Genome Rearrangements Detected by SNP Microarrays in Individuals with Intellectual Disability Referred with Possible Williams Syndrome

Intellectual disability (ID) affects 2-3% of the population and may occur with or without multiple congenital anomalies (MCA) or other medical conditions. Established genetic syndromes and visible chromosome abnormalities account for a substantial percentage of ID diagnoses, although for approximately 50% the molecular etiology is unknown. Individuals with features suggestive of various syndromes but lacking their associated genetic anomalies pose a formidable clinical challenge. With the advent of microarray techniques, submicroscopic genome alterations not associated with known syndromes are emerging as a significant cause of ID and MCA.High-density SNP microarrays were used to determine genome wide copy number in 42 individuals: 7 with confirmed alterations in the WS region but atypical clinical phenotypes, 31 with ID and/or MCA, and 4 controls. One individual from the first group had the most telomeric gene in the WS critical region deleted along with 2 Mb of flanking sequence. A second person had the classic WS deletion and a rearrangement on chromosome 5p within the Cri du Chat syndrome (OMIM:123450) region. Six individuals from the ID/MCA group had large rearrangements (3 deletions, 3 duplications), one of whom had a large inversion associated with a deletion that was not detected by the SNP arrays.Combining SNP microarray analyses and qPCR allowed us to clone and sequence 21 deletion breakpoints in individuals with atypical deletions in the WS region and/or ID or MCA. Comparison of these breakpoints to databases of genomic variation revealed that 52% occurred in regions harboring structural variants in the general population. For two probands the genomic alterations were flanked by segmental duplications, which frequently mediate recurrent genome rearrangements; these may represent new genomic disorders. While SNP arrays and related technologies can identify potentially pathogenic deletions and duplications, obtaining sequence information from the breakpoints frequently provides additional information

BAC-FISH refutes report of an 8p22–8p23.1 inversion or duplication in 8 patients with Kabuki syndrome

BACKGROUND: Kabuki syndrome is a multiple congenital anomaly/mental retardation syndrome. The syndrome is characterized by varying degrees of mental retardation, postnatal growth retardation, distinct facial characteristics resembling the Kabuki actor's make-up, cleft or high-arched palate, brachydactyly, scoliosis, and persistence of finger pads. The multiple organ involvement suggests that this is a contiguous gene syndrome but no chromosomal anomalies have been isolated as an etiology. Recent studies have focused on possible duplications in the 8p22–8p23.1 region but no consensus has been reached. METHODS: We used bacterial artificial chromosome-fluorescent in-situ hybridization (BAC-FISH) and G-band analysis to study eight patients with Kabuki syndrome. RESULTS: Metaphase analysis revealed no deletions or duplications with any of the BAC probes. Interphase studies of the Kabuki patients yielded no evidence of inversions when using three-color FISH across the region. These results agree with other research groups' findings but disagree with the findings of Milunsky and Huang. CONCLUSION: It seems likely that Kabuki syndrome is not a contiguous gene syndrome of the 8p region studied

Down-Regulation of Replication Factor C-40 (RFC40) Causes Chromosomal Missegregation in Neonatal and Hypertrophic Adult Rat Cardiac Myocytes

BACKGROUND: Adult mammalian cardiac myocytes are generally assumed to be terminally differentiated; nonetheless, a small fraction of cardiac myocytes have been shown to replicate during ventricular remodeling. However, the expression of Replication Factor C (RFC; RFC140/40/38/37/36) and DNA polymerase δ (Pol δ) proteins, which are required for DNA synthesis and cell proliferation, in the adult normal and hypertrophied hearts has been rarely studied. METHODS: We performed qRT-PCR and Western blot analysis to determine the levels of RFC and Pol δ message and proteins in the adult normal cardiac myocytes and cardiac fibroblasts, as well as in adult normal and pulmonary arterial hypertension induced right ventricular hypertrophied hearts. Immunohistochemical analyses were performed to determine the localization of the re-expressed DNA replication and cell cycle proteins in adult normal (control) and hypertrophied right ventricle. We determined right ventricular cardiac myocyte polyploidy and chromosomal missegregation/aneuploidy using Fluorescent in situ hybridization (FISH) for rat chromosome 12. RESULTS: RFC40-mRNA and protein was undetectable, whereas Pol δ message was detectable in the cardiac myocytes isolated from control adult hearts. Although RFC40 and Pol δ message and protein significantly increased in hypertrophied hearts as compared to the control hearts; however, this increase was marginal as compared to the fetal hearts. Immunohistochemical analyses revealed that in addition to RFC40, proliferative and mitotic markers such as cyclin A, phospho-Aurora A/B/C kinase and phospho-histone 3 were also re-expressed/up-regulated simultaneously in the cardiac myocytes. Interestingly, FISH analyses demonstrated cardiac myocytes polyploidy and chromosomal missegregation/aneuploidy in these hearts. Knock-down of endogenous RFC40 caused chromosomal missegregation/aneuploidy and decrease in the rat neonatal cardiac myocyte numbers. CONCLUSION: Our novel findings suggest that transcription of RFC40 is suppressed in the normal adult cardiac myocytes and its insufficient re-expression may be responsible for causing chromosomal missegregation/aneuploidy and in cardiac myocytes during right ventricular hypertrophy

BAC-FISH refutes report of an 8p22–8p23.1 inversion or duplication in 8 patients with Kabuki syndrome

