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CLIN. CHEM. 41/10, 1407-1409 (1995) Direct DNA Sequencing in the Clinical Laboratory The article by Berg et al. (1) in this issue touches on of issues investigators are currently dealing with in seeking robust application of direct DNA sequencing to clinical specimens. These include automation, optimal methods to obtain high-quality data, efficient multiplex amplification strategies, quality control, and procedures to deal with very small samples. Not discussed but inherent in cost-effective application of sequencing technology are efficient means for handling the primary sequence data generated. Direct DNA sequencing will be a routine procedure in specialized clinical laboratories within 10 years. The pace of discovery for disease-causing genes and underlying mutations is accelerating, and a number of autosomal genes, such as major ones associated with familial breast cancer (BRCA1, BRCA2, and possibly ATM), cumulatively affect large numbers of individuals in the general population (2-4). Significant challenges confront the transition of DNA sequencing from the research to clinical laboratory, and this editorial focuses on background and general issues in this area. Single nucleotide substitutions leading to amino acid changes (missense mutations), translation stop signals (nonsense mutations), or aberrant exonhintron splicing are responsible for most disease-causing alterations of genes. Whereas the latter two types of change are usually pathogenic, single amino acid changes range from a host of innocent polymorphisms without functional effect on the gene product to devastating instability as wrought by the glutamic acid-to-valine change between hemoglobins A and S. In disorders in which one or relatively few known mutations cause disease, forward and reverse dot-blot hybridization, allele-specific amplification, amplification with restriction endonuclease digestion, or heteroduplex analyses are used as relatively simple and rapid methods to look for specific changes (5, 6). Otherwise, screening for single nucleotide changes and less frequent frameshift losses or gains of one to a few nucleotides is most easily accomplished by direct DNA sequencing. Sequencing generally begins with polymerase chain reaction (PCR) amplification of a patient’s DNA directed at gene regions of interest, including exonhintron boundaries, followed by sequencing reactions on the PCR products. RNA may also be used as starting material in some circumstances. Attractive targets for direct sequencing include genes such as the 13chain of class II histocompatibility molecules, in which numerous nucleotide changes occur in several hundred nudeotides of exon 2 (7). A larger region encompassed by one or perhaps two exons, such as connexin 32 in a range X-linked Charcot-Marie--Tooth disease (8), is also attractive because much information is obtained from a few sequencing reactions. Next in order of appeal are genes with clinical significance, such as p53, in which many mutations occur in a relatively defined area of the gene. The large number of mutations eliminates practical forward or reverse hybridization approaches to detection. DNA sequencing may require PCR amplification of several exons. There is more work and modest additional expense associated with sequencing such genes, but multiplex amplification schemes such as that described by Berg et al. (1 ) frequently allow this to be done economically. Least attractive in terms of effort are relatively large genes in which disease-causing mutations are spread throughout the gene, usually over many exons. Sequencers usually approach candidate samples for these mutations first by screening for nucleotide substitutions by a method such as single-strand conformation polymorphism analysis or denaturing gradient gel electrophoresis. Protein-truncation approaches are becoming more popular to screen for nonsense and frameshift mutations (9). Here, cDNA for the gene of interest is generated from sample mRNA PCR-amplified for different areas of the gene and translated in vitro into peptides; peptides of sizes less than expected indicate the region and approximate location within the gene of a mutation. Despite their greater complexity, these more-complex genes are likely to be a major group analyzed in the future, as many important diseases will fall into this category, including familial breast cancer (10), neurofibromatosis type 1 (11), Duchenne/ Becker muscular dystrophy (12), and hereditary nonpolyposis colon cancer (HNPCC) (13). With good sequence data-and methods must be adequate to detect heterozygotes-sample interpretation is generally straightforward for nonsense or splicesite mutations. Missense nucleotide changes in genes in which many mutations may occur are another matter. Are these polymorphisms or disease-causing mutations? Rules based on conservative vs nonconservative amino acid changes will almost certainly need clinicopathologic confirmation for individual disorders. For an inherited disease in a large pedigree, it may be possible to examine whether a putative mutation segregates as expected to affected and unaffected individuals, although this may raise difficult individual issues of privacy, consent, and family loyalty if analysis constitutes presymptomatic testing. For spontaneous or acquired mutations, as in neoplasms, the significance