rs25531 — SLC6A4 A>G

The Serotonin Transporter's Hidden Switch — rs25531 and SSRI Response

Important Limitation: According to 23andMe's own geneticists, rs25531 is not reliably genotyped on consumer platforms¹¹ not reliably genotyped on consumer platforms
David Hinds of 23andMe reported that "nearly everyone (99.97%) is getting called as CC, and there is no clear heterozygote cluster" and "the genotype calls for rs25531 on our platform are not meaningful". This SNP requires specialized genotyping methods. If your 23andMe data shows AA for rs25531, it is likely a technical artifact rather than your true genotype. This article is included for scientific completeness and for users who have obtained clinical genotyping.

The serotonin transporter gene SLC6A4²² serotonin transporter gene SLC6A4
encodes the protein responsible for clearing serotonin from synapses is one of the most studied genes in psychiatry. The well-known 5-HTTLPR insertion/deletion polymorphism in the gene's promoter has been linked to depression, anxiety, and antidepressant response³³ depression, anxiety, and antidepressant response
though results have been inconsistent across studies for decades. But rs25531, a single nucleotide A→G substitution located within the long (L) allele of 5-HTTLPR, adds a critical layer of complexity: it effectively converts an L allele to function like the short (S) allele⁴⁴ it effectively converts an L allele to function like the short (S) allele
when G is present at rs25531, the L allele has transcriptional activity comparable to the S allele rather than high activity.

The Mechanism

The 5-HTTLPR polymorphism consists of a 44-base-pair insertion/deletion in the SLC6A4 promoter region, creating short (S, 14 repeats) and long (L, 16 repeats) variants. Early research suggested the L allele produced 2-3 times more serotonin transporter mRNA than the S allele⁵⁵ Early research suggested the L allele produced 2-3 times more serotonin transporter mRNA than the S allele
Lesch et al. Science, 1996. However, rs25531 revealed that not all L alleles are functionally equivalent⁶⁶ rs25531 revealed that not all L alleles are functionally equivalent
Hu et al. identified that an A→G substitution at rs25531 within the L allele creates a binding site for the AP-2 transcription factor.

The result is a triallelic system: L_A (L allele with A at rs25531) has high transporter expression, while L_G (L allele with G at rs25531) has low expression similar to the S allele⁷⁷ L_G (L allele with G at rs25531) has low expression similar to the S allele
the G variant disrupts transcription factor binding, reducing promoter activity. This creates a functional hierarchy: L_AL_A > L_AS > L_AL_G > L_GL_G ≈ L_GS > SS in terms of serotonin transporter expression.

The Evidence

The G allele frequency varies dramatically by ancestry⁸⁸ The G allele frequency varies dramatically by ancestry
European-Americans: 7.5%, African-Americans: 21%, reflecting significant population stratification. In the largest study to date of 954 African-American and 2,622 European-American subjects⁹⁹ the largest study to date of 954 African-American and 2,622 European-American subjects
Odgerel et al. Translational Psychiatry, 2013, the G allele was nearly three times more common in African-Americans, and when 5-HTTLPR and rs25531 were combined into high- and low-transcription haplotypes, African-Americans showed significantly fewer low-transcription variants overall.

The clinical significance remains controversial and inconsistent¹⁰¹⁰ controversial and inconsistent
multiple meta-analyses have produced conflicting results. Some studies suggest that individuals with L_AL_A genotypes respond better to SSRIs¹¹¹¹ individuals with L_AL_A genotypes respond better to SSRIs
particularly in Caucasian populations, though effect sizes are modest, while others find no association between rs25531 and treatment outcome¹²¹² no association between rs25531 and treatment outcome
four large studies including STAR*D analyses found no predictive value.

One study found that SSRI serum concentrations correlated with response only in L_A carriers¹³¹³ One study found that SSRI serum concentrations correlated with response only in L_A carriers
suggesting dose-dependent effects specific to the high-expression genotype. Intriguingly, rs25531 also influences opioid analgesia¹⁴¹⁴ rs25531 also influences opioid analgesia
individuals with low-expression genotypes (SA/SA or SA/L_G) showed significantly better pain relief from remifentanil than L_AL_A individuals, suggesting the variant affects multiple neurotransmitter-related drug responses.

Practical Implications

The primary clinical question is whether rs25531 genotyping improves antidepressant selection beyond 5-HTTLPR alone. Current evidence suggests limited additional predictive value¹⁵¹⁵ Current evidence suggests limited additional predictive value
CPIC does not include rs25531 in its recommendations, and multiple studies found no added specificity. The GeneSight pharmacogenomic test¹⁶¹⁶ GeneSight pharmacogenomic test
a commercial panel for antidepressant selection notes that "more data is needed before the rs25531 SNP can be recommended for use in treatment selection."

However, for individuals with clinically obtained rs25531 genotyping, there are some tentative guidelines: Those with G alleles may experience more side effects from SSRIs¹⁷¹⁷ G alleles may experience more side effects from SSRIs
particularly gastrointestinal symptoms and headaches and may benefit from starting at lower doses or considering non-SSRI alternatives. Medications like mirtazapine, which has minimal serotonin transporter affinity¹⁸¹⁸ Medications like mirtazapine, which has minimal serotonin transporter affinity
showed no impact or even improved response in low-expression genotypes, making them reasonable alternatives.

Interactions

Rs25531 must be interpreted together with 5-HTTLPR, as they are in tight linkage disequilibrium and the G allele is almost always found on the L allele background¹⁹¹⁹ the G allele is almost always found on the L allele background
rarely occurring with the S allele. The triallelic classification (L_A, L_G, S) provides more accurate functional prediction than the biallelic (L, S) system alone. Other SLC6A4 polymorphisms including rs2020933 and STin2²⁰²⁰ Other SLC6A4 polymorphisms including rs2020933 and STin2
also affect transporter expression and may compound with rs25531, though the clinical utility of multi-variant haplotypes remains uncertain.

Gene-environment interactions are also critical²¹²¹ Gene-environment interactions are also critical
the combination of low-expression genotypes and stressful life events appears to increase depression risk more than either factor alone, though this finding has been challenged by large meta-analyses. Epigenetic modifications including DNA methylation of the SLC6A4 promoter²²²² Epigenetic modifications including DNA methylation of the SLC6A4 promoter
may interact with rs25531 genotype to affect both expression and treatment response.