Skip Navigation

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (28)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Frints, S. G. M.
Right arrow Articles by Froyen, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Frints, S. G. M.
Right arrow Articles by Froyen, G.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Human Molecular Genetics, 2003, Vol. 12, No. 13 1463-1474
DOI: 10.1093/hmg/ddg165
© 2003 Oxford University Press

CALL interrupted in a patient with non-specific mental retardation: gene dosage-dependent alteration of murine brain development and behavior

Suzanna G. M. Frints1,2, Peter Marynen1, Dieter Hartmann1, Jean-Pierre Fryns2, Jean Steyaert2, Melitta Schachner3, Bettina Rolf3, Katleen Craessaerts1, An Snellinx1, Karen Hollanders1, Rudi D'Hooge4, Peter P. De Deyn4 and Guy Froyen1,*

1Flanders Interuniversity Institute for Biotechnology (VIB) and 2University Hospital Leuven, Center for Human Genetics, Leuven, Belgium, 3Zentrum für Molekulare Neurobiologie, Eppendorf Clinic, University of Hamburg, Hamburg, Germany and 4Laboratory of Neurochemistry and Behavior, Born-Bunge Foundation, Department of Biomedical Sciences and Department of Neurology/Memory Clinic, Middelheim Hospital, University of Antwerp, Antwerp, Belgium

Received November 12, 2002; Revised April 22, 2003; Accepted April 30, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Investigation of MR patients with 3p aberrations led to the identification of the translocation breakpoint in intron five of the neural Cell Adhesion L1-Like (CALL or CHL1) gene in a man with non-specific mental retardation and 46,Y, t(X;3)(p22.1;p26.3). The Xp breakpoint does not seem to affect a known or predicted gene. Moreover, a fusion transcript with the CALL gene could not be detected and no mutations were identified on the second allele. CALL is highly expressed in the central and peripheral nervous system, like the mouse ortholog ‘close homolog to L1’ (Chl1). Chl1 expression levels in the hippocampus of Chl1+/- mice were half of those obtained in wild-type littermates, reflecting a gene dosage effect. Timm staining and synaptophysin immunohistochemistry of the hippocampus showed focal groups of ectopic mossy fiber synapses in the lateral CA3 region, outside the trajectory of the infra-pyramidal mossy fiber bundle in Chl1-/- and Chl1+/- mice. Behavioral assessment demonstrated mild alterations in the Chl1-/- animals. In the probe trial of the Morris Water Maze test, Chl1-/- mice displayed an altered exploratory pattern. In addition, these mice were significantly more sociable and less aggressive as demonstrated in social exploration tests. The Chl1+/- mice showed a phenotypic spectrum ranging from wild-type to knockout behavior. We hypothesize that a 50% reduction of CALL expression in the developing brain results in cognitive deficits. This suggests that the CALL gene at 3p26.3 is a prime candidate for an autosomal form of mental retardation. So far, mutation analysis of the CALL gene in patients with non-specific MR did not reveal any disease-associated mutations.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Subtelomeric deletions usually result in contiguous gene syndromes with mental retardation (MR) as a clinical feature (1). More than 70 patients with 3p- syndrome (2,3) or partial 3p trisomies (4) have been reported. Molecular analysis shows that the deleted or duplicated regions vary in size (3). It is thus assumed that in the 3p chromosomal region a number of genes contribute to the MR phenotype (5,6). MR can be classified as either syndromic or non-specific. In patients with syndromic forms of MR, the clinical manifestations include MR associated with other physical features (such as neurological symptoms, skeletal defects or facial dysmorphism), whereas in patients with non-specific MR no distinctive clinical or biochemical manifestations occur, apart from the cognitive impairment. In the last 6 years, several genes have been identified in non-specific X-linked MR (reviewed in 7). Recently, the neurotrypsin (PRSS12, MIM no. 606709) gene at 4q24 has been found to be responsible for an autosomal recessive form of non-specific MR (8). We present data on a patient with non-specific MR and an interruption of one allele of the neural cell adhesion L1-like (CALL or CHL1; hereafter referred to as CALL) gene. The CALL gene has already been reported as a candidate for autosomal-recessive non-specific MR (9). Moreover, it is proposed as a gene which may be associated with schizophrenia (10). Furthermore, CALL is most frequently deleted in patients with the 3p- syndrome (5,11,12). We provide evidence that the CALL gene might be involved in cognitive and behavioral functions, through CALL and Chl1 mRNA expression studies in brain combined with histology and behavioral studies in Chl1-/- and Chl1+/- mice. Finally, a mutation analysis was performed in patients with non-specific MR and with no family history for the disease.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
FISH analysis of a t(X;3)(p22.1;p26.3)
We investigated patients with 3p chromosomal aberrations in order to identify autosomal candidate genes for non-specific MR in the 3p subtelomeric region. A 3p25.3->pter map was constructed in silico (Fig. 1). This region contains several brain-expressed genes such as CALL (GenBank accession no. NM_006614), contactin 6 (CNTN6), contactin 4 [an axonal-associated (AXCAM-related) cell adhesion molecule], a novel neuronal leucine-rich repeat (NLRR-1)-like, inositol 1,4,5-triphosphate receptor type 1 (ITPR1, MIM no. 147265), a Class B basic helix–loop–helix 2 (BHLHB2/DEC1, MIM no. 604256) and a KIAA0212 transcript. FISH analysis was performed with four BAC clones and one cosmid clone (Fig. 1, P1–P5). In one patient with non-specific MR and translocation 46,Y,t(X;3)(p22.1;p26.3), signals of probe P2 were present on either side of the breakpoint, indicating that the CALL gene is affected in this patient.



View larger version (41K):
[in this window]
[in a new window]
  		Figure 1. Schematic representation of the 3p25.3->pter chromosomal subtelomeric region, with the locations of the probes P1–P5 used for FISH analysis with the distance in Mb between brackets. Probe P1: RP11-731E12, including the 5' region of the CALL gene; probe P2: RP11-114K9, including the 3' region of CALL; probe P3: RP11-497I24 (centromeric to CNTN6); probe P4: CTC-243A6, including D3S18; and probe P5: a cosmid clone containing the Von Hippel Lindau (VHL) locus. The arrow indicates the 3p breakpoint in the patient with translocation 46,Y,t(X;3)(p22.1;p26.3). The dotted line represents the interstitial deletion flanked by markers D3S3630 and D3S1304 of a child reported with MR and 3p syndrome (6). The vertical line on the right corresponds to the linkage interval with flanking markers D3S3525 and D3S1304 of the family with an autosomal recessive form of non-specific MR, reported by Higgins (9), and with an asterisk indicating the maximum LOD-score of 9.18 and 8.27 at marker D3S3050 and D3S1560, respectively. The genes reported to be expressed in brain are indicated in bold. STSs are indicated as D3S numbers.

 
Cloning of the breakpoints
We used standard positional cloning strategies to clone the breakpoints in the patient with translocation 46,Y, t(X;3)(p22.1;p26.3). The Xp22.1 breakpoint was mapped to PAC clone RP6-102C13 (Fig. 2A). On 3p, the CALL gene was interrupted between exon 4 and exon 16 since a split signal was observed by FISH with BAC RP11–166F3 (Fig. 2B and C). The aberrant HindIII band detected by Southern blot analysis with a probe derived from RP6-102C13 (Fig. 2D), was cloned and sequenced. A 26 bp insertion, ACATATATAT ATATATCCTT ATATAT was identified at the breakpoint in CALL intron 5 (BAC RP11-114K9 nucleotide position 16068). PCR with primer pairs (XpF1+3pR1 and 3pF1+XpR1), annealing at either side of the breakpoints, revealed the expected PCR products with gDNA of the patient but not with normal controls (Fig. 2E). RACE experiments starting from CALL exon 3 or 5 (3'-RACE) and exon 6 or 7 (5'-RACE) resulted in the exclusive amplification of the CALL gene derived from the intact second allele, indicating that no fusion transcript was present. Sequence analysis of the open reading frame (cDNA position 272–3946) of the CALL cDNA of the patient did not reveal any mutation of the CALL gene on the second, wild-type allele. Finally, position effects by the 3p breakpoint were not expected since the nearest gene, contactin 6 (CNTN6) (13), was at 1 Mb from the CALL gene. So far, CALL seems to be the most telomeric gene on chromosome 3p.



