FMR2 expression in families with FRAXE mental retardation
FMR2 expression in families with FRAXE mental retardationJozef Gécz1,2,*, Ben A. Oostra5, Athel Hockey6, Pablo Carbonell7, Gillian Turner8, Eric A. Haan9, Grant R. Sutherland1,3 and John C. Mulley1,4
1Centre for Medical Genetics, Department of Cytogenetics and Molecular Genetics and 9Department of Genetics and Epidemiology, Women's and Children's Hospital, Adelaide, Australia, 2Department of Genetics, Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Bratislava, Slovakia, 3Department of Paediatrics and 4Department of Genetics, University of Adelaide, Adelaide, SA, Australia, 5Department of Clinical Genetics, Erasmus University, Rotterdam 3015GE, The Netherlands, 6Genetics Service, Princess Margaret Hospital for Children, Perth, Australia, 7Unidad de Genética Humana, Centro de Bioquímica y Genética Clínica, Espinardo 301 00, Spain and 8Department of Medical Genetics, Prince of Wales Children's Hospital, Sydney, Australia
Received October 3, 1996;Revised and Accepted December 6, 1996
Normal individuals express the two alternative transcripts, FMR2 and Ox19, from the FRAXE-associated CpG island. Molecular analysis of the Ox19 transcript suggests that it is a truncated isoform of the FMR2 gene with an alternative 3' end. Both isoforms showed a similar pattern of expression, with the Ox19 isoform expressed at a much lower level. Fibroblasts, chorionic villi and hair roots showed the highest level of FMR2 expression, whole blood cells and amniocytes showed very low expression, and the transcript was not detected in lymphoblasts. Fibroblasts of 11 individuals from seven families segregating FRAXE were assayed for FMR2 expression and FRAXE CpG island methylation. A man with an unmethylated expansion of 0.6 kb expressed FMR2 and represents a pre-mutation carrier. All chromosomes with FRAXE CCG expansions of 0.8 kb or greater were fully methylated and did not express the FMR2 gene, analogous to the mechanism of silencing the FMR1 gene in carriers of the FRAXA full mutation. The boundary between FRAXE pre-mutation and FRAXE full mutation is between 0.7 and 0.8 kb. Two men with absence of FMR2 expression in fibroblasts were not mentally impaired, suggesting that IQ in some men with FRAXE full mutation may remain within the normal range. Although molecular tools to study FRAXE non-specific mental retardation are now available, further psychometric and molecular studies are needed to characterize the effect of the FRAXE full mutation for the purpose of genetic counselling.
FRAXE is a folate-sensitive fragile site in Xq28 ~600 kb distal to the FRAXA (1 ,2 ). Molecular characterization revealed that individuals expressing FRAXE had amplifications of a CCG repeat adjacent to a CpG island (3 ). Normal individuals showed 4-39 (3 -5 ) copies of the polymorphic FRAXE CCG repeat, while individuals expressing the fragile site had >200 copies and their CpG island was fully methylated (3 ). These findings were similar to those found for another cloned folate-sensitive fragile site FRAXA (6 -8 ).
In the first family reported with FRAXE (1 ,9 ), the fragile site was not clearly segregating with mental retardation (MR). Further studies on additional families co-segregating mental impairment and the FRAXE fragile site suggested that an aetiological relationship may exist between FRAXE and mild non-specific X-linked mental retardation (XLMR) (9 -12 ). This relationship was difficult to establish in the absence of a phenotype other than mild (or borderline) non-specific MR or a gene associated with FRAXE. Recently, the gene FMR2 associated with the FRAXE fragile site was cloned (13 ,14 ). This was accomplished by positional cloning of a candidate FRAXE gene interrupted by two overlapping submicroscopical deletions in Xq28 (15 ) and a large-scale sequencing approach (14 ). FMR2 was confirmed as the FRAXE-associated gene when cytogenetically positive individuals with 100% methylation of the FRAXE CpG island were found to have transcription of the gene silenced (13 ,14 ). Chakrabarti et al. (16 ) simultaneously reported a candidate gene at the FRAXE fragile site (Ox19, ~1495 bp transcript) which overlapped with the FMR2 gene but was considerably truncated and possessed a different 3' end. No expression studies of Ox19 in FRAXE individuals were presented (16 ).
