00019606-201109000-00007ArticleDiagnostic Molecular PathologyDiagnostic Molecular Pathology© 2011 Lippincott Williams & Wilkins, Inc.20September 2011
p 175–179High-throughput Genotyping Robot-assisted Method for
Mutation Detection in Patients With Hypertrophic CardiomyopathyOriginal ArticlesBortot, Barbara PhD*; Athanasakis, Emmanouil MSc†; Brun, Francesca MD‡; Rizzotti, Diego BSc*; Mestroni, Luisa MD§,∥; Sinagra, Gianfranco MD‡; Severini, Giovanni Maria PhD**Department of Molecular Medicine and Laboratory†Medical Genetics Laboratory, Institute of Maternal and Child Health, IRCCS Burlo Garofolo‡Cardiovascular Department, Ospedali Riuniti and University of Trieste, Trieste, Italy§University of Colorado Cardiovascular Institute∥Adult Medical Genetics Program, University of Colorado Denver, COSupported by grants from Assicurazioni Generali S.p.A., the American Heart Association (0150453N and 0250271N), and the Muscular Dystrophy Association (PN0007-056 to LM.)The authors declare no conflict of interest.Reprints: Giovanni Maria Severini, PhD, Department of Molecular Medicine and Laboratory, Institute of Maternal and Child Health IRCCS Burlo Garofolo, Via dell'Istria 65/1, 34137 Trieste, Italy (e-mail:
[email protected]).AbstractHypertrophic cardiomyopathy (HCM) is the most frequent autosomal dominant genetic heart muscle disease and the most common cause of sudden cardiac death in young people (under 30 y of age), who are often unaware of their underlying condition. Genetic screening is now considered a fundamental tool for clinical management of HCM families. However, the high genetic heterogeneity of HCM makes genetic screening very expensive. Here, we propose a new high-throughput genotyping method based on a HCM 96-well sequencing plate for the analysis of 8 of the most frequent HCM-causing sarcomeric genes by automating several processes required for direct sequencing, using a commercially available robotic systems and routinely used instruments. To assess the efficiency of the robot-assisted method, we have analyzed the entire coding sequence and flanking intronic sequences of the 8 sarcomeric genes in samples from 18 patients affected by HCM and their relatives, which revealed 9 different mutations, 3 of which were novel. The automated, robot-assisted assembling of polymerase chain reaction, purification of polymerase chain reaction products, and assembly of sequencing reactions resulted in a substantial saving of time, reagent costs, and reduction of human errors, and can therefore be proposed as a primary strategy for
mutation identification in HCM genetic screening in many medical genetic laboratories.Hypertrophic cardiomyopathy (HCM) is a complex and relatively common form (1:500 of the general population) of autosomal dominant genetic heart muscle disease typically diagnosed by an echocardiogram that produces ultrasound images of the thickened wall of the heart muscle (hypertrophy of the left ventricle). The clinical spectrum of HCM is various, ranging from asymptomatic individuals to those with disabling symptoms of heart failure, exercise intolerance, arrhythmias, and chest pain.1 Moreover, HCM is the most common cause of sudden cardiac death (SCD) in young people (under 30 y of age), including trained athletes, who are often unaware of their underlying condition. Early diagnosis of HCM is important, as at-risk individuals may be advised not to participate in competitive sports and should undergo regular cardiac screening to assess the risk ofSCD.2,3 Moreover, although HCM is a chronic disease without a known cure, a number of treatments are now available to alter its course.HCM is caused by a variety of mutations localized mainly in 10 genes encoding contractile proteins of the cardiac sarcomere,4–7 but mutations in 3 of these genes (MYH7, MYBPC3, and TNNT2) are estimated to account for approximately 60% of all familial cases of HCM (Table 1). Among the approximately 900 individual mutations identified until now (Human Gene
