A transgenic cell line with inducible transcription for studying (CGG)n repeat expansion mechanisms.

There are more than 30 inherited human disorders connected with repeat expansion (myotonic dystrophy type I, Huntington's disease, Fragile X syndrome). Fragile X syndrome is the most common reason for inherited intellectual disability in the human population. The ways of the expansion development remain unclear. An important feature of expanded repeats is the ability to form stable alternative DNA secondary structures. There are hypotheses about the nature of repeat instability. It is proposed that these DNA secondary structures can block various stages of DNA metabolism processes, such as replication, repair and recombination and it is considered as the source of repeat instability. However, none of the hypotheses is fully confirmed or is the only valid one. Here, an experimental system for studying (CGG)n repeat expansion associated with transcription and TCR-NER is proposed. It is noteworthy that the aberrations of transcription are a poorly studied mechanism of (CGG)n instability. However, the proposed systems take into account the contribution of other processes of DNA metabolism and, therefore, the developed systems are universal and applicable for various studies. Transgenic cell lines carrying a repeat of normal or premutant length under the control of an inducible promoter were established and a method for repeat instability quantification was developed. One type of the cell lines contains an exogenous repeat integrated into the genome by the Sleeping Beauty transposon; in another cell line, the vector is maintained as an episome due to the SV40 origin of replication. These experimental systems can serve for finding the causes of instability and the development of therapeutic agents. In addition, a criterion was developed for the quantification of exogenous (CGG)n repeat instability in the transgenic cell lines' genome.

Derivation of iPS cell line (ICGi032-A) from a patient affected with fragile X syndrome.

Trinucleotide repeat expansion diseases such as fragile X syndrome are of great interest to study since the mechanism of its development is still unknown. IPS cell lines are some of the most convenient models for studying. The ICGi032-A iPS cell line was obtained from the peripheral blood mononuclear cells of the patient affected with fragile X syndrome. ICGi032-A iPS cell line have a normal karyotype, expression of pluripotency markers and can differentiate in vitro into the cells of three germ layers.

Establishment of an induced pluripotent stem cell line (ICGi026-A) from peripheral blood mononuclear cells of a patient with fragile X syndrome.

Expansion over 200 CGG repeats in FMR1 gene causes inherited intellectual disability or autism spectrum disorder named as fragile X syndrome. Despite the known cause fragile X syndrome pathogenesis has not been specified yet. The ICGi026-A iPSCs line was obtained by the reprogramming of the peripheral blood mononuclear cells from a 9-year-old boy with fragile X syndrome. The ICGi026-A iPSCs expressed pluripotency markers, had a normal male karyotype (46, XY) and had the capacity to in vivo differentiate into the cells of three germ layers.

Induced pluripotent stem cell line, ICAGi001-A, derived from human skin fibroblasts of a patient with 2p25.3 deletion and 2p25.3-p23.3 inverted duplication.

Skin fibroblasts from a patient with developmental delay and chromosome 2p25.3 deletion syndrome were reprogrammed into induced pluripotent stem cells (iPSCs) and the clonal stem cell line ICAGi001-A (iTAF9-11) was established. ICAGi001-A pluripotency was demonstrated in vitro by three germ layer differentiation capacity. This line is a good model for studying of the developmental delay and brain disorder.

HUMAN KARYOTYPE ANALYSIS IN DIAGNOSIS VERIFICATION.

When analyzing a patient's karyotype using classic cytogenetic tools, clinical cytogeneticists frequently face a problem of whether the observed morphological variant of a chromosome is the norm or pathology. Here we present three cases, when the use of additional approaches allowed us to accurately and reliably describe the chromosomal abnormalities and to provide a substantiated medical and genetic prognosis. Translocations were preliminary diagnosed in the first two patients. This opinion was subsequently challenged, as these patients were the carriers of rare variants of normal chromosome polymorphisms (21pstkstkpss and 20cenh+). Thus, these diagnostic measures helped the wife of the first patient to maintain the pregnancy, whereas the second patient was referred for IVF. In the third case, the preliminary diagnosis trisomy of chromosome 22 has not been confirmed. This patient turned out to be a carrier of a supernumerary marker chromosome invdup(15)(q13), which offers a much more favorable medical prognosis.