Identification of an Elevated Incidence of Chromosomal Rearrangements in the Centromeric

IDENTIFICATION OF AN ELEVATED INCIDENCE OF CHROMOSOMAL REARRANGEMENTS IN THE CENTROMERIC REGIONS OF SCHIZOPHRENIC SUBJECTS

P.Manikantan, V.BALACHANDAR, k.sasikala

Division of Human Molecular Genetics, School of Life Sciences, Bharathiar University, Coimbatore, Tamilnadu, India-641046

ABSTRACT

Introduction: The focal aim of the present investigation was to study the foremostchromosomal aberrations (CA) like deletion, translocation, inversion and mosaic inschizophrenic subjects of Tamilnadu, Southern India. Totally 65 blood samples werecollected from various hospitals in Tamilnadu, Southern India.Materials and methods: Equal numbers of physically and mentally healthy subjectswere serve as a control. In the present study chromosomal examinations were carry outby using the GTG banding technique on 65 schizophrenics and finally the results wereensured by spectral karyotyping (SKY) technique.Results: All the patients had random numerical and structural aberrations were identified.Structural aberrations predominated and usually consisted of deletions, translocation,inversion and mosaicism of various chromosomes. Present study has detected 1, 7, 9, 11,21, 22 and X, suggested that these chromosomal scratches are prevalent inschizophrenics. In comparison with experimental subjects, the control subjects exhibitedvery low (p<0.05) levels of major CA.Conclusion: In the present study, the high frequency of chromosomal rearrangementsdesignates a potential role for mitotic indiscretion coupled with the centromericdisruption. The reason for this might be that these anomalies increase risk forschizophrenia in a relatively nonspecific way, such as contributing to disruption ofnormal biological system. Identification of these chromosome alterations may be helpfulto understanding further molecular basis research of the disease in better way.

Key words: Schizophrenia; chromosome aberration; 1, 7, 9, 11; Spectral karyotyping (SKY).

1. INTRODUCTION

Chromosomal rearrangements revise boast been helpful in identifying diseasegenes that may be helpful to decision a candidate regions. There are numerous accountsof links among psychotic disorders and (Peter Falkai et al. 2005) chromosomalaberrations. “Schizophrenia” is a relatively common debilitating, chronic, psychoticdisorder. Its lifetime prevalence is approximately 0.85% in the general and asianpopulation. Its etiology is unknown, but appears to have a strong root were geneticcomponents are incredibly elevated task in schizophrenia by adoption studies (Crow et al.1995). Genetic factors play an important role in the pathogenesis of schizophrenia(Gottesman, 1991). According to Sebastian Rudolf (2004) atypical lymphocytes areessential morphological role in schizophrenia. Cytogenetic investigation in peripherallymphocytes has identified chromosomal aberrations associated with schizophrenia.In order to this investigation, Identification of several chromosomal aberrationsmay be especially important given the unknown pathophysiology. Several candidatechromosomes such as deletion translocation 1, 1q21, 7q23, inv (9), 9qh+, 11q23, 21q22,22q11-13 and Xp11-q13 are known to play a vital role in the progress of schizophrenia(Osman Demirhan et al. 2003). In addition to infrequent cases showing the partialtrisomic of 5q11-13 (Bassett et al. 1988), specific translocations such ast(18;21)(p11.1;p11.1) (Smith et al. 1996) and t(1;7)(p22q22) (Gordon et al. 1994),inversions such as inv(9)(p11q13) (Toyota et al. 2001; Kunugi et al. 1999) andinv(4)(p15q21.3) (Palmour et al. 1994), trisomies of 5p14.1 (Malaspina et al. 1992) and 8(Ong and Robertson, 1995) deletions at 22q11.1 (Karayiorgou et al. 1995; Bassett et al.2000; Arinami et al. 2001) and 5q21-23.1 (Bennett et al. 1997) and sex aneuploidies(DeLisi et al., 1994) have been reported in schizophrenic subjects. Prefrontal cortexvolume (AFC) loss also biological marker for schizophrenia (William et al. 2002).This suggests that genetic imprinting may be an important observable fact in somechromosomal abnormalities. In order to search for similar or other chromosomalaberrations (CA), at this juncture here reported chromosomal findings by spectralkaryotyping for total of 130 subjects aged 20-60 years old including 65 schizophrenicssubjects (SZS) and 65 physically and mentally healthy Controls in Tamilnadu, southIndia.

