Background: Porokeratosis is a rare chronic progressive hypokeratotic skin disease, possibly related to the mevalonate pathway. Variations in four enzymes, including phosphomevalonate kinase (PMVK) may alter this pathway, ultimately leading to porokeratosis. Objectives: The aim of the study was to identify the causative gene variant of porokeratosis in a Chinese family and investigate its population frequency and pathogenicity. Method: In this study, Sanger sequencing was used to identify the gene variant causative of porokeratosis; its population frequency was investigated by polymerase chain reaction-restriction fragment length polymorphism in 4 patients and three normal individuals as well as in 100 normal unrelated controls; finally, the pathogenicity of the mutation and the associated structural changes were predicted. Results: We identified a novel heterozygous missense variant, c.207G>T (p. Lys69Asn) in the PMVK gene. This variant was found in all patients but not in the normal individuals in this family or in the 100 controls. In silico analysis indicated that the variant was pathogenic; p.Lys69Asn changed the length of the α-helix and the hydrogen bond pattern compared with the wild-type protein. Conclusions: The novel variant c.207G>T (p. Lys69Asn) in the PMVK gene was the causative variant in this porokeratosis family. This finding provides further evidence for the genetic basis of this disease.

Porokeratosis (PK) is a rare chronic progressive hypokeratotic skin disease. Its clinical manifestations include ≥1 annular skin lesions, showing atrophy and depression in the center and a keratinized border [1]. Histologically, it is characterized by a keratinous-like layer [2]. According to its clinical manifestations, it can be divided into six types: porokeratosis of Mibelli, disseminated superficial porokeratosis, disseminated superficial actinic porokeratosis (DSAP), linear porokeratosis, porokeratosis palmaris et plantaris disseminate, and porokeratosis punctata palmaris et plantaris [3‒8]. However, there are >10 other clinical subtypes rarely reported, such as eruptive pruritic papule, ptychotropica, or reticulated PK [9‒11]. Among these clinical types, DSAP is the most common [11]. Most PK-affected individuals show no pruritus, but eruptive pruritic papule PK is accompanied by pronounced itching. PK is considered as a precancerous lesion. In fact, 7.5–11% of patients with PK will develop malignant tumors at hypokeratotic lesions; squamous cell carcinoma being the most common type [7].

The pathogenesis of PK remains unclear. Previous studies indicated its relationship to the mevalonate pathway [2, 12], a crucial metabolic pathway in skin biology, which plays a significant role in the regulation of cell growth, division, and differentiation. Disruption of this pathway may affect keratinocyte formation; in addition, the abnormal apoptosis of keratinocytes is considered an important cause of PK [13, 14]. Mutations in any of four mevalonate pathway genes, namely, FDPS at 1q22, MVD at 16q24.2, MVK at 12q24.11, and PMVK at 1q21.3, have been linked with PK. Among these genes, the most frequently mutated in PK is MVK [2, 12, 15, 16]. In this study, we recruited a Chinese PK pedigree including four PK patients from different generations; the coding exons and flanking introns of the four genes previously related to PK were sequenced by Sanger sequencing; the population frequency of the variant was detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method; finally, the harm of this variant was explored by pathogenicity prediction and protein three-dimensional structure analysis.

Sample Information

A Chinese family with PK was recruited for this study (Fig. 1a). Peripheral venous blood from 4 patients and three healthy individuals was collected in EDTA anticoagulation tubes and stored in a freezer at −80°C. The study was approved by the Ethics Committee of Shenyang Sport University and carried out in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants in the study, who were adults.

Fig. 1.

Pedigree and clinical features of the investigated family with PK. a Pedigree of the four-generation family with PK; white circles and squares indicate healthy females and males, while black circles and squares indicate female and male patients, respectively; slashes indicate deceased, diamonds indicate unknown sex, and the arrow indicates the proband. The subjects of this study are marked by five-pointed stars. b Clinical features of the proband with PK.

Fig. 1.

Pedigree and clinical features of the investigated family with PK. a Pedigree of the four-generation family with PK; white circles and squares indicate healthy females and males, while black circles and squares indicate female and male patients, respectively; slashes indicate deceased, diamonds indicate unknown sex, and the arrow indicates the proband. The subjects of this study are marked by five-pointed stars. b Clinical features of the proband with PK.

