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A novel FZD6 mutation revealed the cause of cleft lip and/or palate in a Chinese family

Cleft lip and/or palate (CL/P) is a most common craniofacial birth defect which has multifactorial etiology. In our study, we aimed to discover the underlying etiological gene variation in a Chinese family diagnosed as non-syndromic CL/P (NSCL/P). The blood sample of the proband and her parents were detected by whole exome sequencing. The Mendelian inheritance pattern, allele frequency, variation location, function analysis and literature search were applied to filtrate and screen the mutation. Besides, the candidates were confirmed by Sanger sequencing. We meanwhile explored the conservative analysis and protein homology simulation. As a result, a start-lost mutation c.1A > GAtg/Gtg in the Frizzled-6 (FZD6) gene predicting p.Met1 was detected. The variation has not been reported before and was predicted to be harmful. The alteration caused missing of two starting amino acids that are evolutionarily conserved for FZD6 protein. Moreover, the specific structure of the mutant protein obviously changed according to the results of the homologous model. In conclusion, the results suggest c.1A > GAtg/Gtg in the FZD6 (NM_001164616) might be the genetic etiology for non-syndromic CL/P in this pedigree. Furthermore, this finding provided new etiologic information, supplementing the evidence that FZD6 is a strong potential gene for CL/P.


Introduction
Cleft lip and/or palate (CL/P) is a most common craniofacial birth defect. CL/P patients usually require long-term and multidisciplinary therapy to solve the abnormality of swallowing, feeding, speaking, malocclusion and mental health. 1e3 CL/P influences about 1/700 births all over the world with a reported prevalence of 1 in 500 births in Asia, higher than other regions. 4,5 CL/P has a complicated etiology including genetic, environmental, geographic factors and so on. 4,6 The infected patients could be classified as syndrome type (SCL/P) which accounts for approximately 30% and non-syndrome type (NSCL/P) accounting for 70% according to whether they also have other system defects. 7 Genome-wide association and linkage studies (GWASs) have been applied to detect the genetic factors for CL/ P. Numerous researches have been performed to detect the underlying molecular causes. 8e10 However, GWASs identified characterization of a limited proportion of the genetic variation mainly with following limitations. The GWASs usually investigated variants with relatively higher allele frequencies. 11 However, candidate variants occur less frequently in healthy populations. As a result, it is complicated to identify the relationship between NSCL/P and the rare variation at a low allele frequencies. 12 Besides, GWASs could easily lead to false positive and false negative data as it relied primarily on the statistical analysis. Thus, most of the genetic factors underlying NSCL/P is unexplained. 1 Recently the next-generation sequencing technology including the whole-genome and the whole exome sequencing (WES) were applied to identify candidate mutation and further explain etiologic mechanisms of NSCL/P, complementing the spectrum of NSCL/P mutations. 13 Many candidates of causal variants of CL/P were recently reported using WES which was more efficient, complete and specific, and capable of identifying rare or de novo variants. 14e17 This could explain a portion of the heritability of NSCL/P. 18 In our study, WES was used to examine a Chinese family diagnosed with NSCL/P. The genetic variants were filtered and screened to identify the underlying causal factors. Sanger sequencing confirmed the mutation and recessive inheritance pattern in this pedigree. Besides, species conservative analysis, variation function prediction, and protein homologous simulation were meanwhile used to analyze the variant. We aimed to identify potential genetic variants that might cause NSCL/P for this pedigree and to expand the pathogenic spectrum of NSCL/P.

Human subjects
In this study, a Han Chinese family with NSCL/P was recruited at Peking University Hospital of Stomatology. We collected the peripheral blood samples and performed clinical examination for the patient and her parents. This study and related studies were ethically approved by the ethics committee of Peking University Hospital of Stomatology and the ethics approval number is PKUSSIRB-20150012. In addition, each recruited subject agreed and signed a written informed consent.
The clinical examination was performed by two maxillofacial surgeons. To confirm the absence of other organ abnormalities, the patient also underwent a complete physical examination, including eye and vision, ear form, craniofacial development, cardiovascular system, skeletal development and neuromuscular system. The oral examination consisted examination of the location and degree of cleft, morphology of palate, number of teeth, enamel hypoplasia and occlusion relationship. Besides, we collected a detailed history of the exposure of the proband's mother during her pregnancy, such as smoking, alcohol consumption, medical history, drug and supplementary intake, exposure to radiation and poisons and so on. The other family members without phenotype were checked to exclude occult submucous CL/P.
After clinical examination, approximately 4 ml peripheral blood samples were obtained from the proband and her parents. Genome DNA was then obtained using the QIAamp DNA Blood Mini kit (Qiagen, Hilden, Germany) following the instructions.

