The PER3rs772027021 SNP induces pigmentation phenotypes of dyschromatosis universalis hereditaria

Dyschromatosis universalis hereditaria (DUH) is a pigmentary genodermatosis characterized by a mixture of hyperpigmented and hypopigmented macules distributed randomly over the body. Although Sterile Alpha motif- and SH3 domain-containing protein 1 (SASH1) and ATP-binding cassette subfamily B, member 6 (ABCB6) have been identified as causative genes for this disorder, some cases involve unknown pathogenic genes. In this study, whole-exome sequencing, data analysis, and Sanger sequencing were utilized for a four-generation extended Chinese family with DUH. A single-nucleotide polymorphism (SNP) (c. 517C > T (p.P173S), rs772027021) variant in exon 5 of Period Circadian Regulator 3 (PER3) (NM_001289861) was detected in each affected individual of the DUH family; the c. 517C > T SNP of PER3 (PER3rs772027021 SNP) and a novel mutation in exon 14 of SASH1 (c. 1574C > G (p.T525R)) were both found in the proband. The affected individuals carrying PER3rs772027021 SNP in this family demonstrated mild-pigmented phenotypes compared to those of the proband carrying PER3rs772027021 SNP and SASH1T525R mutation. Increased melanin synthesis was induced by PER3rs772027021 SNP in the melanocytes of affected epithelial tissues. Mutated SASH1 or PER3rs772027021 SNP alone or cooperation of mutation of SASH1 and PER3rs772027021 SNP synergistically led to increased melanin synthesis and enhanced proliferation of melanoma cells in vitro. We also phenotypically characterized a commercially available zebrafish mutant line harboring the PER3rs772027021 SNP to induce melanocyte proliferation in vivo. Our results are the first to reveal that this PER3 SNP may be pathogenic for a novel DUH subtype with mild hyperpigmented and/or hypopigmented phenotypes and that mutation of SASH1 and PER3 cooperatively promotes hyperpigmentation phenotypes. PER3 rs772027021 SNP is identified to be associated with hyperpigmentation and/or hypopigmentation phenotype and the novel pathogenic variant of PER3 rs772027021 SNP probably contributed the pathogenesis of DUH. SASH1T525R mutation is confirmed to associate with DUH. A novel autosomal dominant inheritance DUH subtype with mild pigmentated phenotypes is caused by the PER3rs772027021 SNP. PER3 rs772027021 SNP is identified to be associated with hyperpigmentation and/or hypopigmentation phenotype and the novel pathogenic variant of PER3 rs772027021 SNP probably contributed the pathogenesis of DUH. SASH1T525R mutation is confirmed to associate with DUH. A novel autosomal dominant inheritance DUH subtype with mild pigmentated phenotypes is caused by the PER3rs772027021 SNP.

Circadian rhythms are implicated in the mechanisms of refractive development. In chicks, expression of the two clock and circadian rhythm-related genes Per3 and Cry1 is influenced by altered visual input [19]. PER3 is a member of the period family of genes and is expressed in a circadian pattern in the suprachiasmatic nucleus, which is the primary circadian pacemaker in the mammalian brain. Downregulation of PER3 and three other circadian entrainment pathwayrelevant genes is involved in psoriasis [20].
In this study, the SNP PER3 rs772027021 was found to be associated with pigmentation in an extended DUH family with mild pigmentated phenotypes, possibly constituting a novel causal gene that induces hyperpigmentation and/or hypopigmentation phenotypes. The hyperpigmented and/or hypopigmented phenotypes appeared in the affected individuals carrying the SNP PER3 rs772027021 in the extended DUH family, which suggested that a novel DUH subtype with mild phenotypes is caused by the PER3 rs772027021 SNP. PER3 rs772027021 and the SASH1 c. 1574 C>G (p.T525R) variant synergistically induced pigmentation phenotypes in the proband.

Materials and methods
The detailed protocol of methods and the detailed information of materials used in the study were indicated in the Supplementary Materials and methods.

Clinical features
We characterized a four-generation extended Chinese family with DUH in Zhaoyang, Hubei, China; the disease was found to be transmitted in an autosomal dominant manner (Fig. 1A). The proband (III-6) was a woman in her early 30 s who had normal skin at birth. The lesions occurred in a symmetrical pattern and were most obvious on the face, neck, trunk, and dorsa of her hands and buttocks. Hyperpigmented macules appeared initially on her face as a toddler, the hyperpigmented macules became larger as evenly distributed freckles, and the color of these macules deepened. Hypopigmented macules began to appear on her neck, elbows, knees, and phalangeal joints of the proband in early adolescence. Her palms and soles, oral mucosa, hair, nails, and teeth were normal. In adulthood, irregularly shaped, asymptomatic hyper, and hypopigmented macules were present over her face, neck, abdomen and back, dorsal aspects of the hands and arms, thighs, calves, and hips ( Fig. 1B and Table 1).
