A New Intronic Variant in ECEL1 in Two Patients with Distal Arthrogryposis Type 5D

Distal Arthrogryposis type 5D (DA5D) is characterized by congenital contractures involving the distal joints, short stature, scoliosis, ptosis, astigmatism, and dysmorphic features. It is inherited in an autosomal recessive manner, and it is a result of homozygous or compound heterozygous variants in the ECEL1 gene. Here, we report two patients of Sardinian origin harboring a new intronic homozygous variant in ECEL1 (c.1507-9G>A), which was predicted to affect mRNA splicing by activating a cryptic acceptor site. The frequency of the variant is very low in the general human population, and its presence in our families can be attributed to a founder effect. This study provides an updated review of the known causative mutations of the ECEL1 gene, enriching the allelic spectrum to include the noncoding sequence.


Introduction
Arthrogryposis multiplex congenital (AMC) refers to the congenital joint contractures or movement restrictions involving multiple areas of the body. It occurs in between 1/3000 and 1/5000 live births [1] in association with several distinct disorders, generally as a consequence of fetal akinesia. In fact, the restriction of intrauterine movements and fetal joint mobility may lead to an excessive connective tissue deposition around the joints, resulting in arthrogryposis. Environmental and genetic factors have been reported, spanning from intrauterine space limitations or drug exposure to the fetal brain, and neuromuscular, skeletal, and connective tissues abnormalities [2].
Distal arthrogryposis (DA) is a clinically heterogeneous subgroup of AMC. It is mostly inherited following an autosomal dominant pattern and is characterized by nonprogressive, congenital contractures involving the distal joints, without any primary neurological or muscular disease. The primary diagnostic criteria are camptodactily, hypoplastic or absent flexion creases, overriding fingers, ulnar deviation of the wrist, talipes equinovarus, calcaneovalgus, and vertical talus.
Distal arthrogryposis type 5D (DA5D) is the only DA type inherited in an autosomal recessive fashion, and it is characterized by severe camptodactily of the hands and wrists, milder camptodactily of the toes, limited knee flexion, talus or varus deformity of the ankles, short stature, scoliosis, ptosis, ophtalmoplegia, astigmatism, micrognathia, tongue groove, and cleft palate [3]. It is due to homozygous or compound heterozygous variants in ECEL1. ECEL1 codifies for the endothelin-converting enzyme-like 1, a transmembrane zinc metalloprotease mostly localized in the endoplasmic reticulum with a slight presentation on the cell surface [4]. Even if little is known about its biological function, it is thought to have a role in the development of the neuromuscular junctions during prenatal life [5,6].
Here, we report a new causative intronic variant detected in two patients presenting with the classical DA5D phenotype (Table 1).

Clinical Features
Patient 1 is a 13-year-old female, the first daughter of a nonconsanguineous Caucasian couple who were referred to our genetic counseling service for increased risk of age-related aneuploidies in their ongoing pregnancy.
The patient was born after two miscarriages. She was naturally delivered at term with a birth weight of 2910 g (23rd percentile) and a length of 49 cm (47th percentile). During pregnancy, the mother reported decreased fetal movements. Two weeks after birth, the patient was admitted to the neonatal intensive care unit for feeding difficulties that required nasogastric tube feeding. At that time, the physical examination revealed symmetrical knee and elbow arthrogryposis, pterigium colli, and eyelid ptosis, all features that led to the clinical diagnosis of multiple distal arthrogryposis. At ten months of age, she underwent hip surgery for a congenital hip dislocation. Her language and development milestones were reported to be normal except for walking, which was attained at two years of age. At six years of age, the severe arthrogryposis of her knees required a hamstring elongation.
Presently, the patient has mild dysmorphic features ( Figure 1) with micrognathia, unilateral ptosis, mouth held open and nasal voice, strabismus, and a short neck. Moreover, she has widespread muscle skeletal abnormalities with contractures limiting the extension of the neck, shoulders, wrists, and knees. She also has axyllae pterygia, scoliosis, hyperlordosis, severe camptodactyly, adducted thumbs, clubfoot, and calcaneovalgus deformity.

