A novel 1-bp deletion mutation and extremely skewed X-chromosome inactivation causing severe X-linked hypohidrotic ectodermal dysplasia in a Chinese girl

X‐linked hypohidrotic ectodermal dysplasia (XLHED), the most common type of hypohidrotic ectodermal dysplasia (HED), is caused by mutations of the gene for ectodysplasin A (EDA) gene, which encodes a protein belonging to the tumour necrosis factor family.1 The resultant phenotype ranges from classic HED to nonsyndromic hypodontia.2 Female patients usually exhibit milder symptoms and present later.3 A 2‐year‐old Chinese girl presented with severe XLHED. She was the first child of unrelated parents. Dysmorphic features were noted at birth, including fragile‐appearing and peeling skin, sparse hair and a flat nasal bridge. Hypohidrosis, oligodacrya, photophobia, chronic eczema, recurrent respiratory infections and pneumonias developed over time. On physical examination, the patient was found to have thin and dry skin, with the skin in the periorbital area being wrinkled, and she also had sparse hair, eyelashes and eyebrows (Fig. 1d), frontal bossing, pointed chin, nail deformities (Fig. 1e) and no deciduous teeth. (a) Sequencing of the entire ectodysplasin A (EDA) coding exon and flanking intron identified a heterozygous mutation of c.954del C in a patient with X‐linked hypohidrotic ectodermal dysplasia. (b) The mother carried the same heterozygous mutation as her daughter, while (c) the father had the normal allele. (d) Sparse hair, lack of eyebrows and (e) nail deformity in the girl at the age of 2 years. Written informed consent was obtained from the parents for genetic study. EDA gene sequencing identified a novel heterozygous c.954del C mutation (Fig. 1a), which causes a frameshift mutation starting from amino acid 319, resulting in a truncated EDA protein. Amino acid 319 is placed at one of the seven receptor binding sites in the conserved tumour necrosis factor (TNF) domain (http://www.ncbi.nlm.nih.gov/NP_001390.1). The novel mutation abrogates the site of trimer interface and receptor binding, therefore abolishing the key function of the TNF homologous domain. The mutant allele was inherited from the patient's mother (Fig. 1b) and the normal allele from her father (Fig. 1c). However, there is a phenotypic difference between the patient and her phenotypically normal mother, which interfamilial variability in clinical expression does not explain. Severe manifestations of an X‐linked recessive disorder in a female may be related to skewing of X‐chromosome inactivation (XCI). The XCI pattern and cDNA expression was analysed. After digestion with the methylation‐sensitive restriction enzyme HpaII, only the inactive X chromosome gave PCR products. The origin of the inactivated X chromosome was determined by segregation analysis. The undigested PCR product of the patient had two peaks of 287 and 290 bp, respectively, while the HpaII‐digested product had only one peak of 290 bp (Fig. 2a). The HpaII‐digested product of the mother showed only one peak of 287 bp, because the CAG repeat region was homozygous, while that of the father had one peak of 290 bp (Fig. 2b,c). These results showed that the patient had complete (100%) skewing of XCI. Segregation analysis showed that the normal X chromosome from her father was completely inactivated. cDNA expression analysis showed a preferential expression of the mutant allele in the girl (Fig. 2d) and the normal allele in her mother (Fig. 2e), who also demonstrated extreme skewing in her XCI pattern. The cDNA expression of the father was normal (Fig. 2f). (a) X chromosome inactivation (XCI) pattern analysis was based on the polymorphic CAG repeat in exon 1 of the gene for androgen receptor (AR). The undigested PCR product of the patient had two peaks of 287 and 290 bp, respectively, while the HpaII‐digested product had only one peak of 290 bp. The girl had 100% skewing of XCI. (b,c) In the undigested PCR products of the parents, (a) only one peak of 287 bp was visible in the mother because the CAG repeat region was homozygous, while (c) the father had one peak of 290 bp. (d) The splicing site of transcript variant 2 of EDA is near to the site of the mutation c.954del C. (d–f) Electropherograms of sequencing by reverse primer showed that in the leucocytes of (a) the patient, the mutant allele was the main peak, while the normal allele was the minor peak, while (e) in those of the mother, the normal allele was mainly expressed and the mutant allele was weakly expressed, and (f) in those of the father, the normal allele was expressed. Skewed XCI patterns have previously been observed in female individuals with XLHED.4 5 It was interesting in our case that both girl and her mother had their XCI skewed, but in opposite directions. In conclusion, we report a novel 1‐bp deletion mutation located in TNF homologous domain of EDA resulting in a severe phenotype of XLHED, with skewed XCI in both the patient and her mother, which is the first report of such a case, to our knowledge. We thank the patient and her family for their participation in this study. Conflict of interest: the authors declare that they have no conflicts of interest. The first two authors contributed equally to this work and should be considered joint first authors.