2. SUBJECT RECRUITMENT AND SAMPLE COLLECTION

The study group was preferred indiscriminately and, total of 65 SZS wereassessed according to the criteria described in the Diagnostic and Statistical Manual ofMental Disorders, fourth edition (DSM-IV) (American psychiatric Association. 1994). Atotalnumber of 130 subjects aged 20-60 years old (mean age and SD was 39.15±08.21),including 65 SZS (among 65 schizophrenic cases, 47 were men, while 18 were women)and 65 physically and mentally healthy controls were recruited and Experimental subjectssubdivided into 4 age groups, namely Group I (20-30 years), Group II (31-40 years), group III (41-50 years) and Group IV (51-60 years). The purpose of groups alienated foronly identification purpose, not any other intention. Five milliliters of peripheral bloodsamples were collected from 65 SZS (different age groups) in various hospitals andrehabilitation centres of Tamilnadu, South India. The study was approved by the localhealth committee.

3. CHROMOSOMAL ABERRATION ASSAY

With written informed consent, each sample was examined for detecting CA. All chemical reagents were purchased from Sigma Chemicals (USA), except colcemid that was obtained from Gibco Laboratory (USA). (Hoyos et al. 1996). Blood samples were set up to establish leukocyte cultures following standard procedures (slightly modified by Sasikala et al. 1996). Briefly, 0.5 ml blood was added to 4.5 ml RPMI 1640 medium supplemented with 10 % fetal bovine serum, 2 mmol/L L-glutamine, 1% streptomycin- penicillin, 0.2 ml reagent grade phytohemagglutinin, and was incubated at 37 °C. After 52 h, cultures were treated with 0.1 g/ml colcemid to block cells in mitosis. Standard Cytogenetic techniques were used for harvesting and slide preparation. Lymphocytes were harvested after 52 h by centrifuging cells to remove culture medium (800~1000 r/min), added with hypotonic solution (KCl, 0.075 mol/L) at 37 °C for 20 min to swell the cells, and treated twice with Carnoy’s fixative (3:1 (v/v) ratio of methanol: acetic acid). Slides were carefully dried on a hot plate (56 °C, 2 min), and then stained using the Giemsa-banding (G-banding) technique. Hundred (100) GTG banded metaphases were scored for chromosome or chromatid sort rearrangements, from each of the patient and control groups per individual by a person with 25 years of experience. All chromosomal rearrangements were recorded and localized according to the International System for Human Cytogenetic Nomenclature (ISCN). Mosaicism was resolute if the fraction of the inconsequential cell population was more than 18% (i.e., 7 or more in 35 cells), in view of the fact that low-frequency of mosaicism has a smaller amount significance in affecting phenotype, and it may also be due to object.

