Premenstrual dysphoric disorder is characterized by recurrent psychological and/or somatic symptoms occurring specifically during the luteal phase of the menstrual cycle and resolving during menstruation. It is estimated that 5% to 10% of regularly ovulating women experience this condition,1 which requires precise diagnostic criteria outlined in the Fourth Edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV).2
The cause of premenstrual dysphoric disorder is unknown. It is unlikely that an ovarian hormone imbalance per se is a major factor, because circulating levels have repeatedly been shown to be normal throughout the menstrual cycle. Several lines of evidence suggest that an underlying dysregulation of serotonergic neurotransmission is involved, with ovarian hormones providing the cyclical trigger. Falling levels of ovarian hormones during the luteal phase of the menstrual cycle have been associated with decreased serotonergic activity.3 Studies indicate that serotonin (5-hydroxytyptamine, 5-HT) exerts an inhibitory effect on core symptoms of premenstrual dysphoric disorder such as irritability, affect lability, and depression.4,5 Treatment with the serotonin precursor tryptophan has been reported to reduce premenstrual symptoms,6 whereas its depletion seems to exacerbate premenstrual irritability.7 Low luteal-phase whole blood and platelet serotonin levels have been found in women with premenstrual syndrome.8–10 Furthermore, selective serotonin reuptake inhibitors (SSRIs), which specifically block serotonin reuptake into the presynaptic terminal, rapidly control the somatic and psychological symptoms of premenstrual dysphoric disorder.11
In the central nervous system, there is evidence of region-specific effects on serotonin synthesis, turnover, uptake, release, and receptors function by estradiol and progesterone.12,13 The rate of serotonin release from raphe neurons is modulated by presynaptic serotonin 1A (5-HT1A) autoreceptors, by way of a negative feedback mechanism. Postsynaptic 5-HT1A receptors are also strongly expressed in brain regions that modulate mood and emotion.14,15 Selective serotonin reuptake inhibitors are thought to enhance serotonergic neurotransmission in part by desensitizing 5-HT1A autoreceptors.
A functional variant within the promoter region of the human 5-HT1A gene consists of a C to G substitution located at nucleotide position 92,928 (Genome Database: AC008965), and is 1019 base-pairs downstream of the transcription initiation site.16 This C(-1019)G polymorphism binds two repressors known as Deaf-1 and He517 and has been reported to be involved in modulating the rate of transcription of the 5-HT1A gene. The G allele fails to bind to these repressors, with a consequent upregulation of 5-HT1A autoreceptor expression and a reduction of serotonergic neurotransmission.18 Several studies have reported an association between the (-1019)G allele and major depression, panic disorder, and suicide and a decreased response to SSRIs in both white and Chinese subjects.19–22
Evidence from family and twin studies suggests that there is a significant genetic contribution to premenstrual dysphoric disorder.23–25 Because low serotonin has been implicated in the pathogenesis of premenstrual dysphoric disorder, we decided to target the 5-HT1A receptor C(-1019)G polymorphism. We postulated that women carrying at least one G allele would be at increased risk of premenstrual dysphoria.
MATERIALS AND METHODS
The study was approved by the Staffordshire and Shropshire Ethics Committee, and informed written consent was obtained from each participant. Between June 2001 and February 2004, women were recruited from a specialized premenstrual syndrome clinic, general gynecology clinics, or following advertisement on the hospital intranet system. A total of 104 white European women between the ages of 18 and 48 years were enlisted from the local population and categorized into two groups: premenstrual dysphoric disorder and controls. All subjects reported regular menstrual cycles (28±4 days), and none was taking oral contraceptives, hormone replacement therapy, or psychotropic drugs. Any woman known to have an existing or previous psychiatric disorder was excluded from the study. Clinical diagnosis was determined by prospective symptom rating using the Daily Record of Severity of Problems (DRSP) scale,26 based on self-assessment reports spanning two consecutive menstrual cycles. Day 1 of each cycle was identified by the first day of menses. Symptom ratings of days 6–12 and the 7 days immediately before the next menstrual period were used to calculate the mean follicular and mean luteal scores, respectively. Women were diagnosed with premenstrual dysphoric disorder if there was a 200% or more increase in severity of one or more, or a 100% or more increase of two or more of the DSM-IV premenstrual dysphoric disorder-defining symptoms (marked depression, anxiety-tension, affect lability, and irritability) during the luteal phase compared with the follicular phase, in both menstrual cycles. The control group comprised women who reported no significant premenstrual symptoms; the difference between their mean luteal and mean follicular scores was less than 100% for any of the DRSP-rated symptoms in the two monitored cycles.