View larger version (32K):
[in this window]
[in a new window]
  		Figure 2. Cloning of the 3p26.3 and Xp22.1 breakpoints. (A) Schematic representation of the X-chromosome with the Xp22.1 breakpoint located in genomic clones RP6-102C13 and RP11-617O8. Both clones form a contig with GS1-466O4 and RP11-702C7. The two flanking genes, MAGE-B6 and NA88-A (pseudogene) are 87 and 148 kb away from the Xp breakpoint, respectively. B, H2 and H3 indicate the restriction sites BamHI, HindII and HindIII, respectively. The gray box represents the Xp probe, used for Southern hybridization. STSs are indicated as DXS-numbers. (B) Schematic representation of chromosome 3 with the 3p26.3 breakpoint situated in BAC contig including clones RP11-166F3 and RP11-114K9, which nearly cover the complete Neural Cell Adhesion L1-like (CALL) gene. The 3p breakpoint interrupts the gene in intron 5. Exons are indicated as black boxes (not drawn to scale) and cover 86 kb of genomic DNA. (C) FISH analysis in the patient with lysamine-labeled alpha-satellite probes of centromere 3 and X, and biotin-labeled RP11-166F3 (3p subtelomeric region), which spans the Xp22.1 and 3p26.3 breakpoints (arrowheads). (D) Southern blot analysis of genomic DNA (HindII and HindIII) of the patient (P), and man (M) and woman (F) controls with probe Xp, derived from PAC RP6-102C13. Size indications of the bands are shown on the side, with arrows pointing at the aberrant bands in the patient. (E) PCR on gDNA with inter-chromosomal primers pairs XpF1+3pR1 and 3pF1+XpR1, situated at either site of the breakpoints showed only the corresponding 716 and 460 bp PCR product in the patient (P) but not in normal men (M1 and M2) or woman (F1). (—) The negative control; SL indicates the 1 kb DNA ladder.

 
A contig map was constructed at the Xp breakpoint combining our data with information available via public (NCBI, Sanger) and private (Celera) databases. The Xp breakpoint was situated in a THE1-internal LTR/MaLR-class repeat (RP11-617O8 nucleotide position 85696). Using various gene prediction programs such as BLAST/unigene, GRAIL-1.3, GENSCAN, Fgene, Genefinder, Hexon and GeneMark, the first predicted genes are located 87 kb upstream (MAGE-B6) and 148 kb downstream (NA88-A pseudogene) with respect to the breakpoint. None of the two predicted genes are expressed in human brain (14–16). However, expression of both genes could not be investigated in the presented patient since they are not expressed in peripheral blood leukocytes (PBLs) or fibroblasts. Our data strongly suggest that no gene on the X-chromosome is involved and that CALL is the only affected gene in this patient.

CALL and Chl1 expression
The CALL gene has been proposed as a candidate gene for non-specific MR because it is highly expressed in brain (11). Real-time quantitative PCR demonstrated high CALL expression in total fetal and adult brain, and in a Schwann cell culture, compared to other human tissues or cell lines (Fig. 3). mRNA expression levels of the CALL gene in PBLs, Epstein–Barr virus (EBV)-transformed cell lines or fibroblasts were very low to undetectable so we were unable to evaluate relative CALL expression levels in the only available material obtained from the patient.



View larger version (31K):
[in this window]
[in a new window]
  		Figure 3. Quantification of CALL mRNA expression by real-time PCR in human adult tissues normalized to ß-ACTIN (black bars) and relative to heart (CT=32.0), indicating that highest CALL expression levels occur in total adult brain (CT=21.1). The gray bars show CALL expression in human neural cell lines and fetal tissues, which was normalized to GAPDH and relative to the glioma HTB-138 cell line (CT=28.1), indicating that the highest expression is in total fetal brain (CT=21.6).

 
CALL has been identified as the mouse Chl1 ortholog in humans (11,17). Chl1 shows high expression levels in brain regions such as cerebral cortex, thalamus, hippocampus and amygdala (18,19). The Chl1-/- mouse (20), allowed us to investigate a Chl1 gene dosage effect in the hippocampus. Using real-time quantitative PCR, we could demonstrate that, in one-day-old (P1) Chl1+/- mice, the expression levels were half of those present in Chl1+/+ littermates. As expected, Chl1-/- mice did not show any Chl1 mRNA expression (Fig. 4A). Furthermore, we could detect a 2-fold decrease of Chl1 expression in the hippocampus of 6-day-old (P6) Chl1+/+ littermates compared to P1 Chl1+/+ mice (Fig. 4B). The observed Chl1 mRNA expression differences between the P1 and P6 littermates were very constant; a finding not observed for another L1 family member, Ncam180 (data not shown).



View larger version (14K):
[in this window]
[in a new window]
  		Figure 4. Analysis of Chl1 mRNA expression levels in hippocampus of Chl1+/- mice normalized to Gapdh (black bars) or ß-Actin (gray bars). (A) Chl1 expression in Chl1+/+ (lane 1), Chl1+/- (lanes 2–7) and Chl1-/- (lanes 8–11) littermates of one-day-old (P1), relative to wild-type P1 expression. The Chl1 expression of wild type (CT=20.5) is about double that of heterozygotes. Chl1-/- mice do not show any expression. (B) Chl1 expression in 6-day-old (P6) wild-type littermates (lanes 1–4), relative to wild-type P1 (indicated with an asterisk) expression. The Chl1 expression in P6 is about half that obtained in P1.

 
Ectopic bundles of mossy fibers in Chl1-/- and Chl1+/- mice
Mouse hippocampi were dissected, fixed and embedded for histological and immunochemical examination. No morphological abnormalities were noticed using markers GFAP and F4/80 in P6 and adult brains of Chl1-/- and Chl1+/- mice (data not shown). However, in Chl1-/- and Chl1+/- adult brains, between one and three focal groups of bundles of mossy fiber synapses per section were situated outside of the trajectory of the infra-pyramidal mossy fiber bundles of the lateral CA3 region of the hippocampus, as detected by Timm staining. These ectopic mossy fiber synapses were detected in seven out of 10 Chl1-/- (Fig. 5E and J) and in three out of nine Chl1+/- mice (Fig. 5B, D, G and I), compared to one in seven Chl1+/+ mice. Synaptophysin staining in the same brains confirmed these ectopic mossy fiber synapses.



View larger version (135K):
[in this window]
[in a new window]
  		Figure 5. Ectopic mossy fiber synapses in the hippocampal formation of Chl1+/- and Chl1-/- mice. Mossy fiber synapses were traced by histochemical visualization of Zn2+ using the Timm sulphide silver technique (A–E) and by antibodies against synaptophysin (F–J). Pictures were taken with a Zeiss Axioplan Imaging 2e combined microscope and camera at primary magnifications of 5x and 10x with the same exposure time. In the wild-type hippocampal formation (A, C, F, H) both techniques reveal the trajectory of mossy fibers originating as axons from the granule cells of the dentate gyrus (DG) and passing through the cornu ammonis fields CA4 and CA3 to stop abruptly at the border between CA3 and CA2. Within the CA3 region, the mossy fibers form discrete suprapyramidal (SMF) and infrapyramidal (IMF) bundles, whereby the latter typically terminates within the medial third of the CA3 region, thereby partly trespassing the pyramidal cell layer. In both Chl1+/- and Chl1-/- mice (B, D, G, I and E, J, respectively) additional infrapyramidal mossy fiber synapses are present in an ectopic position further laterally in the CA3 region. These ectopic mossy fibers (EMF, arrows) form small multifocal groups that can be traced over short series of sections corresponding to a depth of about 50–100 µm each. Zn2+- and synaptophysin-positive structures indicative of synapses are both situated in an infrapyramidal position, possibly contacting basal dendrites (I, J), as well as within the pyramidal cell layer itself (D).