Close modal

Sanger Sequencing and PCR-RFLP

Genomic DNA was extracted from peripheral venous blood using a Universal Genomic DNA Extraction Kit Ver. 3.0 following manufacturer’s instructions; the purity of the extracted DNA was standardized. DNAMAN 8.0 software was used to design the primers for all exon regions and the flanking sequences of the four genes MVK, PMVK, MVD, and FDPS. The polymerase chain reaction products of all seven members of the PK-affected family were detected by 2% agarose gel electrophoresis and directly sequenced by Sanger sequencing (outsourced to BioMed, Beijing, China) to screen for variations in these coding exons and flanking intronic regions.

To confirm the pathogenicity of the novel identified variation, a BspHI restriction site was introduced into the mutant allele by polymerase chain reaction using mismatched primers (PMVK-F: 5′-CTG​GAC​ACC​AGC​ACC​TAT​CA-3′, PMVK-R: 5′-GAG​CTA​GGG​AAA​CAG​GTG​TTC​TT-3′). Then, RFLP analysis was performed on all available family members and 100 unrelated Han Chinese individuals, considered as healthy control individuals.

Bioinformatic Analysis

The conservation of the identified variant, PMVK p.Lys69, among species was analyzed by MEGA software. The pathogenicity of the suspected causative variation of PK in this family was predicted by SIFT, PolyPhen-2, PROVEAN, MutationTaster, and FATHMM. The protein structure stability of PMVK associated with p.Lys69Asn was calculated by mCSM, SDM, and DUET. The three-dimensional models of wild-type and mutant PMVK proteins were generated using the SWISS-MODEL online server. In addition, the hydrophobicity of wild-type and mutant proteins was analyzed by ProtScale.

Clinical Manifestations

The phenotypes of the 4 patients in this family were very similar, with most having multiple irregular circles with both swollen and flat parts inside keratotic lesions, predominantly in typically sun-exposed parts of the body, such as the face, neck, upper chest, back, and distal extremities (Fig. 1b). However, the phenotype of 3-1 was milder than that of the other affected family members, including only keratotic lesions on the legs and upper limbs. The proband and her younger sister first presented annular papules in elementary school, while their father was diagnosed with PK at the age of 17 years old.

Mutant Analysis

After sequencing all exons and flanking intronic regions of the four genes MVK, MVD, PMVK, and FDPS, a novel heterozygous variation, c.207G>T (p. Lys69Asn), was identified in exon 3 of the PMVK (Fig. 2a). The sequencing results were analyzed using Chromas software. This variant was classified as a pathogenic in accordance with the 2015 guidelines of the American College of Medical Genetics and Genomics (ACMG) [17]. Conservation analysis showed that the 69th amino acid of the PMVK is highly conserved among various species, including humans, cows, cats, dogs, rabbits, sheep, chimpanzees, mice, rats, and pigs (Fig. 2b). The mutation c.207G>T (p. Lys69Asn) is located on the acceptor substrate binding region of this protein, which can bind with the substrate and ATP (Fig. 2c). Using PCR-RFLP analysis, the mutation c.207G>T (p. Lys69Asn) of PMVK was found in all affected individuals in the investigated family but not in healthy family members or in 100 control individuals (Fig. 2d).

Fig. 2.

Mutation analysis of the family with PK. a Sequencing chromatogram showing the heterozygous missense mutation c.207G>T in PMVK; the base including the variation is indicated by a red arrow (WT, wild type; MT, mutant type). b Conservation analysis showing conservation of the 69th amino acid of PMVK among species; a red arrow indicates the position of the variation. c Schematic showing the encoded domain structure of PMVK. Previously reported mutations are shown in black characters, while the mutation identified in this study is highlighted with a red character. d Results of PCR-RFLP, lanes 1–4 show the electrophoresis results of the patients in this family, while lanes 5–7 are the electrophoresis results of other healthy individuals in the family.

Fig. 2.

Mutation analysis of the family with PK. a Sequencing chromatogram showing the heterozygous missense mutation c.207G>T in PMVK; the base including the variation is indicated by a red arrow (WT, wild type; MT, mutant type). b Conservation analysis showing conservation of the 69th amino acid of PMVK among species; a red arrow indicates the position of the variation. c Schematic showing the encoded domain structure of PMVK. Previously reported mutations are shown in black characters, while the mutation identified in this study is highlighted with a red character. d Results of PCR-RFLP, lanes 1–4 show the electrophoresis results of the patients in this family, while lanes 5–7 are the electrophoresis results of other healthy individuals in the family.

