Brief Report: Aggression and Stereotypic Behavior in Males with Fragile X Syndrome—Moderating Secondary Genes in a “Single Gene” Disorder
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- Hessl, D., Tassone, F., Cordeiro, L. et al. J Autism Dev Disord (2008) 38: 184. doi:10.1007/s10803-007-0365-5
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Although fragile X syndrome (FXS) is a single gene disorder with a well-described phenotype, it is not known why some individuals develop more significant maladaptive behaviors such as aggression or autistic symptoms. Here, we studied two candidate genes known to affect mood and aggression, the serotonin transporter (5-HTTLPR) and monoamine oxidase A (MAOA-VNTR) polymorphisms, in 50 males with FXS ages 8–24 years. Mothers and fathers of participants reported the frequency and severity of aggressive/destructive, self-injurious, and stereotypic behaviors. Polymorphism genotypes were unrelated to age and IQ. Results showed a significant effect of 5-HTTLPR genotype on aggressive/destructive and stereotypic behavior; males with FXS who were homozygous for the high-transcribing long (L/L) genotype had the most aggressive and destructive behavior, and individuals homozygous for the short (S/S) genotype had the least aggression. Those with the L/L genotype also had the highest levels of stereotypic behavior. There was no effect of MAOA-VNTR on behavior; however those with the high-activity, 4-repeat genotype were more likely to be taking SSRI or SNRI medication. This preliminary study prompts consideration of secondary genes that may modify behavioral phenotype expression in neurodevelopmental disorders, even those with a single gene etiology such as FXS.
KeywordsSerotonin transporterMonoamine oxidase APolymorphism5-HTTLPR, MAOAFMR1 geneSelf-injurious behavior
Fragile X syndrome (FXS) is caused by a mutation in a single gene on the long arm of the X chromosome at Xq27.3. The fragile site on the X chromosome typically results from the presence of more than 200 trinucleotide (cytosine-guanine-guanine or CGG) repeats within the promoter region of the FMR1 gene, which prevents normal transcription. This transcriptional silencing of the gene and the subsequent diminished or absent production of FMR1 protein (FMRP) results in aberrant brain development and function (Devys, Lutz, Rouyer, Bellocq, & Mandel, 1993; Tamanini et al., 1997). As such, FXS is a “single gene” disorder with a well described behavioral phenotype, characterized by autistic symptoms including social and communication deficits, and stereotypic behavior; social anxiety and withdrawal; hyperarousal; unusual responses to sensory stimuli; gaze aversion; inattention; and impulsivity and hyperactivity (Reiss & Dant, 2003).
Aggression and self-injurious behavior (SIB) are significant problems for at least 50% of males with FXS (Hagerman & Hagerman, 2002; Symons, Clark, Hatton, Skinner, & Bailey, 2003). When it occurs, SIB in FXS, mainly hand-biting, has a typical onset early in development, at 12–15 months, with little increase past 25 months. In contrast, aggression appears to worsen in the pubescent and post-pubescent period, when the combination of aggression, increased physical strength and development of teens and young adults can pose a serious threat to peers, family members, and other care providers. From the standpoint of many clinicians and parents, aggressive outbursts, SIB, and stereotypic behaviors such as repetitive hand movements are often precipitated by sensory stimuli or unexpected changes in the physical or social environment that the patient is overwhelmed by, leading to hyperarousal (Miller et al., 1999) and stress (Hessl, Glaser, Dyer-Friedman, & Reiss, 2006).
Despite the now well-known behavioral phenotype, there is considerable variability in the severity of symptoms. The variability in behavioral outcomes of children with FXS can be attributed in part to variation in FMRP expression (Hatton et al., 2006; Hessl et al., 2001; Sullivan et al., 2006), quality of educational services and the home environment (Glaser et al., 2003; Hessl et al., 2001), and even to neuroendocrine systems regulating stress (Hessl et al., 2002). However, secondary genes also may help to explain why some individuals experience more severe emotional and behavioral disturbances than others. Despite symptom variability, FXS is a single gene disorder and thus a relatively homogeneous model condition that provides a unique opportunity to study how secondary genes affect behavior and emotion regulation. For the current study, we considered two candidate genes known to affect mood and behavior, especially impulsive aggressive behavior, the serotonin transporter and monoamine oxidase A gene polymorphisms.
