FMRP expression as a potential prognostic indicator in fragile X syndrome

FMRP Expression as a Potential Prognostic Indicator in Fragile X Syndrome Flora Tassone,1,2 Randi J. Hagerman,2,3 David N. Ikle´,4 Pamela N. Dyer,1 Megan Lampe,2 Rob Willemsen,5 Ben A. Oostra,5 and Annette K. Taylor1 1Kimball Genetics Inc., Denver, Colorado 2Child Development Unit, The Children’s Hospital, Denver, Colorado 3Department of Pediatrics, University of Colorado Health Sciences Center, Denver, Colorado 4Division of Biostatistics, National Jewish Medical Research Center, Denver, Colorado 5Department of Clinical Genetics, Erasmus University, Rotterdam, The Netherlands Absence or deficit of FMR1 protein (FMRP) resulting from methylation of full mutation genes is the fundamental defect in fragile X syndrome. We used FMRP immunocyto- chemistry and detailed phenotypic assess- ment to investigate the relationship be- tween degree of FMRP expression and the broad clinical spectrum of impairment in 80 individuals affected with fragile X syn- drome. FMRP expression correlated with IQ in mosaic males (P=0.043), males with a par- tially methylated full mutation (P=0.0005), and females with a full mutation (P=0.046). In the females, FMRP expression also corre- lated with the number of fragile X physical features (P=0.0003). Even modest deficits in FMRP result in some manifestations of frag- ile X syndrome. In this initial study of 53 males, FMRP expression testing had a very high positive predictive value (100%, confi- dence interval of 29-100%) for a nonretarded IQ among males with expression of FMRP in ‡ 50% of lymphocytes (3 males), suggesting that FMRP expression may have potential as a prognostic indicator in males with frag- ile X syndrome. Am. J. Med. Genet. 84:250– 261, 1999. © 1999 Wiley-Liss, Inc. KEY WORDS: fragile X syndrome; FMR1 mutation; FMRP; mosaic; partial methylation; activa- tion ratio; immunocytochem- istry; clinical correlation INTRODUCTION A wide spectrum of clinical involvement is seen in both males and females with fragile X syndrome. Al- though the disorder is well recognized as a cause of mental retardation, it can also manifest as learning disabilities and a variety of characteristic physical fea- tures and behavioral problems in nonretarded indi- viduals [Hagerman, 1996]. Approximately 13% of males with fragile X syndrome are “high functioning” with an IQ of 70 or above [Hagerman et al., 1994]. Most high-functioning males have a partially methylated full mutation or are mosaic for an FMR1 full mutation and premutation. This finding and other studies indi- cate that variation in the degree of clinical impairment is related in part to variation in the characteristics of the FMR1 mutation responsible for fragile X syndrome [McConkie-Rosell et al., 1993; Loesch et al., 1993a, 1993b; Abrams et al., 1994; Merenstein et al., 1996; Steyaert et al., 1996; de Vries et al., 1996a]. It is the absence or deficit of the FMR1 protein (FMRP) that leads to fragile X syndrome. Early studies by Western blot analysis of lymphoblastoid cell lines demonstrated absence of FMRP in males with a full mutation [Verheij et al., 1993; Siomi et al., 1993; Devys et al., 1993] and a normal level of FMRP in males with a premutation [Devys et al., 1993; Feng at al., 1995a]. FMRP is an RNA-binding protein [Siomi et al., 1993, 1994; Ashley et al., 1993; Verheij et al., 1995], binding to the 60S ribosomal subunit [Khandijan et al., 1996; Siomi et al., 1996] with association via RNA [Tamanini et al., 1996; Eberhart et al., 1996]. FMRP is found pre- dominantly in the cytoplasm [Devys et al., 1993; Ver- heij et al., 1993] but it is also present in the nucleolus [Willemsen et al., 1996a]. Although the function of FMRP is unclear, it has been suggested that its ab- sence may disrupt the translation of numerous other proteins and so lead to the pleiotropic phenotype of fragile X syndrome. A role for FMRP in synaptic matu- ration has also been suggested [Comery et al., 1997; Weiler et al., 1997]. Contract grant sponsor: the March of Dimes; Contract grant number: 12-FY96-0492; Contract grant sponsor: the Children’s Hospital Research Institute; Contract grant sponsor: the Ketter- ing Family Foundation; Contract grant sponsor: Maternal Child Health; Contract grant number: MCJ-089413. *Correspondence to Annette K. Taylor, M.S., Ph.D., Kimball Genetics Inc., 101 University Blvd., Suite 350, Denver, CO 80206. E-mail: [email protected] Received 10 June 1998; Accepted 5 November 1998 American Journal of Medical Genetics 84:250–261 (1999) © 1999 Wiley-Liss, Inc. In individuals with a full mutation, presence of vari- able numbers of cells with an unmethylated FMR1 gene allows for FMRP expression of variable degrees. In males, this occurs if some cells have either a premu- tation (as seen in mosaic males) or an unmethylated full mutation (as seen in males with a partially meth- ylated full mutation). In females with a full mutation, the normal FMR1 allele allows for FMRP expression in cells with the normal gene on the active X chromosome. Substantial information has been gathered on DNA measures relating to the FMR1 mutation and how they correlate with the degree of phenotypic involvement in these categories of fragile X subjects. In males with partial methylation of a full mutation, the degree of methylation influences the fragile X phe- notype. There have been several reports of apparently normal or mildly affected, nonretarded males with a complete or predominant lack of methylation of a full mutation [McConkie-Rosell et al., 1993; Loesch et al., 1993a; Merenstein et al., 1994; Hagerman et al., 1994; Rousseau et al., 1994b; Feng et al., 1995b; Smeets et al., 1995; Lachiewicz et al., 1996, Steyaert et al., 1996; Wang et al., 1996; Wo¨hrle et al., 1998]. In a study of 20 males (nine of which were also used for this study) with partially methylated full mutations, we found that all individuals with 7 cm), high arched palate, hyperex- tensible finger joints (metacarpal phalangeal joint ex- tension >90 degree), double-jointed thumbs, hand cal- luses, single palmar crease, flat feet, and heart mur- mur. Each feature observed is assigned 1 point, and the FMRP/Clinical Correlations in Fragile X Syndrome 251 number of features per subject is calculated (maximum of 10 points). Several cognitive measures were used because of the wide age range of the study subjects. One subject was given the Leiter International Performance Scale, 19 subjects were given the Wechsler Intelligence Scale for Children WISC-R or WISC-III, 19 were given the Wechsler Adult Intelligence Scale-Revised (WAIS-R), 14 were given the Kaufman Assessment Battery for Children (K-ABC), and five were given the Stanford- Binet. When a standardized IQ test could not be ob- tained because of behavioral interference or lack of co- operation, the Vineland Assessment Battery was given to subjects (n 4 11), and the overall Adaptive Behavior Composite was used instead of an IQ test. For subjects under age 3 years (n 4 6), the Bayley Scales of Infant Development was given and the Mental Developmental Index (MDI) was used for the cognitive equivalent score. The cognitive scores are summarized in Table I. We do not have an IQ for five of the subjects because they were either too young or did not cooperate with testing. For the behavioral assessment, 10 behaviors found to be typical in subjects affected by fragile X syndrome were assessed to gain an overall Behavior Index (BI) score [Hagerman, 1996]. These behaviors include hand flapping, hand biting, poor eye contact, tactile defen- siveness, preservation of language or behavior, hyper- activity, attention problems consistent with Attention Deficit/Hyperactivity Disorder (ADHD), violent out- bursts, tantrums, and shyness or social anxiety. A score of 1 is given for each behavior present, and the maximum score is 10 (Table I). The parent or caregiver reported the behaviors. In addition, because symptoms associated with ADHD are very common in both males and females affected by fragile X syndrome [Hager- man, 1996], we have also used a previously published ADHD Checklist [Barkley, 1990] that has been useful for assessing ADHD behaviors. This checklist includes the DSM-IV Diagnostic Criteria for ADHD and has 18 items scored by the parent, caretaker, or the adult sub- ject. There is a four-point scale to describe the severity of each behavior on the checklist. FMR1 DNA Studies DNA studies were performed at Kimball Genetics, Inc. (Denver, CO). Genomic DNA was isolated from 5 ml of peripheral blood samples using standard meth- odology (Puregene kit, Gentra Systems, Inc., Minne- apolis, MN). Both Southern blot and polymerase chain reaction (PCR) analysis were performed on each sample as detailed in Taylor et al. [1994a]. For South- ern blot analysis, 5 mg DNA was digested with EcoR I and Nru I followed by electrophoresis in a 1% agarose/ Tris acetate gel and transferred to nylon membrane. The blots were hybridized with the FMR1-specific probe, StB12.3 [Oberle´ et al., 1991]. The CGG repeat number of full mutations (and premutations with more than approximately 180 repeats) was calculated from the Southern blot autoradiogram images. PCR analysis was performed using primers 1 and 3 described by Brown et al. [1993]. PCR products were separated by 6% denaturing polyacrylamide gel electrophoresis, transferred to nylon membrane, and hybridized with oligonucleotide probe (CGG)5. The CGG repeat number of premutations with less than approximately 180 re- peats was calculated from the PCR autoradiograms. Table