Whole‐exome sequencing identified first homozygous frameshift variant in the COLEC10 gene in an Iranian patient causing 3MC syndrome type 3

Abstract Background 3MC syndrome type 3 is an autosomal recessive disorder caused by mutations in the COLEC10 gene besides other genes like COLEC11 and MASP1. This disorder is characterized by facial dysmorphism, cleft lip and palate, postnatal growth deficiency, cognitive impairment, hearing loss, craniosynostosis, radioulnar synostosis, genital and vesicorenal anomalies, cardiac anomalies, caudal appendage, and umbilical hernia. Methods In the present study, whole‐exome sequencing was performed in order to identify disease causing variant in an Iranian 7‐year‐old affected girl with craniosynostosis, dolichocephaly, blepharoptosis, clinodactyly of the 5th finger, high myopia, long face, micrognathia, patent ductus arteriosus, downslanted palpebral fissures, telecanthus, and epicanthus inversus. Identified variant confirmation in the patient and segregation analysis in her family were performed using Sanger sequencing method. Results A novel homozygous frameshift deletion variant [NM_006438.5: c.128_129delCA; p.(Thr43AsnfsTer9)] was identified within the COLEC10 gene. Up to now, only three 3MC syndrome patients with mutations in the COLEC10 gene have been reported, and here, we report the fourth patient and the first homozygous frameshift variant. Conclusion Other genes and factors responsible for 3MC syndrome occurrence are remained to be discovered. We believe further investigation of the genes in the lectin complement pathway is needed to be done for the identification of other causes of this disease.


| INTRODUCTION
3MC syndrome is a rare developmental disorder, which accounts for four separate autosomal recessive conditions with considerable overlapping clinical features, previously known as Mingarelli, Malpuech, Michels, and Carnevale syndromes (Gardner et al., 2017). 3MC syndrome is characterized by variable developmental anomalies including craniosynostosis, unilateral or bilateral cleft lip and/ or palate, dysplastic ears, hypertelorism, blepharoptosis, downslanting palpebral fissures, postnatal growth retardation, hearing loss, skeletal anomalies, cognitive impairment, and other less common features, such as cardiac anomalies, hypospadias, and omphalocele (Urquhart et al., 2016). These phenotypes are probably due to defects in the induction, differentiation, and migration of cranial neural crest cells (CNCC) during embryonic development (Rooryck et al., 2011;Urquhart et al., 2016).
This syndrome can be caused by mutations in genes involved in lectin complement pathway comprised of MASP1 (OMIM# 600521), COLEC11 (OMIM# 612502), or COLEC10 (OMIM# 607620) genes (Atik et al., 2015;Munye et al., 2017). Studies have shown that the lectin complement pathway components are integrated in the migration of CNCCs during embryonic development, in order to form several organs and tissues. Proper migration of CNCC lead to the morphogenesis of many facial and cranial tissues, hormone-producing glands (endocrine glands), and parts of the nervous system. After birth, this pathway acts as a part of the innate immune system (Atik et al., 2015;Munye et al., 2017).
Collectins, as other members of the innate immune system, have a carbohydrate recognition domain (CRD) and a collagen-like region in their structures. The more well-known parts of this group are collectin liver 1 (CL-L1, also known as CL-10), and collectin kidney 1 (CL-K1, also known as CL-11), which are encoded by COLEC10 and COLEC11 genes, respectively (Selman & Hansen, 2012). CL-K1 protein is present in the serum, while CL-L1 protein is restricted to the cytosol of hepatocyte cells. By binding to antigens such as bacteria, fungi, and viruses through CRDs and also interacting with MASP proteins, CL-L1, and CL-K1 are able to activate the lectin complement pathway (Munye et al., 2017). Mutations in the COLEC11 gene, located on chromosome 2p25.3, reduce the normal serum level of CL-K1 and possibly disrupt its interaction with MASP and CL-L1. This defect results in 3MC syndrome type 2 (OMIM# 265050), which is clinically indistinguishable from 3MC syndrome type 1 (Selman & Hansen, 2012).
In 2017, Munye and colleagues, identified three compound heterozygous mutations [p.(Arg9Ter), p.(Gly-77GlufsTer66), p.(Cys176Trp)] in COLEC10, as a novel gene in three Pakistani patients with 3MC syndrome (Munye et al., 2017). Through functional studies and gene expression evaluation in the mouse embryo, they demonstrated that mutations in the COLEC10 gene and CL-L1 deficiencies as a result are consistent with 3MC syndrome (Munye et al., 2017). Online Mendelian Inheritance in Men (OMIM) classifies this syndrome as 3MC syndrome type 3 (OMIM# 248340).
To the best of our knowledge, only three 3MC syndrome patients with mutations in the COLEC10 gene have been reported, all of whom were compound heterozygous. In the current study, we described the fourth patient with 3MC syndrome type 3 and the first patient with homozygous frameshift variant in this gene.

