Subtelomeric Research Articles

Identification of subtelomeric cluster-genes associated to sexual stage in Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite with sexual reproduction in the intestinal epithelium of felines. The depletion of two gene repressors, AP2XI-2 and AP2XII-1, induces merozoite formation and gene expression towards sexual commitment. Based on RNA-seq datasets of AP2XI-2 and AP2XII-1 knock downs we identified subtelomeric (ST) TgB12 and hypothetical (HP) genes upregulated. Some of the differentially expressed genes (DEGs) are arranged in ST clusters. These DEG products are characterized by high isoelectric points (pI) and may encode small proteins. The potential roles of these clusters of DEG ST genes in environmental resistance or parasite sexual development of T. gondii is discussed.

Translating Single Cell Secrets to Cancer Evolution Accelerates Personalized Therapy

Translating Single Cell Secrets to Cancer Evolution Accelerates Personalized Therapy

Architecture, Chromatin and Gene Organization of Toxoplasma gondii Subtelomeres.

Subtelomeres (ST) are chromosome regions that separate telomeres from euchromatin and play relevant roles in various biological processes of the cell. While their functions are conserved, ST structure and genetic compositions are unique to each species. This study aims to identify and characterize the subtelomeric regions of the 13 Toxoplasma gondii chromosomes of the Me49 strain. Here, STs were defined at chromosome ends based on poor gene density. The length of STs ranges from 8.1 to 232.4 kbp, with a gene density of 0.049 genes/kbp, lower than the Me49 genome (0.15 kbp). Chromatin organization showed that H3K9me3, H2A.X, and H3.3 are highly enriched near telomeres and the 5′ end of silenced genes, decaying in intensity towards euchromatin. H3K4me3 and H2A.Z/H2B.Z are shown to be enriched in the 5′ end of the ST genes. Satellite DNA was detected in almost all STs, mainly the sat350 family and a novel satellite named sat240. Beyond the STs, only short dispersed fragments of sat240 and sat350 were found. Within STs, there were 12 functional annotated genes, 59 with unknown functions (Hypothetical proteins), 15 from multigene FamB, and 13 from multigene family FamC. Some genes presented low interstrain synteny associated with the presence of satellite DNA. Orthologues of FamB and FamC were also detected in Neospora caninum and Hammondia hammondi. A re-analysis of previous transcriptomic data indicated that ST gene expression is strongly linked to the adaptation to different situations such as extracellular passage (evolve and resequencing study) and changes in metabolism (lack of acetyl-CoA cofactor). In conclusion, the ST region of the T. gondii chromosomes was defined, the STs genes were determined, and it was possible to associate them with high interstrain plasticity and a role in the adaptability of T. gondii to environmental changes.

Reactivation of human herpesvirus 6 and its clinical significance

Human herpesvirus 6 (HHV-6) is recognized as a ubiquitous dsDNA virus in human with widespread cell tropism in vivo, which could induce lifelong latent infection and be reactivated in some immunocompromised individuals leading to serious diseases. With the increasing application of hematopoietic stem cell transplantation (HSCT), it is obvious that HHV-6 reactivation has a close relationship with several complications including encephalitis after transplantation. On the other hand, among about 1 % infected people, HHV-6 and subtelomeric region of cell chromosomes in the state of endogenous persistent infection play a part in activated virus infection. As the detection methods make a great progress in HHV-6 detection, clinical data reveals the relationship between HHV-6 reactivation and lots of chronic diseases, especially lymphoma, leukemia as well as some chronic hematopoietic and immunological diseases. Key words: Herpesvirus 6, human; Hematopoietic stem cell transplantation; Lymphoma; Leukemia

6p25 Deletion and Anesthetic Implications During Growing Rod Insertion for Early Onset Scoliosis

6p25 is a rare yet clinically identifiable disorder that results from the deletion of the short arm of chromosome 6. Due to the craniofacial, central nervous system, renal, cardiac, and skeletal abnormalities, anesthetic care may be required during various surgical interventions. The associated structural anomalies and end-organ involvement may impact the perioperative care. We present a 4-year-old girl with subtelomeric 6p25 deletion who required anesthetic care during placement of growing rods to treat progressive scoliosis. A review of literature was performed, the end-organ involvement was discussed, and options for anesthetic care were presented. J Med Cases. 2017;8(3):73-76 doi: https://doi.org/10.14740/jmc2748w

Analysis of Copy Number Variations in Patients with Autism Using Cytogenetic and MLPA Techniques: Report of 16p13.1p13.3 and 10q26.3 Duplications.