 
Altered exploratory and social adaptive behavior in Chl1-/- and Chl1+/- mice
Male Chl1-/-, Chl1+/- and Chl1+/+ mice were subjected to a whole set of behavioral assessment studies (Table 1) and alterations were observed in the Chl1-/- mice. In the probe trial of the Morris Water Maze test, Chl1-/- mice displayed an altered exploratory pattern. In addition, the Chl1-/- mice were significantly more sociable and less aggressive, as demonstrated in the social exploration tests. The Chl1+/- mice showed a phenotypic spectrum ranging from wild-type to knockout behavior. A detailed description of the methods and results obtained with these studies can be found online, in the Supplementary Material.


View this table:
[in this window]
[in a new window]
  		Table 1. Summary of observations from behavioral studies performed with Chl1-/-, Chl1+/- and Chl1+/+ male mice (for details see Supplementary Material)
 
Screening for disease-associated mutations
We screened for mutations in all 26 coding exons (exons 3–28) and intronic boundaries in 20 patients with non-specific MR and with no family history for the disease. In comparison to the reference sequences, eight variants (Table 2) were identified of which five occurred at high frequency (>30%). These include one missense alteration in exon 3 (c.320C>T; p.L17F) with an allele frequency of 0.75/0.25, and four intronic variants namely IVS4+10G>C (0.83/0.17), IVS8-25C>T (0.70/0.30), IVS9+127 C>T (0.30/0.70) and IVS9-41G>A (0.73/0.27). The intronic variation (IVS27+18T>C) was detected in two patients only (0.95/0.05) while IVS12-33G>A was restricted to one single patient (0.98/0.02). Finally, an interesting missense variant in exon 14 (c.1711A>T, p.K480N) was noticed in one patient leading to heterozygosity at this position (0.98/0.02). This nucleotide change affects a non-conserved residue in the Ig5 domain of CALL. This variation was not found in 100 control samples (200 chromosomes) as revealed by sequence analysis of this exon, but was detected in the normally intelligent mother of this patient, indicating it to be a rare polymorphism.


View this table:
[in this window]
[in a new window]
  		Table 2. Genotype and allele frequencies of the 8 polymorphisms of the CALL gene in 20 patients with non-specific MR
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Most patients with partial 3p monosomy show a contiguous gene deletion syndrome with constant clinical findings of severe MR, pre- and postnatal growth retardation and facial dysmorphism. Additional clinical features include deafness, digital abnormalities, renal and gastrointestinal anomalies and congenital heart defects, depending on the extent of the deletion (21). Patients with partial 3p trisomy are usually moderately to severely mentally retarded, show facial dysmorphism and often include clinical features of the associated partial monosomy (22–24). However, in some patients the phenotype is not associated with major dysmorphic features (25). Patients with MR and apparently balanced translocations involving the 3p chromosomal region have not been reported so far. Taken together with various observations (3,6,26,27), this study shows that the 3p chromosomal region is a ‘hot-spot’ in the etiology of autosomal forms of MR.

Several patients with MR and 3p aberrations were investigated by fluorescent in situ hybridization (FISH) analysis. Through positional cloning of a translocation t(X;3)(p22.1;p26.3), we provide strong evidence that the CALL gene is involved in non-specific MR. With real-time quantitative PCR we demonstrated that the CALL gene is highly expressed in the central and peripheral nervous systems, confirming that it is a candidate gene for a functional role in the nervous system. The CALL gene was cloned and mapped (11) based on knowledge of the mouse ortholog Chl1 (17). CALL belongs to the L1 family of immunoglobulin (Ig)-like neural cell adhesion molecules. It has several isoforms and contains six Ig-like domains, four fibronectin type III-like domains, a single-pass transmembrane domain and a short cytoplasmic tail (11). CALL is most closely related to L1CAM and shows an overall 32% identity (49% similarity). Like L1CAM, CALL has recognition sites for integrins, fibroblast growth factor receptors and phosphotyrosine kinases (28). Neural cell adhesion molecules, such as L1, are involved in neurite outgrowth (29,30), axonal guidance (31), cell migration and adhesion (32). These actions are processed via specific signal transduction pathways (reviewed in 33,34). Mutations in neural cell adhesion molecules have been implicated for several neurological disorders. L1CAM mutations, responsible for the ‘L1-syndrome’, mainly affect the central nervous system. This allelic X-linked MR (XLMR) disorder includes X-linked SPG1, MASA syndrome and X-linked hydrocephalus (35–39). Mutations in L1CAM have been found to interfere with ligand interactions and cell-surface expression (40). Heterozygous L1CAM mutations in women can be associated with a variable phenotype including MR, abnormal brain development, spastic paraplegia and adducted thumbs (41). Occasionally, MR is the only clinical feature in woman carriers (28,42). Functional analysis in mice and humans have been reported, including promoter (43), ligand interaction (44) and genotype–phenotype correlation studies (41,45,46).

Since one allele of the CALL gene is interrupted in the patient, and no mutation was found on the second allele, we propose that the pathogenic mechanism in this patient is haplo-insufficiency. Angeloni et al. (5) already suggested that CALL haplo-insufficiency could be the pathogenic mechanism in 3p- syndrome based on its location in the deleted regions. Presumably, the MR phenotype of patients with partial 3p trisomy could also be the result of disturbed CALL gene dosage. Because of the very low expression levels in PBLs, EBV-transformed or fibroblast cell lines we could not investigate a possible gene dosage effect of CALL mRNA expression. For the same reason, potential position effects of the nearest flanking genes at the Xp breakpoint could not be investigated. The nearest gene, CNTN6, is located at ~1 Mb of the 3p breakpoint and it is thus unlikely that it is affected by the rearrangement (47,48). This can not be excluded, however, as a long-range effect of a chromosomal anomaly at a distance of 1 Mb has recently been reported (49).

The phenotype of the Chl1-/- mice shows subtle brain abnormalities in the form of aberrant projection of mossy fibers in the CA3 subfield of the hippocampus (20) and an altered exploratory behavior, suggesting that cognitive processes involving spatial memory are disturbed. Since Chl1+/- mice were not included in their study (20), we initiated tests in Chl1-/- and Chl1+/- mice in order to support the hypothesis of haplo-insufficiency by analyzing the Chl1 expression levels and studying their behavior. Chl1 has an overall identity of 83% with CALL (97% identity in the C-terminal 105 amino acids). Their mRNA expression patterns are similar with highest expression in brain and in neural cell cultures (11,17). Chl1 promotes neurite outgrowth in vitro (19,50) and prevents neuronal death (51). In contrast to L1, Chl1 does not bind to itself, nor does it bind heterophilically to L1 (19). However, both Chl1 and L1 show overlapping expression patterns (19) and their expression is up-regulated in axonal regeneration (52,53).

By real-time quantification we could clearly demonstrate a gene dosage effect in mouse hippocampi. The Chl1 mRNA levels of Chl1+/- mice were ~50% of those found in Chl1+/+ controls. Liu et al. (18) demonstrated a strictly regulated gradient of Chl1 expression with a high-caudal to low-rostral pattern in rat brain during embryonic development and noticed highest Chl1 levels at P1–P7. Thereafter, the expression drops significantly but mRNA levels sustain during adulthood. Both findings support the importance of the spatial and temporal availability of the correct amount of Chl1 during brain development. Indeed, we also find highly constant expression levels at the time points investigated (Fig. 4). The question then arises whether a 50% reduction of Chl1 mRNA expression could result in a disturbed development of neurons leading to abnormal cognitive function and behavior.

Histological examination of the brains of Chl1-/- and Chl1+/- mice demonstrated an aberrant mossy fiber organization in the CA3 region of the hippocampus. However, the aberrant pattern seen in the Chl1-/- mice was different from the one reported by Montag-Sallaz (20). They described a disorganized CA3 region of the hippocampus with individual thin mossy fibers or some axons forming small bundles, which traveled in a disorganized way through the CA3 region between supra and infra-pyramidal mossy fiber bundles. We detected significantly more bundles of ‘ectopic’ mossy fiber synapses in the lateral CA3 region of the hippocampus of Chl1-/- and Chl1+/- mice. Ectopic mossy fibers have already been reported in Ncam180-/- mice (54). However, in other Ncam180 null mutant mice, a reduction of mossy fibers in the CA3 subregion was noticed (55). Similarly, the Gdi1-/- mice, the only knockout mouse model for X-linked non-specific MR so far, did not show gross brain abnormalities (56), but a trilamination of the infra-pyramidal mossy fibers and disorganized CA3 pyramidal cell layer in the hippocampus of these mice was reported. Moreover, they showed decreased synaptic potential of hippocampal neurons in these knockout mice. Further experiments are necessary to define the situation in Chl1-/- and Chl1+/- mice.

