Indian J Pediatr DOI 10.1007/s12098-016-2035-0
EDITORIAL COMMENTARY
Next Generation Sequencing in the Clinic Ratna Dua Puri 1
Received: 11 January 2016 / Accepted: 11 January 2016 # Dr. K C Chaudhuri Foundation 2016
Next generation sequencing (NGS) has heralded a new era in the diagnosis of genetic disorders. Over the years as the cost of sequencing has plummeted, it is increasingly being used in clinical practice. Many studies highlighting the utility of NGS for diagnosis of monogenic genetic disorders including neuromuscular diseases (NMDs) have been published [1, 2]. NMDs include a vast number of phenotypically and genetically heterogeneous conditions, diagnosis of which is a challenge even for an experienced clinician. Congenital muscular dystrophies (CMDs) are a group of distinct NMDs that present with congenital hypotonia, severe muscle weakness and atrophy, contractures and delayed motor milestones. With improved understating of their genetic basis, they are sub classified based on the gene that harbors the causative mutations, these being identified in about 25–50 % cases. A lot still needs to be understood and unraveled about CMDs. Though Sanger sequencing has been available in India since long, genetic heterogeneity and the large size of some genes, involves huge costs of testing. This is a major limitation in a country where most health expenditure is “out-of pocket”. This greatly limits the uptake of genetic testing. However, making a genetic diagnosis is important for ending the diagnostic odyssey for patients and their families, allowing the family to comprehend why it happened, providing prognostic information, permitting genetic counseling and prenatal diagnosis, and at times leading to
* Ratna Dua Puri [email protected]
1
Center of Medical Genetics, Sir Ganga Ram Hospital, New Delhi 110060, India
treatment and search for new research initiatives in disease management [3]. Laminin alpha-2 (merosin) deficiency [LAMA2-related CMD (MDC1A)], manifests due to mutations in the LAMA2 gene and is also referred to as merosin deficient congenital muscular dystrophy.It is an autosomal recessive disorder and accurate molecular diagnosis is essential to confirm the diagnosis in the proband and offer families counseling and prenatal testing.In India, this is a common reason for families to seek consultation for genetic disorders, as was also in the case reported by Bhowmik et al. [4]. Next Generation Sequencing (NGS) is a high-throughput sequencing technology for comprehensive analysis of the coding region or the whole genome of an individual. It involves massively parallel DNA-sequencing that produces many short reads (25–500 bp) for a relatively low cost and in a short time [5]. NGS has slowly made its way into diagnostic testing and is now available in India as well. In this issue of the journal, Bhowmik et al. demonstrate the impact of this new high throughput technology with perspective of a large gene, LAMA2. They demonstrate the utility of using a targeted panel of 40 genes relating to the phenotype being evaluated. Although the diagnosis of merosin congenital muscular dystrophy was made on clinical, neuroimaging and muscle immunohistochemistry in this case, molecular diagnosis was essential to offer prenatal diagnosis. NGS technology is a cost effective technique for sequencing large genes such as LAMA2, as shown by Bhowmik et al. Once the mutation is confirmed in the proband, simpler and cheaper tests are used to confirm the fetal status. In this paper, authors confirmed the fetal status by Sanger sequencing for the specific familial exon 50 deletion. It is very possible that a practicing pediatrician may never have seen even a single case of some rare disorders during his/ her lifetime. Therefore, confirming diagnosis of rare disorders
Indian J Pediatr
can be a challenge, involving multiple consultations, many diagnostic tests that are expensive and protracted time spent for many hospital visits. Next generation sequencing addresses most of these challenges [6], and the targeted panel testing commonly used in clinical practice involves testing a limited number of genes associated with specific phenotypes, such as muscle dystrophy and myopathy, microcephaly, Charcot Marie Tooth disease etc. In cases where the phenotype does not suggest a specific diagnosis, exome sequencing is preferred and that includes sequencing of about 1 % of the genome that contains exons which code for monogenic disorders. This technology is commercially available in India and is being used widely for diagnoses of many monogenic disorders. When a physician orders the test it is important to give detailed clinical phenotype, as this is an integral part of data interpretation and reporting on NGS analysis [7]. The clinician and laboratory have to work together for optimal outcomes with this technology, as well as understand its limitations. Guidelines for NGS testing mandate that qualified personnel obtain a written informed consent after appropriate genetic counseling [8]. How will this new generation sequencing impact clinical care in India? The burden of genetic disorders in India is high and with control of infectious diseases, these are contributing to the neonatal and infant morbidity and mortality. The system of marriages, consanguineous as well as endogamous, also contribute to the load of genetic disorders. Laboratory testing cannot replace a detailed family history and clinical evaluation of the patient, and this holds true for NGS based tests as well. Correlation of clinical findings, imaging, enzyme testing etc., as relevant, is of utmost importance, and currently prenatal testing should only be performed once the familial disease causing mutation is identified and confirmed. Targeted panel testing by NGS and exome sequencing will change the algorithms for testing and diagnosis of monogenic disorders with an ultimate goal to enhance patient care. The role of clinical and molecular geneticists
enlarges in this era of evolving technology to encompass this field of genomics in clinical practice. Even a general pediatrician should learn how to interpret the data of NGS and take appropriate action. Compliance with Ethical Standards Conflict of Interest None. Source of Funding None.
