1. Clinical features and radiographic parameters of the cohort
The study cohort consisted of 33 unrelated Han Chinese patients. There were 28 cases of occipitalization of the atlas and 5 cases, defined as the control group, were cerebellar tonsillar hernia patients without impaired bony development. There were 6 males (21.4%) and 22 females (78.6%) with a mean age at diagnosis of 45.5 ± 14.3 years. Of the 28 AO patients, a mean of 1.6 levels was congenitally fused (range C0-C7), including two different regions of the cervical spine: C0-C1 and C2-C3. 16 cases (57%) were found to present occipitalized atlas and fused C2-C3 vertebrae, diagnosed as KFS type I according to the Samartzis’s classification[11]. Twelve cases (43%) presented only fusion of occipital bone and atlas.
The AO patients presented with a variety of extraskeletal and intraspinal anomalies. In the cohort in the present study, atlantoaxial dislocation was the most common clinical feature. There were 21 patients (75%) manifesting atlantoaxial dislocation, followed by cerebellar tonsillar hernia (14 patients, 50%), basilar invagination (17 patients, 53.5%), syringomyelia (10 patients, 35%). Almost all the patients presented with neck pain and numbness in their limbs. The detailed clinical features and radiographic parameters are shown in Table 1.
Table 1
Demographic and clinical characteristics of the cohort
Parameter | | AO cohort(n = 28) |
Age in years, mean(range) | | 45,(25–69) |
SEX,n(%) | | |
| Men | 6(21.4%) |
| women | 22(78.6%) |
Level of fusion,n(%) | | |
| C0-C1 | 12 |
| C0-C1,C2-C3 | 16 |
Comorbbidities,n(%) | | |
| atlantoaxial dislocation | 21–75% |
| cerebellar tonsillar hernia | 14 |
| basilzr invagination | 15 |
| syringomyelia | 10 |
| scoliosis | 2 |
Clinical manifestations,n(%) | | |
| dizziness | 8 |
| Numbness/weakness/paresthesia of extremities | 19 |
| limited cervical ROM | 10 |
2. Variants of known KFS-related genes in AO patients
Following the processing of the WES data and its interpretation relating to the variants, filtered rare variants were first checked for their potential to be causative for AO in the reported KFS gene. As a result, 1 variant was found in MYO18B (Table 2) and 1 in MEOX1 (Table 2). In MEOX1, a nonsynonymous SNV (c.G20A:p.S7N) was identified in patient AO751123, a 64 year-old female suffering occipitalization of the atlas (vertebral fusion: C0-C1), tonsillar hernia, basilar invagination, syringomyelia, stiff neck, limited cervical ROM but no rib or other vertebral malformations. The patient underwent posterior atlantoaxial fusion with satisfactory results.
MEOX1 (Mesenchyme Homeobox 1), a protein-coding gene, plays a key role in somitogenesis and is also required for the maintenance of sclerotome polarity and the formation of craniocervical joints. Mohamed et al. found that a frameshift mutation in MEOX1, a 1-bp deletion (94delG) in exon 1, was predicted to result in premature termination in a large consanguineous Saudi family with Klippel-Feil syndrome in 2013[9]. Bayrakli et al. also found Klippel-Feil syndrome resulted from MEOX1 homozygous c.670 G-A transition in a consanguineous Turkish family[12]. Susan et al. reported that the loss of MEOX1 gene function in a mouse model caused defects in the development of the axial skeleton during embryogenesis, especially craniocervical joint malformations, including occipitalization of the atlas[13]. Consistent with the reported phenotype of craniocervical joint malformations in a MEOX1-deficient mouse model, the patient in the present study suffered from occipitalization of the atlas. However, there remain insufficient studies of pathogenic variants of MEOX1 associated with AO due to the limited numbers of samples.
A synonymous SNV (c.T1581G:p.A527A) in MYO18B was identified in patient AO763937, a 33 year-old male with occipitalization of the atlas and vertebral fusion at the C2-C3 level, tonsillar hernia, and syringomyelia. This variant of MYO18B was first reported in a Saudi boy and girl that were unrelated and exhibited KFS with facial dysmorphism and nemaline myopathy[14]. There is still insufficient evidence in terms of its relevance to AO disease.
