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J Lab Med 2010;34(6) 2010 by Walter de Gruyter • Berlin • New York. DOI 10.1515/JLM.2010.056et Molecular genetic and cytogenetic diagnostics Next generation sequencing in genetic diagnostics 1)
Saskia Biskup *
“ variation ” , in order to do better justice to the different types of variations. There are pathogenic variants that are certain to Praxis f ü r Humangenetik und CeGaT GmbH, T ü bingen, Germany cause disease, but there also are variants whose signifi cance is not explicitly clear. These include the “ probably pathogen-ic ” , the “ probably non-pathogenic ” , the “ probably benign ” , Abstract
the “ certainly benign ” and fi nally an ever increasing group of variants of uncertain signifi cance (VUS). The introduction of next-generation sequencing technolo- The fi rst large catalog of genes that, when changed, are gies in human genetic diagnostics is a challenge to many of associated with a disease was compiled into a database its aspects. It is mainly positive, even revolutionary, which named OMIM (Online Inheritance In Man) by the American will be discussed as well as its critical aspects. It used to take human geneticist Viktor McKusick (1921 – 2008). As a con- several months or years to complete genetic testing. This al- sequence, he is seen as the founder of medical genetics. The ready belongs to the past. As sequencing technologies are data base grows larger every day and is administered by the progressing at an extremely high speed, Germany recently Johns Hopkins University in Baltimore, USA. So far varia- introduced the new Gen Diagnostics Law in February 2010. tions in 10 % of all human genes could be associated with Four common diseases with genetic contribution (demen- known phenotypes. That is a comparatively large number tia, Parkinson’s syndrome, epilepsy, and hereditary eye dis- and yet does not explain the majority of genetic variability eases) will be used to exemplify the latest development of Genetic diagnostics is useful if it brings about consequenc- es from a therapeutic or prophylactic perspective or eases the Keywords: diagnostic panels; next-generation sequencing;
burden on the individual concerned and the family. The right not to know exists at any time and this should be emphasized during genetic counseling before as well as after testing. What follows are examples from the day-to-day practice that illus- Introduction
trate why human genetics rightly sees technological progress Genetic or molecular genetic diagnostics refers to the study as a quantum leap for its own discipline, for the patient and of individual genes that, when changed, most probably lead to the manifestation of a disease or are the cause of a disease. A genetic cause must be considered especially when the af- From exomes to diagnostic panels in human
fl icted persons are of young age. The sum total of hereditary genetics
material in humans is stated to be approximately 3 billion DNA base pairs and codes for 20 to 30,000 genes. A change By means of the sequencing machines available today, high- in the hereditary material may be transmitted from generation throughput sequencing permits the simultaneous sequencing to generation or it may arise “ de novo ” . A distinction is made of 100 billion basepairs within one week, i.e., the haploid between changes in germ cells, which are hereditary, and so- human genome with an average coverage of 30. Because matic changes, e.g., tumor cells. In this paper, the term “ mu- of higher accuracy and higher coverage and for reasons of tation ” (change in the hereditary material) is replaced with better understanding of variation it has advantages to focus on the coding regions of the human genome. This is called exome-sequencing; the exome is the totality of all coding 1)Original German online version at: http://www.reference-global.
