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Introduction

Lipoid proteinosis (LP) was first described in 1929 by Wiethe and Urbach as a rare autosomal recessive inheritance disease (1). The major clinical features of LP include intracranial calcification, infiltration and thickening of skin mucosal tissues, hair loss, recurrent parotitis, papules in the eyelids, dental underdevelopment, hoarseness, and even suffocation and mortality (2,3). Certain patients with LP also exhibit mental and psychiatric symptoms (4). These pathological features are now standard for clinical LP diagnosis (5).

Although the molecular mechanism of occurrence and development of LP require further elucidation, it is generally considered that the gene mutation of extracellular matrix protein 1 (ECM1) forms the pathological basis of LP (6,7). Hamada et al (6) performed the first linkage analysis and survey of the ECM1 gene in patients with LP, and identified the localization of the ECM1 gene at human chromosome 1q21 locus Further studies indicated the role of an ECM1 gene mutation in LP. Systematic surveys across different countries and regions demonstrated variable mutation points of the ECM1 gene in individual patients with LP, suggesting the potential gene polymorphism of the ECM1 gene in patients with LP (7,8).

The ECM is a group of glycoproteins that are secreted from animal cells to form the complex matrix between cells and exert critical functions during the modulation of intra-cellular transport, signal transduction and energy exchange (9–11). ECM1 has been demonstrated to possess important roles in the differentiation of cellular epidermal cells, and the connection between glycoproteins of the epidermal layer and collagen (12,13). Among the 10 exons of ECM1 gene, frequent mutations occur between exon 6 and exon 7 (7).

Due to its rare incidence, clinical reporting of LP is sparse in China. The diagnosis of LP in China relies mainly on clinical features. With the rapid progression of molecular biology at the clinical level, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis together with DNA sequencing have now become important methods to study the existence of ECM1 gene mutation, thus providing evidence to improve LP diagnosis (6,7). Such a combination of RT-qPCR and gene sequencing can also provide important information for genetic counseling, prenatal diagnosis and prepotency (14).

The present study collected clinical cases for diagnosing LP based on its pathological features. RT-qPCR was performed to detect the presence of mutation in the ECM1 gene of patients. Common gene mutation loci and allele frequencies were further analyzed in patients with LP. The present study aimed to investigate the clinical features and inheritance pattern of ECM1 gene mutation in Chinese LP patients, thus providing useful information for the diagnosis and treatment of LP in China.

Materials and methods

Reagents and drugs

PCR kits (SanTaq PCR Master Mix and Taq Plus DNA Polymerase) were purchased from Shanghai Shenggong Biology Engineering Technology Service, Ltd. (Shanghai, China). Primers were synthesized by Beijing Sanbo Yuanzhi Biotechnology Co., Ltd. (Beijing, China). Universal DNA Purification Kit for PCR products was purchased from Tiangen Bio (Beijing, China). Sequencing of PCR products was performed by Beijing Sanbo Yuanzhi Biotechnology Co., Ltd.

Patients

From November 2016 to September 2017, 4 patients (2 females and 2 males with a median age of 49, ranging from 39–65) with LP in the department of dermatology of Yantai Yuhuangding Hospital (Yantai, China) were recruited for the present study. Inclusive criteria were (15): Intracranial calcification, infiltration and thickening of skin mucosal tissues, hair loss, recurrent parotitis, papules in the eyelids, dental underdevelopment and hoarseness. The present study was approved by the ethical committee of Yantai Yuhuangding Hospital and obtained written consents from patients or families. Family surveys were also performed if possible.

Histopathological examination

Pathological examination was performed on patients with LP, as previously described (16,17). The keratinization degree of epidermis was examined to see if atrophy or atypical hyperplasia of the spinous layer existed. Dermis was then examined for any notable thickening, vessel dilation, as well as thickening of vascular walls and the existence of extracellular translucent layer of eosinophilic cells at the superficial layer of the dermis. Periodic acid-Schiff (PAS) staining performed for this translucent layer of eosinophilic cells and observed under an Olympus BH2 light microscope.

