Molecular Genetics and Metabolism 114 (2015) 380–381
Contents lists available at ScienceDirect
Molecular Genetics and Metabolism journal homepage: www.elsevier.com/locate/ymgme
Commentary
Hyperphenylalaninemia and the genomic revolution Farrah Rajabi a, Harvey L. Levya,b,⁎ a b
Division of Genetics and Genomics, Boston Children's Hospital, Boston, MA, USA Department of Pediatrics, Harvard Medical School, Boston, MA, USA
A R T IC L E
IN F O
Article history: Received 11 December 2014 Accepted 12 December 2014 Available online 20 December 2014 Keywords: Newborn screening Next-generation sequencing Hyperphenylalaninemia Phenylketonuria Cofactor deficiency Genomic revolution
Political revolutions are marked by the dates of their cataclysms, be they major battles or campaigns that shifted momentum: the American Revolution of 1776, the French Revolution of 1789, and the Russian Revolution of 1917. But each of these and every other revolution were preceded by many smaller but very significant events which culminated in the cataclysm. And each revolution was succeeded by changes that had lasting effects which made the revolution meaningful. So it has been with medical revolutions that have shifted the momentum of clinical practice. Immunization is said to have originated in the late 18th century with Jenner's demonstration of cowpox inoculation producing immunity to smallpox. Antisepsis is believed to have originated in the mid-19th century with Semmelweis's insistence that washing the hands before delivery reduces puerperal sepsis [1]. Anesthesia is also thought to have originated in the 19th century with the demonstration of ether administration in Boston [2]. However, each of these medical revolutions, just as political revolutions, had extensive preceding and succeeding histories. And so it is with the medical revolution in which we are now living, the genomic revolution, which may be the most revolutionary of all. Perhaps the “cataclysm” of the genomic revolution will be the demonstration of the double-helical structure of DNA in 1953, or maybe the mapping of the human genome in 2003, or perhaps a development in the future not yet foreseen [3]. What is certain is that there has been and will continue to be a long history of genetic developments that are already beginning to radically change medicine. DOI of original article: http://dx.doi.org/10.1016/j.ymgme.2014.10.004. ⁎ Corresponding author at: Boston Children's Hospital, One Autumn Street, Room 526.1, Boston, MA 02115.
http://dx.doi.org/10.1016/j.ymgme.2014.12.303 1096-7192/© 2015 Elsevier Inc. All rights reserved.
The molecular basis of the genomic revolution is the ability to isolate specific genes in the human genome and identify mutations within those genes. The promise is that these mutations are the ultimate cause of disease. The application of this technology at its simplest level is targeted second-tier molecular testing in newborn screening (NBS) in which the NBS specimen is tested for common mutations when the NBS results indicate a positive screen for certain conditions. This type of second-tier testing is currently in use for conditions such as medium chain acyl-CoA dehydrogenase deficiency (MCADD), galactosemia, and cystic fibrosis. These results have been very effective in confirming the specific disorder and potential severity prior to clinical assessment of the infant [4]. Cao et al. [5] could carry this process a step further. Cao and coworkers are reporting promising results using Ion Torrent Personal Genome Machine (PGM) sequencing that can identify mutations in a panel of five genes associated with neonatal hyperphenylalaninemia (HPA) using a single assay [5]. These include the gene for phenylalanine hydroxylase (PAH) and four genes that encode enzymes which can result in a deficiency of tetrahydrobiopterin (BH4), the cofactor required for PAH activity [6]. In testing 37 patients with HPA of unknown genotype Cao et al. correctly identified mutations in 71 of 74 alleles (95.9%), all confirmed by the “gold standard” of Sanger sequencing [5]. Other groups have demonstrated similar rapid diagnosis with panel testing for HPA-related genes, using either the same Ion Torrent PGM system or high-throughput multiplex-targeted sequencing [7,8]. The turnaround time for the assay described by Cao et al. is 3–4 days, adequate for confirmation of a NBS result [5]. Thus, this methodology might not only confirm the presence of a disorder but also specify the precise disorder in a hyperphenylalaninemic neonate within a few
F. Rajabi, H.L. Levy / Molecular Genetics and Metabolism 114 (2015) 380–381
