Identification of a novel pathogenic variant in the MYH3 gene in a five‐generation family with CPSFS1A (Contractures, Pterygia, and Spondylocarpotarsal Fusion Syndrome 1A)

Abstract Background Distal arthrogryposis (DA) is a group of rare Mendelian conditions that demonstrate heterogeneity with respect to genetics and phenotypes. Ten types of DAs, which collectively involve six genes, have been reported. Among them, the MYH3 gene causes several types of arthrogryposis conditions and therefore has a pivotal role in the skeletal and muscle development of the fetus. For this study, we recruited a five‐generation Chinese family with members presenting DA features and phenotypic variability. Further clinical study characterized it as CPSFS1A (Contractures, Pterygia, and Spondylocarpotarsal Fusion Syndrome 1A). Methods Genomic DNA was extracted from eight family members, including one fetus. Whole‐exome sequencing (WES) was then conducted on the proband's sample, followed by Sanger sequencing as validation for each of the participants. In silico analysis was performed. Western blotting (WB) detection and pathological staining were conducted on skeletal muscle tissue of the induced fetus after prenatal diagnosis. Results A novel heterozygous pathogenic variant, namely NM_002470.3: c.3044_3047delinsTCAATTTGTT: p.E1015_D1016delinsVNLF in the MYH3 gene, was identified and shown to be cosegregated with the condition in the subject family. This variant resulted in the replacement of amino‐acid residues E1015 and D1016 by a string of VNLFs. The pregnancy was selectively terminated because the fetus was genetically affected. However, the WB and pathological results did not indicate a significant change in the norm. Conclusions Our study expanded the variant spectrum of CPSFS1A, in addition to which it provided solid evidence for the appropriateness of genetic counseling and pregnancy management for the family. The results may also provide further insight into the molecular mechanism of MYH3.

gene, was identified and shown to be cosegregated with the condition in the subject family. This variant resulted in the replacement of amino-acid residues E1015 and D1016 by a string of VNLFs. The pregnancy was selectively terminated because the fetus was genetically affected. However, the WB and pathological results did not indicate a significant change in the norm. Conclusions: Our study expanded the variant spectrum of CPSFS1A, in addition to which it provided solid evidence for the appropriateness of genetic counseling and pregnancy management for the family. The results may also provide further insight into the molecular mechanism of MYH3.
Next-generation sequencing (NGS), particularly whole-exome sequencing (WES), has been widely used in the diagnosis of fetal and neonatal malformations and prenatal diagnosis, particularly those in which the phenotypes are not distinguishable (Bae et al., 2016;Fu et al., 2018;Monaghan, Leach, Pekarek, Prasad, & Rose, 2020;Todd et al., 2015). In our previous study, the trio-WES strategy has been demonstrated to be effective in elucidating the etiology of fetal skeletal dysplasia and in providing solid evidence for the genetic counseling of affected families (Yang, Shen, et al., 2019).
As part of this study, we investigated the genetic cause of an inherited CPSFS1A and conducted prenatal diagnosis for the suspected fetal involvement, doing so for the first time with respect to a five-generation Chinese family. Additionally, the potential impact of a detected variant on protein function was discussed in the context of the in silico survey and review of the literature.

| Ethical compliance
This study was approved by the Research Ethics Committee of the Shijiazhuang Obstetrics and Gynecology Hospital (approval no. 20200043). Each of the participants signed an informed consent.

| Subjects
A five-generation Chinese family with inherited DA, including one pregnant proband was referred to the Prenatal Diagnosis Center at the Shijiazhuang Obstetrics and Gynecology Hospital. An autosomal dominant genetic pattern was determined based on the family history survey ( Figure 1). The proband and her mother both had distinct facial features, including down-slanted palpebral fissures, ptosis of the eyelids, a long nasal bridge, a small mouth with downturned corners, neck webbing, and congenital limb contractures (Figure 2b-g). Meanwhile, the mental, intellectual, menstruation, and fertility development of them was normal. The x-ray results indicated that the proband had severe scoliosis ( Figure 2a) and carpal fusion and dysmorphia ( Figure 2b). There were twenty-four members in this family, and eight of them suffered from DA, while the other 15 were of normal phenotype. In total, 12 members (Ⅱ:2; Ⅱ:3; Ⅱ:5; Ⅲ:2; Ⅲ:3; Ⅲ:4; Ⅲ:5; Ⅲ:7; Ⅳ:1; Ⅳ:2; Ⅳ:3; V:1) were recruited (detailed information shown in Table  1) and eight were submitted to genetic testing (except for Ⅱ:3; Ⅱ:5; Ⅲ:4; Ⅲ:7).