Autism is a common neuropsychiatric disorder affecting 1 in 68 children. Copy number variations (CNVs) are known to be major contributors of autism spectrum disorder (ASD). There are different whole genome or targeted techniques to identify CNVs in the patients including karyotyping, multiplex ligation-dependent probe amplification (MLPA) and array CGH. In this study, we used karyotyping and MLPA to detect CNVs in 50 Iranian patients with autism. GTG banding and 4 different MLPA kits (2 subtelomeric and 2 autism kits) were utilized. To elevate our detection rate, we selected the sporadic patients who had additional clinical features including intellectual disability, seizure, attention deficit hyperactivity disorder, and abnormal head circumference. Two out of 50 patients (4%) showed microscopic chromosome abnormalities and 5 out of 50 (10%) demonstrated copy number gains or losses using MLPA kits. Including one overlapping result between karyotype and MLPA techniques, our overall detection rate was 6 out of 50 (12%). Three out of 6 CNVs were de novo and three others were paternally inherited. Two of CNVs detected by karyotyping and MLPA tests were 16p13.1q13.3 and 10q26.3 duplications, respectively. For these two CNVs genotype and phenotype of the patients were compared with other studies. Although the pathogenicity of cytogenetic results was certain, most of MLPA results needed to be better refined using other more accurate techniques such as array CGH. Our findings suggest that it might be possible to obtain some useful information using MLPA technique but it cannot be used as a single diagnostic tool for the autism.

Study of copy number variations in children with unexplained mental retardation/brain development delay

Objective By using array-based single nucleotide polymorphisms comparative genomic hybri-dization (SNP-aCGH) to detect and fine mapping copy number variations (CNVs) in children with unexplained mental retardation/brain development delay(MR/BD), then the CNVs were analyzed to determine diagnosis and offer genetic counseling, finally to discuss the application of SNP-aCGH in genetic diagnosis of MR/BD with unknown causes. Methods Ninety-two children with unexplained MR/BD were recruited.SNP-aCGH was used to get CNVs from the whole genome-wide, and the correlation of CNVs and phenotype was analyzed to definit disease genes or pat-hogenic fragment.Statistics was performed to analyze the common phenotype between positive cases(case with CNVs) and negative cases. Results (1) The CNVs were detected in 10 cases with a detection rate of 10.86%, from which 8 cases showed subtelomeric aberration, 5 cases without subtelomeric aberration, and the rate was 8.70%, 5.40%, respectively.The CNVs related to MR/BD involved 10 different subtelomeric regions (9p, 21q, 3p, 2p, 15q, 4p, 12p, 22q, 16p, 17p), and 7 different regions without subtelomeric (1p, 4q, 2p, 14q, 15q, 12q, 22q). The deletions involved 11 zones (size: 1.05-8.80 Mb), and duplications referred to 8 zones(size: 1.33-31.25 Mb). (2)One case was diagnosed as 9p duplication syndrome, for candidate genes: DOCK8, VLDLR.A case was detected with a gene fracture(CRBN). One case was diagnosed as Coffin-Siris syndrome combined with a deletion of 15q26.3-qter, for candidate genes: SOX11 and LINS1, respectively.One case referred to 12p13.3 deletion syndrome, for candidate genes: ELKS, ERC1.One case referred to 22q13.2-qter deletion, for candidate genes: SHANK3.Two cases were diagnosed as ATR-16 syndrome with 17p13.3 deletion syndrome, their candidate genes: HBA1, HBA2, SOX8 for the former, YWHAE, LIS1 for the latter.(3)There were statistically significant differences in comparison of positive cases to the negative ones for growth delay, internal organs deformity, low birth weight infant(LBW) and premature infant(all P<0.05). Conclusions (1)Besides MR/BD in different degrees in all the positive cases, they also showed growth delay, a portion of them with internal organs deformity, low birth weight infant and premature infant.(2)Subtelomeric aberrations are related to MR/BD, while the submicroscopic rearrangement in regions without subtelomeric is suspiciously pathogenic, and need to be further studied.(3)SNP-aCGH can fine mapping the region of CNVs by high resolution from the whole genome-wide, which does contribute to limit the zones for finding pathogenic region and candidate genes, as well as to offer a technology platform for investigating about the correlation of phenotype and genes or CNVs. Key words: Mental retardation/brain development delay; Single nucleotide polymorphisms comparative geno-mic hybridization; Phenotype analysis

