Introduction

Nail patella syndrome (NPS, OMIM 161200), or onychoosteodysplasia, is a pleiotropic birth defect exhibiting an autosomal dominant pattern of inheritance. Congenital manifestations include dysplastic nails, absent or hypoplastic patellae, bony projections (‘horns’) of the ilia and dysplasia of the elbows (reviewed in Carbonarna and Alpert,1 Bongers et al2 and Sweeney et al3). The obvious orthopedic and ectodermal issues have resulted in NPS being regarded as primarily a disorder of connective tissue. However, it has become apparent that proteinuria, resulting from structural abnormalities of the glomerular basement membrane and open-angle glaucoma are constituent parts of the syndrome and may occur at any age.3, 4, 5 The observation of a phenotype similar to NPS in lmx1b−/− mice6 prompted Dreyer et al7 to consider LMX1B as a candidate gene for NPS. Subsequent work has identified approximately 120 distinct loss-of-function mutations.8 LMX1B is a member of the LIM-homeodomain family of transcription factors that play a variety of roles during development to determine body pattern in vertebrates and invertebrates.9, 10 Both the expression of Lmx1b during development and the spectrum of abnormalities observed in the lmx1b−/− mouse correlate with the NPS phenotype in terms of the ocular, kidney and limb abnormalities.3, 7

Recently, in a large clinical study of 123 NPS patients, peripheral neurological symptoms were recognized, described as numbness, tingling or burning sensations of the distal limb, in 25% (28/110) of patients.3 The neurological problems may be related to abnormalities of the dorsal–ventral patterning of the developing limb including abnormal neuronal migration. Cases in which orthopedic abnormalities may have resulted in nerve entrapment were excluded.3 Alternatively, the neurological symptoms may result from a primary dysfunction of LMX1B within the sensory circuit. Lmx1b expression has been employed as a marker of neuronal differentiation in the developing spinal cord.11, 12

To further investigate the role of LMX1B in the neurological symptoms in NPS, the spatiotemporal pattern of Lmx1b was studied using a mouse model with an internal ribosomal entry site (IRES)-LacZ cassette inserted into the 3′UTR of the gene. The IRES allows for the translation of both the Lmx1b and β-galactosidase proteins from a single transcript. A nuclear localization signal is included to direct β-galactosidase to the nucleus, thus replicating Lmx1b expression. Using β-galactosidase staining as a marker of Lmx1b, a dynamic pattern of expression was observed within the developing spinal cord, with expression persisting through adulthood. Analysis of the spinal cord of lmx1b−/− mice revealed deficit in migration of postmitotic neurons to the superficial layers of the dorsal spinal cord, and inability of afferent fibers to penetrate these layers. Detailed neurological examination revealed neurological deficits in 11 of 16 individuals with NPS. A pathophysiological mechanism linking Lmx1b expression to a neurological phenotype in NPS is proposed.

Methods

Construction of the lmx1b3′LacZ mouse

An IRES-LacZ-floxed PGK-neo cassette13 with a nuclear localization signal was inserted into a unique HindIII site in the 3′UTR of lmx1b via homologous recombination in AB1 ES cells. Two independent lines were injected into C57Bl/6 blastocysts and the resulting chimeras assayed for germline transmission by mating to C57Bl/6 females. The presence of the LacZ cassette was determined by PCR followed by Southern analysis using a probe external to the targeting vector. The neo cassette was removed by mating with CMV-cre mice and homozygous lines were established by intercrossing the resultant progeny and selecting for cre-negative homozygotes. The presence of the LacZ cassette does not affect normal function of Lmx1b, since the LacZ homozygotes are viable and fertile, as were mice that were compound heterozygotes containing the lacZ allele over the null allele (data not shown). The generation of the lmx1b−/− mice was described previously.6

β-galactosidase staining, histology and immunocytochemistry

CD1 females, used because of their large litter sizes, were crossed with either an lmx1b3′LacZ heterozygote or homozygote. The day the plug was seen was designated E0.5. Embryos were collected from E9.5 to E15.5 at one-day intervals. Embryos at E14.5 and E15.5 were partially dissected prior to staining. Adult spinal cord was dissected from postnatal day 21 (P21) mice. Tissues were fixed and stained as described.14 Transverse sections of the cervical region of the spinal cord were made with a razor blade and digital photographs were taken under a Nikon dissecting microscope. Immunocytochemistry was performed on six lmx1b−/− embryos at each developmental stage, as described previously.15, 16

