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Human Molecular Genetics Pages 1063-1070  

doublecortin is the major gene causing X-linked subcortical laminar heterotopia (SCLH)
Introduction
Results
   Gene structure
   Mutation analysis
   Familial case
Discussion
Materials And Methods
   Family material
   Gene structure
   Mutation analysis
Acknowledgements
References

doublecortin is the major gene causing X-linked subcortical laminar heterotopia (SCLH)

doublecortin is the major gene causing X-linked subcortical laminar heterotopia (SCLH)

Vincent des Portes1,+, Fiona Francis1,*,+, Jean-Marc Pinard2, Isabelle Desguerre3, Marie-Laure Moutard3, Irina Snoeck4, Linda C. Meiners5, François Capron6, Raffaella Cusmai7, Stefano Ricci7, Jacques Motte8, Bernard Echenne9, Gérard Ponsot3, Olivier Dulac3, Jamel Chelly1, Cherif Beldjord1

1INSERM U129, Institut Cochin de Génétique Moléculaire, 24 rue du Faubourg Saint-Jacques, 75014 Paris, France, 2Department of Paediatric Neurology, Hôpital R. Poincaré, Garches, France, 3Department of Paediatric Neurology, Hôpital Saint Vincent-de-Paul, Paris, France, 4Department of Child Neurology and 5Department of Radiology, University Hospital, Utrecht, The Netherlands, 6Department of Paediatrics B, Hôpital Bel Air, Thionville, France, 7Department of Neurophysiology, Bambino Gesù Hospital, Rome, Italy, 8Department of Paediatric Neurology, American Memorial Hospital, Reims, France and 9Department of Paediatric Neurology, Hôpital Saint-Eloi, Montpelliers, France

Received February 17, 1998; Revised and Accepted April 21, 1998

DDBJ/EMBL/GenBank accession nos AJ005591-AJ005597

Subcortical laminar heterotopia (SCLH), or `double cortex', is a cortical dysgenesis disorder associated with a defect in neuronal migration. Clinical manifestations are epilepsy and mental retardation. This disorder, which mainly affects females, can be inherited in a single pedigree with lissencephaly, a more severe disease which affects the male individuals. This clinical entity has been described as X-SCLH/LIS syndrome. Recently we have demonstrated that the doublecortin gene, which is localized on the X chromosome, is implicated in this disorder. We have now performed a systematic mutation analysis of doublecortin in 11 unrelated females with SCLH (one familial and 10 sporadic cases) and have identified mutations in 10/11 cases. The sequence differences include nonsense, splice site and missense mutations and these were found throughout the gene. These results provide strong evidence that loss of function of doublecortin is the major cause of SCLH. The absence of phenotype-genotype correlations suggests that X-inactivation patterns of neuronal precursor cells are likely to contribute to the variable clinical severity of this disorder in females.

INTRODUCTION

In the embryonic development of the mammalian brain, neurons migrate long distances to form the complex laminar structures of the cerebral cortex. The molecular mechanisms which guide the neuronal cells to their final destinations are not yet well understood; however, the study of disorders of neuronal migration can help to identify and characterize the genes and pathways involved. Such cortical malformation disorders, which are generally assessed by magnetic resonance imaging (MRI) or brain autopsy, are believed to be responsible for a significant proportion of the severe cases of epilepsy and cognitive impairment in children (1-3).

