*9.1. Genetic Variants of CYP4B1*

The first screening study for genetic polymorphism of*CYP4B1* was performed in French Caucasians and identified the new *CYP4B1* alleles *CYP4B1\*2*, *\*3*, *\*4*, and *\*5* based on the P450 Nomenclature Committee [132]. Among them, *CYP4B1\*2* caused a frameshift and premature stop codon, resulting in complete loss of CYP4B1 function. Two more alleles with frequencies <1%, *CYP4B1\*6* and *CYP4B1\*7*, were identified using a denaturing high-performance liquid chromatography method for 192 Japanese individuals [133]. Since CYP4B1 is involved in the metabolism of pro-carcinogens, its association with bladder cancer was investigated in a Japanese population, and subjects carrying the *CYP4B1\*1*/*\*2* or *CYP4B1\*2*/*\*2* genotypes exhibited a 1.75-fold increased risk of bladder cancer [27]. This finding might be explained as the loss of function allele *CYP4B1\*2* providing lower capacity for activation of carcinogenic compounds. However, a lung cancer risk study of *CYP4B1\*1–\*7* showed no association with lung cancer in a Japanese population [83]. Further studies are needed to determine its association with lung cancer using a large cohort. Study of structure–function relationships has been essential to understanding the efficiency of catalytic activity as well as to explaining the varying degrees of molecular defectiveness of the protein mutants. Investigation of local peptide structures on the CYP4B1 protein and their roles in heme stability with catalytic function has been reported [134–136], and these data will be important to understand inter-individual variations in the activity of CYP4B1 coding variants.

### *9.2. Genetic Variants of CYP4A11, CYP4F2, 4F11, and CYP4F22*

Among *CYP4* family genes, *CYP4A11* and *CYP4F2* have been extensively studied in association with warfarin dosage and the cardiovascular complex. Genetic variants of *CYP4F2* and *CYP4A11* genes are reportedly associated with cardiovascular diseases such as hypertension [137–139]. More than 3400 single nucleotide polymorphisms (SNPs) of human *CYP4A11* and 5900 SNPs of the *CYP4F2* gene have been reported in the NCBI database to date. However, only a small number of the SNPs have been shown to have clinical associations with functional changes. One of the most extensively studied SNPs of *CYP4A11* is a variant of rs1126742 that causes an amino acid change of Phe434 to Ser, leading to reduced 20-HETE synthesis from arachidonic acid [140,141]. Since the discovery of the functional role of CYP4A11 in the synthesis of 20-HETE, the association of *CYP4A11* polymorphisms with cardiovascular risk has been studied extensively in humans [142–148]. The US Food and Drug Administration recommends genotyping of *CYP4F2* variants for determination of warfarin doses [149,150]. The *CYP4F2* genetic variant rs2108622 is a non-synonymous variant that causes a change in the amino acid sequence of valine to methionine and exhibits reduced enzymatic activity toward the metabolism of vitamin K [62]. Since individuals with reduced activity of CYP4F2 for vitamin K inactivation may have higher levels of warfarin than individuals with *CYP4F2\*1*/*\*1*, higher maintenance dosages of warfarin have been recommended for individuals with reduced *CYP4F2* alleles [149]. Many studies have attempted to develop an accurate warfarin dosing algorithm using multiple genes, such as *CYP2C9*, *VKORC1*, and *CYP4F2* [151–155]. Studies regarding *CYP4A22* genetic polymorphisms have been limited to certain populations, such as Japanese and French populations [25,156]. The association of *CYP4A22* variants with human diseases has still not been investigated, which might be due to low expression levels of the *CYP4A22* gene. The *CYP4F3* gene undergoes alternative splicing to form the CYP4F3A and CYP4F3B enzymes, depending on the cell type [157]. Genome-wide investigation showed that the functional SNP *CYP4F3* rs4646904 was associated with lung cancer, especially in smokers [30]. However, the functionality of this SNP in lung cancer pathology remains unidentified. In addition, a high intake of polyunsaturated fatty acids was associated with reduced risk of ulcerative colitis in patients with *CYP4F3* rs4646904 GG/AG, but not those with the AA genotype [158]. Regarding the *CYP4F11* gene, Yi et al. found through in vitro methods that CYP4F11 D315N protein showed approximately 50% and 32% decreases in intrinsic clearance of erythromycin and arachidonic acid, respectively, compared to the wild type [37]. The *CYP4F11* variant (rs1060463) was associated with small bowel bleeding risk induced by aspirin [159]. Seven variants with amino acid changes in the *CYP4F12* gene were identified and functional changes were investigated using ebastine as a substrate [160]. In their report, two coding variants, Val90Ile and Arg188Cys, exhibited significantly decreased activity toward ebastine hydroxylation. The intronic variant *CYP4F12* rs11085971, which contains a nucleotide substitution of guanine to thymine, was identified as a candidate oxidative-stress-related genetic marker for the development of type 1 lesions in cerebral cavernous malformation, and could serve as an early objective predictor of disease outcome [161]. Since the discovery of *CYP4F22* was linked to its association with lamellar ichthyosis [18], genetic studies of *CYP4F22* polymorphisms have been undertaken. A *CYP4F22* variant, CYP4F22 Arg243Leu, was associated with lamellar ichthyosis in a Tunisian family [131], and further genetic studies should be conducted in clinical settings.