regions in the genome. Sequencing an exome is useful if (i) there are strong indications for a genetic disease, (ii) all The German article was translated by Compuscript Ltd. and autho- known genes associated with the disease at issue have been ruled out and (iii) this approach is applied and evaluated by *Correspondence: Dr. med. Dr. rer. nat. Saskia Biskup, Praxis experts in the particular fi eld within the scope of a research f ü r Humangenetik und CeGaT GmbH, Paul-Ehrlich-Str. 17, In contrast, a diagnostic panel is the targeted simultaneous Tel.: + 49 7071-5654400Fax: + 49 7071-5654422 screening of a list of known genes that have already been des- E-Mail : saskia.biskup@humangenetik-tuebingen.de cribed as the cause of a specifi c disease. Up to now genetic 2010/561
diagnostics in such cases was not only very time-consuming, of sight as a rule progresses slowly and can take decades. The but was frequently not performed because of high costs. loss of sight may occur in isolation or in connection with other A diagnostic panel clearly differs from the purely scientifi c symptoms. There is no treatment in most cases. As of now a and explorative approach of exome-sequencing. A diagnostic total of more than 180 genes have been described that, when panel is ordered by the physician. Only the genes whose con- defective, can cause hereditary ophthalmic disease [ 1 ]. The nection with the disease has been established are investigated. description of this list of genes, which has grown so markedly Finally, a fi nding is made and forwarded to the physician. This in just the last few years, has provided the fi rst substantial fi nding contains detailed information about detected variants clues for understanding the pathogenesis of the disease and that are validated through Sanger sequencing and interpreted thus has contributed considerably to understanding the dis- ease itself. The gene or its product is not directly suffi cient for Using a diagnostic panel on a newest generation high- developing any new medication, but it is the central starting throughput sequencer makes sense only if several or very large genes can be considered for the disease. Some examples At present approx. 50 % of all familial ophthalmic dis- are hereditary tumor diseases with the known breast cancer- eases are clarifi ed genetically. Diagnosing frequently takes causing genes BRCA1 and BRCA2 , cardiomyopathies, car- several years and, due to the investigation of many large diac dysrhythmias, familial hypercholesterolemias, mental genes, is very expensive. Such considerations as well as the retardation, epilepsies, hereditary ophthalmic diseases and need to clarify the other 50 % have resulted in the transfer neurodegenerative diseases. Collaboration with clinical ex- of hereditary ophthalmic disease diagnostics to the high- perts is indispensable when compiling the gene list of a panel throughput sequencing arena. The Retina-All-Panel was and also later when interpreting the data. Since gene lists can developed in collaboration with the ophthalmic geneticists be from two to more than one hundred genes in length, ad- Prof. Wolfgang Berger and Dr. John Neidhardt (University vance clinical delimitation is important. After the patient has of Z ü rich, Institute for Medical Genetics) and Prof. Bernd given his written consent his DNA is enriched and sequenced Wissinger and Dr. Susanne Kohl (University of T ü bingen, with all genes contained in the gene list. The clinical expres- Molecular Genetic Laboratory of the Eye Clinic). It contains sion of the disease then determines the sequence of the genes all presently known genes with a connection to hereditary ophthalmic disease. Sequencing this large panel of genes Before a diagnostic panel can be offered commercially takes about one to two weeks. Detected variants are issued for diagnostic purposes it must fi rst be validated. Within in a list, classifi ed according to their importance (pathogen- the framework of such validation patients whose variations ic, benign or VUS) and then verifi ed via the Sanger method. are already known are “ post ” -sequenced on the new panel. Finally, the results are evaluated, summarized and interpret- This requires that 100 % of the known variations are found ed. Altogether the diagnosis at this time requires no more by means of the panel. Furthermore, the enrichment of the genomic regions must be effi cient, specifi c and reproduc- A simultaneous study of more than 180 genes does not ible. Only then can a diagnostic panel be used for a clinical make sense from a clinical point of view. The list of genes can be divided into smaller groups for the ophthalmic ge- But is it then actually useful? We shall closer examine neticist and clinician. This allows for the simultaneous study this question with the help of three examples. Detlef Boehm of 26 genes in a case of autosomal dominant pigmentary (CeGaT GmbH), a pioneer in establishing new methods in retinopathy, of 28 genes in the case of autosomal recessive human genetic diagnostics [ 2 ] and the Practice for Human pigmentary retinopathy, of 10 genes in the case of Usher ’ s bingen together with clinical partners and syndrome, 11 genes in the case of congenital stationary with Applied Biosystems/Life Technologies have put special blindness, 14, 9 or 5 genes, respectively in cases of Bardet- emphasis on hereditary ophthalmic diseases, epilepsies and Biedl, Joubert or Refsum syndrome. A current and compre- neurodegenerative diseases when developing diagnostic pan- hensive article on this subject provides more information on els. The aim here is to clarify the genetic cause in affected the subdivision of ophthalmic diseases and a description of families and thereby to (i) secure a clinical diagnosis, (ii) be able to offer a targeted examination of other family mem- Research in the fi eld of hereditary ophthalmic diseases bers, (iii) make possible an early therapeutic intervention, also gains substantially from the Retina-All-Panel. With (iv) provide a prognostic assessment of the course of the dis- the use of the Retina-All-Panel many more patients carry- ease and (v) provide the basis for new therapeutic methods ing a pathogenic variant in one of the candidate genes are being identifi ed. Hence, the pool of individuals affected by a certain variant is growing worldwide and the clinical obser-vation of the course of the disease in these patients allows Diagnostic panel and hereditary ophthalmic
