A 7-year and 8-month-old girl, with a history of multiple fractures over the past 2 years, was admitted to our hospital due to lower limb deformity after fracture surgery more than 1 year ago. Despite self-reports and physical examinations, the cause could not be determined. Subsequently, the proband underwent whole-exome sequencing analysis.

Peripheral blood samples (2-4 mL) were collected from the patient and her parents. DNA extraction and WES analysis were performed by Berry Genomics (Beijing, China). WES was conducted using the Illumina NovaSeq 6000 platform (San Diego, CA) with PE150 mode, ensuring a sequencing depth of over 20× for more than 99.5% of the target regions. After obtaining raw data, quality control was conducted to remove reads that did not meet the requirements. The Burrows-Wheeler Aligner (BWA) software was used for alignment against the human reference genome sequence (hg19/GRCh37) available at the UCSC Genome Browser (https://genome.ucsc.edu/). Variant calling was performed using the Verita Trekker^® Variants Detection System (Berry Genomics) (Wang et al., 2010) and GATK v3.70. Annotation of variants was carried out using databases and tools such as RefSeq, dbSNP, 1,000 Genomes, ExAC, gnomAD, and the Enliven^® Variants Annotation System (Berry Genomics). Pathogenicity of variants and interpretation of data followed the guidelines of the American College of Medical Genetics and Genomics (ACMG) (Richards et al., 2015).

For predicting the impact of this mutation on splicing at the mRNA level. The pathogenicity of missense variants was predicted using three algorithms: SpliceAI (https://spliceailookup.broadinstitute.org/), Varseak (https://varseak.bio/index.php), and the CBS algorithm.

Two sets of recombinant vectors were constructed: pcMINI-NTRK1-wt/mut and pcDNA3.1-NTRK1-wt/mut. The minigene fragments inserted in the pcMINI-NTRK1-wt/mut vectors consisted of Intron6 (223bp)-Exon7 (133bp)-partial Intron7 (477bp). The minigene fragments inserted in the pcDNA3.1-NTRK1-wt/mut vectors consisted of Exon7 (133bp)-Intron7 (1877bp)-Exon8 (327bp). The construction process involved designing two pairs of nest primers: 10003-F/13460-R and 10214-F/13300-R. Nest PCR (PrimerSTAR MAX DNA Polymerase, R045A, TaKaRa) was performed using normal human DNA as a template. The second-round amplicons from the nest PCR were used as templates for amplification with pcMINI-NTRK1-KpnI-F/pcMINI-NTRK1-EcoRI-R and pcDNA3.1-NTRK1-KpnI-F/pcDNA3.1-NTRK1-EcoRI-R primers, respectively. This resulted in the fragments of pcMINI-NTRK1-wt (853 bp) and pcDNA3.1-NTRK1-wt (2,533 bp). For pcMINI-NTRK1-mut, the left and right halves of the fragment were separately amplified using pcMINI-NTRK1-KpnI-F/NTRK1-mut-R and NTRK1-mut-F/pcMINI-NTRK1-EcoRI-R primers, resulting in fragments of 386 bp and 498 bp, respectively. The left and right halves of the pcMINI-NTRK1-mut fragment were mixed in a 1:1 ratio and used as a template for amplification with pcMINI-NTRK1-KpnI-F and pcMINI-NTRK1-EcoRI-R primers, resulting in a fragment of 853 bp. The amplification steps for the pcDNA3.1-NTRK1-mut fragment were the same as for pcMINI-NTRK1-mut, but the primers used were pcDNA3.1-NTRK1-KpnI-F, NTRK1-mut-R, NTRK1-mut-F, and pcDNA3.1-NTRK1-EcoRI-R (the primers used for constructing the minigene vectors are shown in Supplementary Table S1). The sizes of the pcDNA3.1-NTRK1-mut left, right, and complete fragments were 166 bp, 2,227 bp, and 2,362 bp, respectively. The pcMINI-NTRK1-wt/mut and pcDNA3.1-NTRK1-wt/mut fragments were cloned into the pcMINI and pcDNA3.1 vectors, respectively, using KpnI/EcoRI restriction enzymes. Sequencing verification was performed to confirm the final constructs of the four vectors: pcMINI-NTRK1-wt/mut and pcDNA3.1-NTRK1-wt/mut.

Human embryonic kidney cells (HEK293T; ATCC) and cervical cancer cells (HeLa; ATCC) were transfected with the four vectors separately (pcMINI-NTRK1-wt/mut and pcDNA3.1-NTRK1-wt/mut). The detailed transfection protocol could be referred to in a previous study (Miao et al., 2023). The cells were harvested 48 h post-transfection for further analyses.