Few families co-segregating FRAXE and a mild form of XLMR have been ascertained. This suggests a low incidence of FRAXE MR, difficulty of ascertainment, or both. Screening programs in different candidate populations of mentally impaired boys detected no (4 ,17 ) or only a few new cases of FRAXE MR (18 -21 ). Based on these studies, the prevalence of FRAXE was estimated to be ~4% of FRAXA, or ~1/50 000 males (22 ). However, if FRAXE results in a shift of the distribution of IQ into the mild MR or borderline MR range, leaving a significant overlap between IQ in FRAXE males and normal males, then carriers of FRAXE full mutation may remain unidentified within the community.
Summary of the molecular studies on FRAXE families
Family
Status
[Delta] (kb)
Methylationa
IQ/mental statusb
FMR2
X-inact
Reference
1.
1.1
M, Affected
1.2-1.8
100%
53
-
NA
9, 13
1.2
F, Affected
0.7
505
73
+
R
9, 13
2.
2.1
M, Affected
1.3
100%
special school
-
NA
9, 13
2.2c
M, Molecularly affected,
1.1
100%
uncompleted secondary
-
NA
9, 13
clinically normal
schooling
3.
3.1
M, Affected
1.0
100%
behavioural difficulties
-
NA
9, this study
3.2
F, Premutation
0.8
100%
normal
+
R
9, this study
4.
M, Premutation
0.6
0%
normal
+
NA
this study
5.
M, Affected
1.3-3.1
100%
intellectually disabled
-
NA
9, this study
6.
6.1
M, Affected
1.6
100%
MR, macroorchidism
-
NA
12, this study
6.2c
M, Molecularly affected,
0.8-4.3
100%
normal
-
NA
12, this study
clinically normal
7.
F, Premutation
2.5
100%
normal
+
NR
this study
aThe methylation status was determined on fibroblast DNA in all individuals tested.bIQ is presented only for those individuals tested, otherwise any major mental problems or just MR are noted.cClinically assessed as unaffected.NA, not applicable; R, random inactivation; NR, non-random inactivation; M, male; F, female.
Transcription of full-length and truncated FMR2 in normal individuals
Full-length (13 ,14 ) and truncated (16 ) isoforms of the FMR2 gene have been isolated from the region of the FRAXE CpG island. The Northern blot analysis of the Ox19 cDNA showed a 9.5 kb transcript (16 ) identical in size to that of FMR2 (13 -15 ). Sequence comparison between the small 1.5 kb, Ox19 polyadenylated cDNA clone (16 ) and FMR2 (13 ,14 ) revealed sequence identity in the 5' ends of the two transcripts. However, starting from position 1367 onwards until reaching the 3' end (position 1495), the Ox19 transcript diverged from FMR2. In order to demonstrate that the Ox19 transcript was a true isoform of FMR2, genomic sequence was generated around the point of the divergence and RT-PCR performed to demonstrate transcription in different tissues.
The genomic sequence was generated from the YAC D49G8 (13 ,23 ) employing a modified IRE-bubble approach (24 ) (see Materials and Methods) using FMR2-Ox19-specific oligonucleotides 37 and 38 (Fig. 1 ). The extra sequence of the FMR2-Ox19 transcript (position 1367-1495) is co-linear with that of genomic DNA (Fig. 1 ). At the point of divergence of the two transcripts, there is an exon-intron boundary in the FMR2 pre-mRNA transcript with a relatively weak 5' splice donor site [GUGAA/guaag versus consensus A(C)AG/gugag (25 )]. As a consequence this weaker 5' donor splice site may cause alternative splicing of the FMR2 pre-mRNA and so generate the truncated FMR2-Ox19 isoform. The existence of this short isoform was assayed by RT-PCR using primers 37 (Fig. 1 ) and 10 (a primer upstream from exon 3, ref. 13 ). From a variety of different tissue RNAs tested (adult brain, liver and lung; fetal brain, muscle; mature placenta; chorionic villi; fibroblasts and lymphoblasts), only lymphoblasts and liver were negative (results not shown). RT-PCR analysis using the reverse primer (primer 38; Fig. 1 ) and downstream primers 7 or 43 yielded no detectable PCR products in all tissues examined. This would indicate that the FMR2-Ox19 transcript either terminates at the point detected by Chakrabarti et al. (16 ) or possesses a completely different 3' end from that of FMR2. However, attempts to clone it using a 3' RACE procedure resulted in no sequence additional to that of Chakrabarti et al. (16 ) (results not shown).
