Identification of RESP18 Gene Mutations Linked to Hereditary Non-Syndromic Cleft Lip and Palate in a Southern Chinese Family

Introduction

Non-syndromic cleft lip with cleft palate (NSCLP) is one of the most common congenital birth defects worldwide, and occurs in approximately 1 in 1000 live births [1–3]. NSCLP can be classified into 5 types – (A) Cleft lip and alveolus, (B) Cleft palate, (C) Incomplete unilateral cleft lip and palate, (D) Complete unilateral cleft lip and palate, and (E) Complete bilateral cleft lip and palate – all of which are caused by mesenchymal tissues disorders in cell migration, growth, differentiation, and apoptosis at 4–10 weeks of human embryonic development [4]. Although it is not a main cause of death, this serious malformation has a lifelong impact on health and causes substantial financial and social burdens to patients and their families, and is challenging to treat [2,3,5,6]. Previous studies have shown that the etiology of NSCLP is heterogeneous and closely related to genetic and environmental factors [1,3,4,7]. However, a single-gene mutation may contribute more significance in a hereditary NSCLP family [8]. There have been few studies on shared related mutant genes in NSCLP families. Thus, identification of susceptible variants plays an important role in prevention, treatment, and prognosis of people with NSCLP.

Whole-genome sequencing (WGS) and whole-exome sequencing (WES) are the most extensively used and effective genomics methods in medical genetics, which can more comprehensively identify single-nucleotide variation (SNV), single-nucleotide polymorphism (SNP), insertion deletion mutation (INDEL), copy number mutation (CNV), and structural variation (SV) in the whole genome of hereditary diseases [9,10]. Whole-genome sequencing sequences all genes, detected exons, introns, and regulatory sequences, and screens the genetic variation in the genome, allowing genotype diversity analysis, genetic evolution analysis, disease pathogenesis, and susceptibility gene screening [2,9,11,12]. WGS is increasingly utilized in the research, screening, and monitoring of various diseases. Turro et al used WGS to identify the associations between genes and diseases and Wong et al discovered an actionable target for high-risk childhood cancer by using WGS [11,12]. Whole-exome sequencing (WES) is a more efficient, complete, specific, and inexpensive sequencing technology for studying the etiology of hereditary diseases [13–16]. Basha et al used whole-exome sequencing (WES) to identify variant genes in NSCLP, and found that the susceptibility genes are not only helpful in revealing the etiology of NSCLP, but also help in diagnosis and treatment of patients with NSCLP [17–19]. Many studies have explored the causes of NSCLP [18,20–22]. However, these observations suggested that different populations have different causal variants due to different genetic backgrounds., Leslie and Marazita reviewed orofacial clefts with the many error genes that contributed to the etiology of birth defects [23]. Few studies have explored genomic loci mutation related to NSCLP among Chinese people [24].

In this study, we performed genomic analysis through sequencing technique and identification of de novo RESP18 rs2385404 and rs2385405 gene polymorphisms associated with NSCLP in a southern Chinese family. Our findings provide important insights into the etiology of NSCLP.

Material and Methods

PARTICIPANTS:

A 5-member family with hereditary NSCLP was recruited by the Cleft Lip and Palate Treatment Center of Shantou University Medical College. The adults and the parents/guardians of the children provided informed consent for genetic testing. Three plastic surgeons with attending physician qualification were invited for clinical examination and diagnosis. Complete physical examinations were performed to exclude other organ malformations, deformities, and to identify CL/P. Considering that 5 subjects from the 1 hereditary family lived in the same conditions, variables were deemed to be identical. This study has been checked by the second affiliated hospital of Shantou university medical ethics examination department (2020–24).

We obtained clinical information and peripheral blood samples from each recruited family member. Five members participated in the study (Figure 1, black dotted rectangle). Three members had complete cleft lip and palate and 2 were normal controls (Normal 1 and Normal 2). The parents were II3 (Cleft 1) and II4 (Cleft 2) and both had unilateral complete cleft lip and palate, while III2 (Cleft 3) was the son with bilateral complete cleft lip and palate. This study was approved by the ethics committees of the Shantou University Medical College.

