International Medical Journal Vol. 17, No. 2, pp. 135 - 139 , June 2010
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Divergence in Cytogenetic and Molecular Genetic Analysis Results: A Case Report of Fragile X Syndrome among Siblings Elias Marjanu Hikmah1), Mamat Norhashimah1), Ismail Siti Mariam1), Salmi Razak2), Pornprot Limprasert3), Wanna Sudhikaran3), Ravindran Ankathil1), Bin Alwi Zilfalil1,2) ABSTRACT Background: Fragile X Syndrome (FXS) is the most prevalent inherited cause of mental retardation. The prevalence of FXS in males and females are approximately 1 in 4000 and 1 in 8000 respectively. It is caused by CGG repeat instability in the FMR1 gene, located on chromosome Xq27.3. Normal individuals have CGG repeats ranging from 5 to 53. In premutation carriers, the CGG repeats range from 60 to 200 and shall be more than 200 repeats for full mutation patients. FXS patients have variable clinical features and because of that, an accurate clinical diagnosis is always a problem. Currently, Cytogenetic, PCR and Southern Blot Techniques are widely used for diagnosis of FXS. Case Report: Here we report a pair of brothers suspected to be FXS patients with similar clinical features. However, the cytogenetic result for younger brother did not show fragile site at Xq27.3 of the X chromosome while molecular result was confirmatory for FXS. Conversely, the elder brother showed confirmatory results for Fragile X mutation in both cytogenetic and molecular analysis. Conclusion: We therefore conclude that patient 1 confirms for Fragile X mosaic and patient 2 for Fragile X full mutation. From the result, cytogenetic analysis alone cannot be dependable for the confirmatory diagnosis of FXS.
KEY WORDS fragile X syndrome, cytogenetic analysis, southern blot, FMR1 gene and CGG repeats
INTRODUCTION The Fragile X Syndrome (FXS), OMIM#300624, is the most common form of inherited mental retardation. The prevalence of Fragile X syndrome in males and females are approximately 1 in 4000 and 1 in 8000 respectively (Crawford et al., 2001). Approximately 1 in 700 females are carriers and the frequency of premutation in general population is approximately 1 in 259 females and 1 in 813 males (Rousseau et al., 1995; Turner et al., 1996). However, recent population studies have indicated a much higher prevalence
of the Fragile X Syndrome. Hagerman (2008) reported that the best current estimate for the frequency of the FM in females is 1 in 2500 and 1 in 3600 males. This syndrome was the first genetic disease identified that is caused by CGG repeat instability. This unstable repeat is in the FMR1 gene, located on the Xq27.3 (Verkerk et al., 1991) of the X chromosome. FMR1 gene consists of 17 exons and is roughly 38 kb in size. Normally, the CGG repeats are stable with a range of 5 to 53 repeats. In premutation, the CGG repeats are between 60 and 200 and the instability is emphasized as the number of repeats increases. When 200 or more CGG repeats are pre-
Received on September 14, 2009 and accepted on November 30, 2009 1) Human Genome Center, School of Medical Sciences, Health Campus, Universiti Sains Malaysia 16150 Kubang Kerian, Kelantan, Malaysia 2) Peadriatric Department, Universiti Sains Malaysia Hospital 16150 Kubang Kerian, Kelantan, Malaysia 3) Human Genetic Division, Department of Pathology, Songklanagarind Hospital Hat Yai, Songkhla 90110 Thailand Correspondence to: Elias Marjanu Hikmah (e-mail:
[email protected])
C 2010 Japan International Cultural Exchange Foundation
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Figure 1. Pedigree of the case study
Figure 2. Karyotype of elder brother showing 46, Y, fra(X) (q27.3)[31%]
sent, FMR1 gene is usually hypermethylated, which is associated with transcriptional silencing of the gene that is commonly referred to as the FMR1 full mutation (FM) (Reiss et al., 1995). The FM prevents transcription of the FMR1-gene, resulting in the absence of the Fragile X Mental Retardation Protein (FMRP) production that will lead to the clinical features of the FXS patients. The clinical features for Fragile X Syndrome are variable, ranging from hyperactivity, learning disabilities and behavioral problems associated with mental retardation. Because of this, an accurate diagnosis of fragile X syndrome is usually a problem. The grown up males often exhibit characteristic features like long, thin faces with prominent jaws, large, protuberant ears and post pubertal macroorchidism (Hagerman et al., 1991). The disabilities are substantial and therefore an early diagnosis is mandatory for counseling of families in which Fragile X Syndrome has been observed. Earlier, the diagnosis was based on cytogenetic analysis for the expression of a folate sensitive fragile site at Xq27.3 (Sutherland, 1977). But cytogenetic analysis lacks in sensitivity. Currently the gold standard method for Fragile X diagnosis is by using the PCR followed by Southern Blot Analysis. Here we present the results observed through cytogenetic and molecular analysis in two siblings presented with clinical features of Fragile X Syndrome.
