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X-linked mental retardation is a very common condition that affects approximately 1 in 600 males. Despite recent progress, in most cases the molecular defects underlying this disorder remain unknown. Recently, a study using the candidate gene approach demonstrated the presence of mutations in PAK3 (p21-activating kinase) associated with nonspecific mental retardation. PAK3 is a member of the larger family of PAK genes. PAK proteins have been implicated as critical downstream effectors that link Rho-GTPases to the actin cytoskeleton and to MAP kinase cascades, including the c-Jun amino-terminal kinase (JNK) and p38. We screened 12 MRX pedigrees that map to a large region overlying Xq21-q24. Mutation screening of the whole coding region of the PAK3 gene was performed by using a combination of denaturing gradient gel electrophoresis and direct sequencing. We have identified a novel missense mutation in exon 2 of PAK3 gene (R67C) in MRX47. This confirms the involvement of PAK3 in MRX following the report of a nonsense mutation recently reported in MRX30. In the MRX47 family, all affected males show moderate to severe mental retardation. No seizures, statural growth deficiency, or minor facial or other abnormal physical features were observed. This mutation R67C is located in a conserved polybasic domain (AA 66-68) of the protein that is predicted to play a major role in the GTPases binding and stimulation of Pak activity.

A gene for X-linked congenital cerebellar hypoplasia was recently localized to chromosome Xp11.21-q24. This region comprises several brain-specific genes responsible for various neurological disorders, including the proteolipid protein (PLP), doublecortin, and PAK3 genes. We screened these genes for mutations in patients with X-linked congenital cerebellar hypoplasia and found no pathogenic nucleotide changes or gene dose alterations. These findings allow the ruling out of PLP, doublecortin, and PAK3 as the disease-causing genes in this hereditary neurological syndrome.

Malformations of cortical development are increasingly recognized as causes of mental retardation and epilepsy. However, little is known about the molecular and biochemical signals that control the proliferation, migration, and organization of the cells involved in normal cerebral cortical development. Analysis of genes required for cortical development will help elucidate the pathogenesis of some epilepsies. In humans, two striking examples of abnormal cortical development, with varying degrees of epilepsy and mental retardation, are 'double cortex' and lissencephaly. Double cortex (DC), also known as subcortical band heterotopia, shows an abnormal band of neurons in the white matter underlying a relatively normal cortex. In pedigrees, DC often occurs in females, whereas affected males show more severe lissencephaly (XLIS), i.e. an abnormally thick cortex with decreased or absent surface convolutions. We and others have identified a novel brain specific gene, doublecortin, that is mutated in Double Cortex/X-linked lissencephaly (DC/XLIS) patients. Although the cellular function of doublecortin (DCX) is unknown, sequence analysis reveals a cytoplasmic protein with potential MAP kinase phosphorylation sites, as well as a site that is likely to be phosphorylated by c-Abl, suggesting that doublecortin functions as an intracellular signaling molecule critical for the migration of developing neurons. Interestingly, the scrambler mouse mutant demonstrates abnormal lamination with some similarity to lissencephaly and reflects a mutation in the murine homolog of the Drosophila disabled gene, mdab1, which binds c-Abl. Although a direct interaction between doublecortin and mDab1 has not been demonstrated, it is plausible that these two proteins may be part of a common signaling pathway. Therefore, abnormalities in signal transduction may be an underlying mechanism for the neuronal migration defects in DC/XLIS and the scrambler mouse, but further research is necessary to determine how such abnormalities give rise to cortical malformations and epilepsy.

Mutations in the X-linked gene doublecortin, which encodes a protein with no dear structural homologues, are found in pedigrees in which affected females show "double cortex" syndrome (DC; also known as subcortical band heterotopia or laminar heterotopia) and affected males show X-linked lissencephaly. Mutations in doublecortin also cause sporadic DC in females. To determine the incidence of doublecortin mutations in DC, we investigated a cohort of eight pedigrees and 47 sporadic patients with DC for mutations in the doublecortin open reading frame as assessed by single-stranded conformational polymorphism analysis. Mutations were identified in each of the eight DC pedigrees (100%), and in 18 of the 47 sporadic DC patients (38%). Identified mutations were of two types, protein truncation mutations and single amino acid substitution mutations. However, pedigrees with DC displayed almost exclusively single amino acid substitution mutations, suggesting that patients with these mutations may have less of a reproductive disadvantage versus those patients with protein truncation mutations. Single amino acid substitution mutations were tightly clustered in two regions of the open reading frame, suggesting that these two regions are critical for the function of the Doublecortin protein.