Chromosomal Microarray Analysis and Exome Sequencing
SNP-array analysis was carried out on Patient 1 and Patient 2, who both originated from a village of 5000 inhabitants on the island of Sardinia in Italy and share one common ancestor ( Figure 2).
Two different copy number variants (CNVs) were detected: a 153 Kb duplication at 16q23.1 in Patient 1 (arr[GRCh37] 16q23.1(74186894_74340431) ×3) and a 202 triplication at 5q32 in Patient 2 (arr[GRCh37] 5q32(146407693_146610003) ×4). Both CNVs were classified as VoUS (variant of unknown significance) and were not considered responsible for the common phenotype as they were present only in one of the two affected family mem- Patient 2 is a 39-year-old female, the second daughter of a Caucasian consanguineous couple (i.e., her grandfather and grandmother are first cousins). Furthermore, Patient 2 is distantly related to Patient 1 (whose great-grandfather is the brother of both of Patient 2's great-grandfathers, Figure 2). Patient 2 was born after a natural delivery with normal growth parameters: 2950 g in weight (26th percentile) and 49 cm in length (47th percentile).

Chromosomal Microarray Analysis and Exome Sequencing
SNP-array analysis was carried out on Patient 1 and Patient 2, who both originated from a village of 5000 inhabitants on the island of Sardinia in Italy and share one common ancestor ( Figure 2).
Two different copy number variants (CNVs) were detected: a 153 Kb duplication at During the pregnancy, decreased and abnormal fetal movements were reported. At ten days, she was admitted to the neonatology unit for the presence of knee, elbow, and ankle contractures that led to the clinical diagnosis of multiple distal arthrogryposis. At the age of one, she had surgery for a correction of bilateral calcaneovalgus clubfoot deformity, and at 18 months, she had surgery for a correction of bilateral hip dislocation. Patient 2's walking was delayed until the age of two and a half. At five years, she received further surgery for an elongation of the tendons of the fingers. Patient 2's language and cognitive development were reported normal. At seven years, scoliosis became evident and was treated conservatively with an orthopedic bust. Recently, she had pneumonia followed by respiratory failure that required tracheostomy.
Presently, the patient has similar but more severe dysmorphic features to Patient 1, with the addition of central tongue atrophy and a furrowed tongue, which is likely responsible for her speech difficulties. Even the pattern of joint and muscle anomalies is similar to Patient 1, with the addition of elbow contractures and decreased muscle build.

Chromosomal Microarray Analysis and Exome Sequencing
SNP-array analysis was carried out on Patient 1 and Patient 2, who both originated from a village of 5000 inhabitants on the island of Sardinia in Italy and share one common ancestor ( Figure 2).
Two different copy number variants (CNVs) were detected: a 153 Kb duplication at 16q23.1 in Patient 1 (arr[GRCh37] 16q23.1(74186894_74340431) ×3) and a 202 triplication at 5q32 in Patient 2 (arr[GRCh37] 5q32(146407693_146610003) ×4). Both CNVs were classified as VoUS (variant of unknown significance) and were not considered responsible for the common phenotype as they were present only in one of the two affected family members. Despite the pedigree of the two families not being suggestive of shared parental inbreeding, the presence of long contiguous stretches of homozygosity (LCSH) in both girls and the common place of origin of the patients' parents drove the hypothesis of a recessive condition. In particular, a common LCSH was detected in the two patients on the long arm of chromosome 2, at 2q36.3q37.3 (arr[GRCh37] 2q36.3 q37.3(229890930_238851442) ×2 hmz), 9 Mb in size and including 16 OMIM disease causing genes. Among the involved genes, ECEL1 appeared to be a strong candidate because it has already been described in association with distal arthrogryposis (DA5D, OMIM # 615065). Considering the ongoing pregnancy of Patient 1's mother and the need for a rapid diagnosis, the possible presence of a homozygous sequence variant within ECEL1 was tested by exome sequencing. A biallelic new intronic variant was detected in intron 8-9: NM_004826: c.1507-9G>A. No other known pathogenic variants were detected elsewhere. Sanger sequencing on the parents of both patients revealed the heterozygous presence of the same variant. The analysis of the mutation on the DNA extracted from the amniotic fluid of the mother of Patient 1 was negative. Her pregnancy resulted in the birth of a healthy boy.

