A for Freckles Maps to 4q32–q34

Xue-Jun Zhang,Ãz1 Ping-Ping He,Ãwz Yan-Hua Liang,Ãwz Sen Yang,Ãz Wen-Tao Yuan,w Shi-Jie Xu,w and Wei Huangw1 ÃInstitute of Dermatology & Department of Dermatology at No.1 Hospital, Anhui Medical University, Hefei, China, wChinese National Center at Shanghai, Shanghai, China, zKey Laboratory of Genome Research at Anhui, Hefei, China

Freckles are numerous pigmented macules on the face commonly occurring in the Caucasian and Chinese population. As freckling is considered as an independent trait, no gene or locus for it has been identified to date. Here we performed genome-wide scan for linkage analysis in a multigeneration Chinese family with freckles. A maximum LOD score of 4.26 at a recombination fraction of 0 has been obtained with marker D4S1566. Haplotype analysis localized the freckles locus to a 16 Mbp region flanked by D4S2952 and D4S1607. We have thus mapped the gene for freckles to chromosome 4q32–q34. This will aid future identification of the responsible gene, which will be very useful for the understanding of the molecular mechanism of freckles. Key words: freckles/gene mapping/genome-wide scan/linkage analysis. J Invest Dermatol 122:286 –290, 2004

Freckles, or ephelides are numerous pigmented spots of the tion study with the melanocorin-1-receptor (MC1R) gene, skin commonly occurring in the Caucasian population freckles, and solar lentigines, and found that the MC1R (Bastiaens et al, 1999). They are mainly confined to the gene variants could play an important part in the develop- face, even arms and back. Freckles first appear at about 5 y ment of freckles and solar lentigines, but most of the as light-brown pigmented macules on light-exposed skin. freckling subjects who attended the study are affected with They increase in number, size, and depth of pigmentation melanoma or nonmelanoma skin cancer. As an independent during the summer months, and are smaller, lighter, and trait, freckles have not been mapped to any chromosome fewer in number in the winter. Histologically, the freckles region and even recorded into the OMIM up to now. To show increased production of melanin pigment by a normal localize the gene responsible for freckles, we performed a number of melanocytes. Freckles commonly stop spreading genome-wide scan and first mapped the freckle gene to before adolescence and last for life, but could sometimes chromosome 4q32–q34. be subtle in adulthood. They are transmitted as an autosomal dominant pattern (Brues, 1950). Several pig- Results mented skin disorders such as dyschromatosis symmetrica hereditaria (Online Mendelian Inheritance in Man (OMIM) Clinical findings The subjects are from a four-generation 127400), xeroderma pigmentosum (OMIM 278700), and family with freckles consisting of 42 individuals (Fig 1). The cutaneous malignant melanoma (OMIM 155600) are asso- family was of Chinese Han origin. The mode of inheritance is ciated with freckles; however, freckles mainly occur as an clearly autosomal dominant with complete penetrance. The independent trait, and most cases with freckles have no affected individuals had small brown macules on the face, other abnormalities. which became prominent during the summer when exposed Freckles are cosmetically disfiguring and treatment may to sunlight and fade during the winter (Fig 2). In this family, be requested on cosmetic grounds. This disorder is also freckles usually appeared at about age 5 and have been very common in the Chinese population. There are differentiated from solar lentigo that appears at a later age, approximately 3000 Chinese individuals that have ever mostly in persons with long-term sun exposure. Skin biopsy sought treatment for freckling (Ma, 1998; Wang and Li, of pigmented macules was performed on the proband. 1998; Liang and Luo, 1999; Chen et al, 2000). In Hematoxylin and eosin staining showed abundant melanin Caucasians, freckling is most frequent in individuals with pigment in the basal layer of the epidermis (Fig 3). All red hair and fair skin (Azizi et al, 1988; Bastiaens et al, 1999). affected individuals had no hypopigmented macules or Based on the close relationship between red hair, fair skin, other common associated disorders. None of the affected and freckles, Bastiaens et al (2001) performed an associa- members in this family was found to have skin cancer.

