A neuropsychological investigation of male premutation carriers of fragile X syndrome

Neuropsychologia 42 (2004) 1934–1947

A neuropsychological investigation of male premutation carriers of fragile X syndrome Caroline J. Moore a,∗ , Eileen M. Daly a , Nicole Schmitz a , Flora Tassone b , Carolyn Tysoe c , Randi J. Hagerman d , Paul J. Hagerman b , Robin G. Morris e , Kieran C. Murphy a,f , Declan G. M. Murphy a a

Division of Psychological Medicine, Section of Brain Maturation, Institute of Psychiatry, King’s College London, DeCrespigny Park, London, UK b Department of Biological Chemistry, University of California, Davis, CA, USA c Medical Genetics Service for Wales, University Hospital of Wales, Heath Park, Cardiff, UK d M.I.N.D. Institute and Department of Pediatrics, UC Davis Medical Center, Sacramento, CA, USA e Institute of Psychiatry, King’s College London, DeCrespigny Park, London, UK f Department of Psychiatry, Royal College of Surgeons in Ireland, Education and Research Centre, Beaumont Hospital, Dublin 9, Ireland Received 18 December 2003; received in revised form 10 May 2004; accepted 11 May 2004

Abstract It is currently thought that fragile X syndrome (FraX; the most common inherited form of learning disability) results from having more than 200 cytosine–guanine–guanine (CGG) trinucleotide repeats, with consequent methylation of the fragile X mental retardation (FMR1) gene and loss of FMR1 protein (FMRP). It was also considered that premutation carriers (with 55–200 CGG repeats) are unaffected, although a tremor/ataxia syndrome has recently been described in older adult male carriers. We reported that premutation expansion of CGG trinucleotide repeats affects brain anatomy, which, together with other studies, indicates that the molecular model for FraX needs modification. However, there are few studies on the cognitive ability of adult male premutation carriers. Thus, we selected 20 male premutation carriers on the basis of their genetic phenotype, and compared them to 20 male controls matched on age, IQ and handedness. We investigated intellectual functioning, executive function, memory, attention, visual and spatial perception, and language and pragmatics. The premutation carriers had significant impairments on tests of executive function (Verbal Fluency, Trail Making Test and Tower of London) and memory (Names sub-test of the Doors and People, Verbal Paired Associates Immediate Recall and Visual Paired Associates Delayed Recall sub-tests of the WMS-R, and Category Fluency Test for natural kinds). We therefore suggest that CGG trinucleotide repeats in the premutation range affect specific neuronal circuits that are concordant with specific neuropsychological deficits; and that these deficits reflect an emerging neuropsychological phenotype of premutation FraX. © 2004 Elsevier Ltd. All rights reserved. Keywords: Trinucleotide repeats; X chromosome; Executive function; Memory

1. Introduction Fragile X syndrome (FraX) is the most common form of inherited learning disability, affecting approximately 1:4000–6000 males (Turner, Webb, Wake, & Robinson, 1996; Youings et al., 2000). FraX is associated with an expansion of cytosine–guanine–guanine (CGG) trinucleotide repeats in the 5’ untranslated region of the fragile X mental ∗ Corresponding author. Tel.: +44 207 848 0349; fax: +44 207 848 0650. E-mail address: [email protected] (C.J. Moore).

0028-3932/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropsychologia.2004.05.002

retardation 1 (FMR1) gene on the X chromosome. Expansion of the FMR1 gene to more than 200 CGG repeats (full mutation) is generally accompanied by methylation of the FMR1 gene and loss of FMR1 protein (FMRP) production (Verkerk et al., 1991; Yu et al., 1991). The cognitive and behavioural phenotype of the full mutation of FraX has been described by many authors (reviewed in Turk, 1992; Bennetto & Pennington, 2002). Features include autistic-like symptoms; maladaptive behaviours such as unusual speech patterns and hand flapping; and neuropsychological deficits in attention, processing and remembering sequential information, short-term memory,

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visuo-spatial ability, visual-motor co-ordination, and pragmatic language. Approximately 1:813 men are carriers of premutation expansions of the FMR1 gene (with 55–200 CGG repeats) (Dombrowski et al., 2002). The prevalence for females is much higher; perhaps as high as 1:100 (reviewed in Hagerman & Hagerman, 2002). It was originally thought that premutation carriers of FraX had normal FMRP production, implying that they were clinically unaffected. However, recent molecular genetic investigations have demonstrated diminished production of FMRP (Kenneson, Zhang, Hagedorn, & Warren, 2001; Tassone, Hagerman, Taylor, & Mills, et al., 2000; Tassone, Hagerman, Taylor, & Gane, et al., 2000) and elevated levels of FMR1 messenger RNA (mRNA) (Hagerman et al., 2001; Tassone, Hagerman, Taylor, & Gane, et al., 2000; Tassone, Hagerman, Chamberlain, & Hagerman, 2000) in men who are premutation carriers.These findings are supported by an increasing literature suggesting a clinical affect of premutation expansions of CGG repeats (reviewed in Hagerman & Hagerman, 2002). For example, female premutation carriers may have; (1) a mild form of the physical phenotype of FraX (Hull & Hagerman, 1993; Riddle et al., 1998); (2) elevated levels of follicle-stimulating hormone (Braat, Smits, & Tomas, 1999; Hundscheid, Braat, Kiemeney, Smits, & Thomas, 2001); (3) premature ovarian failure (Allingham-Hawkins et al., 1999; Conway, Hettitarachein, Murray, & Jacobs, 1995; Giovannucci Uzielli et al., 1999; Murray, Webb, Grimley, Conway, 1998; Partington, Moore, & Turner, 1996; Schwartz et al., 1994; Syrrou et al., 1999; Turner, Robinson, Wake, & Martin, 1994; Vianna-Morgante, Costa, Pares, & Verreschi, 1996; Vianna-Morgante, 1999); and (4) neuroanatomical abnormalities (Murphy et al., 1999). Behavioural abnormalities, such as social anxiety (Sobesky, Hull, & Hagerman, 1994; Franke, Barbe, Leboyer, Maier, 1996, 1998), psychosis (Fryns, 1986) and affective disorders (Franke et al., 1996, 1998; Reiss, Hagerman, Vinogradov, Abrams, & King; Thompson et al., 1994; Sobesky, Porter, Pennington, & Hagerman, 1995, 1996) have also been reported in females, with a recent study (Johnston et al., 2001) demonstrating that female carriers with >100 CGG trinucleotide repeats have more emotional problems than those with <100 CGG trinucleotide repeats. Neuropsychological studies of female carriers, on the other hand, have reported that their performance does not significantly differ from controls (Bennetto, Pennington, Porter, Taylor, & Hagerman, 2001; Franke et al., 1999; Mazzocco, Pennington, & Hagerman, 1993; Mazzocco & Holden, 1996; Myers, Mazzocco, Maddalena, & Reiss, 2001; Reiss, Freund, Adams, Boehm, & Kazazian, 1993; Simon et al., 2001). However, studies of female premutation carriers are confounded by Lyonisation (i.e. random inactivation of one of the two X chromosomes) and consequent variation in activation ratios (i.e. the proportion of active X chromosomes that have an affected allele). The range of activation ratios is great; for example there is more variability in the degree