References 1.
Chae JH, Vasta V, Cho A, et al. Utility of next generation sequencing in genetic diagnosis of early onset neuromuscular disorders. J Med Genet. 2015;52:208–16. 2. Todd EJ, Yau KS, Ong R, et al. Next generation sequencing in a large cohort of patients presenting with neuromuscular disease before or at birth. Orphanet J Rare Dis. 2015;10:148. 3. Bainbridge MN, Wiszniewski W, Murdock DR, et al. Wholegenome sequencing for optimized patient management. Sci Transl Med. 2011;3 87re3. 4. Bhowmik AD, Dalal AB, Matta D, Sundaram C, Aggarwal S. Targeted next generation sequencing identifies a novel deletion in LAMA2 gene in a merosin deficient congenital muscular dystrophy patient. Indian J Pediatr. 2015. doi:10.1007/s12098-015-1822-3. 5. Delio M, Patel K, Maslov A, et al. Development of a targeted multidisorder high-throughput sequencing assay for the effective identification of disease-causing variants. PLoS ONE. 2015;10:e0133742. 6. Iglesias A, Anyane-Yeboa K, Wynn J, et al. The usefulness of wholeexome sequencing in routine clinical practice. Genet Med. 2014;16: 922–31. 7. Richards S, Aziz N, Bale S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American college of medical genetics and genomics and the association for molecular pathology. Genet Med. 2015;17:405–24. 8. ACMG Board of Directors. ACMG policy statement: updated recommendations regarding analysis and reporting of secondary findings in clinical genome-scale sequencing. Genet Med. 2015;17:68–9.
EDITORIAL COMMENTARY
Next Generation Sequencing in the Clinic Ratna Dua Puri 1
Received: 11 January 2016 / Accepted: 11 January 2016 # Dr. K C Chaudhuri Foundation 2016
Next generation sequencing (NGS) has heralded a new era in the diagnosis of genetic disorders. Over the years as the cost of sequencing has plummeted, it is increasingly being used in clinical practice. Many studies highlighting the utility of NGS for diagnosis of monogenic genetic disorders including neuromuscular diseases (NMDs) have been published [1, 2]. NMDs include a vast number of phenotypically and genetically heterogeneous conditions, diagnosis of which is a challenge even for an experienced clinician. Congenital muscular dystrophies (CMDs) are a group of distinct NMDs that present with congenital hypotonia, severe muscle weakness and atrophy, contractures and delayed motor milestones. With improved understating of their genetic basis, they are sub classified based on the gene that harbors the causative mutations, these being identified in about 25–50 % cases. A lot still needs to be understood and unraveled about CMDs. Though Sanger sequencing has been available in India since long, genetic heterogeneity and the large size of some genes, involves huge costs of testing. This is a major limitation in a country where most health expenditure is “out-of pocket”. This greatly limits the uptake of genetic testing. However, making a genetic diagnosis is important for ending the diagnostic odyssey for patients and their families, allowing the family to comprehend why it happened, providing prognostic information, permitting genetic counseling and prenatal diagnosis, and at times leading to
* Ratna Dua Puri [email protected]
1
Center of Medical Genetics, Sir Ganga Ram Hospital, New Delhi 110060, India
treatment and search for new research initiatives in disease management [3]. Laminin alpha-2 (merosin) deficiency [LAMA2-related CMD (MDC1A)], manifests due to mutations in the LAMA2 gene and is also referred to as merosin deficient congenital muscular dystrophy.It is an autosomal recessive disorder and accurate molecular diagnosis is essential to confirm the diagnosis in the proband and offer families counseling and prenatal testing.In India, this is a common reason for families to seek consultation for genetic disorders, as was also in the case reported by Bhowmik et al. [4]. Next Generation Sequencing (NGS) is a high-throughput sequencing technology for comprehensive analysis of the coding region or the whole genome of an individual. It involves massively parallel DNA-sequencing that produces many short reads (25–500 bp) for a relatively low cost and in a short time [5]. NGS has slowly made its way into diagnostic testing and is now available in India as well. In this issue of the journal, Bhowmik et al. demonstrate the impact of this new high throughput technology with perspective of a large gene, LAMA2. They demonstrate the utility of using a targeted panel of 40 genes relating to the phenotype being evaluated. Although the diagnosis of merosin congenital muscular dystrophy was made on clinical, neuroimaging and muscle immunohistochemistry in this case, molecular diagnosis was essential to offer prenatal diagnosis. NGS technology is a cost effective technique for sequencing large genes such as LAMA2, as shown by Bhowmik et al. Once the mutation is confirmed in the proband, simpler and cheaper tests are used to confirm the fetal status. In this paper, authors confirmed the fetal status by Sanger sequencing for the specific familial exon 50 deletion. It is very possible that a practicing pediatrician may never have seen even a single case of some rare disorders during his/ her lifetime. Therefore, confirming diagnosis of rare disorders