days after birth, before or at the time the infant is seen for initial clinical visit. A major limitation of this methodology, however, is the need for peripheral blood leukocytes to obtain the required genomic DNA. The authors are currently examining the possibility of obtaining sufficient DNA for the assay using the dried blood NBS specimen [Song F, personal communication]. Indeed, isolation of DNA from the dried blood spot was recently demonstrated to result in sufficient DNA for next generation sequencing (NGS) [9,10]. If the NBS specimen can be utilized and if the turnaround time can be maintained at 3–4 days or even less, NGS technology such as the Ion Torrent PGM system could be added as one of the second-tier screens in the NBS laboratory and cover almost all disorders responsible for increased phenylalanine in the neonate. Why is this of potential importance? HPA in a neonate is not a disorder but an indication of any one of several conditions. The most frequent of these will be PAH deficiency, i.e., phenylketonuria (PKU). However, even PAH deficiency is not a single clinical entity. There are at least three forms, characterized by the degree of PAH deficiency: classic PKU, moderate or mild PKU, and non-PKU mild HPA (MHP). Each of these has important implications for outcome and treatment. Classic PKU is the most severe with the possibility of long term problems and the need for the most stringent dietary treatment; moderate or mild PKU may have a better outcome and require a less stringent diet; and MHP is probably benign, which is likely to have normal outcome without treatment. HPA could also represent BH4 deficiency with its several enzymatic causes, any one of which, with profound implications for outcome and requiring a very different treatment than PKU [6]. Neonatal HPA may also be transient and not indicative of a metabolic disorder. This is most likely in a premature infant receiving total parenteral nutrition (TPN) but also may be seen in infants with liver disease or congenital heart disease [11]. It is important to sort through the possibilities and identify the neonate with a specific genetic cause for HPA or rule it out, in which case it is likely that the increased phenylalanine will be transient. Identification of the genetic etiology for the HPA upon presentation of the infant to a clinical program can allow tailored genotype specific information for the family and for management options. The immediate confirmation of results can also reduce false positives and parental anxiety [12]. The turnaround time for current commercial genetic testing may take weeks or months before the result is known. Ideally, second-tier genetic panel testing could be done reflexively and quickly after screen positive HPA results using the same dried blood spot. Perhaps all second-tier molecular testing in NBS will one day be replaced by NGS as a primary screen [4,13]. The efficiency and the reliability of high quality NGS are rapidly improving, possibly reducing or eliminating the need for time consuming Sanger confirmation of variants in clinical testing [14]. Once the sensitivity and the specificity of NGS can be reliably established, extending this technology to NBS offers an appealing and exciting potential advance in NBS disease detection, allowing NBS to cover not only a limited number of inborn errors of metabolism but also many other genetic disorders. Thus adapting genetic sequencing as part of NBS has broad appeal. Nevertheless, there are
381
major concerns about readiness for implementation. The greatest current limitations include turnaround time for validated results, reliable use of the dried blood spot, and cost [10]. In addition there are major ethical, societal, and even legal concerns [4,13]. Improving the speed of the test must not be done at the expense of accurate mutation detection. Additionally, molecular genetic testing cannot replace biochemical testing without reducing the sensitivity of screening since not all individuals affected by disease will have an identifiable genetic etiology. Further work is required to establish that sequencing technologies can be sufficiently reliable and sensitive for rapid genetic diagnosis. Stay tuned! Conflict of interest The authors declare no conflict of interest.