The proximal FMR2 probe (exons 2-5, ref. 13 ) hybridized to a Northern blot detected at least four smaller size transcripts (3, 1.8, 1.2 and 1.0 kb) in addition to the ~9.5 kb transcript (Fig. 2 and Gu et al. 14 ). Among them, the 1.8 kb transcript might correspond to FMR2-Ox19. These transcripts were not detected with several other FMR2 probes from the region of overlap between FMR2 and FMR2-Ox19 (14 -16 ).They may thus represent other cross-reacting or closely related transcripts such as LAF-4 (26 ) or MLLT2 (27 ,28 ).
Genomic DNA used for methylation and X-inactivation studies was isolated from cultured fibroblast cells. DNA from all FRAXE and control individuals was digested with HindIII alone and then with HindIII and NotI together. The Southern blot was probed with OxE20 (3 ). All affected FRAXE males tested showed 100% methylation of the CpG island at the NotI site (Fig. 3 , lanes 3.1, 5, 6.1). In addition, another boy clinically assessed as unaffected with an expansion [Delta] = 0.8-4.3 kb (6.2; maternal nephew of individual 6.1, ref. 12 ) showed 100% methylation of his mosaic FRAXE allele. Methylation was absent however in man 4 (sib of two FRAXE brothers) with a [Delta] value of 0.6 kb. The existence of a rare HindIII polymorphism which would mimic FRAXE CCG expansion was excluded by haplotype analysis. The three brothers inherited from their mother the same region of the X chromosome surrounding FRAXE, with different FRAXE CCG expansions (B. Oostra, unpublished results). We reported (9 ) a similar size unmethylated expanded allele in a carrier female (Table 1 , individual 1.2). Two additional carrier females tested (Table 1 , individuals 3.2 and 7) showed 100% methylation of the expanded allele ([Delta] = 0.8 kb for individual 3.2 and [Delta] = 2.5 kb for 7, respectively).
Southern blot analysis of two phenotypically normal females carrying full mutations (3.2, [Delta] = 0.8 kb and 7, [Delta] = 2.5 kb) suggested skewed X-inactivation (Fig. 3 ; intensity of the 2.7 kb HindIII-NotI fragment versus 5.2 kb HindIII product, respectively). Female 1.2 carrying a [Delta] = 0.7 kb pre-mutation showed mild mental retardation. To assess the involvement of skewed inactivation to their phenotypes, an X-inactivation study was carried out. X-inactivation was studied in all three females (Table 1 , individuals 1.2, 3.2 and 7) using a differential methylation assay of HpaII sites in the FMR1 (30 ) and androgen receptor (AR) (31 ) genes, respectively. Results are summarized in Table 1 . Only one female (number 7) showed a convincingly skewed pattern of inactivation as determined from the AR assay. The FMR1 assay was not informative as she is homozygous for the FMR1 (CGG)n repeat.
Present knowledge about the pattern of expression of the FMR2 gene is derived from Northern blot hybridization on adult (14-16) and fetal (14) tissue mRNA. The suitability of other cellular mRNAs was evaluated for detecting the presence of the FMR2 transcript in cell types usually available in a diagnostic laboratory. Results of this work are summarized in Table 2 . Fibroblasts and cultured chorionic villi cells expressed the highest levels of FMR2, while no expression was detected in lymphoblasts. FMR2 was detected in whole blood cells, hair roots and cultivated amniocytes, however at a very low level. All expression studies were carried out by standard RT-PCR. Usually 35-40 cycles were required to detect the PCR product on an ethidium bromide-stained agarose gel. A number of FMR2-specific primers were used both from the 5' and 3' ends (see Materials and Methods).
FMR2 expression as assayed by RT-PCR.Abundance was classified according to the intensity of the RT-PCR product as: +++, abundant; ++, present; +, very low, -, not detected.aAfter a 24 h stimulation with PHA.
Expression analysis of FMR2 in individuals from FRAXE families was carried out on mRNA isolated from cultured skin fibroblasts. The same fibroblast cultures as for genomic DNA isolation (methylation and X-inactivation study) were used. First strand cDNA was amplified either in a standard PCR or PCR with a fluorescent label (Fig. 4 ). Results of the expression study are summarized in Table 1 . All methylated FRAXE chromosomes showed no expression of the FMR2 gene, while all unmethylated FRAXE chromosomes did express the gene. In concordance with the methylation studies, individual 6.2 (assessed as unaffected) did not express the FMR2 gene.
