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Editor—Fragile X syndrome is the most common chromosomal cause of inherited mental retardation. At the chromosome level, this syndrome is characterised by the presence of a fragile site at Xq27.3.1 The incidence of this disorder is approximately 1 in 4000 and 1 in 7000 in males and females, respectively.2 3 In most cases, the mutation responsible for fragile X syndrome is a CGG repeat expansion in the 5′ untranslated region (UTR) of exon 1 of the FMR-1 gene. People in the normal population have six to approximately 50 repeats.4 5 Those with 50 to 200 repeats correspond to the premutation class. Repeats in this class are meiotically unstable and can expand to a full mutation.4 The premutation class encompasses the “grey area” of 45-55 CGG repeats for which there is a variable risk of repeat expansion.6 Subjects with a full mutation have repeat lengths in excess of 200, which are associated with hypermethylation of the CpG island immediately upstream of theFMR-1 gene.7-9 This methylation correlates with transcriptional suppression of theFMR-1 gene, while the repeat expansion has been suggested to cause translational suppression by impeding the migration of the 40S ribosomal subunit along the 5′ UTR of theFMR-1 gene transcript.9-11
Fragile X syndrome has also been found to occur in a few patients without CGG repeat expansions. These mutation events fall into two classes, intragenic point mutations12 13 and deletion events.14-22 Of the latter class, five patients with microdeletions in the 5′ UTR of the FMR-1gene transcript have been described.23 24
We report here a patient referred for fragile X testing who was found to carry an apparent null allele by PCR amplification of the CGG repeat region of the FMR-1 gene. This patient was analysed further using a combination of primers flanking the CGG repeat region, together with FMRP studies, in order to characterise the nature of the molecular defect underlying this apparent null allele.
The proband was born to healthy, non-consanguineous parents at 40 weeks of gestation. There was no significant family history. He weighed 4500 g (>90th centile), head circumference was 37.5 cm (>90th centile), and length was 57.5 cm (>90th centile). There was aspiration of meconium at delivery necessitating assessment in the neonatal unit. He appeared well initially but on the following day was noted to be irritable and hypotonic with an abnormal Moro reflex. A cranial ultrasound scan was normal. He required an inguinal hernia repair at a few weeks of age. His early development was felt to be normal. He had gastro-oesophageal reflux diagnosed at 8 months and was treated with ranitidine. He had mild plagiocephaly and a torticollis that required surgical correction at 18 months. He had persistent problems with drooling of saliva and tends to have an open mouthed expression. In the second year of life he had problems with recurrent ear infections requiring insertion of grommets and adenoidectomy. An assessment at the age of 3 years showed his speech and language development to be significantly delayed. His parents felt his comprehension was limited and he had difficulty retaining information. The delay had been noted earlier but had been attributed to his recurrent ear infections. Full assessment at that time showed that he had developmental delay in all areas. He had some behavioural problems with trichotillomania and obsessive traits. He did not play well with other children.
On examination by a clinical geneticist, the proband was found not have any phenotypic features suggestive of fragile X syndrome, although he did have early features of joint laxity. His head circumference was on the 50th-90th centile, his height on the 75th centile, and weight on the 50th centile. He had mild clinodactyly and fetal pads. He had mild facial asymmetry and a deep crease between his first and second toes. Examination was otherwise unremarkable. The case was referred to the laboratory for fragile X screening.
Materials and methods
CYTOGENETIC AND DNA ANALYSIS
Cytogenetic analysis of a folate deprived culture of lymphocytes was performed as previously described.25 An estimation of the length of the CGG repeats, together with an analysis of the methylation status of the CpG island of theFMR-1 gene, were performed by PCR amplification and Southern blot analysis, respectively. In the case of the latter, 5 μg of genomic DNA was digested withEcoRI and NruI, electrophoretically separated, blotted onto a positively charged nylon membrane, and hybridised with approximately 10-20 ng of probe StB12.3, as described previously.26 The hybridisation solution contained herring sperm DNA at 75 μg/ml to prevent non-specific binding of the probe. The blots were washed finally in 0.2 × SSC plus 0.1% SDS at 60°C. DNA controls included a normal male, a male with a full mutation (expanded CGG repeat with hypermethylation of the CpG island), a female with a premutation, and a normal female control. A radioactively labelled 1 kb ladder was included for sizing purposes.
