The PER3 rs772027021 SNP induces pigmentation phenotypes of dyschromatosis universalis hereditaria

doi:10.21203/rs.3.rs-1965349/v1

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 ( PER3 rs772027021 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 PER3 rs772027021 SNP in this family demonstrated mild pigmented phenotypes compared to those of the proband carrying PER3 rs772027021 SNP and SASH1 T525R mutation. Increased melanin synthesis was induced by PER3 rs772027021 SNP in the melanocytes of affected epithelial tissues. Mutated SASH1 or PER3 rs772027021 SNP alone or cooperation of mutation of SASH1 and PER3 rs772027021 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 PER3 rs772027021 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.

Dyschromatosis universalis hereditaria (DUH)

Hyperpigmentation phenotype

PER3 SNP

SASH1 mutation

Dyschromatosis universalis hereditaria [DUH; Online Mendelian Inheritance in Man (OMIM) 127500] is a rare autosomal dominant genodermatosis initially described by Ichikawa and Hiraga in two generations of two families in 1933[1]. This disorder is characterized by asymptomatic hyperpigmented and hypopigmented macules extensively distributed over the trunk, limbs, and sometimes face[2, 3]. Lesions of irregular size and shape begin to appear in infancy or early childhood[2, 4, 5]. Lesions predominantly in the skin appear on the face, trunk and extremities in affected individuals, but the palms and soles are not involved[2]; abnormal lesions of hair and nails have also been reported[5]. We discovered two Chinese DUH pedigrees with dyschromatosis symmetrica hereditaria (DSH) in 2003[6] showing autosomal-dominant inheritance and diagnosed patients with DUH [3]. Three SASH1 variants (c.2000G>A, p.E509K; c.2019T>C, p. L515P and c. 2126T>G, p.Y551D) associated with DUH in two Chinese families and one American family were identified by our team and found to increase migration in melanoma cells[7], to mediate skin melanogenesis through the p53/α-MSH/POMC/Gαs/SASH1 cascade[8] and to engage in crosstalk with the ERK1/2-CREB cascade through MAP2K2 to promote melanogenesis[9]. Additionally, increasing evidence suggests that SASH1 variants including a c.1761C>G (p.Ser587Arg) variant [10],a c.1553A > C (p.Q518P) one [11], and a c.1556 G->A(p. S519N) one[12] are associated with genodermatosis or DUH[10-13]. Recently, the SASH1 variant p.T525R was reported in a sporadic case with lentiginous phenotypes[14]. ABCB6 had also been identified to be the causal genes responsible for DUH and a c.1067T4C (p.Leu356Pro) variant in exon3, a c.508A4G (p.Ser170Gly) one in exon1, a c.1736G4A(p.Gly579Glu) one in exon 12[15], a c.1358C>T( p.Ala453Val) one and a c.964A>C ( p.Ser322Lys) one [16] , a c.1663 C>A( p.Gln555Lys) one and a c.459 delC one[17], and a c.964A>C (p.S322R) one in exon4 and a c.1270T>C one (p.Y424H) in exon6 [18] were associated with DUH .

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 pathway-relevant 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.

Human subjects

All biological samples were obtained after receiving written informed consent from the subjects. The study was performed in accordance with the Declaration of Helsinki protocols and approved by the ethics committee of the respective institution.

Exome sequencing and data analysis

1. DNA extraction and exome sequencing Genomic DNA extracted from peripheral blood was fragmented to an average size of 180~280 bp and used to prepare a DNA library using established Illumina paired-end protocols. An Agilent SureSelect Human All ExonV6 Kit (Agilent Technologies, Santa Clara, CA, USA) was used for exome capture according to the manufacturer’s instructions. The Illumina NovaSeq 6000 platform (Illumina Inc., San Diego, CA, USA) was utilized for genomic DNA sequencing by Novogene Bioinformatics Technology Co., Ltd. (Beijing, China) to generate 150-bp paired-end reads with a minimum coverage of 10× for ~99% of the genome (mean coverage of 100×).

