Written informed consent was obtained from patients and individuals involved in the present study. The present study was approved by the ethics committee of Nanfang Hospital of Southern Medical University (Guangzhou, China). A total of 5 ml of peripheral blood was obtained from one KP and two NC probands, family members and 100 unrelated healthy controls (aged 18–28 years old, 44 males and 56 females, recruited in December 2015). The KP proband was a 12-year-old female (recruited August 2013). The NC probands were an 8-year-old male (recruited May 2014) and a 17-year-old male (recruited December 2015). Genomic DNA was extracted using the Blood DNA Mini kit (cat. no. 3001050; Hangzhou Simgen Biotechnology Co., Ltd., Hangzhou, China) according to the manufacturer's protocol. The genomic sequences of 147 genes associated with 143 skin diseases were initially analyzed in the KP proband using a custom-designed GeneChip [Skin single-gene genetic disease detection package, Illumina High-throughput sequencing platform (Illumina, Inc., San Diego, CA, USA), Beijing Genomics Institute, Guangdong, China] (P<0.05). The details of the gene and skin disease analysis were not presented.

Specific primers for the ABCA12 gene were designed using the web-based tool Primer 3 (http://primer3.ut.ee/; ABCA12 primer sequences available on reasonable request). All exons and exon-intron boundaries of the ABCA12 gene were amplified using PCR with a 20 µl total reaction volume containing 1 µl genomic DNA with a final concentration of 30 ng/µl, 10 µl 2X Taq PCR StarMix (Taq DNA polymerase, Mg²⁺, dNTPs and reaction buffer solution; cat. no. A112-05, GenStar Biosolutions Co., Ltd.), 1 µl (10 µM) forward and reverse primers, and 7 µl ddH₂O. The thermocycling conditions were as follows: Initial denaturation at 94°C for 5 min, 35 cycles of 94°C for 30 sec, annealing at 57°C for 30 sec and 72°C for 15 sec, followed by a final extension at 72°C for 15 sec. PCR products were confirmed using 1% agarose gel electrophoresis and sequenced. The sequencing results were analyzed on an ABI 3130 genetic analyzer (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The functional alterations in the protein were predicted by web-based tools PolyPhen-2 (genetics.bwh.harvard.edu/pph2/) and SIFT release 63 (sift.jcvi.org/). Sequence conservation surrounding the mutated region was verified using ClustalX version 2.1 (http://www.clustal.org/).

Skin lesions from two patients with NC and the patient with KP were collected (one per patient; ~0.8×1 cm). The lesion from the KP proband was obtained from the left leg. NC samples were obtained from the waist and neck. Samples were fixed at room temperature by immersion in 10% formalin solution and, after 12 h the samples were processed into paraffin blocks. Sections (~3–4 µm) were obtained, and the prepared paraffin sections were placed on adhesive slides and incubated overnight at 37°C. Immunofluorescence staining was performed as described below. The slides were de-waxed in 100% dimethylbenzene twice and subsequently dehydrated via a descending alcohol (100, 95, 85 and 70%) gradient. The antigen was retrieved via incubation in a water bath (95°C) for 6 min and then cooled to room temperature. The samples were blocked with 3% peroxide-methanol for 20 min at room temperature to quench endogenous peroxidase activity and then rinsed in PBS three times. The following steps were conducted in a moisture chamber: i) Samples were incubated with rabbit anti-ABCA12 antibody (cat. no. bs-11906R; BIOSS, Beijing, China) diluted 1:50 in antibody diluent (cat. no. IH0340; Beijing Leagene Biotech Co., Ltd., Beijing, China) at 37°C for 1 h and then 4°C overnight. ii) Following rinsing with PBS three times, DyLight goat anti-rabbit green fluorescent protein (cat. no. 4412; CST Biological Reagents Co., Ltd., Shanghai, China) diluted 1:500 in antibody diluent was applied; the samples were then incubated at room temperature without light for 90 min. iii) Following three rinses with PBS, the slides were stained with DAPI at room temperature for 10 min. iv) After washing with PBS for 5 min, the slides were sealed with glycerin. v) The slides were allowed to dry naturally and then visualized under a fluorescence microscope at 360 and 488 nm (magnification, ×200). A total of 5 samples of normal skin were also analyzed which was obtained from a normal skin biopsy. Immunohistochemistry was conducted in the Central Laboratory of Nanfang Hospital of the Southern Medical University.