In Silico Analysis
The new variant detected in our two patients is located in intron 8-9 and is predicted to activate a cryptic splice acceptor site (Human Spicing Finder predictive software, http://www.umd.be/HSF/HSF.html (accessed on 19 February 2021)). The probability of the sequence variant affecting the splicing is also supported by its ADA (adaptive boosting) and RF (random forest) score from dbscSNV, which are 1 and 0.99, respectively [7,8]. When used, the new acceptor site would lead to a 7 bases extension of exon 9, causing a frameshift and introducing a premature termination codon (p.Leu503GlyfsX174) in the luminal domain.

Discussion
DA5D is the only type of distal arthrogryposis presenting with an autosomic recessive segregation pattern. It is caused by homozygous or compound heterozygous variants in the ECEL1 gene, which codifies for a member of the neprilysin family of zinc metalloendopeptidases. It is highly expressed in brain and peripheral nerves during early stages of intrauterine development, suggesting that abnormal neuronal development might be involved in the pathogenesis of DA5D. ECEL1 biallelic deficiency has been shown to affect neuromuscular junctions, resulting in poor contractility [5].
Genetic analysis showed in both the presence of the intronic homozygous variant c.1507-9G>A (rs797045548) in ECEL1. The detected variant has a very low frequency in human population (MAF 0.00004), has not been previously reported in homozygous status and is classified as an uncertain significance variant according to ACMG (American College of Medical Genetics and Genomics and the Association for Molecular Pathology) criteria [9]. The same variant was detected in their parents at the heterozygous status. Three of the four carrier parents shared a common ancestor (Figure 2), while the fourth one was not apparently related with the others. Notably, on GnomAD population databases, no variants in position -9 are reported, while there are several variants in nearby positions (-5, -10), indicating the presence of a conserved sequence.
The new variant detected in our two patients is located in intron 8-9. It is predicted to activate a cryptic splice acceptor site leading to a frameshift and a premature stop codon. As this variant maps in C-terminal domain upstream the active site, if expressed, it would yield a truncated protein devoid of its catalytic domain. Alternatively, the presence of a premature stop codon could lead to a non-sense-mediated mRNA decay process. In both these cases, the variant would result in ECEL1 deficiency.
Unfortunately, ECEL1 is not expressed in blood or in other accessible tissues and the predicted effects of the variant on mRNA could not be verified. However, our patients' phenotype and the segregation analysis within the families suggests its causative association with the disease. (Figure 2).
The c.1507-9G>A variant is 5 times more frequent in the panel of 3514 Sardinian sequences (ProgeNIA project) than in human population (MAF 0.0002846 in the Sardinian Pheweb database at https://pheweb.irgb.cnr.it (accessed on 19 February 2021), compared to a MAF 0.00004 in the general population, DbSNP https://www.ncbi.nlm.nih.gov/snp/ (accessed on 19 February 2021)), suggesting that it can be present in our families as a consequence of a founder effect.
Our results expand the genotypic spectrum of mutations in the ECEL1 gene associated with DA5D to include previously unrecognized intronic variants.

Materials and Methods
The clinical data were obtained in accordance with the ethical standards of Brotzu Hospital (Cagliari, Italy) review board. Informed consent for the case report and the publication of the pictures was obtained.
DNA was extracted from the peripheral blood of Patient 1 and Patient 2 by means of QIAsymphony automatic extractor (QIAGENE, www.qiagen.com (accessed on 19 February 2021)).
Chromosomal Microarray Analysis (CMA) and confirmation tests. The CMA for Patient 1 and Patient 2 was performed using the Infinium CytoSNP-850K BeadChip (SNP-array, Illumina, San Diego, CA, USA) according to the manufacturer's protocol. Array scanning data were generated on the Illumina iScan system, and the results were analyzed by Bluefuse Multi 4.4 software.
The confirmation tests on the patients' DNA were performed by Real-Time PCR on LOC101928035 (Patient 1) and PPP2R2B (Patient 2) using a SYBR Green assay (Livak and Schmittgen, 2001).
The variants, identified as pathogenic, were confirmed by Sanger sequencing following a standard protocol (BigDye Terminator v3.1 Cycle Sequencing Kit, Applied Biosystems by Life Technologies). The same Sanger sequencing test was extended to the DNA from the peripheral blood of the patients' parents and the DNA extracted from the amniotic fluid of Patient 1's mother.  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.