better classifi cation and prognosis assessment in additional diseases
patients with the same variant. In the future, it would also be useful from a therapeutic perspective to group patients based Worldwide hereditary ophthalmic diseases affect several mil- on their genetic background when testing new medications, lion people. The disease usually starts during adolescence and in order to better interpret positive effects as well as side initially often appears as night blindness. Thereafter the loss Next generation sequencing also has an additional scien- Diagnostics panel and Parkinson ’ s disease and
tifi c aspect. From a purely technological standpoint it makes dementia
no difference whether two, one hundred or several thousand patient genes are enriched and sequenced. With a clear diag- Parkinson ’ s disease together with Alzheimer ’ s disease is one nostic objective this would be senseless, since data are gener- of the most frequently occurring neurodegenerative diseases ated whose required evaluation and validation is diffi cult and worldwide. Both diseases most often occur sporadically and very time-consuming. The situation is different with a family as a rule manifest themselves in individuals above 65 years where no genetic cause can be found in the known genes. This of age. With a steadily rising life expectancy Parkinson ’ s and presents a possibility that genes as yet not associated with the Alzheimer ’ s represent one of the greatest medical and socio- disease could be studied for the fi rst time and named as the economic challenges of the future. In most cases the cause of new cause for the disease. In the case of ophthalmic diseases the death of nerve cells remains a mystery. At the present time candidate genes are genes that have an important function in it is impossible to predict whether and when an individual the eye but that have not yet been studied in the patients. They will be affected by the breakdown of nerve cells. Once symp- are candidates for the cause of ophthalmic disease. This is toms occur, however, the majority of affected nerve cells have where the enormous potential of next generation sequencing, already died. Hence, current treatment concepts have little or that would further advance the knowledge about the causes of no effect, since the time of intervention is years too late. A hereditary ophthalmic diseases, is to be found. molecular genetic examination does not immediately offer itself. Why would an individual want to know whether he or she has a predisposition for a neurodegenerative disease as Diagnostic panel and epilepsy
long as no therapies are available? Genetic causes for both Parkinson ’ s and Alzheimer ’ s have been described for slightly Epilepsies affect 1 % to 3 % of the population in the course of more than 10 years. These genetic studies have made impor- a lifetime. The various expressions of the disease are differ- tant contributions to understanding the breakdown of nerve entiated depending on age and the form of progression. A cells. Intensive research is being done on the gene products genetic cause is probable if a symptomatic cause from brain in order to speed up new and innovative therapy concepts. damage, from a tumor, an infection or a metabolic disturbance The identifi cation of mutated genes has for the fi rst time made can be ruled out. Large families with frequently occurring ep- it possible to describe biomarkers, in this case “ genetic bio- ileptic disease have contributed to the identifi cation of genes markers ” , that can predict the occurrence of the disease at a and crucially also to the clarifi cation of the pathogenesis of future point in time with a high degree of probability. This the disease. Identifi ed genes include above all voltage-de- allows us to defi ne a group of individuals who could get ac- pendent ion channels and the receptors of neurotransmitters. cess to therapies decades before any manifestation of disease. Conceptually a change in the neural transmission of nerve Even though at present such therapies are not yet available, cell to nerve cell, caused by defective ion channels or neu- it seems safe to say that the changed gene products will most rotransmitter receptors, fi ts the cause of a convulsive disorder. probably represent the points of action for the therapies of Specifi c therapies, e.g., the targeted attack of a medication on a defective sodium channel (valproic acid, carbamaze- The key to the changed genes were families in which de- pine, oxcarbazepine and phenytoin) or on a defective receptor mentia or Parkinson ’ s disease occurred with great frequency. (GABA receptor, phenobarbital), provide effi cient treatment In the case of Parkinson ’ s disease 16 gene locations for fa- of a patient. The main goal therefore is the molecular ge- milial autosomal recessive and dominant forms have so far netic discovery of the cause in as many cases as possible, in been described in the hereditary [ 3 , 4 ]. Since only a small order to create a specifi c individual treatment of the disease. part of approx. 