A defect in the TUSC3 gene is associated with autosomal recessive mental retardation

Abstract

Recent studies have shown that autosomal recessive mental retardation (ARMR) is extremely heterogeneous, and there is reason to believe that the number of underlying gene defects goes into the thousands. To date, however, only four genes have been implicated in nonsyndromic ARMR (NS-ARMR): PRSS12 (neurotrypsin), CRBN (cereblon), CC2D1A, and GRIK2. As part of an ongoing systematic study aiming to identify ARMR genes, we investigated a large consanguineous family comprising seven patients with nonsyndromic ARMR in four sibships. Genome-wide SNP typing enabled us to map the relevant genetic defect to a 4.6 Mbp interval on chromosome 8. Haplotype analyses and copy-number studies led to the identification of a homozygous deletion partly removing TUSC3 (N33) in all patients. All obligate carriers of this family were heterozygous, but none of 192 unrelated healthy individuals from the same population carried this deletion. We excluded other disease-causing mutations in the coding regions of all genes within the linkage interval by sequencing; moreover, we verified the complete absence of a functional TUSC3 transcript in all patients through RT-PCR. TUSC3 is thought to encode a subunit of the endoplasmic reticulum-bound oligosaccharyltransferase complex that catalyzes a pivotal step in the protein N-glycosylation process. Our data suggest that in contrast to other genetic defects of glycosylation, inactivation of TUSC3 causes nonsyndromic MR, a conclusion that is supported by a separate report in this issue of AJHG. TUSC3 is only the fifth gene implicated in NS-ARMR and the first for which mutations have been reported in more than one family.

Figure 1

Pedigree and Facial Aspects of Affected Family Members Filled symbols indicate severe MR, and three-quarter-filled symbols depict moderate MR.

Figure 2

Results of Parametric Linkage Analysis with Merlin Software A single significant peak (LOD score 6.26) was observed on chromosome 8p22 between rs613566 and rs11203893.

Figure 3

Copy-Number Analysis and Haplotyping of MR Patients (A) Results of nonpaired DNA copy-number analysis (CNAG2 tool for copy-number variations): Copy-number state (upper) and log2 ratios (lower) for Nsp-array SNP markers located inside the first ∼30 Mb of chromosome 8 are displayed for one individual per branch, showing a 120 Kbp homozygous deletion of 8p22 comprising the first exon of TUSC3. (B) The markers bordering the deletion as well as the first and last two deleted markers are shown, revealing that all the affected members are homozygous for the same haplotype, whereas parents and healthy sibs are heterozygous carriers.

Figure 4

Deletion Encompassing the First Exon of TUSC3 in MR Patients (A) Schematic representation of TUSC3: Arrowheads represent exons, and gray boxes mark the positions of simple tandem repeats that could be causatively involved in the genesis of the deletion. The positions of the borders of the 121,595 bp deletion (based on NCBI Build 36.1) are indicated. The sequence chromatogram shows part of the PCR amplicon covering the junction of the deletion borders. (B) Cosegregation analysis by PCR: Results of a deletion-specific PCR (I) and a deletion-spanning PCR (II) are shown for all the available family members. All homozygous carriers show only amplification of the junction fragment (II), heterozygous carriers show both amplicons, and noncarriers show only amplicon I. (C) RT-PCR results from an experiment with cDNA derived from RNA preparations of one patient (IV:5) and one control lymphoblastoid cell line sample. With a sequence of primer pairs specific for amplicons covering two to three consecutive exons each, the complete TUSC3 transcript was detected in the control but could not be amplified from patient cDNA. The results of an agarose gel electrophoresis of 5 μl from a 25 μl RT-PCR reaction (primers and conditions are available upon request) are shown. Patient and control products for a specific amplicon (the exons covered by each amplicon are indicated) were loaded on neighboring lanes in ascending order of the amplified exons. As positive control, a PCR specific for the X-chromosomal HUWE1 gene (MIM 300697) was performed (lane 15 and 16). Filled squares represent the patient and open squares the control, “B” marks the lane loaded with the negative control, and “M” indicates the marker lane (HyperLadder IV, Bioline).

Figure 5

Schematic Representations of Predicted Functional Domains in the TUSC3 Gene Product The 348 amino acid TUSC3 protein is shown as an open box. Different functional domains are indicated. Differently colored shading marks their extent and position within the protein. The deletion encompasses the first 46 amino acids and is indicated by gray shading.