Diagnosis – yes, mutation – no
In 2014, the long-awaited first child was born in the family of Kamilla Schuajpowa and Magomed Schuajpow from the small village Subutli-Miatli in Dagestan. The boy was completely healthy. At the age of six months, he was hospitalized with a suspected intestinal infection. The treatment was unsuccessful and the condition deteriorated, so that the child was transferred to the Russian Clinical Children’s Hospital in Moscow. But even there the doctors didn’t know what to do – there was no clear diagnosis and it was also unclear which therapy could lead to success.
“The child’s physical development stopped at the age of six months. At the age of one it weighed only five or six kilograms, it was very lean and did not grow. The symptoms of an intestinal infection did not improve. In the Russian Children’s Clinical Hospital, we were advised to contact the Weltischtschew Science and Research Institute for Pediatrics. We were immediately told that it was Fanconi syndrome and probably nephropathy cystinosis. Treatment was prescribed that finally brought results, ”says Kamilla.
However, investigations could not confirm the diagnosis. No cystine crystals were found in the cornea of the one-year-old child, which are considered to be one of the main features of the disease. And DNA analysis didn’t reveal any mutations. So the child should be healthy, but it wasn’t.
“Such stories are not uncommon. Some of our patients have a whole range of laboratory clinical manifestations of an autosomal recessive inheritance disorder (they only show if the child has a mutated gene variant from both parents), but when the molecular genetic tests are carried out, no genes are revealed Mutations associated with this pathology, or only one mutation is fixed. This means we cannot make a definitive diagnosis, ”says Svetlana Papisch, the senior researcher at the Weltischtschew Science and Research Institute for Pediatrics.
She was the first to conclude that the boy might have a rare inherited disorder and insisted on immediate pathogenetic therapy. Six months after the first examination, the doctors repeated all the examinations and found cystine crystals in the cornea, which, despite the negative DNA test, suggested nephropathy cystinosis. The pathogenetic therapy proved to be effective, the boy’s condition improved. In the case of this disease, the quality of the patient’s further life depends on early diagnosis and timely treatment.
At the same time, Swetlana Papisch was looking for research groups that would help solve the difficult case. The team of gene researcher Mikhail Skoblow from the Medical-Genetic Science Center showed interest, which among other things dealt with the analysis of matrix RNA – a molecule that played an important role in the expression of the genes.
“A fairly large proportion of mutations that are detected in DNA diagnostics result in the RNA structure being injured. Such an RNA is considered non-functional, which is why an incorrect protein is formed and an illness develops as a result. There are quite a few such cases – up to 30 percent. We have decided to freely analyze the RNA structure and find mutations that are not visible in DNA diagnostics. This makes it possible to identify the broken gene that is not fixed by a simple genetic analysis, ”says the head of the functional genome research laboratory at the medical-genetic research center, Mikhail Skoblow.
The scientist shows a small petri dish where fibroblasts – the patient’s connective tissue cells – are grown, waiting for the diagnosis to be confirmed. A year ago, the cells of Schuajpow, which were removed from a small piece of skin, were multiplied. RNA was removed from the fibroblasts and after it was determined that its structure was not correct, a hidden mutation was discovered that triggered the development of nephropathy cystinosis in the child.
“The greatest difficulty we faced was the very rapid decay of the RNA extracted. Because of a special ferment – the ribonuclease – it is unstable to external effects. Even in the cell, ribonucleic acid only survives for a few minutes and sometimes only seconds. When the RNA has done its job (protein has been made), it is broken down. To prevent this, we destroy cells with a solution that contains inhibitors of ribonuclease, and we take a good RNA that has not decayed. After that it’s easier. We get a cDNA from the ribonucleic acid, in which we observe how the gene structure changes, whether there are any mutations. It takes about a week on average, ”says Skoblow.
Others followed the Schuajpow case. This is how scientists managed to create an unknown mutation in a couple determinewho, because of this genetic mutation, could not have a child for a long time.
“When we did some of these studies and saw changes at the RNA level that were not discovered during DNA analysis, we realized that it was an alternative method of diagnosing hereditary diseases. It is very informative and popular, but very moody. There are only a few laboratories in the world where it can be carried out. We now have four completed patient stories. Some have been published, some are still being reviewed. There is still material from 15 patients in the work. Sometimes we make finds that nobody expected, ”says the scientist.
No analogues worldwide
To date, researchers have already fixed 400 genes, the mutations of which can be determined using RNA diagnostics. Individual systems of RNA analysis were developed for each section of the genome. There is no comparable database worldwide.
“I simply cannot remember a laboratory that has brought such advanced methods into circulation. The Skoblow team is undoubtedly in top positions. You work with RNA analysis, a product of gene transcription. So do not simply predict possible effects, but look directly at the result of the activity of the broken gene. Without such analyzes, the cases could not be deciphered and a correct diagnosis could not be made. Such an approach is already becoming an international trend in several cases, but the Skoblows team has taken the lead, ”said RIKEN (Japan), leader of the Institute for Physical and Chemical Research (RIKEN) in Japan and head of the Laboratory for Extreme Biology at the Federal University of Kazan, Oleg Gussew.
According to Svetlana Papisch, the RNS analysis in the Shuajpov case helped clarify the causes of the disease and confirmed the merits of the pathogenetic therapy. Legal questions can often be solved by this investigation. Very expensive medication is usually required to treat rare diseases. According to the law, they should be provided free of charge by the state. This requires a diagnosis that is confirmed, among other things, by genetic tests.
Kamilla Schuajpowa, whose son was kept alive thanks to the early detection of the illness, is already taking her son to pre-school classes. She is happy about his success and thanks the doctors and scientists who helped her to diagnose the disease and who decided on treatment in good time. Today, her six-year-old son is no different from his peers.