Mutation Database, http://www.hgmd.cf.ac.uk/), very few are recurring mutations in a small number of families, and therefore most of them are “private” mutations with very low frequency.7 In conjunction with the genetic heterogeneity of HCM, phenotypic expression of HCM also exhibits a high level of variability. Members of a single family, who share the same causal
mutation, exhibit considerable differences in the severity of cardiac hypertrophy or the risk of SCD. Variability in the phenotypic expression of HCM is due to multiple factors, including heterogeneity of the causal genes and mutations, activity of modifier genes, presence of multiple functional variants in the sarcomeric proteins, posttranslation modifications of the proteins, and, probably, environmental factors.6 However, genetic testing in HCM has a shown value in establishing a correct diagnosis, and such testing has also helped disclose genotype-phenotype relationships, which have in turn been used to refine the diagnostic algorithm.JOURNAL/dimp/04.03/00019606-201109000-00007/table1-7/v/2021-02-17T200035Z/r/image-tiffGene Symbol, Proteins, Reference Sequences, Loci, and Frequency of
Mutation Detection of the 8 Sarcomeric Genes Analyzed with the HCM 96-well Sequencing PlateHere, we propose a new high-throughput genotyping robot-assisted method based on a HCM 96-well sequencing plate (Fig. 1), for the analysis of 8 of the most frequent HCM-causing genes [myosin-binding protein C (MYBPC3), β-myosin heavy chain (MYH7), regulatory and essential light chains of myosin (MYL2 and MYL3, respectively), cardiac troponin T and I (TNNT2 and TNNI3, respectively), α-tropomyosin (TPM1), cardiac actin (ACTC)] (Fig. 2, Table 1),8 and we assess its efficiency by analyzing DNA obtained from 18 patients affected by HCM and their relatives.JOURNAL/dimp/04.03/00019606-201109000-00007/figure1-7/v/2021-02-17T200035Z/r/image-jpegSchematic representation of the robot-assisted sequencing methods, including PCR assembling, sequencing reactions, and purification steps. PCR indicates polymerase chain reaction.JOURNAL/dimp/04.03/00019606-201109000-00007/figure2-7/v/2021-02-17T200035Z/r/image-jpegSarcomeric genes involved in HCM. Underlined proteins have been included in the HCM 96-well sequencing plate method. (Adapted with permission from N Engl J Med. 1997;336:775–785). HCM indicates hypertrophic cardiomyopathy.MATERIALS AND METHODSA total of 18 unrelated, diagnosed HCM index patients referred to the Cardiovascular Department, the University and Hospital of Trieste, Italy, were included in genetic screening. After informed consent was obtained, index cases were clinically evaluated including physical examination, echocardiogram, and family history. All 18 index patients fulfilled the classical diagnostic criteria for HCM.2 Whenever possible, first-degree and second-degree relatives were subsequently identified and included in the study, yielding a total of 34 index patients and relatives. Relatives were clinically assessed, similar to probands, before genetic testing.Isolation of DNAGenomic DNA was extracted from peripheral blood leukocytes by the EZ1 DNA Blood 350 μL kit (Qiagen, Hilden, Germany) using the EZ1-Biorobot Extraction Workstation (Qiagen, Hilden, Germany). The quantity and quality of the DNA were determined using the nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Inc., Wilmington, DE).Primer DesignTo adapt the gene-sequencing strategy to the analysis of single samples, a rapid protocol was developed, allowing polymerase chain reaction (PCR) amplification and sequencing reactions to be carried out under identical conditions for all reactions in a 96-well reaction plate for each sample. All coding regions and exon-intron boundary regions were amplified using 95 primer pairs achieved by the Primer3 on-line software (BROAD Institute, Cambridge, UK; http://frodo.wi.mit.edu/primer3/). The primer design was made to a melting temperature of 58°C to 62°C and 40% to 60% of GC. Primer sequences are available on request. To obtain a specific HCM-sequencing 96-well plate, for genes with multiple isoforms, primers were designed only for those cardiacs. The primers were purchased from Primm (Milan, Italy).Touchdown PCRThe initial sequencing strategy involved an automated set-up, using a Biomek NX MC liquid-handling platform (Beckman Coulter, Fullerton, CA). The robot was programmed to aspirate, dispense, mix, and apply reagents according to the manufacturer's instructions for a 96-well plate. All PCR primers were distributed in two 96-well plates, with 2 μM of each primer, one for the forward primers and the other for the reverse. For the automated protocol, PCR was carried out in 10 μL of total reaction volume. Each reaction contained 25 ng of genomic DNA and Kapa 2G Fast Hot Start ReadyMix 2X (KapaBiosystems, Cape Town, South Africa). PCR amplification was carried out using a common annealing temperature in a touchdown thermocycler protocol shared in a 2-cycle step: primary, initial denaturation step at 95°C for 3 minutes with next touchdown step planning to decrease the temperature by 0.5°C/cycle, through 9 cycles: 95°C for 15 seconds (denaturation), 62°C for 15 seconds (annealing), and 72°C for 1 second (elongation); secondary, classic PCR step through 26 cycles: 95°C for 10 seconds (denaturation), 58°C for 10 seconds (annealing), and 72°C for 1 second (elongation). The PCR products were purified with 2 μL of ExoSAP (USB Corporation, Cleveland, OH) by incubation at 37°C for 15 minutes and 85°C for 10 minutes. All thermal cycle steps, PCR amplifications, ExoSAP purifications, and sequencing reactions were carried out on a GeneAmp PCR System 9700 Thermocycler (Applied Biosystems, Foster City, CA).SequencingThe PCR and sequencing steps were carried out at 2 different moments to avoid cross-contamination. The 96-well sample plate volume was divided into 2 equal parts and these were transferred directly to another 96-well plate that contained PCR primers at 2 μM and 0.4 μL BigDye v3.1 Terminator Cycle Sequencing mixture (Applied Biosystems, Foster City, CA), for a total volume of 10 μL. The thermal cycle protocol consisted of an initial denaturation step at 96°C for 3 minutes, and then 25 cycle compounds by a step at 96°C for 10 seconds (denaturation), 55°C for 6 seconds (annealing), and 60°C for 90 seconds (elongation). After completion of the thermal cycling, the 96-well sequence products were purified with 48 μL of BigDye XTerminator Purification Kit (Applied Biosystems, Foster City, CA) by vortexing for 15 minutes. Sequence products were sequenced bidirectionally on an ABI PRISM 3130xl sequencer (Applied Biosystems, Foster City, CA), able to read two 96-well plates sequence products in approximately 7 hours. All identified mutations were confirmed by repeated sequencing of both DNA strands of the affected exon on a second PCR product.
Mutation Data HandlingSequencing data collected were imported to the ABI PRISM SeqScape v2.5 software and compared with the published gene sequence database National Center for Biotechnology Information Database (Table 1).Polyphen (Polymorphism Phenotyping), a web tool for the prediction of potential impact of an amino acid substitution on the structure and function of a human protein, using straightforward physical and comparative considerations, was used to test the pathogenicity of all novel missense variants. A sequence variant was considered a disease-causing
mutation according to the following 4 criteria: (1) the nucleotide variation resulted in a missense
mutation, a frameshift, and/or abnormal splicing; (2) the variation affected a conserved amino acid among species; (3) the variation cosegregated with the disease in affected family members; (4) the variation was not identified among 50 ethnically matched control samples. In the absence of available family members for cosegregation studies, disease association was presumed if criteria (1), (2), and (4) were fulfilled.Assessment of EfficiencyTo examine the efficiency of the HCM 96-well sequencing plate, time and cost of reagents required only for automated work (PCR, sequencing reactions and purification steps) were compared with that required for manual processing of the same number of samples. Time required for sequencing, separation, and sequence