[Causes and Consequences of Genome Instability in Psychiatric and Neurodegenerative Diseases].

Each neuron has 100-10000 connections (synapses) with other neural cells, therefore genome pathologies affecting a small proportion of brain cells are capable of causing dysfunction of the entire central nervous system (CNS). Recently, genome and chromosome instability has been uncovered in neurodegeneration (Alzheimer's disease, ataxia telangiectasia). Somatic tissue-specific mosaicism was observed in the brain of individuals with neuropsychiatric diseases including schizophrenia, autism, intellectual disability, and epilepsy. The study of genetic processes in neurons allows determination of a certain number of genetic pathways and candidate processes, modifications of which can cause impaired genome stability. Brain-specific somatic mutations generally occur at the earliest stages of development. Accordingly, genome variability and somatic mosaicism are expected to be mediated by cell cycle regulation, DNA repair, DNA replication, and programmed cell death in the brain. Endomitosis, endoreduplication, and abortive entrance to the cell cycle are also commonly observed in neurodegeneration. Brain-specific genome instability maybe a key element in the pathogenic cascade of neurodegeneration. Here we review the current state of knowledge concerning somatic genome variations in neurodegenerative and psychiatric diseases and analyze the causes and consequences of genomic instability in the CNS.

Somatic cell genomics of brain disorders: a new opportunity to clarify genetic-environmental interactions.

Recent genomic advances have exacerbated the problem of interpreting genome-wide association studies aimed at uncovering genetic basis of brain disorders. Despite of a plethora of data on candidate genes determining the susceptibility to neuropsychiatric diseases, no consensus is reached on their intrinsic contribution to the pathogenesis, and the influence of the environment on these genes is incompletely understood. Alternatively, single-cell analyses of the normal and diseased human brain have shown that somatic genome/epigenome variations (somatic mosaicism) do affect neuronal cell populations and are likely to mediate pathogenic processes associated with brain dysfunctions. Such (epi-)genomic changes are likely to arise from disturbances in genome maintenance and cell cycle regulation pathways as well as from environmental exposures. Therefore, one can suggest that, at least in a proportion of cases, inter- and intragenic variations (copy number variations (CNVs) or single nucleotide polymorphisms (SNPs)) associated with major brain disorders (i.e. schizophrenia, Alzheimer's disease, autism) lead to genetic dysregulation resulting in somatic genetic and epigenetic mosaicism. In addition, environmental influences on malfunctioning cellular machinery could trigger a cascade of abnormal processes producing genomic/chromosomal instability (i.e. brain-specific aneuploidy). Here, a brief analysis of a genome-wide association database has allowed us to support these speculations. Accordingly, an ontogenetic 2-/multiple-hit mechanism of brain diseases was hypothesized. Finally, we speculate that somatic cell genomics approach considering both genome-wide associations and somatic (epi-)genomic variations is likely to have bright perspectives for disease-oriented genome research.

Trisomy 21 mosaicism: we may all have a touch of Down syndrome.

Ever increasing sophistication in the application of new analytical technology has revealed that our genomes are much more fluid than was contemplated only a few years ago. More specifically, this concerns interindividual variation in copy number (CNV) of structural chromosome aberrations, i.e. microdeletions and microduplications. It is important to recognize that in this context, we still lack basic knowledge on the impact of the CNV in normal cells from individual tissues, including that of whole chromosomes (aneuploidy). Here, we highlight this challenge by the example of the very first chromosome aberration identified in the human genome, i.e. an extra chromosome 21 (trisomy 21, T21), which is causative of Down syndrome (DS). We consider it likely that most, if not all, of us are T21 mosaics, i.e. everyone carries some cells with an extra chromosome 21, in some tissues. In other words, we may all have a touch of DS. We further propose that the occurrence of such tissue-specific T21 mosaicism may have important ramifications for the understanding of the pathogenesis, prognosis and treatment of medical problems shared between people with DS and those in the general non-DS population.

Classification and monomer-by-monomer annotation dataset of suprachromosomal family 1 alpha satellite higher-order repeats in hg38 human genome assembly.

