Atypical Presentation of Congenital Insensitivity to Pain With Anhidrosis Leading to Diagnostic Odyssey

ABSTRACT

Background

Congenital insensitivity to pain with anhidrosis (CIPA) (OMIM 256800) is a rare autosomal‐recessive condition, also known as hereditary sensory and autonomic neuropathy type IV (HSAN‐IV). The most commonly reported features include anhidrosis, intellectual disability, self‐mutilation, febrile episodes, impaired temperature perception, recurrent infections and/or autonomic nervous system impairment. Major joint destruction and joint deformity known as Charcot (neuropathic) joints are also seen in CIPA patients attributed to insensitivity to joint pain.

Methods

We present a case of a 46‐year‐old female affected with CIPA with a known NTRK1 variant and previously unidentified variant. Minigene reporter constructs were generated encompassing the exon 8 to exon 13 of the NTRK1 gene using the reference sequence and one harboring c.1483 + 5G > A variant identified in our proband. Minigene constructs were transfected into HEK293T cells, and the transcript was analysed for splicing to evaluate the effect of this variant in splicing.

Results

The patient (46‐year‐old female) exhibited right ankle joint deformity around 5 years of age. Patient also experienced lumbar compression and knee damage in adulthood. She had undergone a significant number of evaluations without clear diagnosis. Her presentation lacked many of the common clinical presentations of CIPA, and therefore, the focus of her evaluation was directed towards her unexplained joint deformities. Exome sequencing revealed a known pathogenic variant in NTRK1 (c.851 ‐ 33T > A:p.? [Intron 7]) and a novel NTRK1 variant (c.1483 + 5G > A:p.? [Intron 11]), which was later re‐classified as likely pathogenic. The patient was started on a biologic disease‐modifying anti‐rheumatic medication (bDMARD) due to a possible inflammatory etiology of her joint deformity. Molecular diagnosis allowed for modification of her treatment and surveillance strategies. Our minigene splicing assay demonstrated that the presence of the c.1483 + 5G > A variant has a negative effect on splicing, supporting the pathogenicity of this novel variant.

Extensive joint destruction from the patient with compound heterozygous variants in the NTRK1 gene with diagnosis of congenital insensitivity to pain with anhidrosis (CIPA).

1. Introduction

Congenital insensitivity to pain with anhidrosis (CIPA) (OMIM 256800) is a rare autosomal‐recessive condition also known as hereditary sensory and autonomic neuropathy type IV (HSAN‐IV) (Indo 1993). It is due to pathogenic variants in the neurotrophic tyrosine kinase receptor type I (NTRK1) gene (Echaniz‐Laguna et al. 2021; Hashimoto et al. 2022; Indo 1993; Li et al. 2021). Pathogenic variants in the NTRK1 gene result in aberrant extracellular nerve growth factor (NGF)‐dependent neurons composed of mainly unmyelinated C‐fibers and sympathetic postganglionic neurons (Echaniz‐Laguna et al. 2021; Lopez‐Cortes et al. 2020). This can present in patients with NTRK1 pathogenic variants as complete insensitivity to superficial and deep painful stimuli, absence of sweating and recurrent episodes of fever (Echaniz‐Laguna et al. 2021; Indo 1993; Lopez‐Cortes et al. 2020). These patients are also prone to recurrent infections, and most common pathogen reported has been Staphylococcus aureus (Fruchtman, Perry, and Levy 2013).

Major joint destruction and joint deformity known as Charcot (neuropathic) joints can often be seen in CIPA patients early on in life, which is attributed to insensitivity to joint pain (Echaniz‐Laguna et al. 2021; Hashimoto et al. 2022; Indo 1993; Kayani et al. 2017). Other commonly reported features include, but are not limited to, anhidrosis, intellectual disability, self‐mutilation, febrile episodes, impaired temperature perception and/or autonomic nervous system impairment (Echaniz‐Laguna et al. 2021; Indo 1993; Indo 2018; Lopez‐Cortes et al. 2020). Here, we report a 46‐year‐old female who was referred to the adult genetics clinic with unexplained right ankle swelling and deformity that started around 5 years of age, with subsequent compression fracture of her spine in her 30s and left knee swelling and pain diagnosed at 44 years of age. Molecular genetic testing via exome sequencing identified one pathogenic variant in NTRK1 inherited from her mother and a previously unreported variant of uncertain significance, later re‐classified as likely pathogenic, in NTRK1 inherited from her father.

This case report details the diagnostic odyssey of a 46‐year‐old woman with an atypical presentation of CIPA.