Total RNA from the HEK293T and HeLa cells transfected with the recombinant vectors was extracted using TRIzol reagent (RNAiso plus, Takara Bio Inc., Shiga, Japan). The concentration and purity of the extracted RNA samples were assessed. cDNA synthesis was performed using the HifairTM first Strand cDNA Synthesis Kit (11123ES70, YEASEN, Shanghai, China). PCR amplification was carried out using the flanking primers pcDNA3.1-F/pcDNA3.1-R (Supplementary Table S1) targeting the minigene vectors. The amplified gene transcribed bands were detected by agarose gel electrophoresis. The respective PCR bands were recovered and subjected to Sanger sequencing.

A set of expression vectors, p3×Flag-CMV-7.1-wt/mut1/mut2, was constructed. Mut1 and mut2 corresponded to c.826_850del (p.Val276Ser fs*180) and c.838_850del (p.Val280Ser fs*180), respectively, representing two alternative splicing events. The EcoRI-wt-SalI fragment was amplified using a synthetic full-length NTRK1 CDS as a template and primers p3×Flag-CMV-7.1-NTRK1-EcoRI-F/p3×Flag-CMV-7.1-NTRK1-SalI-R. The EcoRI and SalI double-digested fragment was then inserted into the p3×Flag-CMV-7.1 vector, resulting in the p3×Flag-CMV-7.1-wt vector, which was validated by sequencing. Using p3×Flag-CMV-7.1-wt as a template, mut1-1 and mut1-2 fragments were separately amplified with primers p3×Flag-CMV-7.1-NTRK1-EcoRI-F/NTRK1-mut1-R and NTRK1-mut1-F/p3×Flag-CMV-7.1-NTRK1-SalI-R, respectively. The obtained amplification products were mixed in a 1:1 ratio and subjected to a second-round PCR amplification using primers p3×Flag-CMV-7.1-NTRK1-EcoRI-F and p3×Flag-CMV-7.1-NTRK1-SalI-R, resulting in the EcoRI-mut1-SalI fragment. The mut1 fragment was double-digested with EcoRI and SalI, and ligated with the p3×Flag-CMV-7.1 vector, followed by sequencing validation, to generate the p3×Flag-CMV-7.1-mut1 vector. Similarly, the p3×Flag-CMV-7.1-mut2 vector was constructed using different primers (Supplementary Table S2) with the same procedure.

The constructed p3×Flag-CMV-7.1-wt/mut1/mut2 eukaryotic expression vectors were transfected into the HEK293T cell line using the previously described transfection method. After transfection, the cells were incubated for 48 h before collecting the samples for further experiments. The plasmid pCMV6-AC-GFP were co-transfected and subsequently detected to assess transfection efficiency.

To synthesize cDNA, SuperMix for qPCR (gDNA digester plus) (11123ES70, YEASEN, Shanghai, China) kit was used following the previously described method. The cDNA samples were then subjected to qPCR analysis to determine the expression levels of NTRK1 gene corresponding to p3×Flag-CMV-7.1-wt/mut1/mut2 vectors. The qPCR was performed using primers NTRK1-CMV-QPCR-F/NTRK1-CMV-QPCR-R (Supplementary Table S2).

Total protein from 293T cells was extracted using RIPA lysis buffer, and the protein concentration was determined using a BSA assay kit. The proteins were then denatured, and equal amounts of total protein were subjected to SDS-PAGE electrophoresis. Western blotting was performed using an anti-flag antibody, with GFP and GAPDH serving as internal references. This was done to detect the expression levels of wild-type and mutant target proteins of the p3×Flag-CMV-7.1 vectors.

Sanger sequencing was utilized for validating the mutations identified by whole exome sequencing (WES). Simultaneously, it was also employed for verifying the constructs required for vector construction.

The intergroup data labeling was conducted using analysis of variance (ANOVA), and intergroup comparisons were tested using Dunnett’s test. p < 0.05 was considered statistically significant. Graphs were visualized and data were analyzed using GraphPad Prism software.

A 7-year and 8-month-old female pediatric patient, who had previously undergone fracture surgery more than 1 year ago resulting in bilateral lower limb deformities, was admitted to our hospital. Over the course of the past 2 years, the patient experienced multiple fractures, initiating with a right lower limb fracture in July 2020 necessitating surgical intervention at an external medical facility. Subsequent to the surgery, a mild internal rotation deformity of the right knee manifested. In March 2021, a left lower limb fracture occurred, revealing pronounced external rotation of the left lower limb and inversion of the left ankle joint upon postoperative assessment. In January 2022, the patient underwent epiphyseal blocking procedures for the distal end of the left femur, proximal end of the left tibia, and distal end of the left tibia. Regrettably, postoperatively, deformities persisted in both lower limbs. Consequently, the patient underwent left femoral internal rotation osteotomy in our hospital in June 2022, with the potential extension of intramedullary nail fixation, removal of left femoral internal fixation, and removal of left tibial internal fixation. Unfortunately, postoperative complications included incision infection and challenges in wound healing. Clinical evaluation, coupled with narratives from the child and her parents, unveiled that she is the firstborn in the family, demonstrating slightly delayed growth and intellectual development, inadequate development of fingernails and toenails, and a history of recurrent fractures. The patient exhibits reduced sweating and diminished pain sensitivity since childhood. Additionally, she displays a proclivity for self-harm, frequently biting fingers and toenails, leading to multiple bite scars on the arms. The patient was clinically diagnosed as CIPA. Furthermore, irregular dental alignment and poor enamel development are evident. X-ray images revealed partial absence of the distal phalanges in digits 1-4 f the left hand. There was significant limb deformity, with the left knee showing an inward rotation of approximately 40° and the right knee showing an outward rotation of approximately 10°. The hip was dislocated, along with bilateral femoral abnormalities and old surgical scars. The left lower limb was approximately 2 cm longer than the contralateral side (Figure 1).