SUBJECTS AND METHODS The cases included are a pair of biological brothers (patient 1 aged 12 years old and patient 2 aged 9 years old)
born to parents (father aged 46 and mother aged 34 years) of a non consanguineous marriage. Both the patients have prominent ears, long and narrow face, flat feet, high arched palate and joint hyper mobility. However, the elder brother (patient 1) has milder joint hyper mobility compared with his younger brother. Reported that both the patient have delayed speech, attention deficit, shyness, poor eye contact and hyperactivity with the younger brother (patient 2) is more active and aggressive. Patient 1 had start talking at 7 years old but patient 2 still cannot talk at all even he is now 9 years old. Another sibling (female) in the family appeared to be normal. These two brothers were suspected as cases of FXS by a pediatric neurologist. In order to confirm the diagnosis, peripheral blood samples from these two patients (samples 01/08 from patient 1 and 02/08 from patient 2) were studied at Human Genome Center, University Sains Malaysia, Kelantan, by conventional cytogenetic and molecular analysis. From each patient, 10 ml blood was collected, of which 5 ml was transferred to EDTA tube for molecular analysis and another 5 ml to Sodium Heparin tube for cytogenetic analysis. For conventional cytogenetic analysis, 0.5 ml blood was cultured in folate deficient culture media, for 72 hours at 37 ℃. The culture medium used comprised of 28.5 ml of RPMI1640 medium without folic acid, 1.5 ml of FBS, 1.5 ml of Penicillin or Steptomycin and 900 μl Phytohaemagglutinin (PHA). At the 70th hour of incubation, the dividing cells were blocked in the metaphase stage by adding Cocemid (10μg/μ l). After 2 more hours of incubation, the cultures were harvested by standard cytogenetic procedures. Slides were prepared, dried and stained. Half the numbers of slides prepared were stained with GTG banding staining and the remaining slides were stained without banding. For each sample, 20 GTG
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banded metaphases were screened for constitutional chromosome abnormalities and another 100 unbanded metaphases were screened for fragile site on X chromosome at q27.3 region. For molecular analysis, DNA of both patients was extracted from the blood collected in EDTA using GENTRA PUREGENE Blood Kit with slight modifications. Then, the specific target of the 5’ untranslated region (5’UTR) of the FMR1 gene was amplified using PCR. The primers used for the amplification were 5’- CTC CGT TTC GGT TTC ACT TC-3’ as forward primer and 5’-GTA CCT TGT AGA AAG CGC CAT T-3’ as reverse primer. Agarose gel electrophoresis was done to analyze the result. The sizes of the CGG repeats were calculated according to band size. The samples were also analyzed using real time PCR with the Taqman Probe to identify its CGG repeats more precisely. The extracted genomic DNA of both the patients was also sent for Southern Blot Analysis and Methylation Specific PCR (MS-PCR) analysis at the Department of Pathology, Prince of Songkla University, Thailand.