One such developmental disorder is subcortical laminar heterotopia (SCLH), or `double cortex' syndrome (3,4). Interestingly, a skewed sex ratio has been observed in sporadic cases of SCLH: mainly female individuals are affected with very few male cases identified (5). In addition, several pedigrees have been identified in which affected males have lissencephaly or `smooth brain' and affected females exhibit SCLH, which suggests that the same X-linked gene causes both diseases [X-SCLH/LIS (5,6)]. Lissencephaly is associated with profound mental retardation and epilepsy with many types of seizure, whereas heterozygous females with SCLH are generally less severely affected (7). Lissencephaly is defined by an absence (agyria) or a decrease (pachygyria) in surface convolutions of the cortex, which is disorganized and abnormally thick. Children with lissencephaly, in addition to severe mental retardation and epilepsy, often have microcephaly, marked hypotonia, no visual contact or speech and a shortened lifespan (8). SCLH presents with bilateral plates of heterotopic grey matter located between the cortex and the cerebral ventricles, below a thin band of white matter. The true cortex appears normal in lamination and is formed with correctly migrated neurons, whilst the heterotopic band consists of scattered neurons which have failed to migrate properly (3). Clinical manifestations are variable and seem to be related to the thickness of the heterotopic layer: intelligence can be normal or patients can exhibit varying degrees of mental retardation. Similarly, SCLH patients exhibit either occasional seizures or more severe forms of epilepsy and, in addition, there is often a delay in speech development and the ability to walk. Identification of the gene involved in this disorder is expected to lead to a more accurate classification of cortical dysgenesis conditions and to contribute to our understanding of the pathogenic mechanisms involved in epilepsy and mental retardation.

We and others have recently identified a novel CNS gene, doublecortin, and demonstrated its involvement in unrelated familial and sporadic cases of atypical SCLH (9,10). The predicted gene product shares significant homology over its whole length with the N-terminal segment of another protein, which is predicted to have a protein kinase domain at its C-terminus. Besides this, the function of doublecortin is unknown. Itis highly expressed during brain development, mainly in fetal neurons, including precursor cells (9). It seems to be most highly expressed at mouse embryonic day 11, compared with later stages (10). Thus, the complete disorganization observed in lissencephaly and SCLH seems to reflect a failure of early events associated with neuronal dispersion.

In order to assess more fully the involvement of doublecortin in SCLH, we have now performed an extensive mutation analysis in a further 11 affected individuals. We have determined the intron-exon boundary sequences of doublecortin and have screened the splice sites and exonic sequences of all coding exons by a combination of denaturing gradient gel electrophoresis (DGGE) (11) and direct sequencing. In summary, nonsense, missense or splice site mutations were found in 10 of the 11 unrelated affected females. The high percentage of mutations detected confirms the major role played by this X-linked gene in SCLH.

RESULTS

Gene structure

The doublecortin gene has been shown to contain nine exons in total, although three exons at the 5[prime]-end, 1A, 1B and 1C, are alternatively used. A putative start methionine in the first common exon (exon 2) has been identified, although exon 1C, if used, may extend the open reading frame at the 5[prime]-end (9). The transcript size is 9.5 kb; however, a large proportion of this sequence is 3[prime]-untranslated region (UTR). Intron-exon boundary sequences for all exons were determined by sequencing cosmid and phage genomic clones which encompass the gene. Sequences corresponding to the splice sites preceding and following each exon are presented in Table 1. These were found to adhere to consensus sequences with an invariant GT and AG in the splice donor and acceptor sites respectively.

Mutation analysis

In order to assess the degree of involvement of doublecortin in SCLH (which is observed mainly as female sporadic cases), we performed a mutation analysis on a collection of 11 independent patients with wide geographical origins. The clinical features of the patients are presented in Table 2. One family with SCLH (family 4) and 10 sporadic cases were examined in all potential coding regions of the gene. Genomic DNA from subjects was screened initially using DGGE. Primer pairs were designed either in intronic sequences to amplify whole exons (exons 1C and 4-6) or, alternatively, the exons were amplified in several overlapping pieces to generate products which had a suitable melting temperature (Tm) for the DGGE analysis (Table 3). Using this method, PCR fragments with an aberrant migration pattern, representing potential mutations, were identified and sequenced using direct automated sequencing. All coding sequences were examined in this way apart from PCR products corresponding to the 3.3 fragment of exon 3, which were directly sequenced and not analysed by DGGE.

