Clinical Data
65 Patients with laterality defects and complex CHD were recruited from Shanghai Xinhua Hospital and Shanghai Children’s Medical Center. The patients’ ages ranged from 12 days to 11 years; 44 patients were male (67.7%) and 21 were female (32.3%). One hundred unrelated individuals without major birth defects were used as controls. The controls were 66 male (66%) and 34 Female (34%) with the age range from 10 months to 11 years.
Basic Information of Candidate Sites
We found three non-synonymous mutations of ROCK2 in patients with laterality defects and these mutations were not detected in the control group(Fig 1). Basic Information of these mutations are listed in Table 1. Clinically, the patient 1 and the patient 3 presented situs inversus, while the patient 2 presented HTX. The other cardiac clinical phenotypes and extracardiac abnormalities discovered in patients with ROCK2 mutations re listed in Table 1.
Table 1 Basic Information of Candidate Sites
Patient
ID
|
Basic information
|
MAF (minor allele frequency)
|
Cardiac abnormalities
|
Extracardiac abnormalities
|
CDS AA
|
PROT AA
|
dp SNP
|
1000G
|
ExAC
|
ESP
|
1
|
c.345G>C
|
p.Q115H
|
NF
|
NF
|
NF
|
NF
|
D,ASS, TGA,VSD,PS,ASD
|
RSS,LSL
|
2
|
c.1247A>G
|
p.Y416C
|
NF
|
NF
|
NF
|
NF
|
L,IRAA,SV,TAPVC,PA,PDA,CAVC
|
Asplenia, RSS, BRB
|
3
|
c.3037G>A
|
p.A1013T
|
rs372763637
|
NF
|
0.000042
|
0.000085
|
L,ASS,SV, TGA,PS
|
RSS, LSL
|
ASD Atrial septum defect, ASS Atrial situs inversus, BRB bilateral right bronchi (short), CAVC common atrioventricular canal, D dextrocardia, IRAA isomerism of right atrial appendages, L levocardia, LI Lack of relevant information, LSL left-sided liver ,NF not found, PA pulmonary atresia, PDA patent ductus arteriosus PS pulmonary stenosis, TGA translocation of great arteries, RSS right-sided stomach, SV single ventricle , TAPVC total anomalous pulmonary venous connection, VSD ventricle septum defect.
The 1000G, the EXAC and the SNP database were applied to see the mutation frequency. The mutations c.345G>C in patient 1 and c.A1247A>G in patient 2 were newly discovered. The mutation rs372763637(c.3037G>A) found in patient 3 was described in ExAC database with an allele frequency of 0.000042 in all populations
In Silico Analysis of ROCK2 Mutations
Cross-species sequence alignment of RCOK2 suggested that the amino acid Q115 was highly conserved in vertebrates and the amino acid A1013 were conserved in mammals(Fig 1), which suggested that the sites may have irreplaceable functions in mammals. The amino acid Y416 was only conserved in Chimpanzee and Pig(Fig 1)
The software Sift, Polyphen-2, PROVEAN and Mutation Taster were applied to predict the pathogenicity of the mutants(Table 2). The results predicted that the ROCK2 p.Q115H variant would be damaging and disease causing in Sift and Mutation Taster. The ROCK2 p.Y416C variant was predicted as “dangerous” among all programs, while the ROCK2 p.A1013T variant may have benign effect on the RCOK2 protein. According to the analysis, p.Q115H and p.Y416C are more likely to be pathogenic mutations.
Table 2 Results of software predictions
Mutations
|
Sift (score)
|
PolyPhen-2 (score)
|
PROVEAN
|
MutationTaster
|
HOPE
|
Amino acid propertie
|
Domain functions
|
p.Q115H
|
Damaging (0.04)
|
Benign (0.097)
|
Neutral(-1.933)
|
disease causing
|
bigger
|
Yes
|
p.Y416C
|
Damaging (0.01)
|
possibly damaging (0.751)
|
Deleterious(-3.099)
|
disease causing
|
smaller, more hydrophobic
|
Yes
|
p.A1013T
|
Tolerated (0.58)
|
Benign (0.006)
|
Neutral(0.061)
|
polymorphism
|
Bigger, more hydrophobic
|
Yes
|
Sift predicts whether the mutations would be tolerated(>0.05)or damaging. (≤0.05). PolyPhen-2 shows that if the mutation is possibly damaging (score> 0.5) or benign (score < 0.5). polymorphism. Mutation Taster provides predictions whether the mutation is disease causing or polymorphism. Yes means that the mutation might impact the domain functions.
Then HOPE software was used to estimate the effect of mutations on physicochemical properties and domain functions. The mutation p.Q115H is located within a domain annotated as ‘Protein kinase’. The mutation introduces a bigger residue at this position and disturb this domain functions. Moreover, the residue is located on the surface of the protein, so that the mutation p.Q115H might disturb interactions with other molecules or other parts of the protein. The mutation p.Y416C is located within a domain annotated in UniProt as “AGC-kinase “. The mutant residue is more hydrophobic and smaller in size than the wild-type residue. According to the analysis of the HOPE online program, the mutation p.Y416C is located within a stretch of residues annotated in a special region which can interact with MYPT1(myosin phosphatase-targeting subunit 1). The differences in amino acid properties may disturb its region function.
With the ROCK2 protein sequence obtained from the Swiss-Model website, the software Swiss-Pdb Viewer was applied to construct the spatial conformation for the mutants and wild type (Fig 2).115Gln connects to 111His and 112Gly with a hydrogen bond, while the mutation 115His abolish the connection with 112Gly. Similarly, 416Tyr is linked with 86Gln in normal condition, and the mutant 416Cys unusually links with 417Arg and disconnects with 86Gln. Changes of hydrogen bonds and connection objects might influence the functions of ROCK2.