DISCUSSION In Fragile X Syndrome, the cognitive, behavioral and physical phenotype varies by sex, with males being more severely affected because of the X-linked inheritance of the mutation. Based on the clinical characteristics recorded, both the patients included in the current report have delayed speech, attention deficit, hyperactivity and mental retardation. All these clinical characteristics were strongly suspicious of a clinical diagnosis of Fragile X Syndrome. However, the patient 2 is more hyperactive and aggressive. In order to confirm the diagnosis, both the patients were screened for fragile Xq27.3 site employing cytogenetic and molecular techniques. Cytogenetic screening for fragile site in the lymphocytic chromosomes prepared from peripheral blood cultured in folate deficient culture medium showed 46, Y, fra (X) (q27.3) keryotype pattern, for the patient 1 (01/08) indicating Fragile Xq27.3 site in 31% of the metaphases. This was consistent with a clinical diagnosis of Fragile X Syndrome. However, the fragile Xq27.3 site could not be detected in the X chromosome of patient 2 (02/08) through conventional cytogenetic analysis. Karyotype analysis employing GTG banded metaphases revealed no other constitutional chromosome abnormalities of either numerical or structural types, in both the patients. In molecular analysis employing PCR technique, the efficiency of the PCR amplification will decrease with the increase of the CGG repeats. In this case report, molecular analysis for FMR1 gene mutation, utilizing Polymerase Chain Reaction was also done for both the patients. The results of molecular analysis showed patient 1 have a permutation band and patient 2 do not have any normal or permutation band pattern. The absence in normal band of patient 2 sample might be due to the limitation of the PCR for detecting the full mutation. This was strongly indicative of an aberrant FMR1 gene in patient 2, which most probably might be a full mutation of the FMR1 gene. For patient 1, even though permutation band do appear, the presence of full mutation allele might also happen. So, patient 1 cannot be categorized as non fragile X patient. Figure 2 shows the result for PCR analysis. The first row is the negative con-
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trol, followed by control with 30 CGG repeats, control with 45 CGG repeats, 60 CGG repeats, 200 CGG repeats, sample of elder brother (01/08) and sample of younger brother (02/08). Correspondingly, when the genotyping of the FMR1 alleles was performed using the fluorescent primers FRAXA-F AND FRAXA-R (Sripo et al., 2007) and the PCR products were determined using ABI PRISM 3130 and Gene Mapper Software (Perkin Elmer), the fragment analysis on sample 01/08 showed the permutation allele with 62 CGG repeats (Figure 3). So, in order to categorize the mutation further, two other confirmatory tests were also done on both the patients. The extracted genomic DNA of both the patients were sent for Southern Blot Analysis and Methylation Specific PCR (MS-PCR) analysis at the Department of Pathology, Prince of Songkla University, Thailand. The results reconfirmed both the patients as having Fragile X Syndrome. An interesting observation in the molecular analysis was that patient 1 (01/08) was diagnosed as a Fragile X mutation mosaic patient. Figure 4 shows the methylation specific PCR and Southern Blot analysis results of this patient which revealed that this patient had one FMR1 allele of abnormal size of 62 CGG repeats (FMR1 premutation allele) and also full mutation alleles. In Fragile X mosaic cases, some cells in their body show full mutation and some show premutation. So, mosaic patients are also able to produce FMRP. However, the amount of the protein being produced is much lower than in normal persons. In 1999, Tassone et al. have shown a positive correlation between IQ testing and degree of protein expressed in mosaic males and partially methylated full mutation males. Thus, mosaicism as observed in this case (01/08) appears to be associated with a better prognosis. Even though conventional cytogenetic analysis showed a negative result in patient 2 (02/08), the diagnosis of Fragile X was made by molecular analysis. Methylation specific PCR and Southern Blot analysis revealed full