II summarizes the molecular measures used in this study and how they were calculated. A phospho- rimager (Molecular Dynamics, Inc., Sunnyvale, CA) was used to accurately quantitate the radioactive in- tensity of chosen Southern blot bands. This data was used to calculate the percent premutation (for mosaic males with a full mutation and premutation), the per- cent lack of methylation (for males with a partially methylated full mutation), and the activation ratio (for females with a full mutation) as shown in the table. Immunocytochemistry For interlab validation, immunocytochemistry on 15 samples from the study was performed at both Eras- mus University and Kimball Genetics, after which analysis on all additional samples were performed at Kimball Genetics. Blood smears were made using 20 ml of peripheral blood on each microscope slide. Several smears were made for each subject. The maximum in- terval between the blood draw and making the smear was 24 hr. Slides were air dried for 15 min, wrapped in cellophane, and stored either at room temperature (for up to 3 weeks) or at 70°C (for up to 3 months). For FMRP immunostaining, an indirect alkaline phosphatase technique was used according to Willem- sen et al. [1995]. Slides were mounted in a disposable coverplate system (Shandon, Inc., Pittsburgh, PA). Cells in the blood smears were fixed with 3% paraform- aldehyde in phosphate buffer (pH 7.4) and permeabi- TABLE I. Clinical Measures (Means/Range) for the Four Categories of Subjects* Subject categories Clinical measures (means/range) Age IQ PIQ VIQ PI BI ADHD Mosaic males (n 4 12) 17.4 60.3 61.0 64.3 4.0 6.0 25.1 (3.8–60) (20–103) (48–75) (56–71) (0–7) (3–9) (7–39) Males with a partially methylated 13.5 66.2 64.5 61.9 4.0 7.0 24.6 full mutation (n 4 13) (4.8–37) (40–106) (46–84) (46–89) (2–6) (4–10) (5–33) Males with a fully methylated 12.8 51.5 na na 4.5 6.7 26.3 full mutation (n 4 36) (1.1–48.8) (36–70) (2–8) (0–10) (11–46) Females with a full mutation 18.7 78.9 85.6 82.3 2.61 4.2 18.1 (n 4 19) (2–43) (48–93) (50–99) (52–94) (0–4) (2–9) (5–39) *The mean age in years and the age range (in parentheses) in each category is indicated. na, not applicable. 252 Tassone et al. lized with methanol. Immunoincubation was accom- plished in three steps. The first incubation was for 16 hr at 4°C with mouse FMRP-specific monoclonal anti- body from hybridoma clone 1C3-1a [Devys et al., 1993]. Antibodies were prepared in a Tecno-mouse system [Willemsen et al., 1995, 1997b; Oostra, 1996]. The next incubation lasted for 60 min at room temperature with goat anti-mouse immunoglobulin conjugated with bio- tin (Dako Corporation, Carpinteria, CA). The final in- cubation was for 45 min at room temperature with streptavidin-biotinylated alkaline phosphatase (Dako). Visualization of FMRP was accomplished by exposing the slides twice for 25 min each in a fuchsin substrate chromagen system (Dako) at room temperature. Nuclei were counterstained blue with Gill’s hemotoxylin. The cytoplasm appears red for FMRP-positive lymphocytes and colorless for FMRP-negative lymphocytes. Slides were analyzed under the microscope, distin- guishing lymphocytes from other blood cell types by morphology. Granulocytes stain nonspecifically [Wil- lemsen et al., 1995] and therefore only lymphocytes are counted. For each slide, 200 lymphocytes were scored, and the percent of lymphocytes expressing FMRP was determined. Scoring was performed in blinded fashion with respect to DNA results. Statistical Analysis This study has a cross-sectional, parallel categories design with four categories. Measures include FMRP expression (percentage of FMRP-positive lympho- cytes), FMR1 mutation DNA parameters relevant to each subject category, and clinical measures of cogni- tive ability and physical and behavioral features. The objectives were to assess differences between catego- ries, relationships between variables within categories, and the predictive value of FMRP expression with re- spect to IQ. Because it was not possible to evaluate all subjects using the same IQ instrument, results are re- ported on analyses using standard scale scores from the different IQ test types. As all these test types use the same standardized mean of 100, the Stanford Binet has a standard deviation of 16 and the remaining tests have a standard deviation of 15. To check whether this difference in standardized scale scores significantly af- fected results, all analyses involving IQ tests were per- formed both on the standardized scale scores and on z-scores computed using the mean and the standard deviation of the test the subject actually performed. Use of z-scores eliminates any potential effect because of the slightly different standard deviations of the tests. Comparisons of continuous measures between