2. Data analysis After sequencing, base call file conversion and demultiplexing were performed with bcl2fastq software (Illumina). In-house quality control software was employed to remove low-quality reads from the resulting fastq data, followed by alignment to the human reference genome GRCh37 (hg19 /NCBI build 37) using Burrows–Wheeler Aligner (bwa)[21]. Duplicate reads were marked using sambamba tools[22].

3. SNP/INDEL calling Single-nucleotide variants (SNVs) and indels were called with sam tools to generate gVCF[23]. The raw calls of SNVs and INDELs were further filtered with the following inclusion thresholds: 1) read depth > 4; 2) root-mean-square mapping quality of covering reads > 30; and 3) variant quality score > 20.

4. CNV calling Copy number variants (CNVs) were detected with CoNIFER software (V0.2.2)[24].

5. Annotation Annotation was performed using ANNOVAR (2017June8)[25]. Annotations included minor allele frequencies from public control datasets as well as deleteriousness and conservation scores enabling further filtering and assessment of the likely pathogenicity of variants.

6. Rare variants filtering Filtering of rare variants was performed as follows: (1) variants with an MAF less than 0.01 in 1000 genomic data (1000 g_all)[26], esp6500 siv2_all, gnomAD data (gnomAD_ALL and gnomAD_EAS) and in house Novo-Zhonghua exome database from Novogene were considered; (2) SNVs occurring in exons or splice sites (splicing junction 10 bp) were further analyzed because they sought to explore amino acid changes; (3) synonymous SNVs not relevant to the amino acid alteration predicted by dbscSNV were discarded, and the small fragment nonframeshift (<10 bp) indel in the repeat region defined by Repeat Masker was discarded; (4) variants were screened according to SIFT scores[27], Polyphen[28], MutationTaster [29] and CADD[30] software, and potentially deleterious variations were reserved if the score of more than two of the four software programs supported the harmfulness of the variant[31], and sites (>2 bp) that did not affect alternative splicing were removed.

7. ACMG classification To better predict the harmfulness of variants, the classification system of the American College of Medical Genetics and Genomics (ACMG) was utilized. The variations are classified into pathogenic, likely pathogenic, uncertain significance, likely benign and benign[32].

8. Genetic linkage analysis To identify DUH loci, we performed an independent genome-wide scan for linkage in the multigenerational family. For linkage analysis, merlin tools and Perl, combined with high-throughput sequencing data for the family and allele frequency of the HapMap database of the Chinese population (CHB), were used with a known SNP as a marker.

9. Pedigree analysis Given the characteristics of the pedigree, heterozygous, homozygous and compound heterozygous variants were considered to be candidate causal variations.

10. Kinship analysis Kinship analysis for the proband and parents was carried out using the pairwise identity-by-descent (IBD) calculation in PLINK[33]. IBD sharing between the proband and parents in all trios was calculated to be between 45% and 55%.

Sanger sequencing

The primers used for PER3, SASH1, DPP10, KYNU, CELSR1, GLMN and SFTA3 variant validation and mutation analysis are listed in Supplemental Table S1. The PCR products from genomic DNA were sequenced using an ABI3730XL DNA Analyzer (International Equipment Trading, Vernon Hills, IL, Applied Biosystems). Sanger sequencing was used to confirm the presence and identity of variants in the candidate gene identified by exome sequencing and to screen the candidate gene in affected and unaffected individuals in the DUH family.

Immunohistochemistry and multi-color IHC(mIHC)

To examine expression of SASH1 and PER3 in skin, normal skin epithelium samples from 14-year-old control subjects and skin epithelial tissues of the hyperpigmented and hypopigmented lesional areas on the thigh and elbow of the proband and shanks of the other affected individuals were stained using a rabbit anti-PER3 antibody (ab224594, Abcam, USA) and rabbit anti-SASH1 polyclonal antibody (pAb) (NBP-26650, Novus Biologicals, LLC, USA). Skin epithelial tissues were fixed in 10% formalin at 4 °C for 24 hr and embedded in paraffin. Paraffin sections (5 µm‑thick) were incubated at 60 °C for 3 hr and then deparaffinized and rehydrated using xylene and an ethanol gradient. The immunohistochemical analysis procedure mainly referred to our previous reports[8, 9].

