The Human Research Ethics Committee from Universidad San Francisco de Quito (No. 2018-127E) approved all experimental protocols. The methods were carried out in accordance with the relevant guidelines and regulations. Lastly, written informed consent to participate was obtained from all of the participants in this study. In case of CIPA patient, a written informed consent to participate was obtained from their parents.
The case of a 9-year-old girl who was born in the community of Piaba (2418 m above sea level, MASL), in Cotacachi, located in the north of Ecuador, is presented. She is diagnosed with CIPA, which begins to be suspected after 72 h of life, where she developed fever of unknown origin; consequently, she was admitted to the hospital, she stayed there for 26 days and she was discharged without specific diagnosis. After 1 month of age, she presented recurring episodes of fever.
When she was 4 months old, she was diagnosed with pneumonia; while she stayed at the hospital, her neurodevelopment was examined by means of the Denver test which provided the following result: unusual for her age. The neurological examination showed generalized hypotonia, active symmetrical movements, incomplete cephalic support, absence of pain sensitivity during peripheral line placement, normal deep tendon reflex, and absence of corneal reflex. Additionally, it was observed that there was a 1-cm dermal ulcer, with regular edges on the proximal and distal phalange of the first finger of the right hand, and lesions in healing process on the second finger (Fig. 1a); consequently, with general anesthesia, a skin biopsy of the sternal region was taken to analyze presence or absence of nerve terminals by immunohistochemical staining for S100 protein (Fig. 1b). The negative results showed superficial and deep dermis with some cutaneous appendices constituted by hair follicles and sweat glands that in multiple cuts have no innervation zones, which can be corroborated since the mother presents absence of perspiration.
When she was 16 months old, she was diagnosed with Riga-Fede disease because she presented ulcerative plaques in the oral mucosa and tongue deformities (Fig. 1a). Furthermore, due to previous ulcers, the patient developed osteomyelitis on the distal phalanges of first and second fingers of the left hand (positive culture for Staphylococcus aureus sensitive to cephalexin). At 2.5 years old, and later at 3 years and 2 months old she presented bilateral corneal ulcers of traumatic origin. At 6 years and 4 months old she suffered a tibia fracture caused by falling (Fig. 1c). At 6 years and 7 months she presented a distal tibial fracture without a determined cause. During the consolidation process a mass was found in the fracture area; a biopsy was carried out and osteochondroma was diagnosed. At 8 years and 1 month she broke her left femur because of a fall. Finally, at 8 years and 5 months old she suffered a subtrochanteric fracture of the right femur, requiring surgery (Fig. 1c). As for the family background, a sister of the patient passed away at 18 months old after developing fever of unknown origin. Figure 1d details the genealogical tree, and consanguinity between relatives of both parents. In addition, the patient presented a normal karyotype 46,XX (Figure S1).
Regarding methodology, peripheral blood samples were extracted from CIPA patient and her parents using FTA buffer (GE Healthcare, UK). Genomic DNA was extracted from whole blood using the PureLink Genomic DNA Kit (Invitrogen, Carlsbad, CA), and purified using Amicon Ultra centrifugal filters (Merck, Darmstadt, Germany). Genomic DNA presented a concentration of 45 ng/μL (mother), 27 ng/μL (father) and 36 ng/μL (patient), using a Qubit 4 (Thermo Scientific, Waltham, MA).
Genotyping was performed through PCR reaction of 54 SNPs located into 14 regions of NTRK1 and Sanger sequencing analysis. Table S1 details features of primers and all 54 variants analyzed. Figures S2a and S2b detail the PCR and the sequencing analysis protocols for all genetic variants, respectively. After carrying out the sequencing analysis of the NTRK1 gene, the patient was observed to have the homozygous mutant genotype T/T of the missense mutation rs80356677 (Asp674Tyr). At the nucleotide level there is a change of the guanine (G) to thymine (T) in DNA. Regarding parents, both have the heterozygous genotype G/T of the mutation rs80356677 (Figure S2c).
Furthermore, a trio analysis of genomic DNA of mother, father and patient was done by using the TruSight One (TSO) Next-Generation Sequencing (NGS) Panel (Illumina, Inc. San Diego, CA, USA), which includes 125,395 probes targeting a 12-Mb region spanning ~ 62,000 target exons of 4811 genes, and sequenced on the Illumina MiSeq platform. Raw sequence reads were processed and aligned against the human NCBI GRCh37 hg19 reference genome assembly using the BWA software. The 80-mer probes target libraries with ~ 500 bp mean fragment sizes and ~ 300 bp insert sizes, enriching a broad footprint of 350–650 based centered symmetrically around the midpoint of the probe. Therefore, in addition to covering the main exon regions, the panels cover exon-flanking regions, which can provide important biological information such as splice sites or regulatory regions. The TSO coverage was ≥20x on 95% of the target regions in the panel, and the TSO full gene list is detailed in the Table S2.
