Mutations in mammalian tolloid-like 1 gene detected in adult patients with ASD

Abstract

Atrial septal defect (ASD) is an incomplete septation of atria in human heart causing circulatory problems. Its frequency is estimated at one per 10 000. Actions of numerous genes have been linked to heart development. However, no single gene defect causing ASD has yet been identified. Incomplete heart septation similar to ASD was reported in transgenic mice with both inactive alleles of gene encoding mammalian zinc metalloprotease a mammalian tolloid-like 1 (tll1). Here, we have screened 19 ASD patients and 15 healthy age-matched individuals for mutations in TLL1 gene. All 22 exons were analyzed exon by exon for heteroduplex formation. Subsequently, DNA fragments forming heteroduplexes were sequenced. In four nonrelated patients, three missense mutations in coding sequence, and one single base change in the 5'UTR have been detected. Two mutations (Met182Leu, and Ala238Val) were detected in ASD patients with the same clinical phenotype. As the second mutation locates immediately upstream of the catalytic zinc-binding signature, it might change the enzyme substrate specificity. The third change, Leu627Val in the CUB3 domain, has been found in an ASD patient with interatrial septum aneurysm in addition to ASD. The CUB3 domain is important for substrate-specific recognition. In the remaining 15 patients as well as in 15 reference samples numerous base substitutions, deletions, and insertions have been detected, but no mutations changing the coding sequence have been found. Lack of mutations in relation to ASD of these patients could possibly be because of genetic heterogeneity of the syndrome.

Figure 1

Heterozygous mutations detected in ASD patients. (a) A change G:G/T at position 779 detected in patient no. 17 from group 2 with ASD, aneurysm of interatrial septum and other defects. (b) A change A:A/C at position 122 433 (1191 in cDNA) detected in patient no. 10 from group 1 with ASD symptoms alone. In the presented case, the sequence was obtained with the use of reverse primer to sequence the fragment starting at the position 121 553 (exons 4 and 5). (c) A change T:C at position 130 814 (1360 in cDNA) detected in the patient no. 1 from group 2 with ASD, aneurysm of intersinal septum and other defects. (d) A change A:A/G at position 187 409 (1885 in cDNA) detected in the patient no. 9 from group 2 with ASD, aneurysm of intersinal septum and other defects. Arrow in each panel indicates the detected changes.

Figure 2

Comparison of conserved amino acid sequence domains mutated in TLL1 with the corresponding domains in selected metzincins. (a) N-terminal end of the catalytic domain, (b) active centers of selected metalloproteases from astacin family and other selected metzincins. (c) N-terminal end of the CUB3 domain. Symbols: H, amino acid sequence of human polypeptides; M, mutated TLL1; dashes, identical residues as in the polypeptide aligned at the top of the alignment. Colors: yellow, conserved cysteines; pink, histidines coordinating zinc in the active center of the enzyme; red, amino acid sequence in extended catalytic center; green, CUB3 domains in different enzymes; blue, mutations detected in ASD patients.