Neonatal genodiagnostics in St. Petersburg: details
A new stage in the development of neonatal screening in Russia
Maxim Russo, <url>
Since February 2015, during neonatal screening, the Medical and Genetic Diagnostic Center of St. Petersburg will begin using the VariFind™ solution for genetic analysis Neoscreen assay, based on a new generation of high-performance sequencing technology. The solution was developed by Parseq Lab with the support of the Skolkovo Foundation.
"The pilot clinical trial will last approximately 9 months and will affect about 1,000 children with testing. At the end of this stage, a feasibility study will be created for the application of the new method and the question of further use of the new approach will be raised both in the Medical and Genetic Center itself and in other regions of Russia," said Alexander Pavlov, head of Parseq Lab (pictured).
Such an approbation is necessary in order to demonstrate in a real clinical protocol on the basis of an existing medical institution (Medical and Genetic Center) that the application of this approach gives better results than the methods currently used. The diagnostic properties of the solution used have already been preliminarily determined in multicenter clinical trials, on the basis of which a conclusion was made about the applicability of the method in medical practice.
We will try to figure out how the new solution for detecting severe hereditary diseases in newborns differs from previous diagnostic methods.
Since the beginning of the 2000s, the introduction of neonatal screening has begun in Russia - a mass examination of all newborns for the early detection of certain hereditary diseases. In 2006, the mandatory testing program included three diseases: adrenogenital syndrome, galactosemia and cystic fibrosis. Later, phenylketonuria and congenital hypothyroidism were added to them.
Why is this necessary? The earliest possible diagnosis in the case of these diseases can be crucial for the life and health of the child. The fact is that clinical manifestations, noticeable to specialists, do not occur immediately, but perhaps months after birth. At the same time, early initiation of treatment can significantly reduce the severity of the disease, improve the prognosis and quality of life.
Let's look at specific examples. If a child has phenylketonuria, his body cannot normally metabolize a molecule of phenylalanine – one of the amino acids that come with food. Accumulating in the body, phenylalanine derivatives have a negative effect on the nervous system, which can eventually lead to serious mental disability. If we detect the disease immediately after birth, then in order to avoid the consequences, we only need to make sure that there are no products containing large amounts of phenylalanine in the food. The patient will have to follow such a diet at least until puberty. But he will avoid negative health consequences (although the carrier of mutations associated with phenylketonuria will remain and may be passed on to offspring in the future).
Galactosemia is characterized by another metabolic disorder. The body cannot convert galactose coming from milk into glucose. As a result, galactose accumulates and leads to disorders in the nervous system and liver, and can also cause changes in the lens of the eye. As a consequence, a child in the first weeks of life may develop severe liver damage and homeostasis, sometimes with fatal consequences. Early detection of galactosemia will also allow you to choose the right diet, in particular exclude milk, which will reduce the risk of serious complications.
The most common monogenic human disease – cystic fibrosis – is caused by mutations in the CFTR gene located on the seventh chromosome. This gene and the protein synthesized from it are responsible for the transport of chlorine ions across the cell membrane. Depending on which mutation has occurred, the disease may have different degrees of severity. In the case of cystic fibrosis, early initiation of therapy helps to reduce the severity of its course. Largely due to neonatal screening in Western Europe, the average life expectancy of patients with cystic fibrosis has now exceeded 40 years, and in Canada it has reached 50. In Russia, it is still much lower.
How does screening work? Usually, the analysis is carried out in the first week of a child's life. In the maternity hospital, a baby takes a little blood from the heel on a special paper form (therefore, screening is often called a "heel test"). Further, in the laboratory, markers of the listed diseases are determined from a blood sample – characteristic substances that are present in the body of both healthy and sick people, only in different quantities. In Russia, immunochemical research methods are used for this. The method is good because a small amount of blood from a dry spot is sufficient for analysis, but a number of problems are associated with its use.
Let's choose cystic fibrosis as an example. Immunoreactive trypsinogen in blood plasma is usually used as its marker. Actually, the first methods of mass neonatal screening in the 1980s were based on the determination of the increased content of trypsinogen. However, an increase in the concentration of this biochemical parameter in the blood of a newborn is observed not only in cystic fibrosis. This also happens with neonatal jaundice, deep prematurity, trisomy of the 13th and 18th chromosomes, in children with congenital infections, renal insufficiency and atresia of the small intestine, as well as in the case of nephrogenic diabetes insipidus. Moreover, if the mother violates the doctors' prescription: do not breastfeed the baby for three hours before taking blood from the heel, then an increased content of trypsinogen may also be found in the blood. As a result, the test will give a false positive result, which will cost a considerable number of gray hairs to parents until a second analysis is carried out. The number of false-positive cases of primary testing reaches 2% of the total number of newborns, despite the fact that the frequency of the disease (true-positive result) in our country is about 0.01%.
What to do? Molecular genetics comes to the rescue. All of these diseases are caused by mutations in specific genes. This means that determining the presence or absence of a mutation in the patient's genome will provide additional valuable information for removing or confirming the diagnosis. In the 1990s, a testing method based on polymerase chain reaction (PCR) was used for diagnostics, when a large number of copies of it can be obtained from a certain DNA fragment using a DNA polymerase enzyme. Multiple multiplication of the amount of the target DNA region allows you to overcome the detection limits of mutation detection methods (in other words, so that the amount of this DNA fragment is sufficient for its detection).