References [1] M. Jackson (Ed.), The Oxford Handbook of the History of Medicine, Oxford University Press, Oxford, UK, 2012. [2] J.M. Fenster, Ether Day: The Strange Tale of America's Greatest Medical Discovery and the Haunted Men Who Made It, HarperCollins, New York, NY, 2001. [3] F.S. Collins, The Language of Life, HarperCollins, New York, NY, 2010. [4] Y.E. Landau, U. Lichter-Konecki, H.L. Levy, Genomics in newborn screening, J. Pediatr. 164 (2014) 14–19, http://dx.doi.org/10.1016/j.jpeds.2013.07.028. [5] Y. Cao, Y. Qu, F. Song, T. Zhang, J. Bai, Y. Jin, H. Wang, Fast clinical molecular diagnosis of hyperphenylalaninemia using next-generation sequencing-based on a custom AmpliSeq™ panel and Ion Torrent PGM sequencing, Mol. Genet. Metab. 113 (2014) 261–266. [6] N. Blau, F.J. van Spronsen, H.L. Levy, Phenylketonuria, Lancet 376 (2010) 1417–1427, http://dx.doi.org/10.1016/S0140-6736(10)60961-0. [7] Y. Gu, K. Lu, G. Yang, Z. Cen, L. Yu, L. Lin, et al., Mutation spectrum of six genes in Chinese phenylketonuria patients obtained through next-generation sequencing, PLoS One 9 (2014) e94100, http://dx.doi.org/10.1371/journal.pone.0094100. [8] D. Trujillano, B. Perez, J. González, C. Tornador, R. Navarrete, G. Escaramis, et al., Accurate molecular diagnosis of phenylketonuria and tetrahydrobiopterindeficient hyperphenylalaninemias using high-throughput targeted sequencing, Eur. J. Hum. Genet. 22 (2014) 528–534, http://dx.doi.org/10.1038/ejhg.2013.175. [9] T.L. Klassen, J. Drabek, T. Tomson, O. Sveinsson, U. von Döbeln, J.L. Noebels, et al., Visual automated fluorescence electrophoresis provides simultaneous quality, quantity, and molecular weight spectra for genomic DNA from archived neonatal blood spots, J. Mol. Diagn. 15 (2013) 283–290, http://dx.doi.org/10.1016/j.jmoldx.2013.01.003. [10] A. Bhattacharjee, T. Sokolsky, S.K. Wyman, M.G. Reese, E. Puffenberger, K. Strauss, et al., Development of DNA confirmatory and high-risk diagnostic testing for newborns using targeted next-generation DNA sequencing, Genet. Med. (2014), http://dx.doi.org/10.1038/gim.2014.117. [11] S.J. Evans, T.C. Wynne-Williams, C.A. Russell, A. Fairbrother, Hyperphenylalaninaemia in parenterally fed newborn babies, Lancet 2 (1986) 1404–1405. [12] S.E. Waisbren, S. Albers, S. Amato, M. Ampola, T.G. Brewster, L. Demmer, et al., Effect of expanded newborn screening for biochemical genetic disorders on child outcomes and parental stress, JAMA 290 (2003) 2564–2572, http://dx.doi.org/ 10.1001/jama.290.19.2564. [13] H.L. Levy, Newborn screening: the genomic challenge, Mol. Genet. Genomic Med. 2 (2014) 81–84, http://dx.doi.org/10.1002/mgg3.74. [14] S.P. Strom, H. Lee, K. Das, E. Vilain, S.F. Nelson, W.W. Grody, et al., Assessing the necessity of confirmatory testing for exome-sequencing results in a clinical molecular diagnostic laboratory, Genet. Med. 16 (2014) 510–515, http://dx.doi.org/10.1038/ gim.2013.183.
Contents lists available at ScienceDirect
Molecular Genetics and Metabolism journal homepage: www.elsevier.com/locate/ymgme
Commentary
Hyperphenylalaninemia and the genomic revolution Farrah Rajabi a, Harvey L. Levya,b,⁎ a b
Division of Genetics and Genomics, Boston Children's Hospital, Boston, MA, USA Department of Pediatrics, Harvard Medical School, Boston, MA, USA
A R T IC L E
IN F O
Article history: Received 11 December 2014 Accepted 12 December 2014 Available online 20 December 2014 Keywords: Newborn screening Next-generation sequencing Hyperphenylalaninemia Phenylketonuria Cofactor deficiency Genomic revolution
Political revolutions are marked by the dates of their cataclysms, be they major battles or campaigns that shifted momentum: the American Revolution of 1776, the French Revolution of 1789, and the Russian Revolution of 1917. But each of these and every other revolution were preceded by many smaller but very significant events which culminated in the cataclysm. And each revolution was succeeded by changes that had lasting effects which made the revolution meaningful. So it has been with medical revolutions that have shifted the momentum of clinical practice. Immunization is said to have originated in the late 18th century with Jenner's demonstration of cowpox inoculation producing immunity to smallpox. Antisepsis is believed to have originated in the mid-19th century with Semmelweis's insistence that washing the hands before delivery reduces puerperal sepsis [1]. Anesthesia is also thought to have originated in the 19th century with the demonstration of ether administration in Boston [2]. However, each of these medical revolutions, just as political revolutions, had extensive preceding and succeeding histories. And so it is with the medical revolution in which we are now living, the genomic revolution, which may be the most revolutionary of all. Perhaps the “cataclysm” of the genomic revolution will be the demonstration of the double-helical structure of DNA in 1953, or maybe the mapping of the human genome in 2003, or perhaps a development in the future not yet foreseen [3]. What is certain is that there has been and will continue to be a long history of genetic developments that are already beginning to radically change medicine. DOI of original article: http://dx.doi.org/10.1016/j.ymgme.2014.10.004. ⁎ Corresponding author at: Boston Children's Hospital, One Autumn Street, Room 526.1, Boston, MA 02115.
http://dx.doi.org/10.1016/j.ymgme.2014.12.303 1096-7192/© 2015 Elsevier Inc. All rights reserved.