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of FMR1 mRNA elevation for females than males (Tassone, Hagerman, Taylor, & Mills, et al., 2000). Thus neuropsychological performance may be differentially affected at the individual subject level, resulting in random group effects. Investigations of male premutation carriers provide a clearer picture of the effect of premutation expansions on neuropsychological performance. There have been relatively few studies of male premutation carriers, who, as grandfathers and uncles of individuals affected by the full syndrome, are less likely to be identified than female carriers. Studies suggest that male premutation carriers may have (1) the facial characteristics of FraX (Hagerman et al., 1996; Hull & Hagerman, 1993;Loesch, Hay, & Mulley, 1994); (2) an increase in behavioural abnormalities, such as alcohol use and dependence, and obsessive-compulsive behaviours (Dorn, Mazzocco, & Hagerman, 1994); (3) learning disability (Hagerman et al., 1996; Murray et al., 1996; Rousseau et al., 1994; Tassone, Hagerman, Taylor, & Mills, et al., 2000; Teague et al., 1998); (4) deficits in vocabulary and block design IQ subtests (Loesch et al., 1994), attention (Dorn et al., 1994), and executive function (Hagerman et al., 2001; Jacquemont et al., 2003). In contrast, a recent study found no significant differences in the cognitive ability of boys who were premutation carriers as compared to controls (Myers et al., 2001). Neuroantomical abnormalities associated with a tremor/ ataxia syndrome in older male carriers (FXTAS) have also been described (Brunberg et al., 2003; Greco et al., 2002; Hagerman et al., 2001; Jacquemont et al., 2003) and we have found neuroanatomical abnormalities in carriers who are clinically unaffected (Moore et al., 2004). Thus, there is evidence that premutation expansion of CGG repeats may affect brain function. However, prior studies are difficult to interpret because many were affected by ascertainment bias; they included people who were identified by their learning or physical disabilities (Hagerman et al., 1996; Murray et al., 1996; Rousseau et al., 1994; Tassone, Hagerman, Taylor, & Mills, et al., 2000; Teague et al., 1998) or neurological features (Brunberg et al., 2003; Greco et al., 2002; Hagerman et al., 2001; Jacquemont et al., 2003). Further, those studies not confounded by ascertainment bias have been relatively small, with groups of ten or fewer individuals (Loesch et al., 1994, 1984; Myers et al., 2001), and/or did not include control subjects of similar IQ (Loesch et al., 1994). No comprehensive neuropsychological profile has yet to be reported for a large cohort of adult male premutation carriers in the absence of ascertainment through learning disabilities or clinical features. To address this issue, we recruited twenty carriers selected on the basis of their genetic phenotype only, via UK genetic services. Participants were given an intellectual assessment and all had IQs in the normal range (96–142). Performance of the carriers on an additional battery of tests was compared to male controls who did not differ significantly in age, IQ, or handedness. The neuropsychological features of the full mutation of FraX have

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been demonstrated extensively in the neuropsychologoical literature, but it is not known whether these features would be seen in milder form in adult male premutation carriers of FraX, or whether more subtle characteristics would emerge. Hence there was a need to provide a broad neuropsychological assessment in order to cover the main areas of function in an exploratory fashion, with a view to further studies following up any deficits that may be revealed. Specifically, we investigated overall level of intellectual functioning, executive function, memory, attention, visual and spatial perception, and language and pragmatics. The purpose of an assessment of intellectual function was to determine an overall level of functioning against which to compare other functions. By definition, the participants were likely to be of relatively normal intelligence because they were not ascertained through having any learning disabilities. The assessment of intelligence in this study contrasts specifically verbal and spatial functioning. Previous studies of subjects with the full mutation of FraX have shown relative weaknesses in individual subtests from the Performance Scale, such as Block Design (Crowe & Hay, 1990). Studies of small groups of premutation carriers of FraX have shown Block Design impairment (Loesch et al., 1994). There was a need for a comprehensive test of executive function, covering the main features, including: generativity, mental flexibility, problem solving, working memory and strategy formation. Executive dysfunction has been demonstrated in subjects with the full mutation of FraX who have difficulties in inhibition (Cornish, Munir, & Cross, 2001; Munir, Cornish, & Wilding, 2000) and in a subgroup of neurological patients who are premutation carriers of FraX (Hagerman et al., 2001; Jacquemont et al., 2003). In relation to these findings, there are reports of deficits in measures of attention for subjects with the full mutation of FraX, with particular effects in selective attention (Cornish et al., 2001; Munir et al., 2000). Thus, our test battery included tests of attention. Memory functioning was assessed using tests that measured the main facets of long-term memory; including recognition versus recall, and verbal versus visual based encoding, with additional tests of prose recall and verbal learning. The study also tested working memory, which has consistently been found to be impaired in subjects with the full mutation of FraX, where there is a dissociation between intact memory for meaningful verbal information but impaired memory for meaningless information, or abstract visual information (Dykens, Hodapp, & Leckman, 1987; Hodapp et al., 1992; Kemper, Hagerman, Ahmad, & Mariner, 1986; Munir, Cornish, & Wilding, 2001). Memory loss has also been demonstrated in the subgroup of neurologically affected premutation carriers of FraX (Hagerman et al., 2001; Jacquemont et al., 2003). Visual and spatial perception was assessed because, although no deficits have been demonstrated for premutation carriers of FraX, subjects with the full mutation have been shown to have difficulties in tests of visuo-spatial contruc-

