Japanese scientists identify earwax gene

[Angel]

Earwax may not play a prominent part in human history but at least a small role for it has now been found by a team of Japanese researchers.

Earwax comes in two types, wet and dry. The wet form predominates in Africa and Europe, where 97% or more of the people have it, and the dry form among East Asians, while populations of Southern and Central Asia are roughly half and half.

By comparing the DNA of Japanese with each type, the researchers were able to identify the gene that controls which type a person has, they report in the Monday issue of Nature Genetics.

They then found that the switch of a single DNA unit in the gene determines whether a person has wet or dry earwax. The gene's role seems to be to export substances out of the cells that secrete earwax. The single DNA change deactivates the gene and, without its contribution, a person has dry earwax.

[Rajiv]

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How did they do this? Scroll down to methods.

Human earwax consists of wet and dry types. Dry earwax is frequent in East Asians, whereas wet earwax is common in other populations. Here we show that a SNP, 538G A (rs17822931), in the ABCC11 gene is responsible for determination of earwax type. The AA genotype corresponds to dry earwax, and GA and GG to wet type. A 27-bp deletion in ABCC11 exon 29 was also found in a few individuals of Asian ancestry. A functional assay demonstrated that cells with allele A show a lower excretory activity for cGMP than those with allele G. The allele A frequency shows a north-south and east-west downward geographical gradient; worldwide, it is highest in Chinese and Koreans, and a common dry-type haplotype is retained among various ethnic populations. These suggest that the allele A arose in northeast Asia and thereafter spread through the world. The 538G A SNP is the first example of DNA polymorphism determining a visible genetic trait.


Methods
SNP identification, genotyping, case-control study and haplotyping.
Study protocols were approved by the Institutional Review Boards of each institution joining the study. Genomic DNA was extracted from whole blood of volunteers using the standard method or from their fingernails with ISOHAIR (Nippon Gene). Volunteers gave written informed consent. Other DNA samples were purchased from the Coriell Institute for Medical Research. We searched for CA repeat sequences and SNPs around the 5.9-cM segment containing D16S3093 and D16S311 and withdrew coding SNP (cSNP) data from the JSNP database because the earwax type is a single-gene trait, and the JSNP data are constructed from the Japanese, in which the dry type is known be the major phenotype2. As we had previously found a shortage of SNPs in this region17, 18, we identified 15 new SNPs by sequencing the region among eight individuals with wet earwax. A total of 134 CA repeat polymorphisms from the region and 15 new SNPs (three already deposited in dbSNP) as well as 21 registered SNPs were used as markers for the first-step case-control studies by genotyping 118 Japanese volunteers (64 individuals with dry earwax and 54 controls with wet earwax) whose DNA samples were collected in an anonymous manner and whose earwax types were self-declared. We found two SNPs, B81540.1 and IMS-JST141676, that were homozygous in 64 individuals with dry earwax. This led us to focus on a 600-kb region between the two SNPs, especially on a four-gene (ABCC12, ABCC11, LONPL and SIAH1) interval, on which we performed PCR-based genome sequencing of genomic DNA except on long repetitive elements in the eight wet-type individuals. If a given SNP within a gene is close to the earwax locus, these individuals are expected to show 80–90% heterozygosity, in view of the previously reported earwax phenotype data in the Japanese2, 4. In this interval, we found 37 SNPs (15 registered and 22 novel SNPs) and used them for another association study among the same 118 samples above and for the construction of LD blocks to confirm the 600-kb region as a candidate region. LD blocks were constructed using the Haploview program25. Because none of the SNPs above completely explained the earwax phenotype as a mendelian trait, we collected blood from another series of 126 Japanese volunteers (88 dry-type and 38 wet-type individuals) from Nagasaki City, whose earwax types were identified by a medical practitioner, and used them for the second-step association study.

Using the Haploview program25, we constructed haplotypes including 24 SNPs and 27 in the 600-kb interval and its flanking region in Native Americans, Native Bolivians and Southern Han Chinese individuals. Primer sequences for construction of haplotypes in various populations are listed in Supplementary Table 4 online.

Complete genome sequencing of the individuals with dry earwax and wet earwax.
We adopted two strategies, PCR-based complete genome sequencing in eight wet-type individuals and BAC clone–based sequencing in a wet/dry-type heterozygote for the genomic regions that could not be amplified from the eight samples. Comparisons between the PCR-based sequence and the complete contig sequence, and between two BAC clones containing the allele responsible for the wet- or dry-type phenotype showed no structural abnormality or any base changes other than SNPs found among the eight wet-type individuals. The complete sequence consisted of 341,290 bp and contained four genes (ABCC12, ABCC11, LONPL and SIAH1) spanning an LD block.

LLC-PK1 cell lines expressing allele A or G of human ABCC11, preparation of plasma membrane vesicles from these cells and detection of ATP-dependent transport of cGMP.
Full-length cDNA carrying allele A (dry-type allele) and that carrying allele G (wet-type allele) of human ABCC11 were inserted into pcDNA3.1-Hygro (Invitrogen) and transfected into pig-derived LLC-PK1 cells (Health Science Research Resources Bank (HSRRB) cell bank) using Lipofectin reagent (Invitrogen). Total RNA was prepared from ABCC11-expressing cell lines (LLC-PK1-A and LLC-PK1-G carrying alleles A and G, respectively) and was used for reverse transcription (RT). Real-time PCR quantification was carried out using a 7900HT Sequence Detection System (Applied Biosystems) as described previously26. Expression of ABCC11-mRNA was compared with that of GAPDH-mRNA.

Plasma membrane vesicles were prepared from LLC-PK1 cells as described previously27. The standard incubation medium contained the plasma membrane vesicles (50 g protein), 500 M 3H-labeled cGMP (Amersham), 250 mM sucrose, 10 mM Tris HCl (pH 7.4), 10 mM MgCl2, 1 mM ATP, 10 mM creatine phosphate and 100 g ml-1 creatine kinase in a final volume of 100 l. Plasma membrane vesicles were incubated at 37 °C for 20 min with 3H-labeled cGMP at different concentrations (0, 5, 10, 20, 50, 100, 250 and 500 M) in the absence (data not shown) or presence of 1 mM ATP. The amount of 3H-labeled cGMP incorporated into the membrane vesicles was measured by a rapid filtration technique28. ATP-dependent cGMP transport was measured by the difference in the radioactivity incorporated into the vesicles in the presence and absence of ATP.