A novel STS mutation and an Xp22.3 microdeletion from one Chinese family with X-linked ichthyosis

Background X-linked ichthyosis (XLI, OMIM# 308100) is a relatively common type of skin disorder, characterized by widespread, dark brown, polygonal scales and generalized dryness. Most patients (90%) are contributed to genomic deletion of the entire STS (steroid sulfatase) gene encoding steroid sulfatase, with the remaining cases being caused by point mutations or partial deletions. The patient carrying genomic deletion comprising the entire STS gene and adjacent genes often manifests neurological symptoms. Methods Herein, we reported a large four-generation Chinese family with 5 individuals with XLI with/ without mental delay. Genetic analysis was conducted by whole exome sequencing and conrmed by Sanger sequencing and quantitative Real-time PCR. Results These patients present generalized dryness and scaling of the skin with dark scales of the skin on trunk, and limbs. Two pathogenic variants, a novel STS mutation (c.1532A > G, p.E511G, NM_000351) and a ~ 2 Mb genomic microdeletion at Xp22.3 (6,451,785-8,434,424) embracing entire STS gene were identied in this family. In addition, an individual with Xp22.3 deletion has mental delay. Conclusion Our study expands the genetic prole of the STS gene in XLI patients and highlights the clinical application of exome sequencing.


Background
The ichthyoses are a heterogeneous group of dermatological conditions characterized by widespread, dark brown, polygonal scales and generalized dryness. Cutaneous manifestations are frequently present few weeks after birth, usually do not improve with age but affect a person throughout life [1]. The majority of ichthyoses is inherited and can be pinpointed to characteristic genetic mutations. X-linked recessive ichthyosis (XLI, OMIM#308100) is a relatively common type of ichthyosis with an estimated prevalence ranging from 1:1500 to 1:6000 worldwide [2]. XLI was rst described in 1927 by Lundborg [3]. It may occur solely as a skin disorder or may be associated with other physical ndings such as corneal opacities (up to 50% of cases), cryptorchidism (20% of cases), chondrodysplasia punctata, and nephritic syndrome [4]. X-linked ichthyosis is caused by the deletion of or mutations in the STS (steroid sulfatase) gene encoding steroid sulfatase. The STS gene is located on chromosome Xp22.3, containing 10 exons and spans about 146 kb [5]. No homologous sequences were detected on the Y chromosome [6] and non-X-inactivation was reported in this gene [7]. Most patients (85%-90%) have a complete or partial deletion of the STS gene, while point mutations within STS gene account for about 10% of cases [8]. The protein encoded by STS gene is a steroid sulfatase enzyme, a membrane-bounding protein present in most organs and tissues including the skin, breast, blood and brain [9]. Patients with mutations involving solely the STS gene may present with attention de cit hyperactivity disorder (ADHD), while those with deletions including neighboring genes such as neuroligin 4 (NLGN4) may be associated with multiple neurodevelopmental disorders, including mental retardation, autism spectrum disorders (ASDs), ADHD, and seizures [4]. In addition, a recent study showed adult males with XLI disease-causing deletions are apparently at increased risk of cardiac arrhythmias and self-reported mood problems [10].
In this study, we report a four-generation Chinese family with XLI and identi ed a novel hemizygote point mutation (c.1532 A > G, p.E511G, NM_000351) in one branch and a recurent ~ 2 Mb genomic deletion (Xp22. 3: 6,451,785-8,434,424) comprising the entire STS gene in other branch. These variants were absent in healthy family members and cosegregated with XLI in this pedigree. Our study extended the knowledge of mutation spectrum in XLI patients and highlighted the clinical application of exome sequencing.

Subjects
The XLI family in this study has 47 members across four generation and 5 patients were diagnosed with ichthyosis. The family pedigree is shown in Fig. 1A. Written informed consents were obtained from all subjects or the patient (III:6)'s guardian, and the study was approved by the institutional review board of

Whole-exome Sequencing
Whole exome sequencing was carried out by a commercial company (Chigene, Beijing, China) following standard experimental procedures. The exome was captured using IDT the xGen Exome Research Panel v1.0 Kit. The high-throughout sequencing was based on Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA). The read length of the sequencing was paired-end 150 bp, and the read depth was 120×. Trio whole exome sequencing was carried out, respectively, on the proband family (III:1, II:1, and II:2) and his cousin family (III:6, II:7, and II:8).

Bioinformatics Analysis
The possible effects of the variant on the function and structure of protein, and likelihood of pathological damage were analyzed by SIFT, MutationTaster, PolyPhen2-HDIV, Provean, PolyPhen2-HVAR, M-CAP and REVEL. The sequences of steroid sulfatase protein from zebra sh, trubripes, Mmulatta, human and chimpanzee were aligned using Clustal Omega Sanger sequencing.

Clinical information
The proband (III:1, male, 32 years old, shown in Fig. 1A) came to our genetic clinic for counseling as his sister (III:3) was pregnant. The proband complained scattered ichthyosis all over the body, which became more severe in winter with dry and cold weather (shown in Fig. 1B). Our patient has one aunt and three uncles from his mother's side, and his aunt's son (III:5) also displayed similar symptom. In addition to the classic feature of ichthyosis, the son (Ш:6, 37 years old) of his youngest uncle (П:7) exhibited intellectual disability and speech delay, with a history of febrile convulsion (shown in Fig. 1A). Three daughters (III:7, III:9, III:11) of this uncle did not show signs of ichthyosis. Interestingly, the branch of his aunt (II8)'s family also have another two individuals with ichthyosis and barriers to effective communication (П:11 and Ш:19), with normal intelligence (Fig. 1A).