2. Materials and Methods

2.1. Cloning of the Minigenes

Sequences spanning from exons 8 through 13 were synthesised flanked by XbaI and EcoRI sites by Genscript (Piscataway, NJ) and cloned into their pUC19 vector. Minigene with our variant of interest was also synthesised and cloned into the pUC19 vector. Insert was excised by XbaI and EcoRI (NEB Biolabs) and ligated with the FLAG‐HA‐pCDNA3.1 vector (kind gift of Dr. Donald Small) digested with XbaI and EcoRI sites using the DNA Ligation Kit, Version 2.1 (Takara), as described by the manufacturer.

2.2. Cell Culture and Transfection

293T cells were cultured as a monolayer at 37°C (95% air, 5% CO₂) in DMEM supplemented with 10% fetal calf serum and penicillin streptomycin. The cells were plated 1 day before transfection onto six‐well plates at a density of approximately 5 × 10⁵ cells/well.

Transfection was performed using FuGENE HD transfection reagent, according to the manufacturer's instructions, using 1 μg/well of plasmid DNA and 4 μL of FuGENE HD transfection reagent/well. Empty vector, reference, and variant constructs were transiently transfected into 293T cells alongside each other. Cells were harvested 48 h post‐transfection.

2.3. Reverse Transcription–Polymerase Chain Reaction (RT‐PCR) Analysis

Total RNAs were isolated from transfected cells using the TRIZol Reagent (Invitrogen), according to the manufacturer's instructions. Total RNAs were quantified by NanoDrop (Thermo Scientifiec).

cDNAs were synthesised from one microgram of each total RNA sample using high‐capacity cDNA reverse transcription kit (Thermo Fisher Scientific), as described by the manufacturer. Synthesised cDNA was diluted 10‐fold in nuclease‐free water.

PCR amplifications were performed from two microliters of the first‐strand cDNA reaction mixture using primers P1 and P2 located, respectively, in exons 8 and exon 12 of the minigene (Figure 3A). PCR amplifications using primers for the Lamin gene were used as internal control for amplification.

FIGURE 3.

Minigene reporter assay developed for detection of the splicing defect for the novel NTRK1 splice variant identified in our proband. (A) Schematic diagram of the minigene expression vector (not drawn to scale). (B) Representative electrophoresis gels from the minigene splicing assay for detection of the splicing variant.

PCR reaction was performed in a 20 μL volume, using GoTaq Master Mixes (Promega), as described by the manufacturer. PCRs are performed as follows: initial denaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 10 s, 60°C for 20 s and 72°C for 50 s, with a final elongation step at 72°C for 10 min.

RT‐PCR products are separated, alongside the DNA size marker, by electrophoresis through an agarose gel (1%) containing ethidium bromide (0.5 μg/mL) in 1× TAE buffer and visualised by the exposure to ultraviolet light.

3. Case Presentation

A 46‐year‐old woman presented for medical genetics evaluation of idiopathic Charcot joint and longstanding history of right ankle pain and deformity with onset at age 5 (Figure 1A,B). It was reported that she was born at term with normal birth parameters, no developmental delays or intellectual disability. She has completed an undergraduate degree, and she remains completely independent. She has had extensive evaluation since her initial onset of right ankle deformity at multiple children's hospitals without diagnosis.

FIGURE 1.

Representative photographs and X‐ray images of the patient's Charcot joints. (A) Photograph of patient's right ankle (left) and X‐ray of the same ankle (right). (B) Photograph of patient's left knee (left) and X‐ray of the same knee (right). (C) X‐rays of the patient's lumbar compression fracture.

The patient recalls crying after vaccinations and falls due to pain in childhood. She is able to sense pain today with vaccinations or when she stubs her toes by accident. She senses pain during dental procedures, especially when receiving local anaesthesia injections. There were no reports of self‐mutilating injuries to her lips, tongue or fingertips. Due to her astigmatism, she receives regular ophthalmologic evaluations but does not recall history of keratitis or other eye pathology. She has not had Staphylococcus aureus infections to her knowledge.

In her 30's, the patient reports that she lost balance and heard a cracking sound in her back. No significant painful sensation was reported during this incident. Imaging studies were performed on her spine including X‐ray, computed tomography (CT) and magnetic resonance imaging (MRI), which identified a lesion that appeared to be diskitis at L3‐L4 and compression fracture of her lumbar spine (Figure 1C). Initial working diagnosis was of infectious nature, but no infectious etiology was identified. She has kyphosis in the lower back where she should otherwise have had a lordosis.

Patient developed swelling and pain in her left knee when she was 44 years old without any known inciting events (Figure 1B). She was found to have a large joint effusion and destructive changes in her knee after orthopaedic evaluation. She did not complain of pain at the time of her diagnosis. She was subsequently evaluated by orthopaedic oncology and arthroscopic biopsy was performed, which showed chronic reactive inflammation without a clear etiology. She has had an extensive infectious evaluation of her left knee, including two aspirations without identification of infectious agents.