To date we have identified 10 mutations in the 11 patients examined (Table 4 and Fig. 1).In case T.M. it was not possible to identify a mutation. Nine distinct point mutations were found, one of which was recurrent in two unrelated individuals: the C->T mutation at nucleotide position 971 in the cDNA sequence (DDBJ/EMBL/GenBank accession no. AJ003112)">http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?db=n&form=6&uid=AJ003112&dopt=g">AJ003112) causes a predicted R186C missense mutation which segregates with the phenotype in family 4 and is present in case J.F. Three different nonsense mutations were observed, R39X (Fig. 2), Q235X and R303X, occuring respectively in exons 2, 3 and 5. In addition, one of the point mutations alters the splice consensus sequence at position -2 of exon 3 (AG->GG, case S.D.).

A total of five different missense mutations were observed confined to exons 3 and 4 (Fig. 1). One tyrosine residue at amino acid position 125 was affected by a T->G mutation (case O.D.), which is predicted to change the tyrosine to an aspartic acid residue. We previously found a mutation in this same tyrosine residue in an X-SCLH/LIS family (9). There are two predicted isoleucine to threonine changes at residues 214 and 250 which occur in cases J.A. and M.L. respectively. Case B.T. has a nucleotide alteration predicted to change Gly223 to a glutamic acid residue. Each of these mutations is predicted to change the Doublecortin protein significantly.

Table 1. Exon-intron boundary sequences
Exon Exon/SD site SA site/exon Exon
1A ACCAGCAGAAAACCG gtgagtggggctttt gtcaaacgggaaaag CACAAGGCAAAGCCT 1B
1B TTTTCCTTTGGAAAA gtgagtttgatgttc cttttttccccccag CTTATTTTTTATGAA 1C
1C TAGGAGCCACTGTCA gtaagtctcaggatt ttctcccacgctcag GTCTCTGAGGTTCCA 2
2 ATGAACTGGAGGAAG gtaatttaaatagtg tatttcttgccttag GGGAAAGCTATGTTC 3
3 CTGGATGGAAAACAG gtaggtactttttca ctgctttctccatag GTAACTTGTCTCCAT 4
4 CTCTGGATGAAAATG gtaagcataaccact gtccttttgccccag AATGCCGAGTCATGA 5
5 CTCCAGCTGACTCAG gtaacgaccaagacg cttgctttgggctag CAAACGGAACCTCCA 6
6 CTCCGGAAGCACAAG gtattatgtctctta cttctcttcttatag GACCTGTACCTGCCT 7