We subsequently investigated whether the histological aberrant brain organization resulted in altered behavior of Chl1-/- and Chl1+/- mice. In the probe trial of the Morris Water Maze test, the Chl1-/- mice were less efficient in exploratory searching behavior, a result in agreement with the observations by Montag-Sallaz (20). However, the swimming patterns in their study were different from the ‘retention’ pattern we observed. Impaired probe trial performance could be indicative of alterations in exploratory behavior or behavioral flexibility that are not due to hippocampal defects (57). In the resident-intruder test, Chl1-/- mice were less aggressive, more sociable and showed mating behavior, a finding that was not observed in Chl1+/+ mice. Interestingly, in all social exploration and interaction tests, the Chl1+/- mice showed a behavioral spectrum ranging from wild-type to knockout, resulting in a mean that reflects the intermediate score. Behavioral studies with Ncam-/- and Ncam+/- mice also demonstrated altered aggressive responses in these animals compared to wild-type mice (58), indicating that the phenotypic outcome of other genes of the immunoglobulin superfamilies could be gene dosage-dependent and that these genes affect social interactions, which often involve the amygdala and the hippocampus. Moreover, increased social and mating behavior has also been described for the Gdi1-/- mouse (heterozygous mice not tested) in the resident–intruder test (56).

The discrepancies in mossy fiber projections and in the behavior in the Morris Water Maze as well as in open field experiments between our mice of a mixed Sv129/C57BL/6J background and mice having an almost pure C57BL/6J background (20) illustrate the importance of the genetic background (59,60). However, in both mouse strains an aberrant mossy fiber projection and an altered exploratory behavior of Chl1-/- mice was observed. Amongst many other examples, genetic background differences were also observed for mossy fiber terminal configuration in Fmr1-/- mice (61,62) as well as for the extent of ventricular enlargement in L1-/- mice (45,63,64). As mentioned earlier, more than one gene has been proposed as a candidate gene for the MR phenotype in the 3p- syndrome. Centromeric to the CALL gene, several other candidate genes for MR can be predicted based on their relatively high expression in the (developing) brain and their functional involvement in signaling pathways and/or presumed role in growth regulation of neurons. Two genes, the CNTN4 and NLRR-l-like genes, are predicted in the 3p25.3 chromosomal region. These genes are promising candidates for autosomal dominant and/or recessive forms of MR. Both genes (but not CALL) are deleted in a child with developmental delay and a 3p25.3–p26.2 interstitial deletion (6), and both are linked to the mapped interval in a six-generation family with autosomal-recessive MR described by Higgins and co-workers (9). In this family, the CALL gene was excluded by single-strand conformation polymorphism analysis (J. Higgins, personal communication). Further investigations on patients with MR and apparently balanced 3p translocations might lead to the discovery of novel MR genes in the proximal 3p21–p25 chromosomal region. Indeed, the novel gene MEGAP/srGAP3 has recently been proposed as one candidate gene for MR associated with the 3p- syndrome (65), but others might still be discovered.

Mutation analysis of the CALL gene in 20 MR patients with non-specific MR did not reveal any disease-associated mutations. Eight polymorphisms were identified (six intronic and two missense changes) including one rare missense change (p. K480N). Recently, sequence analysis of the CALL gene in 24 schizophrenia patients reported only four nucleotide variants (66), of which two were also identified by our screen. In their study, the polymorphic missense alteration in exon 3 (n.320C>T, p.L17F) was correlated with schizophrenia (note that they use a different exon numbering). The Leu allele was present at higher frequencies in schizophrenic patients (0.71/0.29) compared with controls (0.63/0.37). The frequency of the Leu17 allele in our (small) MR subpopulation was even higher (0.75/0.25).

In conclusion, we provide evidence that the CALL gene is involved in non-specific MR. In Chl1+/- mice a gene dosage effect was clearly demonstrated. Since the strictly regulated spatial and temporal Chl1 expression is important for axonal development, abnormalities in Chl1 expression may lead to the subtle histological and behavioral phenotypes in Chl1-/- and Chl1+/- mice. This suggests that CALL haplo-insufficiency is a likely mechanism contributing to the MR phenotype in the reported patient with translocation 46,Y,t(X;3)(p22.1;p26.3). Although it is likely that there are other MR genes in the 3pter chromosomal region, we propose the CALL gene as a first candidate gene for an autosomal dominant form of non-specific MR. The detection of mutations in other patients will have to prove this hypothesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Patients with 3p aberrations
The local Research and Medical Ethical Committee of the University Hospital of Leuven approved the study and informed consent was obtained from the participants or their guardians. Fourteen patients with MR and 3p aberrations were investigated by FISH after conventional G-, T- and high resolution banding was performed on blood lymphocytes.

FISH analysis
FISH analysis was performed on metaphase spreads of blood leukocytes or of EBV-transformed blood lymphocytes, following standard procedures (67). A lysamine-labeled alpha-satellite probe of chromosome 3 (Appligene Oncor, Darra, Australia) was used with the 3p25.3->pter probes RP11-731E12 (GenBank accession no. AC055763), including the 5' region of the CALL gene at 3p26.3 (probe P1), RP11-114K9 (GenBank accession no. AC011609), including the 3' region of CALL (probe P2), RP11-497I24 (GenBank accession no. AC018833), close and centromeric to CNTN6 at 3p26.3 (probe P3), CTC-243A6, partially including the OXTR gene at 3p25.3 (probe P4) and a cosmid clone containing the Von Hippel Lindau (VHL) locus at 3p25.3 (probe P5) (GenBank accession no. NT_005927). Both breakpoint regions of the patient with 46,Y,t(X;3)(p22.1;p26.3) were further analyzed with the 3p26.3 BAC clones RP11-306H5 (GenBank accession no. AC026187) and RP11-166F3 (GenBank accession no. AC026171) and with the Xp22.1 BAC/PAC clones GS1-466O1 (GenBank accession no. AC005297), RP6-102C13, RP11-617O8 (GenBank accession no. AC073614) and RP11-702C7 (GenBank accession no. AC079178).

Clinical report of the patient with 46,Y,t(X;3)(p22.1;p26.3)
One of the eight investigated patients with non-specific MR is a 76-year-old man, who is a resident in the Psychiatric Institute in Geel (Belgium). He was the second child of non-consanguinous Caucasian parents. Two younger brothers and one sister died during early childhood of unknown causes, and the mother had had three miscarriages. Two other brothers have normal intelligence and a normal 46,XY karyotype. Physical measurements of the patient are all normal. Neurological examination showed overall bradykinesia. Tendon reflexes were brisk and there were no pathological reflexes. His spontaneous hand position was with his thumbs adducted, but no contraction of the thumbs, or spasticity of the hands or legs was present. Coordination was intact. Because of his low mental level, psychiatric assessment was obtained from the nursing personnel and through clinical observation. We saw an emotionally dull and passive old man with adequate social contact, orientation appropriate for his mental level, bradyphrenia and poor attention span. He spoke in short sentences and displayed mild echolalia. The subject's history mentions several episodes of anxiety with agitation that responded well to haloperidol. However, a psychiatric diagnosis for these past episodes was not available. A brain CT-scan showed non-specific and age-related findings. Intelligence tests indicated that he was moderately mentally retarded (50<TIQ<70), with a developmental age equivalent of 4–5 years and a balanced developmental profile on McCarthy's Developmental Scale (68). The level of adaptive functioning (AAA test) (69) was situated 2 SD below the mean scores of the standard population with MR. His weakest characteristics (>2 SD below the mean) were community use and home-living.