Table 2
MEOX1 and MYO18B variants and clinical features of patients
Identifier | AO751123 | AO763937 |
Sex/age(years) at diagnosis | F/34 | M/33 |
Mutation | MEOX1 | MYO18B |
Variant type | nonsynonymous SNV | nonsynonymous SNV |
Zygosity | het | het |
Chr_Position | 17_41738883 | 22_261166840 |
Variant nomenclature | c.G20A:p.S7N | c.T1581G:p.A527A |
Exac AF | novel | novel |
GnomeAD AF | novel | novel |
Clinical feature | limited neck ROM, numbness of extremities | dizziness |
Fused levels | C0-C1 | C0-C1 |
Clinical manifestations | occipitalization of atlas,tonsullar hernia, basilzr invagination༌syringomyelia | occipitalization of atlas,tonsullar hernia༌syringomyelia |
3. Variants identified in potential AO-associated genes
Based on rare variant filtering of the AO-associated list of candidate genes (Table S1), the functional prediction tools SIFT, Polyphen-2, Mutation taster, GERP, and CADD were combined to identify candidate genes which were relevant to the etiology of AO[15–20]. Furthermore, we compared the Meta-SVM score of rare coding variants for each candidate gene in the AO group with controls, finding 6 genes with a high Meta-SVM score that were related to vertebral malformation (Table 3)[21].
A reported nonsynonymous SNV (NM_001308022 c.2191T > A:p.S731T) in TNS1 was identified in patient AO757897, a 21 year-old female complaining of extremity adynamia. Fusion of the craniocervical joint at C0-C1 and C2-C3 was identified by radiological scanning, combined with cerebellar tonsillar hernia, basilar invagination, and syringomyelia. TNS1 codes for a protein localized to focal adhesions, regions of the plasma membrane where the cell attaches to the extracellular matrix. This protein crosslinks actin filaments and contains an Src homology 2 (SH2) domain, often found in molecules involved in signal transduction[22]. Mutations of this gene have been reported in Cowden syndrome and Proteus syndrome[23]. Cowden syndrome is a genetic condition characterized by a large head, hamartomatous lesions affecting derivatives of ectodermal, mesodermal, and endodermal layers, macrocephaly, facial trichilemmomas, and scoliosis[24, 25]. The association of TNS1 with AO or KFS has not been previously reported, thus the present findings may represent a novel disease association[26].
A heterozygous variant of c.1052T > A (p.V351D) in the FGFR2 gene was detected at an ExAC allele frequency of 0.000025. This variant has been predicted as a pathogenic mutation using the functional prediction tools MutationTaster, SIFT, and Polyphen-2, with a CADD score of 33 and GERP score of 6.03. FGFR2 was reported to be associated with Pfeiffer and Crouzon syndromes, related mostly to the premature fusion of particular skull bones and cervical spine abnormalities[27–29].
The variant c.619C > T(p.H207Y) in gene TBX2 was detected in patient AO798026. TBX2 is a member of a phylogenetically conserved family of genes that share a common DNA-binding domain, the T-box. T-box genes encode transcription factors that regulate developmental processes[30, 31]. This gene mutation can cause vertebral fusion and scoliosis[32, 33]. Therefore, a significant association of TBX2 with AO may represent an expansion of diseases known to be associated with this gene mutation.
In AGAP1, LRP5, and TONSL, two other rare heterozygous variants were identified in different patients. It has been reported that these genes associate with vertebral or Chiari malformations[34–39]. The related information is displayed in Table 3.
