Balanced chromosomal abnormalities such as translocations and inversions have been identified in many genetic diseases. Cloning of the breakpoints involved in these abnormalities has led to the identification of the disease-related genes. Recent reports suggest the presence of a mental retardation locus at Xq11-12. We have identified a female patient with a balanced translocation t (X;12) (q11;q15) associated with mild mental retardation. We identified a yeast artificial chromosome spanning the X-chromosome breakpoint by using fluorescent in situ hybridization techniques. A cosmid library of this YAC has been constructed and the search for candidate genes is in progress.
A major challenge for human genetics is the identification of new causes of mental retardation, which, although present in about 3% of individuals, is unexplained in over half of all cases. X-linked mental retardation is acknowledged to be a major cause of severe learning difficulties. Surveys have shown an excess of males over females with severe mental retardation and later studies suggested that the excess was the result of an X-linked condition. X-linked mental retardation (XLMR) is a vastly heterogeneous group of disorders which can be roughly categorized as syndromic (MRXS) or nonspecific (MRX). Families with syndromic disorders usually have a quite distinct phenotypic presentation whereas families with nonspecific disorders present no distinctive somatic features. Despite recent advances in identifying genes such as FMR1 , FRAXE [2, 3], L1-CAM , FGD1 and XH2 , involved in MRXS conditions, so far no gene involved in MRX has yet been identified or cloned. Compilation of the literature and McKusick’s catalogue data revealed at least 95 X-linked disorders in which mental retardation appears as the main feature. Of these 95, 40 have been regionally mapped on the X chromosome by conducting linkage studies using DNA markers in single large families or in a collection of families with the same XLMR syndrome. Several loci appear to be located in the proximal Xq region. However, it is impossible to evaluate how many MR genes there are in reality, partly because of the broad localisation and the presence of several overlaps between intervals of assignment. Thus, fine mapping and identification of genes implicated in nonspecific X-linked MR will essentially depend on thorough investigation of molecular abnormalities such as balanced translocation, inversion or contiguous gene deletion associated with MR.
Here, we report molecular cytogenetic investigations of a de novo balanced translocation t(X;12) (q11;q15) observed in a female patient with mild mental retardation. The investigation of the X-breakpoint in Xq11-q12 was undertaken because convergent strong arguments suggest the presence of a non-specific X-linked mental retardation locus in Xq11-q12. These arguments can be summerized as follows: (i) linkage data on individual large MRX families showed an assignment in several families to Xq11-Xq12 [6, 7; Partington et al., pers. commun.]; (ii) identifications of contiguous genes syndromes associated with MR resulting from deletion in Xq11-q12 encompassing the X-breakpoint region described in this report [8, 9; Gustavsson et al., pers. commun.]. We identified a yeast artificial chromosome (YAC) clone spanning the X-chromosome breakpoint by using fluorescent in situ hybridization (FISH) techniques. A cosmid library of this YAC has been constructed and the search for candidate genes is in progress.
Materials and Methods
J.P. is a 12-year-old female, the child of healthy, normal unrelated parents (father 38 years and mother 37 years), born after pregnancy with placenta praevia by delivery at full-term with Apgar scores of ten at 1 min and ten at 10 min. At birth weight was 3.10 kg, height 47.5 cm, and occipitofrontal circumference (OFC) 33.5 cm. Her mother had had two abortions previously. Infancy was complicated by status epilepticus, following by complex partial seizures. Electroencephalography was consistent with left centrotemporal spike waves. Seizures were successfully treated with carbamazepine (Tegretol®) therapy. Walking started at the age of 31 months. Developmental delay was recognized at 4 years. The patient presents severe learning difficulties. She now attends a special school for the retarded. Presently, the WIRC-R (Weschsler Intelligence Scale revised) exhibits a score of 58 (IQ). Verbal skills are seriously affected (verbal IQ 53). She also has neuromotor coordination troubles and dyspraxy without cerebellar and pyramidal tract involvement. Physical examination at 12 years shows a coarse face with proeminent nose and bilateral divergent strabismus. Fundoscopy was normal. Her height, weight and stage of puberty were normal for age. A computed tomography was performed at the age of 3, showing dilated ventricles with moderate cerebral atrophy. Magnetic resonance imagery scan performed at the age of 12 confirmed the dilated lateral ventricles and also exhibited a dilated fourth ventricle without vermis hypoplasia.