This method gives a fairly accurate answer, but, unfortunately, only to a very specific question: is there a certain mutation in the patient's DNA. At the same time, there may be other mutations in the DNA, testing of which was not provided for under this method or, in principle, cannot be carried out. If each of the genetic diseases was caused by a single mutation, PCR would be enough. But in reality, there are, for example, more than a thousand mutations of the CFTR gene that are associated with the development of cystic fibrosis, while hundreds of them are "responsible" for various forms of the disease. In certain regions and in certain ethnic groups, specific mutations are most common. For example, there is a mutation that causes about 75% of cases of cystic fibrosis in European children. And in South America, it determines only 25% of cases. The use of the PCR method for research makes it possible to detect only one or a countable number of different mutations. If we want to test a wide range of mutations associated with the development of cystic fibrosis, then we need to produce and conduct hundreds of different PCR studies, which will take too much time and will be quite expensive.
Therefore, usually a set of the most frequent mutations in a given population is chosen for genetic analysis. But when the ethnic composition is heterogeneous, the accuracy and reliability of diagnosis drops sharply. Studies have been conducted in Europe that have shown that conventional PCR tests detect only half of cases of cystic fibrosis if children come from families of North African or Turkish origin. It is clear that this problem is very relevant for such a vast and multiethnic country as Russia.
A systematic solution to this problem is precisely proposed in the method, which was created by specialists of the St. Petersburg company Parseq Lab. It is able to detect a wide range of mutations in the genes of cystic fibrosis, phenylketonuria and galactosemia, and consists in direct "reading" – sequencing the entire DNA sequence of the patient in the genome of interest. By determining the DNA sequence, you can immediately see whether there are disorders in it that lead to the development of genetic diseases. At the moment, the VariFind™ system Neoscreen assay can detect more than 460 clinically significant mutations, of which 320 are associated with the development of cystic fibrosis.
Also, this system is distinguished by the possibility of a simple expansion of the base of detected mutations. If in the future, in the course of research, it is shown that cystic fibrosis is caused by some other previously unaccounted-for mutation of the CFTR gene, it can be quickly added to the test system so that it is no longer missed during analysis.
Thus, the inclusion of genetic analysis in neonatal screening increases the overall accuracy of the study, and the use of the so-called "next-generation sequencing" (NGS) method for this allows you to simultaneously check almost any number of mutations and much faster.
Alexander Pavlov, CEO of Parseq Lab, noted that at the moment parents do not pay anything for neonatal screening: "For newborns, all studies are done for free, since this all happens within the framework of the state program. Last year, the purchase, which was made by the Medical and Genetic Center, was carried out as part of a subsidy from the government of St. Petersburg for the modernization and development of the diagnostic service of the city. The results of this testing should convince specialists and officials that the use of modern diagnostic methods can improve the effectiveness of the adopted scheme and this type of research will be subsidized in the same way as the entire program as a whole."
According to him, in one launch of the device, an analysis can be done for 48 patients. "A group of patients who have a suspected hereditary disease is recruited and tested together as part of a single device launch. The whole study takes about three days," Pavlov said.
The head of the company also notes that the algorithm and procedure for obtaining permission to use medical devices are not always clear in Russia: "This concerns modern technologies, innovative developments, diagnostic solutions – if such products have no analogues, this can become an obstacle to the rapid introduction of novelties into clinical practice. Often even the experts themselves admit that they have not yet encountered such technologies in their practice. We, one way or another, overcome such obstacles, but not everyone can go through this stage. If the solution is not certified, then it will not be able to get widespread use. Moreover, over the past 3 years, the rules for the registration of medical devices have changed several times, which is why the time for obtaining a permit stretches for years."
Skolkovo helps to cope with the difficulties of Parseq Lab: previously, the team was already a resident of the foundation, now it has submitted a new application related to the development of bioinformatic solutions and a system for processing big data in genomics. "Initially, the Skolkovo Foundation allowed us to enter a certain "ecosystem", where we established a large number of contacts, partnerships, participated in various events and were involved in the development and further commercialization of innovative developments. We have participated in a large number of various initiatives and programs of development institutions, at the moment Skolkovo looks the most professional in this regard," Alexander Pavlov said.
According to Yuri Nikolsky, Director of Science of the Biomedical Technologies Cluster of the Skolkovo Foundation, the BMT cluster enthusiastically supported the company's innovative project. "Firstly, timely and accurate diagnosis of congenital diseases is one of the foundations of modern medicine, and we are happy to see the "state of art" solution developed entirely by a Russian company. Secondly, assay and the Parseq lab service are based on NGS sequencing, a relatively new technology that is just beginning to enter the clinic. NGS technology provides high accuracy and potentially low cost of the test, which is important for our healthcare. We would like to note the high level of bioinformatic analysis carried out in the test and the uniqueness of the database of congenital mutations collected by Parseq lab," Yuri Nikolsky emphasized.
Portal "Eternal youth" http://vechnayamolodost.ru30.01.2015