Biallelic Missense Mutation in the ECEL1 Underlies Distal Arthrogryposis Type 5 (DA5D)

Distal arthrogryposis (DA) is a heterogeneous sub-group of arthrogryposis multiplex congenita (AMC), mostly characterized by having congenital contractures affecting hands, wrists, feet, and ankles. Distal arthrogryposis is mostly autosomal dominantly inherited, while only one sub-type DA type 5D is inherited in an autosomal recessive manner. Clinically, DA5D is described having knee extension contractures, micrognathia, distal joint contractures, clubfoot, ptosis, contractures (shoulders, elbows, and wrists), and scoliosis. Using whole exome sequencing (WES) followed by Sanger sequencing, we report on a first familial case of DA5D from Pakistani population having a novel biallelic missense mutation (c.158C>A, p.Pro53Leu) in the ECEL1 gene. Our result support that homozygous mutations in ECEL1 causes DA5D and expands the clinical and allelic spectrum of ECEL1 related contracture syndromes.


INTRODUCTION
Arthrogryposis multiplex congenita (AMC) is a heterogeneous disorder mainly characterized by congenital contractures of two or more than two joints. AMC incidence rate is 1/3,000 live births affecting both sexes equally (1,2). Distal arthrogryposis (DA) is defined as the condition with nonprogressive contractures and affecting the distal joints (i.e. hands, feet, wrists, and ankles) with limited proximal joints involvement. DA is caused by pathogenic mutations in the genes encoding the contractile apparatus of myofibers (2). DA overlaps clinical features with the other syndromes having both variable inter and intra-familial clinical features (1,3,4).
Herein, we describe two affected siblings with the clinical features of DA type 5D, in whom a homozygous missense variant has been identified in the ECEL1 gene.

Ethical Approval
We investigated a Pakistani family recruited from remote FR Bannu region of KPK province with distinctive distal arthrogryposis like phenotypes. The authors obtained written informed consent agreements from the affected, patients and other family members (both English and local language) for publication of this case report, photographs and radiographs in compliance with the Helsinki Declaration.

Blood Sampling and DNA Isolation
The family was examined for all the primary and secondary features, pedigree ( Figure 1A) was constructed and blood samples were collected in EDTA tubes (BD, Franklin Lakes, NJ, USA) from all the available affected and normal individuals ( Figure 1A). A detailed interview with the parents/elders highlighted different prenatal, postnatal, and neonatal problems with affected individuals. Subsequently, DNA extraction and quantification was performed using standard methods.

Whole Exome Sequencing
DNA of the affected individual (IV-3) was subjected to whole exome sequencing (WES) using the Illumina platform following standard methods (Illumina, Inc., San Diego, Calif, USA). After exome enrichment, all the reads obtained were aligned in contradiction with the human assembly hg19 (GRCh37) using Burrows-Wheeler Aligner (BWA v 0.7.5) and variants were called using different tools such as PINDEL (v 0.2.4t), SAM tools (v 0.1.18) and Exome Depth (v1.0.0). The final VCF file (variant calling format) was analyzed using BaseSpace (Illumina; https:// basespace.illumina.com/) and variant filtration was performed step-by-step using standard methods as described earlier (11,12).

Sanger Sequencing
The variant identified after WES filtering were bi-directionally Sanger sequenced using standard methods (13). The complete gene sequence was retrieved from Ensembl genome browser (https://asia.ensembl.org/index.html) and primers were designed using primer3 (http://bioinfo.ut.ee/primer3/; primers sequences available on request). Sanger sequencing was performed in all the available affected and unaffected individuals of the family.

In silico Analysis
The primary sequence of ECEL1 was retrieved from Uni Prot protein database (https://www.uniprot.org/uniprot/O95672). Retrieved sequence was used to predict the 3D protein structure using I-TASSER server (15). Hydrogen atoms were added and all the water molecules were removed. Five structures were generated by server which were validated by PROCHECK, ERRAT, and VERIFY 3D. Subsequently, the refined structure were chosen for protein modeling. The three-dimensional model of mutated ECEL1 protein (p.Pro53Leu) was generated by MODELER 9.17. Structural analysis was performed using PyMOL and Chimera (1.12). Analysis of both normal and mutant ECEL1 was performed using ProSA plot, which showed Z scores between 0.5 and 1.0, indicating no significant deviation from the scores determined for proteins of similar size. The CUPSAT, SDM, and mCSM were employed to study and predict the impact of single-point mutations on protein stability.