In the latest hg38 human genome assembly, centromeric gaps has been filled in by alpha satellite (AS) reference models (RMs) which are statistical representations of homogeneous higher-order repeat (HOR) arrays that make up the bulk of the centromeric regions. We analyzed these models to compose an atlas of human AS HORs where each monomer of a HOR was represented by a number of its polymorphic sequence variants. We combined these data and HMMER sequence analysis platform to annotate AS HORs in the assembly. This led to discovery of a new type of low copy number highly divergent HORs which were not represented by RMs. These were included in the dataset. The annotation can be viewed as UCSC Genome Browser custom track (the HOR-track) and used together with our previous annotation of AS suprachromosomal families (SFs) in the same assembly, where each AS monomer can be viewed in its genomic context together with its classification into one of the 5 major SFs (the SF-track). To catalog the diversity of AS HORs in the human genome we introduced a new naming system. Each HOR received a name which showed its SF, chromosomal location and index number. Here we present the first installment of the HOR-track covering only the 17 HORs that belong to SF1 which forms live functional centromeres in chromosomes 1, 3, 5, 6, 7, 10, 12, 16 and 19 and also a large number of minor dead HOR domains, both homogeneous and divergent. Monomer-by-monomer HOR annotation used for this dataset as opposed to annotation of whole HOR repeats provides for mapping and quantification of various structural variants of AS HORs which can be used to collect data on inter-individual polymorphism of AS.

Dynamic mosaicism manifesting as loss, gain and rearrangement of an isodicentric Y chromosome in a male child with growth retardation and abnormal external genitalia.

Isodicentric chromosomes are considered the most common structural abnormality of the human Y chromosome. Because of their instability during cell division, loss of an isodicentric Y seems mainly to lie at the origin of mosaicism in previously reported patients with a 45,X cell line. Here, we report on a similar case, which, however, turned out to be an example of dynamic mosaicism involving isodicentric chromosome Y and isochromosome Y after FISH with a set of chromosome Y-specific probes and multicolor banding. Cytogenetic analyses (GTG-, C-, and Q-banding) have shown three different cell lines: 45,X/46, X,idic(Y)(q12)/46,X,+mar. The application of molecular cytogenetic techniques established the presence of four cell lines: 45,X (48%), 46,X,idic(Y)(q11.23) (42%), 46,X,i(Y)(p10) (6%) and 47,X,idic(Y)(q11.23),+idic(Y)(q11.23) (4%). According to the available literature, this is the first case of dynamic mosaicism with up to four different cell lines involving loss, gain, and rearrangement of an idic(Y)(q11.23). The present report indicates that cases of mosaicism involving isodicentric and isochromosome Ys can be more dynamic in terms of somatic intercellular variability that probably has an underappreciated effect on the phenotype.

Molecular cytogenetics and cytogenomics of brain diseases.

Molecular cytogenetics is a promising field of biomedical research that has recently revolutionized our thinking on genome structure and behavior. This is in part due to discoveries of human genomic variations and their contribution to biodiversity and disease. Since these studies were primarily targeted at variation of the genome structure, it appears apposite to cover them by molecular cytogenomics. Human brain diseases, which encompass pathogenic conditions from severe neurodegenerative diseases and major psychiatric disorders to brain tumors, are a heavy burden for the patients and their relatives. It has been suggested that most of them, if not all, are of genetic nature and several recent studies have supported the hypothesis assuming them to be associated with genomic instabilities (i.e. single-gene mutations, gross and subtle chromosome imbalances, aneuploidy). The present review is focused on the intriguing relationship between genomic instability and human brain diseases. Looking through the data, we were able to conclude that both interindividual and intercellular genomic variations could be pathogenic representing, therefore, a possible mechanism for human brain malfunctioning. Nevertheless, there are still numerous gaps in our knowledge concerning the link between genomic variations and brain diseases, which, hopefully, will be filled by forthcoming studies. In this light, the present review considers perspectives of this dynamically developing field of neurogenetics and genomics.

Unexplained autism is frequently associated with low-level mosaic aneuploidy.