The mIHC assays were performed as per the protocols provided by the manufacture（RS0035-15test，ImmunoWay，Suzhou, Jiangsu, China）. The epithelial tissues were incubated with mouse anti-Ki67 polyclonal antibody (Proteintech Group, Inc., Wuhan, China) for 1.5hr at 37 °C and followed by three times washing with phosphate buffered saline(PBS). The epithelial tissues were incubated with HRP polymerized anti rabbit / mouse secondary antibody(ImmunoWay，Suzhou, JiangSu, China) for 30min at room temperature and followed by five times washing with PBS and stained with Alexa Fluor 594(ImmunoWay，Suzhou, JiangSu, China). The epithelial tissues were then washed with PBS and treated with antibody stripping solution and followed by applying the microwave performs the antigen recovery. The epithelial tissues were then incubated with anti-Mitf antibody (ab224594, Abcam, Cambridge, UK) and HRP polymerized anti rabbit / mouse secondary antibody(ImmunoWay，Suzhou, JiangSu, China) and stained with Alexa Fluor 488(ImmunoWay，Suzhou JiangSu, China) and followed with nucleus staining with DAPI. Staining with Alexa Fluor 594（594nm）, Alexa Fluor 488 (488 nm) and DAPI were visualized as green, red and blue signals, respectively. Fluorescence signals were observed by forward Laser Scan Microscope (Carl Zeiss Observer A1，Zeiss，Germany).

Melanin staining

Melanin staining in the skin epithelial tissues of healthy controls and affected individuals in the DUH family was performed as previously described[7, 34].

Gene cloning, site-directed mutagenesis and virus packaging

The myc-DDK-tagged human period homolog 3 (PER3) expression plasmid (RC217739, OriGene Technologies Inc. Ltd) was sequenced to confirm the integrity of the PER3 sequence. Nucleotide 517 “C” of PER3 was mutated to “T” by site-directed mutagenesis using a KOD-Plus mutagenesis kit (TOYOBO, Japan). The cDNA sequence of human PER3 (reference sequence Homo sapiens PER3: NM_ 001289861) was synthesized, and 3×HA sequences were added after the initiation codon of the PER3 CDS. Nucleotide 517 “C” of PER3 was mutated to “T” by site-directed mutagenesis technology. Wild-type PER3 and the PER3^C517TSNP mutant were cloned into the Adeasy 014-pAdEasy- EF1-MCS- CMV-EGFP adenovirus vector. Sanger sequencing was performed to verify the correctness of site-directed mutagenesis of PER3 mutant. The cDNA sequence of SASH1 (reference sequence of Homo sapiens SASH1: NM_015278.5) was synthesized, and 3×Flag sequences were added after the initiation codon of the CDS. Nucleotide 1574 “C” of SASH1 was mutated to “G” by site-directed mutagenesis technology. Wild-type SASH1 and the SASH1 mutant were cloned into the adeasy014- pAdEasy-EF1-MCS-CMV-EGFP adenovirus vector via the restriction enzyme sites EcoRI and BamHI. Sanger sequencing was also performed to verify the correctness of site-directed mutagenesis of SASH1 mutant. The recombinant adeasy014-pAdEasy- EF1-MCS-CMV-EGFP adenovirus was packaged by HanBio Biological Technology Co., Ltd., China.

Cell culture, transfection , infection and Cell cycle and apoptosis assays.

B16 and SK-MEL-1 cells were obtained from the Procell Procell Life Science&Technology Co., Ltd. (Wuhan, China). All cell lines were maintained in Dulbecco’s modified Eagle’s medium (HyClone Laboratories, Inc., Logan, UT, USA) supplemented with 10% BI fetal bovine serum (Bioind, Israel) and cultured at 37 °C with 5% CO₂. The transfection procedure for the GFP-SASH1 and myc-PER3 plasmids mainly referred to our previous reports[8, 9]. Adenovirus infection was performed according to the procedure provided by the manufacturers.