Cloning of the 46,Y,t(X;3)(p22.1;p26.3) breakpoints
A map of the Xp22 breakpoint region was constructed with obtained sequences and sequences available from human genome databases. For Southern blotting, genomic DNA was digested with BamHI, HindII or HindIII. 32P-labeled probes were used for hybridization. The hybridized membranes were exposed to Biomax hypersensitive films (Kodak) at -70°C. High molecular weight genomic DNA of the patient was digested with HindII or HindIII and used for long distance inverse PCR (70). First round PCR was performed with primers LDI2-F1 and -R1, and second round with nested primers LDI2-F2 and -R2, using buffer 3 of the Expand Long Template PCR System (Roche Applied Science, Basel, Switzerland). PCR conditions were as follows: denaturation at 94°C for 4 min, annealing at 63°C for 30 s and elongation at 68°C for 14 min for 30 cycles. The PCR products were cloned and sequenced. A map of the 3p26 breakpoint region was constructed in silico using the available databases. (All primer sequences are available online, see Supplementary Material Table 3.)

RACE experiments and mutation analysis in the patient
Total RNA for rapid amplification of cDNA ends (RACE) was extracted from an EBV-transformed cell line of the patient using Trizol (Life Technologies, Gaithersburg, MD). RACE experiments were performed as described earlier (71). Briefly, first-strand cDNA was generated with the oligo (dT)-adaptor primer 465 (for 3' RACE) or the CALL-specific primers CALL-ex9R1 and -ex7R1 (for 5' RACE). The latter first-strand product was extended with an A-tail using the Terminal Deoxynucleotide Transferase kit (Roche), followed by the second-strand synthesis with the primer mixture 1748+1749+1750 (dT8)V. A first round of PCR was performed with the RACE adaptor primer 467 in combination with the CALL-specific primer CALL-ex3F1 or -ex5F1 for 3' RACE, or CALL-ex7R1 or -ex9R1 for 5' RACE. Nested PCR was performed with the RACE adaptor primer 468 and other internal CALL-specific primers. RACE PCR products were ligated in pGEM-T Easy (Promega, Madison, WI) and sequenced.

For mutation screening of the second CALL allele, cDNA synthesis was performed with random hexamer primers (Life Technologies) starting from total RNA of an EBV-transformed cell line of the patient. Overlapping PCR products for the CALL open reading frame were generated, using primer sets (CALL-ex1F1+-ex5R1, -ex5F1+-ex10R1, -ex10F1+-ex14R1, -ex14F1+-Tmr, -Tmf+-ex23R1, -ex20F1+-ex27R1 and -ex26F1+-ex28R1) for 40 cycles with annealing at 63°C. PCR products were directly sequenced on an automated ABI-PRISM 310 sequencer (Applied BioSystems, Foster City, CA).

Real-time quantitative PCR
Total RNA from human adult tissues was commercially obtained (BD BioSciences Clontech, Palo Alto, CA); total RNA from human fetal brain, lung, liver and muscle was a kind gift of Dr V. Kalscheuer (Max-Planck-Institute for Molecular Genetics, Berlin, Germany). Total RNA from a human Schwann cell line (kindly provided by Dr E. Legius, Leuven, Belgium), the glioma HTB-138 and neuroblastoma CCL-127 cell lines, and from mouse hippocampi was extracted with the RNeasy® mini kit (Qiagen) according to the manufacturer's recommendations. First-strand cDNA synthesis was performed on 1 µg total RNA with 80 U Superscript II using random hexamer primers (Life Technologies) in a final volume of 20 µl. Real-time quantitative PCR was performed with CALL and Chl1 (GenBank accession no. NM_007697) gene-specific primers on an ABI PRISM 7700 SDS apparatus (Applied BioSystems) as described elsewhere (72). The mRNA levels were normalized to the housekeeping genes GAPDH and/or ß-ACTIN and relative differences were calculated according to the comparative CT method (SDS User Bulletin 2; Applied Biosystems).

Immunohistochemistry
The mice were deeply anesthetized and perfused transcardially with 60 ml 1 : 4 Bouin solution in phosphate buffered saline pH 7.4 (PBS) or alternately with 20 ml 1% Na2S, NaH2PO4 and 20 ml 4% paraformaldehyde in PBS solution, twice each, for Timm staining. Brains were fixed overnight at room temperature in the respective perfusion solution. One hemisphere was cryo-preserved in deep-frozen 2-methyl butane and the other was paraffin-embedded after dehydration in ethanol using methyl benzoate as an intermediate. For immunohistochemistry, paraffin-embedded brain sections were used as described previously (73), with primary antibodies directed against glial fibrillary acid protein (GFAP as a marker for astroglia; Sternberger Inc., Lutherville, MD, USA), F4/80 (a marker for microglia and macrophages, obtained from ATCC number HB-198) or synaptophysin (a marker for presynaptic boutons; Synaptic Systems, Göttingen, Germany). Timm staining was performed as described (74) for either cryo-preserved or paraffin-embedded brain sections.

Behavioral studies in mice
Chl1-/- mice were generated and genotyped as described previously (20). In contrast to the mice described in their study (almost pure C57BL/6J background), we obtained mice that were crossed in a mixed Sv129/C57BL/6J background. The behavioral studies focused on hippocampal functions in ten Chl1-/-, 15 Chl1+/- and 10 Chl1+/+ male mice using spatial, exploratory and contextual memory tasks, and tests of anxiety and social exploration. Statistical analysis of the behavioral data was performed using one- and two-way analysis of variance (ANOVA) with Tukey HSD test for pair wise comparison, and the Fisher's exact test. To examine the effect of genotype and trial on escape latency, path length and proportional search times in the Morris Water Maze task, a two-way ANOVA for repeated measures (RM) was used. Data are reported as mean values±SEM, and P-values below 0.05 were considered significant. More details on the methods used are available as Supplementary Material.

Mutation screening of the CALL gene
Mutation screening of CALL was performed by sequence analysis of all 26 coding exons with their flanking intronic sequences. The PCR primers (in Supplementary Material, Table 4) were designed by Vector NTi (Informax, Bethesda, MD) and PCR was performed on 50 ng genomic DNA for 35 cycles on a 9700 thermal cycler (Applied Biosystems). PCR products were directly sequenced with either of both PCR primers using the BigDye v3.1 sequencing kit and ran on an ABI-PRISM 3100 capillary sequencer (Applied Biosystems). Sequences were assembled and analyzed with the ContigExpress sequence assembly software (Informax) towards the genomic (GenBank accession no. AC011609) and cDNA (GenBank accession no. NM_006614) reference sequences. Identified nucleotide changes were verified by sequencing the opposite strand from a new PCR product.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We would like to thank all the patients and family members, which were involved in this study, as well as the medical staff including Rudy Theulinx, from the Psychiatric Institute of Geel (Belgium) for their cooperation. Furthermore, we want to acknowledge Phillipe Demaerel for his expert opinion on the CT-scan, Martine Borghgraef and Bernice de Vos for the IQ testing, Lut van den Berghe for clinical assistance, Myriam Welkenhuysen for psychological support, Reinhilde Thoelen, Giselle Degeest and Bernadette Vanderschueren for technical assistance and Frieda Franck for excellent assistance with the behavioral studies in mice. This project was supported by Research grants (G-0299-01 and G-0027-97) of the Fund for Scientific Research, Flanders (FWO, Vlaanderen), Belgium and the German Research Society.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Human Genome Laboratory, VIB4, Center for Human Genetics, Herestraat 49, B-3000 Leuven, Belgium. Tel: +32 16345948; Fax: +32 16347166; Email: guy.froyen{at}med.kuleuven.ac.be Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

  1. Riegel, M., Baumer, A., Jamar, M., Delbecque, K., Herens, C., Verloes, A. and Schinzel, A. (2001) Submicroscopic terminal deletions and duplications in retarded patients with unclassified malformation syndromes. Hum. Genet., 109, 286–294.[CrossRef][Web of Science][Medline]

  2. Conte, R.A., Pitter, J.H. and Verma, R.S. (1995) Molecular characterization of trisomic segment 3p24.1->3pter: a case with review of the literature. Clin. Genet., 48, 49–53.[Web of Science][Medline]

  3. Drumheller, T., McGillivray, B.C., Behrner, D., MacLeod, P., McFadden, D.E., Roberson, J., Venditti, C., Chorney, K., Chorney, M. and Smith, D.I. (1996) Precise localisation of 3p25 breakpoints in four patients with the 3p syndrome. J. Med. Genet., 33, 842–847.[Abstract/Free Full Text]

