Asynchronous Replication in Lymphocytes from Patients with Inflammatory Bowel Disease and Primary Sclerosing Cholangitisby Ido Laish, Tal Biron-Shental, Hila Katz, Meytal Liberman, Yona Kitay-Cohen, Fred Meir Konikoff, Aliza Amiel

Cytogenetic and Genome Research


Molecular Biology / Genetics (clinical) / Genetics


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Original Article

Cytogenet Genome Res

DOI: 10.1159/000381406

Asynchronous Replication in Lymphocytes from

Patients with Inflammatory Bowel Disease and

Primary Sclerosing Cholangitis

Ido Laish  a, e Tal Biron-Shental  b, e Hila Katz  c Meytal Liberman  c

Yona Kitay-Cohen  a Fred Meir Konikoff  a, e Aliza Amiel  c, d   a   Institute of Gastroenterology and Hepatology, b   Department of Obstetrics and Gynecology, and c   Institute of

Genetics, Meir Medical Center, Kfar Saba , d   Faculty of Life Sciences, Bar Ilan University, Ramat Gan , and e   Sackler

School of Medicine, Tel Aviv University, Tel Aviv , Israel control of structural homologous loci in PSC, and that this phenomenon may be correlated with the inflammation-induced malignant potential of this condition. © 2015 S. Karger AG, Basel

Primary sclerosing cholangitis (PSC) is a chronic, cholestatic disorder that involves both intra- and extrahepatic bile ducts. A combination of inflammatory and fibrotic changes leads to a typical cholangiographic pattern of multiple, widespread bile duct strictures and dilatations, as well as histological features of periportal inflammation and a fibro-obliterative process [Boberg and Lind, 2011].

The pathogenesis of PSC remains poorly understood, but up to 80% of patients have concomitant inflammatory bowel disease (IBD), mostly ulcerative colitis (UC); fewer have Crohn’s disease (CD). One of the hallmarks of PSC, a consequence of the continuous inflammation, is the increased tendency to develop hepatobiliary and extrahepatic malignancies [Bergquist et al., 2002]. The risks of colorectal cancer and dysplasia are also higher in IBD with PSC than in IBD alone [Bergquist et al., 2002; Soetikno et al., 2002; Broomé and Bergquist, 2006].

One of the assumptions in Mendelian genetics is that structurally homologous chromosomal segments in

Key Words

Asynchronous replication · Inflammatory bowel disease ·

Monoallelic expression · Primary sclerosing cholangitis ·

Replication pattern


Primary sclerosing cholangitis (PSC) and inflammatory bowel disease (IBD) are associated chronic inflammatory diseases with malignant potential. Loss of replication synchrony during the S-phase of the cell cycle has been shown to be linked to several malignant and premalignant states. This study evaluated temporal differences in replication timing between these diseases. The replication pattern of peripheral blood lymphocytes obtained from patients with PSC and IBD and healthy individuals was analyzed by fluorescence in situ hybridization (FISH) in 2 pairs of alleles, in 15qter and 13qter. Asynchrony was determined by the presence of 1 single and 1 set of double dots in the same cell. Samples from subjects with PSC showed significantly greater temporal differences in replication timing, in contrast to the high level of synchrony observed in samples from healthy individuals (p = 0.045). Samples from IBD patients exhibited a nonsignificant increase in replication asynchrony. We believe that these results reflect impairment in the replication

Accepted: February 19, 2015 by M. Schmid

Published online: April 11, 2015

Aliza Amiel, PhD

Institute of Genetics, Meir Medical Center 59 Tschernihovsky St.

Kfar Saba 44281 (Israel)

E-Mail amielaliza   @ © 2015 S. Karger AG, Basel 1424–8581/15/0000–0000$39.50/0

D ow nl oa de d by :



M ed ica l C en te r L ib ra ry 12 8. 12 2. 25 3. 21 2 - 5 /2 9/ 20 15 1 0: 59 :0 4




Cytogenet Genome Res

DOI: 10.1159/000381406 2 mammals usually maintain functional symmetry, and the 2 alleles are expressed concomitantly in what is called biallelic expression. However, a subset of the genes shows allele-specific expression (monoallelic expression), in which only 1 allele retains expression capability while the other is silent [Ohlsson et al., 1998; Mostoslavsky et al., 2001; Goldmit and Bergman, 2004]. Monoallelically expressed genes include imprinted genes [Feinberg, 1998], genes subjected to X-chromosome inactivation [Lyon, 1988; Heard et al., 1997], and genes displaying allelic exclusion [Chess, 2005]. The mechanisms involved in the selection of an allele for monoallelic expression are largely unknown, but the asymmetry clearly results from the 2 alleles retaining different epigenetic profiles, in which asynchronous DNA replication plays a central role [Goldmit and Bergman, 2004].

We used the fluorescence in situ hybridization (FISH) replication-assay technique, described by Selig et al. [1992], to study the replication pattern of a given DNA sequence. Accordingly, an unreplicated DNA sequence reveals a single fluorescent signal (singlet; S) at interphase, while a replicated sequence gives rise to a doubled signal (doublet; D). Thus, in a population of replicating cells, a high frequency of nuclei with 2 similar hybridization signals, either 2 singlets (SS) or 2 doublets (DD), would indicate a pair of allelic loci that replicates synchronously. On the other hand, allelic loci that replicate asynchronously are revealed by nuclei containing 2 different hybridization signals, a singlet and a doublet (SD) ( fig. 1 ). In an unsynchronized population of replicating cells, the frequency of cells at a given stage expresses the relative duration of that stage. Thus, a high frequency of

SD cells shows asynchrony in replication timing of the 2 allelic counterparts. A high frequency of DD cells indicates early replication of the identified locus, and a high frequency of SS cells points to late replication.

Using this method to estimate the replication timing of an allele relative to its counterpart within the same cell, several studies have clearly shown close association between the replication timing of a given chromosome region during S-phase and the transcription activity of genes in this region. In biallelically expressed genes, the 2 alleles replicate synchronously, while in monoallelic expression, an asynchronous pattern of allele replication occurs: an early replication of the potentially active allele and late replication of the silent one [Kitsberg et al., 1993; Boggs and