GENOMIC ANALYSIS:

Whole-genome sequencing and whole-exome sequencing were performed by LC-Bio Co., Ltd. (Hangzhou, China, www.lc-bio.com). Genomic DNA was extracted from patients’ peripheral blood. We used RNA 6000 Nano LabChip Kits (Agilent, CA, USA) to ensure the genomic DNA samples were of high quality, with OD 260/280 ratio 1.8–2.0. The DNA library constructed from human samples using the High Sensitivity DNA Chip Kit (Agilent, CA, USA) and Agilent SureSelect Human All Exon V6 (Agilent, CA, USA) was sequenced and run with the Illumina HiSeq2500/4000 sequence platform.

For the alignment step, sequenced reads were aligned to the reference genome (UCSC Genome Browser hg19) by Burrows-Wheeler Aligner (BWA, Oxford, UK), and then sorted (Samtools v1.3.1) and repeated markers (Picard v1.141) [25]. In the second post-alignment processing step, a local read realignment is performed to correct potential alignment errors around indel. Mapping of reads around indel edge usually results in misaligned bases, creating false-positive SNP calls. Local realignment uses these mismatched bases to determine whether sites should be realigned, and applies a computation-intensive algorithm to determine the most consistent position of reads relative indel and to remove misalignment artifacts. More details are available from LC-Bio Co., Ltd. (Hangzhou, China, www.lc-bio.com).

VARIANT CALLING:

Each cardinality read has an associated quality score corresponding to the probability of sequencing error. Due to systematic biases, the reported quality scores are known to be inaccurate, so it must be recalibrated prior to genotyping. After recalibration, the recalibrated quality score in the output BAM will be closer to the probability of a sequencing error [25].

Variant calls can be generated with the Genome Analysis Toolkit (GATK, https://www.broadinstitute.org/gatk/guide/best-practices), which examine the evidence for variation from reference via Bayesian inference. The Gaussian mixture model is suitable for assigning an accurate confidence score to each putative mutation call and for evaluating new potential variants.

VARIANT ANNOTATION:

Biological functional annotation is a crucial step in finding the links between genetic variation and disease. SnpEff is utilized to add biological information to a set of variants. ANNOVAR software tool and in-house codes were used to annotate all of the mutations with Clinical Variation Data Base (https://www.ncbi.nlm.nih.gov/clinvar/). For further screening, the 1000 Genomes Project (http://www.1000genomes.org/), the Exome Aggregation Consortium (ExAC) Browser, SIFT (http://sift.jcvi.org), and PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) were used to predict mutation function.

SANGER SEQUENCING:

Sanger sequencing was performed by SJ-Bio Co., Ltd. (Guangzhou, China, www.saikabio.com). Premier 3.0 was used for primer design; the primer sequences are listed in Supplementary Table 1. Sanger sequencing products were separated using an ABI 3730 genetic analyzer, and the data were analyzed using Chromas V1.0.0.1.

Results

CLINICAL CHARACTERISTIC:

The subjects were a southern Han Chinese family with familial non-syndromic cleft lip and palate (Figure 1). In this family, II3, II4, and III2 were the probands with NSCLP phenotype. I1, I2, I3, I4, II1, II2, II5, II6, III1, III3, and III4 were members of the pedigree without NSCLP phenotype. In this study, 5 members were enrolled. Three had complete cleft lip and palate and 2 were normal controls. All of them were diagnosed and treated at the Cleft Lip and Palate Treatment Center of Second Affiliated Hospital of Shantou University Medical College. Among these patients, II3 (Figure 2A) and II4 (Figure 2B) were the parents, with unilateral complete cleft lip and palate after surgery, III2 (Figure 2C) was the son, with bilateral complete cleft lip and palate before surgery. Figure 2D shows the clinical feature of the proband 1 year after surgery. According to these clinical features, whole-genomic sequencing and whole-exome sequencing was used to detect possible genetic lesions to explain these abnormal symptoms.