  4. Reiss, J.A., Sheffield, L.J. and Sutherland, G.R. (1986) Partial trisomy 3p syndrome. Clin. Genet., 30, 50–58.[Web of Science][Medline]

  5. Angeloni, D., Lindor, N.M., Pack, S., Latif, F., Wei, M.H. and Lerman, M.I. (1999) CALL gene is haploinsufficient in a 3p- syndrome patient. Am. J. Med. Genet., 86, 482–485.[CrossRef][Web of Science][Medline]

  6. Cargile, C.B., Goh, D.L., Goodman, B.K., Chen, X.N., Korenberg, J.R., Semenza, G.L. and Thomas, G.H. (2002) Molecular cytogenetic characterization of a subtle interstitial del(3)(p25.3p26.2) in a patient with deletion 3p syndrome. Am. J. Med. Genet., 109, 133–138.[CrossRef][Web of Science][Medline]

  7. Frints, S., Froyen, G., Marynen, P. and Fryns, J.P. (2002) X-linked mental retardation: vanishing boundaries between non-specific (MRX) and syndromic (MRXS) forms. Clin. Genet., 62, 423–432.[CrossRef][Web of Science][Medline]

  8. Molinari, F., Rio, M., Meskenaite, V., Encha-Razavi, F., Auge, J., Bacq, D., Briault, S., Vekemans, M., Munnich, A., Attie-Bitach, T. et al. (2002) Truncating neurotrypsin mutation in autosomal recessive nonsyndromic mental retardation. Science, 298, 1779–1781.[Abstract/Free Full Text]

  9. Higgins, J.J., Rosen, D.R., Loveless, J.M., Clyman, J.C. and Grau, M.J. (2000) A gene for nonsyndromic mental retardation maps to chromosome 3p25-pter. Neurology, 55, 335–340.[Abstract/Free Full Text]

  10. Sakurai, K., Migita, O., Toru, M. and Arinami, T. (2002) An association between a missense polymorphism in the close homologue of L1 (CHL1, CALL) gene and schizophrenia. Mol. Psychiat., 7, 412–415.

  11. Wei, M.H., Karavanova, I., Ivanov, S.V., Popescu, N.C., Keck, C.L., Pack, S., Eisen, J.A. and Lerman, M.I. (1998) In silico-initiated cloning and molecular characterization of a novel human member of the L1 gene family of neural cell adhesion molecules. Hum. Genet., 103, 355–364.[CrossRef][Web of Science][Medline]

  12. Angeloni, D., Wei, M.H. and Lerman, M.I. (1999) Two single nucleotide polymorphisms (SNPs) in the CALL gene for association studies with IQ. Psychiatr. Genet., 9, 165–167.[CrossRef][Web of Science][Medline]

  13. Kamei, Y., Tsutsumi, O., Taketani, Y. and Watanabe, K. (1998) cDNA cloning and chromosomal localization of neural adhesion molecule NB-3 in human. J. Neurosci. Res., 51, 275–283.[CrossRef][Web of Science][Medline]

  14. Lucas, S., De Plaen, E. and Boon, T. (2000) MAGE-B5, MAGE-B6, MAGE-C2, and MAGE-C3: four new members of the MAGE family with tumor-specific expression. Int. J. Cancer, 87, 55–60.[CrossRef][Web of Science][Medline]

  15. Lurquin, C., De Smet, C., Brasseur, F., Muscatelli, F., Martelange, V., De Plaen, E., Brasseur, R., Monaco, A.P. and Boon, T. (1997) Two members of the human MAGEB gene family located in Xp21.3 are expressed in tumors of various histological origins. Genomics, 46, 397–408.[CrossRef][Web of Science][Medline]

  16. Moreau-Aubry, A., Le Guiner, S., Labarriere, N., Gesnel, M.C., Jotereau, F. and Breathnach, R. (2000) A processed pseudogene codes for a new antigen recognized by a CD8(+) T cell clone on melanoma. J. Exp. Med., 191, 1617–1624.[Abstract/Free Full Text]

  17. Holm, J., Hillenbrand, R., Steuber, V., Bartsch, U., Moos, M., Lubbert, H., Montag, D. and Schachner, M. (1996) Structural features of a close homologue of L1 (CHL1) in the mouse: a new member of the L1 family of neural recognition molecules. Eur. J. Neurosci., 8, 1613–1629.[CrossRef][Web of Science][Medline]

  18. Liu, Q., Dwyer, N.D. and O'Leary, D.D. (2000) Differential expression of COUP-TFI, CHL1, and two novel genes in developing neocortex identified by differential display PCR. J. Neurosci., 20, 7682–7690.[Abstract/Free Full Text]

  19. Hillenbrand, R., Molthagen, M., Montag, D. and Schachner, M. (1999) The close homologue of the neural adhesion molecule L1 (CHL1): patterns of expression and promotion of neurite outgrowth by heterophilic interactions. Eur. J. Neurosci., 11, 813–826.[CrossRef][Web of Science][Medline]

  20. Montag-Sallaz, M., Schachner, M. and Montag, D. (2002) Misguided axonal projections, neural cell adhesion molecule 180 mRNA upregulation, and altered behavior in mice deficient for the close homolog of L1. Mol. Cell. Biol., 22, 7967–7981.[Abstract/Free Full Text]

  21. Lukusa, T., Devriendt, K. and Fryns, J.P. (1999) A 3p deletion syndrome in a child with both del(3)(p25->pter) and dup(17)(q23->qter). Ann. Genet., 42, 91–94.[Web of Science][Medline]

  22. Kleczkowska, A., Fryns, J.P. and Van den Berghe, H. (1984) Partial trisomy of chromosome 3(p14->p22) due to maternal insertional translocation. Ann. Genet., 27, 180–183.[Web of Science][Medline]

  23. Parloir, C., Fryns, J.P. and Van den Berghe H. (1979) Partial trisomy of the short arm of chromosome 3 (3p25 to 3pter). A distinct clinical entity. Hum. Genet., 47, 239–244.[CrossRef][Web of Science][Medline]

  24. Fryns, J.P., Kleczkowska, A., Casaer, P. and Van den Berghe, H. (1986) Double autosomal chromosomal aberration (3p trisomy/9p monosomy) and sex-reversal. Ann. Genet., 29, 49–52.[Web of Science][Medline]

  25. Smeets, E., Vandenbossche, L. and Fryns, J.P. (2001) Partial distal trisomy 3p. A partial autosomal trisomy without major dysmorphic features. Genet. Couns., 12, 85–89.[Web of Science][Medline]

  26. Neri, G., Reynolds, J.F., Westphal, J., Hinz, J. and Daniel, A. (1984) Interstitial deletion of chromosome 3p: report of a patient and delineation of a proximal 3p deletion syndrome. Am. J. Med. Genet., 19, 189–193.[CrossRef][Web of Science][Medline]

  27. Naritomi, K., Hirayama, K., Sameshima, K. and Ohdo, S. (1988) Proximal 3p deletion: case report and review of the literature. Acta Paediatr. Jpn., 30, 78–83.[Medline]

  28. Kenwrick, S., Watkins, A. and De Angelis, E. (2000) Neural cell recognition molecule L1: relating biological complexity to human disease mutations. Hum. Mol. Genet., 9, 879–886.[Abstract/Free Full Text]

  29. Walsh, F.S. and Doherty, P. (1997) Neural cell adhesion molecules of the immunoglobulin superfamily: role in axon growth and guidance. A. Rev. Cell Dev. Biol., 13, 425–456.[CrossRef][Web of Science][Medline]

  30. Haspel, J., Friedlander, D.R., Ivgy-May, N., Chickramane, S., Roonprapunt, C., Chen, S., Schachner, M. and Grumet, M. (2000) Critical and optimal Ig domains for promotion of neurite outgrowth by L1/Ng-CAM. J. Neurobiol., 42, 287–302.[CrossRef][Web of Science][Medline]

  31. Tessier-Lavigne, M. and Goodman, C.S. (1996) The molecular biology of axon guidance. Science, 274, 1123–1133.[Abstract/Free Full Text]