To analyze data generated from targeted sequencing, the BaseSpace Variant Interpreter software (Illumina), the BaseSpace Interpreter (Illumina), Sorting Intolerant From Tolerant (SIFT) (http://sift.bii.a-star.edu.sg/) [35], Polymorphism Phenotyping v2 (PolyPhen-2) (http://genetics.bwh.harvard.edu/pph2/) [36], ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) [33], The Human Gene Mutation Database (http://www.hgmd.cf.ac.uk/ac/index.php) [34], Leiden Open Variation Databases (LOVD) (http://www.lovd.nl/3.0/home) [37], and the Database for Annotation, Visualization and Integrated Discovery (DAVID) (https://david.ncifcrf.gov/) [38] were implemented in the fully detailed bioinformatics pipeline (Fig. 2a).
As results, the total aligned reads was 14,356,459 (father), 17,975,032 (mother) and 20,225,887 (patient). The percentage of reads passing filter that aligned to the reference was 99.9% for all samples. The percentage of targets with coverage greater than 20X was 27.3% for the father, 30.3% for the mother, and 30.4% for the patient with CIPA. Additionally, the analysis of 4811 genes and 18,933 variants was performed in the BaseSpace Variant Interpreter software (Illumina).
After filtering the genomic variants through four steps fully detailed in Fig. 2b, the patient showed a total of 69 pathogenic and/or likely pathogenic variants (Table S3), and 76 variant of uncertain significance (VUS) in 106 genes. The consequence of these 145 variants is fully detailed in Table S4. As results after the DNA genomic analysis, the patient showed a clearly autosomal recessive inheritance pattern with the pathogenic mutation rs763758904 (Arg602*). The other 69 pathogenic and/or likely pathogenic variants in 46 genes were analyzed to better understand the phenotypic heterogeneity of CIPA.
Consequently, the enrichment analysis of GO terms related to biological processes, cellular components and molecular functions was carried on in 46 genes with pathogenic or likely pathogenic variants using DAVID Bioinformatics Resource in order to better understand the phenotypic heterogeneity of the patient [38]. Only 28 of 46 genes were involved in almost one of the categories showed as a heatmap in Fig. 3. The most significant biological processes (BPs) with a Benjamini-Hochberg false discovery rate (FDR) < 0.01 were muscle contraction, maintenance of gastrointestinal epithelium, reverse cholesterol transport, phospholipid translocation, skeletal muscle contraction, cytoskeleton organization, sarcomere organization and visual perception. The most significant cellular components (CCs) with a FDR < 0.01 were late endosome, myosin filament, muscle myosin complex, neuronal cell body, photoreceptor disc membrane, integral component o plasma membrane, high-density lipoprotein particle, myofibril and cell junction. In addition, the most significant molecular functions (MFs) with a FDR < 0.01 were calmodium binding, phospholipid binding, ATP binding, apolipoprotein binding, microfilament motor activity, histone-lysine N-methyltransferase activity and ATPase activity [38] (Fig. 3). The function of all these genes is detailed in the Table S5.
Additionally, a PPi network with a high confidence of 0.7 (p < 0.001) was created using String Database [39, 40]. This network was made up of known and predicted interactions between proteins with pathogenic and/or likely pathogenic variants and the pain matrix proteins proposed by Foulkes et al (2008) [10], and detailed in Figure S3 and Table S6. As results, three proteins (NTRK1, GRM6, and SPTBN2) with pathogenic and/or likely pathogenic variants interact with several proteins of the pain matrix. NTRK1 interacts with NGF (peripheral modulation), BDNF (microglia modulation) and TRPV1 (heat transduction). GRM6 interacts with CNR1, OPRD1, OPRK1, OPRM1 (central modulation), CNR2 (peripheral modulation), BDKRB2, BDKRB1 (damage transduction) and CX3CR1 (microglia modulation). Lastly, SPTBN2 interacts with KCNQ3, KCNQ2 (conduction by K+ channels), SCN1A, SCN11A, SCN10A, SCN8A and SCN9A (conduction by Na+ channels) (Fig. 4).