mutation size (more than 200 CGG repeats) in this patient. The prognosis of full mutation patient appears to be not as good as the mosaics patients as the full mutation patients are not able to produce the FMRP protein at all. So, the aggressive behavioral problem of patient 1 could be well explained by his full mutation status compared with patient 2 who is a Fragile X mosaic mutation patient. From our results, it can be observed that both the cytogenetic and molecular analysis results were confirmatory in only one of the cases whereas in the other case, the results did not correspond to each other. These contradictory results can be explained by lack of sensitivity of the cytogenetic analysis compared to the molecular analysis, with a false-negativity rate of approximately 20%. This may be because of the high degree of variability of the fragile site at Xq27.3 expression between individuals, which results in the fragile site being undetected by cytogenetic techniques. Normally, only 2% to 60% of metaphases will express their fragile site in cytogenetic analysis. So, there are high chances of incidentally missing the metaphases which show fragile sites. Under-diagnosis of Fragile X Syndrome using Cytogenetic technique alone can also be because of interindividual variability among the screening technologists, because fragile site analyses need considerable experience. Even though cytogenetic technique can be used to identify the Fragile X Syndrome, not all fragile X positive samples could be detected cytogenetically. Cytogenetic analy-
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Figure 3. Results of agarose gel electrophoresis for the PCR analysis for both patients
Figure 4. Fragment analysis of Sample 01/08 showed the permutation allele with 62 CGG repeats detected
Figure 5. The Methylation Specific PCR and Southern Blot Analysis of both the patients
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sis is labor intensive, time consuming and less dependable. Moreover, cytogenetic analysis has limitation especially for detecting the mosaic mutation status as observed in case (01/08) reported here and also the premutation carriers. So, cytogenetic analysis supplemented with molecular analysis using PCR or Southern blot analysis is essential for an accurate diagnosis of Fragile X Syndrome.
ACKNOWLEDGEMENT This study is supported by Universiti Sains Malaysia Short Term Grant 304/PPSP/6131490 and 304/PPSP/6131471. We would like to thank the Department of Pathology, Prince of Songkla University, Thailand and all staffs from Human Genome Center, Universiti Sains Malaysia, Health Campus for their valuable contribution in this project. We also like to thank all the patients and their family members who have contributed to this project.
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359-371. 2) Rousseau F, Rouillard P, Morel ML, Khandjian EW, Morgan K. (1995). Prevalence of carriers of permutation size alleles of the FMRI gene and implications for the population genetics of the Fragile X Syndrome. Am J Hum Genet, 57, 1006-18. 3) Turner G, Webb T, Wake S, Robinson H. (1996). Prevalence of fragile X syndrome. Am J Med Genet, 64, 196-7. 4) Hagerman RJ, Silverman AC. (1991). Fragile X Syndrome: diagnosis, treatment, and research. Baltimore: Johns Hopkine University Press. 5) Verkerk AJ, Pieretti M, Sutcliffe JS, Fu YH, Kuhl DP, Pizzuti A, Reiner O, Richards S, Victoria MF, Zhang FP. (1991). Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in Fragile X Syndrome. Cell, 65, 905-914. 6) Reiss AL, LS Freund, TL Baumgardner, MT Abrams, MB Dencla. (1995). Contribution of the FMR1 gene mutation to human intellectual dysfunction. Nat Genet, 11, 331-334. 7) Sutherland GR. (1977). Fragile site on human chromosome: demonstration of their dependence on the type of tissue culture medium science. 265-266. 8) Sripo T, Tandviriyapaiboon D, Limpasert P. (2007). A simple method of DNA isolation from dried blood spot for screening Fragile X Syndrome using multiplex PCR. The 23rd Annual meeting of the Faculty of Medicine, Prince of Songkhla University, Thailand. 15-17. 9) Tassone F, Hagerman RJ, Ikle DN, Dyer PN, Lampe M, Willemsen R, et al. (1999). FMRP expression as a potential prognostic indicator in Fragile X Syndrome. Am J Med Genet, 84, 250-61.