The patient was also evaluated by neurology to determine whether her symptoms were due to an underlying neurological etiology. A notable finding on her neurological evaluation was absent vibration sensation at bilateral toes with decreased sensation in bilateral medial malleoli, bilateral patella and distal interphalangeal joint (DIP) of index fingers. Her electromyogram was notable for mild bilateral median mononeuropathy at the wrist and left ulnar mononeuropathy at the elbow. These findings were consistent with early mild sensorimotor polyneuropathy, although they were not severe enough to explain her joint deformities. Quantitative sensory testing was not completed during her neurological evaluation. Due to her joint pathology, the patient had multiple rheumatological evaluations over the course of her medical journey. Her rheumatological laboratory studies were consistent with elevated inflammatory markers (Table 1 and S1). Given these findings and concerns for inflammatory monoarthritis, she was started on empiric treatment with adalimumab every other week. Although her serologic inflammatory markers (C‐reactive protein and sedimentation rate) improved (Table 1), she did not gain improvement in knee discomfort, function or swelling.

TABLE 1.

Inflammatory markers obtained during patient's evaluations which were notable for mild elevations ESR and CRP.

	14 February 2023	27 September 2022	26 July 2022	3 June 2022	1 April 2022	9 April 2021
RBC sedimentation rate (Ref. Range: 0–20 mm)	35	37	42	84	30	21
CRP (Ref. Range: 0–0.6 mg/dL)	0.7	0.5	2.3	5.3	1.1	N/A

The patient was subsequently referred to the genetics clinic. Her evaluation was completed via a virtual visit, and thus sensory examination was not performed. After our visit, she consented for germline genetic testing via exome sequencing initially completed as proband only. Exome sequencing identified a c.851 ‐ 33T > A:p.? (Intron 7) variant of the NTRK1 gene, which was reported as pathogenic. A second variant c.1483 + 5G > A:p.? (Intron 11) in the NTRK1 gene was also identified, initially reported as a variant of uncertain significance. This variant had not been reported in patients diagnosed with CIPA prior to this report. This was interpreted based on the common interpretation guidelines provided by ACMG (Richards et al. 2015) and was initially classified as having uncertain significance with evidence of PM2 and PP3. There is only one entry in the ClinVar database, and it is absent from gnomAD database (PM2). Using the computational model for prediction of the effect on splicing, it was found that MaxEntScan, a variant identified in our patient, has a deleterious effect on splicing, supporting her diagnosis of CIPA (PP3). Given that the patient's presenting symptoms were suspicious for CIPA, parental samples were provided to determine the phase of the identified variants and to further confirm autosomal recessive inheritance. Evaluation of her parental samples noted that c.851‐33T > A was of maternal origin and the c.1483 + 5G > A variant was of paternal origin (Figure 2). At this time, the second variant from her father has been re‐classified as likely pathogenic with additional evidence of PM3 and PP4.

FIGURE 2.

Pedigree of the proband's family.

As the c.1483 + 5G > A variant had not been previously reported, we further investigated whether this variant had a deleterious effect on splicing. We generated a minigene with a reference sequence from exon 8 to exon 13 of the NTRK1 gene and cloned into a mammalian expression vector (Figure 3A). A second minigene construct harboring the variant identified in this patient was also generated (Figure 3A). Minigene reporter assay showed notable reduction in the correctly spliced transcripts in the presence of the c.1483 + 5G > A variant compared to the reference sequence when transfected into the 293T cells (Figure 3B). Minigene splicing assay indicated that the variant identified in our proband likely causes alternative splicing and reduction in splicing efficiency in vitro, as noted by the presence of a smaller transcript compared to the reference sequence and increased level of the unspliced RNA product (Figure 3B).

4. Discussion

Here, we reported a 46‐year‐old woman with multiple Charcot joints that initially started in her right ankle with progression to multiple joint involvement. Given the unclear and non‐diagnostic evaluations by multiple specialists, she was referred to the genetics clinic, which subsequently led to the identification of compound heterozygous pathogenic variants in the NTRK1 gene, consistent with a diagnosis of CIPA.