4. SPECTRAL KARYOTYPING (SKY)

The SKY™ kit probe cocktail from Applied Spectral Imaging (ASI, Carlsbad,CA, USA) was hybridized to metaphase spreads from each schizophrenia slidesaccording to standard protocols (Garini et al. 1996; Schrock et al. 1996; Veldman et al.1997) and as per the manufacturer’s instructions (ASI, Carlsbad, CA, USA). Afterdestaining the G-banded slides with methanol for 10 min, the slides were rehydrated in adescending ethyl alcohol series (100%, 90%, 70%), and fixed with 1% formaldehyde in50 m mol/L MgCl2/phosphate buffer solution for 10 min. The slides were thendehydrated using an ascending ethyl alcohol series and denatured for 30~45 s in 70%formamide/2× SSC at 75 °C. The SKY probe was denatured for 7 min at 75 °C,reannealed at 37 °C for 1 h, placed on the slide and covered with a glass coverslip. Thecoverslip was sealed with rubber cement and the slides placed in a damp container in a 37°C incubator. After overnight hybridization, the post-hybridization washes wereperformed as per manufacturer’s instructions (ASI, Carlsbad, CA, USA). The metaphaseimages were captured using an SD 200 spectral bio-imaging system attached to a Zeissmicroscope and stored on a SKY image-capture workstation. The images were analyzedusing the SKY View software version 1.2 which resolves individual fluorochrome spectraby Fourier spectroscopy and distinguishes the spectral signatures for each chromosome toprovide a unique pseudocolour for each chromosome (classified image).

STATISTICAL ANALYSIS

All statistical analyses were performed using software SPSS for Windows,version 13. To assess the differences between patients and controls, variables expressedby the mean±SE were analyzed using paired t-tests. P<0.05 was used as the criterion forsignificant difference between the groups.

5. RESULTS

Identifying chromosomal abnormalities (CA) occurring in patients withschizophrenia may be a useful method of establishing loci that contribute to illnesssusceptibility. Selected SZS with CA are shown in Table 1. The majority of the patientswere male (72.30%; 47/65) and female (27.69%; 18/65). A total of 65 SZS haverandomly numerical and structural aberrations were detected and ensured by spectralkaryotyping (SKY). Structural aberrations predominated then numerical aberrationsusually consisted of deletions, translocation, inversion and mosaicism of variouschromosomes. Selected SZS with Chromosomal aberrations are shown in Table. Resultsshowed that CA (8.63±0.82) has increased in subjects with respect to their controls(2.17±0.62). In Table 1, the CA was frequently exhibited in chromosomes 1, 7, 9, 11, 21,22 and X. From the above data, it can be inferred that significant elevation in CA wasobserved in SZS with increased when compared with that of control group. Statisticallysignificant results were obtained from paired t-test (P<0.05). From the above data, it canbe inferred that significant elevation in CA was observed in schizophrenia patients withincrease of CA when compared with that of control age groups.

6. DISCUSSION

The principle relevance of diagnostic (HansJurgen Moller. 2001) systems forbiological psychiatry and the relevance of biological psychiatry in the past and future forthe development of psychiatry in general. This study suggest that nine broadchromosomal regions meet up adequate and capriciously defined consequence criteriawhich show capable for localizing susceptibility genes for schizophrenia by SpectralKaryotyping. Although earlier studies have revealed the presence of chromosomeinstability schizophrenia (Toyota et al. 2001; Maier et al. 1999; Ekelund et al. 2000;Gurling et al. 2001; Murphy et al. 1998; Bassett et al. 2000; Kunugi et al. 1999; Arinamiet al. 2001) and this is the first molecular cytogenetic study to investigate in theperipheral blood lymphocytes of untreated schizophrenia patients from Tamilnadu,Southern India.

In the present study, chromosome 1 showed the deletion and translocation incopious subjects. Lately published consequences studies of schizophrenia have payingattention on chromosome 1. The incidence of a susceptibility locus on chromosome 1q issustained by linkage analysis and by the association of schizophrenia with cytogeneticabnormalities (Brzustowicz et al. 2000; Millar et al. 2000). Kosower et al. (1995)published a combined allelic association, cytogenetic and family study ofheterochromatic c band variants in the q22.1-23 region of chromosome 1. However,Ekelund et al. (2000) found some evidence for a susceptibility locus on chromosome 1.This present study also identified one (1.53%) patient with deletion 1q21 in metaphases.Therefore, the chromosome 1q21 region could play a role in the pathogenesis ofschizophrenia. It is notable that the 1q21 region contains the locus of the dopaminereceptor (DS) pseudogene 2 (Grandy et al. 1992). A balanced reciprocal translocation t(1;11) was found to co-segregate with schizophrenia, which has recently been reported todirectly disrupt two genes, of unknown function, on chromosome 1 (St. Clair et al. 1990).The beyond consequences of the present study have been supplemented with severalreports relating to chromosome 1 and it is involved in schizophrenia whether through thepresence of disease susceptibility genes or through the disruption of common pathwaysinvolved in disease development.

