A novel ABCA12 pathologic variant identified in an Ecuadorian harlequin ichthyosis patient: A step forward in genotype‐phenotype correlations

Abstract Background Autosomal recessive congenital ichthyoses (ARCI) have been associated with different phenotypes including: harlequin ichthyosis (HI), congenital ichthyosiform erythroderma (CIE), and lamellar ichthyosis (LI). While pathogenic variants in all ARCI genes are associated with LI and CIE phenotypes, the unique gene associated with HI is ABCA12. In HI, the most severe ARCI form, pathogenic variants in both ABCA12 gene alleles usually have a severe impact on protein function. The presence of at least one non‐truncating variant frequently causes a less severe congenital ichthyosis phenotype (LI and CIE). Methods We report the case of a 4‐year‐old Ecuadorian boy with a severe skin disease. Genetic diagnosis was performed by NGS. In silico predictions were performed using Alamut software v2.11. A review of the literature was carried out to identify all patients carrying ABCA12 splice‐site and missense variants, and to explore their genotype‐phenotype correlations. Results Genetic testing revealed a nonsense substitution, p.(Arg2204*), and a new missense variant, p.(Val1927Leu), in the ABCA12 gene. After performing in silico analysis and a comprehensive review of the literature, we conclude that p.(Val1927Leu) affects a well conserved residue which could either disturb the protein function or alter the splicing process, both alternatives could explain the severe phenotype of our patient. Conclusion This case expands the spectrum of ABCA12 reported disease‐causing variants which is important to unravel genotype‐phenotype correlations and highlights the importance of missense variants in the development of HI.

ABCA12 encodes a keratinocyte-associated lipid transporter. Pathogenic variants in ABCA12 are known to cause the three major phenotypes of ARCI: HI, LI, and CIE. Genotypephenotype correlations have been established in ABCA12 associated disorders: homozygotes or compound heterozygotes with truncating ABCA12 variants generally lead to an HI phenotype while homozygous missense variants usually cause a milder phenotype (Akiyama, 2010).
Here we report a boy suffering from HI with compound heterozygous disease-causing variants in ABCA12, one truncating mutation: nonsense variant c.6610C>T, p.(Arg2204*), and a novel missense variant, not previously reported: c. 5779G>T, p.(Val1927Leu). The location of the new disease-causing variant (first nucleotide of exon 39) suggests it can potentially alter the splicing process. In order to understand the effect of ABCA12 splice-site and missense pathogenic variants, a literature search was performed.

| CASE REPORT
The patient is a 4-year-old boy who was the third child of apparently non consanguineous parents from Manta, Manabí, Ecuador. There was no family history of congenital ichthyosis. Gestational age was approximately 7 months. After delivery the baby was placed in an incubator, where he spent one month. His mother mentioned that at birth he had several characteristics related to a harlequin fetus: thick large fissures over the whole body, flattened nose and ears, respiratory distress and feeding difficulties that required supplemental tube feeding; although he suffered from these complications he was able to breastfeed when he left the hospital. He also had toe blisters soon after birth that converted in toes synechia, affecting his gait. During the neonatal period the patient only received topical treatments.
Physical examination revealed: ectropion, eclabium, nasal hypoplasia, rudimentary external ears, dental hypoplasia, erythema, inflammation of the gums, and almost complete alopecia (Figure 1a). He presented generalized scales on an erythrodermal background with abundant fissures ( Figure  1c). Upper-extremities showed a high degree of retraction at finger joints, giving a claw hand aspect (Figure 1d). There were nail deformities, abundant fissures in bending sites and palmoplantar hyperkeratosis (Figure 1b). During the clinical examination the patient showed sensitivity and irritability, due to the pain caused by the fissures, when he moved. After obtaining informed consent, blood extraction was performed in the affected child, his parents, and his healthy sisters. Genomic DNA was isolated from peripheral blood cells using standard procedures in the Biomolecular Laboratory located in Cuenca, Ecuador and sent to the Fundación Pública Galega de Medicina Xenómica in Spain, where genetic diagnosis analysis and a comprehensive review of the literature, we conclude that p.(Val1927Leu) affects a well conserved residue which could either disturb the protein function or alter the splicing process, both alternatives could explain the severe phenotype of our was carried out. Ethical approval was obtained and all research was performed in accordance with the principles of the Declaration of Helsinki. Three micrograms of patient´s genomic DNA were enriched using SureSelect (Agilent Technologies) following the manufacturer's protocol. The target resequencing library was then sequenced on a SOLiD 5500xl (Life Technologies). Color space reads were mapped to the GRCh37/hg19 reference genome using LifeScope software version 2.5.1 (Life Technologies). Finally, variants were identified using GATK version 2.1 (Genome Analysis Toolkit, Broad Institute) and LifeScope version 2.5.1 and annotated with ANNOVAR version 2012Mar08. In silico prediction of potential variant effects on splicing were computed by using MaxEnt, NNSPLICE, and Splice Site Finder. Missense prediction analyses were performed by using Align GV-GD, SIFT, and Mutation Taster. All these algorithms are integrated in the Alamut® Visual 2.11 software (Interactive Biosoftware, Rouen, France). The review of the existing literature on splice-site and missense ABCA12 mutations was carried out by taking into consideration each of all carrier patients reported to date.

