Deficiency in nucleotide excision repair (NER) family gene activity, especially ERCC3, is associated with non-pigmented hair fiber growth

The hair follicle bulb is the only site of pigment production for the hair shaft and melanogenically active melanocytes are located in the upper hair matrix. We conducted a microarray study to discover gene expression patterns that may be implicated in the lack of melanogenesis in gray hair follicles (HF). Pigmented and non-pigmented HFs collected from the same individuals were micro-dissected into the lower one third including the hair bulb (HB) and the upper hair shaft and sheaths (HS) including the bulge region. Microarray data was verified with qPCR and immunohistochemistry. Target effects were evaluated in vitro on human epidermal melanocytes (HEMs). In comparison to pigmented HS and HBs, several nucleotide excision repair (NER) family genes exhibited statistically significant lower expression both in non-pigmented HS and non-pigmented HB. These genes were identified as ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, ERCC6, XPA, NTPBP, HCNP, DDB2 and POLH. Immunohistochemistry showed consistent results. By siRNA interference we also detected that a deficiency in ERCC3 in melanocytes reduced the ability to produce melanin in vitro. Our results suggest that loss of NER gene function may lead to DNA damage and mutation accumulation in melanocytes, which may possibly lead to cell death. Further, a loss of ERCC3 function may lead to reduced gene transcription and this in turn may lead to reduced melanin production ability. These results offer a new insight into the molecular changes that occur in non-pigmented HF and may also provide novel information with regard to melanogenesis and its regulation. Samples of human hair follicles were collected from 4 mm scalp biopsies of normal individuals who presented with a combination of pigmented and non-pigmented gray hair. All samples were provided through the Department of Surgery and the Department of Dermatology and Skin Science, University of British Columbia, with approval from the University Clinical Research Ethics Board. Hair follicles (n=10-20 per sample group per subject) were microdissected to remove the sebaceous gland and upper hair follicle infundibulum. Hair follicles were separated into two sample groups; those with pigmentation, where the dermal papilla was hidden in a strongly pigmented hair bulb and pigment-activity was evident in the root sheath; and those where macroscopically no pigmented melanocytes could be detected in the hair bulb and the hair shaft appeared fully unpigmented. Pigmented and non-pigmented hair follicles were then sub-divided into the lower one third including the hair bulb, and into the keratinized hair shaft and root sheaths including the bulge region, for analysis. Human Operon v.2.1 (21K) glass arrays were produced (based on human 70mers from Operon Biotechnologies Inc, Huntsville, AL) by the Microarray Facility of the Prostate Centre at Vancouver General Hospital, Vancouver, Canada. RNAs were amplified using the SenseAmp Plus kit (Genisphere Inc, Hatfield, PA). The calculated A 260/280 ratio was used to determine the appropriate amount of sense RNA for labeling. Total RNA from test samples and Universal Human Reference RNA (Stratagene, Cedar Creek, TX) were differentially labeled with Cy5 and Cy3, respectively, with the 3DNA array detection 350 kit (Genisphere Inc, Hatfield, PA) and cohybridized to cDNA microarrays. Following overnight hybridization and washing, arrays were imaged using a ScanArray Express scanner (PerkinElmer, Boston, MA).