GENOMIC SEQUENCING:

Whole-genome sequencing (WGS) and whole-exome sequencing (WES) were employed to evaluate 5 people of the 2 generations in the NSCLP family. WGS quality summary for the raw data is summarized in Table 1 and Figure 3. The Phred quality score (Q), which is the logarithm of the base error rate P, describes how reliable each sequencing base is. For example, if a base is 99% correct, the mass value is 20 (Q20), and if a base is 99.9% correct, the mass value is 30 (Q30). In the WGS result, the total effective data ranged from 92.56 billion base pairs (Gb) with 31.98 depth (x) to 108.5 billion base pairs (Gb) with 37.49 depth (x). On average, the ratio of valid data was 99.9%. The average ratio of Q20%, Q30%, and GC% were 97.64%, 93.70%, and 41.31%, respectively. WES quality for the raw data is summarized in Table 2. The average total effective data were 12.60 billion base pairs (Gb) with 217.24 depth (x). On average, the ratio of valid data was 98.2%. The average ratio of Q20%, Q30%, and GC% were 98.35%, 95.47%, and 53.44%, respectively. When measured at a minimum depth of 2×, 97.61% of the target region was covered. Likewise, when measured at 10×, 20×, and 30×, 96.04%, 92.15%, and 85.00% of the intended target was covered, respectively (Figure 4). Thus, the depth and coverage should be adequate to reliably detect DNA variants within the majority of the targeted regions.

GENETIC FINDINGS:

The 1000 Genomes Project, the Exome Aggregation Consortium (ExAC) Browser, SIFT, and PolyPhen-2 were used to annotate the structure mutation function. As a result, a total of 7624 mutations were screened, including 1754 mutations in the WGS cohort (23.0%) and 123 mutations in the WES cohort (1.6%). The combination analysis of WGS, WES, and Clinical Variation Data Base showed that a total of 9 variant positions (RESP18, LSR, SPATS2L, ZNF277, IHH, C2orf69, KIR3DL1, MUC3A, and PRKAG3) was overlap between the 3 study cohorts (Figure 5). Among these variant positions, 2 mutation sites (rs2385404 and rs2385405) related to cleft lip and palate in the RESP18 gene were found, and the homozygous mutant was companied with more severe symptoms in NSCLP patients, which were non-synonymous single-nucleotide variations (SNVs) and resulted in functional changes. Eight mutations were excluded because no similar missense variants were found; for example, some are synonymous variants or are less related to lateral or bilateral compared with regulated endocrine-specific protein 18 (RESP18). To verify the DNA sequence variation detected by WES, the criterion standard method Sanger sequencing was employed.

SANGER SEQUENCING:

Using Sanger sequencing, 2 variant positions (rs2385404 and rs2385405) of the RESP18 gene (NM_001007089.4) were detected in all 5 family members. Sanger sequencing validation showed that both rs2385404 and rs2385405 had mutations consistent with the WGS and WES results (Figure 6). Sanger sequencing results indicated that the mutation of RESP18 (rs2385404, c.90G>T, p. Glu30Asp) resulted in the transformation of Glu into Asp in the amino acid sequence of RESP18 protein. Sanger sequencing results indicated that the RESP18 variant (rs2385405, c.157G>A, p. Glu53Lys) resulted in the transformation of Glu into Lys in the amino acid sequence of the RESP18 protein. Both rs2385404 and rs2385405 were missense variants. Therefore, this result confirmed that the RESP18 gene variant was the causal variation in this pedigree.

