A novel variant in the GJB6 gene in a large Chinese family with a unique phenotype of Clouston syndrome

Abstract

Clouston syndrome (OMIM #129500), also known as hidrotic ectodermal dysplasia type 2, is a rare autosomal dominant skin disorder. To date, four mutations in the GJB6 gene, G11R, V37E, A88V, and D50N, have been confirmed to cause this condition. In previous studies, the focus has been mainly on gene sequencing, and there has been a lack of research on clinical manifestations and pathogenesis. To confirm the diagnosis of this pedigree at the molecular level and summarize and analyse the clinical phenotype of patients and to provide a basis for further study of the pathogenesis of the disease, we performed whole-exome and Sanger sequencing on a large Chinese Clouston syndrome pedigree. Detailed clinical examination included histopathology, hair microscopy, and scanning electron microscopy. We found a novel heterozygous missense variant (c.134G>C:p.G45A) for Clouston syndrome. We identified a new clinical phenotype involving all nail needling pain in all patients and found a special honeycomb hole structure in the patients’ hair under scanning electron microscopy. Our data reveal that a novel variant (c.134G>C:p.G45A) plays a likely pathogenic role in this pedigree and highlight that genetic testing is necessary for the diagnosis of Clouston syndrome.

Keywords

Clouston syndrome / whole exome sequencing / GJB6 gene / novel variant / unique phenotype

1 Introduction

Ectodermal dysplasias (EDs) are a group of genetic conditions that affect the development and/or homeostasis of two or more ectodermal derivatives, including hair, teeth, nails, and certain glands. Clouston syndrome (OMIM #129500) is a common type of ED [1]; also known as hidrotic ectodermal dysplasia type 2, it is a rare autosomal dominant skin disorder that is characterized by three major clinical signs: nail dystrophy, partial to complete alopecia and palmoplantar hyperkeratosis without sweat gland or tooth abnormalities. Clouston syndrome occurs rarely worldwide, affecting 1 out of 100 000 individuals [2]. A few cases have been reported to include hearing loss, ophthalmologic defects, oral leucoplakia, and diffuse eccrine poromatosis [3,4]. Clouston syndrome was originally systematically described in a French Canadian family in 1929 [5]. Later reports identified a large French family with Clouston syndrome, followed by reports in China, Malaysia, Spain, the UK, and Africa. Based on linkage analysis of four European and American families, the pathogenic gene of Clouston syndrome was mapped to the 13q11–q12.1 region [6]. In 2000, Lamartine et al. [7] first identified a mutation in the gap junction beta 6 gene (GJB6) that segregated with a typical form of Clouston syndrome in two unrelated French families by recombination mapping and further detected two new mutations (G11R and A88V) by direct sequencing. Subsequently, new Clouston syndrome mutations (V37E, D50N) in the GJB6 gene were found by gene sequencing technology. To date, four mutations in the GJB6 gene, G11R, V37E, A88V, and D50N, have been confirmed [7–9].

At present, G11R and A88V are the most common mutations reported in pedigree studies of Clouston syndrome patients in China. Considering the rarity of Clouston syndrome and the difficulty in diagnosing this syndrome on the basis of clinical symptoms alone, it is necessary to carry out a clinical diagnosis combined with genetic testing for each Clouston syndrome family. Here, we conducted a systematic study of a large Chinese Clouston syndrome pedigree. In contrast to previous studies, which mainly focused on gene sequencing, we included a detailed clinical phenotypic analysis of the patients. We summarized the clinical phenotypes of all patients in this family and found that the patients’ clinical phenotypes were diverse and that all patients had a unique phenotype. Subsequently, we used scanning electron microscopy to observe the microstructure of the patients’ hair and identified a novel structure. Furthermore, we performed whole exome sequencing (WES) and Sanger sequencing in some patients and found a novel likely pathogenic variant according to the American College of Medical Genetics and Genomics (ACMG) Standards and Guidelines [10].

2 Material and methods

2.1 Sample collection

We assembled a large Chinese Han pedigree consisting of 60 individuals across five generations, including 22 patients (10 males and 12 females). Written informed consent was obtained. This study conformed to the guidelines for human research as stated in the Declaration of Helsinki and was reviewed and approved by Anhui Medical University (IRB #20150051). The autosomal dominant inheritance pattern was confirmed (Fig.1). The proband (V:1) in this study was a 20-year-old male who had sparse, soft and absent hair, eyebrows and eyelashes from birth. The severity of the phenotype intensified for all hair types (scalp hair, eyebrows, eyelashes, pubic hair, armpit hair) as he aged. His nail curvature increased at 5–6 months, and the nails became thickened, shortened and slow-growing at approximately 1 year old. After puberty, palmoplantar hyperkeratosis began to develop gradually.