Table 2. Clinical data
No. Family Patient Sex(M/F) MRI: global aspect Heterotopiathickness Cortex Age of walking(months) Age of first speech(years) Neurological examination DQ/IQ Epileptic syndrome
1a 1a Moi.A M Agyria (CT) None Thick, agyric No walking No speech Bedridden, little eye contact, spasticity of all 4 limbs NA (<20) Infantile spasms, symptomatic generalized epilepsy
1b 1 Mav.A F SCLH ++++ Pachygyric NA NA Spastic gait on the left side NA (severe MR) Partial epilepsy, symptomatic generalized epilepsy
1c 1 Mag.A F SCLH (CT) NA NA NA NA Normal NA Partial epilepsy
2a 2a O.H. M Agyria None Thick, agyric No walking No speech Bedridden, pyramidal syndrome, nystagmus, axial hypotonia NA (<30) Infantile spasms, symptomatic generalized epilepsy
2b 2 V.H. F SCLH + (frontal) Normal NA NA Normal NA (low level) Symptomatic generalized epilepsy
3a 3a B.M. M Agyria-pachygyria None Thick, agyric (frontal) Pachygyric (posterior) No walking No speech Bedridden, hypotonia NA (<20) Infantile spasms, symptomatic generalized epilepsy
3b 3 K.M. F SCLH ++ Moderately pachygyric (Frontal) 24 >3 Pyramidal syndrome, tremor, dyspraxia NA (<60) Symptomatic generalized epilepsy
3c 3 S.M. F SCLH ++ Normal 30 >3 Normal 66 Symptomatic generalized epilepsy
3d 3 I.M. F SCLH ± Normal 30 NA Normal NA Fits with fear, without loss of consciousness
4a 4 K.G. F SCLH +++ Numerous gyri, malformation of insula <18 NA Normal 35 Partial epilepsy
4b 4 M.G. F SCLH ++++ Numerous gyri, malformation of insula <18 NA Axial hypotonia, limb hypertonia NA Partial epilepsy
4c 4 C.G. F SCLH ++ Normal NA (normal) NA Normal NA Occasional seizures
5   S.D. F SCLH +++ Pachygyric, numerous gyri, malformation of insula 16 2.5 Normal GIQ = 52 (VIQ = 61, PIQ = 50) Symptomatic generalized epilepsy, Lennox-Gastaut syndrome
6   O.D. F SCLH ++++ Pachygyric, numerous gyri, malformation of insula 36 4 Dysarthria, strabismus 37 Infantile spasms, symptomatic generalized epilepsy
7   J.F. F SCLH +++ Numerous gyri, malformation of insula No walking No speech Axial hypotonia, strabismus NA (severe MR <50) Partial epilepsy
8   S.K. F SCLH ++ Normal 18 3 Normal NA Symptomatic generalized epilepsy
9   M.L. F SCLH +++ Numerous gyri, malformation of insula 24 2 Manual dyspraxia 74 Occasional seizures
10   T.T. F SCLH +++ Pachygyric 24 NA RAS NA (severe MR) Partial epilepsy
11a   J.M. F agyria-pachygyria ++++ Mixed agyria-pachygyria, malformation of insula 42 4.5 Ataxia, dyspraxia, visual and motor disturbance (left side), strabismus 30 Partial epilepsy (frontal?)
12   J.A. F SCLH ++ Pachygyric 18 >2 Dyspraxia 50 Infantile spasms, symptomatic generalized epilepsy; Lennox-Gastaut syndrome
13   T.M. F SCLH +++ Pachygyric, numerous gyri, malformation of insula 16 2 Normal 50 Symptomatic generalized epilepsy
14   C.R. F SCLH ++ Pachygyric, numerous gyri, malformation of insula 18 >3 Normal 50 Symptomatic generalize epilepsy
15   B.T. F SCLH +++ Numerous gyri 19 NA Manual dyspraxia and gait disturbance NA Partial epilepsy
aFamilies or individuals studied previously (9).
++++, very thick; +++, thick; ++, moderately thick; +, slightly thick; ±, thin; NA; not available; DQ, developmental quotient; IQ, intelligence quotient; GIQ, global IQ; VIQ, verbal IQ; PIQ, performance IQ; MR, mental retardation.

Table 3. Conditions for PCR amplification and DGGE
Fragment Sequence of primers Length (bp) Annealing temperature (°C) Gradient (%) Running time (h) at 160 V
Exon 1C 1C F, 5[prime]-TTT TAT GAA TGT CGG ATA GCT GC-3[prime]
1C R, 5[prime]-Pso-TA- GAG GAG AAG GGG AGA TTT TG-3[prime]		 433 55 20-70 11.5
Exon 2.1 2.1 F, 5[prime]-TCC CTT CTT TTT TCC CTT CTC C-3[prime]
2.1 R, 5[prime]-Pso-TA- TGA GGC AGG TTG ATG TTG TC-3[prime]		 394 55 30-80 7
Exon 2.2 2.2 F, 5[prime]-ATC CAG GAA CAT GCG AGG CT3[prime]
2.2 R = 2.1 R		 255 55 40-90 9
Exon 2.3 2.3 F, 5[prime]-TGA CCT GAC GCG ATCT CTG T-3[prime]
2.3 R, 5[prime]-Pso-TA- ACC TCC CAC CAA CGG CCA CC-3[prime]		 148 55 30-80 6.5
Exon 3.1 3.1 F, 5[prime]-Pso-TA- CCT AAT CAC TTA TTT CTT GC-3[prime]
3.1 R, 5[prime]-CTT GTT CTC CCT GGC CTG TG-3[prime]		 183 55 30-80 6.5
Exon 3.2 3.2 F, 5[prime]-TTG GCT AGC AGC AAC AGT GC-3[prime]
3.2 R, 5[prime]-Pso-TA- AGT TTG ATG GCT TCT GTG AT-3[prime]
3.3 R, 5[prime]-TAG AAA AAA CGG GAA AAG TAC TT-3[prime]		 176 55 30-80 6.5
Exon 3.3 3.3 F = 3.2 F 260 50 a a
Exon 4 4 F, 5[prime]-Pso-TA- TCA CAG GAC CAT CAT ATA CA-3[prime]
4 R, 5[prime]-ACC CAT GGA AAT CCT AAA GG-3[prime]		 219 55 5-55 5.5
Exon 5 5 F, 5[prime]-Pso-TA- CCT CTA CTA AGC TGT CTG TG-3[prime]
5 R, 5[prime]-TTG TCC TCC ATA AAT GAA GTC AG-3[prime]		 225 50 40-90 9
Exon 6 6 F, 5[prime]-Pso-TA- TTT ATC CCT TCC TTT TCT CT-3[prime]
6 R, 5[prime]-AAG AGG TTT AGT AAG GTA TA-3[prime]		 161 50 40-90 9
Exon 7 7 F, 5[prime]-Pso-TA- AAC TTT GTC TCT TCT CTT CT-3[prime]
7 R, 5[prime]-GGA TTT GTA CTC TGG ACT CTG A-3[prime]		 119 55 30-80 6
aPCR products were sequenced directly.