Two-point linkage analysis Initially, a supportive LOD Abbreviations: HAND2, heart and neural crest derivatives ex- score was obtained with marker D4S415 (Zmax ¼ 1.92, pressed 2; OMIM, Online Mendelian Inheritance in Man; PLDN, y ¼ 0.00). For fine mapping of the gene, 17 additional pallidin homolog (mouse); SNARE, soluble N-ethylmaleimide- sensitive factor attachment receptors. polymorphic microsatellite markers at 4q were further 1These two authors contributed equally to the paper. tested. Significant evidence for linkage was observed with

Copyright r 2004 by The Society for Investigative Dermatology, Inc. 286 122 : 2 FEBRUARY 2004 GENE FOR FRECKLES MAPS TO CHROMOSOME 4Q32–Q34 287

Figure 1 Haplotype analysis of the family. Black symbols denote affected individuals, whereas white symbols denote unaffected individuals. Haplotypes are shown for all available members with marker names at the left of each generation. Black bars represent disease-carrying haplotypes, and the gray bar denotes noninformative regions adjacent to critical recombination events. the maximum two-point LOD scores of 4.26 (y ¼ 0.00) at markers D4S2952 and D4S1566. The telomeric recombina- D4S1566. LOD scores 43 were also obtained for the other tion events could not be defined with certainty in our markers: D4S2414, D4S2914, D4S3028, and D4S2427. The pedigree because the affected individual II:4 was inferred pairwise LOD scores between the relevant markers and the homozygous for alleles of D4S415, D4S1552, and D4S2417, locus for freckles were given in Table I. and hence the telomeric crossover in one of the descen- dents (individual III:6) could be present between any Haplotype analysis To determine the smallest interval neighboring markers of D4S2427, D4S415, D4S1552, containing the freckles locus, we constructed haplotypes D4S2417, and D4S1607. Thus the most distal border of with Cyrillic Version 2.02 software (Sobel and Lange, 1996). the critical region was defined by a recombination between The proximal border of the critical region was defined by a D4S2417 and D4S1607 in normal individual IV:10. These recombination event, present in individual IV:8, between results suggest that the gene responsible for freckles in this 288 ZHANG ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

determined and may recur in successive generations in similar locations and patterns. Some cases with pigmented diseases such as dyschromatosis symmetrica hereditaria, xeroderma pigmentosum, and cutaneous malignant mela- noma also show freckle features and the for these disorders have been mapped and even cloned. Recently, dyschromatosis symmetrica hereditaria loci have been localized to 1q11–q21 and 6q24.2–q25.2 by Zhang et al (2003) and Xing et al (2003), respectively, but we failed to confirm the linkage to these two regions using this freckle family. In the pigmented macules of freckles, there is no increase in the number of melanocytes but their melano- somes are long and rod-shaped, which form melanin more rapidly after exposure to sunlight, like those found generally in dark-skinned people (Breathnach, 1964). Therefore, genes involved in melanosome formation or movement at 4q32–q34 are attractive candidates. This 16 Mb critical region contains 24 known genes, 23 predicted genes/ expressed sequence tags, and three predicted pseudo- genes. The PLDN gene (OMIM 604310) can be considered as an attractive candidate. The product of the PLDN gene is one of a number of genes that in mice are associated with pigmentation defects and platelet dense granule deficiency (Falcon-Perez and Dell’Angelica, 2002). PLDN encodes a novel, approximately 20 kDa protein that is expressed ubiquitously in mammalian tissues (Huang et al, 1999). The Figure 2 pallidin protein was found to bind to syntaxin 13, a member Numerous pigmented macules on the face of the proband in this of the syntaxin family of SNARE. As SNARE family. mediate fusion of intracellular membranes, pallidin may play a part in membrane fusion events required for family is located within the 16.78 cM interval between melanosome biogenesis (Falcon-Perez et al, 2002). The D4S2952 and D4S1607 (Fig 1), which represents approxi- HAND2 gene (OMIM 602407) whose transcripts have been mately 16 Mb physical distance on chromosome 4q32–q34. isolated from pooled human melanocytes cDNA library could also be selected for further investigation by mutation Discussion analysis. The protein encoded by this gene belongs to the basic helix–loop–helix family of transcription factors (Yama- Freckles are characterized by pigmented macules (1–3 mm) gishi et al, 2000). This gene product is one of two closely on the face, even on the neck, shoulders, and backs of the related family members, The HAND proteins, which are hands (Wilson and Kligman, 1982). They are genetically asymmetrically expressed in the developing ventricular