CLINICAL CHEMISTRY, Vol. 41, No. 10, 1995 1407 of missense mutations will be particularly problematic until we have a larger database of information. It is essential for clinical laboratories that sequencing methods be efficient and robust, with few or no repeat analyses required, since considerable time goes into sample handling, sequencing gels, and data analysis with both standard and automated sequencing. That is the utility of studies such as that of Berg et al. (1), involving a-thio-dNTPs or other methods as general strategies or to approach difficult-to-sequence areas. Improvements in thermal cycler-based sequencing, which improve accuracy and eliminate the need for strand separation, are also being studied. The prospects for non-gel-based DNA sequencing by hybridization to oligonucleotide arrays on silicon chips (14) is particularly appealing because of the potential to design disease-specific sequencing cassettes and the elimination of time-consuming sequencing gels. The ability to apply direct sequencing to a variety of sample types, including small areas of tumor samples (15) or archival paraffin tissue blocks using multiplex approaches such as those described by Berg et al. (1), is also an important feature of robust clinical methods. The impetus of the Human Genome Project has also spurred many technical improvements in DNA sequencing (16) for research, including the development of high throughput, automated DNA sequencing protocols, instrumentation, and interfaced data analysis programs, which may eventually find applicability in clinical laboratories. The ability to effectively manage the large amounts of raw information produced by direct sequencing is crucial to the efficient adaptation of direct sequencing to the clinical laboratory. One program currently being evaluated for HLA allele assignment clinically shows promise but may require significant time for manual checking (17). Given the implications for patient management along with direct repercussions on individual lifestyle and long-term and reproductive planning, direct sequencing must prove itself a reliable technique. Current studies in histocompatibility analysis and familial cancer mutations will provide important benchmarks on the promise and possible pitfalls of real-time direct DNA sequencing for clinical analysis. Laboratory protocols will clearly need to incorporate rigorous qualitycontrol stratagems, perhaps sequencing both DNA strands as for Berg’s p53 studies (1), as well as possible sequencing of the same region with different primer sets or even cross-confirming methodologies. Concurrent sequencing of identity genes such as HLA alleles (1) is an interesting idea to monitor possible sample contamination or carryover, particularly in laboratories in which large numbers of cancer specimens are analyzed and DNA sequence data are used for immediate therapeutic and prognostic decisions. In addition to many newly cloned genes and associated disease mutations each month, several clinical applications highlight the potential benefit of direct DNA sequencing. High-resolution HLA typing appears to be beneficial in allogeneic bone marrow transplan1408 CLINICAL CHEMISTRY, Vol. 41, No. 10, 1995 tation, a technique becoming more and more common for treating cancer and certain inherited diseases (18). Mutations associated with HNPCC and familial breast and ovarian cancer also appear to occur in sporadic cases of these neoplasms (19, 20). Direct sequencing of genes with broad mutational heterogeneity obviates the need for familial linkage studies (which may sometimes be uninformative) and permits prenatal and direct carrier detection for near and distant relatives. Databases of such information offer potentially important prognostic and therapeutic information to other patients by better defining the biology of a particular disorder (21). Selected patients who lack common diagnostic changes in the genome but exhibit clinical features strongly suggestive of a particular inherited disorder may benefit from direct DNA sequencing of particular genes known to be associated with that disorder (22). Sequence information from specific genes may also be useful in certain clinical contexts to predict the likely phenotype and course of individuals with craniofacial dysmorphic syndromes (23) in the case of fibroblast growth-factor receptor mutations. Similarly, a diverse set of outcomes, from conditions such as Hirschsprung disease to aggressive hereditary cancers, appear associated with specific mutations of the RET protooncogene (24). Direct sequencing of specific genes in mycobacterial isolates to detect antimicrobial resistance properties offers significant benefit in choosing appropriate therapy (25). The relevance of direct sequencing is thus clear and certain to grow. Its full impact in the near term on clinical samples may depend on providers of managed care. Direct sequencing is moderately expensive now, a combination of instrument, reagent, and personnel costs along with limited sample volumes. On the other hand, current US Medicare reimbursement rates for molecular Current Procedural Terminology codes are unrealistically low (26) and are often used as guidelines by other payors. Organizational efficiencies and future methodologic advances could lead to substantial savings, particularly for personnel. However, studies documenting the financial value of direct sequencing in overall patient management are sorely needed, since this technology lacks the encrusted precedent that many other laboratory tests enjoy. Given that patients have shown a willingness to pay out-of-pocket for genetic tests and may exhaust all possibilities when cancer is the issue, there may be some wild cards in the financial equation. I would echo the call by many others for timely legislation to prevent discrimination and to protect individual privacy for genetic information derived from such testing. References 1. Berg C, Hedrum A, Homberg A, Pontdn F, LJhl#{233}n M, Lundeberg J. Direct solid-phase sequence analysis of the human p53 gene by use of multiplex polymerase chain reaction and a-thiotriphosphate nucleotides. Clin Chem 1995;41:1461-6. 