PCR amplification of the CGG repeat region of theFMR-1 gene using primers FMRA and FMRB was carried out in 15 μl reactions. Each reaction comprised 10% DMSO, 50% w/v glycerol, 60 pmol of each primer, 0.4 U ofTaq DNA polymerase, 1 × PCR buffer with 0.32 mmol/l of dCTP, dATP, dTTP, and 1.5 mmol/l deaza GTP, 0.25 μl of 10 μCi μl α32P dCTP, and 0.6 mg/ml genomic DNA. Non-radioactive PCR amplification using primers FMR1 and FMR2 was carried out using the GC rich kit of Roche Diagnostics Ltd according to the manufacturer's instructions. The sequences of the primers used in the amplification reactions were FMRA (5′-GACGGAGGCGCCCGTGCCAGG-3′), FMRB (5′-TCCTCCATCTTCTCTTCAGC CCT-3′), FMR1 (5′-ATAACCGGATGCA TTTGAT-3′), and FMR2 (5′-AGGC CCTAGCGCCTATCGAAATGAGAGA-3′). Primers FMR1, FMRA, FMRB, and FMR2 were designed using the FMR-1gene sequence deposited in GenBank (Accession number X61378), with their 5′ ends at base positions 2271, 2684, 2844, and 3106, respectively. The PCR cycling conditions comprised 95°C for two minutes followed by 30 cycles of 97°C for 30 seconds, 55°C for one minute, and 72°C for one minute. The reactions were held at 4°C following a final extension of 72°C for 10 minutes. Amplification products were electrophoresed in a 1% agarose gel, together with a 100 bp DNA ladder. In the case of radioactive amplification, the products were electrophoresed in a denaturing sequencing gel using a radioactively labelled M13 sequencing ladder for sizing purposes.
Amplification products were purified for sequencing using a PCR purification kit (Roche Diagnostics). Each amplicon was sequenced using the forward and reverse amplifying primers and an Applied Biosystems (ABI) sequencing kit. DNA was recovered by ethanol precipitation and subsequently washed in 70% ethanol before the addition of denaturation buffer and loading in an ABI PRISMTM 377 DNA sequencer. The electropherograms were subsequently assembled using SeqMan DNA software.
An EBV transformed B lymphoblastoid cell line was established from a peripheral blood sample of the proband. FMRP and eIF4e levels were determined in whole cell lysates in a slot-blot based assay, using purified flag tagged murine Fmrp27 and purified human eIF4e28 as standards. Sample proteins and standards were applied to nitrocellulose membranes with a Bio-Rad slot blot apparatus. Using standard protocols, FMRP and eIF4e were detected with mouse monoclonal primary antibodies mAb 1C3 for FMRP, kindly provided by Jean-Louis Mandel,29 and anti-eIF4e (Transduction Laboratories) and HRP conjugated goat anti-mouse secondary antibody (Kirkegaard and Perry Laboratories). Signals were generated by Enhanced Chemi Luminescence (Amersham) and detected by exposure to Hyperfilm (Amersham). Signal intensities were quantified by analysis of digital scans using the program NIH Image 1.62b7f to plot signal profiles. Areas under the plot profile were calculated and used as signal intensities after subtracting out signals from the background and from the secondary antibody controls as appropriate. Standard curves were generated using data from the purified proteins, which then allowed the quantitation of protein levels in the samples. Quantitation data was calculated as the molar ratio of FMRP:eIF4e. Purified FMRP was obtained from Keith Wilkinson and eIF4e was from Curt Hagedorn, both of Emory University.
In the case of western blot studies, total proteins were isolated from EBV transformed B lymphoblasts of the proband, as well as from a normal male control. The proteins were electrophoresed in a 7.5% non-denaturing polyacrylamide gel, and transferred to nitrocellulose and hybridised using mAb 1C3 as described above.
In the case of immunohistochemical staining of FMRP from blood smears, a modification of the method of Willemsen et al 30 was used. Blood smears were counterstained with Nuclear Fast Red and 100 lymphocytes were examined for each person, together with positive and negative control blood samples. Less than 42% of lymphocytes are FMRP positive in affected males, whereas for carrier females this figure is 83%; the specificity of this assay is 100% for males and 41% for females.31
Cytogenetic analysis of the proband's chromosomes indicated an apparently normal 46,XY karyotype. Southern blot analysis showed a positively hybridising 2.8 kb DNA fragment, suggesting a normal sized CGG repeat length in the FMR-1 gene (fig1A). PCR amplification of this locus using previously published primers FMRA and FMRB32 yielded no product from the proband's genomic DNA. However, amplification products were obtained using primers FMRA and FMR2 (643 bp) and FMR1 and FMR2 (1 kb, fig 1B). The latter product was sequenced and showed a deletion of a 5 bp direct repeat, GAAGA, either immediately upstream, or encompassing the first base, of the ATG initiation codon of theFMR-1 gene (fig 1C, D). The mother of the proband was found to be heterozygous for this deletion event (data not shown). The deletion leaves the ATG codon unchanged and in phase with the remaining open reading frame of theFMR-1 gene.