| Ethical compliance
Written consent was obtained from parents as legal guardian of the proband. The study was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments (Shrestha & Dunn, 2019).

| Whole-exome sequencing and bioinformatics analysis
The patient's genomic DNA was extracted from her whole blood sample by applying salting-out standard protocol. The Agilent SureSelect Human All Exon V7 Kit was used to enrich human whole exome. The generated library was sequenced on the NovaSeq 6000 platform with the mean coverage of 100X and eight Gb data were generated. An in-house bioinformatics pipeline, Pardis Gene Exomine Technology, was used for bioinformatics analysis including the following steps: 1. Performing quality control (QC) of read length and depth based on GC content (about 50%), Phred value (Phred = 20) to find errors generated during the library preparation and sequencing. We performed this step utilizing the FastP tool which produces an HTML format QC report (Chen, Zhou, Chen & Gu, 2018 . 6. Filtering out was applied on the patient's variants. In this regard, common variants based on population databases including the genome aggregation database (gnomAD v3), the Exome aggregation consortium (ExAC v1) and Iranome (http://www.irano me.ir/), benign annotated ones, considering American college of medical genetics (ACMG) rules, and clinical databases such as the human gene mutation database (HGMD) professional, and ClinVar (https://www.ncbi.nlm. nih.gov/clinv ar/) were excluded from the analysis. Then, based on a similar study (Mohammadi, Heidari, Ashrafi, Mahdieh & Garshasbi, 2021) by paying attention to the patient's clinical information provided to us, the human phenotype ontology (HPO) terms were extracted from the HPO as the most current reliable phenotype-gene association database, in order to find related variants with the disorder. Moreover, OMIM as the most valid gene-disease association database, was used to find the related disease with the altered gene in the patient (Mohammadi et al., 2020).

| Family segregation study and protein analysis
At the first step, genomic DNA of the patient's both parents were extracted. The proband's mother was pregnant at the time of the analysis, so the fetus's DNA was extracted as well. The region including the detected variant in the COLEC10 gene was PCR-amplified considering the standard protocol. At the second step, primers using PrimerQuest tool and UCSC genome browser blat tools (Forward primer: TTCTGGGAGACCCTTTTCTG, Reverse primer: GGGAGGAAACTAGGGAGGAG), were designed and their quality and features were examined by OligoAnalyzer Tool (Rosenbloom et al., 2015).

| Clinical findings
In this study, we described an Iranian 7-year-old affected girl from consanguineous family, who was born with an uneventful pregnancy and delivery at 39th weeks of gestation. Her birth weight and height were normal, and she was admitted to the neonatal intensive care unit (NICU) on day 12 afterbirth due to peripheral cyanosis and respiratory insufficiency. Clinical examination, presented craniosynostosis at birth, therefore, first cranioplasty was performed at 5 months old and second one at 18 months old. At 1 year old, angiography was performed because of congenital heart defect (CHD). At 5 years old, additional clinical manifestations were dolichocephaly, blepharoptosis, blepharophimosis, telecanthus, epicanthus inversus, hypertelorism, downslanting palpebral fissure clinodactyly of the 5th finger, joint laxity, pes planus, loose skin, high myopia, long face, micrognathia, and patent ductus arteriosus (PDA). Height, intelligence, and metabolic tests (including complete blood count (CBC), fasting blood sugar (FBS), liver enzymes activity, low-density lipoprotein (LDL), high-density lipoprotein (HDL) were all in the normal range. The mother had a history of spontaneous abortion, also she had a therapeutic abortion at 17 weeks of pregnancy. The fetus was female and homozygous mutant for the c.128_129delCA variant in the COLEC10 gene, which revealed by prenatal diagnosis (PND), using chorionic villus sampling (CVS).