The B16 cells were infected with the adeasy014- pAdEasy-EF1- MCS- CMV-EGFP adenovirus overexpressing wild type and mutated PER3 and/or wild type and mutated SASH1 and starved and subjected to cell cycle analyses. Cells were harvested and fixed in 70% ethanol at 4°C, then stained with 36mg/ml propidium iodide containing 400mg/ml RNAase (KeyGEN Biotech Ltd, Nanjing, Jiangshu, China) and followed with flow cytometry (Navious, Beckman Coulter) analyses for cell cycle profile determination. The cell cycle results were analyzed by ModFit LT software. The infected and starved B16 cells were trypsinizated and collected and fixed with 95% ethanol at 4℃ for at least 2hr and followed with staining with 7AAD/APC. The stained cells were subjected to flow cytometry for apoptosis analyses and the apoptosis results were analyzed using Flowjo software (CT, USA).

Immunoblotting

Western blotting was performed as previously described[8, 9]. The primary antibodies used for western blotting were as follows: anti-GFP antibody (M20004M, Abmart Shanghai Co. Ltd., Shanghai, China), anti-SASH1 antibody (NBP1-26650, Novus Biologicals, LLC) and anti-PER3 antibody (ab224594, Abcam, Cambridge, UK), p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb (#4695, Cell Signaling Technology ), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Antibody( #9101, Cell Signaling Technology) , CREB (48H2) Rabbit mAb (#9197, Cell Signaling Technology ) and Phospho-CREB (Ser133) (87G3) Rabbit mAb (#9198, Cell Signaling Technology) .

Melanin quantification

At 48 h after transfection or infection, B16 cells infected or transfected with the adeasy014-pAdEasy-EF1-MCS-CMV-EGFP adenovirus overexpressing wild-type and mutant PER3, SASH1 and HA-SFTA3 were washed with GenMed leaning liquid provided by the manufacturer (GenMed Scientifics Inc.), lysed with GenMed lysis buffer at 4 °C for 30 min and subjected to centrifugation at 16,000 × g for 5 min at 4 °C. The B16 cell lysates were centrifuged at 16,000 × g for 5 min at 4 °C, and 10 μl of supernatant was used to determine the protein concentration. The supernatant was removed, and 500 μl of GENMED treatment solution was added to the cell pellet, mixed and centrifuged at 16,000 × g for 5 min. The supernatant was removed, and 500 μl of the dissolved GENMED solution was added to the cell pellets and mixed. Then, 500 μl of GENMED buffer was added, and the sample was stored at 60 °C for 30 min to avoid degradation. The melanin content was determined by measuring absorbance at 360 nm. SK-MEL-1-infected cells (10⁶cells per well) in 10-cm dishes were lysed with 1 M NaOH at 80 ℃ for 2 h. After centrifugation at 12,000× g for 10min at room temperature, the supernatants were transferred to fresh tubes, and the melanin content was determined in triplicate for each sample by measuring absorbance at 405 nm using a spectrophotometer; the results are expressed as micrograms of melanin per mg protein. Synthetic melanin (Sigma, St. Louis, MO, USA) was used to plot a standard curve to calculate the melanin content of SK-MEL-1 cells.

Phenotypic characterization of the PER3^P173S SNP and the GLMN c. 561delT variant in zebrafish embryos.

1. Bioinformatics analysis of human PER3 and zebrafish per3 as orthologous genes.

Whether human PER3 and zebrafish per3 are orthologous genes was analyzed using the online Ensembl database.

2. Zebrafish embryos were used as a reactor to verify the function of the PER3^P173S SNP and the GLMN c. 561delT variant in humans.