tion, visuo-spatial perception, and spatial memory (Crowe & Hay, 1990). We assessed language and pragmatics because subjects with the full mutation of FraX have deficits in rate and prosody of speech similar to the difficulties seem in verbal dyspraxia (Paul, Cohen, Greg, Watson, & Herman, 1984; Vilkman, Niemi, & Ikonen, 1988); impairments in nonverbal pragmatics and pragmatic competence including perseveration, stereotyped and repetitive statements, echolalia, inappropriate remarks (Ferrier et al., 1991; Sudhalter, Cohen, Silverman, & Wolf-Schein, 1990; Wolf-Schein et al., 1987); and impairments in expressive semantics (Sudhalter, Maranion, & Brooks, 1992). Finally, we additionally explored, within premutation carriers, whether there were correlations between neuropsychological performance and genetic abnormalities (expansion of CGG repeats, reduced production of FMRP, elevation of FMR1 mRNA) and/or ageing.

2. Methods 2.1. Subjects Twenty male premutation carriers and twenty male control subjects participated in the investigation. There were no significant group differences in age, full scale IQ or handedness (Table 1). The premutation carriers were recruited from genetic services throughout Britain, on the basis of genetic, not clinical phenotype. All subjects gave informed consent to participate in this study, which has been approved by the ethical committee of the Institute of Psychiatry and the South London and Maudsley NHS Trust, and the individual LRECs attached to the genetic centres where subjects were recruited. Table 1 Matching criteria and genetic variables

Matching criteria Full scale IQ (WAIS-R) Age Genetic variables CGG repeat number

Carriers

Controls

P-value

N = 20 113.30 ± 12.44

115.95 ± 18.98

N = 20 0.60

N = 20 53.25 ± 14.86

49.45 ± 15.79

N = 20 0.44

N = 19 85.6 ± 18.4

%FMRP(+) lymphocytes

N = 18 75.4 ± 5.4

FMR1 mRNA level

N = 17 3.7 ± 1.6

Matching critera: subject numbers (N), mean values and standard deviations for carriers and controls are shown in columns 2 and 3, respectively. The final column shows the P-values resulting from group testing. Genetic variables: subject numbers (N), mean values and standard deviations for carriers.

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All participants underwent routine blood tests, structured physical and psychiatric examination (Murphy et al., 1996, 1997), and clinical MRI to rule out organic brain disease which may affect cognitive function. Full scale IQ was measured using the Canavan (Canavan, Dunn, & McMillan, 1986) short version of the Weschler adult intelligence scale-revised (WAIS-R) (Weschler, 1981), and handedness was determined using Annett’s questionnaire (Annett, 1970).

2.2. Blood testing PCR analysis (Brown et al., 1993) confirmed premutation (55–200 CGG repeats) and control (<50 CGG repeats) status. Further investigation of premutation carriers gave CGG repeat number (N = 19), the percentage of lympocytes staining with anti-FMRP antibodies [%FMRP(+)] (N = 18) and degree of FMR1 mRNA elevation (N = 17), with N variation due to sample decay (Table 1). A ‘Fragile X Size Polymorphism Assay’ kit (Applied Biosystems) measured CGG trinucleotide repeat number and previously published methods were used to measure %FMRP(+) lymphocytes (Tassone, Hagerman, Taylor, & Mills, et al., 2000; Willemsen et al., 1995, 1997), and FMR1 mRNA levels (Tassone, Hagerman, Taylor, & Gane, et al., 2000) in premutation carriers. In brief, %FMRP(+) lymphocytes were determined by immunocytochemistry of blood smears (Tassone, Hagerman, Taylor, & Mills, et al., 2000; Willemsen et al., 1995, 1997), using an indirect alkaline phosphatase-staining technique for detection. For each sample collected, 200 lymphocytes were counted and %FMRP(+) lymphocytes were scored for the presence of a red-staining cytoplasmic ring that indicates the presence of FMRP. It is important to note that this method is not a quantitative measure of FMRP in each cell; rather, it only provides a percentage of the cells expressing FMRP. FMR1 mRNA was quantified using an automated, fluorescence-detection RT-PCR assay (Tassone, Hagerman, Taylor, & Gane, et al., 2000). The extent of fluorescence is directly proportional to DNA copy number and relative mRNA levels were determined by comparison of the cycle numbers for which the relative fluorescence signal exceeded a designated threshold value. In this study, the purpose of obtaining detailed genetic information on premutation carriers was to facilitate an investigation of correlations between genetic and neuropsychological variables (see below). We have previously reported (Moore et al., 2004) that the male premutation carriers investigated here show a significant negative correlation between CGG trinucleotide repeats and %FMRP(+) lymphocytes; as the number of CGG trinucleotide repeats increased, %FMRP(+) lymphocytes was reduced (Kendall’s tau = −0.547, P = 0.002), replicating previous findings (Kenneson et al., 2001; Tassone, Hagerman, Taylor, & Gane, et al., 2000). Whilst we did not demonstrate signif-