BACKGROUND: Autism is a common childhood neurodevelopmental disorder with a possible genetic background. About 5-10% of autism cases are associated with chromosomal abnormalities or monogenic disorders. However, the role of subtle genomic imbalances in autism has not been delineated. This study aimed to investigate a hypothesis suggesting autism to be associated with subtle genomic imbalances presenting as low-level chromosomal mosaicism. METHODS: We surveyed stochastic (background) aneuploidy in children with/without autism by interphase three-colour fluorescence in situ hybridisation. The rate of chromosome loss and gain involving six arbitrarily selected autosomes and the sex chromosomes was assessed in the peripheral blood cells of 60 unaffected children and 120 children with autism. RESULTS: Of 120 analysed boys with autism, 4 (3.3%) with rare structural chromosomal abnormalities (46,XY,t(1;6)(q42.1;q27); 46,XY,inv(2)(p11q13); 46,XY,der(6),ins(6;1)(q21;p13.3p22,1)pat; and 46,XY,r(22)(p11q13)) were excluded from further molecular cytogenetic analysis. Studying <420 000 cells in 60 controls and 116 children with idiopathic autism, we determined the mean frequency of stochastic aneuploidy in control and autism: (1) autosome loss 0.58% (95% CI 0.42 to 0.75%) and 0.60% (95% CI 0.37 to 0.83%), respectively, p = 0.83; (2) autosome gain 0.15% (95% CI 0.09 to 0.21%) and 0.22% (95% CI 0.14 to 0.30%), respectively, p = 0.39; and (3) chromosome X gain 1.11% (95% CI 0.90 to 1.31%) and 1.01% (95% CI 0.85 to 1.17%), respectively, p = 0.30. A frequency of mosaic aneuploidy greater the background level was found in 19 (16%) of 116 children with idiopathic autism, whereas outlier values were not found in controls (p = 0.0019). CONCLUSIONS: Our findings identify low-level aneuploidy as a new genetic risk factor for autism. Therefore, molecular cytogenetic analysis of somatic mosaicism is warranted in children with unexplained autism.

Fluorescence intensity profiles of in situ hybridization signals depict genome architecture within human interphase nuclei.

An approach towards construction of two-dimensional (2D) and three-dimensional (3D) profiles of interphase chromatin architecture by quantification of fluorescence in situ hybridization (FISH) signal intensity is proposed. The technique was applied for analysis of signal intensity and distribution within interphase nuclei of somatic cells in different human tissues. Whole genomic DNA, fraction of repeated DNA sequences (Cot 1) and cloned satellite DNA were used as probes for FISH. The 2D and 3D fluorescence intensity profiles were able to depict FISH signal associations and somatic chromosome pairing. Furthermore, it allowed the detection of replicating signal patterns, the assessment of hybridization efficiency, and comparative analysis of DNA content variation of specific heterochromatic chromosomal regions. The 3D fluorescence intensity profiles allowed the analysis of intensity gradient within the signal volume. An approach was found applicable for determination of assembly of different types of DNA sequences, including classical satellite and alphoid DNA, gene-rich (G-negative bands) and gene-poor (G-positive bands) chromosomal regions as well as for assessment of chromatin architecture and targeted DNA sequence distribution within interphase nuclei. We conclude the approach to be a powerful additional tool for analysis of interphase genome architecture and chromosome behavior in the nucleus of human somatic cells.

Serologic Markers of Autism Spectrum Disorder.

According to WHO data, about 67 million people worldwide are affected by autism, and this number grows by 14% annually. Among the possible causes of autism are genetic modifications, organic lesions of the central nervous system, metabolic disorders, influence of viral and bacterial infections, chemical influence to the mother's body during pregnancy, etc. The conducted research shows that research papers published until today do not name any potential protein markers that meet the requirements of the basic parameters for evaluating the efficiency of disease diagnostics, in particular high sensitivity, specificity, and accuracy. Conducting proteomic research on a big scale in order to detect serologic markers of protein nature associated with development of autism spectrum disorders seems to be highly relevant.

Pericentric inversion inv(7)(p11q21.1): report on two cases and genotype-phenotype correlations.

We report on two unrelated cases of pericentric inversion 46,XY,inv(7)(p11q21.1) associated with distinct pattern of malformation including mental retardation, development delay, ectrodactyly, facial dismorphism, high arched palate. Additionally, one case was found to be characterized by mesodermal dysplasia. Cytogenetic analysis of the families indicated that one case was a paternally inherited inversion whereas another case was a maternally inherited one. Molecular cytogenetic studies have shown paternal inversion to have a breakpoint within centromeric heterochromatin being the cause of alphoid DNA loss. Maternal inversion was also associated with a breakpoint within centromeric heterochromatin as well as inverted euchromatic chromosome region flanked by two disrupted alphoid DNA blocks. Basing on molecular cytogenetic data we hypothesize the differences of clinical manifestations to be produced by a position effect due to localization of breakpoints within variable centromeric heterochromatin and, alternatively, due to differences in the location breakpoints, disrupteding different genes within region 7q21-q22. Our results reconfirm previous linkage analyses suggested 7q21-q22 as a locus of ectrodactily and propose inv (7)(p11q21.1) as a cause of recognizable pattern of malformations or a new chromosomal syndrome.