The consequences of two earlier genome scans would provide evidence for theinvolvement of the 7q22 region. Ekelund et al. (2000) identified a locus on chromosome7q22 for schizophrenia population. Faraone et al. (1998) also reported two markers in 43nuclear families of European–American origin. In our study, chromosome 7 anomalieswere involved in 12.3% (8 of 65) of patients, 5 cases displaying a deletion 7q32 and theother one had del (21) (q22), del (22) (q13). Three patients with a chromosome 7abnormality had a duplication of (7) (p15p21). However, replication of this finding in anindependent study is essential to discern whether this region actually contains aschizophrenia susceptibility gene. Other candidate regions need to be found tocomplement linkage analysis where 7q32 region provide evidence. These results weresupported by other studies on chromosome 7. These results were further strengthened byother reports. Loss of chromosome 7 was identified in schizophrenia using FISH.Recent studies indicated that the pericentric region of chromosome 9 may beetiologically associated to schizophrenia (Kunugi et al. 1999). Pericentric inversion ofchromosome 9 is the most common reciprocal translocation in the general population andthe prevalence of inv (9) varies with ethnicity. It could be predictable that thecommonness in Asian populations is approximately 1.5%. Some studies have indicatedan increased prevalence of inv (9) in schizophrenia. Axelsson and Wahlstro (1984)reported an unusually increased prevalence of inv (9) in male patients with paranoidpsychosis. In distinction, DeLisi et al. (1988) did not find any individual with inv (9)among 46 male schizophrenics. In this study, chromosome 9 seems to be involved moreoften than the other chromosomes. The pericentric region of the chromosome 9 wasimplicated in 12.3% (8 of 65) of patients. In three patients, the abnormality was inv (9)(q11q13), whereas two patients had del (9) (q11q13) and del (9) (q13), and the additionalthree patients had 9qh+. This may point out that the effect of qh region on the expansionof schizophrenia would not be major one, but it may be a risk-increasing aspect. Thelocalization of breakpoints on chromosome 9 may show the way to the replica ofschizophrenia susceptibility genes. Undoubtedly, further studies are necessary tounderstand the role of 9qh+ in schizophrenia.

The present study detected a deletion at 11q23 region of chromosome 11 in threepatients. The other studies have also concerned the chromosomal region 11q21- 22 ascontaining genes increasing liability for schizophrenia. Thus, some balancedtranslocations at q14.3, q21, q22.3 and q25 sites of chromosome 11 were found withschizophrenia and other psychiatric disorders (Millar et al. 2000). In another study, one offour Canadian pedigrees produced an LOD score of 3.41 with D11S35 and q14.3, q21,q22.3, q23 and q25 sites of chromosome 11 suggested that these regions did indeedharbor schizophrenia susceptibility loci (Maziade et al. 1995). The molecular geneticresearch has not been able to identify genes with a major effect on schizophrenia risk.Several genes with gene relative risks of 2–4 have been identified (Riley and McGuffin.2000).

Most analysis revealed a karyotype of 46 chromosomes with an interstitialdeletion from the long arm of chromosome 21, ISCN designation 46, XY, Del (21)(q21.2q22.1) (Rooney. 2001). In this study also identified CA in chromosome 21 inseventeen patients (26.16%). In some cases, however, it is caused by the presence of onlyband 21q22. This band has been called the ‘‘Down syndrome region’’. We havedescribed deletion 21q22 in 10 patients (15.38%), two patient (3.07%) having also 46,XX/47, XX, + 21 mosaicism and five patients (7.69%) with 47,XX, + 21. According toRooney (2001) identify an interstitial deletion of chromosome 21q, del (21) (q21.2;q22.1) in a man with learning disability, dysmorphic features and schizophrenia.