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icant correlations between FMR1 mRNA levels and CGG trinucleotide repeats or %FMRP(+) lymphocytes, previous studies over a broader range of premutation CGG trinucleotide repeat sizes have observed a positive correlation between CGG trinucleotide repeat size and FMR1 mRNA levels (Kenneson et al., 2001; Tassone, Hagerman, Taylor, & Gane, et al., 2000). 2.3. Self-report questionnaires Subjects were asked to complete four self-report questionnaires: the Beck’s anxiety inventory (Beck, 1987a), the Beck’s depression inventory (Beck, 1987b), the Yale Brown obsessive-compulsive scale (YBOC) (Goodman et al., 1989), and the General Health Questionnaire (GHQ) (Goldberg, 1978). 2.4. Neuropsychological testing 2.4.1. Intellectual functioning Intellectual functioning was assessed with the Canavan (Canavan et al., 1986) short version of the Weschler adult intelligence scale-revised (WAIS-R) (Weschler, 1981) which comprises five subtests: vocabulary, comprehension, similarities, block design, and object assembly. IQ scores were obtained from a pro-rating scoring system. 2.5. Executive function 2.5.1. Verbal Initiation and production of verbal information was assessed by verbal fluency (the Controlled Oral Word Association Test) (Benton & Hamsher, 1976). Subjects recalled as many words that they could, beginning with a given letter, in a 60 s time frame. There were three trials, using the letters F, A and S. A score was given for the total responses across all trials. 2.5.2. Mental flexibility Mental flexibility, attention, motor function and speed for visual search were assessed by the Trail Making Test (Reitan, in press; see Lezak, 1995). In Trial A, subjects drew a line connecting the numbers 1–23 in order, displayed spatially in a pseudorandom fashion, as quickly as they could without lifting their pencil from the paper. In Trial B, subjects drew a line connecting numbers and letters consecutively (1 to A, A to 2, 2 to B, B to 3 and so on). Subtracting trial A from trial B (Trail Making B–A) controlled for the motoric and sequencing elements of the test, providing a purer measure of mental flexibility. 2.5.3. Problem solving Problem solving was assessed with a 3D computerised Tower of London Task (Morris, Downes, Sahakian, Heald, & Robbins, 1988; Morris, Downes, & Robbins 1990; Shallice,

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1982). This test required subjects to solve problems in the least number of moves possible. Two arrangements of three different coloured disks on three different sized rods were displayed on a touch sensitive screen. The top row was the target to which subjects matched the second row of disks. The test comprised three-move, four-move and five-move problems (with four trials in each). The number of moves above minimum required to solve each problem was assessed. We report both Total Moves Above Minimum (summed across all problems) and Moves Above Minimum for 5-move problems; the latter was included because a deficit may only be apparent for the more difficult problems. A psychomotor control task requiring subjects to make the same disk movements but without problem solving, was also administered. A Planning Ability Score was obtained by subtracting control-planning time from test-planning time. 2.5.4. Strategy formation Strategy formation and working memory was assessed using the executive golf task (Baker et al., 1996; Morris et al., 1988). The task was presented on a touch-sensitive computer screen displaying a pictorial putting green with a golfer in the distance and an array of golf holes that acted as spatial locations. The subject initially had to guess which hole the golfer was going to putt the ball into, searching holes in turn to reveal which contained the ball. After finding it, the subject had to search again for the next location, with the rule that the golfer will not use the same location twice. The series of searches continued until all of the locations had been targets. After practice trials, the test trials included four attempts with six holes and four with eight holes. Three measures were taken: Within Search Errors, where the same hole was selected more than once in a search; Between Search Errors, where subjects selected holes used as targets before; and Strategy Formation, a measure of the efficiency in which the subject systematically searched the holes for the target location. These measures were summed across the test trials.

2.6. Memory 2.6.1. Short-term memory Verbal and spatial memory span was tested by using sub-tests of the Weschler Memory Scale Revised (WMS-R) (Weschler, 1987). This included the Digit Span test (testing verbal working memory), where subjects repeated increasing amounts of numbers. There were two parts of the test: (i) repetition in the correct order and (ii) repetition in reverse order; these were summed together to give an overall score. In the Visual Memory Span test (testing visual working memory and motor co-ordination), subjects repeated increasing amounts of moves tapped onto an array of coloured squares. As with Digit Span, two parts of the test: (i) repetition in the correct order and (ii) repetition in reverse order, were summed together.

2.6.2. Long-term memory The Doors and People Test of visual and verbal recognition (Baddeley, Emslie, & Nimmo-Smith, 1994) tested verbal and visual recall and recognition memory, with additional measures of forgetting following delays. For verbal recall, the People test, subjects learned the fore and surnames of four people, with visual and verbal aids (photographs and occupations, respectively). A maximum of three trails were given to learn the names. For verbal recognition, the Names test, subjects were shown 12 forename and surname pairs and asked to recognise those they had seen from a set of four names (with one target and three distracters). There were two sets of 12 names, an easy set and a harder set; these were summed together. For visual recall, the Shapes test, subjects copied four simple drawings and then recalled the drawings from memory. A maximum of three trails were given to learn the drawings. For visual recognition, the Doors test, subjects were shown 12 doors and asked to recognise those they had seen from a set of four doors (with one target and three distracters). As with the Names test, two sets of 12, an easy set and a harder set, were summed together. In addition to scores for people, names, shapes and doors, we also reported a total overall aggregate score and additional aggregate scores for verbal memory (people and names), visual memory (shapes and doors), recognition (names and doors). In addition, recall on the People and Shape tests was tested after a further delay; scores were subtracted from immediate recall to produce an aggregate measure of overall forgetting. 2.6.3. Learning Additional assessments of learning were provided by sub-tests of the WMS-R (Weschler, 1987). In verbal paired associates subjects learned a list of eight verbal paired associates spoken by the examiner. The examiner then said one word of each pair and the subject recalled the other word. Scores were given for immediate and delayed recall. In visual paired associates subjects learned a set of six drawings paired with six colours. They were then shown a drawing and asked to point to the relevant colour. Again, scores were given for immediate and delayed recall. 2.6.4. Prose recall Prose recall was assessed by logical memory, a sub-test of the WMS-R (Weschler, 1987). Subjects listened to a short story and were asked to remember as much detail about the story as possible. There were two trials, and scores were given for immediate and delayed recall. 2.6.5. Semantic memory Semantic memory was assessed by category fluency. Subjects named as many words that they could, belonging to a given category in a 60 s time frame. There were four trials, using the categories: animals, fruit and vegetables, furniture, and tools (Mummery, Patterson, Hodges, & Wise, 1996). Scores were summed over animals, fruit and vegetables to

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give a natural kinds score; and over furniture and tools to give a manmade objects score. 2.7. Attention Attention was assessed using the computerised Continuous Performance Test (Conner, 1995). Subjects viewed letters displayed for 250 ms, presented on a computer screen at different speeds (with an inter-stimulus interval of 1, 2 or 4 s). Subjects pressed the space bar following presentation of all letters except the letter X. We report a number of variables. Hit Reaction Times measured the mean response time (ms) for all targets. This score is presented as a t-score, with high scores reflecting fast reaction times. Hit standard error (S.E.) block change measured the slope of change in reaction time standard errors, with a positive slope indicating that reaction times became less consistent as the test progressed. Two types of errors are reported: commission errors show the percentage number of times subjects responded to letters other than the target (i.e. responding to the letter X); omission errors show the percentage number of targets that subjects did not respond to (i.e. letters A–W, Y and Z). A perceptual sensitivity score, presented as a t-score, measured the subject’s ability to discriminate targets from non-targets. A risk taking score, again presented as a t-score, measured subject’s response tendency; low scores represent more frequent responses, indicating increased risk taking and impulsivity. Finally, an overall index score provided a weighted sum of all CPT measures (Conner, 1995), with scores <8 associated with good performance, scores of 8–11 borderline, and scores of >11 indicating attention problems.