Whole-exome Sequencing, Bioinformatics And Segregation Analysis
Through whole exome sequencing, after the ltering steps, a solitary heterozygous missense variant, c.1532A > G (p.E511G), in exon 10 of the STS gene (NM_000351) was identi ed in the proband III:1 based on sequences from NCBI database. Further Sanger sequencing revealed that individual III:5 was hemizygous while II:1, П:5 and III:3 were carriers (shown in Fig. 1C). This mutation is predicted to be pathogenic by bioinformatics tools and conserved (Fig. 1D) among different species, and it is not observed in other family members. The c.1532A > G mutation has not been reported previously, and is not present in any public genomic variants database, including Human Genome Mutation Database, 1000Genomes, ESP6500, ExAC Browser (exome aggregation consortium), and COSMIC (the catalog of somatic mutations in cancer), indicating that c.1532A > G in STS is a novel mutation that may cause ichthyosis.

Discussion
In this study, we reported two variants from one large family, one is a novel missense mutation in the proband with ichthyosis, and the other one is a maternal deletion in Xp22.31 in his cousin (the son of his uncle from his mother's side) with ichthyosis and disability. XLI is a recessive disorder and it almost exclusively affects males. Four XLI female cases have been described, three of these patients were offspring of an affected father and a carrier mother [11,12], and one patient was adopted girl who was homozygous nonsense mutation in the STS gene [8].
To date, in addition intragenic large deletions, a total of 29 STS variants, including the variant (c.1532A > G, p.E511G) reported in this study, have been identi ed. As shown in Fig. 2 and Table 2, of the 29 variants, 5 are truncating, 3 are frameshift, 20 are missense, and 1 is a deletion involving one amino acid, and it seems that exon 7 and 8 are the hotspot variation region.  10 Goodwin et al [36] XLI is characterized by scaly skin on the scalp, trunk and limbs, aggravates in winter and dry climates and alleviate in summer [13,14], which is consistent with the clinical features of our patient and the affected individuals in his family. Previous studies demonstrated that, in addition to skin problems, XLI patients with Xp22.31 deletion usually present with contiguous gene deletion symptoms [15], including short stature(SHOX), Kallmann syndrome(KAL), X-linked chondrodysplasia punctata (ARSE) and X-linked ocular albinism(OA1). In addition, neurodevelopmental conditions, including ADHD, autism, and mental retardation, were also observed in patients with XLI [16,17]. Recently, a study showed adult males with XLI disease-causing deletions are apparently at increased risk of cardiac arrhythmias and self-reported mood problems [10], and altered basal ganglia structure was observed in these patients [10]. In this pedigree in the present study, the patient III:6, who had ichthyosis, mental retardation, delayed speech, and history of febrile convulsion, was found to harbor a Xp22.31 deletion of 1.98 Mb, and the patient has not yet showed heart rhythm problems at the age of 35 years old. Follow-up should be done by regular examination. In addition to STS gene, the deleted region contains 6 neighboring gene (HDHD1, PNPLA4, VX, VCX2, VCX3A, and VCX3B). The VCX3A gene has been reported to be necessary to maintain normal intellectual development and proposed to be responsible for mental retardation in some XLI individuals [18]. However, highly variable phenotypes were observed in individuals with VCX3A deletions, which also happened in our study. Based on the RT-PCR results, it is likely that two patients (II:11 and Ш:19) has Xp22.31 deletion, however they did not present mental retardation but with barriers to effective communication, which further showed that the cognitive behavioral syndromes vary from normal intelligence to mental retardation, even with same size of deletion. Previous report also showed that no difference in deletion size of fragment including VCX-A for XLI patients with and without mental retardation [18]. We also noted that the patient (II:6) harbored a de novo duplication of 2.59 Mb in 11q14.3 containing 12 genes, inducing two protein-coding genes (FAT3 and CHORDC1), 8 noncoding genes (DISC1FP1, NDUFB11P1, OSBPL9P2, OSBPL9P3, PGAM1P9, RPL7AP57, RPS3AP42 and TUBB4BP4) and microRNAs(MIR1261 and MIR4490), it is unclear that whether this duplication is correlated to mental retardation in this patient. Thus, the detailed molecular mechanism underlying mental retardation in patients with XLI and Xp22.31 deletion need further investigation.
Both Xp22.31 deletion and point mutations in the STS gene were also correlated with ADHD, suggesting that STS de ciency has a curial role in the pathogenesis of inattentive/hyperactivity symptoms. Mouse models also support this hypothesis [19]. In addition to ADHD, other symptoms, such as short stature, epilepsy, bone density reduction, and cryptorchidism, were observed in two siblings [20]. However, our XLI patients with point mutation in the STS gene did not display any symptoms of ADHD or other related cognitive behavioral problems.

Conclusion
Through high throughput sequencing of a four-generation Chinese family, we identi ed a novel missense mutation of STS gene in a patient with ichthyosis and a ~ 2 Mb Xp22.3 deletion in his cousin with ichthyosis and mental retardation, expanding the mutation spectrum of XLI. Meanwhile, the study also demonstrates that the genotype-phenotype correlation of XLI is very complicated, further study is needed. Finally, our study emphasizes the importance of whole exome sequencing for genetic diagnosis in rare diseases.

Consent for publication
Written informed consent for publication of clinical details and/or clinical images was obtained from all of the participants or the guardian of one patient who has mental delay.  Identi ed mutations in STS gene in literature and in our study. The mutation in bold was reported in this study.