2. Mild Y, Swensen J, Shattuck-Eidens D, Futreal PA, Harshman K, Tavtigian S, et al. A strong candidate for the breast and ovarian cancer susceptibility gene BRCAI. Science 1994;266:66-71. Wooster R, Neuhausen SL, Mangion J, Quirk Y, Ford D, 3. ollins N, et al. Localization ene, BRCA2, to chromosome of a breast cancer susceptibility 13q12-13. Science 1994;265:2088- )0. I. Savitsky ;elangiectasia K, Bar-Shira A, Gilad S, et al. A single gene with a product similar to P1-3 kinase. ataxia Science 1995;268:749-53. Beutler E, Gelbar T, Kuhl W, Zimran A, West C. Mutations in Eewish patients with Gaucher’s disease. Blood 1992;79:1662-6. . Chehab FF, Wall J. Detection of multiple cystic fibrosis muta;ions by reverse dot blot hybridization: a technology for carrier icreening. Hum Genet 1992;89: 163-8. 7. Spurkland A, Knutsen I, Markussen G, Vartdal F, Egeland T, l’horsby E. HLA matching of unrelated bone marrow transplant airs: direct sequencing of in vitro amplified HLA-DRI31 and .DQf31 genes using magnetic beads as solid support. Tissue tntigens 1993;41:155-64. . Bergoffen J, Scherer SS, Wang S, Scott MO, Bone LI, Paul DL, t al. Connexin mutations in X-linked Charcot-Marie-Tooth lisease. Science 1993;262:2039-42. . Powell SM, Petersen GM, Krush AJ, Booker S, Jen J, Giirdiello FM, et al. Molecular diagnosis of familial adenomatous Dolyposis. N EngI J Med 1993;329:1982-7. LO. Shattuck-Eidens D, McClure M, Simard J, Labrie F, Narod S, ouch F, et al. A collaborative survey of 80 mutations in the RCA1 breast and ovarian cancer susceptibility gene. Implicabions for presymptomatic testing and screening. JAMA 1995;273: . 535-41. 11. Upadhyaya M, Shaw DJ, Harper PS. Molecular basis of rieurofibromatosis type 1 (NFl): mutation analysis and polymorphisms in the NFl gene. Hum Mutat 1994;4:83-101. 12. Prior TW, Bartolo C, Pearl DK, Papp AC, Snyder PJ, Sedra MS, et al. Spectrum of small mutations in the dystrophin coding region. Am J Hum Genet 1995;57:22-33. 13. Wijnen J, Vasen H, Khan PM, Menko FH, van der Klift H, van Leeuwen C, et al. Seven new mutations in hMSH2, an E-INPCC gene, identified by denaturing gradient-gel electrophoresis. Am J Hum Genet 1995;56:1060-6. 14. Pease AC, Solas D, Sullivan EJ, Cronin MT, Holmes CP, E’odor SP. Light-generated oligonucleotide arrays for rapid DNA sequence analysis. Proc Natl Acad Sci U S A 1994;91:5022-6. 15. Finkelstein SD, Sayegh R, Christensen 5, Swalsky PA. Gertotypic classification of colorectal adenocarcinoma. Biologic behavior correlates with K-ras-2 mutation type. Cancer 1993;71: 3827-38. 16 Smith LM. The future of DNA sequencing. Science 1993;262: 530-2. 17. Versluis LF, Rozemuller E, Tonks S, Marsh SGE, Bouwens AGM, Bodmer JG, Tilanus MGJ. High-resolution HLA-DPf3 typing based upon computerized analysis of data obtained by fluorescent sequencing of the amplified polymorphic exon 2. Hum Immunol 1993;38:277-83. 18. Begovich AB, Ehrlich HA. HLA typing for bone marrow transplantation. JAMA 1995;273:586-91. 19. Merajver SD, Pham TM, CaduffRF, Chen M, Poy EL, Cooney KA, et al. Somatic mutations in the BRCAI gene in sporadic ovarian tumours. Nature Genet 1995;9:439-43. 20. Liu B, Nicolaides NC, Markowitz S, Wilson ,JKV, Parsons RE, Jen J, et al. Mismatch repair gene defects in sporadic colorectal cancers with microsatellite instability. Nature Genet 1995;9:4855. 21. Giannelli F, Saad S, Montandon AJ, Bentley DR, Green PM. A new strategy for the genetic counselling of diseases of marked mutational heterogeneity: haemophilia B as a model. J Med Genet 1992;92:602-7. 22. Chance PF, Fischbeck KH. Molecular genetics of CharcotMarie-Tooth disease and related neuropathies. Hum Mol Genet 1994;3: 1503-7. 23. Mulvihill JJ. Craniofacial syndromes: no such thing as a single gene disease. Nature Genet 1995;9:101-3. 24. Smith DP, Eng C, Ponder BA. Mutations of the RET protooncogene in the multiple endocrine neoplasia type 2 syndromes and Hirschsprung disease. J Cell Sci 1994;18(Suppl):43-9. 25. Kapur V, Li LL, Hamrick MR, Plikaytis BB, Shinnick TM, Telenti A, et al. Rapid mycobacterium species assignment and unambiguous identification of mutations associated with antimicrobial resistance in Mycobacterium tuberculosis by automated DNA sequencing. Arch Pathol Lab Med 1995;119:131-8. 26. Kant JA. Molecular diagnosis: reimbursement and other selected financial issues. Diagn Mol Pathol 1995;4:79-83. Jeffrey A. Kant Molecular Diagnosis Laboratory Depart ment of Pathology and Laboratory Medicine University of Pennsylvania Medical Center 3400 Spruce St. Philadelphia, PA 19104-4283 Fax 215-662-7529 CLINICAL CHEMISTRY, Vol. 41, No. 10, 1995 1409