Genomic DNA extraction

With the consent from patients and families, 5-ml peripheral blood samples were collected from veins for extracting genomic DNA as previously described (18). Equal volumes of phenol were added to the blood which was then centrifuged (3,000 × g for 8 min) at room temperature. The upper aqueous phase was collected to a new tube with the addition of equal volume phenol for another centrifugation at 3,000 × g for 8 min at 4°C. The upper aqueous phase was again saved in a new tube with an equal volume of phenol/chloroform (1:1) mixture. Following centrifugation at 3,000 × g for 8 min at 4°C, the upper phase was saved and mixed with equal volume of chloroform. The upper phase was then collected following 3,000 × g centrifugation for 8 min 4°C and mixed with 1/10 volume of sodium acetate (3 M) and absolute ethanol. The white participation was then carefully removed, followed by washing with 75% ethanol. The DNA pellet was dissolved in sterilized water and the genomic DNA was then used in the following experiment.

PCR

PCR primers were designed based on the ECM1 gene sequence. Primer sequence for ECM1 were: Forward, 5′-GGCTTTTGCTTACTCCTTCTACCC-3′; Reverse, 5′-AGTAGCTGGCAGGTTGCGTGG-3′. The primer sequence for b-actin was Forward, 5′-CGTTGCTATCCAGGCTGTGCTAT-3′; Reverse, 5′-CAGCTTCTCCTTAATGTCACGC-3′.

PCR was performed in a 25 µl system containing 1 µl genomic DNA template, 2.5 µl PCR buffer (10X), 2 µl dNTP mixture (1 mM), 1 µl of each primer (20 µM), 1 µl Taq DNA polymerase, 20 mM MgCl2 and 16 µl H2O. PCR was conducted under the following conditions: 95°C denaturing for 5 min, 30 cycles of 95°C for 45 sec, 55°C for 45 sec and finally 72°C for 6 min. Products were kept at −20°C.

Gel electrophoresis

Agarose gel electrophoresis was performed using standard procedure; 5 µl PCR products were mixed with 1 µl loading buffer (6X) and loaded into 0.8% agarose gel. The electrophoresis was performed in a 120 V electrical field for 10 min as previously described (19).

PCR products sequencing

Following agarose gel separation, PCR products were sequenced using directly using Big Dye® Terminator v3.1 cycle sequencing kit (Thermo Fisher Scientific, Waltham, MA, USA). The sequencing was performed by Beijing Sanbo Yuanzhi Biotechnology Co., Ltd. The sequencing analyzer was from Applied Biosystems (3730XLDNA; Thermo Fisher Scientific, Inc.).

Sequence alignment

Sequencing results were aligned with known gene fragments using a Pubmed database as previously described (20).

Results

Pathological examination of patients with LP

In all 4 patients with LP who took part in the present study, similar histopathological features were observed; over-cutinization and a thickening of the spinuous layer with atypical arrangement. Homologous red precipitation was observed in sweat gland, vessels, hair follicles and the middle layer. PAS staining demonstrated a positive reaction, with an onion-shaped arrangement of PAS-positive substances in epidermal layers of the skin (Fig. 1).

Figure 1. Onion-like arrangement of PAS-positive signals in skin epidermal tissues (magnification, ×40). PAS, Periodic acid-Schiff.

Family survey of LP patients

Only 1 out of the 4 patients with LP agreed to have a family survey performed. As demonstrated in Fig. 2, the incidence of LP fitted the autosomal recessive inheritance pattern.

Figure 2. Family tree of 1 patient with LP. Black, LP disease; white, healthy individuals; black/white, mutation carrier. Square, male; circle, female. The proband is the fifth-generation male in the family. LP, lipoid proteinosis.

PCR results

PCR and agarose gel electrophoresis were performed on the genomic DNA of patients with LP. As demonstrated in Fig. 3, the PCR products had a length of ~1,250 bp, consistent with the prediction, suggesting successful amplification.

