Age-dependent cognitive changes in carriers of the Fragile X Syndrome

Introduction

Fragile X syndrome is the most common hereditary cause of developmental delay in males affecting 1 in 4000 live births (de Vries, Halley et al., 1998; Kooy et al 2000; Turner et al 1996). The syndrome is caused by a defect in the Fragile X Mental Retardation 1 (FMR1) gene, located near the end of the long arm of the X chromosome. The FMR1 gene carries a CGG trinucleotide repeat region that in unaffected individuals is usually in the range of 7 – 60 repeats. However, in Fragile X syndrome there is an expansion of these repeats to over 200, known as the full-mutation. At this so-called threshold level, the FMR1 gene is no longer transcribed, resulting in a lack of the encoded protein, the Fragile X mental retardation protein – FMRP. It is the absence of FMRP during early brain development that results in the characteristic cognitive phenotype associated with this syndrome.

Until recently, individuals who carry FMR1 allele expansions with between 55 – ~200 repeats, classified as premutation for Fragile X syndrome (i.e., carrier status), were generally thought to be free from phenotypic effects. However, apparently premutations can be unstable across successive generations eventually giving rise to the Fragile X full-mutation with further expansion past threshold (Kooy, 2003; O’Donnell & Warren, 2002). Both males and females can be carriers of Fragile X syndrome. The absence of observable phenotypic differences in carriers of Fragile X stands in marked contrast the effects of the full mutation (>200 repeats), which results in the moderate to severe mental retardation characteristic of Fragile X syndrome. However, more recent molecular data demonstrate that the moderately expanded CGG repeat region of the gene that occurs in premutation alleles, results in both elevated FMR1 mRNA levels and slight to moderate reductions in FMRP levels (Tassone et al., 2000a; Tassone et al., 2000b). The high prevalence of the Fragile X premutation in the general population, estimated to be 1 in 259 females (Rousseau et al., 1995) and 1 in 813 males (Dombrowski et al., 2002), highlights the necessity of investigating the effect of this condition on cognitive development and functioning. Importantly, there is some evidence that clinically affected carriers display some of the same physical and emotional features typically associated with Fragile X syndrome (Hagerman and Hagerman, 2002; Johnston et al., 2001), albeit with far less severity.

One example of the emerging phenotype in carriers of Fragile X syndrome is the identification of a novel neurodegenerative disorder, the Fragile X-associated tremor/ataxia syndrome (FXTAS), recently identified within a subgroup of older male carriers (> 50 years) (Hagerman et al., 2001; Leehey et al., 2003). FXTAS is specifically associated with the Fragile X carrier status and results in striking neuropathology that include generalized brain atrophy; white matter disease, particularly associated with the middle cerebellar peduncles (MCPs) (Brunberg et al., 2002) as well as eosinophilic intranuclear inclusion bodies in neurons and astroglial cells in broad distribution throughout the CNS (Greco et al., 2002; Hagerman et al., 2003). It is important to note that FXTAS is associated only with carriers of Fragile X and then only within a subset of carrier males aged 50 and over. To date, there is no evidence to suggest that FXTAS is associated with the Fragile X full-mutation.

The pathogenic mechanism of FXTAS is believed to be a toxic “gain of function” resulting from the elevated, abnormally large FMR1 mRNA. Specifically it is thought to result from sequestration of increased amounts of proteins that normally bind to the FMR1 mRNA (Hagerman and Hagerman, 2004). The pathologic mechanisms differ between fragile X syndrome and the premutation condition. Clinical involvement in the premutation condition might arise from two sources, lowered FMRP levels and elevated mRNA levels, with potentially dissociable effects on cognitive functioning. In contrast, clinical involvement in fragile X syndrome arises from the lack of FMRP.