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cards. In position discrimination, subjects were presented with stimuli consisting of two adjacent, horizontal squares with one dot in the centre and another slightly off centre. Subjects pointed to the dot in the centre. In number location, subjects were presented with two squares, the top square containing randomly placed numbers and the bottom square containing a black dot. Subjects identified the number corresponding with the position of the dot. Finally, in cube analysis, subjects were presented with black outline representations of 3D arrangements of square bricks, and identified the number of solid bricks. 2.9. Language and pragmatics 2.9.1. Oral word reading Single word oral reading was assessed by the National Adult Reading Test (NART-R) (Nelson and Wilson, 1991), where subjects read 50 phonetically irregular words that increased in difficulty. 2.9.2. Vocabulary Vocabulary was assessed by the British Picture Vocabulary Scale Second Edition (BPVS) (Dunn et al., 1997). Subjects gave the number pertaining to a word spoken by the examiner. There were four numbered pictures presented for each trial (one target and three distracters). Trials increased in difficulty and the test was discontinued if subjects got eight or more items in each trial of 12 items incorrect. 2.9.3. Naming Naming was assessed by Graded Naming (McKenna & Warrington, 1983), where subjects named 30 pictures that increased in difficulty.

2.8. Visuo-spatial and perceptual processing Visuo-spatial and perceptual processing was assessed by the visual object and space perception battery (VOSP) (Warrington & James, 1991), comprising eight sub-tests (the first four testing object perception and the second four testing space perception). 2.8.1. Object perception In incomplete letters, subjects named a series of letters that were degraded so that 70% of the letter was obliterated. In the Silhouettes test, subjects named a series of objects (15 animals and 15 manmade objects), each presented as a silhouette, and rotated through varying degrees from its lateral axis. In object decision, subjects pointed out the silhouette of a real object from three distracter nonsense objects. Finally, in progressive silhouettes, subjects named a silhouette of an object that became, over ten pages, progressively easier to identify. There were two different objects to identify. 2.8.2. Space perception In dot counting, subjects said how many black dots (arranged in random patterns) were present on a series of white

2.9.4. Comprehension Verbal comprehension was assessed by the Token Test (Spreen & Benton, 1977). Twenty tokens in five colours, two sizes, and two shapes were arranged in a fixed order in front of the subject. Subjects then had to follow 39 commands increasing in complexity (e.g. ‘show me a circle’ and ‘together with the yellow circle, pick up the blue circle’). 2.9.5. Pragmatics Finally, pragmatic language was assessed with the test of language competence extended (TLC-E) (Wiig & Secord, 1989), comprising four sub-tests. In ambiguous sentences, subjects gave two interpretations of ambiguous sentences, then selected two pictures from four depicting possible interpretations. In listening comprehension: making inferences, subjects listened to stories and selected two pictures and corresponding statements from four that best depicted plausible inferences about each story. In oral expression: recreating speech acts, subjects placed propositions into a communication like context that would be a likely accompaniment to a picture of an event. Finally, in figurative language subjects interpreted an idiomatic or metaphoric

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expression, then selected one picture from four that best represented the expression. 2.9.6. Statistical analysis Parametric and non-parametric tests were employed depending on whether or not scores were normally distributed. Group differences for self-report questionnaires and neuropsycological tests were investigated using independent t-tests (two tailed) for scores which were normally distributed, or Mann–Whitney U-tests for scores which were not normally distributed. Post-hoc analysis of significant group effects investigated whether deficits were associated with (i) CGG repeat number, (ii) %FMRP(+) lymphocytes, (iii) FMR1 mRNA level, and/or (iv) age. Within-group correlations were performed on genetic variables, which were not normally distributed, for the premutation FraX carrier group only, using Kendall’s tau. Within-group age-related differences, which were normally distributed, were investigated in both premutation carriers and controls, using Pearson’s r correlations. Between-group differences in ageing were then investigated by transforming Pearson’s r into Fisher’s z-score (zobs ), which assessed whether correlations differed significantly (Pallant, 2001). The Fisher’s z-score transformation was only performed where groups comprised 20 subjects and were normally distributed, as these are assumptions of the test (Pallant, 2001). For differences to be significant, zobs must be ≤−1.96 or ≥1.96. For ageing, we only report correlations where there are significant between-group differences; age effects for many of the neuropsyhcological sub-tests would be expected in both subject groups.

3. Results 3.1. Group differences 3.1.1. Self-report questionnaires There were no significant differences between premutation carriers and controls on any of the self-report questionnaires of mood and general health (Table 2). However, there was a trend for carriers to be more anxious than controls, as measured by the Beck’s Anxiety Inventory (t = 1.89, P = 0.07). 3.1.2. Intellectual functioning There were no significant differences between premutation carriers and controls on verbal IQ, performance IQ, or on any of the sub-tests of the WAIS-R tested (Table 3).

Table 2 Psychiatric self-report questionnaires Carriers

Controls

P-value

N = 20 5.40 ± 5.77

N = 20 2.80 ± 2.09

0.07

N = 20 4.60 ± 5.13

N = 20 3.80 ± 3.56

0.57

YBOC Obsessions # Rituals #

N = 20 1.95 ± 3.52 2.10 ± 3.46

N = 17 1.24 ± 2.61 1.12 ± 2.74

0.61 0.20

GHQ #

N = 20 25.80 ± 4.38

N = 17 24.24 ± 6.42

0.60

Beck’s anxiety inventory Beck’s depression inventory

Psychiatric self-report questionnaires: subject numbers (N), mean values and standard deviations for carriers and controls are shown in columns 2 and 3, respectively. The final column shows the P-values resulting from group testing. The symbol # is used where non-parametric statistical tests were performed.