K E Y W O R D S
CPSFS1A, distal arthrogryposis, MYH3, whole-exome sequencing | 3 of 8 ZHANG et Al.

| Genomic DNA extraction
We collected 3 ml of peripheral blood from each of the eight members, doing so by means of BD Vacutainer™ tubes (BD Biosciences, San Jose, California, USA) for further analysis. Genomic DNA was extracted by means of QIAamp DNA Blood mini-kits (Qiagen Sciences, Inc.) according to the manufacturer's protocol.

| Whole-exome sequencing
Enrichment of the target-region sequences was performed by means of the Agilent Sure Select Human Exon Sequence Capture Kit. The sequencing libraries were quantified using the Illumina DNA Standards and Primer Premix Kit (Kapa Biosystems), massively parallel-sequenced using the Illumina XTEN platform and then massively parallel-sequenced again using the Illumina XTEN. After sequencing and filtering out the low-quality readings, the high-quality readings were compared to the human genome reference sequence [hg19]. The GATK software was used to identify variants (https://softw are.broad insti tute.org/ gatk/). The suspected pathogenic variant was validated by Sanger sequencing using ABI 3730 Automated Sequencer (Applied Biosystems). The mutations were identified by sequence alignment with the NCBI Reference Sequence (NG 011537.1) using Chromas 2.33. Amino-acid sequence conservation analysis of the MYH3 protein was performed using the NCBI BLAST tool (https://blast.ncbi.nlm.nih. gov/Blast.cgi). The pathogenicity of the identified variants was then assessed according to "Standards and Guidelines for the Interpretation of Sequence Variants, Version 2015," issued by the American Association of Medical Genetics and Genomics (Richards et al., 2015).

| Prenatal diagnosis and biochemical analysis
The fetus of the proband was monitored by ultrasonography throughout pregnancy. The thickness of nuchal translucency (NT) was 0.37 cm at 11 +4d gestational weeks (Figure 2h), but no other specific intrauterine phenotype was evident. Amniocentesis was performed at 17 +1d gestational weeks, followed by a combination of genetic detection, including chromosomal karyotyping, chromosome microarray analysis (CMA; Yang, Shen, et al., 2019), and Sanger sequencing.
Subsequent to the receipt of a positive genetic result with respect to the fetus at 21 weeks, the couple decided to terminate the pregnancy at 21 +3d weeks. The appearance of the fetus had no obvious contracture and other abnormalities. Subsequently, we obtained muscle gastrocnemius tissue from the fetus and performed western blotting (WB, with anti-heavy chain myosin/MYH3 antibody, ab124205) along with a control sample from a normal fetus selectively aborted at ~20 gestational weeks, and image data were processed as described in our previous study (Yang et al., 2020). Meanwhile, pathological staining with hematoxylin and eosin (HE), Gomori trichrome (GT), and succinate dehydrogenase (SDH) on it were conducted in accordance with the respective manufacturers' protocols.

| WB and pathological staining
Pathological staining results with all three dyes showed no obvious change in fetal skeletal muscle fibers (Figure 3c; Cuisset et al., 2013). Additionally, the WB result indicated that there was no significant difference in MYH3 expression in the sample from the affected fetus as compared to the control (Figure 3d,e).