Table 4. The cumulative spectrum of nonsense, missense and splice site mutations in the doublecortin gene
Mutation type Position in cDNAa Patient code Mutation Enzyme site change Effect of mutation
Nonsense Exon 2 (530) Case S.K. CGA->TGA None R (39) X
  Exon 3 (1118) Case C.R. CAG->TAG None Q (235) X
  Exon 5 (1322) Case T.T. CGA->TGA None R (303) X
Aberrant splicing Exon 3 (-2 from 780) Case S.D. AG->GG acceptor site StyI (gain) Splice exon 3, premature stop
  Exon 4 (+1 from 1223) Case J.M. GT->AT donor site None Splice exon 4b, premature stop
Missense Exon 2 (599) Family 1 GAC->AAC AvaII (loss) D (62) Nb
  Exon 3 (989) Family 2 CGG->TGG StyI (gain) R (192)Wb
  Exon 3 (788) Family 3 TAT->CAT AluI (loss) Y (125) Hb
  Exon 3 (788) Case O.D. TAT->GAT AluI (loss) Y (125) D
  Exon 3 (971) Family 4 CGC->TGC PstI (gain) R (186) C
  Exon 3 (971) Case J.F. CGC->TGC PstI (gain) R (186) C
  Exon 3 (1056) Case J.A. ATC->ACC EcoRV (loss) I (214) T
  Exon 3 (1083) Case B.T. GGG->GAG MspI (loss) G (223) E
  Exon 4 (1164) Case M.L. ATT->ACT None I (250) T
aNucleotide position in the cDNA sequence according to GenBank database entry AJ003112 starting from the 5[prime]-end of exon 1C.
bMutation reported in a previous study (9).


Figure 1. A schematic representation of the structure of the doublecortin gene. Exons are represented as rectangular boxes and their sizes in bp are indicated inside each box. Exons 1A, 1B and 1C each contain 5[prime]-UTR sequences and have been found to be alternatively spliced to the remaining exons (exons 2-7). Nonsense and splice site mutations are represented above the exons and missense mutations below. Those mutations marked with an asterisk were identified in a previous study (9). The R186C mutation was identified in two unrelated individuals. 5[prime]-UTR (exons 1A and 1B) and 3[prime]-UTR sequences were not screened.


Figure 2. Sequence gel electropherograms showing a C->T point mutation (R39X) in a heterozygous female patient. The region of exon 2 sequence containing the mutation is shown on forward and reverse strands: a genomic DNA PCR product containing the exon 2.2 domain was purified and sequenced using primers 2.2 F and 2.2 R (Table 3). The PCR product contains both the normal and mutated alleles of the affected female patient, hence two bases are called at nucleotide position 530 (GenBank accession no. AJ003112). The C->T mutation (G->A on the reverse strand) changes codon 39 from an arginine to a stop codon.