  32. Kadmon, G. and Altevogt, P. (1997) The cell adhesion molecule L1: species- and cell-type-dependent multiple binding mechanisms. Differentiation, 61, 143–150.[CrossRef][Web of Science][Medline]

  33. Brummendorf, T., Kenwrick, S. and Rathjen, F.G. (1998) Neural cell recognition molecule L1: from cell biology to human hereditary brain malformations. Curr. Opin. Neurobiol., 8, 87–97.[CrossRef][Web of Science][Medline]

  34. Crossin, K.L. and Krushel, L.A. (2000) Cellular signaling by neural cell adhesion molecules of the immunoglobulin superfamily. Dev. Dyn., 218, 260–279.[CrossRef][Web of Science][Medline]

  35. Rosenthal, A., Jouet, M. and Kenwrick, S. (1992) Aberrant splicing of neural cell adhesion molecule L1 mRNA in a family with X-linked hydrocephalus. Nat. Genet., 2, 107–112.[CrossRef][Web of Science][Medline]

  36. Jouet, M., Rosenthal, A., MacFarlane, J., Kenwrick, S. and Donnai, D. (1993) A missense mutation confirms the L1 defect in X-linked hydrocephalus (HSAS). Nat. Genet., 4, 331.[CrossRef][Web of Science][Medline]

  37. Jouet, M., Rosenthal, A., Armstrong, G., MacFarlane, J., Stevenson, R., Paterson, J., Metzenberg, A., Ionasescu, V., Temple, K. and Kenwrick, S. (1994) X-linked spastic paraplegia (SPG1), MASA syndrome and X-linked hydrocephalus result from mutations in the L1 gene. Nat. Genet., 7, 402–407.[CrossRef][Web of Science][Medline]

  38. Vits, L., Van Camp, G., Coucke, P., Fransen, E., De Boulle, K., Reyniers, E., Korn, B., Poustka, A., Wilson, G., Schrander-Stumpel, C. et al. (1994) MASA syndrome is due to mutations in the neural cell adhesion gene L1CAM. Nat. Genet., 7, 408–413.[CrossRef][Web of Science][Medline]

  39. Fransen, E., Van Camp, G., Vits, L. and Willems, P.J. (1997) L1-associated diseases: clinical geneticists divide, molecular geneticists unite. Hum. Mol. Genet., 6, 1625–1632.[Abstract/Free Full Text]

  40. De Angelis, E., Watkins, A., Schafer, M., Brummendorf, T. and Kenwrick, S. (2002) Disease-associated mutations in L1 CAM interfere with ligand interactions and cell-surface expression. Hum. Mol. Genet., 11, 1–12.[Abstract/Free Full Text]

  41. Dobson, C.B., Villagra, F., Clowry, G.J., Smith, M., Kenwrick, S., Donnai, D., Miller, S. and Eyre, J.A. (2001) Abnormal corticospinal function but normal axonal guidance in human L1CAM mutations. Brain, 124, 2393–2406.[Abstract/Free Full Text]

  42. Kaepernick, L., Legius, E., Higgins, J. and Kapur, S. (1994) Clinical aspects of the MASA syndrome in a large family, including expressing females. Clin. Genet., 45, 181–185.[Web of Science][Medline]

  43. Edelman, G.M. and Jones, F.S. (1998) Gene regulation of cell adhesion: a key step in neural morphogenesis. Brain Res. Brain Res. Rev., 26, 337–352.[CrossRef][Medline]

  44. Thelen, K., Kedar, V., Panicker, A.K., Schmid, R.S., Midkiff, B.R. and Maness, P.F. (2002) The neural cell adhesion molecule L1 potentiates integrin-dependent cell migration to extracellular matrix proteins. J. Neurosci., 22, 4918–4931.[Abstract/Free Full Text]

  45. Fransen, E., D'Hooge, R., Van Camp, G., Verhoye, M., Sijbers, J., Reyniers, E., Soriano, P., Kamiguchi, H., Willemsen, R., Koekkoek, S.K. et al. (1998) L1 knockout mice show dilated ventricles, vermis hypoplasia and impaired exploration patterns. Hum. Mol. Genet., 7, 999–1009.[Abstract/Free Full Text]

  46. Fransen, E., Van Camp, G., D'Hooge, R., Vits, L. and Willems, P.J. (1998) Genotype–phenotype correlation in L1 associated diseases. J. Med. Genet., 35, 399–404.[Abstract/Free Full Text]

  47. Kleinjan, D.J. and van Heyningen, V. (1998) Position effect in human genetic disease. Hum. Mol. Genet., 7, 1611–1618.[Abstract/Free Full Text]

  48. Crisponi, L., Deiana, M., Loi, A., Chiappe, F., Uda, M., Amati, P., Bisceglia, L., Zelante, L., Nagaraja, R., Porcu, S. et al. (2001) The putative forkhead transcription factor FOXL2 is mutated in blepharophimosis/ptosis/epicanthus inversus syndrome. Nat. Genet., 27, 159–166.[CrossRef][Web of Science][Medline]

  49. Lettice, L.A., Horikoshi, T., Heaney, S.J., van Baren, M.J., van der Linde, H.C., Breedveld, G.J., Joosse, M., Akarsu, N., Oostra, B.A., Endo, N. et al. (2002) Disruption of a long-range cis-acting regulator for Shh causes preaxial polydactyly. Proc. Natl Acad. Sci. USA, 99, 7548–7553.[Abstract/Free Full Text]

  50. Dong, L., Chen, S., Richter, M. and Schachner, M. (2002) Single-chain variable fragment antibodies against the neural adhesion molecule CHL1 (close homolog of L1) enhance neurite outgrowth. J. Neurosci. Res., 69, 437–447.[CrossRef][Web of Science][Medline]

  51. Chen, S., Mantei, N., Dong, L. and Schachner, M. (1999) Prevention of neuronal cell death by neural adhesion molecules L1 and CHL1. J. Neurobiol., 38, 428–439.[CrossRef][Web of Science][Medline]

  52. Chaisuksunt, V., Campbell, G., Zhang, Y., Schachner, M., Lieberman, A.R. and Anderson, P.N. (2000) The cell recognition molecule CHL1 is strongly upregulated by injured and regenerating thalamic neurons. J. Comp. Neurol., 425, 382–392.[CrossRef][Web of Science][Medline]

  53. Zhang, Y., Roslan, R., Lang, D., Schachner, M., Lieberman, A.R. and Anderson, P.N. (2000) Expression of CHL1 and L1 by neurons and glia following sciatic nerve and dorsal root injury. Mol. Cell Neurosci., 16, 71–86.[CrossRef][Web of Science][Medline]

  54. Cremer, H., Chazal, G., Carleton, A., Goridis, C., Vincent, J.D. and Lledo, P.M. (1998) Long-term but not short-term plasticity at mossy fiber synapses is impaired in neural cell adhesion molecule-deficient mice. Proc. Natl Acad. Sci. USA, 95, 13242–13247.[Abstract/Free Full Text]

  55. Stork, O., Welzl, H., Wolfer, D., Schuster, T., Mantei, N., Stork, S., Hoyer, D., Lipp, H., Obata, K. and Schachner, M. (2000) Recovery of emotional behaviour in neural cell adhesion molecule (NCAM) null mutant mice through transgenic expression of NCAM180. Eur. J. Neurosci., 12, 3291–3306.[CrossRef][Web of Science][Medline]

  56. D'Adamo, P., Welzl, H., Papadimitriou, S., Raffaele, D.B., Tiveron, C., Tatangelo, L., Pozzi, L., Chapman, P.F., Knevett, S.G., Ramsay, M.F. et al. (2002) Deletion of the mental retardation gene Gdi1 impairs associative memory and alters social behavior in mice. Hum. Mol. Genet., 11, 2567–2580.[Abstract/Free Full Text]

  57. D'Hooge, R., Nagels, G., Franck, F., Bakker, C.E., Reyniers, E., Storm, K., Kooy, R.F., Oostra, B.A., Willems, P.J. and De Deyn, P.P. (1997) Mildly impaired water maze performance in male Fmr1 knockout mice. Neuroscience, 76, 367–376.[CrossRef][Web of Science][Medline]