The familial association between the proband in a family with KP was presented in Fig. 1A; that of the two NC families were presented in Fig. 1B and C.

A Chinese pedigree family with three members afflicted with KP across two generations (Fig. 1A) were employed in the present study. The proband, a 12-year-old female, presented symmetric, asymptomatic and rough keratotic follicular papules, and mild perifollicular erythema at the cheek. The extensor of the upper arms and anterior thighs (Fig. 2A-C) were analyzed. The patient had no notable medical or family history and no mental retardation. The father and younger brother of the proband presented similar phenotypes.

Samples were fixed at room temperature by immersion in 10% formalin solution (at room temperature) and after 12 h the samples were processed into paraffin blocks. Sections (~3–4 µm) were obtained, and the prepared paraffin sections were placed on adhesive slides and incubated overnight at 37°C. The slides were de-waxed in 100% dimethylbenzene twice and subsequently dehydrated via a descending alcohol (100, 95, 85 and 70%) gradient. Then the slides were stained with hematoxylin (cat. no. DH0001; Beijing Leagene Biotech Co., Ltd., Beijing, China) for 8 min, then wash in pure water for 3 sec and finally stained with eosin (cat. no. DH0050; Beijing Leagene Biotech Co., Ltd.) for 1 min at room temperature. Finally the slides were visualized under a light microscope (magnification, ×200). The samples were obtained from the left thigh of the proband and revealed that the follicular orifice was distended by a keratin plug; mild infiltration of mononuclear cells in the superficial dermis was observed (Fig. 2D), supporting a diagnosis of KP.

A total of two NC pedigrees were employed in the present study. The proband of the first family was an 8-year-old male with a 4-year medical history. The clinical manifestations of the proband constituted numerous keratotic papules and comedo-like lesions on the waist, buttocks and legs (data not shown). Physical examination revealed symmetric, light brown-colored papules 2–5 mm in size (Fig. 3A). The father and younger brother of the proband (Fig. 1B) exhibited the same symptoms. The second patient, a 17-year-old male, presented aggregated, dilated hair follicles, blackheads with cysts, fistulas and abscesses on the neck (Fig. 3B). Family disease history was not positive for the second NC family (Fig. 1C). The pathology of both patients demonstrated dilated follicular ostia filled with keratin layers (Fig. 3C and D).

High-throughput sequencing revealed a heterozygous missense c.6694G>T (p.Asp2232Tyr) mutation in ABCA12 in the KP proband (Fig. 4A), and direct sequencing of all coding and exon-intron boundaries confirmed that this mutation was present in all of the affected KP family members, but not present in healthy family members, patients with NC or 100 population-matched controls (Fig. 4B). PolyPhen-2 and SIFT-based investigations suggested that this mutation may produce a damaged protein. The region surrounding this mutation is highly conserved among other species, according to ClustalX (Fig. 4C). The aforementioned results support the hypothesis that the p.Asp2232Tyr mutation in ABCA12 may be one of the pathological factors that contribute to KP.

To understand whether ABCA12, which regulates lipid transport, is involved in the development of NC, the expression levels of ABCA12 were examined using immunofluorescence. The results demonstrated that ABCA12 expression was upregulated in the sebaceous glands of patients with NC (Fig. 5B and C) compared with in normal controls (Fig. 5A). Therefore, high expression levels of ABCA12 in the sebaceous gland may be associated with the development of NC.