5 % of familial cases can currently be geneti- Numerous genes that have been described as the cause for the cally clarifi ed, the expectation is that the list of genes causing various forms of familial epilepsy also are possible candidate Parkinson ’ s disease will grow. The greater part of the knowl- genes for non-familial cases. Since ion channel genes in par- edge we possess today about the pathogenesis of Parkinson ’ s ticular represent especially large genes, the development of a disease derives from those genes that have been described in diagnostics panel for high throughput screening seemed the connection with the disease. The gene therefore is the fi rst obvious choice. An epilepsy panel for clinical use was devel- clue concerning the location of the malfunction within the oped by Dr. Johannes Lemke, University of Bern together with diseased cell. Like pieces of a puzzle other genes will add to the groups around Prof. Holger Lerche and Prof. Ingeborg the complex picture of neurodegenerative diseases and – it is Kr ä geloh-Mann (Neurologic and Pediatric University Clinic, hoped – allow a further crucial step in our understanding of T ü bingen) in collaboration with CeGaT GmbH. At present the list of genes for purely diagnostic objectives consists of 55 genes and has been clinically subdivided into generalized/ genes in the case of Parkinson ’ s disease, 19 genes in the myoclonic epilepsies including febrile seizures and absences case of dementia. The panel was created together with Prof. (a total of 24 genes), epileptic encephalopathies (a total of 8 Thomas Gasser (Neurology and Hertie-Institute for Clinical genes) and syndromal diseases with epilepsy (a total of 23 Brain Research, University of T ü bingen). The list of genes genes). The panel contains another 450 candidate genes that will grow rapidly, not least due to the possibility of examin- are being studied for research purposes. ing families affected by the disease for changes in the total genome through a method free of hypotheses and within the caused diseases, but also will establish genetic diagnostics as a quick, effi cient, cost-effective and useful method in the As with a purely diagnostic objective and within the frame- minds of the individuals seeking counseling, of affected indi- work of the genetic diagnostics law the individual seeking viduals, of physicians and scientists. In this regard the diag- advice should also be informed concerning the research ob- nostic panels are a step in the right direction. jective. Most particularly this includes information on the possible handling of incidental fi ndings with relevance for all family members, the possible destruction of the probe after Method/Technology
the examination is completed, the anonymization and use of the probe for further studies, and the right not to know at any Next-generation sequencing stands for high-throughput se- quencing and allows sequencing of up to 100 billion bases within days. A complete human genome can be sequenced with an average coverage of 30 (i.e., each base is read 30 Outlook and open questions
times). Various technology platforms for high-throughput sequencing are commercially available; the main suppli- From the perspective of human genetics the quick and cost ers include Roche, Illumina and Applied Biosystems/Life effi cient sequencing of several thousand human genes within Technologies. While the Roche platform achieves the com- a few days is revolutionary. Also revolutionary is the prospect paratively lowest throughput of 400 million bases per run, of personalized medicine in which each single human and it offers advantages in regard to the length of the sequenced each single tumor can be sequenced. It is hoped that with this fragments (approx. 400 bases). Illumina and Applied knowledge diseases might someday be treated individually, Biosystems/Life Technologies with up to 100 billion bases i.e., much more targeted than today. Each human is unique, achieve far higher throughputs, but with a shorter read length each tumor is unique, each disease with its individual genetic (50 to 100 base pairs). Since all three platforms are based background is unique. Medications therefore have different on different sequencing strategies, which we cannot go into effects in different people. A changed gene is an essential key here, each of them is also suited for different objectives. for understanding a disease. Even if individualized therapy, e.g., with neurodegenerative diseases, lies in the distant fu- Biosystems/Life Technologies on its SOLiD platform deli- ture, the foundation for the therapies of the future is being laid vers the highest throughput with by far the lowest error rate. now. For some time tumor genetics has made targeted therapy This is of great importance for diagnostic applications, since partially possible, other diseases have followed and more will all variants found in high-throughput sequencing are verifi ed follow. With all the euphoria that genetics has been experi- conventionally, i.e., via the Sanger method, and the lower encing for some time it must be remembered that the results the initial error rate the more feasible and safe it will be to of high-throughput sequencing bring up questions that cannot introduce high-throughput sequencing into the human gene- be answered at this time. This includes the identifi cation of as yet unknown variants in the genome of individuals, variants The process of high-throughput sequencing is as follows. A of