X-linked lissencephaly and "double cortex" are allelic human disorders mapping to Xq22.3-Xq23 associated with arrest of migrating cerebral cortical neurons. We identified a novel 10 kb brain-specific cDNA interrupted by a balanced translocation in an XLIS patient that encodes a novel 40 kDa predicted protein named Doublecortin. Four double cortex/X-linked lissencephaly families and three sporadic double cortex patients show independent doublecortin mutations, at least one of them a de novo mutation. Doublecortin contains a consensus Abl phosphorylation site and other sites of potential phosphorylation. Although Doublecortin does not contain a kinase domain, it is homologous to the amino terminus of a predicted kinase protein, indicating a likely role in signal transduction. Doublecortin, along with the newly characterized mDab1, may define an Abl-dependent pathway regulating neuronal migration.

Audiogenic seizure (AGS) susceptibility in mice is a multifactorial behavioral disorder that involves severe generalized convulsions in response to loud, high-frequency sound. The inheritance of AGS susceptibility was examined in crosses between AGS-susceptible DBA/2J (D2) mice and epilepsy-prone (EP) mice. The EP mice were selected for high AGS susceptibility in a BALB/c-derived line. The AGS phenotype was similar in the EP and D2 mice at 30 days of age. The frequency of generalized clonic–tonic AGS was high in both the D2 and the EP mice (53 and 83%, respectively) but was low in the reciprocal EPD2F1 and D2EPF1 hybrids (14 and 19%, respectively). In the backcross to the EP parent, no significant associations were found between AGS susceptibility and microsatellite markers linked to Asp1 or Asp2, AGS genes located on Chromosomes 12 and 4, respectively. Significant associations were found for markers linked to Asp3, which is located in the proximal region of Chromosome 7. The influence of Asp3 on AGS susceptibility was seen in the EP × EPD2F1 backcross but not in the reciprocal EPD2F1 × EP backcross, suggesting that Asp3 expression is influenced by genomic imprinting. A model is proposed where genomic imprinting represses the maternal Asp3 allele, providing an influence largely from the paternal allele.

Mice that are homozygous for the autosomal recessive hydrocephaly with hop gait (hyh) mutation on Chromosome (Chr) 7 have congenital hydrocephalus characterized by an interhemispheric cyst arising from the third ventricle and agenesis of the corpus callosum. Analysis of more than 500 backcross and intercross progeny maps the hyh locus to proximal Chr 7, approximately 13 cM centromeric to its originally reported map position. Analysis of recombinants at several MIT microsatellite markers localized the hyh locus between D7Mit75 and D7Mit56. Development of several new SSLP markers allowed us to refine the hyh candidate interval to a region defined by the cone-rod homeobox ( Crx) gene proximally and D7Mit56 distally. A contig of yeast artificial chromosome (YAC) clones and bacterial artificial chromosome (BAC) clones spanning this entire region has been developed, and a number of potential candidate genes for hyh within this interval have been identified. Gene content is conserved between this region of mouse Chr 7 and human Chr 19q13.3. Physical mapping of the regions around D7Mit75 and D7Mit56 has also determined the order of a number of MIT markers that remain unresolved on the Mouse Genome Database (MGD) map. Our physical map and transcript map may be useful for positional cloning of genes in this unusually gene-rich region of the genome.

Although several genes for mental retardation and epilepsy, including double cortex/X-linked lissencephaly ( DC/XLIS ), have been localized to Xq21.3–q23, there has been no complete physical map of this region available. We constructed a YAC/STS contig map by initiating two yeast artificial chromosome (YAC) walks from the markers that flanked the DC/XLIS candidate gene region. We report an approximately 4-Mb contig extending from DXS287 to DXS8088, encompassing DXS1072 and DXS1059, and composed of 52 YACs identified with 15 previously published STSs and 19 novel YAC-end STSs. This contig also contains two brain-specific genes, doublecortin (HGMW-approved symbol DCX ), responsible for DC/XLIS, and PAK3, which may be responsible for neurological diseases localized to this region. The new contig extends and incorporates several previously published contigs, providing a total overlapping contig extending approximately 34 Mb from DXS441 in Xq13.1 to DXS8088 in Xq23.

Nonsyndromic X-linked mental retardation (MRX) syndromes are clinically homogeneous but genetically heterogeneous disorders, whose genetic bases are largely unknown. Affected individuals in a multiplex pedigree with MRX (MRX30), previously mapped to Xq22, show a point mutation in the PAK3 (p21-activated kinase) gene, which encodes a serine-threonine kinase. PAK proteins are crucial effectors linking Rho GTPases to cytoskeletal reorganization and to nuclear signalling. The mutation produces premature termination, disrupting kinase function. MRI analysis showed no gross defects in brain development. Immunofluorescence analysis showed that PAK3 protein is highly expressed in postmitotic neurons of the developing and postnatal cerebral cortex and hippocampus. Signal transduction through Rho GTPases and PAK3 may be critical for human cognitive function.

The neural function of cystatin B, recently implicated in EPM1, is unclear but may lead to new insights into the mechanisms of epilepsy.