Subtelomeric aberrations in Thai patients with idiopathic mental retardation and autism detected by multiplex ligation-dependent probe amplification

Background: Chromosomal rearrangements involving telomeres account for approximately 1%–30% of causes of mental retardation (MR). It is therefore recommended that all cases of undiagnosed MR be screened for subtelomeric aberration. Nevertheless, resolution of a standard karyotyping using the G-banding technique is limited. Therefore, an additional technique with higher resolution should be performed to detect this type of anomaly. Objectives: To screen for subtelomeric aberration in Thai patients with mental retardation and autism. Methods: Multiplex ligation-dependent probe amplification (MLPA), was used to screen 114 Thai patients with idiopathic MR and 15 patients with autism. All positive results were confirmed by using a different set of MLPA probes or real-time PCR. Results: We identified 5 patients with submicroscopic aberration in patients with MR. One patient had a submicroscopic deletion at the 1p36.33 region, which was confirmed by real-time PCR. There were 2 patients with subtelomeric duplication at the 15q11.2 and 11p15.5 regions sequentially. Two patients had the same duplication at the Xp22.33. region. Conclusions: The present study shows that the incidence of a subtelomeric aberration in Thai patients with idiopathic MR is approximately the same reported previously (3.9%). Identifying these submicroscopic aberrations requires an advanced method with higher resolution than standard karyotyping. Although microarray techniques may be a more informative, they are costly and require an array facility, which are not widely available, especially in developing countries. Thus, MLPA in a routine cytogenetic test for MR patients with normal karyotypes in this setting can help to increase diagnostic yield. Keywords: Autism, mental retardation, MLPA, subtelomere

Personalized evolutionary hypothesis in genomics and auxiliary lymph node through diverse subtelomeric signal profile.

Few available data on the genomic-somatic evolution in breast cancer create limitation to provide the appropriate clinical managements. As an example, human subtelomeres (ST) are diverse-prone and variable targets. STs, as hot spots, have positive and negative impacts on the status of health and malady. We showed higher subtelomere signal copy number (SCN) of specific chromosomes in genomics than in auxiliary lymph node (ALN). Dissimilarity of signal intensity (SI) is found for all chromosomes. Significantly higher SI in genomics than in ALN cells were specified as chromosomes 5, 6, 9-12, 16-19 for weak; 1, 5-9, 19, X for medium; and 2, 5, 9, 10, 16, 18 for strong SI. For lacking, and presence of one and two SCNs; p/q ratio reflected differences for all chromosomes; but, 2, 3, 5, 7, 8, 10, 16, 18, 20, and X chromosomes were involved for three SCN. Chromosomes 1, 4, 9, 12, 17-19 lacked three SCN in ALN and lymphocytes. Weak SI ratio was higher in p- than in q-arm in majority of chromosomes. Manner of evolution and diversity in p- and q-arms is expressive of a novel definition as two diverse domains with a personalized insight. These data have been accompanied by periodic charts as ST array profiles which provide specific and individualized pattern in breast neoplasm. Such profiling at genomics level could be considered as a prediction through the patients' life. Moreover, subtelomere territory by interacting with protein expression of Ki67, cyclin D1, and cyclin E; and molecular targets including telomere length at genomics and somatic level provides package of information to bridge cancer cell biology to the cancer clinic as "puzzling paradigm."