Chimerism and multiple numerical chromosome imbalances in a spontaneously aborted fetus.

We report on a case of chimerism and multiple abnormalities of chromosomes 21, Xand Yin spontaneous abortion specimen. To the best our knowledge the present case is the first documented chimera in a spontaneously aborted fetus. The application of interphase fluorescence in situ hybridization (FISH) using chromosome enumeration and site-specific DNA probes showed trisomy X in 92 nuclei (23 %), tetrasomy X in 100 nuclei (25 %), pentasomy of chromosome X in 40 nuclei (10 %), XXY in 36 nuclei (9 %), XXXXXXYY in 12 nuclei (3 %), XXXXXYYYYY in 8 nuclei (2 %), trisomy 21 and female chromosome complement in 40 nuclei (10 %), normal female chromosome complement in 72 nuclei (18 %) out of 400 nuclei scored. Our experience indicates that the frequency of chimerism coupled with multiple chromosome abnormalities should be no less than 1 : 400 among spontaneous abortions. The difficulties of chimerism identification in fetal tissues are discussed.

[Genomic instability in the brain: chromosomal mosaicism in schizophrenia]. / Genomnaya nestabil'nost' v kletkakh golovnogo mozga: khromosomnyi mozaitsizm pri shizofrenii.

AIM: Experimental verification of the hypothesis about the possible involvement of the mosaic genome variations (mosaic aneuploidy) in the pathogenesis of a number of mental illnesses, including schizophrenia and autism: a genetic study of the level of mosaic genome variations in cells of the brain autopsy tissues in healthy controls and schizophrenia. MATERIAL AND METHODS: Autopsy brain tissues of 15 unaffected controls and 15 patients with schizophrenia were analyzed by molecular cytogenetic methods to determine the frequency of chromosomal mutations (the mosaic aneuploidy) in neural human cells. The original collection of chromosome-enumeration DNA probes to autosomes 1, 9, 15, 16, 18 and the sex chromosomes X and Y was used for the interphase cytogenetic analysis of chromosomes in the cells of the brain. RESULTS AND CONCLUSION: The frequency of low-level aneuploidy per individual chromosome was 0.54% (median - 0.53%; 95% confidence interval (CI) CI - 0.41-1.13%) in controls and 1.66% (median - 1.55%; 95% CI -1.32-2.12%) in schizophrenia (p=0.000013). Thus, the three-fold increase in aneuploidy frequency in the brain in schizophrenia was detected. It is suggested that mosaic aneuploidy, as a significant biological marker of genomic instability, may lead to genеtic imbalance and abnormal functional activity of neural cells and neural networks in schizophrenia.

Segment substitutions in alpha satellite DNA. Unusual structure of human chromosome 3-specific alpha satellite repeat unit.

We have sequenced the full-length copy of the alpha satellite higher-order repeated unit characteristic of human chromosome 3. Its internal structure, the regular alterations of J1 and J2 type monomers, is typical of the alphoid suprachromosomal family 1. This dimeric order is disrupted by the substitution of one J1 unit by an alien dimer which is not clearly related to any of the established monomeric types. We have also observed some other similar cases of segment substitutions in alpha satellite DNA. They probably represent a special type of molecular event which could be generated by gene conversion. Segment substitutions may be one of the important factors responsible for the extreme variability of localization patterns and actual sequences of alpha satellite DNA that should be taken into account in reconstructions of alpha satellite evolution.

Non-disjunction of chromosome 21, alphoid DNA variation, and sociogenetic features of Down syndrome.

The analysis of non-disjunction of chromosome 21 and alphoid DNA variation by using cytogenetic and molecular cytogenetic techniques (quantitative fluorescence in situ hybridization) in 74 nuclear families was performed. The establishment of possible correlation between alphoid DNA variation, parental age, environmental effects, and non-disjunction of chromosome 21 was made. The efficiency of techniques applied was found to be 92% (68 from 74 cases). Maternal non-disjunction wasfound in 58 cases (86%) and paternal non-disjunction - in 7 cases (10%). Post-zygotic mitotic non-disjunction was determined in 2 cases (3%) and one case was associated with Robertsonian translocation 46,XX,der(21;21)(q10;q10), +21. Maternal meiosis I errors were found in 43 cases (64%) and maternal meiosis II errors--in 15 cases (22%). Paternal meiosis I errors occurred in 2 cases (3%) and paternal meiosis I errors--in 5 cases (7%). The lack of the correlation between alphoid DNA variation and non-disjunction of chromosome 21 was established. Sociogenetic analysis revealed the association of intensive drug therapy of infectious diseases during the periconceptual period and maternal meiotic non-disjunction of chromosome 21. The correlation between non-disjunction of chromosome 21 and increased parental age as well as exposure to irradiation, alcohol, tobacco, mutagenic substances was not found. The possible relevance of data obtained to the subsequent studies of chromosome 21 non-disjunction is discussed.