The serotonin transporter polymorphism is found in the promoter region of the gene coding for the 5-HTT receptor at 17q11.2. Two polymorphic regions have been identified: a 44 base pair (bp) insertion/deletion in the promoter region (5-HTT gene-linked polymorphic region, 5-HTTLPR; Heils et al., 1996) and a 17 bp variable number of tandem repeats in the second intron of the gene (VNTR-2; Lesch et al., 1996). Transfection studies demonstrated that long (L) and short (S) variants of the promoter polymorphism differentially modulate transcription of 5-HTT gene, with the S variant resulting in lower transcription and transporter levels and less serotonin reuptake, and the L variant resulting in higher transcription and transporter levels and increased reuptake (Heils et al., 1996). The literature on the association between variants of this polymorphism and behavior is inconsistent, with some studies showing increased rates of aggression, violent behavior and anxiety associated with the S allele (Beitchman et al., 2006; Bellivier et al., 2000; Courtet et al., 2001; Hallikainen et al., 1999; Lesch et al., 1996; Liao, Hong, Shih, & Tsai, 2004; Retz, Retz-Junginger, Supprian, Thome, & Rosler, 2004), other studies showing increased aggression, symptoms of ADHD, and violent behavior associated with the L allele (Han, Park, Na, Kee, & Lee, 2004; Retz, Thome, Blocher, Baader, & Rosler, 2002; Sukonick et al., 2001; Sweet et al., 2001), and studies showing no effect (Baca-Garcia et al., 2004; Courtet et al., 2003; Patkar et al., 2002; Yen, Hong, Hou, Wang, & Tsai, 2003). These same inconsistencies appear across studies of autism (see Sutcliffe et al., 2005), perhaps reflecting differences across disorders, ethnic distribution of study cohorts, or allelic heterogeneity at the 5-HTTLPR locus.
Monoamine oxidase A (MAOA) is a mitochondrial enzyme active in degrading all of the monoamines; namely serotonin, dopamine, and norepinephrine. The gene that encodes it maps to the X chromosome, specifically Xp11.23∼11.4 (Sabol, Hu, & Hamer, 1998). MAOA activity has been repeatedly shown to affect changes in neurotransmitter levels, especially that of serotonin and norepinephrine. A functional repeat polymorphism (VNTR) upstream of the gene in the promoter region has been characterized (Deckert et al., 1999; Sabol, Hu, & Hamer, 1998) and shown to affect MAOA activity by impacting transcriptional efficiency of the gene. The polymorphism consists of a 30 base pair (bp) repeat sequence that can include 2, 3, 3.5, 4 or 5 copies. Genetic association studies have repeatedly implicated the MAOA-VNTR polymorphism in the pathogenesis of aggression and impulse control disorders. The 3-repeat, low activity allele has been associated with greater degrees of aggression and impulsivity in the general population (Manuck, Flory, Ferrell, Mann, & Muldoon, 2000; Swann, 2003). This same low activity allele has been associated with autism symptom severity (Cohen et al., 2003), conduct/aggression disorders comorbid with ADHD (Lawson et al., 2003), as well as unmanageable aggressive behavior in schizophrenia (Jonsson et al., 2003) and Alzheimer’s disease (Swann, 2003).
In the current study, we examined whether the 5-HTTLPR and MAOA-VNTR polymorphisms are associated with severity of behavioral problems, specifically aggression, SIB and stereotypic behavior, in males with FXS.
Fifty males with the fragile X full mutation (13/50 with repeat size or methylation mosaicism) were enrolled in the study, ages 8–24 years (mean = 15.6 ± 4.3), with full scale IQ ranging from 40–75 (mean = 50.0 ± 8.9). The ethnic distribution was 75.4% Caucasian, 19.6% Hispanic, 3.9% Asian, and 2% African-American. Two participants were referred due to concerns about aggression; all others were sequential clinic referrals. Fragile X status was confirmed by FMR1 DNA testing using both PCR and Southern blot analysis as previously described (Saluto et al., 2005; Tassone et al., 2004). Eighty percent of participants were taking psychoactive medication at the time of study (44% SSRI/SNRI; 40% antipsychotics; 36% stimulants; 12% anticonvulsants; 4% tricyclic antidepressants), and 54% were taking two or more types of medication. There were six full sibling pairs in the sample; all other subjects were unrelated. For purposes of reporting the behavioral data descriptive statistics, we retained all 50 subjects in the study. However, to satisfy the assumption of independence in the analyses examining genotype differences, we randomly selected one participant from each of the six sibling pairs, leaving 44 subjects in the sample.
5-HTTLPR genotyping was completed as previously described (Lesch et al., 1996). MAOA-VNTR genotyping was carried out according to procedures described by Deckert and colleagues (Deckert et al., 1999).
Cognitive ability was measured using the Wechsler Intelligence Scales (WISC-III, WAIS-III, or WASI). Intelligence testing was unsuccessful with six of the subjects due to low level of functioning, behavioral noncompliance or severity of autism.