The FMR2 gene is associated with the FRAXE CpG island. The three reported transcripts originate from within the CpG island just distal to the FRAXE CCG repeat. While two of the transcripts (13 ,14 ) were identical, the other (16 ) had a different 3' end. To clarify the relationship between the two different FRAXE transcripts, the origin and expression pattern of the FMR2-Ox19 transcript was investigated. Molecular analysis suggested that the FMR2-Ox19 transcript represents a ~1.5 kb attenuated isoform of the full-length ~9.5 kb FMR2 transcript. A weak 5' donor splice site of exon 9 together with the presence of two polyadenylation-like signals might account for its premature termination and poly(A) addition. Northern blot hybridization with a proximal FMR2 probe (exons 2-5, ref. 13 ) revealed several smaller size transcripts (3, 1.8, 1.2 and 1.0 kb; Fig. 2 and Gu et al., ref. 14 ), suggesting that one of them might correspond to the FMR2-Ox19 isoform. These transcripts were not detected on Northern blots using the genomic VK21A probe (contains exon 3 only), the Ox19.3 cDNA probe (at least exon 3, ref. 16 ) or the B5P6C4 cDNA probe (further downstream exons, ref. 14 ). They may thus represent cross-reacting RNA species probably due to the sequence homology of exons 4 and 5 of the FMR2 gene. Both exons 4 and 5 encode protein domains which show high sequence similarity to those of LAF-4 (26 ) and MLLT2 (27 ,28 ) proteins (CVEEIL for exon 4 and EMTHSWPTPLT for exon 5). Molecular analysis (this work and Chakrabarti et al., ref 16 ) demonstrated the existence of the FMR2-Ox19 isoform, however it seems to be expressed at a very low level, detectable only by PCR and not on a Northern blot. Whether the short FMR2-Ox19 isoform has any physiological significance is yet to be determined. Sequence comparison with cloned and partially characterized FMR2-related proteins LAF-4 (26 ) and MLLT2 (27 ,28 ) suggests such a protein variant (FMR2-Ox19) would lack the nuclear localization signal and lack what is possibly a putative transcription transactivation domain (26 ) (J.G., unpublished observation). It is of interest that at least in LAF-4 a similar isoform has been documented (E41 RAJI, ref. 26 ). Both FMR2 and FMR2-Ox19 isoforms showed similar patterns of expression, with FMR2-Ox19 being much less abundant in all tissues tested.
In this study, we present data from members of seven FRAXE families collected thanks to a wide international collaboration. Altogether we analysed and show here results obtained from six affected FRAXE males, two unaffected sibs of affected FRAXE individuals and three carrier females. As clinical, especially psychometric and familial data have already been or are being presented elsewhere, these are not included. Table 1 gives a summary of individuals examined with references to work describing the clinical data of the corresponding families. Families of individuals 4 and 7 have not been reported yet. Individual 4 has two brothers with an expanded FRAXE CCG repeat (B.A. Oostra, unpublished data), while individual 7 is the mother of a mildly intellectually handicapped male with a FRAXE CCG expansion [Delta] = 1.3 kb (G. Turner, unpublished data).
To generate the genomic sequence around the point of divergence of the FMR2 and FMR2-Ox19 transcripts, the IRE-bubble approach of Munroe et al. (24 ) has been adopted. Briefly, DNA of the YAC D49G8 was digested with three frequent-cutting enzymes, HaeIII, RsaI and AluI, and ligated to NotI-A bubble anchors. IRE-bubble PCR was carried out on the individual ligation products using the FMR2-Ox19-specific oligonucleotides 37 and 38 in combination with the bubble primer. Thirty to 35 cycles of PCR were performed under the conditions described (24 ). Amplification products obtained subsequently were purified by QiaQuick spin columns (Qiagen) and sequenced directly using dye-terminator sequencing chemistry (Perkin Elmer) with the original PCR primers (3.2 pmol per reaction). Sequencing reactions were run and analysed on an ABI373A automated sequencer (Applied Biosystems).
Fibroblast genomic DNA was digested with HindIII alone and HindIII and NotI together at 37oC overnight. Southern blot analysis was performed according to the instructions of the manufacturer of the membranes (Hybond N+; Amersham). The probe OxE20 (3 ) was used in methylation studies. After an overnight hybridization at 65oC, the membranes were washed in medium stringency wash (2* SSC; 0.5% SDS at 65oC) and exposed for 2-4 days at -70oC on a radiographic film (DuPont).
