Utilization of Whole Exome Sequencing in Lethal Form of Multiple Pterygium Syndrome: Identification of Mutations in Embryonal Subunit of Acetylcholine Receptor

Abstract

The acetylcholine receptor (AChR) is a member of the superfamily of transmitter-gated ion channels having a critical role in controlling electrical signals between nerves and muscle cells. Disruptive mutations in genes encoding different subunits of AChR result in multiple pterygium syndrome (MPS), which can be associated with a severe prenatally lethal presentation. This study aimed to investigate the etiology of lethal MPS (LMPS) in two consanguineous families with a history of miscarriages. DNA was extracted from a tissue sample of two aborted fetuses (probands) from two different families with a history of spontaneous miscarriages. Parental peripheral blood samples were collected for confirmatory analysis and follow-up testing. Whole-exome sequencing (WES) was performed on DNA from the probands. The results were confirmed and segregated by Sanger sequencing. Moreover, protein structure evaluations were accomplished. We identified a homozygous frameshift mutation of c.753_754delCT (p.V253fs*44) and a homozygous missense mutation of c.715C>T (p.Arg239Cys) in the CHRNG gene. Both aborted fetuses had pterygium, severe arthrogryposis, and fetal hydrops with cystic hygroma, being compatible with LMPS. The heterozygous state was confirmed in parents for both CHRNG variants. Likewise, CHRNG mutation was predicted to display the damaging effects by lowering the number of helixes and modifying the surface electrostatic potential. The present study identified rare sequence variants in the CHRNG gene in aborted fetuses from consanguineous couples with recurrent miscarriage history. WES is a comprehensive and cost-effective approach to study heterogeneous diseases including MPS. Such findings can improve our knowledge of MPS databases, particularly for genetic counseling of high-risk families and preimplantation genetic diagnosis.

Keywords: CHRNG; Multiple pterygium syndromes; recurrent abortion; whole-exome sequencing.

Fig. 1

Pedigrees for families described in this study.Affected individuals are represented with shaded symbols

Fig. 2

Chromatograms of CHRNG mutations.(A) c.753_754delCT and (B) c.715C>T variants in CHRNG gene in parents of both families. The Sanger sequencing results demonstrated the heterozygous status in father and mother of both fetuses

Fig. 3

Evolutionary conservation of residue V253 relevant for mutation c.753_754delCT (p.V253fs*44).

Fig. 4

Multispecies alignment for the candidate variants of c.753_754delCT [p.V253fs*44] (A) and c.715C>T [p.Arg239Cys] (B) within all species analyzed.Arrows indicate the mutations found in the present study

Fig. 5

Predicted structure of native and truncated protein. (A) Superimposition of the best predicted models of native and truncated protein.The native protein structure was shown in magenta and truncated protein in yellow. Surface electrostatic potential of native protein (B) and mutant proteins (C);blue, red and white colors represent the positive, negative and hydrophobic regions, respectively

Fig. 6

Schematic representation for localization of the identified CHRNG variants in neurotransmitter-gated ion-channel transmembrane region

Fig. 7

The interaction network of CHRNG with other genes, such as CHRNA1 , CHAND , and RAPSN (dark blue circles). Gray circles and green rectanglesindicate the other genes in the same network but not associated with MPS (Gene-gene interactions received from http://phenolyzer.usc.edu/).