categories were made by one-way analysis of variance with multiple comparisons by the Tukey-Kramer pro- cedure. Because for FMRP expression the variances within categories were quite different, an analysis of variance on the ranks was also performed. We discov- ered that the results from analyses using standard IQ scores and z-scores were essentially the same, so we have reported only the results from the analyses with IQ scores. Associations between pairs of variables were inves- tigated using simple linear regression techniques and results are summarized using the square of the corre- lation coefficient. Relationships between more than two variables were assessed using a stepwise multiple regression procedure with FMRP as the dependent variable. The association between high and low FMRP expression (above and below 50% FMRP-positive lym- phocytes, respectively) was determined using 2 × 2 con- tingency tables and the Fisher exact test for comparing independent proportions. The predictive value of the FMRP expression test with respect to IQ was described by the positive and negative predictive values of FMRP expression with 95% confident limits. All tests of hy- potheses were two-tailed at the 0.05 level of signifi- cance, and individual P values are reported. All analy- ses were performed using JMP version 3.2 software (SAS Institute, Inc.) on a PowerPC computer. RESULTS Reproducibility of the Immunocytochemical FMRP Expression Test The FMR1-specific monoclonal antibody and the im- munocytochemistry protocol on blood smears used in TABLE II. Molecular Measures Used in This Study* Measure Molecular measures used Subject category Technique Calculation Percent FMRP-positive lymphocytes All Immunocytochemistry [ of FMRP-positive lymphocytes 200 lymphocytes · 100 Percent premutation Mosaic males Southern blot and phosphorimaging Pre Pre + Full · 100 Percent lack of methylation Males with a partially methylated full mutation Southern blot and phosphorimaging Unmeth full Unmeth + Meth full · 100 Activation ratio Females with a full mutation Southern blot and phosphorimaging Unmeth normal Unmeth normal + Meth normal *The category of subjects for which each measure is relevant is indicated. The technique used to gain data relevant for each measure is also shown as well as the method of calculation for each measure. For the percent of premutation, percent lack of methylation, and activation ratio, the raw data used in the calculation was the radioactive intensity (quantified by phosphorimaging) of the appropriate bands on Southern blots. The fractions show which bands are used for each molecular measure. Abbreviations: pre, premutation; full, full mutation; unmeth, unmethylated; meth, methylated; normal, normal FMR1 band. FMRP/Clinical Correlations in Fragile X Syndrome 253 our studies were the same as described by Willemsen et al. [1995] except that we routinely examined 200 rather than 100 lymphocytes for each individual. Three categories of control samples (normal, premutation, and FMR1 deletion) were used to verify test perfor- mance at Kimball Genetics. The percent of FMRP- positive lymphocytes in 12 individuals without an FMR1 mutation (three males and nine females) ranged from 84 to 97% (mean of 90.2%) and in seven premu- tation carriers (two males and five females) ranged from 84 to 100% (mean of 91.5%). These results are in keeping with those from Willemsen et al. [1997a]. In six independent experiments, we detected no FMRP- positive lymphocytes in blood from a fragile X-affected male with a large FMR1 deletion (deletion described previously by Yanghong et al. [1994]). This latter ob- servation indicates that the false positive rate for de- tection of FMRP-expressing lymphocytes is very low. Reproducibility studies were performed on three sub- jects (a mosaic male, a male with a partially methylat- ed full mutation, and a normal female control). Two separate immunocytochemistry runs were performed for each subject with a total of nine slides per subject. For each subject, variation of results was small be- tween slides run at the same time and between differ- ent experiments. Overall for all slides scored, the stan- dard deviation was 1.7% for the mosaic male (mean of 8.2% FMRP-positive lymphocytes, range 5-11%), 2.7% for the male with a partially methylated full mutation (mean of 26.1% FMRP-positive lymphocytes, range 22- 30%), and 2.5% for the normal female (mean of 87.9% FMRP-positive lymphocytes, range 84-92%). Therefore, results were shown to be reproducible, which is essen- tial for comparing differences in FMRP expression among the fragile X affected individuals in our study. FMR1 DNA Analysis Direct DNA analysis was performed on 80 individu- als with fragile X syndrome in addition to the controls mentioned