the neuronal migration disorders , x - linked lissencephaly syndrome ( xlis ) and subcortical band heterotopia ( sbh ) , also called " double cortex " , have been linked to missense , nonsense , aberrant splicing , deletion , and insertion mutations in doublecortin ( dcx ) in families and sporadic cases ( 1 - 8 ) .
most dcx mutations identified to date are located in two evolutionarily conserved domains ( 7 , 9 ) .
the prevalence of dcx mutations is 38 - 85% in sporadic sbh patients and 100% in all multiplex families with xlis / sbh ( 7 , 10 ) . because sbh is typically seen in women with heterozygous dcx mutations and is a less severe phenotype than lissencephaly , which is a typical phenotype of men with hemizygous mutations , sbh is considered a mosaic phenotype that involves lyonization ( 5 ) .
these findings suggest that the conserved domains in dcx play a major role in neuronal migration during neural system development .
the precise explanation for the function of the dcx protein in neuronal migration , however , remains unknown .
we performed mutation analysis of dcx in two korean patients with sbh and report a novel mutation of dcx that is responsible for sbh .
two unrelated korean patients with epilepsy and sbh ( patients 1 and 2 ) and their clinically unaffected family members were studied after obtaining informed consent .
the sbh patients had mild to moderate developmental delays , drug - resistant generalized seizures , and diffuse thin ( fig .
dna was extracted from peripheral blood using a standard protocol , and all coding regions of dcx ( exons 1 to 7 ; exon numbering according to refseq aj003112.1 , gen - bank ) were amplified by pcr .
pcr assays were performed by using 1.25 u of amplitaq polymerase gold ( applied biosystem , foster city , ca , u.s.a . ) , 100 ng genomic dna , 2.0 mm mgcl2 , and a final concentration of 10 m for each set of primers ( table 2 ) .
amplification conditions were as follows : an initial denaturing cycle at 95 for 7 min followed by 35 cycles of denaturation at 94 for 30 sec , annealing at 62 for 30 sec , and extension at 72 for 1 min . a final extension step of 72 for 7 min was used .
the pcr products were electrophoresed in a 1.2% agarose gel , and the amplified genomic dna fragments were extracted and purified using the manufacturer 's recommended protocol ( qiaquick gel extraction kit ; qiagen , germany ) .
direct sequencing of both strands was performed with bigdye terminator chemistry ( pe biosystems , foster city , ca , u.s.a . ) . as the mutation found in patient 1
destroys an mnl1 restriction site , restriction fragment length polymorphism ( rflp ) analysis was performed using mnl1 enzyme ( new england biolabs , beverly , ma , u.s.a . ) for the pcr products from both the family members of patient 1 and 60 unaffected , unrelated korean women .
two unrelated korean patients with epilepsy and sbh ( patients 1 and 2 ) and their clinically unaffected family members were studied after obtaining informed consent .
the sbh patients had mild to moderate developmental delays , drug - resistant generalized seizures , and diffuse thin ( fig .
dna was extracted from peripheral blood using a standard protocol , and all coding regions of dcx ( exons 1 to 7 ; exon numbering according to refseq aj003112.1 , gen - bank ) were amplified by pcr .
pcr assays were performed by using 1.25 u of amplitaq polymerase gold ( applied biosystem , foster city , ca , u.s.a . ) , 100 ng genomic dna , 2.0 mm mgcl2 , and a final concentration of 10 m for each set of primers ( table 2 ) .
amplification conditions were as follows : an initial denaturing cycle at 95 for 7 min followed by 35 cycles of denaturation at 94 for 30 sec , annealing at 62 for 30 sec , and extension at 72 for 1 min . a final extension step of 72 for
the pcr products were electrophoresed in a 1.2% agarose gel , and the amplified genomic dna fragments were extracted and purified using the manufacturer 's recommended protocol ( qiaquick gel extraction kit ; qiagen , germany ) .
