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Review articles

Cytogenetic studies of chromosomal breakage diseases

Eid, Maha M.a; Temtamy, Samia A.b

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Middle East Journal of Medical Genetics: January 2013 - Volume 2 - Issue 1 - p 11-22
doi: 10.1097/01.MXE.0000422776.59740.e2
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Chromosome breaks occur either as a result of damage to DNA (by e.g. radiation or chemicals) or as part of the mechanism of recombination. Chromosome breaks occurring in the G2 phase of the cell cycle are manifested as chromatid breaks, whereas breaks occurring in the G1 phase appear later as chromosome breaks. Cells have enzyme systems that recognize and, if possible, repair broken chromosome ends. The chromosomal breakage may disrupt an important gene or the break may affect the expression of a gene even though it does not disrupt the coding sequence. It may separate a gene from a control element or it may place the gene in an inappropriate chromatin environment.

The aim of this review is to outline the different chromosome breakage disorders.

Numerous genetic syndromes are marked by chromosome instability that is strikingly associated with various cancers. They represent a group of autosomal recessive heritable disorders that share the common characteristics of increased spontaneous or induced chromosomal breakage. Among these diseases are Fanconi’s anemia (FA), ataxia telangiectasia (TS), Nijmegen’s breakage syndrome (NBS), Bloom’s syndrome (BS), Werner’s syndrome (WS), and Rothmund–Thompson’s syndrome.

In addition to the classical chromosomal breakage syndromes, there are several other disorders that show chromosomal breakage and instabilities. Cancer is one of a major group that shows chromosomal instability, and also other diseases such as chronic inflammatory disease and neurodegenerative disorders. Mitochondrial disease (MD) may show instability, but not as a constant feature of the disease.

In conclusion, several cytogenetic techniques have been emerged to detect chromosomal breakage and instabilities, such as sister-chromatid exchanges (SCE), fluorescent in-situ hybridization (FISH), micronucleus (MN), and comet assay. It is important to properly choose the cytogenetic test according to the disease to be investigated. Analysis of the results could be very helpful in the diagnosis and management of cases.

Cytogenetic techniques used to detect chromosome breakage

Refinements in cytogenetic techniques over the past 30 years have allowed the increasingly sensitive detection of chromosome abnormalities. Chromosome aberration (CA) assays are used to detect chromosome breakage in somatic and germ cells by direct observation of the chromosomal damage during metaphase analysis or by indirect observation of chromosomal fragments. Thus, various types of cytogenetic changes can be detected such as structural CAs and SCE (Doherty et al., 2012).

Chromosome banding technique

Different patterns of breakage can be recognized in genetic disorders using this simple technique.

Chromosome breaks

Chromosome or chromatid breaks have been detected in the cultured lymphocyte of diseases with chromosomal breakage such as FA. Analysis of the distribution of the breakage is informative; the distribution of breakage appeared to be nonrandom in many disorders and localized on sites related to fragile sites (FSs), oncogenes, or tumor suppressor genes (Fundia et al., 1994; Fundia et al., 1996; Schoder et al., 2010).

Isochromatid exchange and multiradial configurations

Isochromatid exchange yielding triradial and quadradial figures (Fig. 1) is characteristic for the diagnosis of FA either spontaneously on a few occasions or after induction of breakage by clastogenetic substances such as diepoxybutane or mitomycin C (MMC) (Auerbach et al., 1989). Triradial exchange figures are produced from two breaks on one chromosome and one break on a second chromosome (Danford, 2012). However, multiradial figures are specific for the diagnosis of the immunodeficiency, centromeric region instability, and facial anomalies syndrome (ICF) (Ehrlich et al., 2006).

Figure 1
Figure 1:
Arrow shows the isochromatid exchange giving rise to a triradial figure in a metaphase spread of a patient with Fanconi’s anemia.

Premature centromere separation

The term premature centromere separation (PCS) describes the cytogenetic abnormalities observed in standard cytogenetic preparations with prematurely separating centromeres during metaphase rather than in anaphase. Many chromosomes show a ‘railroad track’ appearance as a result of the absence of the primary constriction and the presence of ‘puffing’ or ‘repulsion’ at the heterochromatic regions around the centromeres and nucleolar organizer regions. It is a diagnostic feature in Roberts syndrome (RBS) (Gordillo et al., 2009).

Sister-chromatid exchange

SCE is defined as a symmetrical exchange at one locus between sister chromatids, which does not alter the overall chromosome morphology (Perry and Wolff, 1974; Jeyapradha et al., 2011). It was discovered originally by Taylor et al. (1957). Normally, five to eight SCE per cell are present (Fig. 2), but this is considerably increased in patients with hereditary disorders such as BS, xeroderma pigmentosum (XP) as well as malignancies and premalignancies (Fig. 3).