| RESULTS
A total of 18 variants were identified in the patient´s ABCA12 gene (NM 173076.2, NP_775099). Sixteen were filtered out while two putative ABCA12 variants in heterozygous state were prioritized by its location in the gene, the type of change they originated, and the frequency in 1000G project (http://www.1000genomes. org/): (a) a transition from C to T in exon 44: c.6610C>T; p.(Arg2204*) that leads to a nonsense substitution; it is located in the second transmembrane domain of the ABCA12 protein (Figure 2b,c). It has been previously identified in homozygous state in an African American patient that was born at 36 weeks of gestation, and died at 6 months of age from septicemia (Kelsell et al., 2005), (b) a transversion from G to T in exon 39: c.5779G>T; p.(Val1927Leu) that leads to a new missense substitution in a highly conserved amino acid Val1927 (Figure 2b,c). This novel variant, previously reported neither in HGMD nor Clinvar nor GnomAD, is located one nucleotide upstream of the canonical splicing acceptor site. The variant was predicted to have a deleterious effect (Align GV-GD: Class C25, SIFT: Deleterious, Mutation-Taster: Disease causing) and to also affect the splicing process (a total decrease in the score of the natural acceptor site of 59.0%, MaxEnt: −41.6%, NNSPLICE: −76.4%, and Splice Site Finder:-8.5%). Taking all the evidence together, we classified ABCA12: c.5779G>T; p. (Val19227Leu) as likely pathogenic according to ACMG guidelines (Richards et al., 2015).
Segregation analysis of the variants in the family shows that the father of the patient is carrier of the ABCA12 c.6610C>T mutation, and the mother of the c.5779G>T mutation. None of the sisters are the carriers of any of these variants (Figure 2a).

