A Causal Treatment for X-Linked Hypohidrotic Ectodermal Dysplasia: Long-Term Results of Short-Term Perinatal Ectodysplasin A1 Replacement

Abstract

X-linked hypohidrotic ectodermal dysplasia (XLHED), caused by a genetic deficiency of ectodysplasin A1 (EDA1), is a rare developmental disorder of ectodermal derivatives such as hair, sweat glands, and teeth. The absence of sweat glands and perspiration can evoke life-threatening hyperthermia. As molecular genetic findings are not always conclusive, the concentrations of circulating EDA1 may help to distinguish between total and partial EDA1 deficiencies. We previously treated nine male patients with obvious signs of XLHED with a recombinant EDA1 replacement protein, Fc-EDA, either shortly after birth (n = 3) or by prenatal administration in gestational week 26 and beyond (n = 6). Here, we present the long-term follow-up for up to six years. In patients who had received Fc-EDA after birth, neither sweat glands nor sweating ability were detected at the age of 12-60 months. In contrast, prenatal EDA1 replacement resulted in ample sweat gland development and pilocarpine-inducible sweating in all treated subjects, who also attained more permanent teeth than their untreated affected relatives. Normal perspiration has persisted for six years in the two oldest boys treated repeatedly with Fc-EDA in utero. When they had a sauna, adequate thermoregulation was evidenced. Lower sweat production after single prenatal dosing may indicate a dose-response relationship. The absence of circulating EDA1 in five prenatally treated subjects proved that these children would have been unable to perspire if they had been left untreated. The sixth infant was shown to produce an EDA1 molecule that, albeit interacting with its cognate receptor, cannot activate EDA1 signaling. In conclusion, a causal treatment of XLHED before birth is feasible.

Keywords: AlphaLisa; ectodermal dysplasia; ectodysplasin A; prenatal therapy; protein replacement; sweat glands; tooth development.

Conflict of interest statement

H.S. and P.S. are holding patents related to the topic of this work and received research funding. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1

Chemically stimulated sweating. (a) Pilocarpine-induced sweat volumes were collected within 30 min in XLHED-affected infants who received prenatal treatment with Fc-EDA (black triangles and black circles) or postnatal injections of this replacement protein (white circles). (b) Pilocarpine-induced sweat production in male control subjects (white boxes; n = 174) and female subjects (striped boxes; n = 105) of different age groups. The edges of the box denote the 25th and 75th centiles. Crosses indicate the minimum and maximum volumes measured in the individual age group. ▫, mean; ─, median.

Figure 2

Documentation of thermal sweating. (a) Whole-body skin thermography of a 5-year-old prenatally treated XLHED patient (subject P1) before and after having a sauna. Efficient perspiration is indicated by skin temperatures lower than 32.5 °C (red to yellow color), for example at the nose, the right arm, both hands, and the lower legs. (b) Visible thermal sweating on the face of this patient when leaving the sauna: the nose and the forehead were particularly covered by beads of sweat.

Figure 3

Panoramic dental radiographs of the untreated XLHED-affected brother (a) of subjects P1 and P2 compared with the radiographs of these Fc-EDA-treated subjects at preschool age (b,c) and of the untreated affected brother of P3 (d) compared with the younger sibling 6 years after his prenatal treatment with Fc-EDA (e). Two primary tooth germs ((f), arrowed) that had been detectable in P1 and P2 in early infancy did not undergo complete mineralization and did not erupt.

Figure 4

Levels of circulating EDA as determined by AlphaLisa. The measurements, always conducted in duplicates, were reproducible in three independent experiments. A representative assay is shown here. Signals below the dashed red line are considered as background. The standard curve was generated with Fc-EDA diluted in serum from an adult male subject with XLHED. FCS, fetal calf serum.

Figure 5

Circulating EDA, as determined by AlphaLisa, following pre-depletion with EctoD2, protein A, or EDAR-Fc. (a) Fetal calf serum (FCS), serum from an adult subject with XLHED, either pure (HED) or spiked with Fc-EDA (50 ng/mL; HED+), and serum from subject P6 were depleted twice with EctoD2 (anti-EDA)-coupled beads (+) or TACI-Fc-coupled beads (-). EDA in flow-through fractions was measured by AlphaLisa. (b) Same as (a) except that serum was depleted four times with Protein A-coupled beads (+) or Sepharose 6B beads (-). The beads were then acid-eluted and released immunoglobulins were quantified. No immunoglobulin was detected in the 4th elution of Protein A, indicating successful target depletion. There was no retention of immunoglobulins on Sepharose 6B. (c,d) Same as (a) except that sera were depleted once with EDAR-Fc (+) or with TACI-Fc (-) and that serum from an adult subject with XLHED was spiked with Fc-EDA at a concentration of 5 ng/mL (HED+). Measurements were conducted in duplicates. Adult, healthy adult control.

Figure 6

Expression of a shortened EDA molecule in an affected relative of P6. Sera from a pool of adult subjects with XLHED who all carry EDA null mutations (no EDA1 expression), from the grandfather of P6 and also from two healthy adults were investigated by immunoprecipitation with a mix of two anti-EDA monoclonal antibodies, EctoD2 and EctoD3, that both recognize the native C-terminal domain of EDA. Subsequent Western blotting with the antibody Renzo-2 recognizing the denatured C-terminal domain of EDA was performed. Increasing amounts of recombinant Fc-EDA were loaded onto the same gel as positive controls. The positions of molecular weight markers (in kDa) are indicated. HC and LC, heavy and light chains of the antibodies used for immunoprecipitation; EDA, processed soluble form of wild-type EDA; EDA Δ, shorter form of EDA in serum from the grandfather of P6. This experiment was conducted twice with similar results.