Whole exome sequencing (WES)
High-throughput sequencing of the DNA library with exon sequence enrichment was performed for each participant. Then the raw sequence data were collected through BGISEQ-500 platform (BGI, Beijing, China). Adapter sequences, undetected and low quality bases were excluded. The rest clean reads were processed using the burrowswheeler Aligner software (Oxford, UK) through variant calls mapped to the human reference genome database (GRCh37/hg19). Mutation analysis including recalibration of base quality score and local rearrangement around indels was conducted to guarantee the accurate variant calling with the Genome Analysis Toolkit (GATK v3.3.0) following the guidelines. Duplicate reads were removed using Picard Tools. Sequencing depth, capture specificity and target coverage were calculated basing on the alignments. Subsequently, single-nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) were varified and screened using HaplotypeCaller (GATK v3.3.0). SnpEff was then used for prediction and annotation.

Candidate variation screening
After annotation was completed, the filtering process was applied to identify the underlying gene variation for the CL/P. Variants were included with the minor allele frequency (MAF) < 0.05 in the ExAC Browser and the public database of 1000 Genomes Project for East Asian population. The Mendelian inheritance pattern was applied to narrow the scope of the candidates. We also selected mutant regions, including splice receptor-site or donor-site mutations, non-synonymous mutations, insertion and deletion. Besides, the literatures were reviewed to identify the potential causal variation. Besides, in silico tools SIFT and PolyPhen-2 were used to predict underlying functional impacts of the variation.

Conservation analysis
The amino acid sequence and the variant mutation position were obtained and conducted for conservative analysis using ClustalX v2.1 and the UniProt Browser.

Homology protein modelling
To get further information of impacts of the variation on the molecular structure of FZD6, an online tool swiss-model was used to build a homologous model.

Description of the pedigree phenotype
We recruited a four-generation Han Chinese family with NSCL/P. The female proband (D1) showed left cleft lip and cleft palate (CLP), and her parents (C1 and C2) had no cleft (Fig. 1). Besides, her cousin uncle, the son of grandpa's sister, exhibited the same phenotype as female proband according to the oral description from the family members but was not available for examination. To eliminate the possibility of syndromic deformities, the proband underwent a general physical examination including craniofacial development, eyes and vision, ear form and hearing, neuromuscular function and cardiovascular system. Therefore, the patient was diagnosed with non-syndromic CLP. Besides, there was no exposure for C1 to smoking, alcohol abuse, diseases, radiation or chemical teratogens during the pregnancy.
A novel FZD6 mutation is identified as potential causal variant in the NSCL/P pedigree In this family, WES was applied for the patient and her parents (D1, C1 and C2) to identify the underlying etiologic variant. Each subject yielded more than 23 Gb raw base reads, and an average sequencing depth was more than 200 times at the target regions. The Q20 of the clean reads for each subject was more than 97% and Q30 was more than 91%, indicating a high sequencing quality. In total, more than 97% of the exon regions were covered at least 20 times (Table 1a).
We respectively identified 125777, 126223 and 125519 variants, which included mononucleotides and polynucleotides, insertion and deletion variants in D1, C1 and C2. There were 74995, 74176 and 69656 heterozygous variants for in D1, C1 and C2. Besides there were 50782, 52047 and 55863 homozygous ones (Table 1b). The sequences were matched and searched to human GRCh37/hg19 reference genome. Both quantity and quality of the sequencing met the criteria for further analysis.
Following the variants annotation, the filtration and screening procedures were applied to confirm the underlying gene mutations (Fig. 2a). The variants with MAF 0.05 in the 1000 Genomes and the ExAC databases were excluded, with 25149, 24863 and 24888 variants respectively left for D1, C1 and C2. Then we mainly focused on the non-synonymous mutation (NSVs), splice acceptor-site or donor-site variants and InDels in coding DNA sequences (CDS). Thus 3147, 2938 and 3065 variants remained. Subsequently, it was considered that the family was likely to exhibit a recessive inheritance pattern, we selected homozygous variants of D1 and heterozygous variants shared by C1 and C2, resulting 32 variants at last. Then bioinformatic tools PolyPhen-2 and SIFT were used to predict the potential functional impacts, obtaining deleterious variants with the moderate and high effects. After that we examined the genes reported in literature with known roles in the pathogenesis of CL/P. Finally we identified c.1A > GAtg/Gtg in the FZD6 (NM_001164616), predicting p.Met1, which led to a start-lost mutation that was evaluated as deleterious.

FZD6 mutation was validated by sanger sequencing
The sanger sequencing was performed to confirm the FZD6 mutation in the proband and her unaffected parents (Fig. 2b). The patient was homozygous and her parents were heterozygous at the variation allele. And this result was consisted with the WES data and conformed to the autosomal recessive inheritance type.

