1 Introduction
Epidermolysis bullosa (EB) is a group of clinically and genetically heterogeneous diseases characterized by trauma-induced mucocutaneous fragility and blister formation. EB can be grouped into categories, including EB simplex (EBS), junctional EB (JEB), dystrophic EB (DEB), and kindler EB (KEB). The EBS is the most common type of EB and is characterized by fragility within the epidermis. The EBS with mottled pigmentation (EBS-MP; OMIM 131960) is a rare subtype of EBS characterized by intraepidermal blistering and progressive reticular hyperpigmentation and is caused by mutations in keratin 5 (
KRT5). The JEB is characterized by blistering within the lamina lucida of the basement membrane zone (BMZ) and can be caused by mutations of genes, including type XVII collagen (
COL17A1), laminin subunit α3 (
LAMA3), laminin subunit β3 (
LAMB3), laminin subunit γ2 (
LAMC2), integrin α6β4 (
ITGA6 and
ITGB4), and the integrin α3 subunit (
ITGA3). The JEB can be broadly divided into Herlitz (OMIM 226700) and non-Herlitz (OMIM 226650) types on the basis of severity and survival years. The Herlitz JEB is severe and usually cannot survive past the first years of life [
1,
2]. The laryngo−onycho−cutaneous syndrome (LOCS; OMIM 245660) is a subtype of JEB that primarily affects the Punjabi Muslim population and is characterized by prominent skin and mucosal granulation tissue, leading to delayed wound healing, laryngeal obstruction, and blindness. The bi-allelic mutations located in the
LAMA3A-specific exon, such as c.169C>T [
3,
4] and c.151dupG, have been reported to cause LOCS [
5]. The DEB (OMIM 226600) is characterized by blisters formed beneath the BMZ and can be caused by mutations in the type VII collagen gene (
COL7A1). The KEB (OMIM 173650), the rarest type of EB, is characterized by blistering on multiple levels within and beneath the BMZ and is caused by mutations in the fermitin family member 1 gene (
FERMT1) [
6].
EB is extremely rare and has a prevalence of less than 1 case per 2000 individuals [
7], and differences about the prevalence of specific EB subtypes among different geographical areas have been revealed. However, the clinical features of EB are not yet fully understood among the Chinese population due to the limited reports of patients with EB [
8,
9].
2 Results
Five Chinese families with EB were investigated. Clinical information was collected, and written informed consent was obtained. Genetic testing was performed in accordance with the Helsinki Declaration and approved by the Peking Union Medical College Institutional Review Board. Variants were screened by whole-exome sequencing (WES) and confirmed by Sanger sequencing. Clinical manifestations of 7 patients with EB and 8 different pathogenic variants of three genes (i.e., LAMA3, COL7A1, and KRT5) are summarized in Table 1.
2.1 Family 1
In Family 1, the proband is a 42-year-old male. He had severe fingernail onychodystrophy (Fig. 1A(a)) ; extensive mutilating erosions and scars involving extremities, elbows, and orofacial regions; and hypertrophic scars in the armpit (Fig. 1A(b)). Intraoral examination revealed severe enamel hypoplasia, which presented with discolored, pitted teeth, hypodontia, and excessive carious lesions in his oral cavity (Fig. 1A(c)). The brother of the proband was also affected (denoted as II-2 in Family 1, Fig. 1B(a)) and had eyelid scarring and symblepharon, severe scars in his extremities and neck, especially the lower limb (Fig. 1A(d–f)). WES revealed a novel homozygous nonsense variant in the exon 1 of LAMA3 (NM_000227.4: c.47G>A; NP_000218.3: p.W16*) in the proband and his affected brother, and the mother of the proband was found to be heterozygous for the nonsense variant (Fig. 1B(b)). Informed consent was obtained first, and the RNA/protein from the skin tissue of the proband and brother of the proband in Family 1 was collected and extracted to elucidate the effect of this nonsense variant on LAMA3 expression level. Real-time quantitative PCR results showed that the LAMA3A (NM_000227.4) mRNA levels of the proband and brother of the proband in Family 1 were reduced to approximately 23% and 28%, respectively (Fig. 1B(c)). The Western blotting results also revealed that the protein levels of LAMA3A (NP_000218.3) in the proband and his brother were reduced remarkably compared with those of the normal individual (Fig. 1B(d)).
2.2 Families 2–4
In Family 2, the proband was an 11-year-old boy (denoted as Family 2-II-1 in Fig. 2A(a)). He suffered from generalized blistering since birth, and the skin of extremities was most severely affected. He presented with serious fingernail dystrophy. Examination revealed extensive blistering, wounds, and scarring in his neck, backs, hands, and elbows (Fig. 2B(a–c)). In Family 3, the proband was a 54-year-old man (denoted as Family 3-II-1 in Fig. 2A(b)). He was referred to the clinic with a chief complaint of nonhealing skin ulceration on the feet. He suffered from predominantly acral blistering since birth, scarring, erythema, ulceration, crust on his hands, feet, and elbows (Fig. 2B(d–f)). He had a history of esophageal strictures. The histopathological examination of the skin biopsy specimen showed the absence of squamous cell carcinoma. In Family 4, the proband was a 5-year-old girl (denoted as Family 4-II-1 in Fig. 2A(c)). Since birth, she suffered from intermittent blistering and excoriated lesions on her neck, abdomen, and extremities after minor trauma and healed with hypertrophic scars. Severe hemorrhagic blisters were present on her neck, and she suffered from dysphagia, nail dystrophy, and partial nail loss (Fig. 2B(g–k)). Novel compound variants in COL7A1, including a novel missense variant (NM_000094: c.2531T>A; NP_000085.1: p.V844E) in exon 19 and a frameshift deletion (c.6811_6814del, p.R2271fs) in exon 86 of COL7A1, were detected in Family 2. These novel variants were not present in population databases (ExAC no frequency). In silico analysis performed using SIFT, PolyPhen-2, Mutation Taster, M-CAP, and CADD predicted them as pathogenic variants. The proband was compound heterozygous for both variants and inherited the variant c.2531T>A from his mother and the variant c.6811_6814del from his father. Four known variants in COL7A1 were detected in Families 3 and 4 (Fig. 2A). These variants included compound variants of a nonsense variant (c.8569G>T, p.E2857*), an 11 bp deletion (c.3625_3635del, p.S1209fs) in the proband of Family 3, and compound variants of a missense variant (c.6187C>T, p.R2063W) in exon 74, which was inherited maternally, and a de novo missense variant (c.7097G>A, p.G2366D) in exon 92 of COL7A1 in proband of Family 4. The locations of variants identified in our study in the domains of COL7A1 protein are shown in Fig. 2C.
