New Mutation Sites of KIF21A Gene in Chinese Patients with Congenital Fibrosis of the Extraocular Muscles

Background Congenital fibrosis of the extraocular muscles (CFEOM) is a rare hereditary nonprogressive disorder characterized by bilateral ptosis, with severely limited ocular motility. We report a new mutation site of KIF21A gene in a Chinese family with congenital fibrosis of the extraocular muscles type 1 (CFEOM1). Methods We conducted a retrospective study of case series. Standard ocular examinations were performed on 10 persons in a CFEOM1 family. Next-generation sequencing (NGS) was performed to sequence 828 genes related to oculopathy. To detect mutations, we determined the splice sites of the KIF21A gene. Results The five patients with CFEOM1 were found in the ten people of a Chinese family on three generations. A new KIF21A pathogenic SNP mutation site KIF21A-ex20 c.2821C> T (p.Arg941Trp) was identified by compared with human genome reference genes and Sanger sequencing. Conclusions A new KIF21A pathogenic SNP mutation site KIF21A-ex20 c.2821C> T (p.Arg941Trp) may be a major disease‑causing gene for the Chinese family with CFEOM1.


Abstract
Background Congenital fibrosis of the extraocular muscles (CFEOM) is a rare hereditary nonprogressive disorder characterized by bilateral ptosis, with severely limited ocular motility. We report a new mutation site of KIF21A gene in a Chinese family with congenital fibrosis of the extraocular muscles type 1 (CFEOM1).
Methods We conducted a retrospective study of case series. Standard ocular examinations were performed on 10 persons in a CFEOM1 family. Next-generation sequencing (NGS) was performed to sequence 828 genes related to oculopathy. To detect mutations, we determined the splice sites of the KIF21A gene.
Results The five patients with CFEOM1 were found in the ten people of a Chinese family on three generations. A new KIF21A pathogenic SNP mutation site KIF21A-ex20 c.2821C> T (p.Arg941Trp) was identified by compared with human genome reference genes and Sanger sequencing.
Conclusions A new KIF21A pathogenic SNP mutation site KIF21A-ex20 c.2821C> T (p.Arg941Trp) may be a major disease-causing gene for the Chinese family with CFEOM1.

Background
Congenital fibrosis of the extraocular muscles (CFEOM) is a rare autosomal dominant hereditary eye disease, which has been reported at a rate of 1/230000 [1] . CFEOM's main clinical manifestation is a hereditary ocular motility disorder characterized by congenital ptosis and non-progressive restrictive ophthalmoplegia, which can be accompanied by abnormalities in other systems throughout the body.
The present study identified a new heterozygous missense mutation in KIF21A p.Arg941Trp by highthroughput second-generation sequencing and Sanger sequencing in one Chinese family with CFEOM1.

Methods
In this retrospective study we reviewed the medical records files of patients with CFEOM1 between January 2018 and July 2019 at Aier Eye Hospital (JinZhong and TaiYuan). Ten people in a family of three generations had was inquired about the current and previous medical history, family history in details, and was subjected to comprehensive ophthalmic examinations including best -corrected vision acuity, Slit lamp and ophthalmoscope. The study was performed, according to the principles of the Declaration of Helsinki. CFEOM1 diagnostic criteria: congenital ptosis and limited eye movement, especially upward rotation restriction. In primary gaze, the eye position is downward. Its upward rotation could not pass the middle line and the horizontal movement was severely limited.
The eye chip from Hangzhou Dean Medical Test Center was used to perform Next Generation sequencing (NGS) technology on the target area for gene sequencing of samples (two patients with

CFEOM1)
The DNA library is first created and it is tested by Agilent 2100 Bioanalyzer and BMG for fragment size and concentration. After passing the test, the library with different data volumes will be pooled and quantified. Then, the pooled library will be cyclized single-stranded. After the cyclized library was used to generate DNA nano balls (DNB), the high-throughput sequencer MGISEQ-2000 was used to perform continuous bidirectional sequencing and read the raw sequencing data. Detection quality control indicators average coverage is 99.29 (20X), and average sequencing depth is 129.33.

Clinical Findings
Family and medical histories were carefully reviewed. In one family of ten in three generations, five patients with typical CFEOM1 (Fig. 1). The pattern of the onset DFEOM1 in this family showed autosomal dominant inheritance. In this family, proband and his mother, sister, uncle, grandmother suffered from CFEOM1. All patients were congenital severe involvement. Figure 2 showed the appearance of the eyes of the mother and child (Ⅱ3 and Ⅲ3 patients): they shared the typical clinical features of CFEOM1 .These features included bilateral congenital non-progressive ptosis, restricted extraocular muscle paralysis, severely impaired vertical motility with an inability to raise either eye above the horizontal midline and variable impaired horizontal motility, fixation of the eyeballs to the 4 lateral subluxation in primary gaze, chin-up position of the head and absence of Bells phenomenon.
Its upward rotation could not pass the middle line and the horizontal movement was severely limited (Fig. 2). The forced induction test demonstrated marked restriction in passive elevation of the globes in all affected patient.