The molecular basis of the genomic revolution is the ability to isolate specific genes in the human genome and identify mutations within those genes. The promise is that these mutations are the ultimate cause of disease. The application of this technology at its simplest level is targeted second-tier molecular testing in newborn screening (NBS) in which the NBS specimen is tested for common mutations when the NBS results indicate a positive screen for certain conditions. This type of second-tier testing is currently in use for conditions such as medium chain acyl-CoA dehydrogenase deficiency (MCADD), galactosemia, and cystic fibrosis. These results have been very effective in confirming the specific disorder and potential severity prior to clinical assessment of the infant [4]. Cao et al. [5] could carry this process a step further. Cao and coworkers are reporting promising results using Ion Torrent Personal Genome Machine (PGM) sequencing that can identify mutations in a panel of five genes associated with neonatal hyperphenylalaninemia (HPA) using a single assay [5]. These include the gene for phenylalanine hydroxylase (PAH) and four genes that encode enzymes which can result in a deficiency of tetrahydrobiopterin (BH4), the cofactor required for PAH activity [6]. In testing 37 patients with HPA of unknown genotype Cao et al. correctly identified mutations in 71 of 74 alleles (95.9%), all confirmed by the “gold standard” of Sanger sequencing [5]. Other groups have demonstrated similar rapid diagnosis with panel testing for HPA-related genes, using either the same Ion Torrent PGM system or high-throughput multiplex-targeted sequencing [7,8]. The turnaround time for the assay described by Cao et al. is 3–4 days, adequate for confirmation of a NBS result [5]. Thus, this methodology might not only confirm the presence of a disorder but also specify the precise disorder in a hyperphenylalaninemic neonate within a few
F. Rajabi, H.L. Levy / Molecular Genetics and Metabolism 114 (2015) 380–381
days after birth, before or at the time the infant is seen for initial clinical visit. A major limitation of this methodology, however, is the need for peripheral blood leukocytes to obtain the required genomic DNA. The authors are currently examining the possibility of obtaining sufficient DNA for the assay using the dried blood NBS specimen [Song F, personal communication]. Indeed, isolation of DNA from the dried blood spot was recently demonstrated to result in sufficient DNA for next generation sequencing (NGS) [9,10]. If the NBS specimen can be utilized and if the turnaround time can be maintained at 3–4 days or even less, NGS technology such as the Ion Torrent PGM system could be added as one of the second-tier screens in the NBS laboratory and cover almost all disorders responsible for increased phenylalanine in the neonate. Why is this of potential importance? HPA in a neonate is not a disorder but an indication of any one of several conditions. The most frequent of these will be PAH deficiency, i.e., phenylketonuria (PKU). However, even PAH deficiency is not a single clinical entity. There are at least three forms, characterized by the degree of PAH deficiency: classic PKU, moderate or mild PKU, and non-PKU mild HPA (MHP). Each of these has important implications for outcome and treatment. Classic PKU is the most severe with the possibility of long term problems and the need for the most stringent dietary treatment; moderate or mild PKU may have a better outcome and require a less stringent diet; and MHP is probably benign, which is likely to have normal outcome without treatment. HPA could also represent BH4 deficiency with its several enzymatic causes, any one of which, with profound implications for outcome and requiring a very different treatment than PKU [6]. Neonatal HPA may also be transient and not indicative of a metabolic disorder. This is most likely in a premature infant receiving total parenteral nutrition (TPN) but also may be seen in infants with liver disease or congenital heart disease [11]. It is important to sort through the possibilities and identify the neonate with a specific genetic cause for HPA or rule it out, in which case it is likely that the increased phenylalanine will be transient. Identification of the genetic etiology for the HPA upon presentation of the infant to a clinical program can allow tailored genotype specific information for the family and for management options. The immediate confirmation of results can also reduce false positives and parental anxiety [12]. The turnaround time for current commercial genetic testing may take weeks or months before the result is known. Ideally, second-tier genetic panel testing could be done reflexively and quickly after screen positive HPA results using the same dried blood spot. Perhaps all second-tier molecular testing in NBS will one day be replaced by NGS as a primary screen [4,13]. The efficiency and the reliability of high quality NGS are rapidly improving, possibly reducing or eliminating the need for time consuming Sanger confirmation of variants in clinical testing [14]. Once the sensitivity and the specificity of NGS can be reliably established, extending this technology to NBS offers an appealing and exciting potential advance in NBS disease detection, allowing NBS to cover not only a limited number of inborn errors of metabolism but also many other genetic disorders. Thus adapting genetic sequencing as part of NBS has broad appeal. Nevertheless, there are
381
major concerns about readiness for implementation. The greatest current limitations include turnaround time for validated results, reliable use of the dried blood spot, and cost [10]. In addition there are major ethical, societal, and even legal concerns [4,13]. Improving the speed of the test must not be done at the expense of accurate mutation detection. Additionally, molecular genetic testing cannot replace biochemical testing without reducing the sensitivity of screening since not all individuals affected by disease will have an identifiable genetic etiology. Further work is required to establish that sequencing technologies can be sufficiently reliable and sensitive for rapid genetic diagnosis. Stay tuned! Conflict of interest The authors declare no conflict of interest.