Patients and neurological examination

A focused neurology history and neurological examination was performed on 16 sequential, volunteer individuals with NPS after obtaining appropriate informed consent. The focused neurology questions included those signs and symptoms that have been reported in individuals with NPS,3 including sensory deficits and neuropathic pain (ie spontaneous or stimulus-induced burning or electric-like pain that is not in a dermatomal or nerve-specific distribution). A comprehensive bedside neurological examination was also performed, including assessment of cranial nerve function, motor function, sensation, tendon reflexes, cerebellar function and gait. Sensation was evaluated by testing pinprick, cold, vibratory and proprioception. Pinprick and cold sensation assess the function of unmyelinated Aδ and C fibers. Pinprick sensation was tested by lightly touching the tips of the subject's fingers or toes with a safety pin. The subject was asked to state when the prick felt sharp as the pinpricks moved proximal up the digits and onto the hands or feet. Cold sensation was tested by asking the subject if a cold tuning fork felt the same on the tips of the digits, the palm or dorsum of the foot, and the proximal extremity. Cranial nerve function was assessed by determination of sensation to pinprick and cold in the face.

Results

Whole mount staining of lmx1b3′LacZ embryos (Figure 1a–e) indicates that the insertion of the IRES-LacZ cassette does not alter the endogenous pattern of Lmx1b expression. At E9.5, strong expression of Lmx1b is observed in the forelimb bud and midbrain–hindbrain junction (Figure 1a). By E10.5, Lmx1b is also expressed in the hindlimb bud, and expression within the periocular mesenchyme is visible (Figure 1c). Lmx1b continues to be expressed in the limb buds and eye up to E15.5, the latest time point examined. Additionally, expression was detected in the kidney at E14.5 (data not shown). As reported previously,17 Lmx1b expression in the cranial mesenchyme is visible by E11.5 (Figure 1c). Other regions of expression include mammary gland primordia, from E11.5 to E15.5, as well as vibrissae and sensory follicle primordia, from E11.5 to E15.5 (Figure 1c and d and data not shown). Hair follicle expression is visible from E14.5 (data not shown). On whole mount, spinal cord expression first becomes visible at E10.5 and by E11.5 extends the entire length of the neural tube.

Figure 1
figure 1

(a–e) Whole-mount β-galactosidase staining of lmx1b3′LacZ embryos at the stages indicated. Transverse sections of the spinal cord are shown in insets. (f) Transverse section of adult spinal cord (P21).

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Transverse sections of the whole mount specimens reveal a dynamic pattern of Lmx1b expression within the spinal cord (Figure 1b–e, insets). From E10.5 to E15.5, Lmx1b was expressed within the floorplate. Lateral neural tube expression initiated at E10.5 (Figure 1b, inset). The fate of these neurons remains unknown. A second region of Lmx1b expression within the neural tube becomes visible at E11.5 (Figure 1c, inset). This population expands and migrates to the dorsal spinal cord through E12.5 and E13.5 (Figure 1d and e insets) and forms the dorsal sensory interneurons.11 Expression of Lmx1b within this population is maintained to adulthood (Figure 1f).

Nissl staining has shown that the laminar boundary is indistinct in lmx1b−/− embryos compared with wild type and development of laminae I and II neurons is impaired.18 The impaired development of laminae I and II neurons in lmx1b−/− embryos appears to delay the innervation of primary cutaneous afferents, as determined by TrkA staining (Figure 2).18 At E14.5, Trk+ fibers have migrated into the dorsal laminae in wild-type embryos but not in lmx1b−/− embryos (Figure 2a and b). By E17.5, TrkA+ fibers have migrated into the dorsal laminae in lmx1b−/− embryos, but have migrated in a disorganized manner (Figure 2c and d).

Figure 2
figure 2

Projection of TrkA+ fibers in wild-type (a, c) and lmx1b−/− embryos (b, d) at E14.5 (a, b) and E17.5 (c, d). The arrow marks the position of the boundary between laminae I–II and III–V neurons visualized by Nissl staining.18

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To determine whether the expression of Lmx1b in the dorsal spinal cord is relevant in consideration of the NPS phenotype, detailed neurological examinations were performed in 16 subjects. The results of the neurological history and examination are shown in Table 1. Numbness or tingling in the face, arms, hands, feet or lower back was reported in 10/16, and 2/16 complained of burning pain. The pain was in the forearm in one individual and in the hands and shins of the other.