Table 3
Information on rare variants of 6 novel associated genes
Patient | Gene symbol | Variant type | Zygosity | Chr_Pos | Ref transcription | Variant nomenclature | ExAC | GERP++ | CADD | SIFT | Metasvm |
AO757897 | TNS1 | nonsynonymous SNV | het | 2_218712674 | NM_001308022 | c.T2191A:p.S731T | 0.0004 | 4.45 | 24.3 | 0.002 | 1.073 |
AO792018 | FGFR2 | nonsynonymous SNV | het | 10_123263355 | NM_001144914 | c.T1052A:p.V351D | 0.00002473 | 6.03 | 33 | 0 | 0.819 |
AO751123 | AGAP1 | nonsynonymous SNV | het | 2_236617859 | NM_001037131 | c.G200A:p.R67Q | 0.000008236 | 3.88 | 29.1 | 0.043 | 0.762 |
AO752619 | AGAP1 | nonsynonymous SNV | het | 2_237032686 | NM_014914 | c.G2335A:p.V779M | 0.0007 | 4.17 | 22.7 | 0.049 | / |
AO798026 | TBX2 | nonsynonymous SNV | het | 17_59479268 | NM_005994 | c.C619T:p.H207Y | / | 4.92 | 27.9 | 0 | 0.647 |
AO761890 | LRP5 | nonsynonymous SNV | het | 11_68153864 | NM_002335 | c.G1096A:p.V366M | 0.00000829 | 2.89 | 23.9 | 0.018 | 0.532 |
AO749294 | LRP5 | nonsynonymous SNV | het | 11_68125147 | NM_002335 | c.C518T:p.T173M | 0.0004 | 1.67 | 18.39 | 0.108 | 0.283 |
AO749294 | TONSL | nonsynonymous SNV | het | 8_145662165 | NM_013432 | c.C1865T:p.A622V | / | 4.73 | 31 | 0 | 0.503 |
AO745185 | TONSL | nonsynonymous SNV | het | 8_145663874 | NM_013432 | c.G1633A:p.V545I | 0.0003 | 4.21 | 26.7 | 0.056 | -0.82 |
4. GO-BP and KEGG enrichment analysis of candidate genes
Table 4 presents the Fanconi anemia-related genes that were identified in 17 patients. Based on the variants of filtered candidate genes, GO-BP and KEGG enrichment analysis was conducted for the AO cohort and controls, respectively. In GO-BP enrichment analysis, the candidate genes of the AO patient cohort were mostly concentrated in terms related to skeletal system development (p = 1.64 x 10–11), epithelial tube morphogenesis (p = 5.54 x 10–10), bone development (p = 3.42 x 10–11), the smoothened signaling pathway (p = 1.14 x 10–10), and epithelial tube formation (p = 9.03 x 10 − 9). In comparison, the controls were significantly enriched in terms related to skeletal system development (p = 1.18 x 10 − 5), embryonic organ development (p = 0.0002), ear development (p = 4.12 x 10 − 5), positive regulation of the transmembrane receptor protein serine/threonine kinase signaling pathway (p = 5.13 x 10 − 6), and chondrocyte differentiation (p = 1.08 x 10 − 5). Additionally, for KEGG enrichment analysis, the AO patient cohort was significantly enriched in pathways related to Fanconi anemia (p = 3.53 x 10–12), lysine degradation (p = 7.28 x 10 − 5), and homologous recombination (p = 0.0006), while the controls were substantially enriched in pathways related to hepatocellular carcinoma (p = 6.88 x 10 − 5), proteoglycans in cancer (p = 0.0002), and the TGF-beta signaling pathway (p = 4.50 x 10 − 5) (Fig. 1 and Fig. 2).
Table 4
FA related genes of 17 patients
Patient | Gene symbol | Variant nomenclature |
AO748528 | FANCF | c.C638T(p.P213L) |
AO792018 | FANCD2 | c.C3335T(p.A1112V) |
AO744313 | FANCM | c.G2563A(p.D855N) |
AO800756 | FANCM | c.C1663T(p.R555C) |
AO757897 | FANCM | c.G1333A(p.E445K) |
AO751841 | FANCA | c.C2873T(p.A958V) |
AO800891 | FANCA | c.G2080A(p.D694N) |
AO752619 | FANCA | c.G3800A(p.G1267D) |
AO735654 | FANCB | c.C13T(p.Q5X) |
AO745185 | FANCB | c.T869C(p.M290T) |
AO797434 | FAN1 | c.1985_1994del(p.G663Ifs*54) |
AO764048 | FANCI | c.C3056T(p.T1019M) |
AO763937 | FANCI | c.A1111G(p.S371G) |
AO800895 | FANCF | c.C638T(p.P213L) |
AO769366 | FANCG | c.A55G(p.K19E) |
AO800905 | FANCE | c.C589A(p.P197T) |
AO742782 | BRCA2 | c.C3883T(p.Q1295X) |