Fern Tsien

Long-term In vitro Expansion Alters the Biology of Adult Mesenchymal Stem Cells

Mesenchymal stem cells (MSC) derived from bone marrow stem cells (BMSC) and adipose tissue stem cells (ASC) of humans and rhesus macaques were evaluated for their cell cycle properties during protracted culture in vitro. Human ASCs (hASC) and rhesus BMSCs (rBMSC) underwent significantly more total population doublings than human BMSCs (hBMSC) and rhesus ASCs (rASC). The cell cycle profile of all MSCs was altered as cultures aged. hMSCs underwent an increase in the frequency of cells in the S phase at P20 and P30. However, rhesus MSCs from both sources developed a distinct polyploid population of cells at P20, which progressed to aneuploidy by P30. Karyotype analysis of MSCs revealed the development of tetraploid or aneuploid karyotypes in the rhesus cells at P20 or P30. Analysis of the transcriptome of the MSCs from early and late passages revealed significant alterations in the patterns of gene expression (8.8% of the genes were differentially expressed in hBMSCs versus hASCs, and 5.5% in rBMSCs versus rASCs). Gene expression changes were much less evident within the same cell type as aging occurred (0.7% in hMSCs and 0.9% in rMSC). Gene ontology analysis showed that functions involved in protein catabolism and regulation of pol II transcription were overrepresented in rASCs, whereas the regulation of I kappa B/nuclear factor-kappaB cascade were overrepresented in hBMSCs. Functional analysis of genes that were differentially expressed in rASCs and hBMSCs revealed that pathways involved in cell cycle, cell cycle checkpoints, protein-ubiquitination, and apoptosis were altered.

DNA hypomethylation and unusual chromosome instability in cell lines from ICF syndrome patients

The ICF syndrome (i̲mmunodeficiency, c̲entromeric region instability, f̲acial anomalies) is a unique DNA methylation deficiency disease diagnosed by an extraordinary collection of chromosomal anomalies specifically in the vicinity of the centromeres of chromosomes 1 and 16 (Chr1 and Chr16) in mitogen-stimulated lymphocytes. These aberrations include decondensation of centromere-adjacent (qh) heterochromatin, multiradial chromosomes with up to 12 arms, and whole-arm deletions. We demonstrate that lymphoblastoid cell lines from two ICF patients exhibit these Chr1 and Chr16 anomalies in 61% of the cells and continuously generate 1qh or 16qh breaks. No other consistent chromosomal abnormality was seen except for various telomeric associations, which had not been previously noted in ICF cells. Surprisingly, multiradials composed of arms of both Chr1 and Chr16 were favored over homologous associations and cells containing multiradials with 3 or >4 arms almost always displayed losses or gains of Chr1 or Chr16 arms from the metaphase. Our results suggest that decondensation of 1qh and 16qh often leads to unresolved Holliday junctions, chromosome breakage, arm missegregation, and the formation of multiradials that may yield more stable chromosomal abnormalities, such as translocations. These cell lines maintained the abnormal hypomethylation in 1qh and 16qh seen in ICF tissues. The ICF-specific hypomethylation occurs in only a small percentage of the genome, e.g., ICF brain DNA had 7% less 5-methylcytosine than normal brain DNA. The ICF lymphoblastoid cell lines, therefore, retain not only the ICF-specific pattern of chromosome rearrangements, but also of targeted DNA hypomethylation. This hypomethylation of heterochromatic DNA sequences is seen in many cancers and may predispose to chromosome rearrangements in cancer as well as in ICF.

Prolonged culture of normal chorionic villus cells yields ICF syndrome-like chromatin decondensation and rearrangements.