Figure 2
Figure 2:
Normal level of sister-chromatid exchange.
Figure 3
Figure 3:
Increased level of sister-chromatid exchange.

By adapting the comet-FISH technique (CO-FISH) for the specific measurement of SCEs in the telomeres, subtelomeres, and body of chromosomes, it was found that SCEs are highly concentrated within the most distal 100 kb of the chromosome (Rudd et al., 2007).

Fluorescent in-situ hybridization

FISH is a straightforward technique that essentially consists of hybridizing a DNA probe to its complementary sequence on chromosomal preparations fixed previously on slides to be visualized in situ by microscopic analysis. It provides an intermediate degree of resolution between DNA analysis and chromosomal investigations (Volpi and Bridger, 2008).

Different FISH techniques have been designed to detect chromosomal breakage as follows.

Quantitative-fluorescent in-situ hybridization

The quantitative-fluorescent in-situ hybridization (Q-FISH) methodology allows the measurement of probe signal intensity. Q-FISH was initially invented by Lansdorp and collaborators (Martens et al., 1998). This method has been used mainly for the measurement of the number of telomere repeats on particular chromosome ends. Q-FISH has become an important tool to study the role of telomeres in aging and cancer.

Telomeres can be visualized directly under the microscope by FISH. Short-for-age leukocyte telomeres were observed in several human diseases, including FA (Fig. 4) (Hanson et al., 2001), aplastic anemia (Calado, 2009), dyskeratosis congenita (DC), pulmonary fibrosis, liver disease (Calado and Young, 2009), and cancer (Murnan, 2010).

Figure 4
Figure 4:
Fluorescent in-situ hybridization using an all-human telomere probe shows telomeric ends without signals in a patient with Fanconi’s anemia.

Split-signal fluorescent in-situ hybridization

Split-signal FISH was developed for the detection of chromosomal breaks. Split-signal or break-apart probes use differently colored probes on both sides of a known break point region, resulting in a fused signal in normal cells, and two different single colors when a chromosomal break occurs. This approach is advantageous in tissue sections as each single-colored signal indicates a specific chromosomal break (Van Rijk et al., 2010). Split-signal FISH was initially introduced for the detection of all types of MLL gene translocations in acute lymphoblastic leukemia and acute myeloid leukemia using only a single FISH test (Van der Burg et al., 2004).

Cytokinesis blocked micronucleus cytom assay

In this method, CAs are detected indirectly through chromatin loss from the nucleus, leading to MN formation in the cytoplasm of the cell (Kirsch-Volders et al., 2003). MN is expressed only in dividing cells by adding cytochalasin-B, an inhibitor of the mitotic spindle that prevents cytokinesis and allows the identification of cells that have completed one nuclear division by their binucleated appearance, to cell cultures (Fenech, 2007). The cytokinesis-blocked micronucleus (CBMN) assay has three main products: MN (Fig. 5), nuclear buds (NBUD) (Fig. 6), and nucleoplasmic bridges (NPB) (Fig. 7) (Fenech, 2006).

Figure 5
Figure 5:
Arrow shows micronucleus.
Figure 6
Figure 6:
Arrow shows nuclear buds.
Figure 7
Figure 7:
Arrow shows nucleoplasmic bridges.

Micronucleus origin

The MN mainly originate from acentric chromosome fragments, acentric chromatid fragments, or whole chromosomes that fail to be included in the daughter nuclei at the completion of telophase during mitosis because they did not attach properly with the spindle during the segregation process in anaphase. These displaced chromosomes or chromosome fragments are eventually enclosed by a nuclear membrane and, except for their smaller size, are morphologically similar to nuclei after conventional nuclear staining (Fenech, 2007).

Origin of nuclear budding

The process of nuclear budding occurs during the S phase and the NBUDs are characterized by having the same morphology as an MN, with the exception that they are linked to the nucleus by a narrow or a wide stalk of nucleoplasmic material depending on the stage of the budding process. NBUD is a biomarker of elimination of amplified DNA and/or DNA repair complexes. The amplified DNA is selectively localized to specific sites at the periphery of the nucleus and is eliminated through nuclear budding during the S phase of the cell cycle (Shimizu et al., 2000).

NBUD have also been shown to be formed when an NPB between two nuclei breaks and the remnants shrink back toward the nuclei (Pampalona et al., 2010).

Nucleoplasmic bridge origin

NPBs occur when centromeres of dicentric chromosomes are pulled to opposite poles of the cell at anaphase. In the CBMN assay, binucleated cells with NPBs are allowed to accumulate because cytokinesis is inhibited and the nuclear membrane is eventually formed around the chromosomes, allowing an anaphase bridge to be observed as an NPB.

Various mechanisms could lead to NPB formation following DNA misrepair of strand breaks in DNA. Typically, a dicentric chromosome and an acentric chromosome fragment are formed that result in the formation of an NPB and an MN, respectively. Misrepair of DNA strand breaks could also lead to the formation of dicentric ring chromosomes that could also result in the formation of NPB (Thomas et al., 2003).