Abstract Background Kabuki syndrome is a multiple congenital anomaly/mental retardation syndrome. The syndrome is characterized by varying degrees of mental retardation, postnatal growth retardation, distinct facial characteristics resembling the Kabuki actor's make-up, cleft or high-arched palate, brachydactyly, scoliosis, and persistence of finger pads. The multiple organ involvement suggests that this is a contiguous gene syndrome but no chromosomal anomalies have been isolated as an etiology. Recent studies have focused on possible duplications in the 8p22–8p23.1 region but no consensus has been reached. Methods We used bacterial artificial chromosome-fluorescent in-situ hybridization (BAC-FISH) and G-band analysis to study eight patients with Kabuki syndrome. Results Metaphase analysis revealed no deletions or duplications with any of the BAC probes. Interphase studies of the Kabuki patients yielded no evidence of inversions when using three-color FISH across the region. These results agree with other research groups' findings but disagree with the findings of Milunsky and Huang. Conclusion It seems likely that Kabuki syndrome is not a contiguous gene syndrome of the 8p region studied.</p

Immunohistochemical analyses revealed RFC40, PCNA, Cyclin A, phosphor-Aurora A/B/C kinase and pHis3 positive CMs nuclei in hypertrophied RV.

<p>Control (Con) and SUHxNx-5 wks (5 wks) RV sections (n = 3) were incubated with polyclonal anti-RFC40 (A) anti-PCNA (B), anti-Cyclin A (C), anti-phosho Aurora A/B/C kinase (p-Aurora-A/B/C; D) and anti-H3P (E) antibodies, respectively, and then with Alexa-568-labeled anti-rabbit and Alex-488-labeled anti-mouse secondary antibodies, respectively. Images were collected using an Olympus Plan x20/NA 0.25 Phi objective. In each experiment, all data were collected at identical imaging settings. Arrows indicates positive CMs.</p

Chromosomal missegregation/Aneuploidy was observed in the CM nuclei from the hypertrophied RVs.

<p>Control (Con) and SuHxNx-5 wks (5 wks) RV sections were used to perform FISH analyses by co-hybridization of the tissues with the Cen12-ROX probe (Red). Nuclei were counterstained with DAPI antifade (Blue) and slides were imaged with Spectral Imaging Software using an Olympus BX61 microscope with 1000X magnification. Two FISH signals for Cen12-ROX was observed in the CM nuclei from the control RV (A-i), however, one (B-i) and three (C-i) signals were observed in the CM nuclei from the hypertrophied RVs. Localization of the CM nuclei in the control (A-ii) and hypertrophied (B–C-ii) RVs were confirmed by performing immunohistochemical analyses for cardiac Troponin I (Green). (D) Graph represents the number of FISH signals for Cen12-ROX observed per CM nuclei in the control and hypertrophied RVs. Fifty CM nuclei were measured from three individual animals in control and hypertrophied RVs. (E) Diving CM nuclei undergoing chromosomal missegregation in the hypertrophied RVs, with one nucleus receiving three sister chromatids while the other receiving only one chromatid.</p

Endogenous knock-down of RFC40 in rat neonatal cardiac myocytes results in chromosomal missegregation/aneuploidy.

<p> Rat Neonatal cardiac myocytes (RNCMs) were isolated as described previously and grown in 12 well plates and 2-chambered slides for 48 hr. RNCMs were then treated with non-targeting-siRNA (NT) and On-Target plus smartpool RFC40-siRNA respectively, for 72 hr. (<b>A–D</b> and <b>F–G</b>) RNCMs grown in 2-chambered slides were subjected to FISH analysis following treatment with RFC40-siRNA. Untransfected (UT) and RFC40-siRNA-RNCM slides were co-hybridization with the Cen12-ROX probe (Red). Nuclei were counterstained with DAPI antifade (Blue). RFC40 knock-down was confirmed by performing immunohistochemical analyses for RFC40 (A–D-ii and F–G-ii; Green) in each sample. Merge images are shown in A–D-iii and F–G-iii. Panels A–D represents RNCM nuclei aneuploidy and panels F–G represent chromosomal missegregation. White arrows point to Cen12-ROX signals. Yellow arrow points to micronuclei in G-i & iii. (E) Graph represents the number of signals for Cen12-ROX observed per RNCM nuclei in the UT and RFC40-siRNA treated samples. Fifty RNCM nuclei in control and RFC40-siRNA treated samples from three individual experiments were measured. (H) Before lysing the cells grown in 12-well plates for western blot analyses (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039009#pone.0039009.s004" target="_blank">Fig. S4</a>), the RNCMs (n = 3) were trypsinized, resuspended in 1X PBS and counted using a hemocytometer. Graph represents the number of RNCMs vs the different RNCM treated samples. Values are mean ± SE. * indicates P<0.05 vs. Untransfected (UT).</p

DNA replication proteins are re-expressed in hypertrophied RV.

<p>Total protein lysates (50 µg) obtained from LV and RV tissues (n = 5) of the control (Con), SUHx-3 wks (3 wks) and SUHxNx-5 wks (5 wks) were analyzed by 12% or 9% SDS-polyacrylamide gels. (<b>A</b>) Expression of RFC37, RFC38, RFC40, RFC140, PCNA and p125 proteins was determined by Western blot analyses. Whole hearts isolated from 15 day-old fetus (n = 5) were used as a positive control (Ft). GAPDH was used as the loading control. (<b>B and C</b>) Graphs represent summary data for the protein expression of RFC40 and p125 proteins in LV (B) and RV (C) normalized by GAPDH (n = 5). Values are mean ± SE. *indicates P<0.05 vs. control.[/LOOSEST]</p

Expression of DNA replication proteins in control and hypertrophied hearts.

<p>All values are mean ±SD; ns-non-significant;</p>*<p>P<0.05 vs control.</p