Diagnosis of MECP2 duplication syndrome with molecular genetic techniques

To investigate whether the four boys with delayed motor development and intellectual disability suffer from MECP 2 duplication syndrome. Blood specimens and clinical data of four patients and mothers of patient 2 and patient 4 were collected. Genomic DNA was extracted from peripheral blood using DNA extraction kit. At first multiplex ligation-dependent probe amplification (MLPA) was employed in 4 patients, two distinct kits SALSA P036 and P070 for sub-telomere screening, and SALSA P245 for the 22 common microdeletion and microduplication syndromes. Then array-CGH analysis was carried out. Two mothers of patients were tested by array- comparative genomic hybridization (CGH) and X chromosome inactivation analysis. All the 4 patients presented with severe hypotonia, delayed motor development, intellectual disability and absent or limited language. Three patients manifested recurrent pneumonia in infancy except patient 2. Four patients had duplication on chromosome Xq28 with MLPA kit SALSA P245. Array-CGH identified the size of each duplication on Xq28. The precise size of each duplication was different in the four patients: patient 1, 14.931 Mb, patient 2, 0.393 Mb, patient 3, 0.482 Mb and patient 4, 0.299 Mb. To compare Xq28 duplications with UCSC database (http://genome.ucsc.edu/) revealed that each duplication harbors the MECP 2 and HCFC 1 gene. Mothers of patient 2 and patient 4 also carried microduplication on Xq28. X chromosome inactivation analysis demonstrated completely skewed inactivation (0: 100) and it is the inactive allele that passed on to the patients. For patients that present with delayed motor development, intellectual disability, hypotonia, absent or limited language and recurrent infection, combination of MLPA and array- CGH is effective and specific diagnostic methods of MECP 2 duplication syndrome.

Study on subtelomeric aberrations in children with mental retardation

Objective To make an etiology study on children with unexplained mental retardation (MR) by using combined multiplex ligation-dependent probe amplification (MLPA), and to explore associations between subtelomeric aberrations and phenotypes in local children. Methods Sixty-seven children with unexplained MR were enrolled in study group from Jul.2009 to Dec.2011 in Guangdong Women and Children's Hospital.Peripheral blood of patients and their parents were collected as samples of subtelomeric test by MLPA.Two kinds of probes of MLPA were combined to verify the aberration results.After confirmed test the parent of positive children were tested by the same way, then to analyze associations between test data and the clinical feature. Results Among sixty-seven children enrolled in the study aged 6 months to 15 years, where were 42 male and 25 female; the intelligence of 47 children belonged to mild degree(IQ≥50 scores), that of 20 children belonged to severe degree(IQ 0.05). The aberration rate in moderate to severe degree group was higher than those mild degree group.The rates of features including physical developmental retardation, malformation and organ aberrations in positive children were higher than those of the negative cases.There were significant differences between the 2 severity groups (all P<0.05). Conclusions Aberrations in subtelomeric region can be one of the important causes of unexplained MR.The MR patients were supposed to have subtelomeric region tested so as to provide the evidence for diagnosis and genetic counseling. Key words: Mental retardation; Subtelomeric region; Multiplex ligation-dependent probe amplification; Child

Novel Evolutionary Models and Periodic Charts in p- and q-Individual Chromosomes of Auxiliary Lymph Node and Buccal Cells

Signal copy number (SCN) and signal intensity (SI) of subtelomeres (ST) are investigated in auxiliary lymph node (ALN) and buccal (BUC) cells by fluorescence in situ hybridization. The extracted total cell of 38256 and 2309 was, respectively, analyzed from the benign ALN- and BUC-cells of an affected breast cancer patient. The Periodic model was based on ST behavior including normal-, down-, and upregulated clones with diverse SCN. The arm-p/q ratio based signature, as a subtelomeric array, reflects discordance and concordance of ST-behavior within individual chromosomes as a concept of “Individualization of Cells” rather than “Global Insight of Cells”. The Periodic charts could be considered as a reliable and refreshable platform through which the cellular evolution could be patterned and characterized. Signature of ST-profile in the BUC and ALN cells and the nature of diverse SCN and SI as quantitative and qualitative value led to modeling the real personalized perspective of cellular evolution. Protein expression of Ki67, Cyclin D1, and Cyclin E was assayed, as a complementary panel. These targets could be applied as the predictive and preventive markers for an early detection at BUC and ALN levels to plan the required managements in the breast cancer patients.