To date, few studies exist that have explicitly examined the pattern of cognitive deficit that may be present in individuals with the fragile X premutation and fewer still that have delineated the premutation male cognitive phenotype (Cornish et al., 2005; Loesch et al., 2003a; Loesch et al., 2003b). Preliminary findings from these studies suggest a neuropsychological profile that includes impairment of social cognition (Cornish et al., 2005), alongside deficits in planning of goal-directed behaviour and in executive functions (Loesch et al., 2003a; Loesch et al., 2003b). In stark contrast, the profile of adult males with the FMR1 full mutation, i.e., Fragile X syndrome, is now well documented with substantial evidence supporting the idea that this condition is not simply characterized by global mental retardation. Rather, the Fragile X neuropsychological profile can be described as comprising uneven abilities within and across cognitive domains that remain stable into adulthood. Relative strengths in vocabulary (Abbeduto et al., 2003), verbal working memory (Jakala et al., 1997), and long-term memory for meaningful and learned information (Cornish et al., 2001; Freund and Reiss, 1991) are accompanied by relative weaknesses in executive control (Cornish et al., 2001; Loesch et al., 2003a), selective and sustained attention (Cornish et al., 2001), visual working memory and visual-motor processing (Crowe and Hay, 1990; Kogan et al., 2004).

The extent to which this profile - or some variation thereof - is present in the carriers of fragile X syndrome is the focus of the present study. As discussed, previous research has demonstrated an association between ataxia/tremor and premutation carriers in older men in the form of FXTAS (Hagerman and Hagerman, 2004; Jacquemont et al., 2003). Previous publications have also reported many cases of mild to severe dementia in older males carriers of the premutation whether these patients were ascertained through neurology clinics (Van Esch et al., 2005), or through family studies of Fragile X syndrome (Hagerman et al., 2001). Here, we examine aspects of cognition known to be affected in full-mutation Fragile X males, through a cross-section of the lifespan of premutation males. We raise the possibility that – as with motor control – there is a trajectory of specific and clinically important cognitive deficits that while initially subtle, progress to culminate in significant functional impairments with increasing age. We also address the extent to which potential FXTAS symptoms impact upon cognitive performance and whether there are specific weaknesses across or within cognitive domains that precede the onset of ataxia/tremor. We also explore whether individuals with the premutation allele may vary in their risk of developing more serious effects of CGG repeat expansion in proportion to the number of repeats they possess. Thus, the aim of the research presented here is to provide new information about the specific cognitive deficits in the premutation population so that clinicians will have a reliable tool to identify patients who may need intervention and who may be at risk for developing FXTAS.

Methods

The study enrolled a total of 107 participants, of whom 40 were pre-mutation males. Recruitment was conducted through the UK Clinical Genetics Services and the UK Fragile X Society. Participants’ ages ranged from 20 to 69 years with a mean age of 46.88 years (SD 14.50). The comparison group comprised 67 non-affected adult males with normal FMR1 alleles and were matched individually on the basis of age to the premutation group. Ages ranged from 20 to 68 years, with a mean age of 45.33 years (SD 14.87). The group means did not differ on socio-economic status or occupational status. The entire sample was Caucasian (indigenous white British). Ethics approval to conduct the study was obtained from regional and local ethics committees across the United Kingdom.

Response inhibition

From a neuropsychological perspective, successful performance on tasks of response inhibition require coordination of several cognitive processes in order to withhold a response that has been strongly triggered by a particular context or cue. Functional imaging studies have generally found increased prefrontal activation during performance on these tasks including activation in the right inferior frontal cortex, right dorso-lateral prefrontal cortex and right inferior parietal cortex [e.g. (Aron et al., 2003; Garavan et al., 2002)]. The following tests were administered:

(1) The Hayling Sentence Completion Test (Burgess and Shallice, 1997). In this two part task, participants had to complete a sentence with a single word, which was either congruous (Section A) or totally incongruous (Section B) with the overall meaning of the sentence. In each case the word is strongly cued by the sentence content with a total of 15 sentences in each section. An error score was obtained which represented the number of inappropriate sentence completion items where the response word is either the word most strongly cued by sentence content or semantically related (see Burgess and Shallice, 1997).

(2) The Stroop Color-Word Test (Trenerry et al., 1989). Participants are presented with lists of color names printed in colored ink. The color name is incongruent with the color of the ink in which it is printed. In the first part, participants must read aloud 112 color words (red, blue, green, tan) within a 2 minute time limit. In the second part, the 112 color words are again presented but this time the participant must say the color of the ink and not the color word itself (e.g., with the word ‘green’ written in blue ink, the participant should say blue). The score is represented by the number of errors made on the interference condition.

