13 common categories were found for the genes higher in the donor scalp, and 8
30 for the bald scalp (Figure 4D). The 4 most populated gene function categories were development, morphogenesis, organ development and organogenesis, with p values ranging from 1.99 x 10"'° to 4.72 x 10'12. In addition to multiple known genes, genes not known to be expressed in HF were discovered.
[00502] Thus, findings of the present invention reveal genes that participate in hair growth, healthy
HF maintenance and cycling, and hair loss.
[00506 ] To test the role of steroid metabolism in aging, the expression of genes involved in steroid metabolism was examined. The androgen receptor (AR) and the enzyme 3-beta-HSD were more abundant in the bald scalp (p < 0.05; Figure 5A), thus corroborating the findings of the present invention.
[00507 ] Immune-related gene expression in bald vs. haired scalp was also examined. Multiple MHC
20 genes, both class I and II, were enriched in the bald scalp (Figures 3 and 5A). In addition, transcripts specific for T cells, Langerhans cells and mast cells were enriched in a statistically significant manner in the bald scalp (CD4 [p=0.017], CdIa [p=0.0087], FCERlA [p=0.046] (Figure 5A). Several genes not known to be expressed in human HF are depicted in Figure 5B. These genes also exhibited a high degree of statistical significance (minimum p value=0.001 ) and minimum fold-enrichment in haired scalp of 4.5.
25 EXAMPLE 5: IN SITU AND IMMUNO-HISTOLOGICAL CHARACTERIZATION OF
NOVEL HF GENES
[00508] In situ hybridization and immuno-histochemistry was next used to determine tissue patterns of expression of significantly enriched transcripts in the haired scalp, using human haired scalp samples from different patients than those used to generate the array and flow cytometry data.
30 [ 00509 ] Microarray showed that LRRC 15 was upregulated 4.5 fold in the haired samples (Figure
5B). LRRCl 5 is a transmembrane glycoprotein with leucine-rich repeats. To determine whether LRRC 15
145
functions in cell migration, LRRC15 expression was measured in scalp samples by immuno- histochemistry. LRRCl 5 was present in Huxley's layer and the cuticle layer of the inner root sheath, especially at the lower follicle (Figure 6A), which is an area of rapid cell movement during hair growth. Thus, LRRCl 5 functions in cell migration necessary for hair growth.
5 [00510 ] Serpin A was up-regulated 5.7 fold in the haired samples. Serpin A is, in another embodiment, a clade A anti-protease in the same family as anti-trypsin and anti-chymotrypsin. Serpin A was expressed in the companion layer of the outer root sheath, as shown by immuno-histochemistry (Figure 6B).
[00511 ] GPR49 (LGF5, HG38), another leucine rich repeat-containing protein, was upregulated 6.8
10 fold in the haired samples, and was expressed in human outer root sheath cells, as shown by immuno- histochemistry (Figure 6C). GPR49 is known to be upregulated in the mouse bulge (outer root sheath), thus further confirming results of the present invention. Enrichment of this G-protein in anagen/terminal follicles show its utility as a drug target for stimulating hair growth.
[00512 ] The Angiopoietin-like gene CDT6 (upregulated 18 fold in the haired samples) is an anti-
15 vascular factor that is also expressed in the cornea (Corneal Derived Transcript 6), and thought to maintain the avascularity of the cornea. CDT6 was expressed in the outer root sheath, as shown by immuno- histochemistry (Figure 6D), which is also avascular.
[00513 ] GPRC5D (upregulated 19.5 fold in haired samples) is a homologue of RAIG-I
(retinoic acid inducible gene-1). GPRC5D was expressed in the inner root sheath and precorneal cells of 20 the hair, as shown by immuno-histochemistry (Figure 6E).
[ 00514 ] FGFl 8 (upregulated almost 6 fold in the haired samples; Figure 5B) was found to be expressed in the inner root sheath, the companion layer, and to a lesser extent in the suprabasal outer root sheath of the bulge area (Figure 6F-G).
[ 00515 ] The genes identified in this Example are all enriched in haired scalp, and are thus
25 therapeutic targets for stimulating hair growth.