The above six affected individuals all had hypopigmented and/or disseminated hyperpigmented macules.  Representative  images of irregularly shaped, asymptomatic hyper, and hypopigmented macules on the extremities and hips of the proband (III-6) are present in a generalized distribution manner. C, D, and E Sporadic or disseminated hyper-and/or hypopigmented macules distributed on the neck and trunk of affected individual II-3, dense or sporadic hypo-or hyper-pigmented macules distributed on the shoulder and dorsum, and trunk of affected individual III-9, shanks of affected individual II-10 and auricles of affected individual II-11. Close-up views of II-3 and II-10 distinctly show hyper and hypopigmented macules. Red arrows indicate hyperpigmented macules, and blue arrows indicate hypopigmented macules ◂ These observed clinical symptoms supported the diagnosis of pigment abnormalities in these six affected individuals. DUH is characterized by asymptomatic hyperpigmented and hypopigmented macules that occur in a generalized distribution over the trunk, limbs, and sometimes face [2,5]. Therefore, these six affected individuals were likely to have DUH. The pigmentation abnormality phenotypes of the proband were significantly more obvious than those of the six DUHaffected individuals (I-2, II-3, II-10, II-11, III-9, and III-12). The family members' clinical characteristics are summarized in Table 1. The DUH pedigree was ascertained by two experienced dermatology doctors of the Affiliated Hospital of Guizhou Medical University and the First Affiliated Hospital of Chengdu Medical College, and all of these patients were diagnosed as having the clinical phenotypes of DUH.
In general, DUH should be considered in differential diagnosis of all cases manifesting as mixed hyper and hypopigmented macules, and biopsy specimens should be obtained to confirm the diagnosis [21]. We further confirmed the phenotypes of increased melanin pigmentation in the affected skin epithelial tissues of II-3 and II-10 and the proband (III-6) of this family using melanin staining.
These melanin staining assays revealed excessive melanin pigmentation in both the basal and suprabasal layers of the proband's hyperpigmented macules (Fig. 2B). Less melanin and a mosaic-like melanin distribution were observed in the hypopigmented macules of the proband (Fig. 2C). Similar to the proband, increased melanin pigmentation and a mosaiclike melanin distribution were found for the basal and suprabasal layers of the hyperpigmented macules of II-3 and II-10 ( Fig. 2D-G).

Whole-exome sequencing identifies candidate genes in the family
We subjected the exomes of five affected (I-2, II-3, II-10, III-6, and III-12) and five unaffected (I-1, II-1, II-4, II-13, and III-5) family members to whole-exome sequencing. Approximately 100 million bases per individual were mapped, and approximately 60 million bases were sequenced. Variants with an MAF less than 0.01 in databases including 1000 genomic data (1000g_all) and the in house Novo-Zhonghua exome database were identified. SNVs occurring in exons or splice sites were analyzed, synonymous SNVs were discarded, and variations were screened according to the scores of pathogenicity prediction programs, including SIFT [22], Polyphen [23], MutationTaster [24], and CADD software [25]. Linkage analysis was performed in the multigenerational family to determine the candidate region using merlin tools and Perl combined with high-throughput sequencing data for the family and the HapMap database of Chinese population (CHB) allele frequency using the known SNP as a marker. A candidate SNP for the disease-related pathogenic mutation was defined as one existing exclusively in the five affected family members with clinical phenotypes but not in the five unaffected individuals without clinical phenotypes. Twenty-eight variants involving multiple chromosomes were identified. According to our previous reports about DUH, the SASH1 variant rs770362998 was included for consideration (Table S2). Nevertheless, Sanger sequencing uncovered no mutation in SASH1 (data not shown). The SASH1 variants including the c.1761C>G (p.Ser587Arg) variant [10], the c.1553A>C (p.Q518P) one [11], and the c.1556 G->A(p. S519N) [12] one of SASH1 which had been reported previously to associate with genodermatosis or DUH were not indicated in the Exome sequencing results. The genetic disease testing report provided by Chigene Translational Medical Research Center Co. Ltd. (Beijing, China) regarding the exome sequencing results of the proband showed that c. 1574C>G (p.T525R) variant in exon 14 of SASH1 (NM_015278) was detected during prenatal diagnosis (Table S2). This variant did not exist in normal controls of Chinese populations as indicated in two times of exome sequencing results provided by Chigene Translational Medical Research Center Co. Ltd and Novagene (Table S2). Sanger sequencing analysis was further performed to identify A Homogeneous melanin-stained epithelial cells of the normal control from foreskin tissues of an early adolescent boy were found in the basal layers. B and C Heterozygous melanin distribution or a mosaic-like melanin distribution was observed in affected epithe-lial layers of hyperpigmented and hypopigmented macules of the proband. Excessive melanin distribution was observed in both the basal and suprabasal layers. D-G Similar to the proband, a mosaiclike melanin distribution and increased melanin synthesis were observed in the affected epithelial layers of hyperpigmented macules II-3 and II-10 the C1574G SASH1 mutation in the proband and four affected (I-2, II-3, II-10, and III-12) and five unaffected (I-1, II-1, II-4, II-13, and III-5) individuals. Based on Sanger sequencing analysis, c. 1574C>G (p.T525R) was only detected in the proband and not in the other five affected and unaffected individuals (Figs. 3C and S1). The c. C1574G SASH1 variant was also found in the aborted fetus of the proband (data not shown). Three unaffected individuals (I-1, II-1, and II-4) and four affected individuals (II-3, II-10, III-6, and III-12) carried SASH1 rs208696 , which suggests that this SNP does not cosegregate with the pigmented phenotype (Table S2).
