Mutations in ROGDI Cause Kohlschütter-Tönz Syndrome

Abstract

Kohlschütter-Tönz syndrome (KTS) is an autosomal-recessive disease characterized by the combination of epilepsy, psychomotor regression, and amelogenesis imperfecta. The molecular basis has not yet been elucidated. Here, we report that KTS is caused by mutations in ROGDI. Using a combination of autozygosity mapping and exome sequencing, we identified a homozygous frameshift deletion, c.229_230del (p.Leu77Alafs(∗)64), in ROGDI in two affected individuals from a consanguineous family. Molecular studies in two additional KTS-affected individuals from two unrelated Austrian and Swiss families revealed homozygosity for nonsense mutation c.286C>T (p.Gln96(∗)) and compound heterozygosity for the splice-site mutations c.531+5G>C and c.532-2A>T in ROGDI, respectively. The latter mutation was also found to be heterozygous in the mother of the Swiss affected individual in whom KTS was reported for the first time in 1974. ROGDI is highly expressed throughout the brain and other organs, but its function is largely unknown. Possible interactions with DISC1, a protein involved in diverse cytoskeletal functions, have been suggested. Our finding that ROGDI mutations cause KTS indicates that the protein product of this gene plays an important role in neuronal development as well as amelogenesis.

Figure 1

Pedigrees of Investigated Families The pedigree for family A shows a consanguineous Moroccan family in which linkage analysis and exome sequencing were performed. The pedigree for family B shows a Tyrolean family affected by KTS, and the parents are not knowingly related. The pedigree for family C shows the newly diagnosed Swiss family (the parents are X:1 and X:2) and its relationship with the distantly related index family reported in 1974 (parents IX-3 und IX-4). Note that the parents in the newly identified family are distantly related to each other, but the affected child is compound heterozygous for two different mutations.

Figure 2

Dental Phenotype in the So Far Unreported Individual C-XI:2 Tooth discoloration due to global enamel defect (amelogenesis imperfecta).

Figure 3

Linkage and Genomic Sequence Analyses (A) Linkage analysis in family A revealed four autozygous regions in chromosomes 3, 11, 16, and 17. (B) Exome sequencing in family A revealed a homozygous 2 bp deletion, c.229_230del, in exon 4 of ROGDI. (C–F) Identification of mutations by Sanger sequencing. Homozygous deletion c.229_230del (C) is present in family A, homozygous nonsense mutation c.286C>T (D) is present in family B, and heterozygous splice-site mutations c.531+5G>C (E) and c.532-2A>T (F) are present in family C.

Figure 4

cDNA Analyses (A) RT-PCR analysis of ROGDI in family C. Note the absence of the wild-type amplicon as well as the presence of two aberrant bands in affected individual C-XI:2. One of the aberrant bands is approximately 100 bp shorter than the wild-type band and is also found in the father (C-X:1) and sister (C-XI:1), who are both heterozygous for c.531+5G>C. The other aberrant band is weak, approximately 80 bp larger than the wild-type band, and is also observed in the mother (C-X:2), who is heterozygous for c.532-2A>T. (B) RT-qPCR analysis of ROGDI in affected individuals, healthy family members, and controls shows markedly reduced mRNA transcript in affected individual B-II:1. Heterozygosity for c.532-2A>T in C-X:2 is associated with a cDNA reduction of approximately 50%, most likely reflecting nonsense-mediated decay of that allele. In contrast, heterozygosity for c.531+5G>C is not associated with the loss of cDNA in C-X:1 and C-XI:1. The fact that affected individual C-XI:2 has half normal cDNA reflects the combination of both alleles. The error bars represent means and standard deviations of three independence measurements of the probands and four controls. (C) cDNA sequence analysis of the RT-PCR product of exons 6–9 in individual C-X:1, heterozygous for c.531+5G>C, shows skipping of in-frame ROGDI exon 7.