Micronucleus-fluorescent in-situ hybridization

FISH analysis of MN is based on the achievements of interphase FISH. The MN test was combined successfully with different kinds of DNA FISH probes that recognize centromeres, other chromosome-specific regions, and whole chromosomes inside micronuclei and the main nuclei. The analysis of MN combined with centromeric DNA probes for all chromosomes allows differentiation between centromere-negative MN and centromere-positive MN. This approach also allows the detection of nondisjunctional events (i.e. unequal distribution of homologous chromosomes in daughter nuclei) in binucleated cells. The application of other chromosome region-specific and whole chromosome paint probes allows the evaluation of their participation in the formation of spontaneous and induced MN (Fig. 8) (Hovhannisyan, 2010).

Figure 8
Figure 8:
Comet assay shows a normal cell without DNA damage.

Comet assay

The comet assay is a rapid and very sensitive fluorescent microscopy-based method for the measurement of DNA damage and repair at the level of individual cells (Collins et al., 2004; Collins et al., 2008). In this assay, cells are embedded in agarose, lysed, and then electrophoresed. Negatively charged broken DNA strands exit from the lysed cell under the electric field and form a comet with a ‘head’ and a ‘tail’ (Figs 8 and 9). The amount of DNA in the tail, relative to the head, is proportional to the amount of strand breaks (Olive and Banáth, 2006). It allows to identify mainly early, still repairable, moderate DNA damage and can be used in almost any tissue (Speit and Hartmann, 2006). The comet assay has been applied to detect DNA damage in some genetic disorders such as FA and Down’s syndrome (Maluf and Erdtmann, 2001; Mohseni-Meybodi et al., 2009).

Figure 9
Figure 9:
Comet assay of cell culture with mercury chloride added to it that induced DNA damage.

Comet-fluorescent in-situ hybridization

The CO-FISH technique is a useful tool to detect overall and region-specific DNA damage and repair in individual cells. The combination of both techniques has been applied in particular for the detection of site-specific breaks in DNA regions that are relevant for the development of different diseases (Glei et al., 2009). Various DNA probes were applied successfully with the comet assay for analysis of damage and repair of specific genome loci (genes, chromosomes, and chromosome regions) (Rapp et al., 2000; Glei et al., 2009). Microscopic evaluation of the CO-FISH images includes a record of the number of probe signals and their localization on the comet. The position of the fluorescence signals indicates whether the sequence of interest lies within the undamaged (head) or within a damaged (tail) region of DNA. Repositioning of gene-specific signals from the tail to the head provides evidence for repair of all the lesions within and around the locus of interest (Shaposhnikov et al., 2009). One of the applications of CO-FISH is the use of directly labeled telomere-specific peptide nucleic acid hybridization probes. Fragmentation of telomeres and subtelomeric regions was highly specifically detected by this approach (Arutyunyan et al., 2004).

Diseases with chromosomal breakage

Many diseases, whether inherited or not, show chromosomal breakage. The main group in these disorders is the chromosomal instability syndromes. Apart from chromosomal instability syndromes, several other diseases show chromosomal breakage, such as cancer, chronic inflammatory disease, MDs, and others.

Chromosome instability syndromes

A defect of DNA repair underlies the chromosome instability syndromes, also known as chromosome breakage syndromes (Taylor, 2001). The instability refers to the predisposition of the chromosome to undergo rearrangement or to show other abnormal cytogenetic behavior, which might need a special cytogenetic technique to identify it. The classic chromosome instability syndromes are FA, BS, AT and NBS, and XP. Susceptibility to cancer is a common feature in these syndromes. Also, chromosome instability has been reported as an occasional observation in quite a number of known conditions; among these are Cockayne’s syndrome (CS), Rothmund–Thomson’s syndrome, and Cornelia de Lange’s syndrome (CDLS) (Gardner et al., 2004).

Classic chromosome breakage syndromes

Fanconi anemia

Clinical findings: The characteristic features of FA (Online Mendelian Inheritance in Man #227650) include short stature, abnormal skin pigmentation, radial ray defects, microphthalmia, and renal anomaly. Hematological findings are thrombocytopenia, leukopenia, and anemia. Occasionally, myelodysplastic syndrome or acute myeloid leukemia is the initial manifestation (Gardner et al., 2004; Shimamura and Alter, 2010).

Genetic background: FA-related genes are inherited in an autosomal recessive manner, except for mutations in FANCB, which are inherited in an X-linked manner. To date, 15 FA genes have been identified, the most frequent being FANCA, FANCC, FANCG, and FANCD2 (De Winter and Joenje, 2009; Soulier, 2011). The gene products of these loci contribute toward the control of cellular DNA repair (Tischkowitz and Hodgson, 2003).