  58. Stork, O., Welzl, H., Cremer, H. and Schachner, M. (1997) Increased intermale aggression and neuroendocrine response in mice deficient for the neural cell adhesion molecule (NCAM). Eur. J. Neurosci., 9, 1117–1125.[CrossRef][Web of Science][Medline]

  59. Wolfer, D.P. and Lipp, H.P. (2000) Dissecting the behaviour of transgenic mice: is it the mutation, the genetic background, or the environment? Exp. Physiol., 85, 627–634.[Abstract]

  60. D'Hooge, R. and De Deyn, P.P. (2001) Applications of the Morris water maze in the study of learning and memory. Brain Res. Brain Res. Rev., 36, 60–90.[CrossRef][Medline]

  61. Ivanco, T.L. and Greenough, W.T. (2002) Altered mossy fiber distributions in adult Fmr1 (FVB) knockout mice. Hippocampus, 12, 47–54.[CrossRef][Web of Science][Medline]

  62. Mineur, Y.S., Sluyter, F., de Wit, S., Oostra, B.A. and Crusio, W.E. (2002) Behavioral and neuroanatomical characterization of the Fmr1 knockout mouse. Hippocampus, 12, 39–46.[CrossRef][Web of Science][Medline]

  63. Dahme, M., Bartsch, U., Martini, R., Anliker, B., Schachner, M. and Mantei, N. (1997) Disruption of the mouse L1 gene leads to malformations of the nervous system. Nat. Genet., 17, 346–349.[CrossRef][Web of Science][Medline]

  64. Demyanenko, G.P., Tsai, A.Y. and Maness, P.F. (1999) Abnormalities in neuronal process extension, hippocampal development, and the ventricular system of L1 knockout mice. J. Neurosci., 19, 4907–4920.[Abstract/Free Full Text]

  65. Endris, V., Wogatzky, B., Leimer, U., Bartsch, D., Zatyka, M., Latif, F., Maher, E.R., Tariverdian, G., Kirsch, S., Karch, D. and Rappold, G.A. (2002) The novel Rho-GTPase activating gene MEGAP/srGAP3 has a putative role in severe mental retardation. Proc. Natl Acad. Sci. USA, 99, 11754–11759.[Abstract/Free Full Text]

  66. Sakurai, K., Migita, O., Toru, M. and Arinami, T. (2002) An association between a missense polymorphism in the close homologue of L1 (CHL1, CALL) gene and schizophrenia. Mol. Psychiat., 7, 412–415.

  67. Kermouni, A., Van Roost, E., Arden, K.C., Vermeesch, J.R., Weiss, S., Godelaine, D., Flint, J., Lurquin, C., Szikora, J.P. and Higgs, D.R. (1995) The IL-9 receptor gene (IL9R): genomic structure, chromosomal localization in the pseudoautosomal region of the long arm of the sex chromosomes, and identification of IL9R pseudogenes at 9qter, 10pter, 16pter, and 18pter. Genomics, 29, 371–382.[CrossRef][Web of Science][Medline]

  68. van der Meulen, B.F. and Smrkovsky, M. (1986) MOS 2 6/12-8 6/12 McCarthy Ontwikkelingsschalen. Handleiding. Swets & Zeitlinger, Lisse.

  69. Bryant, B., Taylor, R. and Rivera, D.P. (1996) Assessment of Adaptive Areas. A Method for Obtaining Scores that Correspond to the American Association on Mental Retardation's Adaptive Areas. Examiner's Manual, PRO-ED Inc., 1–23.

  70. Willis, T.G., Jadayel, D.M., Coignet, L.J., Abdul-Rauf, M., Treleaven, J.G., Catovsky, D. and Dyer, M.J. (1997) Rapid molecular cloning of rearrangements of the IGHJ locus using long-distance inverse polymerase chain reaction. Blood, 90, 2456–2464.[Abstract/Free Full Text]

  71. Cools, J., Bilhou-Nabera, C., Wlodarska, I., Cabrol, C., Talmant, P., Bernard, P., Hagemeijer, A. and Marynen, P. (1999) Fusion of a novel gene, BTL, to ETV6 in acute myeloid leukemias with a t(4;12)(q11–q12;p13). Blood, 94, 1820–1824.[Abstract/Free Full Text]

  72. Giulietti, A., Overbergh, L., Valckx, D., Decallonne, B., Bouillon, R. and Mathieu, C. (2001) An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods, 25, 386–401.[CrossRef][Web of Science][Medline]

  73. Herreman, A., Hartmann, D., Annaert, W., Saftig, P., Craessaerts, K., Serneels, L., Umans, L., Schrijvers, V., Checler, F., Vanderstichele, H. et al. (1999) Presenilin 2 deficiency causes a mild pulmonary phenotype and no changes in amyloid precursor protein processing but enhances the embryonic lethal phenotype of presenilin 1 deficiency. Proc. Natl Acad. Sci. USA, 96, 11872–11877.[Abstract/Free Full Text]

  74. Danscher, G. and Zimmer, J. (1978) An improved Timm sulphide silver method for light and electron microscopic localization of heavy metals in biological tissues. Histochemistry, 55, 27–40.[CrossRef][Web of Science][Medline]

  75. Crawley, J.N. and Paylor, R. (1997) A proposed test battery and constellations of specific behavioral paradigms to investigate thebehavioral phenotypes of transgenic and knockout mice. Horm. Behav., 31, 197–211.[CrossRef][Medline]

  76. Rogers, D.C., Peters, J., Martin, J.E., Ball, S., Nicholson, S.J., Witherden, A.S., Hafezparast, M., Latcham, J., Robinson, T.L., Quilter, C.A. and Fisher, E.M. (2001) SHIRPA, a protocol for behavioral assessment: validation for longitudinal study of neurological dysfunction in mice. Neurosci. Lett., 306, 89–92.[CrossRef][Web of Science][Medline]

  77. D'Hooge, R., Van Dam, D., Franck, F., Gieselmann, V. and De Deyn, P.P. (2001) Hyperactivity, neuromotor defects, and impaired learning and memory in a mouse model for metachromatic leukodystrophy. Brain Res., 907, 35–43.[CrossRef][Web of Science][Medline]

  78. Van Dam, D., D'Hooge, R., Hauben, E., Reyniers, E., Gantois, I., Bakker, C.E., Oostra, B.A., Kooy, R.F. and De Deyn, P.P. (2000) Spatial learning, contextual fear conditioning and conditioned emotional response in Fmr1 knockout mice. Behav. Brain Res., 117, 127–136.[CrossRef][Web of Science][Medline]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Neurosci.Home page
K. Amano, M. Fujii, S. Arata, T. Tojima, M. Ogawa, N. Morita, A. Shimohata, T. Furuichi, S. Itohara, H. Kamiguchi, et al.
DSCAM Deficiency Causes Loss of Pre-Inspiratory Neuron Synchroneity and Perinatal Death
J. Neurosci., March 4, 2009; 29(9): 2984 - 2996.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
S. Kolata, J. Wu, K. Light, M. Schachner, and L. D. Matzel
Impaired Working Memory Duration But Normal Learning Abilities Found in Mice That Are Conditionally Deficient in the Close Homolog of L1
J. Neurosci., December 10, 2008; 28(50): 13505 - 13510.
[Abstract] [Full Text] [PDF]

Home page
 Cereb CortexHome page
R. Batista-Brito, R. Machold, C. Klein, and G. Fishell
Gene Expression in Cortical Interneuron Precursors is Prescient of their Mature Function
Cereb Cortex, October 1, 2008; 18(10): 2306 - 2317.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
I. Jakovcevski, J. Wu, N. Karl, I. Leshchyns'ka, V. Sytnyk, J. Chen, A. Irintchev, and M. Schachner
Glial Scar Expression of CHL1, the Close Homolog of the Adhesion Molecule L1, Limits Recovery after Spinal Cord Injury
J. Neurosci., July 4, 2007; 27(27): 7222 - 7233.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (28)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Frints, S. G. M.
Right arrow Articles by Froyen, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Frints, S. G. M.
Right arrow Articles by Froyen, G.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?