BAK1 Gene Variation: the doubts remain

Dr. Hatchwell [2010] has proposed that the BAK1 gene variants were likely due to sequencing of a processed gene on chromosome 20. However, in response, Dr. Gottlieb and co-authors [2010] have argued that “some but not all of the sequence changes present in the BAK1 sequence of our abdominal aorta samples are also present in the chromosome 20 BAK1 sequence. However, all the AAA and AA cDNA samples are identical to each other and different from chromosome 20 BAK1 sequence at amino acids 2 and 145”. I have been following this discussion because I have independently reached almost the same conclusion as Dr. Hatchwell did [Yamagishi, 2009], and, unfortunately, the response from Dr. Gottlieb and his co-authors seems to me to be unsatisfactory for the reasons listed below

💡 Research Summary

The paper by Michel E. Beleza Yamagishi revisits the controversy surrounding reported BAK1 gene variants in abdominal aortic aneurysm (AAA) tissue versus peripheral blood, a debate sparked by Gottlieb et al. (2009) and subsequently addressed by Hatchwell (2010). Gottlieb’s original study described a set of single‑nucleotide polymorphisms (SNPs) that appeared in cDNA derived from AAA tissue but not in matched blood samples. Hatchwell argued that these apparent “mutations” were likely artifacts caused by inadvertent amplification of a processed, intron‑less copy of BAK1 located on chromosome 20. In response, Gottlieb et al. (2010) claimed that while some of the observed changes overlapped with the chromosome 20 copy, the AAA and AA cDNA sequences were identical to each other and differed from the chromosome 20 reference at amino‑acid positions 2 and 145.

Yamagishi systematically dismantles Gottlieb’s rebuttal on several fronts:

1. Sequence Inconsistencies – By reconstructing the BAK1 sequence inferred from Gottlieb’s Table 2, Yamagishi shows that it does not correspond to the canonical RefSeq NM_001188.3 (chromosome 6) nor to the processed chromosome 20 copy. Four codons (positions 28, 42, 52, 103) contain nucleotides that are incompatible with either reference. For example, codon 28 is listed as GTC (interpreted as alanine) whereas NM_001188.3 contains GCC (alanine); codon 42 is shown as CAG (glutamine) while the reference has CGC (arginine); codon 52 is GTG (valine) in the reference but appears as GCT (alanine) in Gottlieb’s table; codon 103 is ACG (threonine) in the reference but is recorded as GCC (alanine). These discrepancies suggest that the reported cDNA is a chimeric construct comprising fragments from both the chromosome 6 gene, the chromosome 20 processed copy, and additional undefined sequences.

2. Primer Design Flaws – The forward (CAGGCTGATCCCGTCC TCCACTGAG) and reverse (GGGCACCCTTGGGAGTC ATGATTTG) primers employed by Gottlieb et al. match perfectly to both the chromosome 6 and chromosome 20 loci. Consequently, the primers are non‑discriminatory and would preferentially amplify the intron‑less chromosome 20 copy because of its simpler template. Although Gottlieb later claimed to have used intron‑specific primers for tissue samples, the original methods section suggests the use of the non‑specific primers, creating ambiguity about which locus was actually amplified.

3. Misquotation of Hatchwell – Gottlieb et al. incorrectly cited Hatchwell as describing the chromosome 20 copy as a “pseudogene.” Hatchwell explicitly avoided that term, preferring “processed gene” to acknowledge that the locus is transcribed and may produce functional RNA. This misrepresentation weakens Gottlieb’s argument that the chromosome 20 copy is biologically inert.

4. Experimental Recommendations – Yamagishi proposes a definitive experiment: design primers that span intron‑containing regions unique to the chromosome 6 BAK1 gene, amplify AAA and AA tissue cDNA, and publish the full sequence. Such data would unequivocally determine whether the observed variants arise from genuine somatic mutation, transcriptional editing, or merely from co‑amplification of the processed chromosome 20 paralog.

Overall, Yamagishi’s critique underscores that Gottlieb’s response suffers from multiple technical oversights—incorrect codon assignments, ambiguous primer specificity, and inaccurate citation—rendering their claim that the AAA/AA cDNA truly differs from the chromosome 20 copy unconvincing. The paper calls for transparent reporting of the complete BAK1 sequence and the use of locus‑specific primers to resolve the controversy definitively.