FMRP quantitation, western blot analysis, and immunohistochemical studies were undertaken using the patient's lymphoblasts to determine the effect of the deletion event on translation initiation (fig 2). In order to assess the level of FMRP in the patient's lymphoblasts, quantitation studies were undertaken using the protein eIF4e as an internal control. The latter protein is the cap binding protein in eukaryotic translation initiation33 and is the rate limiting factor in translation initiation.34-36 FMRP levels were normalised with respect to eIF4e levels as a loading control. In seven cell lines from males with normal CGG allele lengths, the mean molar ratio of FMRP:eIF4e is 0.218 (standard deviation of 0.009). In the case of the cells from the proband, the molar ratio was 0.214, and thus the level of FMRP is not reduced compared to normal cell lines. In the case of the western blot analysis, normal sized FMRP was detected (fig 2A). Immunohistochemical staining of lymphocytes from the proband and his carrier mother showed FMRP staining in 80% and 98% of 100 lymphocytes examined, respectively (fig2B).
The proband reported here carries an apparent null allele with respect to the primer pair FMRA and FMRB, which are used routinely for amplifying the CGG repeat tract of the FMR-1gene. This case suggests that caution should be exercised regarding predictive testing for fragile X syndrome that relies solely on PCR amplification of the FMR-1 gene using one primer pair only. This reliance has been suggested as a first level predictive screen for fragile X syndrome in the general population.37 The need for caution with respect to single PCR amplifications of trinucleotide repeats has also been described with regard to predictive testing for the Huntington's disease (HD) gene.38 Our data underline the need for complementing PCR analysis with Southern blotting or, at minimum, PCR amplification of the CGG repeat region with two primer pairs.
Direct sequencing of amplification products using primers that map further upstream and downstream of FMRA and FMRB identified a 5 bp microdeletion near, or encompassing, the initiation codon of theFMR-1 gene. It appears that this deletion affects the annealing of the FMRB primer leading to inefficient amplification using this primer. The proband represents one of only a few cases that have been reported to have microdeletions in theFMR-1 gene.23 24 In these other cases, which were found in subjects with fragile X syndrome, the microdeletions ranged from 116 bp to 567 bp and were located in the 5′ UTR of the FMR-1 gene. The deletions were expected to lead to a lack of the FMR-1 gene product, which was confirmed in some patients.23 A mispairing model for the generation of a 486 bp deletion was described by Schmucker et al,24 which involved chi-like elements flanked by direct tandem repeats. In the case reported here, end joining, strand slippage, or indeed homologous recombination are possible molecular mechanisms that could account for the 5 bp deletion event.
Changes in the sequence of DNA upstream of an initiation codon can dramatically influence translation efficiency.39 Fragile X males with a full mutation have complete absence of FMRP. However, in the case described here, FMRP was detected of apparently normal size and at normal levels in the lymphocytes of the proband.
This study leads to the suggestion that the proband does not have fragile X syndrome and that the 5 bp deletion in this patient'sFMR-1 gene is not causative of his phenotype. The FMRP detected in this patient appears to be qualitatively and quantitatively normal. Therefore, the comprehensive screening of genes implicated in disorders that are similar to fragile X syndrome may help resolve the cause of this patient's phenotype.
We report here a case that was referred for testing for fragile X syndrome. The patient was found to carry an apparent null allele by routine clinical PCR, but with CGG repeats that fall within the normal range.
DNA sequencing showed that the patient carried a microdeletion of a 5 bp direct repeat immediately upstream, or encompassing, the translation initiation codon of the FMR-1 gene.
Protein studies indicated that the patient expressed the protein product of the FMR-1 gene (FMRP), and that this expression was at near normal levels in the patient's lymphoblasts.
We acknowledge Dr Hugh Lees of Waikato Tauranga for bringing this case to our attention and the technical assistance of Jane Iber. We further acknowledge the financial assistance of Laboratory Services of Auckland Hospital for running expenses, and the University of Auckland Research Committee and the Lottery Grants Board of New Zealand for funding an Applied Biosystems Model 377 DNA Sequencer.
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