above. DNA measures (determined by phos- phorimaging of Southern blot signals as described in Table II) relevant to each category of subjects are sum- marized in Table III. CGG repeat numbers were also determined (data not shown). For mosaic males, the premutation was the predominant mutation in only two cases (percent premutation of 80 and 83.6%, re- spectively), whereas the other 10 mosaics had percent premutation values of (PIQ), verbal IQ (VIQ), PI, BI, and ADHD) versus the relevant FMR1 DNA measure in mosaic males, males with a partially methylated full mutation, and females with a full mutation. Results are shown in Table V. Results reported in Table IV and V involving IQ tests are based on analyses of the standardized scale scores for those tests. These results were not different when the analyses were repeated using the z-scores from the respective tests. Full scale IQ correlated significantly with each FMR1 DNA measure: the percent premutation in mo- saic males (P40.036); the percent lack of methylation in males with a partially methylated full mutation (P40.032); and the activation ratio in females with a full mutation (P40.02). Performance IQ (P40.047) but not verbal IQ showed a marginal significance with ac- tivation ratio in females with a full mutation. In this category, a strong negative correlation was also seen between the PI and the activation ratio (P40.005). A significant negative correlation was found between the BI and the percent premutation in mosaic males (P40.03). ADHD score did not correlate with any of the FMR1 DNA measures. Phenotypic Variables Versus FMRP Expression Results of simple linear regression analysis to iden- tify phenotypic characteristics that correlate signifi- cantly with FMRP expression are shown in Table IV. In males with a fully methylated full mutation, no signifi- cant correlations were found for FMRP expression with any variables, as expected given the low level of FMRP expression. Results for this category were not included in the table. A highly significant correlation was found between full scale IQ and FMRP expression in males with a partially methylated full mutation (P40.0005). A mar- ginally significant correlation was found for these mea- sures in mosaic males (P40.043) and females with a full mutation (P40.046). For performance IQ, a posi- tive correlation with FMRP expression was evident in males with a partially methylated full mutation (P40.02) and a weak correlation was seen in females with a full mutation (P40.045). In addition, verbal IQ correlated with FMRP expression in males with a par- tially methylated full mutation (P40.045). Therefore, in males with a partially methylated full mutation, FMRP expression correlated with performance IQ and verbal IQ, whereas the DNA measure (percent lack of methylation) did not. Considering full scale IQ vari- ance, FMRP expression explains much of this variance in males with a partially methylated full mutation (68%) but less in mosaic males (38%) and females with a full mutation (24%) as shown by the r2 values in Table IV. In the group of males with a partially meth- ylated full mutation, one observation (an individual with 67% FMRP-positive lymphocytes and a full scale IQ of 106) was a strongly influential point (Fig. 2B). Without this individual, the positive correlation be- tween FMRP and IQ in this group maintained mar- ginal significance (P40.04). A highly significant negative correlation was found between the PI and FMRP expression in females with Fig. 1. Diamond plot of FMRP expression (percent FMRP-positive lymphocytes) in each category of subjects determined by one way analysis of variance. The upper and lower vertices of the diamonds define the 95% confidence limits about the mean. The horizontal line across the diagram represents the mean for all subjects combined. The categories are as follows: A, mosaic males; B, males with a partially methylated full mutation; C, males with a fully methylated full mutation; D, females with a full mutation. FMRP/Clinical Correlations in Fragile X Syndrome 255 a full mutation (P40.0003) but not in the other catego- ries. A marginal negative correlation between the BI and FMRP expression was observed in mosaic males (P40.048) but not in other categories. No significant correlations were found between the ADHD score and FMRP expression in any of the subject categories. Stepwise multiple regression analysis was used to determine if any of the simple linear relationships de- scribed in Table IV could be improved by considering more than one independent variable to explain FMRP variation. A stepwise variable selection procedure was used with a significance level of 0.05 as the criterion for inclusion or exclusion of variables at each step. In ev- ery case, the most significant