Yukako Inoue1, Masaaki Yamamoto1, Yoshiko Sakaguchi1, Orie Jitsukawa1, Katsunori Hayano2#, Masayuki Yamane2, Michiko Sakamoto3 and Kiyofumi Yamanishi1*
1Department of Dermatology, Hyogo College of Medicine, Nishinomiya, 1-1, Mukogawa-cho, Nishinomiya, Hyogo, 663-8501, 2Department of Pediatrics, Saiseikai Hyogo Hospital, Kobe and 3Department of Pediatrics, Takarazuka Municipal Center for Handicapped Children, Takarazuka, Hyogo, Japan. #Present address: Department of Pediatrics, Kita-Harima Medical Center, Ono, Hyogo, Japan. *E-mail: email@example.com
Accepted Feb 4, 2015; Epub ahead of print Feb 6, 2015
Congenital ichthyosiform erythroderma (CIE) is an entity classified in non-syndromic ichthyosis of autosomal recessive congenital ichthyosis (ARCI). Genes responsible for CIE include ABCA12, ALOX12B, ALOXE3, CERS3, CYP4F22, NIPAL4, PNPLA1 and TGM1 (1–3). The genetic diagnosis of CIE could be achieved by conventional Sanger sequencing of multiple PCR-amplified cDNAs and/or genomic DNA fragments, but it is laborious to identify possible mutation(s) among all those genes. Alternatively, target-captured DNA analysis (4) and whole exome analysis (5) of genes using next-generation sequencing (NGS) can efficiently identify mutations involved in ichthyoses. Here we report a case of CIE with an intellectual disability, in which NGS of a panel of 54 genes involved in keratinisation disorders was used to establish an exact diagnosis of ABCA12 deficiency.
A 3-year-old boy was referred to us regarding his erythroderma with severe scales. The initial information was that he was born as a collodion baby, but the record of the NICU was suggestive of harlequin ichthyosis (HI). His parents and older sister were healthy and no other members of his pedigree had similar skin manifestations. On examination, his entire body surface was dry and mildly erythrodermic and was covered diffusely with large whitish scales (Fig. 1a, b). His scalp was covered with erosions and patchy thick scales and/or crusts (Fig. 1a). Scalp hairs were sparse, but there were no hair abnormalities. His eyebrows and eyelashes were also sparse and irregularly grown and mild ectropion and eclabium were observed (Fig. 1a). The nails were thickened and his palms and soles were also as hyperkeratotic as the dorsal sides of his hands and feet. He had just undergone an operation for cryptorchism but no major malformations were found. No neurological symptoms, paraplegia, ataxia, peripheral neuropathies and deafness were evident. However, the overall developmental quotient (DQ) measured using the Kyoto Scale of Psychological Development (6) was 55 at 3 years and 11 months of age (normal DQ = 70 at that age), indicating a delay in intellectual development.
Haematoxylin and eosin staining of a biopsy from his abdominal skin revealed epidermal acanthosis hypergranulosis and a markedly thickened homogeneous stratum corneum (Fig. 1c). Mild inflammatory cell infiltrates were seen in the upper dermis. Ultrastructural analysis of the epidermis showed aggregated abnormal vesicular structures, vesicles containing granules and lipid droplets in the granular layer and stratum corneum (Fig. 1d), comparable to CIE cases reported previously (7).