Higher involvement of subtelomere regions for chromosome rearrangements in leukemia and lymphoma and in irradiated leukemic cell line

Importance of subtelomeric chromosome rearrangements associated with idiopathic mental retardation and with methylation of gene expression in neoplastic cells has been shown. In order to observe incidence of deletions or translocations involving subtelomere region in leukemias and lymphomas, 41 patients were observed precisely by chromosome metaphase fluorescent in situ hybridization (FISH) using subtelomere probes and so on, specially focus on the end of long arm of chromosome 11 or short arm of chromosome 17. The abnormalities of subtelomere region on chromosome 11 were frequently observed in 7 of 17 patients (41.6%) with 11q22-q25 abnormalities, which were 3 of 9 patients with add(11)(q23-q25) and 3 of 7 patients with del(11)(q22-25), and 16 of 24 patients (66.0%) with 17p13 abnormalities, which were 2 of 6 with translocation between 17 and other chromosome, 6 of 10 with add(17)(p13) and all of 8 with del(17)(p13). Lymphoid disease had slightly higher abnormalities of subtelomere region than myeloid disease in patients with 11q22-25 or 17p13 abnormalities. Chemo- or radiotherapy treated patients had also subtelomeric chromosomal rearrangements. These results indicate that leukemia and lymphomas have many abnormalities at subtelomere region and possible association of chromosome instability, relating to pathogenesis of these diseases especially after therapy. Furthermore, FISH and chromosome analyses were performed on long-term cultured HL-60 leukemic cell line after irradiation using three different sources, and the results confirmed the higher induction of chromosome instability involving subtelomere region after α-rays irradiation.

Multiplex ligation-dependent probe amplification analysis of subtelomeric chromosome rearrangements in children with idiopathic mental retardation

To study the rate of subtelomeric rearrangements in patients with idiopathic mental retardation (MR) and to search the cause of MR. DNA was extracted and purified from peripheral blood leukocytes of 180 patients with idiopathic MR. DNA was tested using specific subtelomeric multiplex ligation-dependent probe amplification (MLPA) kits P036B/C and P070 according to manufacturer's instructions. The amplification products were separated by capillary electrophoresis using an ABI 3100 automated sequencer and size standard. MLPA data were extracted by GeneScan Analysis software. Data normalization and analysis were performed with the built-in MLPA application in GeneMarker. Among 180 patients with idiopathic MR, 12 had pathological subtelomeric deletions including 3 cases with a 4p deletion, 2 cases with a deletion at 9q and 22q respectively, 1 case with a deletion at 1p, 7p, 8p, 9q and 12q respectively. Subtelomeric rearrangements were responsible for 7% cases of idiopathic mental retardation. Subtelomeric rearrangement is a common cause of idiopathic mental retardation.

Use of Subtelomeric FISH in the Work-Up of Children with Developmental Delay

Small rearrangements in the telomeric region (the terminal regions of chromosomes) are responsible for many cases of developmental delay with minor

Variation in the chromosomal distribution of amplified esterase (FE4) genes in Greek field populations of Myzus persicae (Sulzer)

In northern Greece, insecticides have been used intensively against aphid populations of the Myzus persicae group, both on its primary host peach, on which annual sexual reproduction occurs, and on secondary host field crops such as tobacco, on which reproduction is entirely parthenogenetic. This has resulted in the selection of high levels of resistance based on the amplification of two genes encoding insecticide-degrading esterases, E4 and FE4. We have used fluorescence in situ hybridization (FISH) to study variation in the number and distribution of loci with amplified esterase genes in clones established from field populations of M. persicae-group aphids sampled on various crops in northern Greece. All clones collected from peach, as well as most of the clones (74%) from tobacco and other secondary host plants, had amplified FE4 genes and were of normal karyotype. Amplicon clusters containing FE4 occurred at multiple sites which varied in number, zygosity and distribution between clones. Most loci were on autosome 1, which also had the only site that was consistently occupied by amplified FE4, situated near subtelomeric repetitive DNA. Possibly this was the original site of FE4 gene amplification, and the location of the single-copy ‘wild type’ esterase genes. The rest of the clones from tobacco and other secondary hosts (26%) had amplified E4 genes, and all those analysed by FISH had an amplicon cluster on autosome 3T at a site close to the breakpoint of an A1,3 translocation, confirming the close linkage of this translocation with, and its probable involvement in, E4-based resistance. Three translocated clones collected on capsicum at one site had both E4 and FE4 amplified, the first time that both esterase gene types have been found together in individual aphids from field populations.