For X-inactivation studies, the differential methylation of the HpaII sites in the FMR1 (30 ) and AR (31 ) genes was studied. Fibroblast genomic DNA (100 ng) either uncut or pre-digested to completion with HpaII was used. PCR was performed under the conditions described (30 ,31 ) using the following primers: FMR1, forward primer 5'-GCGCTCAGCTCCGTTTCGGTTTCA-3' and reverse 5'-CTCCATCTTCTCTTCAGCCCTGCTA-3'; and AR, forward 5'-TCCAGAATCTGTTCCAGAGCGTGC-3' and reverse 5'-GCTGTGAAGGTTGCTGTTCCTCAT-3'. Amplification products were analysed on 5% denaturing polyacrylamide gels. Dried gels were exposed to radiographic films (DuPont) for 1-3 days.
Cytoplasmic mRNA was isolated either by the guanidinium thiocyanate method (34 ) followed by oligo(dT) chromatography (Pharmacia), or by using direct mRNA purification on magnetic beads (Dynal). For cultured cells (fibroblasts, lymphoblasts, chorionic villi and amniotic fluid cells), 1*105-5*106 cells were processed in a single experiment. Hair roots (10-20) were collected into the denaturing buffer of the direct mRNA purification kit (Dynal) and homogenized mechanically in a porcelain mortar. To obtain fresh blood, ~ 100 ml was taken from a finger prick, spun immediately and pellets resuspended in the denaturing buffer (Dynal). Phytohaemaglutinin-stimulated whole blood cells were processed after a 24 h incubation in the same way as fresh blood cells. Approximately 10-200 ng of purified mRNA was random primed for first strand cDNA synthesis using SuperScript II RNase H- reverse transcriptase (BRL). Reverse transcription was carried out for 1 h at 45oC in a water bath. Negative controls where the reverse transcriptase was omitted were run at the same time although the primers were chosen to amplify across large introns. PCR amplifications using either FMR2 gene-specific primers or control esterase D primers were performed on 1/20 of the original reverse transcription reaction. Altogether, 35 cycles of PCR were carrying out (96oC for 30 s; 60oC for 30 s; and 70oC for 1 min) in a final 100 ml volume containing 100 mM Tris-HCl, pH 8.3; 50 mM KCl; 1.5 mM MgCl2; 0.2 mM of each dNTP; 50 pmol of each primer; and 1 U of Taq polymerase (Boehringer). For FMR2 expression studies, the following primers were used: primers 10 5'-GAAAACCCAGTGCAGCCAGTTC-3' (1358-1379, ref. 13 ) and 37 5'-TTTCACATGCATCGAGTCCGTT-3' (Fig. 1 ), amplicon 463 bp and/or 376 bp; primers 38 5'-TTAACGGACTCGATGCATGTGA-3' (Fig. 1 ); and primer 7 5'-CCCTAAT- GAGACTGAGGAGAGGC-3' (2143-2122, ref. 13 ) or 43 5'-CTCAGAGCTGCTCTCCGATTCGC-3' (1848-1815, ref. 13 ). Primers 10 and 37 amplify across the exon 5 which was found to be alternatively spliced (13 ). As a control, esterase D primers 5'-GGAGCTTCCCCAACTCATAAATGCC-3' (423-447; GenBank accession no. M13450) and 5'-GCATGATGTCTGATGTGGTCAGTAA-3' (875-851, idem), amplicon 452 bp were used (35 ). One-third of the PCR product was analysed on 1.5% agarose gels with 0.5 mg/ml of ethidium bromide in 1* TBE.
We greatly appreciate help from Rosalie Smith with the fibroblast lines, Kathie Friend with the GeneScan analysis and Jean Spence with DNA isolation and oligonucleotide synthesis. We are grateful to the families and patients who kindly agreed to donate a skin biopsy and thus participate in this study. We thank K.E. Davies and S.J.L. Knight for the OxE20 probe and L.B.A. de Vries for supplying us with a fibroblast cell line from individual 4 (Table 1 ). This work was supported by the National Health and Medical Research Council of Australia and by an International Research Scholars Award from the Howard Hughes Medical Institute to G.R.S. P.C. was supported by grant 93/00004-00 from FISS.
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