Close modal

Bioinformatic Analysis

The pathogenicity analysis of the identified missense variant c.207G>T (p. Lys69Asn) using four different in silico tools indicated that this variation was “affect protein function,” “probably damaging,” “deleterious,” “disease-causing,” and “damaging,” respectively (Table 1). The protein structure stability analysis showed that p.Lys69Asn PMVK was destabilized with a negative free energy change value (ΔΔG: −1.125 kcal/mol, −0.37 kcal/mol, −0.942 kcal/mol, respectively) (Table 2). The change in the amino acid at position 69 from lysine to asparagine caused by this variant shortens the length of the α-helix where this amino acid is located. Moreover, the native p.69Lys forms a hydrogen bond with the nearby amino acid p.72Phe, which disappears with the mutation. In contrast, the mutant p.69Asn forms an additional hydrogen bond with the neighboring amino acid p.65Thr (Fig. 3a). Thus, the hydrophobicity at position 69 increased in association with the mutation (Fig. 3b).

Table 1.

Prediction of functional effect of mutations by using different algorithms

SIFTPolyPhen-2PROVEANMutationTasterFATHMM
scorepredictionscorepredictionscorepredictionscorepredictionscoreprediction
Affect protein function 1.000 Probably damaging −4.987 Deleterious NA Disease-causing −5.53 Damaging 
SIFTPolyPhen-2PROVEANMutationTasterFATHMM
scorepredictionscorepredictionscorepredictionscorepredictionscoreprediction
Affect protein function 1.000 Probably damaging −4.987 Deleterious NA Disease-causing −5.53 Damaging 

The table enlists the scores from SIFT, PolyPhen-2, PROVEAN, MutationTaster, and FATHMM for the mutation Lys69Asn in PMVK.

Table 2.

Prediction of PMVK protein stability upon mutation

AlgorithmLys69AsnEffect
mCSM −1.125 Destabilizing 
SDM −0.37 Destabilizing 
DUET −0.942 Destabilizing 
AlgorithmLys69AsnEffect
mCSM −1.125 Destabilizing 
SDM −0.37 Destabilizing 
DUET −0.942 Destabilizing 

The table enlists the changes in Gibbs free energy (ΔΔG) in kcal/mol. ΔΔG >0 indicates stabilization while ΔΔG <0 indicates destabilization.

Fig. 3.

Bioinformatic analysis of the mutant PMVK. a Three-dimensional structure of PMVK. The protein structure of the WT protein shows an hydrogen bond between p.69Lys and the nearby amino acid p.72Phe, while that of MT shows that p.69Asn altered the length of an α-helix and formed additional hydrogen bonds with the neighboring amino acid p.65Thr while losing that with the nearby amino acid p.72Phe (WT, wild type; MT, mutant type). b Hydrophobicity prediction of the mutant PMVK indicating increased local hydrophobicity around the variation site Lys69Asn.

Fig. 3.

Bioinformatic analysis of the mutant PMVK. a Three-dimensional structure of PMVK. The protein structure of the WT protein shows an hydrogen bond between p.69Lys and the nearby amino acid p.72Phe, while that of MT shows that p.69Asn altered the length of an α-helix and formed additional hydrogen bonds with the neighboring amino acid p.65Thr while losing that with the nearby amino acid p.72Phe (WT, wild type; MT, mutant type). b Hydrophobicity prediction of the mutant PMVK indicating increased local hydrophobicity around the variation site Lys69Asn.

Close modal

PK is a genetically heterogeneous skin disorder for which the genetic basis and pathological mechanism remain somewhat unclear. Zhang et al. [2] reported that variants in the mevalonate pathway genes MVK, MVD, PMVK, and FDPS were found in 98% of cases of familial PK. In this study, we sequenced four genes previously associated with PK in an affected family and identified a novel mutation, c.207G>T (p. Lys69Asn) in PMVK, classified as pathogenic according to 2015 ACMG guidelines [17]. PMVK is located on chromosome 1q21.3, having five exons and four introns. It is expressed in the human skin, heart, skeletal muscle, liver, kidney, and pancreas, among others [18, 19]. Phosphomevalonate kinase, encoded by PMVK, is composed of 192 amino acids and has a molecular weight of 21 kDa. PMVK is one of the most important enzymes in the mevalonate pathway, playing a significant role in maintaining cell membrane structure and function as well as regulating cell growth and differentiation [20]. PMVK uses mevalonate phosphate and ATP as substrates to synthesize mevalonate pyrophosphate, a crucial step for cholesterol synthesis in vivo [20]. As cholesterol is a key component of eukaryotic cell membranes, this explains the role of PMVK in cell membrane integrity.