Indian J Pediatr
can be a challenge, involving multiple consultations, many diagnostic tests that are expensive and protracted time spent for many hospital visits. Next generation sequencing addresses most of these challenges [6], and the targeted panel testing commonly used in clinical practice involves testing a limited number of genes associated with specific phenotypes, such as muscle dystrophy and myopathy, microcephaly, Charcot Marie Tooth disease etc. In cases where the phenotype does not suggest a specific diagnosis, exome sequencing is preferred and that includes sequencing of about 1 % of the genome that contains exons which code for monogenic disorders. This technology is commercially available in India and is being used widely for diagnoses of many monogenic disorders. When a physician orders the test it is important to give detailed clinical phenotype, as this is an integral part of data interpretation and reporting on NGS analysis [7]. The clinician and laboratory have to work together for optimal outcomes with this technology, as well as understand its limitations. Guidelines for NGS testing mandate that qualified personnel obtain a written informed consent after appropriate genetic counseling [8]. How will this new generation sequencing impact clinical care in India? The burden of genetic disorders in India is high and with control of infectious diseases, these are contributing to the neonatal and infant morbidity and mortality. The system of marriages, consanguineous as well as endogamous, also contribute to the load of genetic disorders. Laboratory testing cannot replace a detailed family history and clinical evaluation of the patient, and this holds true for NGS based tests as well. Correlation of clinical findings, imaging, enzyme testing etc., as relevant, is of utmost importance, and currently prenatal testing should only be performed once the familial disease causing mutation is identified and confirmed. Targeted panel testing by NGS and exome sequencing will change the algorithms for testing and diagnosis of monogenic disorders with an ultimate goal to enhance patient care. The role of clinical and molecular geneticists
enlarges in this era of evolving technology to encompass this field of genomics in clinical practice. Even a general pediatrician should learn how to interpret the data of NGS and take appropriate action. Compliance with Ethical Standards Conflict of Interest None. Source of Funding None.
References 1.
Chae JH, Vasta V, Cho A, et al. Utility of next generation sequencing in genetic diagnosis of early onset neuromuscular disorders. J Med Genet. 2015;52:208–16. 2. Todd EJ, Yau KS, Ong R, et al. Next generation sequencing in a large cohort of patients presenting with neuromuscular disease before or at birth. Orphanet J Rare Dis. 2015;10:148. 3. Bainbridge MN, Wiszniewski W, Murdock DR, et al. Wholegenome sequencing for optimized patient management. Sci Transl Med. 2011;3 87re3. 4. Bhowmik AD, Dalal AB, Matta D, Sundaram C, Aggarwal S. Targeted next generation sequencing identifies a novel deletion in LAMA2 gene in a merosin deficient congenital muscular dystrophy patient. Indian J Pediatr. 2015. doi:10.1007/s12098-015-1822-3. 5. Delio M, Patel K, Maslov A, et al. Development of a targeted multidisorder high-throughput sequencing assay for the effective identification of disease-causing variants. PLoS ONE. 2015;10:e0133742. 6. Iglesias A, Anyane-Yeboa K, Wynn J, et al. The usefulness of wholeexome sequencing in routine clinical practice. Genet Med. 2014;16: 922–31. 7. Richards S, Aziz N, Bale S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American college of medical genetics and genomics and the association for molecular pathology. Genet Med. 2015;17:405–24. 8. ACMG Board of Directors. ACMG policy statement: updated recommendations regarding analysis and reporting of secondary findings in clinical genome-scale sequencing. Genet Med. 2015;17:68–9.