Figure 3 Histologic section stained with hematoxylin and eosin (original magnification 400) showed abundant melanin pigment in the basal layer of the epidermis. 122 : 2 FEBRUARY 2004 GENE FOR FRECKLES MAPS TO CHROMOSOME 4Q32–Q34 289

Table I. Two-point LOD scores for freckles and 18 microsatellite markers on chromosome 4q32–q34

LOD score at h ¼

a Markers Location (cM) 0.00 0.10 0.20 0.30 0.40 Zmax hmax D4S1603 162.47 1 0.55 0.62 0.43 0.19 0.62 0.20

D4S2952 166.85 1 0.61 0.56 0.39 0.18 0.61 0.10 D4S1566 166.85 4.26 3.51 2.68 1.76 0.80 4.26 0.00 D4S620 167.55 1.62 1.31 0.97 0.62 0.30 1.62 0.00 D4S2414 167.55 4.25 3.50 2.67 1.76 0.80 4.25 0.00 D4S2979 169.42 2.04 1.64 1.20 0.73 0.29 2.04 0.00 D4S2910 171.39 1.67 1.33 0.98 0.64 0.31 1.67 0.00 D4S2992 174.9 1.73 1.49 1.13 0.71 0.31 1.73 0.00 D4S2991 175.62 2.10 1.68 1.24 0.78 0.34 2.10 0.00 D4S2431 176.19 2.01 1.67 1.30 0.91 0.47 2.01 0.00 D4S2914 177.33 3.05 2.48 1.86 1.21 0.58 3.05 0.00 D4S3028 177.33 3.66 2.94 2.16 1.31 0.49 3.66 0.00 D4S2427 178.66 3.08 2.50 1.87 1.19 0.52 3.08 0.00 D4S415 181.36 1.92 1.55 1.15 0.76 0.38 1.92 0.00 D4S1552 181.36 1.79 1.46 1.10 0.73 0.37 1.79 0.00 D4S2417 181.93 1.58 1.32 1.04 0.73 0.38 1.58 0.00 D4S1607 183.63 1 1.16 1.03 0.60 0.16 1.16 0.10 D4S2951 185.28 10.09 0.17 0.14 0.05 0.17 0.20 aAccording to the final Marshfield human genetic map (Broman et al, 1998).