| DISCUSSION
Pathogenic variants in ABCA12 have been described in ARCI including HI, CIE, and LI. HI shows the most severe phenotype, associated exclusively with ABCA12 mutations. Homozygous or compound heterozygous missense ABCA12 variants are frequently linked to LI and to a lesser extent CIE, whereas the majority of pathogenic variants associated with HI are homozygous or compound heterozygous nonsense and frameshift substitutions. Missense variants in combination with truncating mutations, including splice-site variants, can be found in both CIE and HI (Akiyama, 2010). In this report we describe an Ecuadorian HI patient who harbors two different types of mutations in ABCA12. One is a nonsense variant which creates a premature codon. The second variant leads to a missense substitution in a conserved residue of the protein that is predicted to alter the splicing process by different algorithms. As both missense and splice-site variants could lead to HI, any of these mechanisms could be affecting the pathogenicity of the variant. To better understand the implication of these type of variants in the development of the different ARCI subtypes, we performed a literature review of all ABCA12 missense and splice-site mutation carrier patients and their associated phenotypes. Thirty patients carrying ABCA12 splice-site variants were found (Table 1). Seven are homozygous carriers, and from these, those with pathogenic variants affecting the consensus splice-sites and its surroundings, are classified as HI (patients 7, 11, 12, 18, 25, and 27) (Akiyama et al., 2005;Goldsmith et al., 2013;Hellström-Pigg et al., 2016;Kelsell et al., 2005;Sheth, Bhavsar, Patel, Joshi, & Sheth, 2018;Thomas et al., 2008). Only two HI patients were compound heterozygous carriers of two different splice-site variants (patients 24 and 28) (Esperón- Moldes et al., 2018;Washio et al., 2017). Interestingly, the homozygous carrier of the synonymous variant c.3456G>A, p.(Ser1152=) (patient 10), located in the middle of the exon 24, shows a CIE phenotype. This milder phenotype could be explained by the fact that this mutation does not alter a consensus site but deregulates the expression of common transcripts; in this case a decrease in the expression of the wild type transcript and an increase in one minor transcript is observed (Goldsmith et al., 2013). Ten out of the 30 patients were compound heterozygous carriers of one ABCA12 splice-site variant affecting the consensus splice-site and a second truncating variant including eight nonsense (patients 1, 4, 8, 9, 13, 17, 22, and 30)   2007; Diociaiuti et al., 2016;Hellström-Pigg et al., 2016;Kelsell et al., 2005;Loo, Batilando, Tan, & Koh, 2018;Scott et al., 2013;Takeichi, Sugiura, Matsuda, Kono, & Akiyama, 2013;Thomas et al., 2006;Tourette et al., 2012;Umemoto et al., 2011) all these patients were diagnosed with HI at birth (with exception of patient 13 of whom there was not available phenotypic information). However, there are still few data of patients carrying a combination of splice-site and missense variants; from the eight patients reported to date, four showed CIE (patients 3, 15, 19, 26) (Bochner et al., 2017;Esperón-Moldes et al., 2018;Fukuda et al., 2012), and one presented HI (patient-16) (Hellström-Pigg et al., 2016). We also identified a total of sixty-three ABCA12 missense carrier patients. As shown in Table 2, most of HI patients bear at least one truncating variant in one of the two alleles (Patients 32,34,35,41,42,50,67,79,80,81,83,86,89,(91)(92)(93) (Akiyama et al., 2006Esperón-Moldes et al., 2018;Hellström-Pigg et al., 2016;Kelsell et al., 2005;Loo et al., 2018;Numata et al., 2015;Peterson, Lofgren, Bremmer, & Krol, 2013;Scott et al., 2013;Tanahashi, Sugiura, Sato, & Akiyama, 2016;Xie et al., 2016). Two HI patients were described as carriers of missense variants in both alleles (Patients 31 and 47), however, the variants identified in patient 31; ABCA12: c.130C>G; p.(Arg44Gly) and c.2033A>G p.(Asn678Ser) (Scott et al., 2013) could be not the causative variants assuming that almost all algorithms predict a nondeleterious effect and considering that a heterozygous known TGM1 mutation: c.401A>G; p.(Tyr134Cys) was also detected in this same patient; in the case of patient 47, described as carrier of a pathogenic variant c.3535G>A; p.(Gly1179Arg) in homozygous state, the zigosity needs to be confirmed. Interestingly, we did not find any difference between the type of mutations in patients with moderate and severe HI phenotypes. As previously reported, CIE patients carry at least one missense variant in combination with other missense, nonsense, splice-site and frameshift mutations, while almost all LI patients are carriers of missense mutations in both alleles. Exceptions are two LI cases (patients 57 and 64) who harbor nonsense and frameshift variants. Interestingly these two patients did not show a more severe phenotype compared to other LI patients who carried missense mutations in both alleles (Akiyama et al., 2008;Bučková et al., 2016;Chao, Aleshin, Goldstein, Worswick, & Hogeling, 2018;Esperón-Moldes et al., 2018;Fukuda et al., 2012;Hellström-Pigg et al., 2016;Israeli et al., 2013;Lefèvre et al., 2003;Loo et al., 2018;Murase et al., 2018;Natsuga et al., 2007;Nawaz et al., 2012;Numata et al., 2015;Sakai et al., 2009;Scott et al., 2013;Shimizu et al., 2013;Sitek et al., 2018;Thomas et al., 2008;Wada et al., 2017;Wakil et al., 2016). The majority of the genotype-phenotype associations found in these patients are in accordance with the correlations previously established by Akiyama, with some few exceptions as previously stated (Akiyama, 2010).
Given the current available data, further characterization of missense variants, including the confirmation of the zigosity in putative homozygous patients and the assessment of their impact in the splicing process, would be needed to better elucidate this genotype-phenotype correlation.
In brief, our case expands the spectrum of ABCA12 reported disease-causing variants. Additionally the literature review of splice-site and missense ABCA12 mutations performed in this study contributes to further understanding of the complex genotype-phenotype correlations in the different subtypes of ARCI.