Wild-type human PER3 and GLMN cDNA were synthesized and cloned into the pXT7 vector. Site-directed mutagenesis to produce the PER3 c. C517T and GLMN c. 561delT mutations was performed per the protocol provided by the KOD-Plus- Mutagenesis kit (TOYOBO, Japan) using the primers PER3 and GLMN (Supplemental Table S1). Site-directed mutation of PER3 and GLMN were verified by Sanger sequencing. Linearization of the expression plasmid for wild-type and mutant PER3 and GLMN was performed by SalI digestion. The linearization products were purified with the PCR clean up kit (Axygen). In vitro transcription was performed using mMESSAGE mMACHINE™ T7 Ultra Transcription Kit (Ambion). The mRNA transcribed in vitro was purified according to the protocol provided by mMESSAGE mMACHINE™ T7 Ultra Transcription Kit (Ambion) by adding lithium chloride precipitation solution (Ambion) and 75% ethanol in RNase-free H₂O. Agarose gel electrophoresis (1%) was used to identify the integrity of the purified mRNA, and the concentrations were measured. Fertilized eggs of zebrafish were collected according to the conventional method. Purified PER3 mRNA (20, 100 and 250 ng/µl) and purified GLMN mRNA (25, 100 and 250 ng/µl) were microinjected into the fertilized zebrafish eggs. The survival rate of the zebrafish fertilized eggs at 24 hpf was assessed, and expression of PER3 and GLMN was detected by qRT–PCR. To characterize the pigmentation phenotype of zebrafish, 72 hpf was selected as a reference time point based on previous reports[35, 36].

Statistical analysis

Data were analyzed using a homogeneity of variance test, and one-way ANOVA was used for multiple comparisons using the least significant difference in SPSS 16.0 to generate the required P values. P values of less than 0.05 were considered statistically significant. Data are presented as means±SDs. Cartograms were generated and plotted using GraphPad Prism 5.

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 30s 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).

Affected individuals in the pedigree, including her grandmother (I-2), father (II-3), fifth aunt (II-10), sixth uncle (II-11) , younger brother(III-9) and younger female cousin (III-12), all showed sporadic or disseminated hyperpigmented and/or hypopigmented macules on the limbs (I-2, II-3, II-10 and III-12), trunk(II-3 and III-9), back (II-3) and III-9 and auricle (II-11) (Fig. 1B-1E, and Table 1). The hypopigmented and hyperpigmented lesions of the II-3 affected individual were concentrated on his trunk, shoulder, and back, and his hyperpigmented lesions were diffusely distributed (Fig. 1C). The hyperpigmented lesions of II-10 were concentrated on both lower limbs (Fig. 1D). The hyperpigmented lesions of II-11 were focused on the auricles (Fig. 1E). The hypopigmented lesions of III-9 were diffusely distributed on right upper chest and back. The dense hyperpigmented lesions distributed on the shoulder and dorsum (Fig. 1F).

The above six affected individuals all had hypopigmented and/or disseminated hyperpigmented macules; there was no skin atrophy, telangiectasia, or inflammation, among others. 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 DUH-affected 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[37]. 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 mosaic-like melanin distribution were found for the basal and suprabasal layers of the hyperpigmented macules of II-3 and II-10 (Fig. 2D-2G).

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[27], Polyphen[28], MutationTaster[29] and CADD software[30]. Variants were classified as pathogenic, likely pathogenic, uncertain significance, likely benign or benign according to the classification system of the American College of Medical Genetics and Genomics (ACMG)[32].

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 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 (Fig. 3C, Fig. 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.1067T4C (p.Leu356Pro) variant of ABCB6 , the c.508A4G (p.Ser170Gly) one, the c.1736G4A(p.Gly579Glu) 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.1270T>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).

A c. 517C>T (p.P173S, rs772027021) variant in exon 5 of PER3 (NM_001289861), a c. 211delC (p.L71fs) variant in exon 3 of surfactant-associated 3 (SFTA3) (NM_001101341), a c. 716C>T (p.A239V, rs199529102) variant in exon 9 of kynurenine (KYNU) (NM_ 001199241.2) and a c. 561delT variant in exon 5 of glomulin (GLMN) (NM_053274.3) were screened as potential pathogenic 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^rs772027021SNP 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 and 3E, Table S4). Pathogenicity prediction of the PER3^rs772027021 SNP was performed with the VarSome tool (https://varsome.com/). PER3^rs772027021 SNPis 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 (Table S8).