3.2.1. Vocabulary Premutation carriers produced significantly fewer responses than controls in verbal fluency (t = 2.68, P = 0.01). 3.2.2. Mental flexibility The time difference between the Trail Making B and the Trail Making A tests (trail making B–A) was significantly longer in carriers (t = −2.56, P = 0.02). 3.2.3. Problem solving On the Tower of London task, premutation carriers required significantly more moves than controls to solve the task on 5-move problems (t = 2.29, P = 0.03). 3.2.4. Strategy formation There were no significant differences between premutation carriers and controls on any measures of the executive golf task.

Table 3 Intellectual functioning Carriers (N = 20) 113.25 51.15 25.70 20.60 111.15 33.55 27.90

± ± ± ± ± ± ±

15.25 8.80 3.95 3.25 11.58 8.33 6.27

Controls (N = 20) 116.60 52.65 25.25 21.10 111.60 33.60 28.45

± ± ± ± ± ± ±

21.10 12.24 4.38 4.72 18.20 10.21 7.07

P-value

3.2. Executive function

Verbal IQ Vocabulary Comprehension Similarities Performance IQ Block design Object assembly

0.57 0.67 0.74 0.70 0.93 0.99 0.80

There were significant differences between premutation carriers and controls on a number of measures of executive function (Table 4).

Intellectual functioning: subject numbers (N), mean values and standard deviations for carriers and controls are shown in columns 2 and 3, respectively. The final column shows the P-values resulting from group testing.

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Table 4 Executive function Carriers

Controls

P-value

Verbal Verbal fluency Total responses

N = 20 35.95 ± 9.76

N = 20 47.00 ± 15.65

0.01**

Mental flexibility Trail Making Test Trail Making B–A

N = 19 54.35 ± 24.24

N = 18 30.22 ± 14.36

0.02*

Problem solving Tower of London Moves above minimum (5-move problems) Total moves above minimum

N = 19 2.55 ± 2.82 5.18 ± 4.47

N = 20 1.04 ± 0.87 3.28 ± 2.64

0.03* 0.12

N = 17 20.05 ± 12.97

N = 20 18.38 ± 8.48

0.64

N = 19 1.62 ± 2.33 8.51 ± 6.86 29.95 ± 4.13

N = 20 1.03 ± 2.18 7.52 ± 5.79 31.65 ± 4.59

0.11 0.94 0.18

Planning ability score Strategy formation Executive golf task Within search errors # Between search errors Strategy formation

Executive function: subject numbers (N), mean values and standard deviations for carriers and controls are shown in columns 2 and 3, respectively. The final column shows the P-values resulting from group testing. The symbol # is used where non-parametric statistical tests were performed; asterisk (*) marks differences significant at P = 0.05; (**) marks differences significant at P = 0.01.

3.3. Memory 3.3.1. Short-term memory There were no significant group differences for the digit span or visual memory Span sub-tests of the WMS-R. 3.3.2. Long-term memory In the Doors and People Test, overall memory was significantly worse in premutation carriers as compared to controls (t = 2.25, P = 0.03); however, the only significant difference at sub-test level was for the Names test (t = 2.23, P = 0.03) (Table 5). Premutation carriers were significantly worse than controls on verbal memory tasks (t = 2.59, P = 0.01), and there a trend for premutation carriers to be worse at Recognition Memory (t = 1.93, P = 0.06). 3.3.3. Learning In the WMS-R, premutation carriers remembered fewer Verbal Paired Associates than controls, although this was only significant for Immediate Recall (t = 1.99, P = 0.05). Conversely, premutation carriers remembered less Visual Paired Associates on the WMS-R, but this was only significant for Delayed Recall (U= 125.5, P = 0.02).

significant group differences for category fluency for manmade objects. 3.4. Attention For the CPT (Table 6), premutation carriers had significantly higher Hit Reaction Time t-scores than controls (t = 2.9, P = 0.006); they took longer to press the space bar after letter presentations. However, there were no significant group differences in Hit Standard Error Block Change, where t-scores were within the normal range, indicating that reaction times were consistent throughout the test (Conner, 1995). Although premutation carriers took longer to react, this improved their accuracy, as carriers made significantly fewer Commission Errors than controls (t = 2.429, P = 0.02). Indeed, there were no significant differences on the CPT between premutation carriers and controls on Omission errors; Perceptual Sensitivity; or Overall Index Scores (these were within the normal range in both groups). Finally, although there were no significant group differences on Risk Taking, both groups had a mean t-score over 60, indicating that both were cautious, choosing not to respond very often to stimuli (Conner, 1995). 3.5. Visual and Spatial Perception

3.3.4. Prose recall There were no significant group differences for Logical Memory sub-tests of the WMS-R. 3.3.5. Semantic memory Significant differences were observed for category fluency for natural kinds, with premutation carriers providing fewer examples than controls (t = 2.04, P = 0.05). There were no

There were no significant differences between premutation carriers and controls on any of the sub-tests of the VOSP (Table 7). 3.6. Language and Pragmatics There were no significant differences between premutation carriers and controls on any tests of language and

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Table 5 Memory Carriers

Controls

P-value

Short-term memory WMS-R Digit span Visual memory span

N = 20 15.42 ± 3.27 15.60 ± 3.03

N = 20 16.00 ± 4.13 15.25 ± 2.55

0.63 0.70

Long-term memory Doors and people People Names Shapes Doors Overall Verbal Visual Recall Recognition Overall forgetting