The c.1067T>C (p.Leu356Pro) variant of ABCB6, the c.508A>G (p.Ser170Gly) one, the c.1736G>A(p.Gly-579Glu) one [15], the c.1358C>T ( p.Ala453Val) one and the c.964A>C ( p.Ser322Lys) one [16], the c.1663 C>A ( p.Gln555Lys) one and the c.459 delC one [17], and the c.964A>C(p.S322R) one in exon4 and the c.1270 T>C (p.Y424H) [18] one were not found in the Exome sequencing results of this DUH family. The exon 1 SNP rs1109866 (c. G117A, p.L39 L) in ABCB6, a synonymous SNV, was detected in five affected individuals (I-2, II-3, II-10, III-6, and III-12); it is predicted to be harmless. ABCB6 rs1109867 was also found in these five affected individuals; however, pathogenicity prediction does not support that this variant is harmful (Table S3).  tion site in SASH1 is in an less evolutionarily conserved region, as framed by a dotted line. A partial sequence of SASH1 was compared with orthologs in other species. E The family diagram to illustrate the affected individuals with the PER3 rs772027021 SNP and/or SASH1 T525R mutation. Among 24 individuals whose blood was drawn for DNA sequencing analyses, the PER3 rs772027021 SNP was found in seven affected (I-2, II-3, II-10, II-11, III-6, III-9, and III-12) but not in unaffected individuals. Both PER3 rs772027021 SNP and SASH1 T525R variant were found in the proband (III-6). *Indicates individuals whose blood was drawn for exome sequencing and/or Sanger sequencing. PM: PER3 mutation, SM: SASH1 mutation mutations based on pathogenicity prediction programs. The DNA of twenty-four individuals in the extended 31-member DUH family was subjected to Sanger sequencing to identify causative genes. The PER3 rs772027021 SNP, which is a missense SNV, was subsequently detected by whole-exome sequencing in 7 affected individuals with hyperpigmented and/or hypopigmented phenotypes. The PER3 rs772027021 SNP is predicted to be harmful (Table S4). To analyze this SNP in more individuals in the extended DUH family, 24 individuals among the 31-member extended family agreed to blood draw to identify the PER3 rs772027021 SNP. Sanger sequencing analyses detected PER3 rs772027021 SNP in seven affected members of the family, including affected individuals II-11 and III-9, but not in any of the seventeen unaffected individuals (Fig. 3A, E and Table S4). Pathogenicity prediction of the PER3 rs772027021 SNP was performed with the VarSome tool (https:// varso me. com/). PER3 rs772027021 SNP is predicted to be damaging or deleterious by six CADD predication software tools, and one individual prediction software tool and one Meta score and pathogenicity score support that the SNP is harmful (Table S5).
The c. 211delC (p.L71fs) variant in exon 3 of SFTA3 was found in six affected individuals (I-2, II-3, II-10, II-11, III-6, and III-12) with hyperpigmented and hypopigmented phenotypes whose DNA was analyzed by whole-exome sequencing. An unaffected individual (II-7) was later identified to harbor SFTA3 c. 211delC (p.L71fs) by Sanger sequencing (Table S6). These results suggest that SFTA3c. 211delC (p.L71fs) does not cosegregate with the pigmented phenotype in this DUH family. The SNP KYNU rs199529102 was detected in affected individuals (I-2, II-3, II-10, III-6, and III-12); however, it was also found in an unaffected individual (II-13), which indicates that this SNP does not cosegregate with the pigmented phenotype (Table S7). The c. 561delT variant in exon 5 of GLMN was detected in the affected individuals (I-2, II-3, II-10, II-11, and III-12) and in one unaffected individual (III-14), though it was not detected in the proband. Thus, the c. 561delT variant of GLMN does not cosegregate with the pigmented phenotype (  (Fig. 3B and 3D).