In terms of its structure, PMVK is folded into three domains: the CORE region (amino acids 1–42, 101–108, 122–132, 166–192), the LID region (amino acids 133–165), and the acceptor substrate binding region (amino acids 43–100, 109–121) [21]. The protein’s secondary structure includes five-stranded parallel β-sheets and eight α-helices [21]. c.207G>T (p. Lys69Asn) is located in the acceptor substrate binding region, drastically altering PMVK’s three-dimensional structure, including the length of an α-helix and hydrogen bonding pattern with the neighboring amino acids. This might affect PMVK’s normal binding to its substrates, leading to disruption of the mevalonate pathway, influencing cell proliferation and differentiation, and ultimately leading to PK. Wang et al. found that the mutation c.412C>T (p. Arg138*) results in a punctuated distribution of the encoded protein in the cytoplasm, along with decreased expression and decreased solubility [22]. Solubility is essential for PMVK’s function; the local hydrophobicity of p. Lys69Asn is predicted to increase, a finding consistent with those of Wang et al. However, given the complicated pathogenesis of PK, further studies are needed to confirm the pathogenicity of this variation.

At present, 10 different PMVK mutations associated with PK are described in the HGMD database, including c.1A>G (p. Met1Val) [2], c.46G>T (p. Gly16Cys) [14], c.65A>G (p. Lys22Arg) [12], c.94A>T (p. Arg32*) [2], c.143A>G (p. Lys48Arg) [23], c.205A>G (p. Lys69Glu) [2], c.312G>A (p. Trp104*) [2], c.314T>C (p. Leu105Pro) [24], c.412C>T (p. Arg138*) [22], and c.550delC (p. Leu184*) [2]. These mutations are distributed throughout the PMVK gene, with no hotspots. According to previous reports, the disease subtypes caused by PMVK mutations include linear porokeratosis, DSAP, and porokeratosis of Mibelli for mutations in the CORE region; genital PK for those in the acceptor substrate binding region; and disseminated superficial porokeratosis for those in the LID region [12, 14, 22‒24]. The patients in this study developed typical DSAP symptoms: small and circular keratinous lesions distributed in the sun-exposed parts of the body, such as the face, neck, upper chest, back, and distal extremities. Notably, although Zhang et al. [2] reported a similar variation, p. Lys69Glu, they did not report its clinical manifestations; thus, whether their patients exhibited similar phenotypes to the ones shown by our patients is unclear. Together with the present mutation, c.143A>G (p. Lys48Arg) is the second identified in PMVK’s acceptor substrate binding region domain, reported in a sporadic patient who showed genital PK, which clearly differs from the DSAP phenotypes in this study [23]. Based on the above, DSAP is likely to be the most common PK phenotype caused by PMVK mutations. As only 10 mutations have been reported so far, genotype-phenotype correlations remain unclear. More cases need to be accumulated to explain these relationships.

In conclusion, this study identified the novel mutation PMVK c.207G>T (p. Lys69Asn) in a Chinese family affected by PK. This finding expands on known mutations associated with PK, providing further evidence for the genetic basis of this disease.

We sincerely thank all the participants in this study.

This study was conducted in accordance with the Declaration of Helsinki and approved by an independent Ethics Committee (the Ethics Review Committee of Shenyang Sport University). The reference number of this project is 2021-3. The samples collected in this study were all from adults, and written informed consent was obtained from all participants.

The authors have no conflicts of interest to declare.

This study was supported by the National Natural Science Foundation of China (81502176, 81670896) and the Liaoning Provincial Education Department’s Science and Technology Research Project (LK201653).

W.Z. conducted the research and wrote the manuscript; X.N. assisted in the bioinformatics analysis and manuscript preparation; L.S. completed the data collection and literature search; L.C. designed the study and revised the manuscript; and F.S. oversaw the research program and reviewed the manuscript. All authors read and approved the final manuscript.

All the data generated in this study are available and included in this article. For further inquiries, please email the corresponding author directly.

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