2.3 Family 5
In Family 5 (denoted as the pedigree in Fig. 3A), the proband (II-1) and her mother (I-2) suffered from blistering since birth, and the tendency to blister decreased with age. The additional mottled and reticulate macular pigmentation increased especially at the previous blistering sites. The variant c.74C>T (p.P25L) in KRT5 (NM_000424.4) was detected in the proband and her mother, and the variant was inherited maternally in the family (Fig. 3B).
3 Discussion
Laminin-332 genes, including
LAMA3,
LAMB3, and
LAMC2, code for heterotrimeric noncollagenous glycoproteins consisting of α, β, and γ subunits that build anchoring filaments in the lamina lucida. To date, 52
LAMA3 (NM_000227.4) mutations of EB (i.e., 16 nonsense mutations, 7 missense mutations, 9 splice site mutations, 19 frameshift mutations, and 1 gross deletion) have been identified (Fig. 1C).
LAMA3, which is located on 18q11.2, contains 76 exons and 3 major alternative transcripts. These transcripts encode three laminin α3 polypeptides, i.e., α3a, α3b1, and α3b2. Two major transcripts, including
LAMA3A and
LAMA3B, encode laminin a3a and a3b1.
LAMA3A is expressed from a promoter within intron 38, and its protein product is encoded by exons 39 to 76 (5175 bp open reading frame, encoding 1724 amino acids).
LAMA3B is long and consists of exons 1 to 38 and the common 3′ exons 40 to 76, and exon 39 is skipped (10 002 bp open reading frame, encoding 3333 amino acids) [
10]. Here, we identified a novel homozygous nonsense variant p.W16* located in the exon specific to the laminin α3a transcript (designated exon 1 of
LAMA3A, NM_000227.4). Clinically, our case harboring this nonsense mutation most closely resembles LOCS particularly the symblepharon, leading to visual impairment, fingernail dystrophy, and laryngeal obstruction in our patients. Moreover, we reveal that the
LAMA3 homozygous nonsense variant remarkably reduces the
LAMA3A mRNA and protein levels. Thus, the reduction in the
LAMA3A may affect its function to the assembly competent and ability of making anchoring filaments extracellularly in the cutaneous BMZ, whereas about 30% of
LAMA3A mRNA with the variant may escape from nonsense-mediated mRNA decay. Considering that the majority of
LAMA3 mutations reported in H-JEB patients are premature termination codon mutations located on
LAMA3A and
LAMA3B transcripts, the loss of laminin a3a and a3b expression is hypothesized to cause H-JEB and that the loss of a3a alone causes LOCS [
5], which can explain the mild phenotype of patients with this homozygous nonsense variant (c.47G>A, p.W16*) that only affects the
LAMA3A transcript.
COL7A1 is a large gene located on chromosome 3p21.31 that comprises 118 exons coding for a pro-a1(VII) procollagen polypeptide consisting of 2944 amino acids. Each pro-a1 (VII) polypeptide chain contains a central triple helical collagenous domain flanked by a large amino-terminal (NC-1) and a small carboxyl-terminal (NC-2) noncollagenous domains. We reported six variants (2 novel and 4 reported variants) in COL7A1 from three patients. The novel missense variant is located at the NC1 domain and may affect its biological function to interact with BMZ components at one end and at the other end with type IV collagen in “anchoring plaques.” The novel frameshift variant (c.6811_6814del, p.R2271fs) may interrupt the Gly-X-Y repeats within the crucial collagenous domain of COL7A1 protein.
EBS-MP is a rare subtype of EBS mostly caused by the missense variant of p.P25L in
KRT5. Several reports about p.P25L in
KRT5 concerning multiple cases with European and Japanese origins are available [
11–
17]. Here, we report two patients with EBS-MP from a Chinese pedigree. A heterozygous p.P25L variant in the first exon of
KRT5 is identified in the proband and the proband’s mother. To our knowledge, this study is the second report of a Chinese family harboring the recurrent variant. Our report extends the limited number of EBS-MP cases and provides further evidence that the
KRT5 variant is also responsible for this rare phenotype within the Chinese population.
Patients with EB show wide interfamilial clinical variability. However, the clinical features of EB are not yet fully understood among the Chinese population due to the rare reports of patients with EB. Here, clinical manifestations of 7 patients with EB and 8 different pathogenic variants of three genes (i.e.,
LAMA3,
COL7A1, and
KRT5) are summarized in Table 1 [
11–
22]. The genotype–phenotype relationship of known variants identified in our patients based on previous reports is summarized in Table S1 [
19–
23]. In this study, we characterize the genotypes and phenotypes in five Chinese families with EB. This study reported the homozygous nonsense variant in
LAMA3 that causes EB within the Chinese population and investigated its molecular characteristics, which will benefit our understanding of genotype–phenotype correlations.
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