PER3 shows differential expression between hyperpigmented and hypopigmented macules
We further investigated expression of PER3 in hyperpigmented macules and hypopigmented macules in affected family members II-3 and II-10 and the proband. In the proband, immunohistochemical analysis revealed a mosaiclike distribution and high expression of PER3 in the affected epidermis of hyperpigmented macules compared to those in hypopigmented macules (Fig. S2A). High expression of PER3 was detected in the affected epidermis of hyperpigmented macules compared to that of hypopigmented macules in II-3 and II-10 (Figs. S2B and 2C). The expression and distribution of SASH1 was also investigated, and a mosaiclike distribution and high expression were detected in the affected epidermis of hyperpigmented macules compared to hypopigmented macules in the proband (Fig. S3A, B). However, no differential expression of SASH1 was observed in affected epithelial layers between the hyperpigmented and hypopigmented macules of II-3 and II-10 ( Fig. S3C-F).

Proliferation of melanocyte were induced by PER3 rs772027021 SNP alone or the cooperation of PER3 rs772027021 SNP and SASH1 T525R variant in affected skin and in vitro
To investigate the effects of PER3 rs772027021 SNP or SASH1 T525R variant on melanocyte proliferation, multi-color IHC(mIHC) of epithelial tissues of normal-pigmented foreskin samples, the proband, and II-3-and II-10-affected individuals was performed using Ki67 marking the proliferative epithelial cells and Mitf marking melanocytes. The Ki67positive cells in the epithelial tissues which can be identified as the proliferative cells and the Mitf-positive cells which can be suggested to be melanocytes were quantified. The melanocytes both with Ki67 and Mitf both being positive were suggested as the proliferative melanocytes. Immunoflourence analyses indicated that increased absolute numbers of Ki67positive or Mitf-positive cells or enhanced ratio of proliferative melanocytes (Ki67 and Mitf-positively merged cells) in the proliferative cells(Ki67-positive cells) in the hyperpigmented macules were induced by PER3 rs772027021 SNP or/ and SASH1 T525R variant, respectively. Oppositely, increased absolute numbers of Ki67-positive or Mitf-positive cells or enhanced ratio of proliferative melanocytes in the hypopigmented macules were not induced (Figs. 4 and S4). The immunoflourence results indicated proliferative melanocytes was induced by PER3 rs772027021 SNP alone or cooperation 1 3 of PER3 rs772027021 SNP or SASH1 T525R variant. Increased numbers of proliferative melanocyte were observed in the affected epithelial tissues of the proband and the II-3 and II-10 affected individuals in the hyperpigmented but not the hypopigmented macules (Figs. 4 and S4).
We further assessed the effects of PER3 P173S SNP and SASH1 T525R variant on the proliferation of B16 cells. Apoptosis assays in B16 cells indicated that increased proportions of apoptotic cells were induced by overexpressed wild-type SASH1 compared to that by negative control adenovirus; however, reduced proportions of apoptotic cells were induced by overexpressed mutant-type SASH1 compared to that by wild-type SASH1 (Fig. S5A).
Obviously, enhanced proportions of apoptotic cells were induced by the combinations of SASH1 T525R variant and PER3 rs772027021 SNP (Fig. S5A). Cell cycle profile indicated that increasing proportions of S-phase of B16 cells was induced by overexpressed wild-type SASH1 compared to those by negative control and caused by overexpressed SASH1 T525R variant compared to those by overexpressed wild-type SASH1 (Fig. S5B).
Reduced proportions of apoptotic cells and increased proportions of G2/M phase were induced by overexpressed mutant type PER3 compared to those by wild-type PER3 (Fig. S5C, D). Decreased proportions of apoptotic B16 cells and increased proportions of S phase cells were induced by the cooperation of overexpressed SASH1 T525R variant and PER3 rs772027021 SNP compared to those by negative control (Fig. S5A, B).

Increased melanin was induced by the PER3 rs772027021 SNP and/or SASH1 T525R variant
We also assessed the effects of PER3 rs772027021 SNP and/or SASH1 T525R on melanogenesis in vitro. Western blot analysis indicated upregulation of mutant SASH1 in B16 cells (Fig. 5A, 5B) and SK-MEL-1 cells (Fig. 5D, 5E). Melanin quantification suggested that increased melanin synthesis was induced by the PER3 rs772027021 SNP in B16 (Fig. 5C) and SK-MEL-1 (Fig. 5F) cells compared to that induced by wild-type PER3. More synthesized melanin was induced through cooperation between the PER3 rs772027021 SNP and the SASH1 T525R variant compared to the SASH1 T525R variant alone (Fig. 5C) in B16 cells. Moreover, enhanced melanin synthesis occurred via cooperation between PER3 rs772027021 SNP and SASH1 T525R variant compared to either alone in SK-MEL-1 cells (Fig. 5F). More melanin was synthesized after overexpression of SASH1 T525R compared to wild-type SASH1 in B16 cells (Fig. 5C) and SK-MEL-1 cells (Fig. 5F). Rescue assays were performed to identify the inducement of PER3 on melanin synthesis. The inducement of increased melanogenesis by PER3 rs772027021 SNP and wild-type PER3 was reversed by the PER3 siRNA knockdown (Fig. 5G and 5H).