Discussion

Non-syndromic cleft lip with cleft palate (NSCLP) is one of the most common congenital malformations in China, almost 20% of which are familial cases [21,26]. NSCLP is associated with various factors, and the pathogenesis of NSCLP is not completely clear. It is believed that susceptibility genes are closely related to NSCLP caused by abnormal palatal morphogenesis and tissue fusion during embryonic development [24,27–29]. A variety of approaches have been used to identify key genes contributing to NSCLP through linkage analysis, genomic rearrangements, candidate genes, and genome-wide association studies (GWAS) [23]. Researchers found several variants on SATB2 and IRF6 genes, which are closely related to the causes of NSCLP [17,30,31]. Yu et al performed GWAS on NSCLP and identified 41 SNPs of achieve genome-wide significance within 26 loci on 14 genes (RAD54B, TMEM19, KRT18, WNT9B, GSC/DICER1, PTCH1, RPS26, OFCC1/TFAP2A, TAF1B, FGF10, MSX1, LINC00640, FGFR1, and SPRY1) [2]. Awotoye et al conducted whole-genome sequencing (WGS) analyses in Africa to identify 162 high-confidence protein-altering de novo mutations (DNMs) that were predicted to be pathogenic [22]. The study of Hoebel et al processed whole-exome sequencing (WES) in individuals from independent families and suggested rare variants of 5 candidate genes (ACACB, PTPRS, MIB1, GRHL3, CREBBP), which might contribute to NSCLP risk [13]. Polymerase chain reaction and Sanger sequencing were used to genotype rs7078160 in Vax1, shown by GWAS to be closely related to NSCLP in European populations, and compared mutation through case–control and family-based associations [32]. Therefore, NSCLP may be associated with the mutation of candidate genes.

In the present study, we used whole-genome sequencing (WGS) and whole-exome sequencing (WES) to identify susceptible variants in a south-eastern Han Chinese NSCLP family. In this family, the parents (II3 and II4) had unilateral complete cleft lip and palate, while III2 had bilateral complete cleft lip and palate. Genomic analysis showed that there are 2 mutations in RESP18 gene in the NSCLP family. Coincidentally, patient II3 and II4 had heterozygous mutations and III2 had homozygous mutations. Our results suggest that the NSCLP phenotypic differences and severity were associated with RESP18 gene mutations. The same result was obtained from further Sanger sequencing. As a result, we consider RESP18 is an important gene in NSCLP development and is related to the severity of cleft palate.

Regulated endocrine-specific protein 18(RESP18) is an 18-kilodalton protein which mainly exists in the endoplasmic reticulum (ER) [33,34]. As an important glucocorticoid-reactive protein in the adrenocorticotropic hormone secretion pathway, RESP18 is regulated by physiological and pharmacological stimuli such as dopamine, glucocorticoids, and insulin [35–37]. Furthermore, RESP18 participates in regulation of the limbic system and autonomic function, regulates the growth of cytoskeleton and neurite, and then affects organ development through post-transcriptional O-glycosylation [38–41]. Therefore, RESP18 may be an important link in development of the maxillofacial region, and its abnormal expression has a great impact on the occurrence of cleft lip and palate [42–44].

In conclusion, the incidence and condition of NSCLP are related to RESP18 gene mutation. We speculate that the RESP18 gene plays an important role in the pathogenesis of cleft lip and palate, and this gene may be relevant to etiology prediction for NSCLP families.

A limitation of the present study is that we only investigated 1 NSCLP southern Han Chinese family. We need to expand the sample to verify this results in further study to eliminate sampling errors. In addition, although RESP18 gene mutation has been found in NSCLP patients, it is still necessary to verify the function and pathogenesis of the gene in future studies.

Conclusions

WGS, WES, and Clinical Variation Data Base (ClinVar) were used to detect the gene variant in a southern Han Chinese family with non-syndromic cleft lip with cleft palate. The combined genomic results showed that 2 mutations (rs2385404 and rs2385405) of the RESP18 gene were found in patients in this NSCLP family. Sanger sequencing validation were consistent with the former results. Moreover, we found that RESP18 gene mutation resulting in amino acid and function changes in proteins. As a result, we speculate the RESP18 gene plays an important role in the etiology and pathogenesis of cleft lip and palate.