Clinical Evaluation
The present family has two affected individuals (IV-3 and IV-4) in the fourth generation and five unaffected individuals (III-1, III-2, IV-1, IV-2, and IV-5). Digital images for both affected individuals were obtained, while radiographs of only one affected individual (IV-3) were obtained (Figures 1B-H). Growth parameters at birth were not available. After birth, both the affected individuals had flexion deformities involving the knees, digits and elbows. The ages of affected individual examined were IV-3:16 years and IV-4:18 years. Both the affected individuals showed features such as mild scoliosis, poor weight/height gain, nasal tone speech, mild facial dysmorphism, strabismus, adducted thumbs, ptosis, camptodactyly, stiff wrists, limited knee flexion, progressive weakness of hip flexors, and pes cavus. They suffered from knees, elbows contractures, with flexion contracture of digits. All phalanges had fixed flexion deformity, although they had a firm grip (Figures 1B,D). Feet were thin, stiff, and the toes had restricted movements (Figures 1C,E).

Radiographic Examination
The affected individual (IV-3) showed flexion deformity of digits in both hands and feet. Knees also revealed flexion contracture at the beginning, later after surgery, he was able to bend the knee and walk without support. Foot radiographic examination revealed vertical talus and fixed flexion deformity (contracture) of the toes.
Both sibs performed very well in school and underwent knee surgery, after which they were able to bend the knee and walk without support. Abnormalities such as hearing, dental anomalies, vision, and intellectual disability were not observed at the time of clinical examination. The affected individuals did not reveal any features such as polydactyly, syndactyly, split hand-foot malformation, or some other related skeletal abnormality since the family was initially examined. Detail clinical evaluation of the two affected individuals has been presented in Table 1.

Whole Exome Sequencing
WES using DNA of the single affected individual (IV-3) was performed using standard Illumina platform, and filtering steps were performed as previously described (11,12). The final VCF file (variant calling format) was uploaded and analyzed online using Illumina BaseSpace (Illumina; https://basespace.illumina. com/) software. As pedigree depicted autosomal recessive inheritance (Figure 1A), so first we screened the VCF file for homozygous variants, although compound heterozygous, de novo and heterozygous variants were not ignored. Filtering process revealed a homozygous missense mutation (c.158C>A; p.Pro53Leu) in the ECEL1 gene (

Pathogenicity Index
The pathogenicity index of the identified mutation was determined using different online tools represented in Supplementary Table 2 and the mutation was classified as damaging by most of the tools.

ECEL1 Sanger Sequencing
Sanger sequencing of the identified mutation was performed using standard methods as described previously (13). The mutation (c.158C>A; p.Pro53Leu) segregated perfectly with the disease phenotype (Figures 1I-K).

In silico Analysis
We performed protein structure prediction for the novel homozygous missense mutation (c.158C>A, p.Pro53Leu) in the ECEL1 gene. The residue Proline53 is located in the cytoplasmic domain and missense mutations at this position might affect the secondary structure of the protein (Figures 2D-F). Our analysis revealed that Pro53 interact with Arg49, Ala48, Ser50, Arg57, and Leu85. Substitution of a non-polar Proline to a non-polar Leucine do not introduce new H-bond interactions but Leucine due to its different structure may interact with surrounding amino acid residues differently and these new interactions in turn might potentially disrupt both protein secondary structure and function (Figures 2G,H).
Using CUPSAT, SDM, and mCSM we predicted that pro53Leu mutation would cause a −0.66, −0.586, and −0.63 kcal/mole change in the G, respectively, indicating that the mutation would greatly destabilize the protein structure and hence disrupted function.