The remaining 24 variants or SNPs screened as potentially pathogenic mutation prediction programs included SNP MYOC^rs74315337, SNP CELSR1^rs374501629, SNP ST3GAL5^rs549326241, SNP DPP10^rs138159056, variant DES c. 740delT, SNP UMP^rs12191789, SNP BCHE^rs537434945, SNP EVC2^rs200140401, SNP ADAMTS6^rs147540204, variant MAK c. A1598G, variant PKHD1 6:51768842-T-A, SNP PHIP^rs200515013, SNP RAD54B^rs114216685, variant HPS6 c. 1687dupC, variant ST14 c. C1819T, SNP ^{Trp4rs757614572}, SNP SF3B2^rs201160612, SNP LRRK2^rs34594498, SNP GCH1^rs770547722, SNP DUOX2^rs180671269, variant DUOX2 c. G943T, variant CRTC3 c. G967A, variant AP2B1 17:34036332-G-A and SNP GSS^rs113191242. The remaining 24 variants or SNPs do not cosegregate with the pigmented phenotype in the affected and unaffected family members who underwent whole-exome sequencing. High conservation among species was show in Thr525 site of SASH1 but not the Pro173 of PER3 (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 mosaic-like 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 (Fig. S2B,2C). The expression and distribution of SASH1 was also investigated, and a mosaic-like distribution and high expression were detected in the affected epidermis of hyperpigmented macules compared to hypopigmented macules in the proband (Fig. S3A -3B). 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-S3F).

Proliferation of melanocyte were induced by PER3^rs772027021 SNP alone or the cooperation of PER3^rs772027021 SNP or SASH1^T525Rvariant in affected skin and in vitro.

To investigate the effects of PER3^rs772027021 SNP or SASH1^T525Rvariant on melanocyte proliferation, mIHC of epithelial tissues of normal pigmented foreskin samples and the proband，II-3 and II-10 affected individuals was performed using Ki67 marking the proliferative epithelial cells and Mitf marking melanocytes. The Ki67- positive 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 Ki67- positive 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^T525Rvariant, 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 (Fig. 4 and Fig. S4). The immunoflourence results of affected epithelial tissues of proband and II-3 and II-10 indicated proliferative melanocytes was induced by PER3^rs772027021 SNP alone or cooperation 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 (Fig.4 and Fig.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 and S5D). 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 and S5B).

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 do 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[38]. PER2 has been suggested to be the target gene of SFTA3 by LncRNA2Target(http://123.59.132.21 /lncrna2target/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 wild-type 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.

In this study, the PER3^P173S SNP is first reported to induce pigmented phenotypes in DUH-affected individuals. The zebrafish is an ideal model for studying melanocyte differentiation[35, 39]. 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 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 [35, 36]. 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 and 6D). 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 and 6F)，which indicated that a PER3-CREB phosphorylation cascade may mediate melanogenesis.

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, 15-18], 6q24.2-q25.2 (SASH1)[6-11, 13, 40] 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, 41-43], 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 increased by PER3^rs772027021 SNP or via cooperation of PER3^rs772027021 SNP and SASH1^T525Rmutation(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^T525Rmutation (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 and 6F), which may interpret the underlying reason that increased melanin was induced by PER3^rs772027021 SNP.

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 [44]. Furthermore, diurnal preference has been associated with the PER3 VNTR, whereas diurnal preference in young people is more closely associated with PER3 polymorphism[45]. 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[46]. 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 [47]. 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/ rs772027021). 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 (Figure 5C and 5F) 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 DUH-affected 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)[34]. 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)[7-9], two identified SASH1 variants (p.Y551H[40] 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 this SASH1 variant is a de novo mutation. 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^T525Rvariant on melanogenesis. Functional analyses in melanoma cells indicated enhanced melanin synthesis induced via cooperation of PER3^rs772027021 SNP and the SASH1^T525Rvariant (Fig. 5C, 5F). 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 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 and S9E).

Acknowledgments We thank the Clinical Research Center at the Affiliated Hospital of Guizhou Medical University for in-house experiments. We thank Dehou Yu at the Department of Dermatovenereology, the Affiliated Hospital of Guizhou Medical University, for the disease diagnosis of DUH family members. We thank Nanjing YSY Biotech, Ltd. at Jiang shu, China for zebrafish husbandry and lines and injection of PER3 mRNA into zebrafish fertilized eggs and other functional assays.

