A, DGAP095 capillary abnormalities on face and limbs. B, Ideogram of t(X;2)(p11.2;q37)dn present in DGAP095. C, Metaphase spread following staining with DAPI and fluorescein-conjugated BrdU antibodies. Note that the normal chromosome X is BrdU-stained, indicating its late-replication status.

Mapping of the breakpoint on chromosome 2. A, Representative metaphase spread following FISH with clone RP11-400N9 (left) and RP11-79G2 (right); both probes are biotin labeled and detected by FITC-conjugated avidin. FISH signals are detected on the normal chromosome 2, der(X), and der(2), indicating that the breakpoint is contained within the same genomic sequence on RP11-400N9 and RP11-79G2. B, Refinement of the breakpoint region, performed using Southern blot hybridization with a 1,363-bp radioactive PCR probe from the chromosome 2 breakpoint region. Red arrows indicate novel aberrant restriction fragments in DGAP095 DNA caused by chromosomal rearrangement. C, Orientation of the RP11-400N9 and RP11-79G2 clones relative to each other (and reflective of uneven split signals on the derivative chromosomes). The defined breakpoint region falls within the first intron of DAG kinase delta (DGKD), as shown on the DGKD structural diagram, where numbered boxes represent exons. Restriction sites of the enzymes used for the Southern blot are indicated by arrows, and the location of the PCR probe is represented by a red rectangle. D, Representative metaphase chromosomes X, 2, der(2), and der(X) after hybridization with DGKD cDNA labeled with FITC. Note the absence of DGKD hybridization signal on the der(2). (Chromosome X subtelomeric probe labeled with SpectrumOrange was used to facilitate identification of the der(X).)

Mapping of the breakpoint on chromosome X. A, Graphic representation of orientation of PCR probes used for FISH and map of restriction sites of the enzymes used for the Southern blot, with the probe location represented by a red rectangle. B, Metaphase chromosomes following FISH with PCR probe 1 (left) and PCR probe 2 (right); both probes are labeled by biotin and detected by FITC-conjugated avidin. Probe 1 hybridizes to the normal chromosome X and to the der(2), whereas probe 2 hybridizes to the normal X and der(X), indicating that the breakpoint is located within the sequence flanked by the probes. C, Southern blot hybridized to the 371-bp radioactive PCR probe from the chromosome X breakpoint region (originally defined by FISH). (No aberrant fragment is present in the XbaI digest because the breakpoint occurs outside of the area flanked by XbaI sites.)

Whole-mount in situ hybridization showing expression of Dgkd orthologs. A, Stage 16 fly embryo hybridized to the Drosophila Dgkd antisense probe. Note expression in brain and ventral nerve cord. B, E12.5 mouse embryo hybridized to the mouse Dgkd antisense probe. Note prominent expression in forebrain and midbrain. C, In situ hybridization performed using a 3′ UTR probe to the Dgkd transcript on adult wild-type mouse neocortex and cerebellum. Dark granules represent Dgkd transcripts. Neocortex: moderate to high Dgkd expression in large pyramidal neurons (pn and arrows) and relatively absent to low in small cells with the morphology of glia (arrowheads). In the cerebellum, expression is relatively low in glia and neurons of the molecular layer (ml), intermediate within the Purkinje cell and Bergmann glial cell layer (pc and arrows point to Purkinje cells), and highest in granule neurons within the internal granule cell layer (igl).

A, Graphic representation of the gene-trap vector insertion within Dgkd intron 1. Numbered boxes represent exons, arrows represent primers used for genotyping and expression assays, and double-sided arrows represent expected amplicons. B, Representative genotyping assay performed on wild-type, heterozygous (het), and homozygous (homo) mice. C, Dgkd expression analysis in brain tissues from homozygous and wild-type mice. D, EEG recording showing epileptiform activity in a Dgkd knockdown mutant mouse. A sudden onset of ictal electrographic discharge is recorded from both hemispheres (upper channel [red] and middle channels [blue] represent left and right hemispheres, respectively), with higher amplitude on the left. The frequency and amplitude of the discharge evolves in a manner typical of seizure activity. Simultaneous EMG recording (lower channel [green]) demonstrates fast, low-voltage frequencies that might reflect fine-motor activity associated with electrographic seizure discharge. (It is possible that the EMG channel is contaminated with EEG activity, as the recording electrode is placed close to the head.) No gross movements are observed, excluding the artifact as a cause of the EEG changes. Time scale is 5 s per major division. Amplitude scale is 800 microvolts per major division.