This is a unique case of a patient affected with CIPA diagnosed in adulthood and highlights the phenotypic diversity of the condition, which may have contributed to the delay in her diagnosis. The commonly reported characteristic features include impaired perception of pain, impaired temperature perception, anhidrosis, impairment of the autonomic nervous system and intellectual disability (Indo 1993). Our patient, however, reports preserved sensation to painful stimuli, and her neurological evaluation was notable for preserved pinprick sensation at her first neurological evaluation at age 45. Her subsequent neurology evaluation, approximately a year after her first visit, was notable for decreased pinprick sensation in bilateral plantar aspects of her feet and fingertips. While she reports ability to sense pain, it is likely severely diminished given her multiple Charcot joints. Our patient does report anhidrosis, although its severity is unclear. To our knowledge, she has not experienced recurrent febrile episodes prompting treatment or evaluation to this date. Our patient also does not have signs suggestive of Horner syndrome, although she has not been fully evaluated for autonomic dysfunction. Most patients affected with CIPA have been reported with varying degrees of intellectual disability, which may help with diagnosis (Indo 1993). She had no concerns with intellectual disability since birth, and she has completed her undergraduate degree without difficulty. She continues to remain independent and maintains her profession. While some of her clinical presentations were suggestive of CIPA, the most striking presentations were her Charcot joints, which likely resulted in delayed diagnosis as her clinical evaluations were focused on her joint deformities.

Her clinical phenotype appears to be milder than the previously reported cases of CIPA, potentially due to both of her variants being that of splicing variants (Baeza‐Centurion et al. 2020). It is likely that her splice variants result in a reduced expression of tropomyosin receptor kinase A (TrkA), but the expressed TrkA retains residual function. Our minigene splicing assay shows the reduction in the correctly spliced RNA in the presence of c.1483 + 5G > A, which suggests that the proband likely has a reduced expression of the correctly spliced transcript (Figure 3B), although we are unable to quantitate the degree of reduction due to the nature of the assay used. Furthermore, it is unknown how much the reduction in the RNA splicing affects TrkA protein expression in our patient, as we are unable to measure the protein expression in this patient. We have reviewed the literature reporting patients with the NTRK1 c.851 ‐ 33T > A pathogenic variant. While there are limitations with the reported data, the majority of the patients reported with this variant have classical clinical phenotype described in CIPA, although there are cases with milder presentation (Table S2). This variabiltiy is likely attributed to the second variant these patients harbor. However, there is variability in clinical phenotypes in the reported cases in patients, who are homozygous for this variant, which would suggest that there are likely other factors that may modulate the severity of the condition.

Here, we discovered a novel likely pathogenic variant in the NTRK1 gene in a patient with atypical clinical features of CIPA. Initially, this variant was classified as a variant of uncertain significance in the absence of a segregation study, as this variant had not been reported previously in patients with CIPA. Currently, there is only one entry in the ClinVar database, which was entered prior to identification of the same variant in our patient. This variant is also absent from the gnomAD database. Using the computational model for the prediction of the effect on splicing, it was found that MaxEntScan, a variant identified in our patient, has a deleterious effect on splicing, supporting her diagnosis of CIPA.

While the diagnostic technology has improved and accessibility to these technologies is increasing, there are still significant unmet needs for the evaluations of patients with rare disease (Aaltio et al. 2022; Balasar and Basdemirci 2023; Chung et al. 2023; Felix et al. 2023). This case highlights the importance of referral to the genetics clinic in adult patients with a previously undiagnosed condition. This case also highlights the importance of a comprehensive diagnostic evaluation utilising exome or genome sequencing in adult patients with a significant diagnostic odyssey. The average time that a patient with rare disease will experience a diagnostic odyssey is commonly reported to be between 4 to 5 years, although it is not uncommon for patients to go undiagnosed for over 20 years (Willmen et al. 2023; Wu, McMahon, and Lu 2020). The availability of molecular testing during this patient's case was limited early on in her disease course, therefore contributing to the 40 years of diagnostic odyssey, while being evaluated by multiple specialists. Not only do patients with rare disease undergo diagnostic odyssey, they may also be misdiagnosed, leading to treatments or surgeries without clinical benefit (Schieppati et al. 2008; Wu, McMahon, and Lu 2020). Here, the patient had undergone at least two knee joint aspirations in adulthood that were negative. Due to the initial suspected diagnosis and labs that were supportive of inflammatory arthritis, a trial of adalimumab was not unreasonable; however, there can be significant side effects with long term use, which may have resulted in adverse events (Bessone and Bjornsson 2023; Patel et al. 2023).

Currently, there are no available therapeutic options for CIPA. However, in establishing a diagnosis for this patient, we were able to alter medical management to prevent possible future iatrogenic harm from ineffective treatment. Accurate diagnosis in our case has reduced follow‐up visits, lab monitoring, medication burden and economic burden to the patient. Her extensive evaluation indicates that the Charcot joints in CIPA patients are independent of inflammatory cytokines despite the elevation in inflammatory markers (Tables 1 and S1). Use of DMARD was ineffective in the treatment of Charcot joints in our proband, although this may have modestly improved the associated reactive inflammation, as evidenced by reduced inflammatory markers (Table 1).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Table S1.

Table S2.

Data S1.

Data Availability Statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.