Figure 3. PCR results of patients with LP. Agarose gel electrophoresis was performed on PCR products of patients with LP. PCR, reverse transcription-quantitative polymerase chain reaction; LP, lipoid proteinosis; ECM1, extracellular matrix protein 1.

PCR product sequencing and alignment

Following the DNA sequencing of PCR products, a novel mutation locus was identified at the 6th exon of the ECM1 gene. Specifically, a CAG-insertion at position 508 bp of the ECM1 gene: 5′-AAGGCCGCCCAG-3′ 3′-TTCCGGCGGGTC-5′ 5′-CTGGGCGGCCTT-3′. From these sequencing results a CTG-insertion mutation at position 508 bp of the ECM1 gene may be identified. This frameshift mutation was designated c.508insCTG for short. Such a mutation caused the transition of the 169th amino acid of the ECM1 protein to proline instead of leucine.

Gene sequencing and alignment results in all 4 patients with LP in the present study are presented in Figs. 4–7. These results suggested point mutations of the ECM1 gene, including one homozygous point mutation (C220G) as previously reported, one novel homozygous mutation (c.508insCTG) and two heterozygous mutations (C220G/P.R481X and c.507delT/c.l473delT).

Figure 4. DNA sequencing of patient number 1. This patient exhibited one homozygous point mutation (C220G). The arrow indicates the mutation site. LP, lipoid proteinosis.

Figure 7. DNA sequencing of patient number 4. This patient exhibited a heterozygous mutation of c.507delT/c.l473delT (arrow). LP, lipoid proteinosis.

Discussion

LP is a rare skin disease with autosomal recessive inheritance. Various studies have indicated that an ECM1 gene mutation underlies the pathology of LP (1,2). In the Chinese population, however, the association between ECM1 mutation and occurrence of LP requires further investigation. The present study discussed the mutation types of the ECM1 gene in Chinese patients with LP.

The present study obtained three main results: i) Histopathological examination revealed the consistent features of LP in the patients; ii) PCR amplification of the ECM1 gene in patients with LP obtained relevant bands; and iii) sequencing of PCR products identified point mutations of ECM1 gene, including one homozygous point mutation (C220G) as previously reported, one novel homozygous mutation (c.508insCTG) and two heterozygous mutations (C220G/P.R481X and c.507delT/c.l473delT).

A previous study demonstrated the existence of LP-associated ECM1 gene mutations mainly in exons 6 and 7 (21). The present study agreed that exons 6 and 7 possessed gene mutations. The difference was that previous reports identified mainly homozygous mutations (8–10), while heterozygous mutations occurred in the present study. The mutation of the ECM1 gene can lead to LP, even by heterozygous mutation.

A previous study suggested that consanguineous marriage is a major reason for LP (22,23). The majority of studies also agree that homozygous mutation of the ECM1 gene is typically observed. The present study identified the existence of heterozygous mutations in patients with LP. These data suggested higher susceptibility even without consanguineous marriage, stressing the importance of premarital examination and prepotency.

Certain weaknesses and limitations also existed in the present study: i) Relatively few cases were included (n=4) and may bias the final results; ii) only the association between the ECM1 gene and LP was analyzed, without considering other possible factors; and iii) the present study did not further investigate the molecular mechanism of the ECM1 mutation for LP, or the normal function of ECM1 protein for inducing LP.

In summary, the present study demonstrated the association between ECM1 gene mutation and patients with LP. Patients with LP exhibited one homozygous point mutation (C220G) as previously reported, one novel homozygous mutation (c.508insCTG) and two heterozygous mutations (C220G/P.R481X and c.507delT/c.l473delT).

Acknowledgements

Not applicable.

Funding

This work was supported by Yantai University (grant no. 2012084).

Availability of data and materials

All data generated or analysed during this study are included in this published article.

Authors' contributions

DG designed the study and wrote the manuscript. DG, XM, PL, SZ and JC performed the experiments and analysed the data.

Ethics approval and consent to participate

All experimental procedures involving animals were approved by the Ethnic Committee of Yantai Yuhuangding Hospital (Yantai, China). Not applicable for consent to participate for human individuals.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References