Mutation analysis of CHRNA1, CHRNB1, CHRND, and RAPSN genes in multiple pterygium syndrome/fetal akinesia patients

Abstract

Multiple pterygium syndromes (MPS) comprise a group of multiple congenital anomaly disorders characterized by webbing (pterygia) of the neck, elbows, and/or knees and joint contractures (arthrogryposis). MPS are phenotypically and genetically heterogeneous but are traditionally divided into prenatally lethal and nonlethal (Escobar) types. Previously, we and others reported that recessive mutations in the embryonal acetylcholine receptor g subunit (CHRNG) can cause both lethal and nonlethal MPS, thus demonstrating that pterygia resulted from fetal akinesia. We hypothesized that mutations in acetylcholine receptor-related genes might also result in a MPS/fetal akinesia phenotype and so we analyzed 15 cases of lethal MPS/fetal akinesia without CHRNG mutations for mutations in the CHRNA1, CHRNB1, CHRND, and rapsyn (RAPSN) genes. No CHRNA1, CHRNB1, or CHRND mutations were detected, but a homozygous RAPSN frameshift mutation, c.1177-1178delAA, was identified in a family with three children affected with lethal fetal akinesia sequence. Previously, RAPSN mutations have been reported in congenital myasthenia. Functional studies were consistent with the hypothesis that whereas incomplete loss of rapsyn function may cause congenital myasthenia, more severe loss of function can result in a lethal fetal akinesia phenotype.

Figure 1

Sequence Traces for Normal Control, Homozygote, and Heterozygote Carrier of RAPSN Frameshift Mutation

Figure 2

Clinical Features of Fetal Akinesia Associated with Homozygous Rapsyn Mutation (A and B) Affected twin fetuses. (C) Affected singleton fetus with clinical features of the fetal akinesia sequence.

Figure 3

Functional Analysis of RAPSN Mutation Associated with Fetal Akinesia Sequence (A) DNA sequences and translations of exon 8 for wild-type and mutant human rapsyn. In the wild-type sequence, the two nucleotides that are deleted in the mutant are shown in red. As a consequence of the frameshift, mutant rapsyn is 62 amino acid residues longer than wild-type (asterisk indicates termination codon). (B) Diagram of wild-type and mutant rapsyn tagged with EGFP at the carboxyl terminus. Wild-type or mutant rapsyn cDNA without a stop codon were cloned into pEGFP-N1 (Clontech) so that EGFP was in-frame with rapsyn. Mutagenesis to delete the two adenine residues was carried out with the QuikChange mutagenesis kit purchased from Stratagene and was confirmed by DNA sequencing. The frameshift in the c.1177-1178delAA mutant extends the length of the protein by 62 amino acid residues, and so the corresponding 3′UTR was included in this construct. (C) Rapsyn mutation c.1177-1178delAA (also referred to as 1177delAA) does not cluster the AChR. TE671 cells were cotransfected with cDNAs encoding rapsyn-EGFP or rapsyn-1177delAA-EGFP and the human AChR subunits. AChR was detected with mAb B3 directed against the AChR β subunit. Cells were fixed with 3% paraformaldeyde at room temperature for 20 min, washed three times with PBS, and incubated with secondary antibody Alexa Fluor 594 goat anti-mouse IgG (H⁺L) diluted 1:1000 in PBS containing 1% BSA (Molecular Probes). Cells were washed 3× in PBS and mounted in fluorescent mounting media (Dako Cytomation). Microscopy was performed on an Olympus BX60 wide-field fluorescence microscope, and images were captured with Openlab software (Improvision). (D) Western blot of rapsyn-EGFP and rapsyn-1177delAA-EGFP expressed in TE671 muscle cells. TE671 cells were transfected with wild-type or mutant rapsyn-EGFP, and 48 hr later, total cell lysate was analyzed by Western blotting. Rapsyn was detected by mAb clone 1234 (Abcam) followed by anti-mouse-HRP and ECL (Amersham). As a control, α-tubulin was detected on the same western blots with a mAb (Sigma-Aldrich) followed by anti-mouse-HRP and ECL. The experiment was performed twice; one example is shown. (E) The ratio of rapsyn:α-tubulin was obtained by densitometric scanning of western blots. Whereas rapsyn-EGFP gave robust expression on days 1 and 2, rapsyn-1177delAA-EGFP was barely detectable throughout the time course.