Attention

Selective attention, as assessed here, refers to a speeded search in a visually noisy environment for targets defined by a certain feature. Such visual selection has been argued to stem from interactions between regions involved in basic visual processing and those involved in storing representations of the target in memory that exert a ‘top-down’ influence on the system. Animal models and human functional imaging studies suggest that a network linking dorso-lateral and ventro-lateral prefrontal cortex and inferior parietal regions are involved in this process (Duncan, 2006). In terms of sustained attention, the processes of actively maintaining attention to tasks that lack inherent interest or reward have also been linked to networks that incorporate the prefrontal (i.e., right dorso-lateral) and parietal cortices (Pardo et al., 1991). The following tests were administered:

(1) For selective attention, The Test of Everyday Attention- Map Search Task (Robertson et al., 1994). This timed visual search task requires participants to identify target symbols (e.g. a knife and fork sign representing restaurant facilities) from competing and irrelevant distracters on a large colour map of Philadelphia. The score is the total number of correctly identified symbols across two timed conditions.

(2) For sustained attention, The Sustained Attention to Response Test (Manly et al., 2000). This computerized test requires participants to make responses to digits (1 – 9) presented on a screen, except when the critical ‘no-go’ digit (i.e., the number 3) appears. The score includes errors of commission and omission.

Visual Functions

Following on from earlier animal studies, recent human functional imaging studies contrasting visual working memory tasks with suitable control conditions have reported increased activation in dorso-lateral and ventro-lateral regions of the right prefrontal cortex as well as with increased activation in posterior parietal cortex (Bor et al., 2003; Owen, 2000; Owen et al., 1996). In contrast, basic visual functions, such as those required for visual-spatial processing, are thought to rely more on posterior systems, and in particular, the occipital-parietal region. The following tests were administered for:

Results

Discussion

Our primary aim was to establish whether individuals who are carriers of the Fragile X premutation show subtle impairments on neuropsychological tasks that assess cognitive domains previously demonstrated to be impaired in individuals with full mutation Fragile X syndrome. In addition, we sought to identify the trajectory and specificity of any such impairment – in particular, whether phenotypic effects of the premutation become more apparent and more severe with increasing age. Finally, we sought to address the extent to which a newly identified neurological disorder associated with the Fragile X premutation, FXTAS, may impact on performance in identified domains of cognitive weakness. Our findings are remarkable in three respects.

First, carrier males with the premutation performed significantly worse than males without the premutation on tasks that require inhibitory and executive control in particular. At a cognitive level, problems in attention switching and control have been reported as a fundamental deficit in the Fragile X full mutation from infancy, through childhood and into adulthood (Cornish, Scerif & Karmiloff-Smith 2006; Cornish, Sudhalter and Turk 2004; Cornish, Munir and Cross, 2001). Our findings extend this characteristic profile to include individuals with the Fragile X premutation. Although carrier males appear to possess a less severe deficit as compared to males with the Fragile X full mutation (who will also have mental retardation), the result is nonetheless strongly suggestive of a significant executive dysfunction in these individuals.

Second, we report here the first time, a correlation between CGG repeats length and inhibitory impairment in premutation males. This finding suggests greater neuropathology in carrier males with larger expansions in FMR1 mRNA transcripts. It is therefore possible that expression of FMR1 mRNAs containing repeat expansions approaching the full mutation range (i.e., 200 repeats) produce exceptionally toxic effects to neurons comprising brain circuits critical to inhibitory control. This result is consistent with previous research suggesting that individuals with larger premutations display clinical features approaching the full mutation condition (Hagerman and Hagerman, 2004; Tassone et al., 2000a; Tassone et al., 2000b).

At the brain level, difficulties in inhibitory control have been associated with disruptions to frontal circuits, in particular to the right inferior frontal cortex (IFC). Evidence of this comes from functional imaging of brains of individuals with Attention-Deficit Hyperactivity Disorder (ADHD), a population in which response inhibition is proposed to be a critical endophenotype (Aron and Poldrack, 2005). Similarly, studies of naturally occurring lesions in humans implicate the IFC in inhibitory control (Aron et al., 2003). The tasks employed in the present study evaluated the domains of attention, perception, and response inhibition, all proposed to engage prefrontal cortical areas as well as regions of the parietal lobe. However, the finding of a selective and worsening deficit with age in response inhibition in the premutation males specifically implicates the right IFC, an area exclusively activated in tasks of response inhibition. Interestingly, results of a functional imaging study of fragile X affected females, who display a broad range of functioning dependent on the degree of activation of the affected FMR1 gene, indicate a correlation between activity in the right IFC during a response inhibition task and FMR1 expression levels (Menon et al., 2004). Taken together, data from full mutation males, fragile X affected females, and now premutation males, strengthens the notion that the right IFC has differential susceptibility to alterations in FMR1 gene function.