Although the KYNU c. C716T variant exhibited a distribution similar to that of PER3 rs772027021 SNP in this family (Fig. S6A), melanin quantification of B16 cells infected with KYNU ADV revealed that no melanin increase was induced by the KYNU mutation (Fig. S6B). The SFTA3 c. 211delC variant also exhibited a familial distribution similar to that of the PER3 rs772027021 SNP (Table S6). SFTA3 is an RNA Gene and is affiliated with the lncRNA class, which is now thought to not encode a protein although previous study suggested that it encoded surfactant protein H which was part of the multifunctional surfactant gene family of the lung [26]. PER2 has been suggested to be the target gene of SFTA3 by LncRNA2Target (http:// 123. 59. 132. 21/ lncrn a2tar get/ index. jsp). So, HA-SFTA3 and myc-PER3 were introduced into B16 cells to assess their combinations on melanin synthesis. However, no increase in melanin synthesis was caused by overexpression of mutant SFTA3 (Fig. S7A, S7B), and enhanced melanin synthesis was not induced by the combination of WT-PER3 + WT-SFTA3 compared to that of wildtype PER3. Additionally, melanin was not increased by the combination of MT-PER3 + MT-SFTA3 compared to that by mutant PER3 (Fig. S7E).

The PER3 rs772027021 SNP is essential for melanocyte proliferation and development in vivo and PER3-CREB cascade may regulate melanogenesis
The zebrafish is an ideal model for studying melanocyte differentiation [27,28]. Homology analyses of the PER3 gene sequence from the Ensembl database suggested that human PER3 and zebrafish per3 are homologous genes, with collinear genes vamp3 (vamp3) and UTS2 (uts2b), among others, which indicates that human PER3 and zebrafish per3 are orthologous genes (Fig. 6A). Homology analyses of the amino acid sequence of PER3 showed 37.1% homology between humans and zebrafish (Fig. S8).
To investigate the in vivo pathogenic effects of the variant in zebrafish, wild-type and mutant PER3 were transcribed into RNA in vitro and injected into fertilized zebrafish eggs. Quantitative RT-PCR detection in fertilized eggs at 24 h postfertilization (hpf) showed significant increases in the Fig. 4 Enhanced ratio proliferative melanocytes in the affected epithelial tissues of hyperpigmented macules were induced by the PER3 rs772027021 SNP or/and SASH1 T525R variant. A mIHC analyses indicated that in the affected epithelial layers of hyperpigmented macules, absolute numbers of Ki67-positive or Mitf-positive cells or enhanced ratio of proliferative melanocytes (positive Ki67 and positive Mitfmerged cells) were induced by PER3 rs772027021 SNP or/ and SASH-1 T525R variant, respectively. The mIHC of epithelial tissues of normal pigmentation foreskin samples, the proband, and II-3-and II-10-affected individuals was performed using Ki67 marking the proliferative epithelial cells and Mitf marking melanocytes. The blue fluorescence indicated the cell nucleus stained with DAPI. The green, red, and yellow fluorescence indicated the subcellular expression of Mitf, Ki67 and the merging of the green and red fluorescence of Mitf and Ki67, respectively. The melanocytes which were indicted with red arrows and merged yellow fluorescence were suggested to be the proliferative melanocytes. NPNC: Normal Pigmentation Negative Control. B Increased quantity of Mitf-positive cells in the affected layers of the hyperpigmented macules in the proband and II-3-and II-10-affected individuals and in those of the hypopigmented macules of II-10 one were induced as compared to those of normal-pigmented epithelial layers of foreskin samples. However, enhanced quantity of Mitf-positive cells in the affected layers of the hypopigmented macules in the proband and II-3 affected individual were not induced by PER3 rs772027021 SNP and /or SASH1 T525R variant, respectively. The absolute numbers of Mitf-positive cells in the hyperpigmented or hypopigmented macules were quantified and statistically analyzed one-way ANOVA. C Enhanced quantity of Ki67-positive cells in the affected layers of the hyperpigmented macules but not the hypopigmented ones in the proband and II-3-and II-10-affected individuals were induced by PER3 rs772027021 SNP or/ and SASH1 T525R variant, respectively as compared to those of normal pigmentation epithelial layers of foreskin samples. D Increased ratio of proliferative melanocytes in the affected layers were induced by PER3 rs772027021 SNP or/ and SASH1 T525R variant. The melanocytes both with Ki67 and Mitf both being positive were suggested to be the proliferative melanocytes, which were then quantified and the ratio of proliferative melanocytes was assessed using the formula: the numbers of melanocytes both with Ki67 and Mitf being positive in each field of vision /the numbers of melanocytes with