DISCUSSION
Mutations in the ECEL1 (NM_004826.3) gene have been previously reported causing distal arthrogryposis phenotypes with no clear genotype-phenotype correlations. Phenotypes such as ptosis, distinct facial features, severe hand camptodactyly, toe and foot contractures, and knee deformities reported earlier (3,(16)(17)(18) were also observed in the present affected individuals. While, Shaheen et al. (19) reported incomitant strabismus in three affected individuals and Ullmann et al. (9) reported the same feature in 1/7 affected individuals, this feature was also observed in both the affected individuals reported here. Moreover, respiratory issues reported earlier were also observed in our affected individuals (9,19). Some of the features such as deep central groove in the tongue, digital webbing, spine issue and cleft Consanguineous marriages have become an alarming issue in a culturally rich and religious country like Pakistan, which increase the likelihood for single gene disorder (autosomal recessive). It has been estimated that almost 82.5% of Pakistani parents are blood relatives (20,21). In such a situation, next generation sequencing technologies such as WES and whole genome sequencing (WGS) is a preferred choice for quick and efficient molecular diagnostic tool in single gene disorders.
Keeping this in mind, we used WES to identify the molecular cause of the disease in both the affected individuals in the family. WES followed by bi-directional Sanger sequencing identified a homozygous missense mutation (c.158C>A; p.Pro53Leu) that segregated perfectly with the disease phenotype. Homology modeling of the normal and mutant ECEL protein revealed substantial changes in the N-terminal of the ECEL1 protein, which might result in the change in the secondary structure of the mutated protein (Figures 2D-H). Structural analysis showed that the mutation (Pro53Leu) would greatly destabilize the protein structure. Amino acid sequence comparison of human ECEL1 protein with other orthologs species showed that the Pro53 residue is highly conserved across different species (Figure 2C).
To date, only 32 mutations have been identified in the ECEL1 gene in patients from different ethical backgrounds causing arthrogryposis multiplex congenital with axoglial defects, contractual syndrome and distal arthrogryposis type 5. These mutations include 13 missense, 6-splice site, 4 nonsense, 4 small deletions, 4 small duplications, 1 regulatory region (Figures 2A,B; Supplementary Table 3). Most mutations in the ECEL1 gene were reported in the exon 2 that corresponds to the transmembrane domain (TM; 59aa-82aa) of the ECEL1 protein. The mutation identified in our patients also lies near to the TM domain, highlighting the importance of this domain in the proper function of ECEL1 protein.
ECEL1 encodes the endothelin-converting enzyme-like 1, a membrane-bound metalloproteinase, which is similar in structure to the endothelin-converting enzyme (ECE) (21). It is highly expressed in neurons within the peripheral and central nervous system and belongs to the neprilysin family of zinc metalloendopeptidases (2, [22][23][24][25]. Ecel1 −/− mice die early due to respiratory failure after birth (22,23). Similarly, homolog knock-in rodent animal model demonstrate a decrease in peripheral motor axons, having a decrease in final branching of the diaphragm, skeletal muscles nerve terminals, and fail to form an adequate number of neuromuscular junctions (NMJs) (22).
To the best of our knowledge, the present study is the first report ECEL1 gene mutation causing rare DA syndrome from the Pakistani population and adds to the limited knowledge of rare DA syndrome related to contractual disorder pathogenesis. The present report further expands the ECEL1 mutation spectrum, which might help in genotype-phenotype correlations and highlights the importance of including DA5D in the differential for multiple pterygium syndromes.

DATA AVAILABILITY
The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

ETHICS STATEMENT
This study was carried out in accordance with Declaration of Helsinki approved by Institutional IRB committee and written informed consent was obtained from all subjects.

AUTHOR CONTRIBUTIONS
MU drafted the manuscript. MU, MY, WA, EM, SN, SK, AA, and AK collected the samples, clinical data, analyzed data, and performed experiments. SA and AH performed the in silico analysis. MU, AA, and AK analyzed the genomic data. MA and FA edited the manuscript. MU, FA, and MA conceived and designed the research study.