| DISCUSSION
The myosin superfamily is a highly conserved versatile group of molecular motors involved in the transport of specific biomolecules, vesicles, and organelles in eukaryotic cells (Syamaladevi, Spudich, & Sowdhamini, 2012). Along with an actin filament, its transport function is achieved by generating physical force from the chemical energy of ATP hydrolysis (Pokrzywa et al., 2015;Syamaladevi et al., 2012). The myosin involved in myofilaments in rhabdomyocytes, smooth muscle, and non-muscle cells is myosin class II, whose heavy chain (MyHC) is encoded by the MYH gene family (Sellers, 2000). The MYH3 gene encodes an embryonic isoform (MyHC-emb) expressed in the fetal musculature, which plays an important role in the formation of normal vertebrae and in the regulation of fetal muscle contraction and relaxation.11 Moreover, it can be mediated by actin-binding protein and connected with the cell membrane to maintain the stability of cytoskeleton (Tajsharghi & Oldfors, 2013  Human MYH3 is located in cytogenetic location 17p13.1, contains 41 exons, and encodes a 1940 aa peptide chain (XP_011522172.1 in NCBI blast). MyHC can be divided into two major domains: the globular amino-terminal head, which is responsible for binding to the myosin light chain and to actin as well as for ATP hydrolysis; and the alpha helical carboxy-terminal rod, which is responsible for the ability of myosin to form filaments (Tajsharghi & Oldfors, 2013). The novel variant identified in this study is located in the coiled-coil domain in the tail, a type of secondary structure composed of two or more alpha helices, which entwines to form a cable structure and serves a mechanical role in forming stiff bundles of fibers (Alva, Syamala Devi, & Sowdhamini, 2008). In previous studies, 50 pathogenic variants in the MYH3 gene associated with various DA disorders have been reported, which were distributed in various structural domains of MyHC-emb as shown in Figure 4 and Table S1 (Bae et al., 2016;Beck et al., 2013;Fu et al., 2018;Hague et al., 2016;Monaghan et al., 2020;Tajsharghi et al., 2008;Todd et al., 2015;Toydemir et al., 2006;Wang, Kong, Zuo, & Kang, 2020;Xu, Kang, & Zhang, 2018). The clinical classification of DA disorders associated with MYH3 variants or the genotype-phenotype correlations, as documented in numerous reports, was unclear and therefore required additional clarification. Overall, however, there are some differences between DA1/2's pathogenic variants and CPSFS1A's distribution within specific domains of MYH3, which may explain the phenotypic differences in these disorders (Figure 4).
Few reports have described the CPSFS1A (Carapito et al., 2016;Chong et al., 2015;Scala et al., 2018) and CPSFS1B (Cameron-Christie et al., 2018) conditions caused by MYH3. However, in this study, we detected a novel heterozygous variant c.3044_3047delinsTCAATTTGTT in MYH3 in a five-generation Chinese family presenting typical features of CPSFS1A. This variant resulted in a change in protein length (but not in reading frames), and the relevant amino acids E1015_D1016 were evolutionarily conservative. This variant meets the evidence level of PM2 (0 frequency in the databases of 1000G (https://www.inter natio nalge nome.org/), gnomAD (http://gnomad.broad insti tute.org/), and Berry Genomics inhouse database), PM4 (resulting in a change in the length of the protein but not in the reading frame), PP1 (cosegregation of variant and phenotype), PP3 (evolutionary conservatism, potential damage to protein), and PP4 (the disease caused by the mutated gene matches the phenotype of this case), so it could be interpreted as "Pathogenic." Presumably, the phenotypic variability of MYH3 related DAs is probably attributable to different polymorphism backgrounds between patients or functional differences in molecules interacting with MyHCemb (Cameron-Christie et al., 2018; In any case, the mechanism behind CPSFS1 needs further delineation in larger groups of study subjects.
The fact that the affected fetus did not show a severe phenotype like any other patient's at the twenty-first gestational week seemed to coincide with the results of biochemical experimentation, suggesting that the manifestations of CPSFS1 might be aggravated dynamically during late pregnancy or perinatal period. However, this inference should be supported by more solid evidence, such as the results of protein function studies.
WES provides an accurate method in the prenatal diagnosis of CPSFS1A. The couple was counseled on management options including chromosomal karyotype、CMA and WES. Prenatal diagnosis indicated that the fetus in this  Table S1.) family carried the same variant as the proband, so the fetus was presumably to be a patient. The parents decided to terminate the pregnancy. Following the termination of the pregnancy, the result of gene analysis of the aborted tissues was consistent with prenatal diagnosis. As can be seen in numerous diagnostic studies (Fu et al., 2018;Monaghan et al., 2020;Wang & Yuan, 2017;Yang, Shen, et al., 2019), on the bases of methodology and time consumption, WES is an effective method for rapid acquisition of pathogenic variants. For any future pregnancy of the proband in this study, the recurrent risk of CPSFS1 condition would be 50%. Given such circumstances, the couple was informed of reproductive options such as prenatal testing and preimplantation genetic diagnosis (PGD).
In conclusion, our study has first detected a novel heterozygous variant c.3044_3047delinsTCAATTTGTT in MYH3 in a five-generation Chinese family presenting typical features of CPSFS1A. Our results expand the variant spectrum of MYH3 CRYGC mutations, which may further be helpful in the molecular diagnosis of CPSFS1A. Additionally, we avoided the birth of the affected fetus by prenatal diagnosis. Additional in-depth research is necessary in order to investigate the pathogenesis of CPSFS1A.