Multiplex ligation-dependent probe amplification detects DCX gene deletions in band heterotopia
ABSTRACT Subcortical band heterotopia (SBH, or double cortex syndrome) is a neuronal migration disorder consisting of heterotopic bands of gray matter located between the cortex and the ventricular surface, with or without concomitant pachygyria. Most cases show diffuse or anteriorly predominant (A>P) migration abnormality. All familial and 53% to 84% of sporadic cases with diffuse or A>P SBH harbor a mutation of the DCX gene, leaving the genetic causes unexplained, and genetic counseling problematic, in the remaining patients. Our purpose was to verify the extent to which exonic deletions or duplications of the DCX gene would account for sporadic SBH with A>P gradient but normal gene sequencing.
We identified 23 patients (22 women, 1 man) with sporadic, diffuse, or anteriorly predominant SBH. After sequencing the DCX gene and finding mutations in 12 (11 women, 1 man), we used multiplex ligation-dependent probe amplification (MLPA) to search for whole-exon deletions or duplications in the 11 remaining women. We used semiquantitative fluorescent multiplex PCR (SQF-PCR) and Southern blot to confirm MLPA findings.
MLPA assay uncovered two deletions encompassing exons 3 to 5, and one involving exon 6, in 3 of 11 women (27%) and raised the percentage of DCX mutations from 52% to 65% in our series. SQF-PCR performed in all three women and Southern blot analysis performed in two confirmed the deletions.
MLPA uncovers large genomic deletions of the DCX gene in a subset of patients with SBH in whom no mutations are found after gene sequencing. Deletions of DCX are an underascertained cause of SBH.
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ABSTRACT: Mutations of the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) cause an X-linked encephalopathy with early onset intractable epilepsy, including infantile spasms and other seizure types, and a Rett syndrome (RTT)-like phenotype. Very limited information is available on the frequency and phenotypic spectrum associated with CDKL5 deletions/duplications. We investigated the role of CDKL5 deletions/duplications in causing early onset intractable epilepsy of unknown etiology in girls. We studied 49 girls with early onset intractable epilepsy, with or without infantile spasms, and developmental impairment, for whom no etiologic factors were obvious after clinical examination, brain magnetic resonance imaging (MRI) and expanded screening for inborn errors of metabolism. We performed CDKL5 gene mutation analysis in all and multiplex ligation dependent probe amplification assay (MLPA) in those who were mutation negative. Custom Array-comparative genomic hybridization (CGH), breakpoint polymerase chain reaction (PCR) analysis, and X-inactivation studies were performed in patients in whom MLPA uncovered a genomic alteration. We found CDKL5 mutations in 8.2% (4 of 49) of patients and genomic deletions in 8.2% (4 of 49). Overall, abnormalities of the CDKL5 gene accounted for 16.3% (8 of 49) of patients. CDKL5 gene deletions are an under-ascertained cause of early onset intractable epilepsy in girls. Genetic testing of CDKL5, including both mutation and deletion/duplication analysis, should be considered in this clinical subgroup.Epilepsia 09/2009; 51(4):647-54. DOI:10.1111/j.1528-1167.2009.02308.x · 4.58 Impact Factor
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ABSTRACT: Classical lissencephaly, or isolated lissencephaly sequence (ILS), and subcortical band heterotopia (SBH) are neuronal migration disorders associated with severe mental retardation and epilepsy. Abnormalities of the LIS1 and DCX genes are implicated in the majority of patients with these disorders and account for approximately 75% of patients with ILS, whereas mutations of DCX account for 85% of patients with SBH. The molecular basis of disease in patients with ILS and SBH, in whom no abnormalities have been identified, has been questioned. We studied a series of 83 patients with ILS, SBH or pachygyria, in whom no abnormalities of the LIS1 or DCX genes had been identified, for intragenic deletions and duplications by multiplex ligation-dependent probe amplification (MLPA). In 52 patients with ILS, we identified 12 deletions and 6 duplications involving the LIS1 gene (35%), with the majority resulting in grade 3 lissencephaly. Three deletions of the DCX gene were identified in the group of nine female patients with SBH (out of 31 patients with DCX-suggestive brain anomalies), ie 33%. We estimate an overall mutation detection rate of approximately 85% by LIS1 and DCX sequencing and MLPA in ILS, and 90% by DCX sequencing and MLPA in SBH. Our results show that intragenic deletions and duplications of the LIS1 and DCX genes account for a significant number of patients with ILS and SBH, where no molecular defect had previously been identified. Incorporation of deletion/duplication analysis of the LIS1 and DCX genes will be important for the molecular diagnosis of patients with ILS and SBH.European journal of human genetics: EJHG 01/2009; 17(7):911-8. DOI:10.1038/ejhg.2008.213 · 4.23 Impact Factor
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ABSTRACT: Lissencephaly spectrum (LIS) is one of the most severe neuronal migration disorders that ranges from agyria/pachygyria to subcortical band heterotopia. Approximately 80% of patients with the LIS spectrum carry mutations in either the LIS1 or DCX (doublecortin) genes which have an opposite gradient of severity. The aim of the study was to evaluate in detail the phenotype of DCX-associated lissencephaly and to look for genotype-phenotype correlations. Of the 180 male patients with DCX-related lissencephaly, 33 males (24 familial cases and nine cases with de novo mutations) were found with hemizygous DCX mutations and were clinically and genetically assessed here. DCX mutation analysis revealed that the majority of mutations were missense (79.2%), clustered in the two evolutionary conserved domains, N-DC and C-DC, of DCX. The most prominent radiological phenotype was an anteriorly predominant pachygyria or agyria (54.5%) although DCX-associated lissencephaly encompasses a complete range of LIS grades. The severity of neurological impairment was in accordance with the degree of agyria with severe cognitive impairment in all patients, inability to walk independently in over half and refractory epilepsy in more than a third. For genotype-phenotype correlations, patients were divided in two groups according to the location of DCX missense mutations. Patients with mutations in the C-DC domain tended to have a less severe lissencephaly (grade 4-5 in 58.3%) compared with those in the N-DC domain (grade 4-5 in 36.3%) although, in this dataset, this was not statistically significant (p = 0.12). Our evaluation suggests a putative correlation between phenotype and genotype. These data provide further clues to deepen our understanding of the function of the DCX protein and may give new insights into the molecular mechanisms that could influence the consequence of the mutation in the N-DC versus the C-DC domain of DCX.Neurogenetics 09/2008; 9(4):277-85. DOI:10.1007/s10048-008-0141-5 · 2.66 Impact Factor