome that codes for TABLE 1 Disease Gene Panels That Use Next- alone, for every real, causative muta- Generation Sequencing (ie, the exome, which com- tion detected, one also finds at least prises ∼230 000 exons). Exome se- Hypertrophic cardiomyopathy 18,000 variants in other genes, most of quencing involves an additional step Dilated cardiomyopathy which are of uncertain clinical signif- Hereditary arrhythmias (channelopathies) beyond those described in the previous Retinitis pigmentosa icance; in the whole genome, the paragraph: an exon-capture step by Albinism number is more like 3 million. In ag- which the coding regions are selected Intellectual disability gregate, these variants have been DNA repair defects “ ”4 from the total genome DNA by means of Skeletal dysplasias dubbed the incidentalome. Obvi- hybridization, either to a microarray or Disorders of sexual development ously no one can go through all of in solution. Because most inherited Hearing loss them manually to decide what they

PEDIATRICS Volume 132, Supplement 3, December 2013 S213 Downloaded from www.aappublications.org/news by guest on October 2, 2021 mean and which ones should be revolutionized our ability to detect available for individuals with disorders reported. Instead, we have to rely on common microdeletion syndromes that are presumed to be genetic in ori- computer software to do the initial such as deletion 22q11.2 (previously gin. However, the effectiveness of this filtering; that is, the discarding of known as DiGeorge syndrome and technology for diagnosis involving a those variants judged, by a number of velocardiofacial syndrome), Prader- single individual, compared with its rules, as likely to be nonpathogenic. Willi syndrome, Angelman syndrome, power when the genomic information Even after that, however, it is not un- and Williams syndrome. from several individuals with the same common to be left with several hun- When chromosomal microarrays be- condition is available, has yet to be de- dred variants that are potentially came clinically available, the floodgates termined. worrisome, and sorting through them opened, and we began to identify As stated previously, next-generation may require the combined efforts of microdeletions and microduplications sequencing is clinically available in molecular , clinical , that were of unknown significance. the form of panels for certain find- and bioinformaticists. Even more con- However, with time, the significance (or ings that are known to have an un- cerning, from an ethical perspective, is insignificance) of some of the micro- derlying genetic basis (Table 1). The “ the incidental discovery of off-tar- deletions and microduplications has use of such panels has proven to ” get mutations. These are deleterious become clearer, as more and more be a cost-effective way to simulta- changes in known genes that are ir- individuals are found to have these copy neously interrogate several genes relevant to the patient’scurrent number variants. For example, micro- from a single patient’ssample.Inthe phenotype and the reason for order- deletions involving 16p11.2 have been past, the genetics provider would ing the test but which may predict identified in individuals with varying often start with 1 gene and have future disease; for example, the degrees of intellectual disability and testing performed by $1 laboratory finding of a pathologic BRCA mutation behavioral issues (eg, autism spectrum on a reflex basis. in a 3-year-old girl undergoing whole- disorder). The penetrance of this exome sequencing for autism or microdeletion, however, seems to be hearing loss. The BRCA mutation has Timing of Primary Care Integration incomplete, because some individuals nothing to do with those conditions, of Genomics with it are reportedly normal. This and we would never deliberately do It is likely that whole-exome and whole- microdeletion may well become 1 of the such predictive testing for an adult- genome sequencing and genomics, in examples of a truly genomic variation; onset disease in a young child; how- the strict sense of the word, will even- that is, it is disease-causing only in ever, what is the liability of failing to tually be integrated into primary care. conjunction with mutations in other disclose this incidentally detected risk, When and how this approach will be genes and/or specific environmental either for the future health of the child integrated, however, is still up for dis- influences. or the present health of the parent who cussion.Somehaveproposedthatitwill transmitted it? This example illustrates The use of whole-exome and whole- be more cost-effective to obtain whole- only 1 of the many difficult ethical genome sequencing has revolutionized genome sequencing data, store that questions raised by this technology the way researchers identify mutations data, and interrogate the sequence as and highlights the need for informed in single genes (eg, Mendelian dis- needed. When that sequencing data consent for this new testing. orders). In the first example, described should be obtained (whether in the in 2009, whole-exome sequencing was newborn period or at some later point used to identify the causative gene for fi INTEGRATION OF GENOMICS INTO when genetic information may rst Freeman-Sheldon syndrome, a disorder PRIMARY CARE become helpful in providing health characterized bymultiple contractures.5 care) is yet to be determined. We must Research Discoveries and Clinical In a groundbreaking study, the authors also determine how that information Advances From Genomic Advances, used whole-exome sequencing and, by will be stored so that it is “safe” and Early and Now comparing sequence variants that were who will be able to interrogate an Therehavebeenmanyclinicaladvances present only in the 4 affected individu- individual’s genome information. Clearly, in the diagnosis of individuals with als, were able to identify the causative the medical will continue to suspected genetic conditions since the gene. Since that time, this technology assist the primary care provider in de- aforementioned technologies became has been used to identify the genes re- ciding what genetic or genomic testing, if available for clinical use. The use sponsible for at least 100 conditions. In any, is indicated and how to interpret the of fluorescence in situ hybridization 2012, this technology became clinically results.