direct sequencing of both strands was performed with bigdye terminator chemistry ( pe biosystems , foster city , ca , u.s.a . ) . as the mutation found in patient 1
destroys an mnl1 restriction site , restriction fragment length polymorphism ( rflp ) analysis was performed using mnl1 enzyme ( new england biolabs , beverly , ma , u.s.a . ) for the pcr products from both the family members of patient 1 and 60 unaffected , unrelated korean women .
sequence analysis of the dcx coding region for patient 1 revealed a c.386c > t change in exon 3 ( fig .
the sequence variation results in a serine to leucine amino acid change at position 129 ( s129l ) , which has not been found in other family members of patient 1 , including maternal grandmother , parents , or siblings .
rflp analysis was conducted to confirm the sequencing result and to exclude the possibility that the genetic variation is a polymorphism .
the heterozygous mutant band pattern was observed in patient 1 , whereas it was not found in other family members of patient 1 or in a control population of 60 healthy , unrelated korean women ( fig .
no genetic variation in dcx coding region was found for patient 2 and her mother .
in the developing cortex , cortical neurons must migrate long distances to reach the site of their final differentiation .
the protein encoded by the dcx gene is a cytoplasmic protein that appears to direct neuronal migration by regulating the organization and stability of microtubules ( 10 ) .
mutation clusters in the dcx gene were previously identified in exons 2 and 3 , and 3 and 4 , overlapping significantly with two proposed evolutionarily conserved domains of the dcx protein ( 7 , 9 ) .
the first conserved domain binds tubulin and enhances microtubule polymerization , and the second binds tubulin less well and does not enhance microtubule assembly .
both domains seem necessary for optimal dcx protein function , and differences in the location and type of mutations in these regions may produce variable functional disturbances . the mutation identified in the present study
is located in the first preserved domain of the dcx protein , which not been reported previously . in the setting of a family with a single girl affected with sbh ,
it is hard to determine the risk for sbh or xlis in future children . because of x - linkage , a male with a dcx mutation
should be clinically affected while a female with the mutation may be unaffected due to germline or somatic mosaicism or skewed x inactivation ( 5 , 11 - 13 ) . in the present study ,
the genetic analysis of the dcx mutation c.386c > t was negative in the maternal grandmother , mother , and two siblings of patient 1 who are free from sbh or xlis .
although patient 2 in the present study had similar clinical and radiological features to those of patient 1 , she did not carry any genetic variation in the coding region of dcx .
this suggests that the non - coding region of dcx , including the promoter region and introns , or other genes implicated in neuronal migration disorders may be responsible for this patient 's condition .
we identified a previously unreported missense mutation in the first preserved domain of the dcx protein .
second , it is absent from a large panel of 120 control x - chromosomes .
third , it occurs in a highly conserved position - the first domain of the dcx protein where the majority of the known dcx mutations cluster .
the discovery of the new mutation reported here supports existing evidence that mutation in the first conserved domain of the dcx protein is important in the pathogenesis of sbh .
 the neuronal migration disorders , x - linked lissencephaly syndrome ( xlis ) and subcortical band heterotopia ( sbh ) , also called " double cortex " , have been linked to missense , nonsense , aberrant splicing , deletion , and insertion mutations in doublecortin ( dcx ) in families and sporadic cases . 
 most dcx mutations identified to date are located in two evolutionarily conserved domains . 
 we performed mutation analysis of dcx in two korean patients with sbh . 
 the sbh patients had mild to moderate developmental delays , drug - resistant generalized seizures , and diffuse thick sbh upon brain mri . 
 sequence analysis of the dcx coding region in patient 1 revealed a c.386 c > t change in exon 3 . 
 the sequence variation results in a serine to leucine amino acid change at position 129 ( s129l ) , which has not been found in other family members of patient 1 or in a large panel of 120 control x - chromosomes . 
 we report here a novel c.386 c > t mutation of dcx that is responsible for sbh . 