Third, we report significant differences in response inhibition between younger (i.e., less than 50 years old) premutation and comparison males. This difference, however, is not evident when performance of older (i.e., greater than 50 years old) premutation and comparison males is contrasted. This unusual finding may be explained by differential survival of subgroups of premutation carriers. Young premutation males are much less likely to manifest FXTAS or other related pathology and would therefore be considered healthy even if a subgroup of them harbours incipient yet undetectable pathology. As individuals transition to the older group it is likely that morbidity and mortality rates increase. This may result in a bias in the older group we define here as not displaying symptoms of FXTAS, because a relatively larger proportion of the sample were never going to develop FXTAS in the first place. The category of older premutation males who were to develop FXTAS are more likely to express symptoms at this age and therefore will be accurately categorized as belonging to the premutation males with FXTAS group. A longitudinal study would be able to address this hypothesis and this could be the basis of a future study.

Given that the inhibition deficits appear to have their onset in younger premutation males before the first signs of FXTAS-related symptoms it may therefore point to disruption of inhibitory control as a useful neurological soft sign preceding more generalized and profound effects associated with FXTAS (i.e., brain atrophy, ataxia, peripheral neuropathy, progressive intention tremor, dementia) reported in older patients (>50 years) (Hagerman and Hagerman, 2004). The present study found inhibitory deficits to have a strong co-occurrence in older males with FXTAS-related symptoms but not in older males without FXTAS symptoms. This finding strongly supports the idea that inhibitory deficits are a defining feature of FXTAS and possibly predictive of at-risk patients. It is therefore possible that inhibitory deficits in premutation males are a marker of an early onset of a pathological process affecting prefrontal cortex.

As a parallel to our findings, early neuropsychological markers have demonstrated clinical utility in identifying patients with so-called mild cognitive impairment (MCI), a precursor condition to Alzheimer’s dementia (AD). Neuropsychological tests of episodic memory, which employ a visual-spatial learning task prove to be good predictors of those patients with MCI who will go on to develop to AD (Blackwell et al., 2004; Swainson et al., 2001). Identification of MCI patients with a specific neuropsychological profile allows for earlier intervention and in turn, a more prolonged period without the onset of frank dementia. The selectivity of the inhibitory deficits identified in the present study of PM males, supports the use of measures of inhibitory control as potential predictors of risk for later development of FXTAS cognitive symptoms. Although there are no known medications to prevent FXTAS, those such as Venlafaxine can be helpful in improving inhibitory deficits.

Performance in other cognitive domains also provides a window to explore the premutation phenotype. The relatively normal performance and age trajectory on tests of visual working memory, visuo-spatial functions and sustained attention also suggest a rather specific effect of the FMR1 premutation within cognitive function. Given previous evidence, the presence of a disproportionate aging effect may itself form a useful marker of functions that are closely related to the premutation condition. In this respect, selective attention performance, while at a lower rate than comparison males overall, showed no such increase with age. This suggests that there are two effects of the premutation on cognition, one present throughout life and another that increases in severity with age. The latter effect is most likely related to the toxic mRNA effect that can cause cell death and brain atrophy over time, and eventually the full clinical picture of FXTAS. The former effect may be related to a mild reduction in FMRP level which results in a subtle yet measurable Fragile X phenotype including attentional problems. Given that the specific difficulties in inhibitory control exhibited in the premutation males appear to become worse with age, it is likely that these are related to the mRNA toxic effect that is known to accrue with age.

Taken together, these findings highlight an important trajectory of cognitive deficit in premutation carrier males of Fragile X syndrome that appears to begin early in adulthood and become progressively more severe across developmental time. Pieces of the puzzle still need to be explored. For example, a careful investigation of the childhood profile is essential and is hitherto unexplored. Yet precursors of cognitive decline may present even earlier than the thirties having their roots in late childhood.

Overall, the pattern of results highlights a distinct inhibitory ‘signature’ in premutation males that corresponds to the well-documented attentional control impairment in full mutation males with Fragile X. Furthermore, our finding that CGG repeat length correlates with the degree of impairment suggests that increasingly large mRNA transcripts of the FMR1 gene damage susceptible neural networks, in particular, those associated with the right IFC.