N = 20 21.40 ± 15.75 ± 31.60 ± 17.70 ± 8.55 ± 7.40 ± 9.95 ± 9.00 ± 8.85 ± 10.80 ±

6.80 3.63 5.07 3.06 3.30 3.07 2.90 2.68 3.48 3.35

N = 20 23.75 ± 18.30 ± 33.05 ± 18.85 ± 11.00 ± 10.50 ± 11.20 ± 10.70 ± 10.95 ± 10.40 ±

10.68 3.61 4.45 2.98 3.58 4.39 2.76 3.56 3.56 2.46

0.41 0.03* 0.34 0.24 0.03* 0.01** 0.17 0.10 0.06 0.67

N = 20 16.80 ± 6.80 ± 12.30 ± 4.75 ±

4.44 1.32 3.60 1.62

N = 20 19.60 ± 7.40 ± 13.30 ± 5.75 ±

4.48 1.05 4.19 0.64

0.05* 0.09 0.42 0.02*

WMS-R Verbal Verbal Visual Visual

paired paired paired paired

associates associates associates associates

immediate delayed # immediate delayed #

Prose recall WMS-R Logical memory immediate Logical memory delayed

N = 20 20.85 ± 6.34 16.40 ± 6.97

N = 20 22.70 ± 5.40 18.95 ± 7.61

0.36 0.28

Semantic memory Category fluency Natural kinds Manmade objects

N = 20 35.85 ± 7.18 24.35 ± 4.13

N = 20 41.30 ± 9.54 25.85 ± 6.89

0.05* 0.41

Memory: subject numbers (N), mean values and standard deviations for carriers and controls are shown in columns 2 and 3, respectively. The final column shows the P-values resulting from group testing. The symbol # is used where non-parametric statistical tests were performed; asterisk (*) marks group differences significant at P = 0.01; (**) marks group differences significant at P = 0.05.

pragmatics (Table 8). However, there was a trend for carriers to be make more errors than controls on the NART (t = 1.87, P = 0.07).

or FMR1 mRNA level); and there were no significant between group differences in ageing. 4. Discussion

3.6.1. Correlations Deficits were not significantly associated with genetic variables (CGG repeat number, %FMRP(+) lymphocytes,

In this study, we investigated the neuropsychological profile of 20 male carriers of premutation alleles of the FMR1

Table 6 Attention Carriers CPT Hit reaction times (t-scores) Hit SE block change (t-scores) Commission errors (percentiles) Omission errors (percentiles) # Risk taking (t-scores) # Perceptual sensitivity (t-scores) Overall index score #

N = 20 37.60 ± 43.85 ± 26.78 ± 69.99 ± 67.05 ± 45.81 ± 2.32 ±

Controls 8.01 5.66 20.93 22.61 18.96 12.07 4.23

N = 20 44.86 ± 47.48 ± 43.04 ± 67.13 ± 63.56 ± 52.48 ± 3.44 ±

P-value 7.82 8.01 23.82 28.96 19.94 10.77 5.71

0.01** 0.11 0.03* 1.00 0.49 0.07 0.51

Attention: subject numbers (N), mean values and standard deviations for carriers and controls are shown in columns 2 and 3, respectively. The final column shows the P-values resulting from group testing. The symbol # is used where non-parametric statistical tests were performed; asterisk (*) marks differences significant at P = 0.05; (**) marks differences significant at P = 0.01.

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Table 7 Visual and spatial perception Carriers

Controls

P-value

Object perception VOSP Incomplete letters # Silhouettes Object decision # Progressive silhouettes

N = 20 19.55 ± 22.00 ± 18.40 ± 9.65 ±

0.60 4.23 2.41 3.13

N = 20 19.40 ± 23.20 ± 18.70 ± 8.70 ±

0.75 2.41 1.66 2.41

0.59 0.32 0.99 0.29

Space perception VOSP Dot counting # Position discrimination # Number location # Cube analysis #

N = 20 9.90 ± 19.40 ± 9.70 ± 10.20 ±

0.31 2.23 0.80 2.14

N = 20 9.80 ± 19.35 ± 9.75 ± 9.75 ±

0.52 2.25 0.44 0.55

0.60 0.97 0.56 0.77

Visual and spatial perception: subject numbers (N), mean values and standard deviations for carriers and controls are shown in columns 2 and 3, respectively. The final column shows the P-values resulting from group testing. The symbol # is used where non-parametric statistical tests were performed.

gene (recruited on the basis of their genotype and not on their physical or cognitive phenotype) compared to controls who did not differ significantly on age, IQ, or handedness. Although there were significant differences between premutation carriers and controls on two measures of the CPT, this reflected a speed accuracy pay-off (premutation carriers were slower to respond to stimuli, but made fewer Commission Errors). We found no significant group differences for psychopathological or general health symptoms; or in tests of overall intellectual functioning; visual and spatial perception; or language. Thus, our findings are unlikely to be explained simply by differences in overall cognitive ability or general health factors. Male premutation carriers performed significantly worse than controls on some measures of executive function and

memory. Verbal executive function deficits were observed in Verbal Fluency, where premutation carriers produced fewer responses. Mental flexibility deficits were found in the Trail Making Test, which premutation carriers took longer to complete. Problem solving was impaired for Tower of London, where premutation carriers required more moves to solve the task. Long-term memory deficits were observed for the Names sub-test of the Doors and People, Verbal Paired Associates Immediate Recall and Visual Paired Associated Delayed Recall sub-tests of the WMS-R, with premutation carriers recognising fewer items. Semantic memory deficits were observed for the Category Fluency Test for natural kinds, where premutation carriers produced fewer examples. Our findings suggest that premutation carriers of FraX have specific cognitive deficits. However, we performed

Table 8 Language Carriers

Controls

P-value

Oral word reading NART-R Number of errors

N = 20 21.6 ± 7.53

N = 20 16 ± 11.10

0.07

Vocabulary BPVS Raw score

N = 20 154.95 ± 8.02

N = 19 156.68 ± 9.06

0.53

Naming Graded Naming Correct Responses

N = 20 22.65 ± 3.83

N = 20 23.65 ± 3.87

0.42

Comprehension Token test Correct Responses #

N = 20 160.95 ± 0.60

N = 20 161.15 ± 2.68

0.54

Pragmatics Test of Language Competence Ambiguous sentences Listening comprehension: making inferences Oral expression: recreating speech acts Figurative language #

N = 20 47.00 ± 39.25 ± 93.80 ± 49.95 ±

N = 18 48.67 ± 41.11 ± 93.89 ± 48.39 ±

0.17 0.45 0.92 0.80

3.85 8.42 2.33 9.36

3.43 6.30 3.03 2.57

Language: subject numbers (N), mean values and standard deviations for carriers and controls are shown in columns 2 and 3, respectively. The final column shows the P-values resulting from group testing. The symbol # is used where non-parametric statistical tests were performed.