variable identified in each category as explaining most of the variation in FMRP was the only variable that remained in the re- gression model (IQ for the mosaic males and males with a partially methylated full mutation and PI for females with a full mutation). Predictive Value of the FMRP Expression Test To explore the value of the FMRP expression test for predicting presence or absence of mental retardation from FMRP expression values, a two-by-two contin- gency table was used (Table VI). We used a cutoff of 70 for IQ to define presence and absence of mental retar- dation and explored a cut-off of 50% FMRP-positive lymphocytes. In a pooled category of all the males stud- ied, an FMRP expression test with this cutoff had a positive predictive value (prevalence of nonretarded IQ with high ($50%) FMRP expression) of 100% and a Fig. 2. (A) FMR1 DNA measures versus FMRP expression (percent FMRP-positive lymphocytes). In the graph on the left, the (`) symbols and bottom curve represent the percent premutation (% pre) versus the percent FMRP-positive lymphocytes in mosaic males (P#0.0001), and the (+) symbols and top curve represent the percent lack of methylation versus the percent FMRP-positive lymphocytes in males with a partially methylated full mutation (P40.0001). In the graph on the right, the curve represents the activation ratio versus the percent FMRP-positive lymphocytes in females with a full mutation (P#0.0001). (B) Full scale IQ versus percent FMRP-positive lymphocytes. In the graph on the left, the (`) symbols and bottom curve are for mosaic males (P40.043), and the (+) symbols and top curve are for males with a partially methylated full mutation (P40.0005). In the graph on the right, the curve is for females with a full mutation (P40.046). 256 Tassone et al. negative predictive value (prevalence of IQ in the re- tarded range with low ( derstanding of the nonretarded end of the clinical spec- trum and identifying possible predictors of this higher level of functioning in this disorder [Loesch et al., 1993a; McConkie-Rosell et al., 1993; Hagerman et al., 1994; Smeets et al., 1995; Lachiewicz et al., 1996; Stey- aert et al., 1996; de Vries et al., 1996a; Wo¨hrle et al., 1998]. Our studies have revealed that FMRP expression correlates strongly with the relevant DNA-related measure in mosaic males, males with a partially meth- ylated full mutation, and females with a full mutation (all P values were 0.0001 or less). FMRP expression also correlated significantly with IQ in these catego- ries. This correlation was the strongest in males with a partially methylated full mutation (P40.0005). In this group, FMRP expression correlated with IQ much more strongly than did the DNA-related measure (percent lack of methylation). This may be due to translation inhibition of transcripts with a high CGG repeat num- ber, which could render some cells with an unmethyl- ated FMR1 mutation incapable of producing FMRP [Feng et al., 1995b]. The repeat number range exerting this effect is not yet clearly defined. In fibroblast cell lines, Feng et al., [1995b] observed strong translational inhibition of transcripts with 285 CGG repeats or greater. In contrast, we have detected substantial FMRP expression (38.5%) in a male with a partially methylated full mutation (81% lack of methylation) and CGG repeat number of the unmethylated mutation ranging from 285-430. Similarly, De Vries et al. [1996a] found 75% FMRP-positive lymphocytes in a male with a 90% unmethylated full mutation and CGG repeat number ranging from 200-800. An interesting finding was that FMRP accounted for 68% of the variance in IQ in males with a partially methylated full mutation, 38% in mosaic males, but only 24% in females. In the females, the activation ra- tio accounted for 31% of IQ variance, which compares closely with 33% reported by Reiss et al. [1995] in an- other group of females with a full mutation. In females, possible differences in the activation ratio and percent FMRP-positive cells in the brain compared with the blood may explain why the correlation is only marginal between FMRP and IQ. It has been shown in female mice that the X inactivation ratio of a given gene (in this case, a transgenic X-linked marker) differs dra- matically in different clonally derived regions of the cortex [Tan et al., 1995]. Binstock [1995] hypothesized that such differences in the FMR1 activation ratio in the cortex of females with fragile X syndrome could explain some of the clinical/molecular observations. All fragile X male and female studies relying on measures in the blood to correlate with cognitive abilities are limited by the possibility that findings in the blood may not always be reflective of the situation in the brain. In males, several cases showing lack of significant