We extracted DNA from leukocytes in ethylenediaminetetraacetic acid-anticoagulated blood using standard techniques. A 169 base-pair (bp) section of the 5-HT1A receptor gene containing the C(-1019)G polymorphism was amplified by polymerase chain reaction (PCR). The penultimate nucleotide at the 3′ end of the reverse primer was deliberately altered from a thymine (T) to an adenine (A, underlined below), to introduce BseGI restriction site in the G allele. The forward and reverse primer sequences were 5′-GTAAGGCTGGACTGTTAGATG-3′ and 5′-GGAAGAAGACCGAGTGTGTCAT-3′, respectively. Polymerase chain reaction amplification was carried out in a total volume of 25 μL containing approximately 100 ng DNA, 1X TaqDNA polymerase buffer (containing 1.5 mmol/L MgCl2), 12.5 pmol of each primer, 5 nmol of deoxynucleotide triphosphates, and 0.5 units of TaqDNA polymerase. Polymerase chain reaction cycling conditions consisted of an initial denaturation at 95°C for 3 minutes, then 35 cycles of annealing at 59.5°C for 1.5 minutes, extension at 72°C for 30 seconds, and denaturation at 94°C for 30 seconds. To confirm successful PCR, the products were electrophoresed on 1% agarose gels containing 0.5 mcg/mL ethidium bromide, and the bands were viewed under ultraviolet light. All samples were then digested in duplicate using the restriction enzyme BseGI (MBI Fermentas, Hanover, MD), which recognizes and cuts the G variant only. Digests were incubated for 4 hours at 55°C in a total reaction volume of 20 μL, containing 5 μL of PCR product, 1X reaction buffer, and 1.5 units of BseGI. Digest products were separated on 4% agarose gels and were visualized under ultraviolet light. This identified two alleles: G (152+17 bp) and C (169 bp). Genotypes were independently determined by two researchers.
The χ2 test was used to assess conformation to the Hardy-Weinberg equilibrium and to detect any association between each genotype distribution and clinical category. To account for low frequencies in some groups, Fisher exact tests were used to compare genotype distributions between the premenstrual dysphoric disorder and control group. Statistical significance was considered at P<.05. Odds ratios were calculated to estimate whether a particular genotype was associated with an increased risk of premenstrual dysphoric disorder.
A total of 104 white European women were categorized into two groups; premenstrual dysphoric disorder (n=53), mean age 37.7 years (range 27–46 years) and controls (n=51), mean age 36.2 years (range 22–48 years). Table 1 shows the genotype distribution in the premenstrual dysphoric disorder and control groups. All genotypes conformed to the Hardy-Weinberg equilibrium (Controls χ12=0.46, premenstrual dysphoric disorder χ12=0.32). The difference in genotype distributions between premenstrual dysphoric disorder cases and controls approached statistical significance (P=.056, χ22=5.66). Compared with the postulated “high-risk” G/G genotype, there was a marked overrepresentation of the C/C genotype in the premenstrual dysphoric disorder group (odds ratio 3.63, 95% confidence interval 1.22–10.78; P=.034). Dichotomizing genotypes into presence or absence of the C allele showed that presence of at least one C allele was associated with a 2.5-fold increased risk of premenstrual dysphoric disorder (odds ratio 2.46, 95% confidence interval 1.03–5.88; P=.053), when compared with the control group. Similarly, allelic distributions showed an increased association of the C variant with premenstrual dysphoric disorder (Table 1).