Cytogenetic findings: The diagnosis of FA rests on cytogenetic testing for increased chromosomal breakage and radial figures in the presence of diepoxybutane or MMC (Auerbach, 1993). Study of the telomeric ends using direct FISH in FA patients indicated excessive telomeric shortening (Hanson et al., 2001). Also, the genomic instability in FA has been assessed by a micronucleus and comet assay (Maluf and Erdtmann, 2001).

Bloom syndrome

Clinical findings: BS (OMIM#210900) is characterized by intrauterine growth retardation, dolicocephaly, bird-like facies, sun-sensitive erythema, skin pigmentation abnormalities, a high-pitch voice, and immunodeficiency associated with repeated infections (German, 1995). Increased susceptibility to development of malignancy is common in BS (German, 1997).

Genetic background: BS is an autosomal recessive disorder characterized by marked genetic instability. The BLM gene is the only gene identified so far (Sanz and German, 2010). It is located on chromosome 15q26.1. The BLM protein interacts with other proteins to maintain genomic integrity and suppress the occurrence of SCE. Mutation in the BLM gene leads to increased SCE (Wang et al., 2000).

Cytogenetic findings: The main cytogenetic features of BS are increased levels of SCE, which is a diagnostic cytogenetic finding for BS with a mean of 40–100 per metaphase (vs. <10 in controls) (Gardner et al., 2004; Sanz and German, 2010).

Ataxia telangiectasia

Clinical findings: AT (OMIM #208900) is suspected in children who show progressive cerebellar dysfunction between the ages of 1 and 4 years. Patients presented with gait and truncal ataxia, head tilting, slurred speech, and oculomotor apraxia. In addition, the patients have oculocutaneous telangiectasia, immunodeficiency, and increased predisposition to cancer (Chun and Gatti, 2004).

Genetic background: AT is an autosomal recessive disorder. It occurs because of homozygosity or compound heterozygosity mutation of the ATM gene, located on 11q22.3. The ATM gene plays a key role in responding to double-stranded DNA damage by halting the cell cycle until the damage is corrected (Gardner et al., 2004; Gatti, 2010).

Cytogenetic findings: Translocation between chromosomes 7 and 14 was found in 5–15% of cells in peripheral blood of individuals with AT. The break points are commonly located at 14q11 (the T-cell receptor-α locus) and at 14q32 (the B-cell receptor locus) (Stumm et al., 2001). Another cytogenetic finding is telomeric fusion in some patients. Also, there is increased sensitivity of the chromosomes when exposed to radiomimetic drugs such as bleomycin (Kojis et al., 1991).

Nijmegen breakage syndrome

Clinical findings: NBS (OMIM #251260) is characterized by progressive microcephaly, intrauterine growth retardation, short stature, immunodeficiency leading to recurrent sinopulmonary infections, an increased risk for cancer, and premature ovarian failure in females. About 35% of those reported may develop malignancies between the ages of 1 and 34 years, with the risk being the highest for B-cell lymphomas (Gardner et al., 2004; Concannon and Gatti, 2011).

Genetic background: It is inherited in an autosomal recessive manner. The causative gene is NBS1; it is located on chromosome 8q21 and interacts with the ATM gene. The NBS1 gene encodes protein that senses abnormal DNA structures and monitors postreplication DNA repair (Gardner et al., 2004).

Cytogenetic findings: Inversions and translocations involving chromosomes 7 and 14 are observed in lymphocytes in 10–50% of metaphases. The break points most commonly involved are 7p13, 7q35, 14q11, and 14q32, which are the loci for immunoglobulin and T-cell-receptor genes. Also, chromosomes from patients with NBS have increased sensitivity to irradiation (Van der Burget et al., 1996).

Immunodeficiency, centromeric region instability, facial anomalies syndrome (OMIM #242860)

Clinical findings: The immunodeficiency always results in severe recurrent infections, and is often seen in early childhood as the presenting finding in this syndrome (De Ravel et al., 2001). The typical facial features are a broad flat nasal bridge, hypertelorism, and epicanthic folds. Patients may also have mental retardation and neurologic abnormalities, intrauterine growth retardation, and skin pigmentation abnormalities (Wijmenga et al., 2000; Ehrlich et al., 2006).

Genetic background: ICF is a rare autosomal recessive disease. It is caused by mutations in DNMT3B, located on 20q11-q13. It seems likely that the DNA hypomethylation is responsible for the disease (Ehrlich et al., 2006).

Cytogenetic findings: These are whole-arm deletions and pericentromeric breaks of chromosomes 1 and 16 and sometimes 9; multibranched chromosomes containing three or more arms of chromosomes 1 and 16 joined in the vicinity of the centromere (mostly at the 1qh or the 16qh region); and occasional isochromosomes and translocations with breaks in the vicinity of the centromere. In addition, prominent stretching (decondensation) in the 1qh and 16qh region can be observed in chromosomes 1 and 16 (Tuck-Muller et al., 2000). Also, there is an elevated level of MN formation (Narayan et al., 2000).