Genetic Test Results
High-throughput sequencing technology was performed on 828 genes related to hereditary ophthalmopathy in this study. Combined with the clinical symptoms of the patients, one pathogenic mutation, KIF21A-ex20 c.2821C > T (p.Arg941Trp) was detected and heterozygous missense mutated (Fig. 3). ClinVar database included and annotated this mutation as a disease-causing gene. The conserved sequencing of its protein by SIFT and Polyphen-2 software showed that the mutation site was detected in both patients, so hit was determined to be a pathogenic mutation. The inheritance of KIF21A gene-related congenital extraocular muscle fibrosis is autosomal dominant inheritance, which is presumed to be the main cause of disease in patients. In this study, genetic analysis documented the presence of a heterozygous mutation, c.2821C > T (p.Arg941Trp), in the KIF21A gene within the two patients of the one family. This mutation was not observed in the unaffected family members or in the unrelated control subjects from the same population.

Discussion
Patients with congenital fibrosis of the extraocular muscles syndrome have non-progressive eye movement disorders, accompanied with various ptosis, eye position fixed on the lateral-downward, and severe bilateral eye movement restrictions. The CFEOM is divided into four types according to its genetic mutations and genetic types, of which CFEOM1 is a rare autosomal dominant genetic disease, and its typical clinical manifestation is congenital non-progressive blepharoptosis, fixation of eyeball at lower index of at least 10 °, the upward movement cannot exceed the midline and the horizontal motion is restricted to varying degrees. CFEOM2 is an autosomal recessive inheritance. Patients with CFEOM2 have congenital blepharotosis, partial or complete fixation of the eyeball in the oblique position, and lack of free eye movement. CFEOM3 is an incomplete autosomal dominant inheritance, and its clinical phenotype varies. The forth type is a typical. In Our study, all five patients are typical 5 CFEOM1. .
In 2003, Uvama et al. [4] reported that KIF21A gene mutation is closely related to CFEOM1, and think that CFEOM1 was caused by KIF21A encoded kinesin missense mutations. Subsequently, researchers found more than 10 new mutation sites in the KIF21A gene from patients with CFEOM1 families in different nationalities and regions. The most popular one was 2860C > G (R954W) in exon 21, with a mutation rate of 72%, followed by the 2861G > A (R954Q) mutation in exon 21 with a mutation rate of 13% and a mutation rate of up to 85%. Tiab [5] studied the R954W mutation in all patients by studying three CFEOM1 families from France, Switzerland, and Turkey and one sporadic case from Iran. Chen [7] found two families of CFEOM1 and CFEOM3 with a heterozygous KIF21A c.2860 C > T mutation In our study, KIF21A-ex20 c.2821C > T (p.Arg941Trp) is detected as a heterozygous missense mutation by studying pedigree. The KIF21A gene was included in the ClinVar database and annotated as a disease-causing gene. Functional tests showed that the impaired function of the gene resulted CFEOM1 phenotype; and other mutations at the same site of the gene (R941L, R941Q, R954W) were reported in patients with CFEOM [6][7][8] . KIF21A-ex20 c.2821C > T (p.Arg941Trp) found in this study belongs to heterozygous missense mutation, and the frequency of carrying this mutation in East Asian populations has not been reported. The conserved sequencing of its protein by SIFT and Polyphen-2 software showed that the mutation site was detected in both patients, so it was determined to be a pathogenic mutation. The inheritance of KIF21A gene-related congenital extraocular muscle fibrosis is autosomal dominant inheritance, which is presumed to be the main cause of disease in patients.
In conclusion, KIF21A-ex20 c.2821C > T (p.Arg941Trp) is detected as a heterozygous missense mutation by studying pedigree, is a new mutation sites of KIF21A gene in Chinese patients with congenital fibrosis of the extraocular muscles.

Conclusion
We conducted a retrospective study of case series. The five patients with CFEOM1 were found in the ten people of a Chinese family on three generations. We found A new KIF21A pathogenic SNP mutation site KIF21A-ex20 c.2821C > T (p.Arg941Trp) in two patients of a family with CFEOM1. The 6 mutation may be a major disease-causing gene for the Chinese family with CFEOM1. Abbreviations CFEOM: Congenital fibrosis of the extraocular muscles; NGS: Next Generation sequencing; Declarations Figure 2 The appearance of the eyes of the mother (Top, II 3 case) and child (Bottom, III 3 case): drooping of the upper eyelids of both eyes, restricted extraocular muscle paralysis, fixation of the eyeballs to the lateral subluxation, but no upward movement of the midline, limited horizontal movement, compensating head position, and jaw lifting.
11 Figure 3 Sequencing and mutation sites of exon 21 of the patient's KIF21A gene in the II 3 and III3 affected members of the family. The position of the mutation is marked by an arrow and the corresponding wild-type sequence is shown as a control.