Use of the «BCR/ABL - multitest¼ kit in the algorithm of laboratory diagnostics of oncohematological diseases: economic aspects.

Abnormal mRNAs of the hybrid BCR-ABL gene in the majority of cases initiate the synthesis of proteins with a mass of 210 kDa (p210), 190 kDa (p190), and 230 kDa (p230). Expression of the p210 variant is most common in CML (95% of cases), while the p190 and p230 variants are less common (1-4%). On the contrary, p190 predominates in ALL. Measurement of BCR/ABL gene expression is included in clinical guidelines for the diagnosis of CML and ALL as sequential tests in accordance with their occurrence. At the same time, in the context of primary patients testing with suspected hematological malignancies with a low prevalence of BCR-ABL positive patients in the cohort of examined individuals, sequential testing is associated with low cost-effectiveness. Purpose: approbation of a parallel algorithm for detecting all three (p210, p190 and p230) using the multiplex RT-PCR format implemented in the «BCR/ABL-MULTITEST¼ reagent kit. We used anonymized blood samples from patients with suspected CML, as well as samples from ALL patients before starting therapy. Testing of blood samples was carried out using two variants of the algorithm: sequential determination of individual BCR-ABL transcripts and parallel determination using the developed set of reagents «BCR/ABL-MULTITEST¼. To detect the p210 transcript, a commercial kit «AmpliSens® Leukemia Quantum M-bcr-FRT¼ (Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) was used. Simultaneously, a test was used to detect all three variants of BCR-ABL transcripts using the «BCR/ABL - MULTITEST¼ reagent kit based on a monochrome multiplex reaction «in one test tube¼. Reverse transcription were carried out using the REVERTA-L reagent kit (Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) in accordance with the manufacturer's instructions. Using the reagent kits «BCR/ABL-MULTITEST¼ and «AmpliSens® Leukemia Quantum M-bcr-FRT¼ there is a high level of correlation of quantitative results of determining the chimeric transcript BCR-ABL р210 (r = 0.99). When using the proposed parallel algorithm with the primary use of the «BCR/ABL-MULTITEST¼ reagent kit, out of 95 patients with suspected CML, 9 samples with p210 transcript were identified, one with p190 BCR / ABL, and in one case a transcript variant characteristic of chronic neutrophilic leukemia - p230 BCR / ABL. The estimated cost for detecting one positive case of BCR-ABL when using the parallel diagnostic algorithm «BCR/ABL-MULTITEST¼ with a focused flow of studies is reduced by about 2 times due to a decrease in the amount of laboratory plastic used and the volume of the reaction mixture, as well as the absence of the need for repeated separate tests to detect p190 and p230. The use of the multiplex PCR-RT test system «BCR/ABL-MULTITEST¼ allows detecting in one test tube all three main variants of BCR-ABL transcripts - p210, p190, p230 and achieving significant resource savings when examining a cohort of patients with suspected CML and ALL and low frequency of positive samples.

[Clinical and molecular genetic features of 3 family cases of the central precocious puberty, due to MKRN3 gene defects].

Gonadotropin-dependent precocious puberty (central) is a condition resulting from the early (up to 8 years in girls and 9 years in boys) reactivation of the hypothalamic-pituitary-gonadal axis. An increase in the secretion of sex steroids by the gonads in this form is a consequence of the stimulation of the sex glands by gonadotropic hormones of the pituitary gland. In the absence of central nervous system abnormalities, CPP is classified as idiopathic and as familial in some cases, emphasizing the genetic origin of this disorder. Loss-of-function mutations in Makorin Ring Finger Protein 3 (MKRN3) are the most common identified genetic cause of central precocious puberty compared to sporadic cases. In the present study we performed the first descrition of 3 family cases of central precocious puberty duo to novel MKRN3 gene mutation detected by NGS in the Russian Federation.

Screening of somatic mutations in the JAK2 and CALR genes by high-resolution melting curve analysis.

Somatic mutations associated with oncological diseases, including Ph-myeloproliferative neoplasms (Ph-MPN), are very diverse, occur with different frequencies and different allelic burden levels. Therefore, at the initial stage of performing molecular-genetic diagnostic procedures, it is desirable to be able to conduct screening tests in the laboratory. This is especially important when analyzing rare and diverse mutations. Analysis of high resolution melting curves (HRM analysis), which has high sensitivity and is suitable for screening all types of mutations, in a number of studies is proposed for the analysis of Ph-MPN associated mutations in the JAK2 and CALR genes. For analysis of somatic mutations in the majority of literature sources that we reviewed, the authors use the LightCycler (Roche) thermocycler and much rarely the CFX96 (Bio-Rad), which is often presented in Russian scientific and practical and medical organizations. The aim of the study was to screen the somatic JAK2 and CALR mutations by HRM analysis using the CFX96 thermocycler and the Precision Melt Analysis software (Bio-Rad, USA) for patients with Ph-MPN. In the present research, HRM analysis was conducted on the DNA samples from patients with mutations in the JAK2 or in the CALR gene. The Precision Melt Analysis software identified all variants of the analyzed mutations, both a single nucleotide substitution in the JAK2 gene (with allelic burden level in the range of 5-40%), and various indel mutations in the CALR gene (with allelic burden level in the range of 40-50%) Therefore, the HRM analysis that was conducted on the CFX96 allows screening of highly specific mutation for the diagnosis of Ph-MPN in the exon 14 of the JAK2 gene and in the exon 9 of the CALR gene. The inclusion of this screening research in the laboratory testing algorithm improves the efficiency and accessibility of molecular genetic technologies in the diagnosis of Ph-MPN.

[Somatic mutations in the androgen receptor gene as the cause of androgen insensitivity syndrome].

Androgen insensitivity syndrome is an X-linked disorder characterized by either complete or partial insensitivity of target tissues to androgens. This disease is caused by mutations in the AR gene located on the Х chromosome. Currently, there are no distinct clinical, biochemical, or hormonal markers that would allow one to differentiate androgen insensitivity syndrome from a number of other forms of 46,XY disorders of sex development. Therefore, final verification of this condition is based on the results of molecular genetic tests. Although more than 1,000 point mutations in the AR gene have been reported, somatic mutations in this gene have been described rather rarely. However, this very type of mutations makes the course of this disease difficult to predict, since various cells in the human body contain both normal and mutant receptors. Somatic mosaicism can cause spontaneous masculization during puberty in individuals born with a completely normal female phenotype. In this case report, we describe the phenotypic and molecular genetic characteristics of eight patients with various forms of androgen insensitivity syndrome caused by somatic mutations in the AR gene.