Xeroderma pigmentosum

Clinical findings: XP (OMIM #278780) is defined by extreme sensitivity to sunlight, which is the first feature of the disease that triggers severe sunburns. This is followed by areas of increased or decreased pigmentation, skin aging, and multiple skin cancers. A minority of patients show progressive neurological abnormalities. Ocular abnormalities occur upon exposure to sunlight (Stefanini and Kraemer, 2008; Ramkumar et al., 2011).

Genetic background: It is a rare autosomal recessive disorder and has been found in all continents and racial groups. There are eight XP complementation groups, corresponding to eight genes, which, if defective, can result in XP. The products of these genes are involved in the repair of ultraviolet (UV)-induced damage in DNA (Stefanini and Kraemer, 2008).

Cytogenetic findings: Chromosomal breakage is not a constant feature for the disease and it is not reliable for the diagnosis (Gardner et al., 2004). The levels of SCE in patients with XP did not differ markedly from normal (Wolf et al., 1975). However, the level of CAs of lymphocytes exposed to irradiation has shown marked elevation than normal (Saraswathy and Natarajan, 2000). Also, cultured fibroblast exposed to UV has shown elevated levels of chromosomal breakage (Ahmad and Hanaoka, 2010).

Cockayne’s syndrome

Clinical findings: It is characterized by developmental and progressive defects. Premature aging, cachectic dwarfism, and neurological abnormalities are hallmark symptoms of CS (OMIM #216400/5q12.1), and this disease is used as a model system in aging research (Berquist and Bohr, 2011). CS spans a spectrum that includes CS type I, the ‘classic’ form; CS type II, a more severe form with symptoms present at birth (also known as cerebro-oculo-facial syndrome or Pena-Shokeir syndrome type II); CS type III, a milder form; and xeroderma pigmentosum-Cockayne syndrome (Nance and Berry, 1992).

Genetic background: CS is a rare autosomal recessive genetic disorder. Mutations within two genes form the genetic basis: CSA (ERCC8) and CSB (ERCC6), found in ∼20 and ∼80% of patients, respectively. CS proteins are involved in DNA excision repair and in mitochondrial function (Berquist and Bohr, 2011).

Cytogenetic findings: Chromosomal breakage is reported occasionally with CS patients; it is not part of the syndrome but it may support the diagnosis (Gardner et al., 2004). A recent study by Abdel Ghaffar et al. (2011) reported an elevated level of chromosomal breakage in CS patients using MMC, and recommended using chromosomal breakage as a criterion for the diagnosis.


This group of diseases is caused by mutations in genes involved in the process of sister-chromatid cohesion (Liu and Krantz, 2008) that includes CDLS, RBS (Vrouwe et al., 2007; Van der Lelij et al., 2009), and the recently described Warsaw’s breakage syndrome (Van der Lelij et al., 2010). Sister-chromatid cohesion is the process of pairing replicated chromosomes during mitosis and meiosis; it is mediated through the essential cohesin complex and a number of nonessential cohesion genes. Mutant forms of these nonessential cohesion genes typically have mild cohesion defects (15–30% premature sister-chromatid separation) at both chromosome arms and centromere regions (Mayer et al., 2004; Warren et al., 2004; Xu et al., 2004). By contrast, essential cohesion mutants show 50–60% premature sister-chromatid separation at these loci (Michaelis et al., 1997). In addition to their roles in cohesion, nonessential cohesion genes also participate in other cellular processes, such as the S phase replication checkpoint (Warren et al., 2004).

Roberts syndrome

Clinical findings: RBS (OMIM #268300/8p21.1) was first identified as a distinct syndrome by Temtamy (1966). It is a rare autosomal recessive disorder characterized by heterogeneous clinical features, the most notable being tetraphocomelia, which was emphasized as a prominent feature of the syndrome, med face defects, prenatal and postnatal growth retardation, and craniofacial anomalies (Freeman et al., 1974; Mc Daniel et al., 2000).

Genetic background: RBS it is an autosomal recessive disorder, caused by mutations in ESCO2, which encodes an acetyltransferase that is involved in sister-chromatid cohesion (Vega et al., 2005).

Cytogenetic findings: Standard cytogenetic preparations stained with Giemsa or C-banding techniques show the characteristic chromosomal abnormality of PCS and separation of the heterochromatic regions (also termed heterochromatin repulsion in most chromosomes in all metaphases) (Gordillo et al., 2008).