For the differential diagnosis of ichthyoses, coding exons and their flanking splice sites of 54 genes (Table SI1) were examined by NGS, after approval of the Institutional Review Boards. Informed consent was obtained from his parents. The 8 genes responsible for ARCI and ALDH3A2 and AP1S1 for syndromic ichthyoses with neurological complications were included in the panel of 54 genes. For target capture, HaloPlex® probes of those 54 genes were obtained from Agilent Technologies Inc. (Santa Clara, CA, USA). A total of 137.419 kbp in 674 regions of those target genes were examined using a MiSeq NGS sequencer (Illumina Inc., San Diego, CA, USA). DNA samples from 10 other cases with keratinisation disorders and one case with pustular psoriasis were also examined. Sequences were analysed using a SureCall (Agilent Technologies Inc.) and human hg19 was used as a reference genome sequence. NGS revealed compound heterozygous mutations chr2: g.215876794_215876795delAA and chr2: g.215823087delG in ABCA12 (Fig. S11). No other SNV (single nucleotide variation) and indels (insertions and deletions) compatible with the phenotype of the present case were found in any of the other genes examined. PCR products from genomic DNAs and/or cDNAs of the patient and his parents were subjected to Sanger sequencing and the mutations detected by NGS were confirmed. The maternal mutation was c.2021_2022delAA in exon 16 leading to p.Lys674Argfs*48, while the paternal mutation was c.6031delG in exon 41 leading to p.Glu2011Asnfs*16.
Immunofluorescence analysis revealed that ABCA12 was stained in the granular layer (Fig. 1e, upper) and glucosylceramide (GlcCer) staining was weak in the upper spinous layer and intense in the granular layer in healthy control skin (Fig. 1f, upper). In contrast, in the skin of the patient, ABCA12 was only faintly stained (Fig. 1e, lower) and weak staining of GlcCer remained in the upper spinous and granular layers of the skin (Fig. 1f, lower).
He was treated with etretinate 1 mg/kg daily, and his ectropion and eclabium had improved by the age of 4.5 years, but whitish scales were still diffusely distributed and he was treated topically with tacalcitol. Heat retention often occurred with a body temperature over 38°C in the summer season. We recommended a cool environment and cooling to avoid heat stress.
The present case corresponds to a survivor case of HI. The survival rates of HI range from 50% to 80% (8, 9). The mutations identified in this case cause compound heterozygous truncation mutations. One mutation is p.Lys674Argfs*48 in the N-terminal domain of ABCA12, and another one is p.Glu2011Asnfs*16 in the second transmembrane domain of the protein (Fig. 1g). Each mutation has been identified in an HI case (10) and a CIE case (11), respectively, but the compound heterozygosity of those truncation mutations have not been reported in survivors of HI with the phenotype of CIE. Using immunofluorescence, ABCA12 was only faintly detected by an antibody raised against residues 93–180 of the protein (Fig. 1e). This might be due to conformational changes of the mutant ABCA12 proteins. The defects in ABCA12 possibly impair the accumulation of GlcCer into lamellar granules in the granular layer (Fig. 1f), as suggested previously (12).
Reportedly, a developmental delay accompanies 32% of survivor cases with HI (8), but ABCA12 mutations associated with an intellectual disability have not been fully delineated. Eilers et al. (13) reported a German boy with severe psychomotor retardation. In that case, at 1 year of age, his mental development corresponded to that of a 3-month-old baby. He could not speak and his auditory skills were equivalent to those of a 6-month-old child. The Griffith-development score was 27, which is much lower than the normal range of 95–115. The mutation of that case was a homozygous mutation of c.7322del1C in exon 49 of ABCA12. That mutation causes p.Val12442Serfs*22, which is also a truncation mutation of the ABCA12 protein, but is more downstream from the mutations found in our case. An intellectual delay is common in childhood and it is unknown whether those truncation mutations are associated with the complication or occurred by chance in those cases with HI. In follow-up of survivors of HI, it is necessary to carefully observe the intellectual development of infants to get help early in life by appropriate care in such cases.
The authors thank Dr. Yoshie Yoshikawa, Department of Genetics, Hyogo College of Medicine, and Mr. Daiki Haruguchi and members of the Joint-Use Research Facilities of the Hyogo College of Medicine for their helpful suggestions and technical assistance for NGS.
This work was supported in-part by JSPS KAKENHI Grant Number 26461668 and by a grant from the “Research on Measures for Intractable Diseases” Project: matching fund subsidy (H23-028) from the Ministry of Health, Labour and Welfare, Japan.