Mutations of MLC1 (KIAA0027), encoding a putative membrane protein, cause megalencephalic leukoencephalopathy with subcortical cysts

Abstract

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is an autosomal recessive disorder characterized by macrocephaly, deterioration of motor functions with ataxia, and spasticity, eventuating in mental decline. The brain appears swollen on magnetic resonance imaging, with diffuse white-matter abnormalities and the invariable presence of subcortical cysts. MLC was recently localized on chromosome 22q(tel). We have narrowed down the critical region by linkage analysis of 11 informative families with MLC to a region of approximately 250 kb, containing four known genes. One family with two patients who were siblings did not display linkage between the MLC phenotype and any of the analyzed microsatellite markers on chromosome 22q(tel), suggesting genetic heterogeneity and the existence of at least a second MLC locus. The maximum two-point LOD score for the 11 families was 6.6 at recombination fraction .02. Twelve different mutations in seven informative and six uninformative families were found in one of the candidate genes, KIAA0027, which we renamed "MLC1." The gene encodes a putative membrane protein with eight predicted transmembrane domains. The patients of one family were compound heterozygotes for mutations that both introduced stop codons. The mutations further included frameshifts, splice-acceptor mutations, a putative splice-donor mutation, and amino acid substitutions of residues in predicted transmembrane domains. These data provide strong evidence that mutations of MLC1 cause the disease.

Figure 1

Localization of the MLC gene on chromosome22q_tel. A, Chromosome 22q_tel haplotypes of three informative families with MLC. Microsatellite and SNP markers are listed on the left, according to their order. The alleles are depicted as PCR fragment length (microsatellites) or DNA sequence at the polymorphic position (SNPs). Haplotypes that are identical by descent in individual patients are indicated by the boxes. In family ML1, there are two patients with parents who are first cousins. The healthy sibling, M6, is a recombinant between markers UT580 and 355c18, indicated by the broken line. Patient M18 also has parents who are first cousins. Together, the two families limit the critical region for MLC between rs4624 and rs8238. The two sibling patients in family ML10 do not share a genotype in the MLC region. B, Physical map of the MLC region. The map is based on the completed and annotated DNA sequence of chromosome 22. Contig numbers and the position of markers are indicated. The gaps between contigs are of unknown length. Vertical lines depict known ESTs and genes. The four candidate EST clusters and genes are numbered: (1) KIAA0027 (“MLC1”); (2) dJ402G11.8 similar to MOV10; (3) dJ402G11.9 similar to MRS1; and (4) dJ402G11.4.

Figure 2

MLC1 mutations in genomic DNA of patients. Patient M31 was a compound heterozygote for a 4-bp deletion at the 3′ end of exon 7 and a point mutation that led to a stop codon. The allele with the deletion was sequenced after subcloning of the PCR products. The GT sequence of the splice donor is marked by an asterisk in this sequence. Lines indicate homologous residues of control and deletion mutant DNA sequences. The electropherograms were obtained by automated DNA sequencing with PCR products from control and patient DNA templates.

Figure 3

Organization of MLC1. A, Conservation of the N-terminal end of the MLC1 gene product. The translated DNA sequences of the Xenopus EST and MLC1 (Genbank accession numbers AW640978 and D25217, respectively) are compared. Vertical lines indicate identical residues, and the plus sign (+) indicates conservative changes. The top line shows part of the 5′ end of the Xenopus EST with a stop codon (underlined) in frame with the coding region. The stop codon that is 132 nt upstream of and in frame with the first methionine of MLC1 in EST AU120203 is not shown. The asterisk (*) indicates the conserved serine residue that was mutated in patients M66, M73, and M75. B, Genomic structure of MLC1. The exons are shown as boxes; introns, as solid lines. The gray parts indicate the eight regions that encode transmembrane domains of the protein. The positions of the mutations that were found in DNA of patients with MLC are shown by arrows. Interrupted lines indicate deviations from scale. M = start codon, X = stop codon, and fs = frameshift.