A novel missence mutation in the transglutaminase-1 gene in an autosomal recessive congenital ichthyosis patient

Autosomal recessive congenital ichthyosis (ARCI) is a rare hereditary disorder of cornification. ARCI is a congenital recessive skin disorder characterized by generalized scaling and hyperkeratosis. Mutations in the transglutaminase-1 (TGM1) gene, which encodes for the epidermal enzyme transglutaminase-1, are one of the causes of ARCI. The transglutaminase 1enzyme, is critical for the assembly of the cornified cell envelope in terminally differentiating keratinocytes. In this study a nine-year old boy who presented with ARCI. The whole exome sequencing WES of transglutaminase-1 gene was investigated. The patient harbored a homozygeous mutation of C.1165T>C transition located in exon8 of TGM1 gene, resulting in the substitution of argenine by serine at amino acid position 389. The parents and one of the sibs were hetrozygeous for the variant and one was homozygeous for wild allele. The mutated allele was not found in controls. This mutation localized in this study correspond to the core catalytic core domain of enzyme, based on provean algorithm, the variant at position level was predicted to decrease TGM1 Enzyme activity by producing an unstable protein.

Large darke, plate-like cutaneous scales with minimal erythema is a feature of LI patients, whereas, in NBCEI patient's erythroderma with overlying white scales is a common feature of the disease [5][6][7].
In this study exome sequencing applied for an Iranian ichthyosis patient, and a novel homozygous causing variant in TGM1 was identified.

Patient and methods
A nine-year-old boy suffering from ichthyosis was the proband of the study. Dry, rough scaly skin all over the body was started at birth. Clinical features include: dark areas over the elbows and knees, scaring deformity of the hands, depressed nasal bridge, non-developed ears are other signs of the disease in the proband, prominent erythroderma and fine white, superficial semi adherent scales nail dystrophy, subungual hyperkeratosis, scalp involvement and loss of eyebrows was observed in the proband in clinical examination. Proband was the yield of first cuisine marriage, the two other children of the parents were normal, and the mother has been experienced a miscarriage.
Whole exome sequencing WES has been used extensively to diagnosis novel disease and find novel causative mutations for known disease phenotypes. In this issue WES is investigated. After taking informed consent, 5 ml peripheral blood sample were drowned from patient and other members of the family. DNA extracted from peripheral blood lymphocytes by BIOGENT, Korean DNA extraction kit for WES using the Maxwell RSC instrument (standard instrument protocol). DNA fragments containing targeted coding sequences were captured using the seqcapEZ Med exome target enrichment kit and sequenced on the illumine Hiss equation 2500 platform. The resulting sequence was analyzed for single nucleotide variants and small insertion and deletions differing from the reference genome (human genome 19 (HG 19). In this study the mutations affected the predominant gene that causes ARCI encoding keratinocyte transglutaminase (TGM1) investigated.

Results and discussion
Clinical characterization of affected individual included a dry rough scaly skin all the body with dark area over the elbow diagnosed as nonbullous congenital ichthyosis form erythroderma (NCIE). A form of the autosomal recessive congenital ichthyosis ARCI. ARCI is a heterogeneous group of congenital keratinization disorders [15,23,24]. The most common Causes of ARCI are the mutations in TGM1, that encodes Transaminase-1 enzyme [18].
To date, more than 115 various mutations in the TGM1 gene in 234 unrelated patients from different racial/ethnic backgrounds have been reported and the common type of the mutation is missense. The most common reported mutations in north America and Norouch patients was the c.877-2 A>G [18].
In this paper, the clinical and molecular aspects of ARCI have been investigated in patients from Iran. The proband showed the typical features of ARCI. Homozygous state of c.1165 C>T was observed in the proband, parents and one of the sibs were heterozygous for the variant and one sib was homozygous for wild allele (Figure 1). The mutated allele was not found in 100 controls. In silico analysis was done by mutation taster 2.0 and FATHMM MKL coding prediction tools and the effect of the variant on protein was evaluated by Poly-phen 2.0, SIFT and PROVEN online soft-wares. Variant calling was done according to American College of Medical Genetics (ACMG) guidelines. The outcome effect is substitution of Arginine by serine in position 389 of the protein, a highly conserved position. Based on conservation scores this novel variant is probably damaging. Substitution of Arginine by serine can cause great effect on protein configuration by loss of one positive charge, because serine is a neutral amino acid, in addition serine is smaller than Arginine in size. It seems that Arginine in position 389 play an important role, two missense mutations in this site have been reported by Akima (p.R389H) and Shevchenkov (p. R389P) in ARCI patients [24,25]. Also, the variant p.R389H in compound heterozygous state with p.R307W in TGM1 has been reported as ARCI causing [26].
In protein level, p.R389C decreases the TGM1 enzyme activity by producing an unstable protein, based on PROVEAN prediction software [18,27]. From the mutational analysis investigated by WES, the proband was confirmed to be a homozygeous c>t transition at position 1165 in the exon 8 region with genotype Missence.Arg of TGM1 gene. The origin wild type residue and novely introduced mutant residue often differ in these properties. The mutant residue (ser.) is smaller than the wild-type residue (Arg.) that the wild-type residue charge is positive while the mutant residue charge is neutral. Sequential arrangement of the domain structure of transglutaminase reveal that the novel mutation localized in this study correspond to the core catalytic domain of this enzyme. The core catalytic domain started at amino acid positioned in 224 to end 577 area. The previous study defined some other mutations in the exone 8 (Table 1) were in the catalytic core domain [18,28]. Definitive and interpretation of the effects of these mutations on the catalytic functions of the TGM1 protein cannot be provided, owing to lack of good models. However, based on provean algoritm, the variant (p. Arg389Cys) at the protein level was predicted to decrease TGM1 enzyme activity by producing an unstable protein.

Conclusion
In conclusion our study revealed one mutation in the TGM1 gene in an Iranian patient. This mutation is a novel variant missence (c.1165C>T) and may also be prevalent among Iranian populations. The results are useful for genetic counseling in the pedigree and applicable for preventing disease recurrence via preimplantation genetic diagnosis.