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multiple statistical comparisons. Therefore, some of our results may be due to Type-I errors associated with multiple outcomes. Nonetheless, we do not believe that this can explain all of our findings. We performed a total of 58 neuropsychological comparisons. At a significance level of P = 0.05, we would expect approximately 3 differences to occur by chance, distributed across different cognitive domains. In contrast, we report 9 ‘clustered’ differences; 3 out of 8 executive function measures, and 6 out of 20 memory measures. Thus, the number of group differences we observed was greater than would be expected by chance, was of a specific nature, and restricted to tests of executive function and memory. Unfortunately, we were unable to identify whether there are inter-task correlations of the tests we used; the small sample size rules out more illuminating statistics, such as Discriminant Analysis or Factor Analysis. However, despite these limitations, our findings add weight to the suggestion that premutation expansion of CGG repeats has specific effects on the brain (Murphy et al., 1999; Hagerman et al., 2001; Brunberg et al., 2003; Greco et al., 2002; Moore et al., 2004). For instance, Hagerman et al. (2001) recently reported that 5 older male premutation carriers, who developed an intention tremor and ataxia, had brain atrophy and cognitive decline. Particular features were executive function deficits as measured by the Wisconsin Card Sort Test (in all five carriers), memory loss and eventual dementia (in two carriers). Similar findings were seen by Jacquemont et al. in 26 patients who were diagnosed with FXTAS (Jacquemont et al., 2003). This disorder is associated with white matter disease and brain atrophy that is more extensive than for age-matched controls who do not have the premutation. The molecular underpinnings of these findings include elevated FMR1 mRNA and the formation of ubiquitin-positive intranuclear inclusions in all brains of FXTAS patients that have been examined to date (Greco et al., 2002; unpublished results); similar inclusions are also seen in the premutation mouse model (Willemsen et al., 2003), suggesting that they may develop in all premutation carriers. An important issue is whether subtle cognitive changes occur prior to the onset of the neurological problems, and our current data suggest that this is the case. The cause of the neuropsychological differences that we report here, in clinically normal premutation carriers, are unknown, but may relate to neurobiological differences. We have reported (Moore et al., 2004) that premutation carriers, recruited on the basis of their genetic phenotype, have significant reductions in prefrontal white matter tracts, and the grey and white matter volumes of cerebellum, amygdalo-hippocampal complex, and inferior temporal cortices. Prefrontal and cerebellar regions are implicated in executive function. For example, a recent study using diffusion tensor MRI, found that prefrontal white matter mean diffusivity was correlated with executive function, as measured by the Trail Making Test (O’Sullivan et al., 2003). In the general population, acquired brain damage in dorsolateral prefrontal cortex, basal ganglia, and cere-

bellum is associated with deficits in executive function, planning and decision making (Shallice, 1988). Likewise, the amygdalo-hippocampal complex and inferior temporal cortices are implicated in memory (Shallice, 1988). For example, functional imaging studies have reported that inferior temporal cortices process semantic information, including natural kinds of objects (Devlin et al., 2002). This study demonstrates that premutation carriers have deficits in executive function and memory; this is consistent with the neuroanatomical differences that we have previously reported (Moore et al., 2004). Neuropsychological deficits other than executive function and memory deficits found in this study have been reported by Loesch et al. (1994). They investigated 10 male premutation carriers considered ‘normal’ by family and employers, and compared them to their non-FraX male relatives of corresponding age. They reported that male premutation carriers had significantly lower scores on a Block Design Test and the Peabody Picture Vocabulary Test. However, subjects were not matched on IQ. In our study the two groups did not differ in age and IQ. We found no significant differences between the Block Design sub-test of the WAIS-R or the BPVS, tests analogous to those employed by Loesch et al. (1994). Similarly, Myers et al. (2001) (who investigated intellectual functioning, academic achievement and visual motor integration, but not executive function or memory) reported no generalised psychological deficit in premutation FraX for seven boys compared to age-matched controls. There have been reports of increased rates of psychopathology in male premutation carriers. For instance, Loesch et al. (1984) reported ‘emotional instability’ (including nervous breakdowns, nervousness, and war neurosis); and Dorn et al. (1994) reported Obsessive Compulsive Disorder-like behaviours, and alcohol abuse/dependence. However, Loesch et al. (1984) only investigated four subjects who were not compared to controls, and Dorn et al. (1994) relied on retrospective observations from the daughters of premutation carriers. In contrast, using self-report measures we found no significant differences on mental health variables. Therefore, there is, to date, little convincing evidence for increased rates of psychopathology in male premutation carriers. Our findings suggest that executive function and memory deficits may comprise an emerging neuropsychological phenotype of premutation expansion of CGG trinucleotide repeats. These deficits are not restricted to a sub-group of carriers with FXTAS (Hagerman et al., 2001; Jacquemont et al., 2003), or to those with a high number CGG trinucleotide repeats (Johnston et al., 2001). This is consistent with neuroantonical deficits that we have reported for premutation carriers of FraX recruited on the basis of their genetic phenotype (Moore et al., 2004). The cause of the subtle cognitive differences that we have demonstrated may be related to a hypothesized “toxic RNA gain-of-function” effect, leading to the gradual accumulation of CNS inclusions (Greco et al., 2002; Hagerman et al., 2001; Jacquemont

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et al., 2003). Thus, the current molecular model proposing that neuropsychological abnormalities in FraX result from significant loss of FMRP production requires revision. This has relevance to the general population because approximately 1:813 men (Dombrowski et al., 2002) and 1:100–260 women (Rousseau et al., 1996; see Hagerman & Hagerman, 2002) are premutation carriers with >54 CGG trinucleotide repeats. Further studies are required investigating the biological basis of our findings, for example by relating executive function to white matter tract abnormalities using diffusion tensor MRI, and/or measuring the genetic effects on brain function directly using functional imaging.

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