Mitf being positive in each field of vision = the ratio of proliferative melanocytes. The ratio of proliferative melanocytes were statistically analyzed with one-way ANOVA. E The ratios of proliferative melanocytes in the affected layers of hyperpigmented macules were much higher than those of hypopigmented one in the proband and II-3-and II-10-affected individuals ◂ Fig. 5 Increased melanogenesis in melanoma cells was induced by the PER3 rs772027021 SNP or SASH1 T525R variant and augmented by their combination. A-B SASH1 T525R was upregulated in B16 cells. Expression of wild-type or mutant PER3 and wild-type and mutant SASH1 in B16-affected cells with combined adenovirus was identified by western blotting. B16 cells were infected with adeasy014-pAdEasy-EF1-MCS-CMV-EGFP adenovirus containing wild-type PER3, PER3 rs772027021 SNP, wild-type SASH1 or the SASH1 T525R variant in different combinations. At 60 h after infection, the cells were lysed and subjected to western blotting. C An increased melanin content in B16 cells was induced by PER3 rs772027021 SNP or SASH1 T525R variant and augmented by their combination. B16 cells were infected with the adeasy014-pAdEasy-EF1-MCS-CMV-EGFP adenovirus expressing PER3 and SASH1 in different combinations.
At 60 h after infection, the cells were lysed, and the melanin content was quantified according to the manufacturer's protocol. D-E T525R SASH1 was upregulated in SK-MEL-1 cells. Expression of wildtype or mutated PER3 and wild-type SASH1 and mutated SASH1 in SK-MEL-1-affected cells was identified by western blotting. F An enhanced melanin content in SK-MEL-1 cells was induced by the PER3 rs772027021 SNP or SASH1 T525R variant and augmented by their combination. G and H The enhanced melanin syntheses caused PER3 rs772027021 SNP were reversed by PER3 silencing in B16 cells. PER3 specific siRNAs was transfected after overexpression of wild-type PER3 and PER3 P173S SNP in B16 cells for 24 h. At 48 h of transfection of PER3 siRNA, B16 cells were lysed for melanin quantification and western blot expression levels of all injection groups, which indicated wild-type PER3 and PER3 rs772027021 SNP to be expressed in this model (Fig. 6B). A concentration of 100 ng/μl was selected as the optimum dose for further studies based on the observed expression of PER3. We selected 72 hpf as a reference time point to characterize the pigmentation phenotype of zebrafish based on previous reports [28,29]. As anticipated, a significantly large number of proliferative melanocytes were induced by the PER3 rs772027021 SNP compared to the control group and wild-type group (Fig. 6C, D). These results indicate that the PER3 rs772027021 SNP plays an important role in melanocyte proliferation and development in zebrafish in vivo. In contrast, no enhanced melanin synthesis was induced by the GLMN c. 561delT variant compared to wild-type GLMN in B16 cells (Fig. S9A). Additionally, no increase in the number of proliferative melanocytes was induced by the c. 561delT variant of GLMN compared to the control group and wild-type group (Fig. S9D, S9E) in vivo. Increase in phosphorylated ERK1/2 and CREB levels is caused by mutated SASH1 alleles [9]. Western blot indicated that over-expression of wild-type and mutant type of PER3 in B16 and SK-MEL-1 cells enhanced the phosphorylation level of CREB, however downregulated that of ERK1/2 (Fig. 6E, F), which indicated that a PER3-CREB phosphorylation cascade may mediate melanogenesis.

Discussion
DUH is a pigmented dermatosis with genetic heterogeneity. A previous study reported three loci and two causal genes responsible for DUH: 2q33.3-q36.1 (ABCB6) [11,[15][16][17][18], 6q24.2-q25.2 (SASH1) [6-11, 13, 30], and 12q21-q23 [3]. In this study, through an integrated approach, we detected a PER3 rs772027021 SNP and/or SASH1 T525R mutation in a DUH pedigree. Our study identifies a disease-causing SNP in PER3 that is responsible for DUH. Our study provides a novel pathogenic variant of PER3 of DUH and contributes to the identification of causal genes for DUH. Different from the DUH clinical phenotypes previously reported, with lesions of irregular size and shape always appearing in infancy or early childhood [4,11,[31][32][33], in our study, clinical symptoms on the bodies of six affected individuals except those on the proband were mild, hyperpigmented phenotypes especially the hypopigmented ones of which were often ignored according to their self-memory and descriptions. Different clinical manifestations were demonstrated in the I-2-, II-3-, II-10-, II-11-, III-9-, and III-12-affected individuals with mild clinical phenotypes This may indicate that a novel DUH subtype with autosomal dominant inheritance and mild pigmentated phenotypes were caused by the PER3 P173S SNP.