Untreated cultures from normal chorionic villus (CV) or amniotic fluid-derived (AF) samples displayed dramatic cell passage-dependent increases in aberrations in the juxtacentromeric heterochromatin of chromosomes 1 or 16 (1qh or 16qh). They showed negligible levels of chromosomal aberrations in primary culture and no other consistent chromosomal abnormality at any passage. By passage 8 or 9, 82 ± 7% of the CV metaphases from all eight studied samples exhibited 1qh or 16qh decondensation and 25 ± 16% had rearrangements in these regions. All six analyzed late-passage AF cultures displayed this regional decondensation and recombination in 54 ± 16 and 3 ± 3% of the metaphases, respectively. Late-passage skin fibroblasts did not show these aberrations. The chromosomal anomalies resembled those diagnostic for the ICF syndrome (immunodeficiency, centromeric region instability, and facial anomalies). ICF patients have constitutive hypomethylation at satellite 2 DNA (Sat2) in 1qh and 16qh, generally as the result of mutations in the DNA methyltransferase gene DNMT3B. At early and late passages, CV DNA was hypomethylated and AF DNA was hypermethylated both globally and at Sat2. DNMT1, DNMT3A, or DNMT3B RNA levels did not differ significantly between CV and AF cultures or late and early passages. The high degree of methylation of Sat2 in late-passage AF cells indicates that hypomethylation of this repeat is not necessary for 1qh decondensation. Sat2 hypomethylation may nonetheless favor 1qh and 16qh anomalies because CV cultures, with their Sat2 hypomethylation, displayed 1qh and 16qh decondensation and rearrangements at significantly lower passage numbers than did AF cultures. Also, CV cultures had much higher ratios of ICF-like rearrangements to heterochromatin decondensation in chromosomes 1 and 16. These cultures may serve as models to help elucidate the biological consequences of cancer-associated satellite DNA hypomethylation.

Chromosome Preparation From Cultured Cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births(1,2), 60-80% of all miscarriages(3,4), 10% of stillbirths(2,5), 13% of individuals with congenital heart disease(6), 3-6% of infertility cases(2), and in many patients with developmental delay and birth defects(7). Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance(8,9). Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents(10-13). Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoys fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)(14,15).

High frequencies of ICF syndrome-like pericentromeric heterochromatin decondensation and breakage in chromosome 1 in a chorionic villus sample

Editor—The immunodeficiency, centromeric region instability, and facial anomalies syndrome (ICF) usually involves mutations affecting the catalytic domain in DNMT3B, one of the three human genes known to encode DNA methyltransferases.1-3 ICF always results in defective immunity, a high frequency of chromosomal abnormalities in the vicinity of the centromere (pericentromeric region) of chromosome 1 and/or chromosome 16 in mitogen stimulated lymphocytes, and hypomethylation of a small portion of the genome.4-6 ICF symptoms are manifested often from infancy and this syndrome can cause early childhood death from infections. The DNA sequences targeted for undermethylation in ICF include the heterochromatin adjacent to the centromeres of chromosomes 1 and 16 (1qh and 16qh), where a high incidence of chromatin decondensation, chromosome and chromatid breaks, and rearrangements to form multiradial chromosomes are characteristically seen in mitogen stimulated ICF blood cultures and in ICF lymphoblastoid cell lines.6-9 These aberrations are more common in chromosome 1 than in chromosome 16 and only infrequently observed in chromosome 9.5 8 9 We describe an unusual primary culture from a chorionic villus (CV) biopsy in which a high frequency of ICF-like chromosomal abnormalities was observed. However, follow up indicated that the infant did not have ICF. A 30 year old, gravida 4, para 0, ab 3 white female was referred for genetic counselling and CV sampling because of a previous pregnancy with trisomy 13. Both the patient and her husband were phenotypically normal and healthy apart from their reproductive history. The patients first two pregnancies ended in spontaneous abortion at 15 and 10 weeks. No fetal studies had been performed; parental chromosomes were analysed and reported to be normal. The third pregnancy was found to be affected with trisomy 13 (47,XY,+13) and was then terminated. During the fourth pregnancy, the patient was offered and …

der(1)t(1;19)(p12;p11): A new nonrandom chromosomal abnormality in myelodysplastic syndrome

The der(1)t(1;19)(p12;p11) has not been previously reported in myelodysplastic syndrome (MDS). Fluorescence in situ hybridization (FISH) using chromosome 1- and chromosome 19-specific probes, performed on the bone marrow (BM) cells of this patient confirmed the initial karyotype, i.e., 47,XY,+der(1)t(1;19)(p12;p11).

Phenotypic features of a boy with trisomy of 16q22-->qter due to paternal Y; 16 translocation.

We report on the phenotypic features of a patient with partial trisomy of the long arm of chromosome 16 due to an unbalanced Y;16 translocation (46,X,der[Y]t[Y;16] [q12;q22]pat). The patient was noted to have craniofacial anomalies and developmental delay, but no other major malformations. The father, a balanced Y;16 translocation carrier, has apparently normal fertility.

Acquisition of an oncogenic fusion protein serves as an initial driving mutation by inducing aneuploidy and overriding proliferative defects.