Metaphase Chromosome Preparations
Metaphase chromosomes were obtained from Epstein-Barr virus immortalized lymphoblastoid cells from the female patient with mental retardation and from phytohemagglutinin-stimulated blood cell cultures of healthy males using the methotrexate-BrdU technique as described previously .
A selection of YAC clones expected to be located in the Xq11-q13 region were used as probes for FISH on the patient’s metaphase chromosomes. We have used ICRF YAC clones ICRFy900HO493 (4690) and ICRFy900DO896 (4565) positive for DXS159 (Xq12) and DXS227 (Xq13.1) markers, respectively.
YAC probes were prepared by Alu-PCR from 100 ng of YAC DNA as reported elsewere . Alu-PCR probes specific for chromosome X or 12 were prepared as described by Cherif et al. . PCR products were ethanol precipitated, dissolved in TE and quantified on a 1.3% agarose gel. PCR products of YAC and cosmid DNAs (1 µg) were labelled by nick translation with biotin-16-dUTP (Boeh-ringer Mannheim) and purified using a Sephadex G-50 Quick spin column (Pharmacia). Chromosome X and 12 painting probes were labelled by nick translation with biotin-16-dUTP (Boehringer Mannheim) and with digoxigenin-11-dUTP (Boehringer Mannheim), respectively.
YAC 4690 Cosmid Library:
A YAC 4690-specific cosmid library was prepared using a partial Mbol digest of YAC 4690 clone, sCos-1 vector  and XL1-Blue MR cells as hosting bacteria (Stratagene). Packaging was performed using freeze thaw lysate and sonic extract (Gigapack Gold II, Strata-gene). The cosmid library was plated on XL1-Blue MR, and filter lifts were prepared on a Hybond N+ membrane (Amersham) according to the manufacturer’s protocol. The cosmid library filters were hybridized with total human DNA to identify the YAC recombinant clones. The DNA of 100 cosmid clones representing approximately 4 YAC equivalents was prepared using a conventional miniprep protocol. Proximal or distal location of different cosmids with respect to the X breakpoint has been performed by FISH analysis.
Fluorescence in situ Hybridization
To exclude a potential complex rearrangement in this X;12 translocation in a female patient, chromosome painting using Alu polymerase chain reaction (PCR) probes was performed as described by Cherif et al. . The chromosome X biotinylated painting probe was detected by avidin-FITC (Vector Laboratories) and amplified once with additional layers of biotinylated goat anti-avidin (Vector Laboratories) and avidin-FITC (Vector Laboratories). The chromosome 12 digoxigenin-labeled painting probe was detected by rho-damine anti-digoxigenin from sheep (Boehringer Mannheim) and amplified with layer of Texas red anti-sheep (Vector laboratories). The slides were observed under a Zeiss fluorescence microscope (Ax-ioplan). Chromosomes were counterstained with DAPI (Sigma).
To determine the chromosomal localization and a possible chi-merism, the YAC probes were initially hybridized to normal human metaphase chromosomes. When biotinylated PCR products of YAC DNA were used in hybridization experiments, 75 ng of probe was precipitated with 35 µg of Cotl DNA (Gibco-BRL), 10 µg of sonicated salmon sperm DNA, and resuspended in 10 µl of hybridization buffer (50% formamide, 10% dextran sulfate, 2 × SSC, pH 7). The cosmid probes were directly hybridized on metaphase chromosomes from patient lymphoblastoid cells. 50 ng of cosmid probe was mixed with 25 µg of Cot1 DNA and 10 µg of sonicated salmon sperm DNA in 10 µl of hybridization buffer. Probe denaturation, prehybridization and hybridization were performed as described by Cherif et al. . After posthybridization washes, the biotinylated probe was detected by avidin-FITC (Vector Laboratories) and amplified once with additional layers of biotinylated goat anti-avidin (Vector Laboratories) and avidin-FITC. Simultaneous localization of probes and R-banding were performed as previously described . The slides were observed under a Zeiss fluorescence microscope (Axioplan). Chromosomes were counterstained with propidium iodide and the fluorescent signal of probes appeared as two symmetrical yellow-green spots on both chromatids.