Other family members of the nine sporadic cases for which a mutation was identified were also tested for the presence of the mutation. In each case at least the mother of the affected individual was examined. PCR products amplified from genomic DNA were analysed by either restriction enzyme digestion, DGGE analysis or direct sequencing. In each case it was not possible to detect mutations in any of the other family members, which confirms the sporadic nature of the mutations.

A control set of >90 unrelated X chromosomes were tested for the presence of all but one of the splice site and missense mutations. With the exception of the I250T missense mutation, all others led to the loss or gain of a restriction enzyme site (Table 4). None of the sequence differences was identified in the 90 chromosomes examined, which further verifies that these are significant disease-causing mutations and not coding polymorphisms.

Familial case

In the case of family 4 it is interesting to compare the clinical phenotype of the mother with those of her daughters (Table 2 and Fig. 3A-C). The mother has a relatively thin heterotopic band and suffers from only occasional seizures and thus appears to have a milder form of this disease. The two affected sisters were found to be heterozygous for the C->T mutation at nt 971 (R186C), which leads to the gain of a PstI site. Interestingly, PCR amplification and digestion of the mother's DNA (case C.G.) was performed and the results suggest that she has only a partial gain of this PstI site (Fig. 3D). Whereas the PCR products of the two affected heterozygous daughters show ~50% digested versus undigested products, the mother's DNA shows an unequal amount of the different amplified alleles (~20% have the PstI site versus 80% without). PCR amplification and digestion were repeated three times for this family with reproducible results. As a further control the PCR products were also digested with another enzyme (AluI), that has a restriction site 16 nt upstream from the mutated nucleotide. A complete digestion of the PCR product was observed for every family member with this enzyme (Fig. 3D). Sequencing of the PCR products reflected the results of the PstI digestions, with both the normal and mutated nucleotide present in the mother's DNA sample in an uneven ratio (data not shown). In an attempt to quantify more precisely the percentage of alleles, hybridizations were performed to a Southern blot of PstI-digested genomic DNA from members of this family. However, despite hybridizing with several different sized probes containing exon 3, it was not possible to obtain a unique hybridization signal, which may reflect the repetitive nature of the region and the presence of other doublecortin-like sequences in the genome. Although using the PCR technique it is not possible to quantify the percentage of the different alleles in this individual, our results suggest that she is mosaic for this mutation and this may explain her milder phenotype.

Figure 3. Clinical severity of SCLH correlated with an uneven proportion of mutated alleles. (A) normal MRI: note the thickness of the white matter (which appears dark grey in the photograph), below the cortical mantle. (B) MRI of affected daughter (case KG) shows a thick heterotopic band of grey matter (arrows) below a thin band of white matter and a true cortex. (C) In comparison, the mother (case CG) has a much thinner heterotopic band (arrows). (D) The R186C mutation identified in family 4 leads to the gain of a PstI site. Two affected daughters in this family (II.2 and II.3) show a reduced amount of the undigested PCR product and the presence of digested products (indicated with arrowheads) compared with their normal sister (II.1). A smaller proportion of the PCR products of their mother (I.2, lane indicated with an asterisk) are digested with PstI. A control digestion with AluI shows complete digestion of all PCR products (ND, undigested material).

DISCUSSION

In a previous study we analysed three X-SCLH/LIS families and two sporadic SCLH cases by nested RT-PCR using lymphoblastoid cell lines (9). Three missense mutations co-segregating with the phenotype and a de novo splice site mutation were detected, demonstrating that defects in doublecortin were responsible for the X-SCLH/LIS syndrome. However, investigation of doublecortin transcripts carried out in one of the sporadic female cases did not reveal any abnormality. We suggested a poor efficiency of the RT-PCR-based approach in detecting heterozygous mutations due to inefficient amplification of the abnormal transcript and have hence implemented the DGGE method using genomic DNA for mutation screening in this new study. We now present the first systematic mutation analysis screen of all coding regions of doublecortin, examining 11 unrelated SCLH cases.

