Combination of linkage mapping and microarray-expression analysis identifies NF-kappaB signaling defect as a cause of autosomal-recessive mental retardation

Abstract

Autosomal-recessive inheritance accounts for nearly 25% of nonsyndromic mental retardation (MR), but the extreme heterogeneity of such conditions markedly hampers gene identification. Combining autozygosity mapping and RNA expression profiling in a consanguineous Tunisian family of three MR children with mild microcephaly and white-matter abnormalities identified the TRAPPC9 gene, which encodes a NF-kappaB-inducing kinase (NIK) and IkappaB kinase complex beta (IKK-beta) binding protein, as a likely candidate. Sequencing analysis revealed a nonsense variant (c.1708C>T [p.R570X]) within exon 9 of this gene that is responsible for an undetectable level of TRAPPC9 protein in patient skin fibroblasts. Moreover, TNF-alpha stimulation assays showed a defect in IkBalpha degradation, suggesting impaired NF-kappaB signaling in patient cells. This study provides evidence of an NF-kappaB signaling defect in isolated MR.

Figure 1

Pedigree of the Family and Brain MRI Features (A) Pedigree of the family. Shaded symbols indicate individuals presenting with MR. (B) Discordance between T2 and FLAIR sequences in patients V-1 and V-4. Coronal T2 weighted image (left) and coronal FLAIR image (right) of patient V-4 (upper panels) and patient V-1 (middle panel) at the level of the third ventricle demonstrate normal myelination on T2 sequence. By contrast, FLAIR sequence shows important white-matter abnormalities (white arrow) at the sus tentorial level compared to a normal control (lower panels).

Figure 2

Genetic Analysis of the Family (A) Results of the linkage analysis with the Merlin software. The y axis represents the LOD score and the x axis represents the genetic distance. (B) Quantitative PCR analysis of TRAPPC9 mRNA. TRAPPC9 expression in fibroblast cells from two controls (black and gray bars) and patient V-1 is shown. Data are normalized to beta2 microglobuline (B2M) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Means ± standard deviation are given (n = 5 independent experiments). ^∗∗∗Significance of difference with control values (Student's test), p < 0.01. (C) Electrophoregrams illustrating the c.1708C>T variant in the TRAPPC9 gene. Data are shown for a control, an affected child (V-1), and a healthy heterozygote parent (IV-1). The position of the single-nucleotide change is shown by a black arrow.

Figure 3

Functional Consequences of the TRAPPC9 Mutation (A) Immunoblot analysis of TRAPPC9 protein. Lysates from controls (C1-2) and patient (V-1) fibroblasts were subjected to immunoblotting with a polyclonal anti-β-actin antibody as a loading control (lower panel) and with a rabbit polyclonal anti-TRAPPC9 antibody (upper panel). Molecular weights are indicated on the left. TRAPPC9, shown as a 140 kD band in controls, is absent in the patient. (B) Time-course analysis of TNF-α-induced IκB-α degradation as detected by immunoblot. Lysates were subjected to immunoblotting with a polyclonal anti-β-actin antibody as a loading control (lower panel) and with a rabbit polyclonal anti-IKB-α antibody (upper panel). IKB-α is shown as a 37 kD band. Results from one representative experiment are shown.