Newborn Screening in Russia By Means of Next-Generation Sequencing
Russia will be one of the first countries in the world to implement the method of high-performance genomic analysis into newborns screening for severe hereditary diseases: cystic fibrosis, phenylketonuria and galactosemia.
At the end of the outgoing year Parseq Lab, an R&D company, specialized in development of integrated genetic diagnostics assays, has signed a contract whereby in winter of 2015 newborn screening for severe hereditary diseases in St. Petersburg, the second most populous city in Russia, will be carried out by means of next generation sequencing (NGS). It is one of the first precedents when genome sequencing technology is included in the diagnostic algorithm and routinely used in clinical practice.
VariFindTM Neoscreen assay, an NGS-based diagnostic solution consists of custom AmpliSeqTM-based targeted enrichment reagents, sequencing procedure protocols (for Ion PGMTM System), specialized software and database for data analysis. The assay is intended for diagnostics of three inborn errors of metabolism: cystic fibrosis, phenylketonuria and galactosemia. It is designed to identify more than 460 clinically significant genetic variants thus being currently the widest assay on the market among the intended for neonatal screening ones. The development of the VariFindTM Neoscreen assay took three years. The process was supported by the "Skolkovo" innovation center, the leading genetic institutions the Russian Academy of Medical Sciences — Medical Genetic Research Center (Moscow) and the Institute of Medical Genetics (Tomsk).
Clinical trials of the assay were held in three European laboratories: Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP, Portugal), the StabVida company (Portugal) and the Centre for Genomic Research University of Liverpool (England). It consisted of multiple independent testing of 576 samples, which included both clinical ones and samples from healthy individuals. Diagnostic characteristics of the assay have been established according to the results of this work: sensitivity — 99.31% and specificity — 100%.
The verification procedure, aimed to confirm the analytic properties of the assay, was performed in a certified Microsynth laboratory in Switzerland. 100 samples were tested both using the VariFindTM Neoscreen assay and by means of Sanger bidirectional sequencing method. High analytical characteristics of the method have been proved at the result of verification: sensitivity — 99.34%; specificity — 97.22%; overall accuracy — 99.99%.
Alexander Pavlov, CEO Parseq Lab: "The development was completed in 2013, yet substantial processing of the assay to match the needs of healthcare institutions took us another year. VariFindTM Neoscreen assay is, in fact, a hardware-software complex consisting of sequencing equipment (Ion PGM, Life Technologies), a server, a set of specific reagents for genome targeted research (Custom AmpliSeq panel, Life Technologies) and an IT-part (VariFindTM Software) for quality evaluation and analysis of data. The software enables the annotation of the sequencing results, offering clinical geneticists all the attendant information on the clinically significant genetic variants found".
460 mutations are currently inherent in the diagnostic assay and are automatically annotated by software in case of their presence (320 of them are associated with cystic fibrosis). At the same time the assays design allows to sequence all coding and some significant non-coding regions of CFTR, GALT and PAH genes, which makes the mutations list extensible. The expansion of it will be performed in accordance with the new data emerge on mutations that lead to the development of cystic fibrosis, phenylketonuria and galactosemia. Such a broad diagnostic panel meets all the requirements of the health care system, including the applicability for use in population with a large genetic diversity. The diagnostic assay is multi-ethnic and can be efficiently used worldwide.
Alexander Pavlov, CEO Parseq Lab: "Not only in Russia but worldwide, VariFindTMNeoscreen assay is one of the first NGS-based assays which made its way from research laboratories to the clinic. We expect to obtain a large volume of results next year when the routine use of the assay will start. It will indicate how the inclusion of high-throughput sequencing based DNA analysis improves the diagnostic efficiency of the existing algorithm of neonatal screening. It's designed and intended to be used as second tier test for cystic fibrosis screening and as a confirming stage for galactosemia and phenylketonuria".
St. Petersburg is one of the first regions where the high-performance genome sequencing method is going to be included in the neonatal screening algorithm. If this innovation shows good results, we can expect a wide spread of new technology utilization in healthcare facilities to deal with routine clinical problems.
Reference: cystic fibrosis is one of the most common severe hereditary diseases. This monogenic disorder is caused by mutations in the CFTR gene, which is known to have more than 1600 genetic variants. However only part of them lead to the development of the disease. Some of the revealed de novo mutations have also been proven to cause the disorder. Such variability makes sequencing a method of choice for genetic testing.
Cystic fibrosis is recommended to be screened in newborns by the World Health Organization, which is mandatory in many developed countries. In Russia, the screening is also performed and includes 4 stages: determination of biochemical markers — immunoreactive trypsinogen (IRT), a re-test of the IRT, sweat test and DNA diagnostics. However, only the first three stages are mandatory. At the same time, non-genetic methods of cystic fibrosis diagnostics are time-consuming and often giving false or questionable results.
Currently there are more than 25 different protocols of neonatal screening for cystic fibrosis worldwide, most of which include imperative DNA testing. This stage is intended to reduce the number of false-positive results and to verify the diagnosis in case of clinical and biochemical evidence of the disease presence.