References [1] M. Jackson (Ed.), The Oxford Handbook of the History of Medicine, Oxford University Press, Oxford, UK, 2012. [2] J.M. Fenster, Ether Day: The Strange Tale of America's Greatest Medical Discovery and the Haunted Men Who Made It, HarperCollins, New York, NY, 2001. [3] F.S. Collins, The Language of Life, HarperCollins, New York, NY, 2010. [4] Y.E. Landau, U. Lichter-Konecki, H.L. Levy, Genomics in newborn screening, J. Pediatr. 164 (2014) 14–19, http://dx.doi.org/10.1016/j.jpeds.2013.07.028. [5] Y. Cao, Y. Qu, F. Song, T. Zhang, J. Bai, Y. Jin, H. Wang, Fast clinical molecular diagnosis of hyperphenylalaninemia using next-generation sequencing-based on a custom AmpliSeq™ panel and Ion Torrent PGM sequencing, Mol. Genet. Metab. 113 (2014) 261–266. [6] N. Blau, F.J. van Spronsen, H.L. Levy, Phenylketonuria, Lancet 376 (2010) 1417–1427, http://dx.doi.org/10.1016/S0140-6736(10)60961-0. [7] Y. Gu, K. Lu, G. Yang, Z. Cen, L. Yu, L. Lin, et al., Mutation spectrum of six genes in Chinese phenylketonuria patients obtained through next-generation sequencing, PLoS One 9 (2014) e94100, http://dx.doi.org/10.1371/journal.pone.0094100. [8] D. Trujillano, B. Perez, J. González, C. Tornador, R. Navarrete, G. Escaramis, et al., Accurate molecular diagnosis of phenylketonuria and tetrahydrobiopterindeficient hyperphenylalaninemias using high-throughput targeted sequencing, Eur. J. Hum. Genet. 22 (2014) 528–534, http://dx.doi.org/10.1038/ejhg.2013.175. [9] T.L. Klassen, J. Drabek, T. Tomson, O. Sveinsson, U. von Döbeln, J.L. Noebels, et al., Visual automated fluorescence electrophoresis provides simultaneous quality, quantity, and molecular weight spectra for genomic DNA from archived neonatal blood spots, J. Mol. Diagn. 15 (2013) 283–290, http://dx.doi.org/10.1016/j.jmoldx.2013.01.003. [10] A. Bhattacharjee, T. Sokolsky, S.K. Wyman, M.G. Reese, E. Puffenberger, K. Strauss, et al., Development of DNA confirmatory and high-risk diagnostic testing for newborns using targeted next-generation DNA sequencing, Genet. Med. (2014), http://dx.doi.org/10.1038/gim.2014.117. [11] S.J. Evans, T.C. Wynne-Williams, C.A. Russell, A. Fairbrother, Hyperphenylalaninaemia in parenterally fed newborn babies, Lancet 2 (1986) 1404–1405. [12] S.E. Waisbren, S. Albers, S. Amato, M. Ampola, T.G. Brewster, L. Demmer, et al., Effect of expanded newborn screening for biochemical genetic disorders on child outcomes and parental stress, JAMA 290 (2003) 2564–2572, http://dx.doi.org/ 10.1001/jama.290.19.2564. [13] H.L. Levy, Newborn screening: the genomic challenge, Mol. Genet. Genomic Med. 2 (2014) 81–84, http://dx.doi.org/10.1002/mgg3.74. [14] S.P. Strom, H. Lee, K. Das, E. Vilain, S.F. Nelson, W.W. Grody, et al., Assessing the necessity of confirmatory testing for exome-sequencing results in a clinical molecular diagnostic laboratory, Genet. Med. 16 (2014) 510–515, http://dx.doi.org/10.1038/ gim.2013.183.