A physical examination revealed the total absence of scalp hair, eyebrows, eyelashes, pubic hair, and armpit hair in the proband (Fig.2). Nail thickening, shortening and yellowing (Fig.2), slight hyperkeratosis of the palms and obvious hyperkeratosis of the soles were observed (Fig.2). Sweat glands, teeth, hearing and cognitive function were normal. The detailed clinical information is summarized and shown in Tab.1 and Fig.3.

2.2 Variant detection and confirmation

2.2.1 Whole exome sequencing (WES)

Blood samples were collected from 11 members (8 affected and 3 unaffected individuals) of the family and stored at −80 °C until DNA extraction. Genomic DNA was extracted from peripheral blood lymphocytes by standard procedures via FlexiGene DNA kits (Qiagen). Four patients (IV:11, IV:15, IV:19, and V:10) and one control (III:5) were selected for WES. Genomic DNA fragments corresponding to all exons in the genome were amplified by polymerase chain reaction and subjected to automatic DNA sequencing after purification. The MGIEasy Exome Universal Library Prep Kit and MGIEasy Exome Capture V5 Probe Kit were used for library preparation and capture, followed by 150 bp pair-end sequencing on the BGISEQ-500 platform according to the manufacturer’s recommendations. Clean reads of each sample were aligned to the human reference genome (GRCh38/hg38) using Burrows–Wheeler Aligner (BWA V0.7.15). After quality control of the raw data, variant comparisons and mutation recognition and annotation, a list of suspected variants was obtained. Screening for disease-associated deleterious mutations was then conducted with emphases on all the possible pathogenic variations in the reported GJB6 gene, according to the frequency in the control database, variant types, and prediction of mutation function, such as the score in the REVEL, SIFT, and PolyPhen2_HVAR databases.

2.2.2 Sanger sequencing

The likely pathogenic variant identified by WES was confirmed by Sanger sequencing in 11 patients and 3 controls (patients marked with a blue pentagram in Fig.1) to detect genotype-phenotype co-segregation. Primers flanking all coding regions of the possible variation in GJB6 were designed using Primer Premier 5.0 software (Primer Biosystems, Foster City, CA, USA). PCR products from genomic DNA were sequenced using an ABI 3730XL DNA Analyzer (ABI, Foster City, CA, USA). The sequencing results were analyzed using Finch TV (Version 1.5).

2.3 Clinical examination

Histopathological examination was performed for the proband’s scalp tissue (location: 15 cm above the brow) and nail bed (location: the second finger of the left hand near the nail root). These specimens were immersed in formaldehyde for preservation and sent to the pathology department of our hospital for observation under a microscope (hematoxylin and eosin staining; original magnification 100× for scalp skin, 40× for nail bed).

Dermoscopic (FotoFinder Systems GmbH, handyscope for iPhone5) examination of the proband’s scalp 15 cm above the brow line showed that the hair was sparse, soft and curly, that the tips of some hairs exhibited thinning and that the hair follicles were reduced (Fig.2).

We used electron microscopy (Zeiss GeminiSEM 500 scanning electron microscope) to observe the microstructure of the hair surface of the proband and control. We used sterile tweezers to pick hair from the hair root on the head. The thinner hair of the proband was labeled sample 1, and the thicker hair of the proband was labeled sample 2. The control hair was labeled sample 3. All samples were evaluated at both 500× and 5000× magnification. To observe the internal structure of the hair, patient hair samples were cut both crosswise and lengthwise using a glass knife and were examined at both 2500× and 5000× magnification.