In addition to the four mutations we previously reported, we have now found mutations in 10 of the 11 new cases explored. Five patients had either nonsense point mutations or aberrant splicing leading to a premature stop codon and the five other cases, including one familial case, had point mutations leading to missense amino acid substitutions. In all of the SCLH individuals we have examined to date, no mutations have been identified in exons 1C, 6 and the coding part of exon 7. Two mutations were observed in exon 2, eight in exon 3, two in exon 4 and one in exon 5. Hence, exon 3, which represents ~29% of the total coding sequence, contains 8/13 (62%) of the different mutations identified. Considering all the missense mutations, none was found in 90 control chromosomes examined, suggesting that they are not common coding polymorphisms. In addition, the missense mutations identified in the familial cases were all shown to co-segregate with the phenotype. Interestingly, only missense mutations have thus far been observed in male individuals with X-LIS, which may indicate that null mutations are embryonic lethal.

In one patient, case T.M., it has not been possible to identify a mutation. In this case mutations may be located in the unexplored 5[prime]-untranslated region, the introns or the promotor sequences of the doublecortin gene. Alternatively, other loci may be involved and a gene which has strong homologies to doublecortin in its N-terminal region, KIAA0369, could be considered as a candidate gene (9,10). In addition, we looked for mutations in three males affected with SCLH. In these cases we did not detect a mutation (data not shown), suggesting that SCLH in males may represent a rare, distinct, perhaps autosomal, locus. Missense and frameshift mutations in doublecortin have also been found by other investigators in four out of nine pedigrees with X-SCLH/LIS and three additional female patients, although this mutation screen was incomplete (10).

The clinical severity of SCLH varies strikingly from asymptomatic clinical presentation with heterotopic bands assessed by MRI, to severe mental impairment with intractable epilepsy. The relative thickness of the heterotopic band seems to correlate with the phenotype, as patients with thicker bands have more severe mental retardation and seizures (12). Furthermore, an SCLH `forme fruste', consisting of bilateral and symmetric bands with a regional distribution, has been described (13), which is associated with a milder phenotype. At first sight, a correlation between the clinical severity and mutation profiles might be suggested by our data. Indeed, four of the five nonsense and aberrant splice mutations leading to a premature stop codon occur in severely affected females with thick laminar heterotopia or pachygyria. However, this correlation remains unclear in other cases: (i) case S.K., who has a stop codon in exon 2, has a normal cortex and milder phenotype; (ii) in families 1 and 3, whereas the mothers and daughters share a constitutive missense mutation, the mothers have a thinner laminar heterotopia and a milder mental retardation than their daughters (14); in family 4 this also seems to be the case (Fig. 3) and we show that this is likely to be attributable to mosaicism; and (iii) missense mutations were observed in SCLH patients exhibiting variable severity and these mutations do not seem to involve functional domains such as putative PKC or CK2 phosphorylation sites, except R192W, which affects a predicted nuclear localization signal [(motif KPRK (15)]. Interestingly, eight of the 13 different mutations were detected within exon 3; however, correlations between the nature or the site of the mutation and the phenotype remain premature, since the critical functional domains of doublecortin are as yet unknown.

Furthermore, since SCLH is an X-linked condition, patterns of X chromosome inactivation in the brain could widely modulate the consequences of doublecortin mutations on cortical development. Indeed, assuming random patterns of inactivation in females with SCLH, functional Doublecortin should be absent only in cells which inactivate the X chromosome bearing the normal allele. This hypothesis is supported by histopathological data on SCLH brains (3) showing two populations of neurons, heterotopic cells lying in the white matter and cells that reach the normal six layered cortex. Strikingly, heterotopic cells are not rescued by neighbouring normal neurons, fitting in with a probable neuronal intracellular localization of doublecortin (9). Thus, since X chromosome inactivation occurs very early in development (16), different patterns of inactivation among neuronal precursors may explain phenotypic heterogeneity, especially laminar heterotopic band thickness and mental impairment. Taking into account this major phenomenon, correlations between mutations and clinical severity could be difficult to assess.