Asymptomatic hyperpigmented and hypopigmented macules extensively distributing over the trunk, limbs, and sometimes face are major characteristics of DUH [2,3]. Increased melanin or increased numbers of melanocytes in the hyperpigmented macules was induced by SASH1 variants through the cascade activation of the p53/α-MSH/POMC/ Gαs/SASH1 and SASH1-MAP2K2-ERK1/2-CREB cascade [8,9]. In this study, the absolute numbers of melanocyte and the ratio of proliferative melanocytes among proliferative cells in the affected layers of hyperpigmented macules were observed (Fig. 4). Although increased numbers of melanocytes were slightly induced in the affected layers of hypopigmented macules, the ratio of proliferative melanocyte were not induced by PER3 rs772027021 SNP or via cooperation of PER3 rs772027021 SNP and SASH1 T525R mutation (Fig. 4B, 4C, 4E and Fig. S4). These results may suggest that increased melanin contents in the affected epithelial layers of hyperpigmented macules may be synthesized by increased proliferative melanocytes and no increased proliferative melanocytes in affected epithelial layers of hypopigmented macules could not synthesize more melanin. Additionally, a PER3-CREB phosphorylation cascade may mediate melanogenesis (Fig. 6E, F), which may interpret the underlying reason that increased melanin was induced by PER3 rs772027021 SNP. PER3-positive cells in the affected hyperpigmented epidermis are not only located in the basal layers but also the horny layers (Fig. S2). So, it is believed that the suprabasal layers of the affected hyperpigmented epidermis contain the PER3positive cells. Both Mitf-and Ki67-positive cells which were actually the proliferative melanocytes were both found in the basal and suprabasal layers in the hyperpigmented epithelial layers of the proband and II-3-and II-10-affected individuals, which indicated the suprabasal layers translocation of proliferative melanocytes from basal layers may result from PER3 rs772027021 SNP (Fig. 4). Additionally, significantly reduced proportions of apoptotic cells resulted from PER3 rs772027021 SNP (Fig. S5C), which indicates that proliferation of melanoma cells are induced by PER3 rs772027021 SNP. Therefore, it is believed that most of PER3-positive cells in the suprabasal layers may be the proliferative melanocytes. We have previously reported that SASH1 mutations induced an increased transferring of melanocytes from basal layers to suprabasal layers [7]. Various growth factors produced by keratinocytes affect the proliferation and differentiation of melanocytes, such as endothelin-1, stem cell factor and basic fibroblast growth factor. Soluble factors derived from keratinocytes regulate melanogenesis of adjacent melanocytes [34]. Therefore, we maintain that increased synthesis in the affected layers of II-3-and II-10-affected individuals may be caused by activation of the PER3-CREB cascade in the proliferative melanocytes and the keratinocytes in the suprabasal layers may trigger proliferation, differentiation, and melanogenesis of adjacent melanocytes.
PER3 was originally described as a core component of the circadian clock and one of the most robustly rhythmic genes in humans and animals. PER3 variants, especially the variable number tandem repeat (VNTR) in humans, are associated with diurnal preference, mental disorders, nonvisual responses to light, and brain and cognitive responses to sleep loss/circadian misalignment. The onset age of 99 affected homogeneous patients with bipolar disorder type I was influenced by a PER3 variable-number tandem-repeat (VNTR) polymorphism [34]. Furthermore, diurnal preference has been associated with the PER3 VNTR, whereas diurnal preference in young people is more closely associated with PER3 polymorphism [35]. These observations indicate that PER3 polymorphism is related to the onset age of mental illness and diurnal preference. Two rare variants (P415A and H417R) in PER3 cause familial advanced sleep phase (FASP) and are associated with elevated Beck Depression [36]. Homozygosity for the longer allele (PER3(5/5)) has a considerable effect on sleep structure, and cognitive performance in response to sleep loss is significantly decreased in PER3(5/5) individuals, revealing that this polymorphism in PER3 predicts individual differences in sleep loss-induced reduction in performance [37]. The proband and other patients in the DUH family reported that they had good sleep quality, even though they carried the PER3 rs772027021 SNP. The PER3 rs772027021 SNP is a missense variant in the coding sequence of PER3 that has not yet been reported to be of clinical significance (https:// www. ncbi. nlm. nih. gov/ snp/ rs772 027021). Indeed, this is the first report that PER3 P173S is associated with skin hyperpigmentation, melanogenesis, and melanocyte proliferation. To identify the functions of the PER3 rs772027021 SNP in increased melanin pigmentation and melanocyte growth, melanoma cells were infected with wild-type PER3 and mutant PER3, and melanin quantification and flow cytometry analyses indicated increased melanin synthesis was induced by the PER3 rs772027021 SNP (Fig. 5C, F) and reduced proportions of apoptotic cells and decreased proportions of G2/M-phase cells were caused by PER3 rs772027021 SNP ( Fig. S5C and 5D). More importantly, in vivo functional analyses of human PER3 in zebrafish verified the increased number of melanocytes induced by PER3 rs772027021 SNP (Fig. 6), suggesting that pigmented phenotypes can be caused by the SNP PER3 rs772027021 in DUHaffected individuals.