Annotation of suprachromosomal families reveals uncommon types of alpha satellite organization in pericentromeric regions of hg38 human genome assembly.

Centromeric alpha satellite (AS) is composed of highly identical higher-order DNA repetitive sequences, which make the standard assembly process impossible. Because of this the AS repeats were severely underrepresented in previous versions of the human genome assembly showing large centromeric gaps. The latest hg38 assembly (GCA_000001405.15) employed a novel method of approximate representation of these sequences using AS reference models to fill the gaps. Therefore, a lot more of assembled AS became available for genomic analysis. We used the PERCON program previously described by us to annotate various suprachromosomal families (SFs) of AS in the hg38 assembly and presented the results of our primary analysis as an easy-to-read track for the UCSC Genome Browser. The monomeric classes, characteristic of the five known SFs, were color-coded, which allowed quick visual assessment of AS composition in whole multi-megabase centromeres down to each individual AS monomer. Such comprehensive annotation of AS in the human genome assembly was performed for the first time. It showed the expected prevalence of the known major types of AS organization characteristic of the five established SFs. Also, some less common types of AS arrays were identified, such as pure R2 domains in SF5, apparent J/R and D/R mixes in SF1 and SF2, and several different SF4 higher-order repeats among reference models and in regular contigs. No new SFs or large unclassed AS domains were discovered. The dataset reveals the architecture of human centromeres and allows classification of AS sequence reads by alignment to the annotated hg38 assembly. The data were deposited here: http://genome.ucsc.edu/cgi-bin/hgTracks?db=hg38&hgt.customText=https://dl.dropboxusercontent.com/u/22994534/AS-tracks/human-GRC-hg38-M1SFs.bed.bz2.

Genomic organization, sequence and polymorphism of the human chromosome 4-specific alpha-satellite DNA.

Two alpha-satellite fragments specific for human chromosome 4 have been cloned and characterized. Under stringent annealing conditions, they hybridized in situ only to the pericentromeric region of chromosome 4, but under non-stringent conditions they hybridized to all chromosomes containing the sequences of alpha-satellite suprachromosomal family 2 (viz., chromosomes 2, 4, 8, 9, 13, 14, 15, 18, 20, 21 and 22). Southern blot analysis reveals the 3.2-kb higher-order repeated unit which exists in two forms: as a single MspI fragment or a combination of the 2.6-kb and 0.6-kb MspI fragments. The two chromosome-4-specific cloned sequences appear to be different parts of this repeated unit. Taken together they constitute about 60% of its length. The primary structure of the higher-order repeated unit is characterized by a dimeric periodicity of the D1-D2 type which is usual to suprachromosomal family 2. At least in one site this regularity is disrupted by monomer deletion leading to the D2-D2 monomeric order. The most likely mechanism of this monomer excision is homologous unequal crossing-over. These sequences may serve as both cytogenetic and restriction-fragment length polymorphism (RFLP) markers for the pericentromeric region of chromosome 4.

Multicolor fluorescent in situ hybridization on post-mortem brain in schizophrenia as an approach for identification of low-level chromosomal aneuploidy in neuropsychiatric diseases.

Fluorescence in situ hybridization (FISH) of DNA-DNA or DNA-RNA using post-mortem brain samples is one approach to study low-level chromosomal aneuploidy and selective expression of specific genes in the brain of patients with neuropsychiatric diseases. We have performed a pilot molecular-cytogenetic analysis of post-mortem brain of schizophrenic patients. Multicolor FISH on two post-mortem brain samples of normal individuals and six schizophrenic individuals (area 10 of cortex) was applied. A set of DNA probes for FISH included: (i) centromeric alphoid DNA probes for chromosomes 7, 8, 13 and 21, 18, X and Y; (ii) classical satellite DNA probes for chromosomes 1 and 16; and (iii) region-specific DNA probes for chromosomes 13, 21 and 22. A statistically significant level of aneuploidy (up to 0.5-4% of neurons) involving chromosomes X and 18 was detected in two post-mortem brains of patients with schizophrenia. These results indicate that low-level chromosomal aneuploidy could be involved in the pathogenesis of schizophrenia. FISH could be applied to extended studies of chromosomal aneuploidy, abnormal patterns of chromosomal organization and functional gene expression in situ in the neurons of the brain in different psychiatric and neurodevelopmental diseases. Schizophrenia and Rett syndrome might be considered as psychiatric diseases of special interest for molecular-cytogenetic analysis as both of them could be associated with mutations in genes involving regulation of neurodevelopmental processes in the brain.