Chromosome painting using Alu PCR probes, specific for chromosomes X and 12 respectively, confirmed the t (X;12) in the female patient (fig. 1). The rearrangement appears as a balanced X;12 translocation and small molecular rearrangement could not be ruled out. This analysis only suggests that there is no complex rearrangement. YAC clones 4565 and 4690 were tested by FISH to normal metaphase chromosomes. They were localized on band Xq13.1 and Xq11-12, respectively, without cytogenetic evidence of chimerism. These YACs were hybridized to the patient’s chromosomes. The YAC clone 4565 showed specific signals on the normal X chromosome and the derivative chromosome 12, placing this YAC distal to the translocation breakpoint on band Xq11. YAC 4690 showed signals on the normal X chromosome and on both the der(X) and the der(12) chromosomes (fig. 2) indicating that this YAC clone spans the breakpoint on Xq11. By CCD camera visualization, the probe signal measurement revealed that the signal on der(12) is at least 3 times stronger than that on der(X). These data suggest that the breakpoint is located approximately 300 kb from the centro-meric YAC’ extremities (YAC 4690 is approximately 850 kb in size). More than 100 YAC 4690-specific cosmid clones have been obtained by subcloning YAC inserts into a cosmid vector. Eleven cosmids were analyzed by FISH to patient chromosomes. All cosmids were localized on Xq11 band of the normal X chromosome. Seven of them showed a signal on the der(12) chromosome and four hybridized to the der(X) chromosome.
X inactivation studies based on an R-banding pattern produced by the incorporation of BrdU after cell synchronization made it evident that the patient’s normal X chromosome was late replicating and the der(X) chromosome earlier replicating in all cells analyzed (data reviewed but not shown).
Balanced X-autosome translocations have been successfully used to identify genes involved in several X-linked diseases. In this study, we report a molecular cytogenetic investigation of an X;12 translocation associated with a nonspecific mental retardation as the first step in identifying a potential gene involved in this phenotype. Although it is difficult to rule out an involvement of the chromosome 12 breakpoint or a spreading of inactivation on the autosome, we thought it reasonable to focus first on the X chromosome breakpoint. As a direct approach towards cloning the t(X;12) (q11;q15) breakpoint, we have used YAC clones as probes for FISH analysis. Our results show that YAC clone 4690 spans the Xq breakpoint of a balanced (X;12) translocation observed in a female patient with mild mental retardation. The other clone tested was localized distal to the breakpoint on Xq. These data suggest that the genomic critical region involved in nonspecific mental retardation observed in this patient is within YAC clone 4690.
This 850-kb YAC has been shown to contain the PGK1P1, DXS133, and DXS159 loci. Recently, EPLG2, a high-affinity binding protein for a member of the eph family of receptor tyrosine kinases (Elk), has also been localized in this YAC clone . Elk is expressed only in brain and testis of the developing and adult rat, and the interaction of elk with its ligand(s) has been suggested to play a role in the development or maintenance of the nervous system. According to Fletcher et al., , EPLG2 maps distal to PGK1P1. Therefore, ELPG2 was investigated as a candidate gene for the condition observed in this female. However, EPLG2 appears to be distal to the breakpoint on Xq. FISH analysis using cosmid clones containing ELPG2 gene produced fluorescent signals on the normal X chromosome and on the der/(12) (data not shown); these results strongly suggest that ELPG2 is not disrupted by the breakpoint.
Fine α mapping of the breakpoint region using pulse field gel electrophoresis and a cosmid contig covering the breakpoint region is currently being assembled using DNA probes corresponding to cosmid ends. STSs flanking the X-breakpoint will be generated and used to look for microdeletions by PCR in about 200 unrelated non-fragile X mental retardation . Also, the cosmid contig covering the breakpoint will be used to search for expressed sequences and genes.
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