Our data show that doublecortin is the major gene involved in SCLH. Since we have identified mutations in atypical cases of SCLH, for example a `forme fruste' (mother in family 4 with mosaicism causing occasional seizures) or severe forms leading to pachygyria and corpus callosum agenesia (case J.M.), it will be of interest to determine whether mutations in doublecortin contribute to other cortical dysgeneses or idiopathic forms of epilepsy. For instance, two previously reported pedigrees of X-linked dominant pachygyria associated with corpus callosum agenesia in males with decreased expressivity in female carriers (17-19) might be allelic disorders of X-SCLH/LIS. In addition, we might expect the involvement of doublecortin in some cortical dysgeneses associated with isolated corpus callosum agenesia. The study of doublecortin and other related genes opens new avenues in understanding the pathophysiological mechanisms underlying cortical malformations and child epilepsy.

MATERIALS AND METHODS

Family material

The phenotype of each family member and routine MRI or CT scans were checked by the same paediatric neurologist. Each family member was included in the genetic study after obtaining informed written consent.

The patients originated from France, Italy, The Netherlands, India, Vietnam, Portugal, North Africa and the West Indies, and hence the dataset involved variable ethnic origins.

Gene structure

Cosmid and phage clones encompassing the doublecortin gene (9) were HindIII digested and subcloned into BluescriptSK (Stratagene). Subclones were end-sequenced and hybridized with gene-specific oligonucleotides to identify exon sequences and further sequenced with exonic oligonucleotides. Sequencing was performed using ABI dye terminator chemistry and an ABI sequencing machine and intron-exon boundary sequences were identified. `Prediction of protein localization sites' (PSORT) was used to screen the amino acid sequence (15).

Mutation analysis

Lymphocyte DNA was obtained from Genethon, generated using standard methods. PCR reactions were performed using oligonucleotides specifically designed for use in the DGGE technique (Table 3). For each primer set one oligonucleotide contained a psoralen clamp at its 5[prime]-end (20). In cases where more than one primer set was used to analyse an exon, primers were designed to amplify overlapping regions. PCRs were performed using a PTC-200 thermal cycler (MJ Research) at the annealing temperatures specified in Table 3, each for 40 cycles of 94°C × 40 s, annealing temperature × 30 s and 72°C × 40 s, with an initial denaturation and final extension of 4 and 5 min respectively. Taq and PCR buffer were obtained either from Eurogentec (Goldstar) or Gibco BRL and PCRs were performed at final concentrations of 0.25 mM dNTPs and 1.5 mM magnesium chloride. PCR products were analysed on agarose gels under standard conditions prior to the DGGE.

Heteroduplexes were allowed to form (94°C × 5 min, 55°C × 30 min) and PCR products were UV cross-linked (365 nm) for 15 min. PCR products were electrophoresed on polyacrylamide gels containing linear gradients of urea and formamide as specified in Table 3. The DGGE conditions for each type of PCR product were determined by assessing their melting profiles, using the Meltmap program (11). PCR products showing an abnormal migration pattern were directly sequenced on both strands.

Family members of individuals with identified mutations were checked where possible by restriction enzyme digestion (as detailed in Table 4). Restriction enzymes were used according to the manufacturers' specifications. In other cases PCR products of family members were checked by DGGE (cases S.K. and T.T.) or direct sequencing (case C.R.). Control individuals were mothers of Duchenne muscular dystrophy patients.

ACKNOWLEDGEMENTS

We thank all patients and other family members for their participation in this study. The authors are grateful to Mrs Boisson, Dr Prudhomme and co-workers for the use of Genethon's facilities for lymphoblastoid cell culture and DNA extraction. We thank Orly Reiner for interesting discussions. This work was supported in part by grants from the Institut National de la Santé et de la Recherche Médicale (INSERM), Assistance Publique-Hôpitaux de Paris (CRC 950164), the Association Française contre les Myopathies (AFM), the Fondation Jérôme Lejeune and the Conseil Régional d'Ile de France.

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*To whom correspondence should be addressed. Tel: +33 1 44 41 24 29; Fax: +33 1 44 41 24 21; Email: ffrancis@infobiogen.fr
+The two first authors contributed equally to this study

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