While many solid tumors are defined by the presence of a particular oncogene, the role that this oncogene plays in driving transformation through the acquisition of aneuploidy and overcoming growth arrest are often not known. Further, although aneuploidy is present in many solid tumors, it is not clear whether it is the cause or effect of malignant transformation. The childhood sarcoma, Alveolar Rhabdomyosarcoma (ARMS), is primarily defined by the t(2;13)(q35;q14) translocation, creating the PAX3-FOXO1 fusion protein. It is unclear what role PAX3-FOXO1 plays in the initial stages of tumor development through the acquisition and persistence of aneuploidy. In this study we demonstrate that PAX3-FOXO1 serves as a driver mutation to initiate a cascade of mRNA and miRNA changes that ultimately reprogram proliferating myoblasts to induce the formation of ARMS. We present evidence that cells containing PAX3-FOXO1 have changes in the expression of mRNA and miRNA essential for maintaining proper chromosome number and structure thereby promoting aneuploidy. Further, we demonstrate that the presence of PAX3-FOXO1 alters the expression of growth factor related mRNA and miRNA, thereby overriding aneuploid-dependent growth arrest. Finally, we present evidence that phosphorylation of PAX3-FOXO1 contributes to these changes. This is one of the first studies describing how an oncogene and post-translational modifications drive the development of a tumor through the acquisition and persistence of aneuploidy. This mechanism has implications for other solid tumors where large-scale genomics studies may elucidate how global alterations contribute to tumor phenotypes allowing the development of much needed multi-faceted tumor-specific therapeutic regimens.

Delayed diagnosis of a patient with Usher syndrome 1C in a Louisiana Acadian family highlights the necessity of timely genetic testing for the diagnosis and management of congenital hearing loss

Advances in sequencing technologies and increased understanding of the contribution of genetics to congenital sensorineural hearing loss have led to vastly improved outcomes for patients and their families. Next-generation sequencing and diagnostic panels have become increasingly reliable and less expensive for clinical use. Despite these developments, the diagnosis of genetic sensorineural hearing loss still presents challenges for healthcare providers. Inherited sensorineural hearing loss has high levels of genetic heterogeneity and variable expressivity. Additionally, syndromic hearing loss (hearing loss and additional clinical abnormalities) should be distinguished from non-syndromic (hearing loss is the only clinical symptom). Although the diagnosis of genetic sensorineural hearing loss can be challenging, the patient’s family history and ethnicity may provide critical information, as certain genetic mutations are more common in specific ethnic populations. The early identification of the cause of deafness can benefit patients and their families by estimating recurrence risks for future family planning and offering the proper interventions to improve their quality of life. Collaboration between pediatricians, audiologists, otolaryngologists, geneticists, and other specialists are essential in the diagnosis and management of patients with hearing disorders. An early diagnosis is vital for proper management and care, as some clinical manifestations of syndromic sensorineural hearing loss are not apparent at birth and have a delayed age of onset. We present a case of Usher syndrome (congenital deafness and childhood-onset blindness) illustrating the challenges encountered in the diagnosis and management of children presenting with congenital genetic sensorineural hearing loss, along with helpful resources for clinicians and families.

Detailed Audiological Evaluation of a Patient with Xeroderma Pigmentosum with Neural Degeneration

Background: Xeroderma pigmentosum (XP) is a rare autosomal recessive condition characterized by extreme sensitivity to ultraviolet light. Individuals with XP lack the ability to repair DNA (deoxyribonucleic acid) damage caused by ultraviolet radiation, leading to sunburn and increased susceptibility to skin cancers. Approximately 25% of patients also exhibit neural degeneration, which includes progressive mental deterioration, cortical thinning, and sensorineural hearing loss. Purpose: Herein, we describe the audiological and genetic findings in a patient with XP subtype D with neural degeneration and hearing loss. Research Design: This is a case report of a patient with XP subtype D, type 1 diabetes, and some clinical features typical of Charcot‐Marie‐Tooth disease. Data Collection and Analysis: We obtained audiological evaluations over a course of 11 yr, including serial audiograms, auditory processing disorders evaluations, and electrophysiological testing. Results: Hearing sensitivity has progressed from a unilateral mild high‐frequency sensorineural hearing loss to a bilateral sloping moderate to severe/profound sensorineural hearing loss. In addition to the dramatic decline in hearing sensitivity, the patient demonstrates global auditory processing deficits, indicating a central component to his hearing loss. Conclusion: These findings emphasize the importance of the contribution of audiological evaluations to the diagnosis of a genetic disorder. Periodic evaluations of hearing sensitivity and auditory processing can provide information on disease progression in patients with XP with neural degeneration.