Author contributions Ding’an Zhou made substantial contributions to the conception and design of the work. Qinghe Xing，Xiaohua Zeng and Dongsheng Wang provided constructive comments for analyzing the results of the exome sequencing and Sanger sequencing，especially Xiaohua Zeng some scientific research funds to ensure the completion of this project and the vision. Hongyu Chen, Pingping Yang, and Dan Yang performed most of the experiments and analyzed and interpreted the data. Yadong Li, Jing Zhang, Zhen Zeng and Zhiqiang Zhong participated in some of the experiments. Zhi Liu and Mao Lu participated in the diagnosis of the pedigree individuals. Ding’an Zhou and Xu Jia drafted and revised the manuscript. All authors read and approved the final manuscript.

Funding This work was supported partly by the following funds: National Natural Science Foundation Project (31860319 and 32060165 to Ding’an Zhou), Guizhou Provincial Science and Technology Department Project (grant no: qian ke he zhicheng [2020]4Y125 and qian ke he jichu-ZK[2021] zhongdian 031 to Ding’an Zhou), and National Natural Science Foundation Project (31371274 to Qinghe Xing).

Ethics approval and consent to participate

All procedures performed in studies involving human participants were approved by the ethics committee of the Affiliated Hospital of Guizhou Medical University, and the ethical approval documents were acquired (No. 2020073K). The study was conducted in accordance with the 1964 Helsinki Declaration. Informed consent to participate in the study was obtained from participants. Written informed consent was obtained from all participants.

Conflicts of interest The authors declare that they have no conflicts of interest.

Availability of data and material I confirm I have included a data availability statement in my main manuscript file. My manuscript has data included as electronic supplementary material.

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Table 1 PER3^rs772027021 SNP and a SASH1 variant in the DUH family individuals and their clinical characteristics.

						Number and distribution of hyperpigmented macules			Number and distribution of hypopigmented macules
Patient	Sex	Age	Inheritance	Pathogenic gene(s)	Mutation site(s)	Face	Limbs	Trunk	Face	Limbs	Trunk
Ⅲ-6	F	32	Familial	PER3	c.C517T, p.P173S	Extensive	Extensive	Extensive	Many	Many	Several
Ⅲ-6	F	32	Familial	SASH1	c.C1574G, p.T525R	Extensive	Extensive	Extensive	Many	Many	Several
Ⅱ-3	M	57	Familial	PER3	c.C517T, p.P173S	No	Several	Several	No	Many	Many
I-2	F	79	Familial	PER3	c.C517T, p.P173S	No	Several	Several	No	Many	Many
Ⅱ-10	F	51	Familial	PER3	c.C517T, p.P173S	No	Many	No	No	Several	No
II-11	M	49	Familial	PER3	c.C517T, p.P173S	Several	No	No	Several	No	No
III-12	F	30	Familial	PER3	c.C517T, p.P173S	No	Several	Several	No	Few	Few
III-9	M	26	Familial	PER3	c.C517T, p.P173S	No	No	Many	No	No	Many
II-1	M	53	No	No	No	No	No	No	No	No	No
I-1	M	79	No	No	No	No	No	No	No	No	No
II-2	F	59	No	No	No	No	No	No	No	No	No
II-4	F	56	No	No	No	No	No	No	No	No	No
II-9	M	51	No	No	No	No	No	No	No	No	No
II-12	F	42	No	No	No	No	No	No	No	No	No
II-13	M	45	No	No	No	No	No	No	No	No	No
II-14	F	35	No	No	No	No	No	No	No	No	No
III-1	M	37	No	No	No	No	No	No	No	No	No
III-11	M	25	No	No	No	No	No	No	No	No	No
III-13	F	14	No	No	No	No	No	No	No	No	No
III-14	M	15	No	No	No	No	No	No	No	No	No
III-16	F	5	No	No	No	No	No	No	No	No	No
IV-1	M	16	No	No	No	No	No	No	No	No	No
IV-2	F	14	No	No	No	No	No	No	No	No	No

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The PER3 rs772027021 SNP induces pigmentation phenotypes of dyschromatosis universalis hereditaria

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