Table 1 Neurological findings in NPS patients
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In most of the individuals examined, finger pinprick sensation was diminished. (In the absence of calluses or trauma, any reduction in response to pinprick is abnormal.) In a minority of individuals, this deficit was associated with impaired toe and foot pinprick sensation and cold sensation in the hands and feet (see Table 1). When pinprick sensation was impaired, the ventral and dorsal surfaces were equally impaired, and all the digits were usually affected equally. Also, the impairment was typically symmetric between the right and left hands or feet. In two cases, a deficit in sensation was observed in the absence of numbness or tingling. There was no apparent association between neurological symptoms and LMX1B mutations: symptoms were noted in individuals with missense, nonsense or frameshift mutations, and symptoms were as variable between individuals with the same mutation as among the cohort as a whole.

Cranial nerve function and cerebellar function were within normal limits. Weakness and hyporeflexia was noted in three individuals, but was likely secondary to joint disease and orthopedic procedures. Gait was abnormal in only one individual who walked with a cane due to severe orthopedic problems. Proprioception in the thumb and great toe was diminished in one individual. Vibration sensation in the great toe was diminished in five individuals. This may be associated with NPS, but the fibers that transmit vibration sensation do not synapse onto laminae I and II (see below). However, this deficit is not rare in the general population, particularly in older individuals.

Discussion

The pattern of lmx1b expression observed in limb, eye and kidney correlates with the recognized signs and symptoms of NPS. The relevance to the NPS phenotype of Lmx1b expression in cranial mesenchyme, mammary gland primordia, hair follicles and vibrissae remains to be determined, although poor breast development has been reported in some women with NPS, and a characteristic hairline described.3 Preliminary evidence suggests that ADHD may be a constituent part of NPS, associated with the requirement for Lmx1b in the development of mesencephalic dopaminergic neurons (McIntosh et al, in preparation).19, 20

TrkA+ fibers are nociceptive sensory neurons with cell bodies located in the dorsal root ganglia and have axons that project into the dorsal laminae of the spinal cord (reviewed by Snider and McMahon21). Although the migration of the TrkA afferents into the spinal cord of lmx1b−/− mice is abnormal, this deficit most likely reflects a noncell autonomous role of Lmx1b in the migration of the TrkA afferents since Lmx1b is not expressed in the dorsal root ganglion. Alternatively, the expression of Lmx1b in the outer laminae of the developing spinal cord may be necessary to establish the proper developmental cues for the projection of TrkA fibers into the dorsal horn.

Pain and temperature modalities are carried by lightly myelinated Aδ and unmyelinated C fibers. 22, 23 The Aδ fibers are responsible for sharp pain (ie pinprick response) and are found in higher numbers in the distal limb relative to the proximal limb.23 Dysfunction of the Aδ and C fibers causes impaired pinprick and cold sensation, and the patient will frequently complain of numbness, tingling and neuropathic pain. With regard to the NPS phenotype, there is a high incidence of tingling or numbness in individuals with NPS (Table 1).3 Two individuals complained of neuropathic pain. Pinprick sensation was diminished in a significant number of individuals examined. The deficit was noted in the hands more than the feet, but not in the arms, legs or face. A minority of these individuals also had deficits in cold sensation in the hands and feet but not in the face. Notably, there was no difference between sensitivity across either the dorsoventral or anterior–posterior axes, which would correlate with the expression pattern of Lmx1b. The degree of impairment may be correlated with age since the mildest symptoms were seen in adolescents, and two of the highest scores in subjects over 50 years, but a larger (preferably longitudinal) study is required to address this issue.

Vibration sensation and proprioception are mediated by myelinated fibers. Since these modalities were preserved in most of the affected individuals, it is likely that the observed neurological deficit results from abnormality of the distal, unmyelinated fibers of the hands and feet. Furthermore, the impaired sensation to pinprick and cold temperature did not follow a specific nerve or dermatome pattern. Therefore, it is unlikely that these findings are due to nerve compression secondary to bone degeneration.

The Aδ and C fibers synapse in laminae I and II within the spinal cord. In Lmx1b mutant mice, differentiation of laminae I and II neurons and cutaneous afferent ingrowth from the dorsal root are impaired (Figure 2).18 The cell bodies of Aδ and C neurons are located in the dorsal root ganglia, where Lmx1b is not expressed, supporting the hypothesis that the phenotype is the result of a requirement of Lmx1b-specified interneurons for TrkA+ fiber innervation and not a defect in the development of the neurons per se. Therefore, in individuals with NPS, deficient Aδ and C fiber projection into laminae I and II may be associated with the neuropathic pain, tingling, and impaired pinprick and cold sensations observed in this study.