Identification and characterization of NF1 and non-NF1 congenital pseudarthrosis of the tibia based on germline NF1 variants: genetic and clinical analysis of 75 patients

Abstract

Background: Congenital pseudarthrosis of the tibia (CPT) is a rare disease. Some patients present neurofibromatosis type 1 (NF1), while some others do not manifest NF1 (non-NF1). The etiology of CPT, particularly non-NF1 CPT, is not well understood. Here we screened germline variants of 75 CPT cases, including 55 NF1 and 20 non-NF1. Clinical data were classified and analyzed based on NF1 gene variations to investigate the genotype-phenotype relations of the two types of patients.

Results: Using whole-exome sequencing and Multiplex Ligation-Dependent Probe Amplification, 44 out of 55 NF1 CPT patients (80.0%) were identified as carrying pathogenic variants of the NF1 gene. Twenty-five variants were novel; 53.5% of variants were de novo, and a higher proportion of their carriers presented bone fractures compared to inherited variant carriers. No NF1 pathogenic variants were found in all 20 non-NF1 patients. Clinical features comparing NF1 CPT to non-NF1 CPT did not show significant differences in bowing or fracture onset, lateralization, tissue pathogenical results, abnormality of the proximal tibial epiphysis, and follow-up tibial union after surgery. A considerably higher proportion of non-NF1 patients have cystic lesion (Crawford type III) and used braces after surgery.

Conclusions: We analyzed a large cohort of non-NF1 and NF1 CPT patients and provided a new perspective for genotype-phenotype features related to germline NF1 variants. Non-NF1 CPT in general had similar clinical features of the tibia as NF1 CPT. Germline NF1 pathogenic variants could differentiate NF1 from non-NF1 CPT but could not explain the CPT heterogeneity of NF1 patients. Our results suggested that non-NF1 CPT was probably not caused by germline NF1 pathogenic variants. In addition to NF1, other genetic variants could also contribute to CPT pathogenesis. Our findings would facilitate the interpretation of NF1 pathogenic variants in CPT genetic counseling.

Keywords: Genomic variation; Genotype; Neurofibromatosis 1; Phenotype; Whole exome sequencing.

Fig. 1

Clinical classification and NF1 pathogenic variants identified in 75 CPT patients. a. The distribution of the number of cases in different onset-age in NF1 CPT patients, non-NF1 CPT patients, NF1⁺ (with NF1 pathogenic variants identified) patients, and NF1⁻ (no NF1 pathogenic variants identified) patients. b. The distribution of the number of cases in four different Crawford types classified when CPT occurred according to age stage. y: year. c. The distribution of the number of NF1⁺ (blue bar) and NF1⁻ (red bar) patients in different clinical classification groups. d. The distribution of exonic functional effect of NF1 pathogenic variants in different Crawford type patients. The majority variants are stop codon (blue bar), InDel (red bar) or splicing (green bar) variants, only three are missense variants (purple bar). e. The inheritance mode distributed in 43 CPT patients (exclude 5B) identified NF1 pathogenic variants. De novo variants show in blue, and inherited variants show in purple which is consist of paternal mode (red bar) and maternal mode (green bar). f. Bar plot of the percentage of rare SNVs and InDels of the NF1 gene in NF1 and non-NF1 CPT patients compared to gnomAD database. Nonsynonymous variants in the coding region of the NF1 gene with MAF < 0.005 were calculated. gnomAD_EAS: East Asian population of gnomAD, gnomAD_all: all population. LoF: loss-of-function associated variants, including stop-gain, splicing changes, startlost, stoplost and InDels

Fig. 2

NF1 pathogenic variants identified by WES in genomic and protein view. NF1 pathogenic variants view from genome to protein secondary structure and domain. Genomic view: showing in the top with black bars marked as the relative position of exons from NF1 gene transcript variant 1 (GenBank: NM_001042492.2). NF1 pathogenic variants map: NF1 pathogenic variants identified in this study are marked at the bottom according to the relative position of protein amino acids. NF1 de novo variants show the amino acid change label in red color; inherited variants show in purple color. Vertical lines show variant position, and Crawford type IV shows in black color, Crawford type II shows in orange color. Protein domains and repeats, homologous superfamilies (InterPro: P21359): Ras GAP domain (1187-1557aa, glaucous bar), CRAL-TRIO lipid-binding domain (1580-1738aa, glaucous bar), Bipartite nuclear localization signal domain (2555-2571aa, green bar), Ploy-Ser domain (1352-1355aa, purple bar), PH-like domain superfamily (1727-1837aa, red bar), Armadillo-type fold superfamily (1849-1886aa, 1920-1984aa, 2200-2420aa and 2613-2676aa, blue bar). Ras GAP and CRAL-TRIO lipid binding domains with PDB structure are marked at the bottom showing amino acid positions and PDB accessions

Fig. 3

X-ray images of four NF1 CPT vs. four non-NF1 CPT patients. Four NF1 CPT patients show at the left column, and four non-NF1 CPT patients show at the right column. Case 71A (NF1) and 60A (non-NF1) are Crawford II type showing cortical thickening and narrowed medullary canal; case 13A (NF1) and 19A (non-NF1) are Crawford III type with cystic lesion; case 47A (NF1) and 70A (non-NF1) were Crawford IV type presenting pseudarthrosis and an abnormality of the proximal tibial epiphysis (APTE); case 18A (NF1) and 16A (non-NF1) are bilateral and are classified as Crawford IV type