3 Results

3.1 GJB6 variant analysis and confirmation

To diagnose the patients at the molecular level, first, we performed WES and obtained 18 candidate variants consistent with co-segregation in pedigree. Besides, nine loci (Nos. 4, 5, 7, 8, 10, 11, 12, 14, and 18) were excluded based on frequency gnomAD_exome_ALL less than incidence. Thirdly, according to the predicted value of “REVEL,” “SIFT score,” and “Polyphen2_HVAR_score,” the remaining 8 sites are excluded. Fourth, based on the information provided by omim_phenotype, the candidate gene GJB6 variant is consistent with the clinical manifestations of the patient (autosomal dominant, nail dystrophy, partial to complete alopecia and palmoplantar hyperkeratosis) (detailed data are in Table S1). Furthermore, we performed Sanger sequencing to verify the GJB6 gene variant in the pedigree. Finally, we found a novel heterozygous missense variant NM_001110221: c.134G>C, NP_001103689: p.(Gly45Ala) in the GJB6 gene. The minor allele frequency (MAF) of GJB6 c.134G>C in the gnomAD-total population and gnomAD-Asian population was “0.” Additionally, Sanger sequencing results were consistent with genotype phenotype co-segregation in the pedigree (as shown in Fig.4 and 4B). The p.G45A variant in GJB6 was predicted to be harmful to protein function according to the REVEL, SIFT, and PolyPhen2 databases.

3.2 Clinical findings

In this family, there were significant differences in the clinical characteristics of different affected individuals. With increasing age, some affected individuals exhibited an exacerbation of symptoms, with abnormalities gradually developing in the hair, nails, and palmoplantar skin. In contrast, other affected individuals tended to experience symptom resolution, with all hair gradually becoming denser and thicker and no palmoplantar abnormalities. However, all affected individuals had nail abnormalities, which demonstrated that the expression of the mutant gene in the nails was stable. An interesting and unique phenomenon was observed in which all affected individuals experienced needling pain (numeric rating scale: 8–9 points) when the nails were exposed to low temperature (below 10 °C). This pain lasted approximately 30 min and could resolve gradually on its own or immediately by submerging the nails in hot water. This symptom is distinct from Raynaud’s phenomenon, which involves three typical changes, pallor, cyanosis, and flushing, accompanied by hand pain and nail dystrophy, when the hands encounter cold.

Histopathological examination showed normal epidermis and sebaceous glands with hair follicles almost absent in the proband (Fig.2). Examination of the proband’s nail bed revealed that small vessels in the superficial dermis were increased in number, dilated, and congested (Fig.2).

According to the results of scanning electron microscopy, compared with that of the healthy control, the patient’s hair, regardless of diameter or size, had different degrees of disorderly scaling, a rough surface, uneven edges, and honeycomb holes (Fig.5–5F). Cross-sections and longitudinal sections of the hair were also observed, and the results were consistent with those of scanning electron microscopy. There were a large number of sieve structures in both the cross-section and longitudinal section in each layer of the hair (Fig.6 and 6B).

4 Discussion

In this study, we conducted a systematic study of a large Chinese Clouston syndrome pedigree that had diverse clinical phenotypes and polarization with age (self-healing and aggravating tendencies). This pattern has rarely been seen in previous studies. Because it is difficult to differentiate Clouston syndrome from congenital pachyonychia and other EDs based on their clinical phenotypes alone, additional genetic analysis is necessary; therefore, we performed WES and Sanger sequencing and found a novel heterozygous missense variant (c.134G>C:p.G45A) in the GJB6 gene. Moreover, we identified a new clinical phenotype in which all patients in the family developed needling pain in the nails after exposure to cold temperature, which has not been previously reported. In addition, we examined the patient’s hair with an electron microscope and found sieve structures both on the surface of and inside the hair, which were not observed in normal hair. The report of this novel structure may provide a basis for further study of the mechanism of hair loss in Clouston syndrome.

The GJB6 gene is located at 13q12.11 and contains 6 exons. Gap junction protein beta 6, also known as connexin-30 (Cx30) and encoded by the GJB6 gene, is preferentially expressed in the human brain and skin and is associated with nonsyndromic severe hearing loss [11]. Atsushi Fujimoto et al. [12] reported that Cx30 is abundantly expressed in the nail matrix and nail bed in both human infants and mouse embryos. When stimulated internally or externally, the protein expression and posttranslational regulation of connexin can be altered, including changes in phosphorylation, oxidoreduction, protein interaction, and other mechanisms to adapt to internal and external changes [13,14]. Therefore, we speculated that the new phenotype of this family, unlike the Raynaud phenomenon, may be due to the decreased threshold and adaptability of gap junction channels to low-temperature stimulation. Na⁺ channels are opened after low-temperature stimulation, and a large amount of Na⁺ flows into the cytoplasm from outside the cell, which leads to an action potential at the cell membrane. The action potential is transmitted to the central pain sensation area through the nerve axon and finally produces a sensation of pain. Then, the gap junction channel is closed slowly, and the pain is relieved. The histopathology of the proband’s nail bed revealed that small vessels in the superficial dermis were increased in number, dilated, and congested. To some extent, this phenomenon can explain the unique phenotype, not only enriching the clinical phenotype spectrum of Clouston syndrome but also providing many new ideas for the further study of the pathogenesis of Clouston syndrome and the role of ion channels therein.