uncertain signifi cance (VUS). Add to this that, in spite of patient ’ s genomic DNA is obtained from blood or tissue, frag- high-throughput sequencing, in many cases the cause of a dis- mented randomly by sonication and subsequently equipped ease cannot be found, be it that no genetic cause exists or that with adaptors. The fragments to be sequenced are fi shed out the cause lies in the non-examined regions of the genome, with complementary RNA baits that are linked to magnetic or that it is the synergy of several changed genes with the beads (SureSelect Method by Agilent). The enriched DNA is environment that results in the disease, or that changed gene fi nally amplifi ed. This is called targeted enrichment. Here as products (RNA or proteins) are the actual cause of the disease. well different technologies are available in the marketplace but cannot be discussed in any detail in this paper. The meth- screening method. Hereditary material is illuminated and it od used to perform a large number of amplifi cations in paral- can happen that variations are found that are characterized lel is called EmulsionPCR. This EmulsionPCR is a special as incidental fi ndings. Dealing with incidental fi ndings, par- kind of PCR that takes place on beads in an aqueous drop- ticularly if they have serious consequences for the patient, is lets separated by an oil phase. These beads, equipped with a considerable challenge for the physician and the individual several ten thousands of universal PCR-primer molecules, seeking counseling. As with the fi ndings of variants of un- are magnetic and can later on separated by magnetic forces. certain signifi cance the patient must be informed about this The enriched DNA is bound via its attached adaptors to the issue in advance. The result of a genetic examination should primers on the beads. One DNA fragment is then multiplied be conveyed within the framework of a consultation. The law by means of PCR in one bead per aqueous droplet at a time. on gene diagnostics, in force since February 2010, established The newly formed fragments bind to additional primers on guidelines for the performance of genetic diagnostics that can the same bead. Following breaking the emulsion, those beads, also be directly applied to high-throughput diagnostics. on which a PCR has successfully occurred, are cross-linked It is hoped that high-throughput diagnostics will arrive in by polystyrene beads and separated through centrifugation many laboratories and as a consequence will not only con- based on their size and weight of the cross-linked beads when tribute to markedly higher clarifi cation quotas of genetically compared to those to which nothing has bound. Then about 700 million beads are deposited on a slide, which in turn is sequence coded by the color. The automatic comparison with clamped into the high-throughput sequencer. the reference sequence converted into color space, in this Sequencing occurs through detection of light signals that case the human genome, takes place in Step 2. The individ- are given off by hybridizing octamers (8 nucleotides with ual 50 color space base pair long fragments are compared to one specifi c fl uorescence signals). The octamers contain two the reference sequence and the color spaces are displayed by nucleotides that code a color space and gives off one of four side-by-side (alignment). This two base pair encoding of the fl uorescent signals immediately after binding, which are pho- SOLiD system allows the quick and easy recognition of mis- tographed by a digital camera with a CCD sensor. The color calls and the clear differentiation between miscalls and actual signals are then separated and a further binding cycle can be sequence variations. All true deviations from the reference initiated until approx. 50 nucleotides can be converted in a sequence are transmitted and verifi ed via Sanger sequencing. series of color signals, whereby one color always corresponds The last and most diffi cult step involves the interpretation of to two nucleotides. The staggered multi-step read of the origi- nal sequence ensures that each base is read at least twice and variations can be identifi ed reliably. This results in the special reliability of the SOLiD system by Applied Biosystems/Life References
Technologies in regard to the interpretation of the data. At the end of the analysis the color signals are converted back into 1. Berger W, Kloeckener-Gruissem B, Neidhardt J. The molecular the nucleotide sequence that then enters into the continuing basis of human retinal and vitreoretinal diseases. Prog Retin Eye analysis. Five to seven days are required for the sequencing step. The subsequent data analysis consists of the following 2. Boehm, D, Herold S, Kuechler A, Liehr T, Laccone F. Rapid de- tection of subtelomeric deletion/duplication by novel real-time three individual steps: Step 1: Initially the data generated by quantitative PCR using SYBR-green dye. Hum Mutat 2004;23: the sequencer arrive in the form of pictures consisting of in- dividual color dots, whereby one particular color dot corre- Biskup S, Gerlach M, Kupsch A, Reichmann H, Riederer P, sponds to two nucleotides. The color dots are examined by Vieregge P, et al. Genes associated with Parkinson syndrome. J the software for their intensity and quality, both necessary for Neurol 2008;255(Suppl 5):8 – 17. Review. defi nitely determining the nucleotides. A software supplied by 4. Gasser T. Mendelian forms of Parkinson ’ s disease [review]. Bio- the manufacturer converts the color dots into the color space

Source: http://www.cegat.de/files/CeGaT_JLM_2010e.pdf

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