We genotyped the 5-HT1A receptor C(-1019)G polymorphism in two groups of regularly ovulating women, one group with clinically diagnosed premenstrual dysphoric disorder and one group of normal healthy controls with negligible symptoms of premenstrual dysphoria. There was a significant excess of the C/C genotype and an increased prevalence of the C allele in the premenstrual dysphoric disorder cohort compared with controls. Our finding is in stark contrast to previous studies of this marker in psychiatric disorders, which showed the G/G genotype and the G allele to be significantly associated with major depression, suicide, panic disorders and agoraphobia.17,27
Given that mood disorders and premenstrual dysphoric disorder share several key symptoms such as irritability, depression, anxiety and affect lability, and the fact that SSRIs comprise standard treatment for both pathologies, our results were unexpected. However, there are two crucial differences between the psychological symptoms of premenstrual dysphoria and those of other mood disorders. First, premenstrual dysphoric disorder symptoms are cyclical, occurring exclusively during the luteal-phase of ovulatory cycles, then dissipating during menses; in contrast to the chronicity of symptoms in major depression. Second, the therapeutic response to SSRIs is more immediate in premenstrual dysphoric disorder, in most cases within one menstrual cycle after starting treatment.28 This characteristic has led to targeted luteal-phase SSRI therapy, which has been shown to be as effective as continuous treatment throughout the menstrual cycle.29,30 In contrast, a lag of 3–6 weeks is required before SSRIs achieve their maximum effect in mood disorders. Recent studies of the 5-HT1A receptor C(-1019)G polymorphism in major depression have reported that female patients with the C/C genotype showed a better response to SSRIs than those with the G variant.19,20 This improved response was independent from clinical variables (P=.036).21 Although the functional characteristics of the C/C genotype do not explain why it would increase susceptibility to premenstrual dysphoric disorder, it may provide insight into the rapid and effective response to SSRIs.
There are limitations to our preliminary findings. First, the clinical diagnosis of women with premenstrual dysphoric disorder can be difficult, due to the subjective nature of symptom interpretation. However, we used a robust methodology to diagnose premenstrual dysphoric disorder, based on well-established DSM-IV criteria.26 Second, when conducting case-control association studies, the possibility of population stratification must be borne in mind. We attempted to minimize this effect by recruiting ethnically matched white European cases and controls from the local female population living in Staffordshire, United Kingdom, a genetically highly homogeneous population. In studies comprising subjects taken primarily from a localized community, it is important to include healthy controls to determine typical genotype and allelic frequencies, although these may not be representative of the wider population. Our control group 5-HT1A receptor C(-1019)G data did not concur with that of any white study reported to date. However, there does seem to be a wide variation in genotype frequencies for this marker between different white control groups, even within the same country.17,27,31,32 Thus, our 5-HT1A receptor C(-1019)G data may represent a local genotypic anomaly. Our previous premenstrual dysphoric disorder study,33 which genotyped eight polymorphic markers in the serotonin transporter, tryptophan hydroxylase 1 and monoamine oxidase A genes (all of which control serotonin metabolism), used the same cohort of women as presented here. The genotype distributions and allelic frequencies in our control group concurred with those in other white European studies for all polymorphisms except the serotonin transporter VNTR-2, which showed an excess of the 12-repeat allele compared with other reports. Third, the association between the 5-HT1A receptor C(-1019)G marker and premenstrual dysphoria may be affected by sample size. Our study population is relatively small, nevertheless the control group statistics from our previous study indicate that the numbers are sufficient to provide reliable initial data for the 5-HT1A receptor C(-1019)G polymorphism.
Cautious interpretation of the present study is warranted both by the preliminary nature of these findings and by their basis in simple association analysis. Confirmation of our findings will require independent validation in a larger group of subjects. Our hypothesis that the high-risk G allele is associated with the occurrence of premenstrual dysphoria was not proved in this study. Instead, we detected a potential link between the 5-HT1A receptor C(-1019) allele and premenstrual dysphoric disorder. Whether the C variant is also associated with clinical response to SSRIs is an intriguing possibility and presents an exciting area for future research.
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