FMR1 mutation variability between tissues have been re- ported [Sutherland et al., 1991; Devys et al., 1992; Wo¨hrle et al., 1992, 1993; de Graaff et al., 1995; Tas- sone et al., 1999], but significant differences between tissues have been found in some cases [Merenstein et al., 1994; Taylor et al., 1994a; Dobkin et al., 1996; Maddalena et al., 1996; Taylor et al., 1999]. Considering subcategories of IQ, FMRP correlated significantly with performance IQ and verbal IQ in males with a partially methylated full mutation and with performance IQ but not verbal IQ in females with a full mutation. This is consistent with a previous re- port by de Vries et al. [1996b], which suggested that the impact of fragile X syndrome in women was greater on performance IQ than verbal IQ. In our female group, the FMR1 activation ratio and FMRP expression were similar in their relationships with these IQ subcatego- ries. A striking discovery was that the physical index is strongly influenced by FMRP expression in females. This is not the case in males, suggesting that there may be a fundamental difference in the impact of fragile X syndrome on males and females. In males a deficit in FMRP most strongly affects IQ, whereas physical fea- tures in females are influenced to a much greater de- gree than cognitive ability. A weakly significant corre- lation was seen between FMRP and the behavioral in- dex in mosaic males. Improved measures of behavioral involvement in fragile X syndrome and more detailed anthropometric measures of physical involvement could lead to stronger correlations with FMRP expres- sion. It would be of interest to explore whether a com- bination of age, DNA measures, and FMRP data would correlate with phenotypic measures more strongly than any one parameter alone. For all phenotypic mea- sures background genetic effects should be taken into account through careful family studies to clarify the effect of the FMR1 mutation alone. The finding that 67% of males studied here with a fully methylated full mutation have limited but detect- able FMRP expression (in and have FMRP expression (70, 67, 60, and 47%) in greater than 42% of lymphocytes. Learning disabilities, emotional problems, and some physical features typical of fragile X syndrome are present in these high func- tioning males although IQ is in the normal range. DNA and clinical data from three of these males (with $50% FMRP-positive lymphocytes) are summarized in Table VII. Similarly, de Vries et al. [1996a] reported facial features of fragile X syndrome in a male with normal IQ and 75% FMRP-positive lymphocytes. It is apparent that even small deficits of FMRP result in some mani- festations of fragile X syndrome. The cut-off of 42% suggested for screening may be appropriate for detect- ing mentally retarded males with fragile X syndrome but would miss mild cases of fragile X syndrome. We have explored a cut-off of 50% FMRP-positive lymphocytes for predicting presence or absence of men- tal retardation in males with fragile X syndrome. We caution that this is an initial study with few individu- als in the FMRP expression range of 30-70% and just three with $50% FMRP-positive lymphocytes. In this small study, the positive predictive value of this test (to predict a nonretarded IQ in males with $50% FMRP- positive lymphocytes) and the negative predictive value (to predict mental retardation in males with sured background gene effects, and most significantly by small sample size in three of the four subject cat- egories. Among the males with a partially methylated full mutation, one individual (with 67% FMRP expres- sion) was strongly influential in analyses by linear re- gression. However, this does not mean that he is un- representative of males with this high percent of FMRP-positive lymphocytes. We emphasize that our results are preliminary and that larger studies are nec- essary to confirm our findings. Another important point is that the immunocytochemistry approach used here measures the percent of lymphocytes expressing FMRP but not the amount of FMRP produced per cell. It is not yet known which measure is the more clini- cally relevant. Direct measurement of the amount of FMRP in blood (e.g., by ELISA) has not yet been dem- onstrated. Our results help to define the role of FMRP in the clinical spectrum of fragile X syndrome and illustrate that mild deficits of FMRP are associated with behav- ioral and physical problems without mental retarda- tion. This preliminary work suggests that the percent of FMRP-expressing lymphocytes potentially may be a useful predictor of long-term nonretarded IQ in males. From these early data, FMRP expression in 50% of lymphocytes or more suggests a high functioning sta- tus. 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