Interestingly, this is not the first report of association between a chromosomal deletion of21q21-q22 and schizophrenia. Takhar et al. (2002) reported a case of an interstitialdeletion (21) (q21; q22.1) in a 16-year-old student originally from Somalia with learningdisability, schizophrenia and dysmorphic features. Facial features included a long facewith high nasal bridge, short ears and exotropia. More recently, using the GTG bandingtechnique, Demihran and Tastemir (2003) identified 4 individuals with deletion 21q22 ina schizophrenia sample. It is not clear how representative their sample is of the generalschizophrenia population. However in this study suggest that Down syndrome (21)significantly increased in patients with schizophrenia. In particular, Down syndromepatients were more likely to have been diagnosed as having depression and dementia(Collacott et al. 1992).

High rates of schizophrenia have been reported in the individuals with velocardio-facial syndrome (Murphy et al. 1999). This condition is associated with smallinterstitial deletions of chromosome 22q11. This region is also indicated in family basedlinkage disequilibrium mapping using SNP markers in a schizophrenic population (Li etal.2000). The relevance of these two conditions is that both these deletions roughlycorrespond to regions, for which there is modest evidence of linkage in schizophrenia.Since the first report by Pulver et al. (1994) implicating 22q11-q13 in schizophrenia,most studies of schizophrenia have found linkage to more distal 22q12-13 region. In thisstudy also identified fifteen patients (23.07%) with 22q11-13 deletions among 65schizophrenics , suggesting that there may be two or more loci on chromosome 22(Schwab et al. 1999). This is maximum number of chromosomal region of this study andresult indicates that the rate of 22q11 deletion in schizophrenia may be approximately100 times more than conservative estimates of the general population prevalence. Finallyconfirming an association between the psychiatric illness and this chromosomal anomaly.In this present study X and YCA such as deletion, translocation, mosaicism inscores of schizophrenic patients. Chromosome Xp11 region is of interest here, because itharbors the monoamine oxidase B gene, which is a potential candidate gene forneuropsychiatric disease. Inversion of X chromosome in patient can lead to local block ofseveral genes in that break point, thus leading to schizophrenia. There were three patientswith an aneuploidy of the X chromosome: and most of the patients with a Y chromosomeaneuploidy. According to Ratcliffe et al. (1986) noted that mosaicism of the Xchromosome was substantially increased in the schizophrenic females in their studies.

This gives additional evidence for the increased incidence of Klinefelter’s syndrome inschizophrenics reviewed by DeLisi et al. (1994). It is also curious that XXY and XXXindividuals are increased among hospitalized patients with schizophrenia (reviewed inDeLisi et al. 1994). Taken together, the above observations could be accounted for by alocus for illness on these chromosomes, although, undoubtedly, there are alternativeexplanations for each. Inversion of X chromosome in our patient can lead to local blockof several genes in that break point, thus leading to schizophrenia.

In conclusion while there is confirmation of chromosomal aberrations inschizophrenia, including reported associations of schizophrenia, it is still not clearwhether there is a relationship between these autosomal and gonosomal anomalies andschizophrenia. We can suggest that schizophrenia, are likely to exist in some or all of thechromosomal regions noted above. These interesting chromosomes may harbor importantgenes for schizophrenia mental psychosis, and such chromosomal abnormalitiesassociated with schizophrenia might provide important implications for further molecularresearch for the chromosomal location of major genes in this disease. But to draw anydefinite conclusion, a large number of patients should be selected, examined and furtherstudy should be needed An identifiable subtype with potential importance for delineatinga genetic etiology and neurodevelopment mechanisms of schizophrenia provides anexciting opportunity for researchers, clinicians and societies.

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