| Exome results
Prioritization and filtering of the most relevant variants, were manually assessed in order to detect those had the matched clinical outcomes and observed in the family by the following steps: A Variants with minor allele frequency (MAF) >0.01 were filtered based on population databases including gnomAD v3, ExAC v1, and Iranome. Purpose: achieving rare variants in the patient. Remaining variants: 9,311 (Fattahi et al., 2019;Koch, 2020  can be identified as cause of the disease in the proband (Figure 1).

| DISCUSSION
For the first time, Munye et al. (2017), revealed that compound heterozygous pathogenic variants in the COLE10 gene, cause autosomal recessive 3MC syndrome type 3. In a study (Munye et al., 2017), they evaluated 43 families of Asia, Middle Eastern and Europe origin, with 3MC syndrome type 3, using WES and Sanger sequencing methods. Mutation in COLEC11 and MASP1 genes was found in 30 families, including 32 patients. Also in two Pakistani families with three patients, WES identified three mutations with compound heterozygous manner in the COLEC10, as a novel gene, including a Golgi apparatus of murine chondrocyte cell line. Also, COLEC10 expression in the epithelium and mesenchyme of the palate shelf and jaw in embryos was detected by expression analysis during murine craniofacial development. Interestingly, they did not find any mutation in the remained 28 families, which means pathogenic genomic alterations in the MASP1, COLEC11, and COLEC10 genes are responsible for the minority of affected individuals with 3MC syndrome phenotypes, and the other genes and factors responsible for this disease occurrence are remained to be discovered (Munye et al., 2017).
Here, we described a seven-year-old girl from an Iranian consanguine family with craniosynostosis, dolichocephaly, blepharoptosis, blepharophimosis, telecanthus, epicanthus inversus, clinodactyly of the 5th finger, high myopia, long face, micrognathia, patent ductus arteriosus (PDA), and downslanting palpebral fissure. The clinical symptoms in our case seem to be more severe than Pakistani patients reported by Munye and colleagues (Munye et al., 2017). Craniosynostosis, dolichocephaly, high myopia, long Face, micrognathia, PDA, and downslanting palpebral fissure are not reported in any of them (Table 2).
In this regard, a novel homozygous frameshift deletion variant, [NM_006438.5: c.128_129delCA;] was identified within the COLEC10 gene, which creates a shift in the reading frame starting at codon 43. The new reading frame ends in a stop codon 9 positions downstream. This is the first time that a homozygous mutation has been reported in this gene. The COLEC10 gene (OMIM# 607620), which spans 3133 bp, has six exons and only one protein coding transcript (ENST00000332843.3). Highly expressed in liver, moderately in lung, and weakly in artery coronary and adrenal gland, the Collectin-10 protein has one isoform with 277 amino acids (Uniprot ID: Q9Y6Z7-1), which contains a signal peptide (residue 1-27), a collagen-like domain (residue: 53-112), a C-type lectin domain (residue: 155-271), two disulfide bond (between Cys176-Cys270 and Cys248-Cys262), and a glycosylation site (Asn258; Figure 2; Selman & Hansen, 2012).
The COLEC10 and COLEC11 genes encode CL-L1 and CL-K1 proteins, respectively. The polypeptide chains (one of CL-L1 and two of CL-K1) join to form heterocomplexes called CL-LK, which binds to MASP 1/3 and MASP 2 genes in the lectin complement pathway (Henriksen et al., 2013). In this way, CL-LK complex by its chemotactic properties participates in the neural crest cells (NCCs) migration in the early stages of embryogenesis. The proper migration of NCCs is crucial for the formation of bone, cartilage, ganglia, and muscles in the head (Kitazawa et al., 2015;Minoux & Rijli, 2010). Mutations in this complex and specifically the frameshift deletion variant in the COLEC10 gene in the present case, which cause premature stop codon and harbor the gene for nonsense-mediated decay, resulting in not secreting and also not generating the protein product (Rooryck et al., 2011). In this regard, NCCs adhesion and migration are affected and there is an increase in NCCs dispersion. As a result, the affected individuals with 3MC syndrome show craniofacial conditions (Jani et al., 2016). In this study, we could find a frameshift deletion variant in the COLEC10 gene in the patient showing 3MC syndrome phenotypes. We believe further investigation of the genes T A B L E 2 Comparison of clinical features and identified variants in the COLEC10 gene for all 3MC three syndrome described patients in the lectin complement pathway is needed to be done for the identification of other causes of this disease.