The Behavioral Problems Inventory (BPI; Rojahn, Matson, Lott, Esbensen, & Smalls, 2001) was used as a parent-report to assess SIB (14 items including self-biting, self-hitting, and self-scratching), stereotyped behavior (24 items including rocking back and forth, pacing, spinning objects), and aggression/destruction (11 items including hitting, kicking, pushing and biting others, and verbal abuse) over the past 2 months. For the BPI, each item is scored on a five-point frequency scale (from never = 0, to hourly = 4), and a four-point severity scale (from no problem = 0, to severe problem = 3). The BPI is a reliable instrument and has been validated against the Aberrant Behavior Checklist and Diagnostic Assessment for the Severely Handicapped-II (Rojahn, Aman, Matson, & Mayville, 2003; Rojahn, Matson, Naglieri, & Mayville, 2004). Mother–father inter-rater agreement was very good to excellent (intraclass correlations: aggression frequency = 0.90; aggression severity = 0.90; SIB frequency = 0.81; SIB severity = 0.77; stereotypy frequency = 0.89; stereotypy severity = 0.79). To increase reliability and for data reduction purposes, mother and father data were averaged when both sets of data were available (n = 33) and otherwise either the mother (n = 6) or father (n = 5) data was used. To further data reduction, and to increase power and sensitivity, aggression, SIB, and stereotypy severity index scores were taken as the multiplication of the frequency and severity scores within each domain. Due to positive skew in the data, log transformations were used to achieve approximate normality.
For descriptive purposes, we first report the frequencies of aggression, self-injury, and stereotypic behaviors. Then, analysis of variance tests were used to determine if 5-HTTLPR or MAOA-VNTR genotype was associated with severity indices for each type of behavior.
The most common SIBs were self-hitting (50% of the sample) and self-biting (30%) with 79% demonstrating some type of SIB during the 2-month period. The most common aggressive behaviors were hitting others (49% of the sample) and kicking others (30%) with 75% demonstrating some aggression toward others during the 2-month period. The most common stereotypic behaviors were repetitive hand movements (50% of the sample), waving or shaking arms (48%), and waving hands (44%), with 98% demonstrating some stereotypic behavior during the 2-month period. Among those with SIB, the average frequency of this behavior was weekly, while the frequency mode was daily. Among those with aggression, the average frequency was weekly, with one-third demonstrating daily, one-third with weekly, and one-third with monthly aggressive behaviors. Finally, the average frequency of stereotypy was daily, with 21% having hourly, 59% having daily, 14% having weekly and 6% having only monthly stereotypic behaviors.
The current study provides evidence that genotype differences in the serotonin transporter polymorphism (5-HTTLPR) modify expression of aggressive and stereotypic behavior in males with FXS. Individuals homozygous for the L genotype had significantly higher levels of aggressive/destructive behavior than those homozygous for the S genotype. Individuals with the L/L genotype also had the highest levels of stereotypic behavior. There was no significant effect of the monoamine oxidase A genotype (MAOA-VNTR) on behavior. Replication studies with larger sample sizes and using alternative methods for measuring these and other maladaptive behaviors, are needed to confirm and specify these findings.
The weight of evidence in the literature suggests susceptibility for aggressive/impulsive behaviors and anxiety in individuals with the 5-HTTLPR S allele. Given the predisposition for these problems in males with FXS, we expected to observe the most significant problems in those with the S/S genotype. However, the established relationship between 5-HT depletion and aggression (Frankle et al., 2005; Lidberg, Belfrage, Bertilsson, Evenden, & Asberg, 2000) may explain our findings. Individuals with the L/L genotype have less 5-HT available at the synapse due to enhanced reuptake. Perhaps it is the relative decrease of synaptic 5-HT in individuals with FXS and the L/L genotype that lowers their threshold for irritability and aggressive behavior. Alternatively, it could be argued that the S/S genotype offers protection against an aggressive/impulsive predisposition, as has been suggested in a study of 5HTTLPR and SIB in rhesus monkeys (Tiefenbacher et al., 2005). In addition, the reported association between genotype differences at the serotonin transporter locus and risk for autism (Sutcliffe et al., 2005) and the current findings suggest that 5-HTTLPR may also play a role in expression of the autism phenotype in FXS.
If the current preliminary results are confirmed, they could have clinical implications for treatment of behavioral problems in individuals with FXS. For example, those with FXS and the 5-HTTLPR L genotype demonstrating serious maladaptive behavior may be more responsive to SSRI medication, as has been shown in studies in multiple clinical groups (Serretti & Artioli, 2004), including children with autism (Sugie et al., 2005). We know that approximately 70% of individuals with FXS respond well to an SSRI (Berry-Kravis & Potanos, 2004). In the present study, the higher rate of SSRI/SNRI medication use among males with FXS having the MAOA-VNTR genotype associated with high serotonin reuptake is intriguing and could have treatment implications. Controlled clinical trials, perhaps taking into account serotonin or other genotype differences, are clearly needed in this population. Finally, this work prompts consideration of secondary genes that can modify behavioral phenotypes of neurodevelopmental disorders, even those with a primary single gene etiology.
We thank the boys and young men with fragile X syndrome and their families for their participation and generous time and effort. We also thank Patrick Adams and Aaron Campbell for their time and dedication to this research. A portion of this work was presented at the 10th International Fragile X Conference, Atlanta, GA, in July 2006. This research was supported by a grant from the National Fragile X Foundation (D. Hessl) and NIH grants MH77554 (D. Hessl) and HD36071 (R. Hagerman).