chambers and play an essential part in cardiac morphogen- showed clear signs of multiple, small (1–3 mm), pale-brown esis. Working in a complementary fashion, they function in macular lesions with a poorly defined margin, which are more the formation of the right ventricle and aortic arch arteries, pronounced in summer, were scored as affected. In the study, participants were asked if they had freckles in early childhood implicating them as mediators of congenital heart disease (p10 y of age) localized in the face, on the arms, and upper back, (Srivastava et al, 1997). Whether or not this gene is related which differentiated from solar lentigo that appears at a later age, to the freckling mechanism will be determined in a future mostly in persons with long-term sun exposure. The youngest study. There are no other mouse pigmentation gene nonaffected individual (IV:12) in this family was 11 y old, which homologs mapped to this 16 Mb region. At this stage, the exceeded the peak age of onset of freckles (about 5 y). Every mapping region is still wide and collecting additional family member had a signed consent form and this study was families with freckles may help to fine-map this gene region approved by the Anhui Medical University Review Board. A total of 42 family members participated in this study, after having given and make freckle gene cloning feasible. informed consent. Blood samples were collected from available In conclusion, we first identify a locus for freckles on family members and genomic DNA were extracted from peripheral chromosome 4q32–q34 by a large Chinese family. Although blood by use of QIAgen blood kit (QIAGEN Inc., Valencia, freckling is not considered as a pathologic disorder, it is California). common in the population and cosmetically disfiguring. This study will produce significant impact on the development of Genotyping We initiated a genome-wide scan in the family using cosmetology of pigmented skin disorders. Further studies 382 polymorphic microsatellite markers with an average marker are continuing, to refine the genetic interval and screen density of 10 cM. The Marshfield linkage map (Broman et al, 1998) was used to establish marker order and intermarker distances. For candidate genes for mutations. The elucidation of the fine mapping, 17 additional microsatellite markers at 4q were molecular mechanisms must await the identification and further genotyped. Polymerase chain reaction reactions were functional analysis of the freckle gene. carried out with a touchdown program in a 5 mL volume, including 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 0.1 mg gelatin per mL, 3.0 mM MgCl2, 0.2 mM deoxyribonucleoside triphosphate (each), 0.2 Materials and Methods U AmpliTaq Gold (Applied Biosystems, Foster City, California), 0.04 mM of each primer, and 10 ng of genomic DNA. Polymerase chain Subjects A large family from the Anhui province of China who had reaction products were electrophoresed on a MegaBACE-1000 typical freckle features was recruited for this work (Fig 1). It showed DNA sequencer (Amersham Bioscience, Piscataway City, New an autosomal dominant inheritance pattern. Freckles were Jersey) The size of allele was determined on the basis of assessed by physical examination by experienced dermatologists MegaBACE ET400-R Size Standard. Following the electrophoresis, using standard clinical criteria (Habif, 1996). Only individuals who data were collected and analyzed with Image Control manager and 290 ZHANG ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Genetic Profiler software (Amersham Bioscience). All genotyping Falcon-Perez JM, Starcevic M, Gautam R, Dell’Angelica EC: BLOC-1, a novel data were verified by PedCheck (O’Connell and Weeks, 1998). complex containing the pallidin and proteins involved in the Genotypes of each locus were further reviewed and scored biogenesis of melanosomes and platelet-dense granules. J Biol Chem independently by two observers in case of discrepancy. 