WES was performed on the proband as well as her parents. The results revealed two variants in NTRK1 in the patient, which were confirmed by Sanger sequencing (Figure 2). The c.851-33T>A variant was inherited from her father and classified as a pathogenic based on the ACMG guidelines (PM3_VeryStrong + PS3 + PM2 + PP1_Moderate). The c.850 + 5G>A variant was inherited from her mother and was classified as a variant of uncertain significance (VUS) as it lacks population frequency data in the ExAC, 1000G, and gnomAD databases (PM2 + PM3 + PP3).

The c.850 + 5G>A variant was located in the fifth bp of intron 7, which was a "class II mutation region" known to affect splicing. SpliceAI, Varseak, and CBS tools were used to predict the impact of this variant on splicing. All three tools suggested that the variant would affect the splicing of the donor site (Table 1). SpliceAI predicted the loss of the original donor site after the variant (score 0.98), and the emergence of a new donor splice site near the variant (score 0.36). CBS predicted that the original donor site would also be lost after the variation. Varseak predicted a reduced score for the original donor site after the variation.

To validate the impact of the c.850 + 5G>A variant on NTRK1 gene pre-mRNA splicing, pcMINI-NTRK1-wt/mut vectors were constructed (Figures 3A, B) and transfected into HeLa and 293T cell lines. In both cell lines, NTRK1-wt showed a single band (a-band) of the expected size (522bp) (Figure 3C). NTRK1-mut also showed a single band (Figure 3C). The splicing pattern of NTRK1-wt (a-band) was ExonA (192bp)-Exon7 (120bp)-ExonB (57bp) (Figure 3D). However, NTRK1-mut band represented two abnormal splicing patterns: b-band showed a 13bp deletion on the right side of Exon7, resulting in ExonA (192bp)-∆ Exon7 (120bp)-ExonB (57bp), while c-band showed a 25bp deletion on the right side of Exon7, resulting in ExonA (192bp)-∆ Exon7 (108bp)-ExonB (57bp) (Figure 3D).

To exclude the influence of the vector, pcDNA3.1-NTRK1-wt/mut vectors were also constructed (Figures 4A, B) and transfected into HeLa and 293T cell lines. The size of the NTRK1-wt (a-band) was consistent with the expected size (647bp) (Figure 4C), and the splicing pattern was Exon7 (133bp)-Exon8 (327bp) (Figure 4D). As expected, the NTRK1-mut band showed two abnormal splicing patterns (Figure 4D). b-band represented a 13bp deletion on the right side of Exon7, resulting in ∆ Exon7 (120bp)-Exon8 (327bp) splicing pattern (Figure 4D). c-band represented a 25bp deletion on the right side of Exon7, resulting in ∆ Exon7 (108bp)-Exon8 (327bp) splicing pattern (Figure 4D).

Therefore, the results from both the pcMINI and pcDNA3.1 minigene constructs demonstrated that the c.850 + 5G>A variant affected normal splicing of NTRK1 mRNA. Both sets of vectors showed consistent results. The 13bp and 25bp deletions on the right side of Exon7 resulted in the production of premature termination codons (PTCs) in Exon11, ultimately leading to the production of truncated proteins of 458aa and 454aa, respectively, denoted as c.838_850del13bp (p.Val280Serfs*180) and c.826_850del25bp (p.Val276Serfs*180).

To investigate the impact of the two aberrant splicing patterns on NTRK1 expression, p3×Flag-CMV-7.1-wt/mut1/mut2 expression vectors were constructed for in vitro expression (Figure 5A). The expression levels of the wild-type and the two mutant transcripts were compared using qPCR. Compared to the wild-type control, the ex-pression of mut1 (c.826_850del,p.Val276Serfs*180) was reduced to 0.83, while the ex-pression of mut2 (c.838_850del,p.Val280Serfs*180) was reduced to 0.44 (Figure 5B).

Similarly, at the protein level, the wild-type protein was predicted to be 90 kDa. However, both mut1 and mut2 variants produced truncated proteins (mut1 predicted size: 53kDa, mut2 predicted size: 53 kDa), and their expression levels were also significantly lower compared to the wild-type (Figure 5C).