Cornelia de Lange syndrome

Clinical findings: The facial features in CDLS (OMIM #122470, #300590, #610759) are the most clinically consistent and recognizable findings in CDLS. Most individuals have a short neck, a low posterior hairline, hirsute forehead, and arched eyebrows. Thick and long eyelashes, low-set ears, flattened midface, a short nose, and a long philtrum are also commonly seen. Typical oral features include a thin upper lip with down-turned corners, a high palate, and a cleft palate. Typical limb findings range from small hands and small feet to more severe reduction defects of the upper limbs. Probands have a proportionate small stature that occurs prenatally. Multiple organ systems are involved in CDLS such as pyloric stenosis, and congenital heart and renal malformations. Thrombocytopenia has also been consistently reported (Liu and Krantz, 2009).

Genetic background: Mutations in three genes, NIPBL on chromosome 5p13, SMC1A on chromosome Xp11, and SMC3 on chromosome 10q25, can be identified in 65% of individuals with clinically diagnosed CDLS. All three of these genes are involved in sister-chromatid cohesion events (Krantz et al., 2004; Musio et al., 2006; Deardorff et al., 2007).

Cytogenetic findings: Studies have reported that an increased sensitivity to MMC for both fibroblasts and B lymphoblastoid cells of CDLS patients leads to premature centromere division and PCS (Vrouwe et al., 2007; Van der Lelij et al., 2010).

Warsaw’s breakage syndrome (OMIM #613398)

Clinical findings: Van der Lelij et al. (2010a) described a patient from Poland with severe prenatal and postnatal growth retardation, microcephaly, facial dysmorphism (small and elongated face, narrow bifrontal diameter, jugular hypoplasia, bilateral epicanthal folds, relatively large mouth, and cup-shaped ears), high-arched palate, coloboma of the right optic disc, deafness, ventricular septal defect, bilateral clinodactyly of the fifth fingers, syndactyly of the second and third toe, and abnormal skin pigmentation.

Genetic background: Compound heterozygosity for mutations in the DDX11 gene located on chromosome 12q11.21 has been described. The DDX11 gene functions at the interface between DNA repair and sister-chromatid cohesion (Van der Lelij et al., 2010a).

Cytogenetic findings: The patient has a unique cellular phenotype. There is increased sensitivity to MMC and spontaneous occurrence of premature centromere division (Van der Lelij et al., 2010a).

Chromosomal breakage disorders with telomeric shortening

Telomeric attrition has been described in several disorders, whether inherited or not, as well as in malignancies (Calado and Young, 2009); among these disorders are FA, DC, WS, and aplastic anemia.

Dyskeratosis congenita

Clinical findings: DC is an inherited bone marrow failure (BMF) and cancer predisposition syndrome caused by defects in telomere biology. The consequences of DC affect all body systems; these may include the diagnostic triad of, abnormal nails, reticular skin pigmentation, and oral leukoplakia. BMF, pulmonary fibrosis, liver disease, neurologic and ophthalmic abnormalities, and increased risk for cancer may occur (Savage and Alter, 2009).

Genetic background: The inheritance of DC can be X-linked recessive (OMIM #305000), autosomal dominant (OMIM #127550), or autosomal recessive (OMIM #224230). There is also a high frequency of sporadic cases, which are presumably because of new mutations in dominant genes. Six genes in the telomere biology pathway have been identified to date as mutated in patients who have DC; these are DKC1 (Xq28), TERC (3q26.2), TERT (5q15.33), TINF2 (14q12), NOLA2 (5q35.3), and NOLA3 (15q14-q15) (OMIM database).

Cytogenetic findings: Telomere shortening can be detected using the FISH technique in DC, and it is presumed to be the cause of the systemic abnormalities and BMF (Alter et al., 2007).

Werner syndrome

Clinical findings: The hallmark feature of WS (OMIM #277700) is premature aging. It is characterized by multiple progeroid features including graying and loss of hair, wrinkling of skin, diabetes mellitus (DM), cataract, osteoporosis, cardiovascular disease, and a high incidence of cancer (Goto et al. 1996; Martin et al., 1999).

Genetic background: WS is a rare autosomal recessive disorder. It is caused by a mutation of the WRN gene that encodes protein WRN and is located on 8p12. Studies have suggested the role of the WRN gene in DNA double-strand break repair and DNA replication (Ariyoshi et al., 2007).

Cytogenetic findings: A study of the telomere using FISH showed abnormal signals, extratelomere signals, and increased signal loss of telomere (Ariyoshi et al., 2007). This finding is suggested to be the major cause of the early onset of age-related symptoms and a predisposition to sarcoma and carcinoma in WS (Ishikawa et al., 2011).

Other diseases with chromosome instability and breakage

Cancer and chromosomal instability

Both genetic instability and impaired DNA restitution have been pointed out as factors underlying increased susceptibility to malignancy (Lengauer et al., 1998; Thompson and Schild, 2002). Genomic instability has also been described for various hereditary cancers such as breast cancer. Genomic instability and DNA repair capacity have been analyzed in numerous population-based studies using a variety of assays that assess CAs, SCE, MN, and DNA fragmentation by means of the comet assay (Djuzenova et al., 2006).