Potentially etiologic impacts of the variation were analyzed by conservation analysis and protein homology modelling
Multiple-sequence alignment of FZD6 showed the residue M was evolutionarily conserved in compared species which were showed in Fig. 2c.
The c.1A > GAtg/Gtg p.Met1 variant affected a highly conserved amino acid which is located in the extracellular region of the FZD6 protein, indicating the variation could be deleterious. Moreover, the homology modelling of FZD6 showed a obvious difference in the mimetic structures between wide-type (Fig. 3a) and mutant proteins (Fig. 3b). For the mutant simulation, the starting two amino acids were missing. Besides, the mutant protein presented an extra a-helix in sequence Val99-Thr102. Moreover, the structures were significantly different for the sequence from Phe105 to Asp124. The starting two amino acids might interact with the sequence from Phe105 to Asp124 in the view of electron cloud. However, the mutant FZD6 did not show this interaction within the biological complex.

Discussion
NSCL/P is a well-known multifactorial disease including genetic and environmental etiology. In clinical examination and history collection, we collected a detailed history of the exposure for the proband's mother during pregnancy, including smoking, alcohol consumption, medical history, drug and supplementary intake, exposure to radiation, poison and chemicals. All of the above factors were excluded. Besides, Family members without phenotype were checked to exclude the possibility of occult submucous CL/P. As a multigenetic disease, NSCL/P could present various genetic patterns including dominant 13,19 and recessive inheritance models. 20 According to the pedigree information of the recruited family for this study, a recessive Mendelian inheritance pattern was thought to be the most suitable.
In this study, we used WES in the patient with left CLP and her unaffected parents in a Han Chinese family. Following the filtering and screening the variants, we detected a novel start-lost variation in the gene of FZD6:  D1  125777 74995  50782  25408 85504 3360  142  11385  10489  86  42  C1  126223 74176  52047  25048 85778 3528  144  11147  10372  86  36  C2  125519 69656  55863  24981 85257 3326  139  11159  10353  94  38 c.1A > GAtg/Gtg predicting p.Met1. This variant has not been reported in current databases and is predicted to be harmful with in silico tools. Besides, sanger sequencing verified the variation and the recessive inheritance pattern of the family. Moreover, conservative analysis and homologous models suggested that the variation could change the wide-type structure and function of the protein and thus is an underlying etiologic mutation. FZD6 is part of the Frizzled gene family, which encodes a set of receptors critical for initiating the wingless-type (WNT) signaling pathway. 21,22 FZD6 encodes three mRNA subtypes, which have been detected in adult and fetal tissues, respectively. 23,24 FZD6 codes for a 706 amino-acid transmembrane protein with seven-pass. 23 Our research identified a start-lost variation in the FZD6 coding area of the proband and predicted that this mutation might be likely harmful to the function of FZD6 protein. Besides, multiple sequence allocated the mutation to a highly conserved amino acid. This amino acid is proved to be highly conserved in the process of evolution. Furthermore, there were significant differences in the local structure of proteins between wildtype and mutant types, suggesting that the amino acid change might be detrimental and could interfere with the WNT signaling pathway.
Craniofacial dysplasia including maxillofacial clefts has been found in WNT knockout mice and zebrafish. 25,26 An association between individual WNT genes and NSCL/P was reported in humans. 27  atypical planar cell polarity (WNT/PCP) pathway. 29 During normal craniofacial development, NCCs migrate to build the facial prominences. 30 Any interference in NCC formation, differentiation, and migration could lead to craniofacial abnormalities. 31 Thus a mutation in FZD6 expression might influence the WNT/PCP pathway and change the migration of neural cell in craniofacial structures and then potentially lead to NSCL/P. Besides, FZD6 is widely expressed in the craniofacial mesenchyme, indicating FZD6 involves the craniofacial development for zebrafish and chick. 32,33 In zebrafish, FZD6 is expressed in pectoral fin buds, phargyngeal arches and the head between 2 and 4 dpf in zebrafish. 34 FZD6 mutants presented underdeveloped Meckel's cartilage and ceratohyals and a smaller palate. 33 Moreover, some relevant studies suggested that alteration of FZD6 expression could result in abnormal craniofacial development. 35,36 And Cvjetkovic et al reported a rare mutation in the 1st intron of FZD6 in a big NSCL/P African-American family and demonstrated loss or excess of fzd6 in zebrafish led to the craniofacial anomalies. 36 Based on the above content, the gene of FZD6 is suggested to be a strong potentially pathogenic gene for the NSCL/P. Certain variation in FZD6 could affect the WNT pathway and in turn result in NSCL/P.
In conclusion, this study identified a novel variation in the coding area of FZD6 using WES for the Chinese pedigree with NSCL/P. The preliminary analysis including functional prediction, variation screening, conservative analysis and protein homology simulation indicated that the mutation c.1A > GAtg/Gtg p.Met1 potentially underlie the cleft in the family. The results further supported that FZD6 mutation played a role in the etiology of NSCL/P.

Authorship statement
Jiuxiang Lin and Feng Chen: study design; Zhibo Zhou: samples collection; Jieni Zhang: experimental studies, data analysis and manuscript preparation; Fengqi Song and Wenjie Zhong: statistical analysis; Mengqi Zhang and Yunfan Zhang: assistance for samples collection.