ABCB1-Related Disorders via the ABCB1 Gene

ATP-binding cassette transporter B1 (ABCB1) is a membrane bound protein which functions to pump molecules from the inside of the cell to the outside. This process is important in facilitating excretion and preventing toxic accumulations of molecules within cells. ABCB1 is mainly found in the epithelial layer of tissues that provide barrier or excretion functions such as the intestines, colon, liver, and kidney. ABCB1 is also a major component of the blood brain barrier and fetal tissues. A wide variety of structurally-diverse molecules, including various metabolites and many different classes of drugs, are able to bind and undergo transport by ABCB1 (Wolking et al. 2015. PubMed ID: 25860377; Chen et al. 2012. PubMed ID: 22119877).   

Studies have shown that expression levels of ABC transporters, including ABCB1, are increased in certain cancers and are one of the major contributing factors of multidrug resistance (MDR) (Sui et al. 2012. PubMed ID: 22613403; Alavarez et al. 1995. PubMed ID: 7738186). MDR is considered a major challenge in cancer treatment and occurs when cancer cells no longer respond to a drug or other unrelated drugs after a short period of treatment (Ye et al. 2019. PubMed ID: 31245292). It is estimated that MDR is responsible for ~90% of cancer-related deaths (Bukowski et al. 2020. PubMed ID: 32370233).  Due to its ability to recognize and efflux a wide-range of molecules, ABCB1 decreases the overall accumulation of anti-cancer drugs within tumor cells, leading to decreased efficacy and overall tumor survival.  Increased expression of ABCB1 has been shown to induce drug resistance >100 times higher than normal cells, leading to resistance to many chemotherapeutic agents such as doxorubicin, colchicine, and imatinib (Breier et al. 2012. PubMed ID: 22931413; Kathawala et al. 2015. PubMed ID: 25554624). Analysis of ABCB1 levels in neuroblastoma patients correlated with the overall success or failure of treatment (Chan et al. 1991. PubMed ID: 1682809).  

In addition to MDR, genetic variation within ABCB1 has also been associated with a variety of other conditions including: differential response to antidepressants (Brückl and Uhr. 2016. PubMed ID: 27918249), Alzheimer’s disease (Chai et al. 2019. PubMed ID: 31750987; van Assema et al. 2016. PubMed ID: 27494062), chronic inflammatory bowel diseases (Brant et al. 2003. PubMed ID: 14610718; Ho et al. 2003. PubMed ID: 12692067) and fetal susceptibility to cleft lip and palate (Bliek et al. 2009. PubMed ID: 19760622; Omoumi et al. 2013. PubMed ID: 23443032). ABCB1 has been reported to play a role in ~30% of epilepsy patients who are treatment resistant (Leschzinger et al. 2006. PubMed ID: 16708052). The molecular details of how genetic variation within ABCB1 impacts these disorders is still emerging.

Although several studies have reported an association between ABCB1 haplotypes with drug response and various other phenotypes, the results from genotyping studies have been largely conflicting and clinical relevance has been limited (Wolking et al. 2015. PubMed ID: 25860377; Brückl and Uhr. 2016. PubMed ID: 27918249).  It is speculated that many of the issues with reproducibility of these studies stem from the functional redundancy within the ABC transporter family. For instance, the products of other genes such as ABCC1, ABCC2 and ABCC5 are capable of transporting similar drugs as ABCB1 and have also been shown to contribute to drug resistance. This redundancy may mask certain ABCB1 variants and may be a confounding factor in previous analyses. Additionally, much of the literature has focused on analyzing common variants within ABCB1 while many rare variants are relatively unstudied.  It is anticipated that deep-sequencing approaches and additional clinical and functional studies will clarify current associations as well as identify additional variants with greater effects on drug response (Wolking et al. 2015. PubMed ID: 25860377).  

Advantages of ABCB1 germline testing include identification of genetic variations, both common and rare, that may be considered in understanding an individual’s response to drugs targeted by ABCB1. According to the Clinical Pharmacogenetics Implementation Consortium (CPIC; https://cpicpgx.org/), there are 12 entries for ABCB1-drug pairs including antidepressants, methotrexate, and digoxin. However, at this point in time there are no established clinical guidelines for genetic-based dosing of ABCB1 targeted drugs.

The ABCB1 gene is found on chromosomal region 7q21 and is composed of 29 exons (NM_0000927) with the first two 5’ exons untranslated. This gene encodes a 1,280 amino acid protein known in the literature by various names such as ABCB1, P-glycoprotein (P-gp), mulitdrug resistance protein 1 (MDR1), and GP170. The expression of ABCB1 is influenced through a number of different mechanisms including use of alternative promoters (~100 kb apart) and the production of alternative transcripts (~11), many of which undergo alternative splicing (search ABCB1 at www.ncbi.nlm.nih.gov/IEB/Research/Acembly; Hodges et al. 2011. PubMed ID: 20216335). Several transcription factors and signaling pathways have been shown to influence ABCB1 expression, some of which are exploited in cancer cells to up-regulate ABCB1 and promote tumor survival (Sui et al. 2012. PubMed ID: 22613403; Scotto 2003. PubMed ID: 14576854).  Studies have also shown the epigenetic events also influence the expression of ABCB1 (Scotto 2003. PubMed ID: 14576854).  