analysis are not influenced by the use of the robot-assisted protocol.RESULTSWe analyzed the 8 sarcomeric genes (Fig. 2, Table 1) in 18 individuals using the HCM 96-well sequencing plate using robot-assisted protocols (Fig. 1). In terms of efficiency, the time required for genotyping of 2 patients, considering manual work only (PCR, sequencing reactions, and purification steps), was reduced from 12 hours for manual processing (according to the manufacturer's instructions) to 6 hours using the new automated HCM 96-well sequencing plate protocol, thus reducing cost of personnel. Moreover, the reduction of the reaction volumes, owing to the use of the robotic system, provided additional savings of reagent costs.Sequencing of the 8 sarcomeric genes in a cohort of 18 patients showed 9 different mutations, 3 of which were novel (Table 2), with a
mutation detection rate of 50%, comparable with that reported in the literature.6 No mutations were intronic or affected splice-site function. None of the index cases were carriers of the two disease associated mutations.JOURNAL/dimp/04.03/00019606-201109000-00007/table2-7/v/2021-02-17T200035Z/r/image-tiffList of the Mutations Identified in 9 of the 18 Index PatientsThe 3 earlier unreported new mutations have been analyzed, and fulfilled at least 3 of the 4 criteria (1, 3, and 4) reported earlier for the disease-causing definition. None of the 3 new mutations were identified in DNA from 50 unrelated controls. All the mutations are nucleotide substitutions, 7 missense and 2 nonsense mutations (stop codons, in MYBPC3 gene). Of the 9 relatives to index patients in whom a
mutation could be identified, 7 were identified as
mutation carriers.DISCUSSIONHCM is caused by approximately 900 individual mutations localized mainly in 10 genes encoding contractile proteins of the cardiac sarcomere.4–7 Genetic screening is now considered a fundamental tool for clinical management of HCM families. However, the presence of such a large number of mutations requires direct sequencing of a large number of putative causal genes for each individual, and hence makes genetic screening expensive, time-consuming, and often confined to research-oriented laboratories, and therefore it cannot yet have a fundamental role in routine management of patients.Array-Based Resequencing Technology9,10 has been proposed for the analysis of these and other genes related to HCM, but these techniques often require dedicated, expensive instruments not always available in general medical genetic laboratories. Other genetic screening methods, such as single-strand conformational polymorphism11 and denaturing high-performance liquid chromatography,12,13 are currently used for detecting HCM-causing mutations, but their use is often limited to the analysis of the most suitable HCM genes (MHY7 and MYBPC3).Here we have proposed a new high-throughput genotyping method based on a HCM 96-well sequencing plate, for the analysis of 8 of the most frequent HCM-causing genes by automating several processes required for direct sequencing, using a commercially available robotic systems and routinely used instruments. To assess the efficiency of the robot-assisted method, we have analyzed DNA derived from 18 patients affected by HCM and their relatives. For the assessment of sensitivity, we have compared our results with those obtained in other studies reported in the medical literature. Although the clinical significance of the 3 novel mutations detected in this study remains to be confirmed, the
mutation detection rate and distribution of mutations suggest that the sensitivity of the method is satisfactory.The new robot-assisted method also resulted in a substantial saving of time and reagent costs. The procedure, including preparation of PCR, sequencing reactions, purification, and automated sequencing of the 8 sarcomeric genes, was reduced to half of the time using the robotic system compared with the manual protocol, and, in reducing the PCR reaction volume, provided an additional savings in reagent costs. Furthermore, human errors leading to DNA contamination, switching of samples, or inaccurate volumes obtained by manual pipetting may have been eliminated by the use of the robotic system. Therefore, we conclude that the proposed genotyping robot-assisted method can be considered as a primary strategy for