FISH analysis of replication and transcription of chromosome X loci: new approach for genetic analysis of Rett syndrome.

Differential replication staining using the 5-bromo-2'-deoxyuridine+Hoechst 33258 technique has been carried out on a series of 28 girls with Rett syndrome (RTT). The results indicated that regions Xq23 and Xq28 of inactive chromosome X could contain early replicating and, therefore, transcriptionally active loci in RTT. Interphase fluorescence in situ hybridization studies of replication timing, using chromosome X-specific genomic DNA probes, was applied to determine the loci with altered replication and transcription in RTT. Randomly selected P1 artificial chromosome (PAC) clones for Xp, Xcen and Xq were used. Two PAC clones from Xq28 (anonymous clone 24.23.0 and 671D9, containing MeCP2 locus) probably escape inactivation in late replicating chromosome X in some RTT patients. Therefore, region Xq28 could contain the genes escaping X inactivation and with expression from the human active and inactive X chromosomes. These results support the hypothesis proposing the disturbances in dosage compensation effect due to aberrant activation of genes in inactive chromosome X in RTT (bi-allelic expression instead of mono-allelic). Our results indicate that the normal allele of the MeCP2 gene could escape X inactivation and reduce the pathogenic effect of mutated allele in RTT.

Molecular-cytogenetic investigation of skewed chromosome X inactivation in Rett syndrome.

We have developed an approach to differentiate homologous X chromosomes in metaphase chromosomes and interphase nuclei by a fluorescence in situ hybridization (FISH) technique with chromosome X-specific alpha-satellite DNA probe. FISH analysis of metaphase chromosomes in a cohort of 33 girls with Rett syndrome (RTT) allowed us to detect eight girls with structurally different X chromosomes, one X chromosome with a large and another one with a small centromeric heterochromatin (so-called chromosomal heteromorphism). Step-wise application of differential replication staining and the FISH technique to identify the inactivation status of paternal and maternal chromosome X in RTT girls was applied. Skewed X inactivation in seven RTT girls with preferential inactivation of one X chromosome over the other X chromosome was detected in 62-93% of cells. Therefore, non-random or skewed X inactivation with variable penetrance in blood cells could take place in RTT.

Cytogenetic and molecular-cytogenetic studies of Rett syndrome (RTT): a retrospective analysis of a Russian cohort of RTT patients (the investigation of 57 girls and three boys).

Rett syndrome (RTT) is a severe neurodevelopmental disorder with an incidence of 2.5% in mentally retarded girls in Russia. We have performed cytogenetic studies of 60 patients (57 girls and three boys) with a clinical picture of RTT, selected according to the criteria for diagnosis of RTT defined by B. Hagberg et al. in 1996. Collection of DNA samples and fixed cell suspensions of RTT patients (37 girls and two boys) and their parents (27 patients) was established for molecular studies, for example analysis of MECP2 mutations in a Russian cohort of RTT patients. Among 60 patients 57 girls with a clinical picture of RTT had normal female karyotype (46,XX), one boy had normal male karyotype in peripheral lymphocytes (46,XY) and two boys had a mosaic form of Kleinfelter's syndrome (47,XXY/46,XY) in peripheral lymphocytes or muscle cells (with MeCP2 mutation R270X). Twenty-four mothers and parents of RTT girls had normal karyotype, two mothers had mosaic forms of Turner syndrome (45,X/46,XX) and one had mosaic karyotype (47,XX,+mar/48,XXX,+mar). We analyzed chromosome X in lymphocytes of 57 affected girls with a clinical picture of RTT using the 5-bromo-2'-deoxyuridine+Giemsa staining technique. A specific type of inactive chromosome X (so-called type 'C') with unusual staining of chromatin in the long arm of chromosome X was found in 55 (from 57) girls with RTT. This technique was positively used for presymptomatic diagnosis of RTT in five girls in earlier stages of the disease. We believe that the phenomenon of altered chromatin conformation in inactive chromosome X could be used as a laboratory test for preclinical diagnosis of the RTT.