We assessed the clinical applicability of whole-exome sequencing in the molecular diagnosis of a series of 5 unrelated families with hereditary ichthyosis and identified 3 variants in TGM1 and 2 variants in ALOX12B, 4 of which were novel. Four out of 5 detected variants were pathogenic/likely pathogenic according to ACMG classification and segregation analysis. We also performed prenatal diagnosis in 2 families whose next generation were at increased risk of ichthyosis.
A definitive diagnosis of ichthyosis requires a multidisciplinary approach involving clinical findings on physical examination, histopathologic assessment, and genetic tests. Accurate genetic diagnosis is crucial to improve patient care by providing useful data for diagnostic classification, clinical management, genetic counseling, and prenatal diagnosis. It also provides insight into the future development of targeted therapeutics.
Preliminary reports indicated that NGS technologies, particularly WES, represent a powerful tool for molecular diagnostics of various genodermatoses (9). An increasing number of WES-based studies have identified the causative variants underlying ichthyosis, suggesting WES as a diagnostic adjunct to clinical testing in these patients (10–12).
Different mutations in these genes have previously been implicated in the etiology of various types of ichthyosis. Mutations in TGM1 are the most common cause for autosomal recessive congenital ichthyosis (ARCI; OMIM: 190195, 242100, 242300), followed by mutations in the two lipoxygenase genes, ALOX12B and ALOXE3 (13, 14).
TGM1 (14q12) encodes transglutaminase 1 (TG1) enzymes, which is responsible for cross-linking cornified epithelium proteins and lipids during formation of the stratum corneum. Mutations in TGM1 are the most common and account for approximately 32% of heritability of ARCI (11). The gene has 15 exons, and codes an 817- amino acid long polypeptide. TGM-1 is composed of 4 functional domains including Transglut N (117–235 aa), Transglutaminase/protease-like homologs domain (TGc) (348–468 aa), Transglut C domain (587–691 aa & 699–796 aa). TGM1 mutations can lead to varying degrees of transglutaminase impairment, which impairs protein crosslinking and esterification of epidermis-specific ceramides during formation of keratinocyte proteins and lipid envelopes and disrupts the skin-barrier function.
Currently, the most reported TGM1 mutation is the c.877-2A > G splice-site mutation (15). Ichthyosis patients can be either homozygous for a single pathogenic mutation or compound heterozygous for two different mutations (15). Besides, it has been determined that some ichthyosis clinical features are associated with particular TGM1 genotypes (10)
In current study three different mutations in TGM1 were identified as genetic causes of ARCI in three unrelated Iranian families (Table 1), two of which (p.Lys219Glu and p.Glu266Gly) were located in highly conserved residues. p.Lys219Glu is located in β-sandwich domain, while p.Glu266Gly is located in catalytic core domain of TGM1. Pathogenic mutations in both domains have been previously reported in Autosomal Recessive Congenital Ichthyosis (15)
Table 1
The summary of whole exome sequencing results in the studied families diagnosed with hereditary ichthyosis.
Family number | Gene name | Detected variants | Variant type | Variant classification | Genotype | Known/Novel |
Family 1 | TGM1 | c.655A > G; p.Lys219Glu | Missense | Likely Pathogenic | Homo | Novel |
Family 2 | ALOX12B | c.527 + 2T > G | Splice site | Pathogenic | Homo | Novel |
Family 3 | ALOX12B | c.1654G > T; Gly552Cys | Missense | Pathogenic | Homo | Novel |
Family 4 | TGM1 | c.797A > G, p.Glu266Gly | Missense | Unknown significance | Homo | Novel |
Family 5 | TGM1 | c.428G > A, p.Arg143His | Missense | Pathogenic | Het | Known |
In case 5, we found c.428G > A pathogenic variant in TGM1 in two affected siblings. The mutation had been reported as pathogenic in ClinVar. We did not find the second allele in this family which could be an undetected single nucleotide variant, located in the regions not covered by WES, or a copy number variant.
ALOX12B gene, located at chromosome 17p13.1, is another significant gene associated with ichthyosis (13, 16). ALOX12B is more than 15 kb, consisting of 14 exons. It encodes a lipoxygenase and is responsible for the conversion of arachidonic acid to 12R-hydroxyeicosatetraenoic acid.
ALOX12B and ALOXE3 were firstly described as causative genes for ARCI by Jobard et al. (17). To date, approximately 88 pathogenic variants have been reported in ALOX12B, among which 64% are missense mutations. Majority of mutations are located in exon 9 (17). Pathogenic mutations in this gene appeared to be responsible for ichthyosis phenotypes in 2 out of the 5 cases presented (Table 1). While a clear genotype-phenotype correlation has not been clearly elucidated yet, it seems that mutations in evolutionarily conserved ALOX12B regions are associated with more severe ichthyosis phenotypes (18).
Here we have reported two pathogenic variants including c.1654G > T (Gly552Cys) and c.527 + 2T > G, located in exon 12 and 4 of ALOX12B respectively. The variant c.527 + 2T > G has been classified as pathogenic in ClinVar (RCV000504018); however, c.1654G > T variant has not been reported previously. The novel mutation was predicted to be damaging by several in-silico prediction tools including BayesDel, Meta SVM, REVEL, EIGEN, PROVEAN, and MutationTaster.
In agreement with available studies, our study supports promising potential of WES in genetic diagnosis of hereditary ichthyosis (19). We showed that WES is a powerful tool that simultaneously identifies previously known mutations and detects novel disease-causing variants.
One of the major benefits of unambiguous identification of ichthyosis-causing mutations in a family is the establishment of fundamental prerequisite to performing DNA-based prenatal or preimplantation genetic tests, when faced with the prospects of risk of recurrence of a genetic skin disease (20). Four out of 5 families presented here, were referred to us either during pregnancy or before reproductive planning since they were at increased risk of ichthyosis in their next generation due to the family history of ichthyosis. Among which, families 1, 2, 3 benefited from WES as it helped with identification of the mutation in the probands followed by carrier detection of at-risk relatives, and prenatal diagnosis, if indicated. Due to the high frequency of consanguineous marriages in Iran (21), it is of paramount importance to choose the proper approach for molecular diagnosis of this disease.
In summary, our findings suggest that WES should be considered in the clinical work-up of patients suspected of having ichthyosis.