To date, four mutations in the GJB6 gene, G11R, V37E, A88V, and D50N, have been reported, summarized in Tab.2. The most commonly reported mutations in Clouston syndrome families in China are A88V and G11R. Patients with the same mutation may have significantly different phenotypes. A mouse model of Clouston syndrome carrying the GJB6 mutation A88V developed enlarged and hyperproliferative sebaceous glands and presented an altered hearing profile [15,16]. However, deafness is rare in families with A88V mutations, which may be related to species differences. In the affected members of the family in this study, we found that the epidermis and the sebaceous glands in the dermis were normal, and hair follicles were almost absent. Similar pathological phenomena in scalp tissue have also been reported [17,18]. It is not clear why these features are inconsistent with those in the mouse model, but it is speculated that this inconsistency may also be related to differences between the species.

The clinical phenotypes of the patients in this family were diverse. There are several potential explanations for this observation. First, polymorphisms in keratins, connexins or other genes encoding epithelial structural molecules are likely to play a role. Unidentified regulatory variants in the untranslated region (UTR) and promoter sequences of the GJB6 gene may affect the expression or function of Cx30. In vitro analyses performed by Atsushi Fujimoto et al. [12] suggest that ΔNp63α might be involved in GJB6 expression by binding to the sequences in intron 1 of the GJB6 gene. Therefore, sequencing of the UTR and promoter region is valuable for further study of GJB6 gene function. Second, Cx30 has a common structure, consisting of four plasma membrane domains, two extracellular domains, and three cytoplasmic domains [19]. The previously identified Clouston syndrome mutations were located in the cytoplasmic N terminus (G11R), the first plasma membrane domain (V37E), the second plasma membrane domain (A88V), and the first extracellular domain (D50N). The novel variant is also located in the first plasma membrane region (Fig.4). Guilherme Munhoz Essenfelder [20], by transfecting cells, showed that the abnormal hemichannel activity caused by GJB6 mutations G11R and A88V may play an important role in the pathophysiological processes resulting in the Clouston syndrome phenotype. The novel variant (G45A) was also located in the adjacent plasma membrane region and therefore might have a similar pathogenetic effect. This pathogenetic mechanism may be related to the diversity of clinical phenotypes in the family. Third, different living and working environments may also lead to the gradual aggravation or remission of the disease. Clouston syndrome exhibits a high variability of clinical manifestations. This suggests that an exact diagnosis will require WES and that the pathogenesis of Clouston syndrome may be caused in part by factors other than GJB6 gene mutation.

Advanced research on the hair structure of the patients revealed a sieve-like structure on the surface and inside of the hair under scanning electron microscopy. This similar structure has been reported in two Thai patients [21,22] with the G11R variation, showing abnormalities of the hair, nails, and teeth, and the absence of palmoplantar keratoderma, different from our case. However, needling pain symptoms in the nails after exposure to cold were not mentioned for either case. Previous studies have found that Cx30 is expressed in the outer root sheath of hair follicles, where hair follicle stem cells and their daughter cells reside [12,23]. We speculated that the decrease in hair follicles and the sieve-like hair structure may be related to the expression of Cx30 in the outer hair root sheath. However, identification of the specific pathogenetic mechanism still needs further study.

5 Conclusions

In this study, a novel heterozygous missense variant (c.134G>C:p.G45A) for Clouston syndrome was found. A new clinical feature of Clouston syndrome patients, the development of needling pain in the nails after exposure to cold, was described for the first time. The hair structure of the family members was further studied by using electron microscopy, and the hair of the patient was found to have sieve-like pores. The results of this study not only reveal a new Clouston syndrome variant but also enrich the clinical phenotypes associated with Clouston syndrome. Unfortunately, we have not been able to further explain the relationship between gene variants and these clinical phenotypes but provide ideas for further research on the role of upstream and downstream cell membrane signaling pathways and ion channels in the pathogenesis of GJB6 gene variants.

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