Western blot analysis of α-dystroglycan. Immunoblot analysis of WGA-enriched homogenates from fibroblasts obtained from Patient 1 and a healthy individual (control). Absent staining of antibodies IIH6 and VIA4 that bind to glycosyl epitopes on α-dystroglycan (α-DG) and a laminin-1 (Lam) overlay assay show abnormalities in αDG glycosylation in patient fibroblasts that is associated with loss of laminin-1 protein binding. Beta-dystroglycan (β-DG) is used as a loading control.

Analysis of the LARGE gene insertion in genomic and cDNA. (a) Agarose gel showing the PCR products from patient (P) and control (C) cDNA generated from cultured lymphoblasts (Lymph) and fibroblasts (Fib) using primers that span from exons 9 to 12 of LARGE (Supplementary E, Table 1). In patient cDNA sample, two abnormally high bands predominate that correspond to insertions of abnormal DNA sequences between exons 10 and 11. A faint band of the normal size is also seen (arrow). (b) Diagram showing the amino acid sequence that is coded by normal and patient cDNA around the exon 10/11 boundary. The abnormal insertion found in patient cDNA results in the introduction of a premature STOP codon, predicted to cause protein termination mid-way through the translation of the LARGE protein. (c) Results from qPCR analysis of genomic DNA (gDNA) for EST DA935254 and the surrounding chromosomal region. gDNA concentrations between samples were adjusted using qPCR results from a distant gene (PTPLA) and results were scaled so that control samples had a mean copy number of 2 at each point (data not shown). In patients' gDNA sample, qPCR analysis of the EST sequence and for five regions, located at variable distances upstream and downstream, shows twice the expected copy number consistent with homozygous duplication of this region. Genomic copy number returns to normal for markers 5b and 3b in the patient gDNA samples, and therefore these markers define the maximum size of the duplication. Analysis of gDNA from both parents shows that both had three copies of the duplicated region, consistent with each having the duplication in a heterozygous state. All results are shown relative to the reverse gDNA strand.

Brain MRI images obtained from Patient 1 performed at an age of 3.5 years. (a) Coronal T1-weighted image showing cerebellar and vermis hypoplasia (white arrows), cerebellar cysts (black arrow), generalised white matter atrophy and increased CSF spaces. (b) Transverse T2-weighted image showing abnormal high white matter signal and dilated lateral ventricles. (c) Sagittal T1-weighted image showing cerebellar (white arrow) and pontine hypoplasia.

Genotyping for the LARGE locus. Family tree showing analysis of microsatellite markers in close proximity to the LARGE locus (22q12.3). Both of the affected children are homozygous for four contiguous markers, consistent with homozygosity by descent for this chromosomal region.

Diagram of the insertion/deletion mutation involving intron 10 of the LARGE gene. The EST DA935254, normally located at 100 kb centromeric of LARGE, is duplicated together with ∼40 kb of flanking DNA and likely inserted into intron 10 of LARGE. Numerous repetitive elements, which may have contributed to the large intra-chromosomal rearrangement, are present in both intron 10 and flanking the EST, including SINEs (green), LINEs (red) and LTRs (blue). bp=base pairs.