Also, cancer cells commonly have a high rate of telomere loss, even when expressing telomerase, contributing toward chromosome instability and tumor cell progression.

The inclusion of genetic biomarkers such as mutagen sensitivity or CBMN end points in risk assessment models allows for a more comprehensive determination of the risk of cancer. The simplicity, rapidity, and sensitivity of the CBMN assay make it a valuable tool for screening of cancer risk. However, cytogenetic assays are classical methods to detect CAs, and generally considered indicative of an increased risk for cancer for those exposed to DNA-damaging agents (Bonassi et al., 2000; El-Zein et al., 2011).

Chronic inflammatory and autoimmune diseases

Rheumatoid arthritis: Rheumatoid arthritis (RA) is a chronic, systemic, inflammatory, autoimmune syndrome that produces degradation of articular cartilage and bone erosion (Goronzy and Weyand, 2005). Oxidants play a significant role in causing oxidative stress, which underlies the pathogenesis of RA (Remans et al., 2005). Genetic factors that predispose individuals to RA are considered to play an important role in the development of the disease.

The extent of DNA damage was evaluated by the comet and MN assay in patients with RA. The results indicated an increased production of reactive oxygen species in RA, as reflected by higher plasma malondialdehyde levels and lower plasma glutathione peroxidase and superoxide dismutase levels, and this condition may impair genetic stability, as shown by a strong response in the comet assay and the MN test in RA patients. This information is potentially important, as it enables understanding the mechanism of action of current therapies and in particular the development of new therapeutic strategies (Karaman et al., 2011).

Systemic lupus erythematosus and systemic sclerosis: Systemic lupus erythematosus (SLE) and systemic sclerosis (SS) are autoimmune diseases characterized by the presence of antibodies against self-antigens. The presence of clastogenic factors (CF) capable of inducing chromosome breakage has also been reported in the plasma of some patients. CFs have been shown to act through oxygen free radical (FR) formation (Emerit, 1994). In addition, evidence indicates that IgG antinuclear antibodies can enter cells and reach their nuclear target, resulting in cell and organ damage (Koren et al., 1995).

In SS, the presence of antibodies to the centromere or to topoisomerase I seems to be associated with either a mild clinical subset or a more severe form characterized by progressive skin and visceral organ involvement. The results of the MN FISH assay for patients with SLE and SS indicated significantly higher MN frequencies in SS patients, but not in SLE patients; however, the data showed a significant prevalence of MN with acentric fragments in SLE more than normal (Migliore et al., 1999).

Celiac disease: Celiac disease (CD) is a unique autoimmune disorder; it is not a rare disease, affecting ∼1% of the population (Fasano et al., 2003). It results from the interaction between gluten, immune, genetic, and environmental factors. CD is developed in genetically predisposed individuals, by the ingestion of gluten, the major storage protein of wheat and similar grains (Green and Jabri, 2003). In patients with CD, immune responses to gliadin fractions promote an inflammatory reaction, primarily in the upper small intestine, characterized by infiltration of the epithelium with chronic inflammatory cells that leads to villous atrophy (Sollid, 2002).

The genetic influence in the pathogenesis of CD is indicated by its familial occurrence. CD does not develop unless an individual has alleles that encode for HLA-DQ2 or HLA-DQ8 protein products of two of the HLA genes (Sollid and Lie 2005).

Infants and young children generally present with diarrhea, abdominal distention, and failure to thrive (D’Amico et al., 2005). The diagnostic standard in celiac serologies is the endomysial IgA antibodies; they are highly specific markers for CD, approaching 100% accuracy (Rostom et al., 2005).

Chromosomal instability was detected in untreated patients with CD. Spontaneous CAs and induced FSs using caffeine were analyzed in adult (CD) patients. The frequencies of spontaneous CAs and induced FS were significantly higher in CD patients (Fundia et al., 1996).

Hepatitis C: The plasma of patients with hepatitis C contains chromosome-damaging substances, the so-called ‘CFs’. These endogenous clastogens can be detected using cytogenetic methods. Certain components of CF have not only superoxide-stimulating properties but also interfere with DNA repair enzymes. CF-induced DNA damage that leads to genomic instability may also be responsible for the high risk of liver cancer in patients with hepatitis C. Chromosomal breakage is an indicator for events occurring in DNA. Thus, the pathway of carcinogenesis through CF formation appears to be of particular interest because superoxide scavengers can be used as anticarcinogens in such patients (Emerit, 2011).

