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Power and Pitfalls of Computational Methods to Identify New Genes Responsible for Acute Liver Failure of Indeterminate Etiology in Adults

We have read with great interest the recent article by Rakela et al. (1) describing genetic alterations detected by whole exome sequencing in a cohort of 26 adult patients with acute liver failure of indeterminate etiology (ALFIE). The authors conclude that 12 variants of 11 genes were significantly associated with (ALF-IE), particularly the stop codon of SEPRINB11 identified in 23 of the 26 patients and the missense variant of CYP2D6 detected in 13 patients. However, we have several key concerns regarding the results of the study. First, Rakela et al. reported that the SERPINB11 (c.268G.T, p.Glu90Ter) variant, recorded as rs4940595 in the database of human single nucleotide polymorphism, was detected in most of the cohort. However, a careful examination of this variant using VarSome (2) and the latest update of the Broad Institute’s gnomAD (3), a large database of human polymorphisms, reveals that this SERPINB11 variant has a global allele frequency (AF) as high as 63% for all ethnic subgroups (Tables 1 and 2). Thus, we cannot conclude that the variant is frequently associated with (ALF-IE). According to the pipeline of analysis described in the study (AF threshold of 0.01 in human polymorphism databases), this result can be explained by the fact that authors probably used the old version of gnomAD, in which all the variants and polymorphisms were not alignedwith the new version of the genome and not the updated version of gnomAD. This update issue is also true for the second missense variant of CYP2D6, which has neither a high frequency nor an overall frequency for all ethnic subgroups in the updated gnomAD database (Tables 1 and 2). Furthermore, SERPINB11 is a unitary pseudogene in human genome (4). The pseudogene refers to a gene that has lost its functional counterpart during evolution because of a mutagenic event resulting in a premature stop codon. These mutagenic effects are often located in the initial portion of the coding gene, as for SERPINB11. Interestingly, SERPINB11 has the particularity of being polymorphic in humans (4). Hence, the rs4940595 (G/T) variant distinguishes between individuals bearing a functional copy of SERPINB11 with a wildtype c.268G allele and those bearing a nonfunctional copy, with the mutated c.268G.T allele, introducing a stop codon. The extremely high frequency of the mutated allele of SERPINB11 in the general population in different ethnic groups makes the link between this variant and ALF-IE in adults highly unlikely. Finally, 5 of the 9 other variants located inMUC6,OR6J1,AADACL3,CYP2D7, and KIAA1161 also occur frequently in the gnomAD genome, with an AF up to 40%. For the remaining 4 variants, the results can be considered inconclusive because they correspond to multiallelic polymorphisms which must be referenced precisely, theirs ID being insufficient (Table 1). For instance, rs200886831 ofRRP36 detected in 9 patients, refers to 4 different alleles and corresponds to in-frame deletion or insertion of either 6 or 12 bp. One of the 4 alleles has an AF of 45% in gnomAD, whereas the other 3 are extremely rare. Thus, the conclusion raised by Rakela et al. on the significant association of the 12 variants with ALF-IE in adults is incompatible with careful examination of recent databases. A careful processing of exome and genome data is necessary to find out genes causing ALF-IE in adults.

We have read with great interest the recent article by Rakela et al. (1) describing genetic alterations detected by whole exome sequencing in a cohort of 26 adult patients with acute liver failure of indeterminate etiology (ALF-IE). The authors conclude that 12 variants of 11 genes were significantly associated with (ALF-IE), particularly the stop codon of SEPRINB11 identified in 23 of the 26 patients and the missense variant of CYP2D6 detected in 13 patients. However, we have several key concerns regarding the results of the study.
First, Rakela et al. reported that the SERPINB11 (c.268G.T, p.Glu90Ter) variant, recorded as rs4940595 in the database of human single nucleotide polymorphism, was detected in most of the cohort. However, a careful examination of this variant using VarSome (2) and the latest update of the Broad Institute's gnomAD (3), a large database of human polymorphisms, reveals that this SERPINB11 variant has a global allele frequency (AF) as high as 63% for all ethnic subgroups (Tables 1 and 2). Thus, we cannot conclude that the variant is frequently associated with (ALF-IE). According to the pipeline of analysis described in the study (AF threshold of 0.01 in human polymorphism databases), this result can be explained by the fact that authors probably used the old version of gnomAD, in which all the variants and polymorphisms were not aligned with the new version of the genome and not the updated version of gnomAD. This update issue is also true for the second missense variant of CYP2D6, which has neither a high frequency nor an overall frequency for all ethnic subgroups in the updated gnomAD database (Tables 1 and 2).
Furthermore, SERPINB11 is a unitary pseudogene in human genome (4). The pseudogene refers to a gene that has lost its functional counterpart during evolution because of a mutagenic event resulting in a premature stop codon. These mutagenic effects are often located in the initial portion of the coding gene, as for SERPINB11. Interestingly, SER-PINB11 has the particularity of being polymorphic in humans (4). Hence, the rs4940595 (G/T) variant distinguishes between individuals bearing a functional copy of SERPINB11 with a wildtype c.268G allele and those bearing a nonfunctional copy, with the mutated c.268G.T allele, introducing a stop codon. The extremely high frequency of the mutated allele of SERPINB11 in the general population in different ethnic groups makes the link between this variant and ALF-IE in adults highly unlikely.
Finally, 5 of the 9 other variants located in MUC6, OR6J1, AADACL3, CYP2D7, and KIAA1161 also occur frequently in the gnomAD genome, with an AF up to 40%. For the remaining 4 variants, the results can be considered inconclusive because they correspond to multiallelic polymorphisms which must be referenced precisely, theirs ID being insufficient (Table 1). For instance, rs200886831 of RRP36 detected in 9 patients, refers to 4 different alleles and corresponds to in-frame deletion or insertion of either 6 or 12 bp. One of the 4 alleles has an AF of 45% in gnomAD, whereas the other 3 are extremely rare.
Thus, the conclusion raised by Rakela et al. on the significant association of the 12 variants with ALF-IE in adults is incompatible with careful examination of recent databases. A careful processing of exome and genome data is necessary to find out genes causing ALF-IE in adults.