Subtelomeric duplications in Saccharomyces cerevisiae chromosomes III and XI: topology, arrangements, corrections of sequence and strain-specific polymorphism.

We have determined the sequence of a 3.42 kb segment from the left arm of chromosome III (coordinates 5394-8815 of Oliver et al., 1992). Instead of four open reading frames (ORFs) listed previously, the verified sequence reveals the presence of only one ORF, renamed YCL070/73c, encoding a protein of 615 amino acids. The putative product of ORF YCL070/73c shows 98.5% identity and 99% similarity with the protein of the same length encoded by ORF YKR106w from the right arm of chromosome XI and displays a topology characteristic for the Major Facilitators Superfamily of membrane proteins. These corrections will be deposited in the EMBL data library under the Accession Number X59720. In strain S288C the subtelomeric sequence 4319-11 215 of chromosome III is 98.3% identical with the subtelomeric sequence of 658 204-665 061 from the right arm of chromosome XI. Using various subtelomeric probes from chromosome III (coordinates 2097-3646 of S288C) we have analysed eight different Saccharomyces cerevisiae strains and the closely related species S. douglasii: some S. cerevisiae strains have additional duplications and longer chromosomes XI; in all strains chromosome III contains the 1200-11 000 segment (strain FL100 is disomic) while S. douglasii does not show any hybridization in this region.

Sequencing of a 9.2 kb telomeric fragment from the right arm of Saccharomyces cerevisiae chromosome XIV.

We report the complete sequence of a 9.2 kb fragment next to and including the right telomere of Saccharomyces cerevisiae chromosome XIV. Four open reading frames (ORFs) longer than 100 amino acids were observed in the sequenced segment. One ORF (378 codons) does not show any significant homology with proteins in the databases and corresponds to a putative new gene. Two ORFs are almost identical to the known YCR007/YKL219 and PAU1-like hypothetical protein families already identified on several S. cerevisiae chromosomes. These ORFs, whose function is unknown, are generally associated with sub-telomeric regions of chromosomes. The fourth one shows significant identities with bacterial mannitol dehydrogenases. It could be a yeast gene implicated in the metabolism of mannitol (or a related substrate). The sequence has been deposited in the EMBL data library under Accession Number X86790.

Sequencing analysis of a 4.1 kb subtelomeric region from yeast chromosome IV identifies HXT15, a new member of the hexose transporter family.

The DNA sequence of a 4.1 kb region of Saccharomyces cerevisiae chromosome IV was determined. This region contains a single open reading frame which codes for a member of the hexose transporter family. This new gene has been named HXT15 according to yeast gene data bases.

Sequencing a cosmid clone of Saccharomyces cerevisiae chromosome XIV reveals 12 new open reading frames (ORFs) and an ancient duplication of six ORFs.

A sequence of 31431 bp located on the left arm of chromosome (chr.) XIV from Saccharomyces cerevisiae was analysed. A total of 18 open reading frames (ORFs) could be identified. Twelve ORFs are new, two of which are most likely ribosomal protein genes, leaving ten ORFs of unknown function. Nine of the 18 ORFs show either at least 20% overall amino acid identity or significant regional homology to other S. cerevisiae ORFs. Additionally, six of these nine ORFs have homologues of similar size and the same transcriptional orientation within a stretch of 50 kb on chromosome IX. The degree of homology ranges from 90% overall identity to 23% in 375 amino acids. The homologues on chromosome IX are grouped in two blocks that are separated by relatively long ORFs. This is the first example of a multi-gene duplication in S. cerevisiae not linked to a centromere or subtelomere region.