Diabetes mellitus

DM is characterized by an elevation in blood glucose concentration. The disease is progressive and is associated with the development of complications (Wolff, 1993). Experimental evidence indicates that these complications occur mainly because of the production of excessive concentrations of FRs, which result in oxidative damage (Dandona et al., 1996). Oxidative damage to the genetic material could cause DNA strand breaks and MN, and these types of damage could have teratogenic or carcinogenic consequences. This damage caused by FRs can be mitigated by antioxidant defense systems such as folic acid evidenced by a reduction in the frequency of MN in DM patients (Zúñiga-González et al., 2007).

Neurodegenerative diseases

The incidence of common neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease is increasing worldwide as a result of the increasing longevity of humans (Sloane et al., 2002). Biomarkers that may identify individuals who are at an early stage of neurodegeneration would be useful as this would allow a potential timely preventative intervention before the severity of the disease advances to more critical stages. There is increasing interest in the evaluation of chromosome damage markers within somatic cells of individuals affected by neurodegenerative diseases that may prove to be predictive of an increased risk (Migliore et al., 1997; Petrozzi et al., 2002).

MN, which are biomarkers of chromosome malsegregation and/or breakage, have been investigated in patients affected by neurodegenerative disorders and in groups of patients at an increased risk for neurodegeneration (Trippi et al., 2001). The first application of the MN assay to peripheral cells of neurodegenerative patients was carried out in 1997 (Migliore et al., 1997). High levels of micronuclei were found in peripheral blood lymphocytes of Alzheimer’s disease patients. Furthermore, after application of the FISH technique utilizing a pancentromeric DNA probe for the detection of the presence of the centromere, it was found that the majority of MN were composed of whole chromosomes (Migliore et al., 1997). Subsequently, using dual-color FISH with differential labeled DNA probes, the increase in spontaneous chromosome loss or gain for both chromosomes 13 and 21 was detected (Trippi et al., 2001; Migliore et al., 2011).

Parkinson’s disease patients showed higher frequencies of MN and a significant increase in the levels of single-strand DNA break (Migliore et al., 2001). Data obtained by FISH analysis showed that the percentage of centromere-negative MN was higher than that of centromere-positive MN, arguing in favor of MN originating following chromosome breakage events (Petrozzi et al., 2002).

In Huntington’s disease, one of the trinucleotide repeat expansion diseases, a study carried out on fibroblast of juvenile Huntington disease patient indicated a high frequency of MN (Sathasivam et al., 2001).

Mitochondrial diseases

MDs are a wide group of disorders often characterized by an impairment in energy production by means of the mitochondrial oxidative phosphorylation process. A common presenting feature of mitochondrial syndromes is the involvement of muscle and the central nervous system; these tissues greatly rely on oxidative metabolism for their energy supply and are susceptible to its impairment (DiMauro and Moraes, 1993). These rare disorders are usually associated with a delayed age of onset, organ target selectivity, and a progressive course.

Endogenous oxidative stress is believed to play a key role in the pathogenesis of MD. The level of nuclear DNA damage was evaluated before and after a 2-week therapy with a coenzyme Q10 analogue. The extent of cytogenetic damage, expressed as chromosome breakage and chromosome loss, was assessed using the cytokinesis block MN method in cultured peripheral blood lymphocytes, coupled with FISH analysis using a labeled pancentromeric DNA probe. The comet assay was used to quantify oxidative DNA damage in leukocytes. In MD patients, an increased level of chromosome damage, expressed as an increased frequency of MN in lymphocytes, was detected. The FISH analysis indicated a preferential occurrence of micronuclei arising from the loss of whole chromosomes. However, the comet assay indicated a slightly higher level of DNA damage in patients compared with controls. Patients receiving the coenzyme Q10 analogue showed a statistically significant reduction in the frequency of micronuclei (Migliore et al., 2004).

Inflammatory bowel diseases

The inflammatory bowel diseases: Crohn’s and ulcerative colitis result from an altered host response to intestinal flora. Recurrent inflammation with ulceration of the tissues confers an increased risk of cancer in both ulcerative colitis and Crohns. Studies have detected chromosomal instability and common genetic mutations (Jawad et al., 2011). The incidence of chromosome breakage was found to be elevated in patients with Crohn’s disease (Emerit et al., 1979). Peripheral blood lymphocytes from patients with chronic ulcerative colitis showed deficient repair of radiation-induced DNA damage. DNA repair was measured indirectly by quantifying chromatid breaks after the irradiation of cells with X-rays or ultraviolet during the G2 phase of the cell cycle (Sanford et al., 1997). Also, telomeric shortening was observed in patients with ulcerative colitis as a cause for the genomic instability in this disease (O’Sullivan et al., 2002).


Cytogenetic tests should be chosen properly according to the situations required to be investigated, and proper analysis of the data could be very helpful in the diagnosis and management of the patients.


Conflicts of interest

There are no conflicts of interest.


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    chromosome breakage; comet assay micronucleus test; comet FISH

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