277:28191–28199, 2002 Habif TP: Light-related diseases and disorders of pigmentation. In Clinical Dermatology, 3rd edn. Mosby-Year Book, Inc., Oxford 1996; p 622 Linkage and haplotype analyses Two-point linkage analysis was Huang L, Kuo YM, Gitschier J: The pallid gene encodes a novel, syntaxin 13- performed with Linkage programs version 5.10 (Lathrop and interacting protein involved in platelet storage pool deficiency. Nat Genet Lalouel, 1984). Autosomal dominant inheritance with 100% 23:329–332, 1999 penetrance was assumed. The affected allele frequency was taken Lathrop GM, Lalouel JM: Easy calculations of lod scores and genetic risks on as 0.01. Marker allele frequencies were counted from founders of small computers. Am J Hum Genet 36:460–465, 1984 Liang YM, Luo XX: Clinical effect of liquid nitrogon therapy in 577 cases with the pedigrees. The recombination frequency was assumed to be freckles and verruca plana. J Guangdong Med College 17:264–265, 1999 equal for both sexes. Haplotypes were constructed with Cyrillic (in Chinese) Version 2.02 software (Sobel and Lange, 1996). Ma WP: Clinical effect of phenol therapy in 400 cases with freckles. Chin J Dermatol 31:258, 1998 (in Chinese) O’Connell JR, Weeks DE: PedCheck: A program for identification of genotype This work was funded by grants from the Chinese High Tech Program incompatibilities in linkage analysis. Am J Hum Genet 63:259–266, 1998 (863) (2001AA227031, 2001AA224021, and 2002BA711A10). We are Sobel E, Lange K: Descent graphs in pedigree analysis: Applications to most grateful to the freckle family for participating in this study. haplotyping, location scores, and marker-sharing statistics. Am J Hum Genet 58:1323–1337, 1996 DOI: 10.1046/j.0022-202X.2004.22244.x Srivastava D, Thomas T, Lin Q, Kirby ML, Brown D, Olson EN: Regulation of cardiac mesodermal and neural crest development by the bHLH Manuscript received September 2, 2003; revised October 6, 2003; transcription factor, dHAND. Nature Genet 16:154–160, 1997 accepted for publication November 4, 2003 Wang JH, Li QH: Clinical effect of liquid nitrogen therapy in 680 cases with freckles. Chin J Dermatol 31:257, 1998 (in Chinese) Address correspondence to: Professor Xue-Jun Zhang, Institute of Wilson PD, Kligman AM: Experimental induction of freckles by ultraviolet-B. Br J Dermatology, Anhui Medical University, 69 Meishan Road, Hefei, Anhui Dermatol 106:401–406, 1982 230032, PR China. Email: [email protected] or Dr Wei Huang, Xing QH, Wang MT, Chen XD, et al: A gene locus responsible for dyschromatosis Chinese National Human Genome Center at Shanghai, 250 Bi Bo symmetrica hereditaria (DSH) maps to chromosome 6q24.2-q25.2. Am J Road, Shanghai 201203, PR China. Email: [email protected]. Hum Genet 73:377–382, 2003 Yamagishi H, Olson EN, Srivastava D: The basic helix-loop-helix transcription factor, dHAND, is required for vascular development. J Clin Invest References 105:261–270, 2000 Zhang XJ, Gao M, Li M, et al: Identification of a locus for dyschromatosis Azizi E, Lusky A, Kushelevsky AP, Schewach-Millet M: Skin type, hair color, and symmetrica hereditaria at chromosome 1q11–1q21. J Invest Dermatol freckles are predictors of decreased minimal erythema ultraviolet 120:776–780, 2003 radiation dose. J Am Acad Dermatol 19:32–38, 1988 Bastiaens M, ter Huurne J, Gruis N, et al: The melanocortin-1-receptor gene is the major freckle gene. Hum Mol Genet 10:1701–1708, 2001 Bastiaens MT, Westendorp RG, Vermeer BJ, Bavinck JN: Ephelides are more Appendix: Electronic-database information The URLs related to pigmentary constitutional host factors than solar lentigines. for data presented herein are as following: Pigment Cell Res 12:316–322, 1999 Online Mendelian Inheritance in Man (OMIM), http:// Breathnach AS: Electron microscopy of melanocytes and melanosomes in freckled human epidermis. J Invest Dermatol 42:389–394, 1964 www.ncbi.nlm.nih.gov/Omim/(for dyschromatosis symme- Broman KW, Murray JC, Sheffield VC, White RL, Weber JL: Comprehensive trica hereditaria, cutaneous malignant melanoma, xeroder- human genetic maps. Individuals and sex-specific variation in recombi- ma pigmentosum, PLDN, HAND2) nation. Am J Hum Genet 63:861–869, 1998 Brues AM: Linkage of body build with sex, eye colour and freckling. Am J Hum Marshfield genetic map, http://www.marshmed.org/ Genet 2:215–239, 1950 genetics Chen L, Chen WY, Xu A: Clinical effect of dye laser therapy in 1072 cases of Genome View, National Center for Biotechnology freckles. Clin J Dermatol 29:214–216, 2000 (in Chinese) Information, http://www.ncbi.nlm.nih.gov/mapview/map_ Falcon-Perez JM, Dell’Angelica EC: The pallidin (Pldn) gene and the role of SNARE proteins in melanosome biogenesis. Pigment Cell Res 15:82–86, search.cgi? 2002 Genebank, http://www.ncbi.nlm.nih.gov/Genebank