There have been >635 missense, >260 synonymous, and 37 loss of function variants described in ABCB1 in the gnomAD public population database (https://gnomad.broadinstitute.org). Variants in ABCB1 are found throughout its entire genomic region with ~97% of variants occurring at an allele frequency of <0.001.

The mode of inheritance is autosomal recessive for ABCB1 variants. Several common variants (i.e. polymorphism) in ABCB1 have been associated with the action of a variety of drugs (Wolking et al. 2015. PubMed ID: 25860377). Variants such as c.3435C>T (p.Ile1145Ile), c.1236T>C (p.Gly412Gly), and 2677T>G/A (p.Ser893Ala/Thr) are considered the three most common ABCB1 variants and have been widely studied. Approximate gnomAD allele frequencies for these variants are ~80-90% in Africans, ~35-63% in Asians, ~55% in Latinos, and ~47-55% in European (non-Finnish). These three variants are in linkage disequilibrium (LD) and have been described as haplotypes in certain ethnic populations (Tang et al. 2004. PubMed ID: 14976162). Additional linkage studies have shown that a high level of LD occurs between the c.3435C>T (p.Ile1145Ile) variant in ABCB1 and the c.504C>T (p.Asn168Asn) variant in the neighboring ABCB4 gene, suggesting that the coding sequence of one ABC transporter may function as a regulatory region for adjacent ABC transporter genes (Leschziner et al. 2006. PubMed ID: 16708052). The functional and clinical impact of rare ABCB1 variants (found in <1% of the population) have largely been unstudied (Wolking et al. 2015. PubMed ID: 25860377).

Copy number variants for ABCB1 are mainly seen as somatic gene duplications in different cancer cell lines (Spitzwieser et al. 2016. PubMed ID: 27689338). Cytogenetic analysis has shown complex translocation events in chemotherapy-treated cancer cell lines involving 4q and 7q, forming a novel hybrid gene on chromosome 4. Other ABCB1 hybrids have been observed with portions of the ABCB1 gene located 3’ to active genes, suggesting that chromosomal rearrangement and over expression of hybrid mRNA function as a mechanism of acquired drug resistance in tumor cells (Mickley et al. 1997. PubMed ID: 9109439). Double minute, or extrachromosomal circular DNA (ecDNA), have also been found in multidrug-resistant human tumor cell lines (Ruiz et al. 1989. PubMed ID: 2648129).  

A canine study analyzing the drug ivermectin, a treatment for worms and parasites, found collie dogs to be sensitive to ivermectin-induced neurotoxicity. This led to the discovery of a 4 base pair deletion causing a frameshift and premature protein termination in canine ABCB1. Dogs homozygous for the deletion experienced ivermectin-induced neurotoxicity, while wild type and heterozygous dogs did not (Mealey et al. 2001. PubMed ID: 11692082). Further analysis of the ABCB1 deletion in canines showed a segregation of the variant in several additional breeds (Neff et al. 2004. PubMed ID: 15289602). Due to its prevalence in canines and the relevance of this variant  to veterinary pharmacology, genotyping assays have been developed to pre-preemptively screen at risk breeds to reduce the risk of toxic effects of ivermectin and other ABCB1-affected drugs in dogs (Silvestro et al. 2019. PubMed ID: 31711409).

In addition to the naturally occurring ABCB1 knockout canine breeds (Mealey et al. 2001. PubMed ID: 11692082), knockout mouse models for ABCB1 have also been developed. One study showed that ABCB1 knockout mice were viable and phenotypically normal. However, when treated with ivermectine and other ABCB1-affected drugs, the mice had an ~100-fold increase in neurotoxcity compared to wild type mice (Schinkel et al. 1994. PubMed ID: 7910522). In a separate study, it was found that ABCB1 homozygous knockout mice kept under pathogen free conditions developed spontaneous intestinal inflammation resembling human irritable bowel diseases. This study suggests that ABCB1 may also be necessary in the maintenance and function of intestinal epithelial (Panwala et al. 1998 PubMed ID: 9820555).

Structural and functional studies have shown that the ABCB1 protein undergoes post-translational modifications (glycosylation and phosphorylation) and is composed of two halves, each consisting of six transmembrane domains and a cytoplasmic ATP-binding site. ABCB1 undergoes a dynamic rearrangement between an inward and outward facing state (Kim and Chen. 2018. PubMed ID: 29371429). In the inward facing state, the two halves of ABCB1 display flexibility within the drug-binding cavity which contributes to the ability of ABCB1 to bind to a large variety of different molecules. After the substrate is bound, ATP binds to its cytoplasmic sites, causing the transporter to isomerize to the outward facing state. This structural rearrangement causes a decrease in binding affinity of ABCB1 with the substrate leading to release and extrusion of the substrate, against the concentration gradient and into the extracellular space. ATP hydrolysis converts ABCB1 back to the inward facing confirmation and the transport cycle continues (Kim and Chen. 2018. PubMed ID: 29371429).   

This test is performed using Next-Gen sequencing with additional Sanger sequencing as necessary.

This test provides full coverage of all coding exons of the ABCB1 gene plus 10 bases of flanking noncoding DNA in all available transcripts along with other non-coding regions in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere. We define coverage as ≥20X NGS reads or 2x Sanger sequencing.

 Dependent on the sequencing backbone selected for this testing, discounted reflex testing to any other similar backbone-based test is available (i.e., PGxome panel to whole PGxome; PGnome panel to whole PGnome).