mutation identification for HCM genetic screening in many medical genetic laboratories.REFERENCES1. Maron BJ, Towbin JA, Thiene G, et al. Council on epidemiology and prevention: contemporary definitions and classification of the cardiomyopathies: an American Heart Association Scientific Statement from the Council on Clinical Cardiology, Heart Failure and Transplantation Committee; Quality of Care and Outcomes Research and Functional Genomics and Translational Biology Interdisciplinary Working Groups; and Council on Epidemiology and Prevention Circulation.. 2006;113:1807–1816[Context Link]2. Taylor MR, Carniel E, Mestroni L. Familial hypertrophic cardiomyopathy: clinical features, molecular genetics and molecular genetic testing Expert Rev Mol Diagn.. 2004;4:99–113[Context Link]3. Maron BJ, Doerer JJ, Haas TS, et al. Sudden deaths in young competitive athletes: analysis of 1866 deaths in the United States, 1980–2006 Circulation.. 2009;119:1085–1092[Context Link]4. Sinagra G, Di Lenarda A, Moretti M, et al. The challenge of cardiomyopathies in 2007 J Cardiovasc Med (Hagerstown).. 2008;9:545–554[Context Link]5. Colombo MG, Botto N, Vittorini S, et al. Clinical utility of genetic tests for inherited hypertrophic and dilated cardiomyopathies Cardiovasc Ultrasound.. 2008;6:62[Context Link]6. Marian AJ. Hypertrophic cardiomyopathy: from genetics to treatment Eur J Clin Invest.. 2010;40:360–369[Context Link]7. Wang L, Seidman JG, Seidman CE. Narrative review: harnessing molecular genetics for the diagnosis and management of hypertrophic cardiomyopathy Ann Intern Med.. 2010;152:513–520[Context Link]8. Spirito P, Seidman CE, McKenna WJ, et al. The management of hypertrophic cardiomyopathy N Engl J Med.. 1997;336:775–785[Context Link]9. Waldmuller S, Muller M, Rackebrandt K, et al. Array-based resequencing assay for mutations causing hypertrophic cardiomyopathy Clin Chem.. 2008;54:682–687[Context Link]10. Fokstuen S, Lyle R, Munoz A, et al. A DNA resequencing array for pathogenic
mutation detection in hypertrophic cardiomyopathy Hum Mutat.. 2008;29:879–885[Context Link]11. Rodríguez-García MI, Monserrat L, Ortiz M, et al. Screening mutations in myosin binding protein C3 gene in a cohort of patients with hypertrophic cardiomyopathy BMC Med Genet.. 2010;11:67[Context Link]12. Yu B, Sawyer NA, Caramins M, et al. Denaturing high performance liquid chromatography: high throughput
mutation screening in familial hypertrophic cardiomyopathy and SNP genotyping in motor neurone disease J Clin Pathol.. 2005;58:479–485[Context Link]13. Girolami F, Olivotto I, Passerini I, et al. A molecular screening strategy based on beta-myosin heavy chain, cardiac myosin binding protein C and troponin T genes in Italian patients with hypertrophic cardiomyopathy J Cardiovasc Med (Hagerstown).. 2006;7:601–607[Context Link]hypertrophic cardiomyopathy; genetic screening; robotic; sarcomeric gene; mutationGene Symbol, Proteins, Reference Sequences, Loci, and Frequency of
Mutation Detection of the 8 Sarcomeric Genes Analyzed with the HCM 96-well Sequencing PlateSchematic representation of the robot-assisted sequencing methods, including PCR assembling, sequencing reactions, and purification steps. PCR indicates polymerase chain reaction.Sarcomeric genes involved in HCM. Underlined proteins have been included in the HCM 96-well sequencing plate method. (Adapted with permission from N Engl J Med. 1997;336:775–785). HCM indicates hypertrophic cardiomyopathy.List of the Mutations Identified in 9 of the 18 Index PatientsHigh-throughput Genotyping Robot-assisted Method for
Mutation Detection in Patients With Hypertrophic CardiomyopathyBortot Barbara PhD; Athanasakis, Emmanouil MSc; Brun, Francesca MD; Rizzotti, Diego BSc; Mestroni, Luisa MD; Sinagra, Gianfranco MD; Severini, Giovanni Maria PhDOriginal ArticlesOriginal Articles320p 175-179
