Phased Haplotype Resolution of the SLC6A4 Promoter Using Long-Read Single Molecule Real-Time (SMRT) Sequencing.

Erick R Scott,Yao Yang,Robert J Desnick,Stuart A Scott,Mariana R Botton

doi:10.3390/genes11111333

Abstract

The SLC6A4 gene has been implicated in psychiatric disorder susceptibility and antidepressant response variability. The SLC6A4 promoter is defined by a variable number of homologous 20–24 bp repeats (5-HTTLPR), and long (L) and short (S) alleles are associated with higher and lower expression, respectively. However, this insertion/deletion variant is most informative when considered as a haplotype with the rs25531 and rs25532 variants. Therefore, we developed a long-read single molecule real-time (SMRT) sequencing method to interrogate the SLC6A4 promoter region. A total of 120 samples were subjected to SLC6A4 long-read SMRT sequencing, primarily selected based on available short-read sequencing data. Short-read genome sequencing from the 1000 Genomes (1KG) Project (~5X) and the Genetic Testing Reference Material Coordination Program (~45X), as well as high-depth short-read capture-based sequencing (~330X), could not identify the 5-HTTLPR short (S) allele, nor could short-read sequencing phase any identified variants. In contrast, long-read SMRT sequencing unambiguously identified the 5-HTTLPR short (S) allele (frequency of 0.467) and phased SLC6A4 promoter haplotypes. Additionally, discordant rs25531 genotypes were reviewed and determined to be short-read errors. Taken together, long-read SMRT sequencing is an innovative and robust method for phased resolution of the SLC6A4 promoter, which could enable more accurate pharmacogenetic testing for both research and clinical applications.

Highlights

Antidepressants are the third most commonly prescribed drug class in the United States, surpassed only by antihyperlipidemics and analgesics [1]
Expression of 5-HTT is directly correlated with 5-HT transporter function, which has been implicated as a susceptibility gene for several psychiatric disorders as well as a pharmacogenetic determinant of interindividual selective serotonin reuptake inhibitors (SSRIs)
Genotype results from the interrogated SLC6A4 promoter region identified by short-read WGS

Summary

Introduction

Antidepressants are the third most commonly prescribed drug class in the United States, surpassed only by antihyperlipidemics and analgesics [1]. Selective serotonin reuptake inhibitors (SSRIs) are used to treat obsessive-compulsive disorder (OCD), panic disorder, posttraumatic stress disorder and anxiety disorders; efficacy among the different SSRIs has been estimated at only ~65% across treated patients [2]. The SLC6A4 gene at chromosome 17q11.2 encodes the serotonin (5-HT) transporter (5-HTT), which mediates 5-HT reuptake and is the major target of SSRI antidepressants. Expression of 5-HTT is directly correlated with 5-HT transporter function, which has been implicated as a susceptibility gene for several psychiatric disorders (e.g., affective disorders, schizophrenia, anxiety, autism, depression, suicide, OCD, and addiction [4]) as well as a pharmacogenetic determinant of interindividual SSRI response variability [5,6,7,8]. The polymorphic SLC6A4 promoter is composed of a variable number (11–24) of tandem repeat units (5-HTTLPR), which are 20–24 bp in length and highly homologous

Methods

Results

Discussion

Conclusion