06 July 2024: Clinical Research
Identification of RESP18 Gene Mutations Linked to Hereditary Non-Syndromic Cleft Lip and Palate in a Southern Chinese Family
Xiaoping Zhong12ACEG, Xiaosha Han12BCD, Qihu Xie12BCD, Wanxian Chen12BCD, Deyi Geng12EF, Genghong Guo12EF, Xuefen Chen12BCD, Wancong Zhang12A, Jiasheng Chen12DE, Shijie Tang12ACE*DOI: 10.12659/MSM.944294
Med Sci Monit 2024; 30:e944294
Abstract
BACKGROUND: Non-syndromic cleft lip with cleft palate (NSCLP) is one of the most common congenital birth defects worldwide; it causes lifelong problems and imposes burdens on patients and their families. This study aimed to describe the genomic analysis and identification of de novo regulated endocrine-specific protein 18 (RESP18) rs2385404 and rs2385405 gene polymorphisms associated with NSCLP in a southern Chinese family and to improve prevention, treatment, and prognosis of NSCLP.
MATERIAL AND METHODS: We performed a genome-wide association study (GWAS) to investigate the association of NSCLP phenotype with gene mutation. We investigated a 5-persons NSCLP family to screen the genetic variation of Han nationality in southern Chinese. Whole-genome sequencing (WGS) was used to detect all candidate genetic variants, and whole-exome sequencing (WES) was implemented to further verify mutations. The Clinical Variation Data Base (ClinVar) was employed for screening gene mutations. Finally, Sanger sequencing was applied to verify gene variations.
RESULTS: The combined analysis of WGS, WES, and ClinVar showed that a total of 9 variation positions overlapped among the 3 study cohorts. Sanger sequencing verified Glu amino acid variation in 2 mutation sites (rs2385404, rs2385405) from the RESP18 gene, which caused abnormal RESP18 function and was associated with hereditary NSCLP.
CONCLUSIONS: The combined genomic results showed that 2 mutations (rs2385404 and rs2385405) of the RESP18 gene were related to NSCLP in the family. The RESP18 gene may play an important role in the etiology and pathogenesis of cleft lip and palate.
Keywords: regulated endocrine secretory protein 18, Whole Exome Sequencing, Cleft Lip with or without Cleft Palate, Nonsyndromic, 7, Whole Genome Sequencing, genetic variation
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,
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,
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 (
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
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
Regulated endocrine-specific protein 18(
In conclusion, the incidence and condition of NSCLP are related to
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
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
Figures
Figure 1. Pedigree of the non-syndromic cleft lip with cleft palate (NSCLP) family. II3, II4, and III2: the proband with the phenotype of NSCLP. I1, I2, I3, I4, II1, II2, II5, II6, III1, III3, and III4: members of the pedigree with no NSCLP phenotype. II3, II4, III2, III3, and III4 represent Cleft_1, Cleft_2, Cleft_3, Normal_1, and Normal_2, respectively. The black symbols represent an affected member, and the arrow indicates the proband. Five members participated in the study (Figure 1, black dotted rectangle). Figure 2. Clinical manifestation of the NSCLP pedigree. (A) (II3) and (B) (II4) were parents with unilateral complete cleft lip and palate after surgery, (C) (III2) was the son with bilateral complete cleft lip and palate before surgery. (D) shows the clinical features of the proband 1 year after surgery. Figure 3. Depth and coverage of whole-genomic sequencing (WGS). (A) The sequence depth and cumulative sequence depth of WGS. (B) The coverage and coverage of depth of WGS. II3, II4, III2, III3, and III4 represent Cleft_1, Cleft_2, Cleft_3, Normal_1, and Normal_2, respectively. Figure 4. Depth and coverage of whole-exome sequencing (WES). (A) The sequence depth and cumulative sequence depth of WES. (B) The depth of coverage in WES. (C) The sequence coverage when depth at 2×, 10×, 20×, and 30×. II3, II4, III2, III3 and III4 represent Cleft_1, Cleft_2, Cleft_3, Normal_1, and Normal_2, respectively. Figure 5. Venn diagram of variant position between WGS cohort (filled in green), WES cohort (filled in yellow) and ClinVar cohort. 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%). Nine variant positions overlapped between the 3 study cohorts (RESP18, LSR, SPATS2L, ZNF277, IHH, C2orf69, KIR3DL1, MUC3A, PRKAG3), and 2 of them were rs2385405 and rs2385404 in RESP18 gene. WGS, whole-gonome sequencing. WES – whole-exome sequencing. ChinVar cohort – Clinical Variation Data Base. NCBI showed that these 2 mutation sites could affect the synthesis and function of RESP18 protein. NCBI, National Center for Biotechnology Information. Figure 6. Sanger sequencing confirmed the variants (rs2385404 and rs2385405) in the RESP18 gene sequence (GRCh38.p14 chr2). (A) Sanger sequencing showed 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. (B) 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. Glu – glutamic acid, Asp – aspartic acid, Lys – lysine.References
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