SASH1 is a signaling adaptor protein of 1,247 amino acids that contains an evolutionarily conserved SLY domain (401-555), an SH3 domain (557-614) and two SAM domains (633-697 and 1177-1241, annotation from the UniProt database) [38]. Increasing numbers of SASH1 variants have been found to be associated with DUH [7-11, 13, 14]. Apart from 3 additional variants in SASH1 reported by us, which are located in the SLY domain of SASH1 (p.E509K, p.L515P, and p.Y551D) [7][8][9], two identified SASH1 variants (p.Y551H [30] and p.Q518P [11]) are associated with DUH and located in the highly conserved SLY domain. All these findings suggest that the SLY domain is functionally critical for skin pigmentation regulation and may represent a mutational hotspot region during development of the DUH phenotype. The p.T525R variant of SASH1, which was reported in a sporadic DUH Japanese patient, causes lentiginous melanoma [14]; in the present study, p.T525R caused DUH hyperpigmentation phenotypes. This mutation was only detected in the proband and not in the other affected or unaffected individuals in the DUH family, which indicates that SASH1T525R mutant cosegregates with the DUH phenotype in the family studied. In vitro assays revealed increased melanin synthesis was induced by the T525R mutation. Our findings enrich the functions of SASH1 variants in hereditary pigmentary diseases, including DUH and lentiginous nevus. To reveal the reasons that the clinical phenotypes of the proband were more serious than those of other affected members of the DUH family, we analyzed the synergistical effects of PER3 rs772027021 SNP and the SASH1 T525R variant on melanogenesis. Functional analyses in melanoma cells indicated enhanced melanin synthesis induced via cooperation of PER3 rs772027021 SNP and the SASH1 T525R variant (Fig. 5C, F). These results explain the underlying reasons that pigmentation phenotypes in the proband were more serious than those of the other affected individuals.
Of the 28 variants that were screened as potential pathogenic mutations based on pathogenicity prediction programs, changes in SFTA3, KYNU, and GLMN showed a strong possibility of causing hyperpigmentation phenotypes in the DUH family (Table S6, Table S7, and Table S8). The SFTA3, KYNU, and GLMN variants displayed a family distribution similar to that of the PER3 P173S SNP, and the in vitro Fig. 6 The PER3 rs772027021 SNP is required for melanocyte proliferation in zebrafish in vivo and PER3-CREB pathway may regulate melangenesis. (A) Human PER3 and zebrafish per3 are orthologous genes. Homology analyses of the gene sequence of PER3 from the Ensembl database indicated that human PER3 and zebrafish per3 are homologous genes; collinear genes are vamp3 (vamp3) and UTS2 (uts2b), among others. B Quantitative RT-PCR showed wild-type and mutant PER3 to be expressed in zebrafish at 24 hpf. C Representative images of 72-hpf zebrafish embryos injected with wild-type PER3 and PER3 rs772027021 SNP or control transcript. D Quantitative measurement shows that highly proliferative melanocytes were induced by PER3 rs772027021 SNP compared with the control group and wild-type PER3. E Increased phospho-CREB and reduced phospho-ERK1/2 levels in B16 cells were caused by overexpression of PER3. B16 cells were infected with the adeasy014-pAdEasy-EF1-MCS-CMV-EGFP adenovirus containing wild-type and mutant type PER3. At 60 h after infection, cells were lysed and subjected to western blot. F Enhanced phospho-CREB and decreased phospho-ERK1/2 levels in SK-MEL-1 cells were resulted from overexpression of PER3 rs772027021 SNP ◂ 1 3 and in vivo functions of these variants were investigated. Melanin quantification analyses indicated that the SFTA3 c. 211delC variant alone or in combination with PER3 P173S SNP had no effect on melanin synthesis compared to control or wild-type SFTA3 or mutant PER3 (Fig. S7). Moreover, no increased melanin synthesis was induced by the KYNU variant (Fig. S6B). No increased melanogenesis was induced by the GLMN c. 561delT variant in vitro (Fig. S9A), nor was melanocyte proliferation increased in vitro (Fig. S9D, E).