Methods: Extraction of the human hair shaft proteins was performed using a newly developed alkaline solubilisation method. The extracts were profiled by 2-dimensional electrophoresis and resolved protein spots were identified by mass spectrometry and queried against the human hair database. The study was then followed-up by immunoblotting of the identified hair shaft keratin of interest using commercially available antibodies.
Results: Separation of the human hair shaft proteins by 2-dimensional electrophoresis generated improved and highly resolved profiles. Comparing the hair shaft protein profiles of 10 female with 10 male subjects and their identification by mass spectrometry and query of the human hair database showed significant altered abundance of truncated/processed type-II keratin peptides K81 (two spots), K83 (one spot) and K86 (three spots). The 2-dimensional electrophoresis profiling of 30 hair shaft samples taken from women of similar age range but from three distinctive ethnic subpopulations in Malaysia further showed significant altered abundance of one type-I and four type-II truncated/processed keratin peptides including K33b, K81, K83 and K86 (2 spots) between at least two of the ethnic groups. When a followed-up immunoblotting experiment was performed to detect the relative expression of the K86 peptides using commercialised antibodies, similar trends of expression were obtained. The present data, when taken together, demonstrated the potential use of keratin peptide signatures of the human hair shaft to distinguish gender and ethnicity although this needs to be further substantiated in a larger scale study.
RESULTS: We analyzed the whole-genome deep sequencing data (~ 30×) of five native trios from Peninsular Malaysia and North Borneo, and characterized the genomic variants, including single nucleotide variants (SNVs), small insertions and deletions (indels) and copy number variants (CNVs). We discovered approximately 6.9 million SNVs, 1.2 million indels, and 9000 CNVs in the 15 samples, of which 2.7% SNVs, 2.3% indels and 22% CNVs were novel, implying the insufficient coverage of population diversity in existing databases. We identified a higher proportion of novel variants in the Orang Asli (OA) samples, i.e., the indigenous people from Peninsular Malaysia, than that of the North Bornean (NB) samples, likely due to more complex demographic history and long-time isolation of the OA groups. We used the pedigree information to identify de novo variants and estimated the autosomal mutation rates to be 0.81 × 10- 8 - 1.33 × 10- 8, 1.0 × 10- 9 - 2.9 × 10- 9, and ~ 0.001 per site per generation for SNVs, indels, and CNVs, respectively. The trio-genomes also allowed for haplotype phasing with high accuracy, which serves as references to the future genomic studies of OA and NB populations. In addition, high-frequency inherited CNVs specific to OA or NB were identified. One example is a 50-kb duplication in DEFA1B detected only in the Negrito trios, implying plausible effects on host defense against the exposure of diverse microbial in tropical rainforest environment of these hunter-gatherers. The CNVs shared between OA and NB groups were much fewer than those specific to each group. Nevertheless, we identified a 142-kb duplication in AMY1A in all the 15 samples, and this gene is associated with the high-starch diet. Moreover, novel insertions shared with archaic hominids were identified in our samples.
CONCLUSION: Our study presents a full catalogue of the genome variants of the native Malaysian populations, which is a complement of the genome diversity in Southeast Asians. It implies specific population history of the native inhabitants, and demonstrated the necessity of more genome sequencing efforts on the multi-ethnic native groups of Malaysia and Southeast Asia.