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DIAGNOSIS, TREATMENT, AND For example, if a couple knows that they ately increased risk,” which individuals PREVENTION are having an infant with a lethal con- in a population challenged by “numer- dition, they can plan to deliver close to acy” may have difficulty understanding Forallmedicalconditionsthathealthcare homewherefamilysupport is nearby.If, and using. Even when posttest coun- providers attempt to diagnose, the goal is however, the fetus is diagnosed with seling is offered, it is often rudimen- to maximize effective treatment and, ul- a that is likely to tary. Unfortunately, the continued timately, to prevent the condition from require multiple evaluations and pro- increases in the speed and efficiency of ever occurring in the first place. There- cedures after birth, delivery at a ter- genomic sequencing, the decreases in fore, although genomics is currently used tiary care center may be indicated. cost, and the ability to perform it on mostly for diagnosis, as we gain a better specimens easily collected at home are understanding of the underlying molec- only going to make DTC genetic test ular mechanisms of various genetic and DIRECT-TO-CONSUMER TESTING offerings more global and prevalent in genomic disorders, we should also be Soon after microarray and sequencing the near future. The American College able to use genomics to tailor our techniques advanced to the point of of Medical Genetics and Genomics has treatment of these conditions. general availability, commercial labo- recommended strongly that “a certi- The use of genomics in the prevention of ratories began to market to the public fied medical geneticist or genetic genetic conditions currently involves pro- “personalized testing” of an individu- counselor be involved in the process of viding accurate to at- al’s genome, regardless of whether ordering and interpreting”7 such tests risk individuals so that they can use this there was a medical indication for such and their results. However, in the ab- information for family planning. Some testing.6 This direct-to-consumer (DTC) sence of some sort of government couples choose not to have a child if there testing may not involve appropriate regulation or oversight, restricting indi- is a risk that that child will have a genetic pretest counseling, which can help the viduals’ access to such services will be condition. Others choose to use currently individual to fully understand the po- difficult, if not impossible. available forms of prenatal diagnosis, tential limitations of the test and, more including chorionic villus sampling and importantly, to recognize the potential CONCLUSIONS amniocentesis, if the mutations are “harms” that may attend testing (the Whole-genome/whole-exome sequencing known. Increasingly, preimplantation previously mentioned incidental find- is a powerful approach to genetic di- genetic diagnosis is being used by cou- ings, off-target mutations, and variants agnosis when used in the appropriate ples for whom familial mutations have of unknown significance within the circumstances and supported by the fi been identi ed. In the near future, in- genome). Interpretation of test results infrastructure of expert clinical and terrogating cell-free fetal DNA from the and posttest counseling are also criti- molecular geneticists, genetic coun- maternal serum will likely be widely cal to the meaningful use of the in- selors, and other patient-care services clinically available and will allow for formation by the individual (and available at tertiary care centers. Se- noninvasive prenatal genetic diagnoses. perhaps other family members). Test quencing should not be viewed as Prenatal genetic diagnosis is used not results provided by DTC laboratories a platform for personal entertainment only to decide whether to continue usually are couched in terms such as or be taken lightly, given the potential apregnancybutalsotoplanfordelivery. “slightly increased risk” or “moder- harms and challenges discussed here.

REFERENCES

1. Manning M, Hudgins L; Professional Practice 3. Bamshad MJ, Ng SB, Bigham AW, et al. Exome of 12 human . . 2009;461(7261): and Guidelines Committee. Array-based tech- sequencing as a tool for Mendelian disease 272–276 nology and recommendations for utilization in gene discovery. Nat Rev Genet. 2011;12(11): 6. Bender L, Silverman LM, Dinulos MB, Nickel medical genetics practice for detection of 745–755 J, Grody WW. Direct-to-consumer : chromosomal abnormalities. Genet Med. 2010; 4. Kohane IS, Masys DR, Altman RB. The inci- are we ready for a brave new world? Clin 12(11):742–745 dentalome: a threat to genomic medicine. Chem. 2010;56(7):1056–1060 2. ten Bosch JR, Grody WW. Keeping up with the next JAMA. 2006;296(2):212–215 7. American College of Medicine Genetics Board of generation: massively parallel sequencing in clin- 5. Ng SB, Turner EH, Robertson PD, et al. Targeted Directors. ACMG statement on direct-to-consumer ical diagnostics. JMolDiagn. 2008;10(6):484–492 capture and massively parallel sequencing genetic testing. Genet Med. 2004;6(1):60

PEDIATRICS Volume 132, Supplement 3, December 2013 S215 Downloaded from www.aappublications.org/news by guest on October 2, 2021 Whole-Exome/Genome Sequencing and Genomics Wayne W. Grody, Barry H. Thompson and Louanne Hudgins Pediatrics 2013;132;S211 DOI: 10.1542/peds.2013-1032E

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Downloaded from www.aappublications.org/news by guest on October 2, 2021 Whole-Exome/Genome Sequencing and Genomics Wayne W. Grody, Barry H. Thompson and Louanne Hudgins Pediatrics 2013;132;S211 DOI: 10.1542/peds.2013-1032E

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