FMR1 allele size distribution in 35,000 males and females: a comparison of developmental delay and general population cohorts.

PURPOSE: Developmental delay phenotypes have been associated with FMR1 premutation (PM: 55-200 CGG repeats) and "gray zone" (GZ: 45-54 CGG repeats) alleles. However, these associations have not been confirmed by larger studies to be useful in pediatric diagnostic or screening settings. METHODS: This study determined the prevalence of PM and GZ alleles in two independent cohorts of 19,076 pediatric referrals to developmental delay diagnostic testing through Victorian Clinical Genetics Service (cohort 1: N = 10,235; cohort 2: N = 8841), compared with two independent general population cohorts (newborn screening N = 1997; carrier screening by the Victorian Clinical Genetics Service prepair program N = 14,249). RESULTS: PM and GZ prevalence rates were not significantly increased (p > 0.05) in either developmental delay cohort (male PM: 0.12-0.22%; female PM: 0.26-0.33%; male GZ: 0.68-0.69%; female GZ: 1.59-2.13-%) compared with general population cohorts (male PM: 0.20%; female PM: 0.27-0.82%; male GZ: 0.79%; female GZ: 1.43-2.51%). Furthermore, CGG size distributions were comparable across datasets, with each having a modal value of 29 or 30 and ~1/3 females and ~1/5 males having at least one allele with ≤26 CGG repeats. CONCLUSION: These data do not support the causative link between PM and GZ expansions and developmental-delay phenotypes in pediatric settings.

ß-glucuronidase use as a single internal control gene may confound analysis in FMR1 mRNA toxicity studies.

Relationships between Fragile X Mental Retardation 1 (FMR1) mRNA levels in blood and intragenic FMR1 CGG triplet expansions support the pathogenic role of RNA gain of function toxicity in premutation (PM: 55-199 CGGs) related disorders. Real-time PCR (RT-PCR) studies reporting these findings normalised FMR1 mRNA level to a single internal control gene called ß-glucuronidase (GUS). This study evaluated FMR1 mRNA-CGG correlations in 33 PM and 33 age- and IQ-matched control females using three normalisation strategies in peripheral blood mononuclear cells (PBMCs): (i) GUS as a single internal control; (ii) the mean of GUS, Eukaryotic Translation Initiation Factor 4A2 (EIF4A2) and succinate dehydrogenase complex flavoprotein subunit A (SDHA); and (iii) the mean of EIF4A2 and SDHA (with no contribution from GUS). GUS mRNA levels normalised to the mean of EIF4A2 and SDHA mRNA levels and EIF4A2/SDHA ratio were also evaluated. FMR1mRNA level normalised to the mean of EIF4A2 and SDHA mRNA levels, with no contribution from GUS, showed the most significant correlation with CGG size and the greatest difference between PM and control groups (p = 10-11). Only 15% of FMR1 mRNA PM results exceeded the maximum control value when normalised to GUS, compared with over 42% when normalised to the mean of EIF4A2 and SDHA mRNA levels. Neither GUS mRNA level normalised to the mean RNA levels of EIF4A2 and SDHA, nor to the EIF4A2/SDHA ratio were correlated with CGG size. However, greater variability in GUS mRNA levels were observed for both PM and control females across the full range of CGG repeat as compared to the EIF4A2/SDHA ratio. In conclusion, normalisation with multiple control genes, excluding GUS, can improve assessment of the biological significance of FMR1 mRNA-CGG size relationships.

Cerebellar volume mediates the relationship between FMR1 mRNA levels and voluntary step initiation in males with the premutation.

Recent evidence indicates that adults with a premutation (PM: 55-199 CGG repeats) expansion in the fragile X mental retardation 1 (FMR1) gene show postural control deficits that may reflect disruption to cerebellar motor regions. Less is known about the influence of reduced cerebellar volume and structural changes, and increase in CGG repeat and FMR1 mRNA levels on the attentional demands of step initiation in PM males. We investigated the effects of a concurrent cognitive task on choice stepping reaction time (CSRT) and explored the associations between CSRT performance, cerebellar volume, CGG size, and FMR1 mRNA levels in blood in PM males. We examined 19 PM males (ages 28-75) and 23 matched controls (CGG <44; ages 26-77), who performed a verbal fluency task during CSRT performance and single-task stepping without a secondary cognitive task. Our results provide preliminary evidence that smaller cerebellar volume (ß = -2.73, p = 0.002) and increasing CGG repeat length (ß = 1.69, p = 0.003) were associated with greater dual-task step initiation times in PM males, but not in controls. There was evidence of a mediating effect of cerebellar volume on the relationship between FMR1 mRNA levels and single-task CSRT performance in PM males (estimate coefficient = 8.69, standard error = 4.42, p = 0.049). These findings suggest increasing CGG repeat and FMR1 mRNA levels have neurotoxic effects on cerebellar regions underlying anticipatory postural responses during stepping. Cerebellar postural changes may be predictive of the increased risk of falls in older PM males.

Use of a Novel Nonparametric Version of DEPTH to Identify Genomic Regions Associated with Prostate Cancer Risk.

BACKGROUND: We have developed a genome-wide association study analysis method called DEPTH (DEPendency of association on the number of Top Hits) to identify genomic regions potentially associated with disease by considering overlapping groups of contiguous markers (e.g., SNPs) across the genome. DEPTH is a machine learning algorithm for feature ranking of ultra-high dimensional datasets, built from well-established statistical tools such as bootstrapping, penalized regression, and decision trees. Unlike marginal regression, which considers each SNP individually, the key idea behind DEPTH is to rank groups of SNPs in terms of their joint strength of association with the outcome. Our aim was to compare the performance of DEPTH with that of standard logistic regression analysis. METHODS: We selected 1,854 prostate cancer cases and 1,894 controls from the UK for whom 541,129 SNPs were measured using the Illumina Infinium HumanHap550 array. Confirmation was sought using 4,152 cases and 2,874 controls, ascertained from the UK and Australia, for whom 211,155 SNPs were measured using the iCOGS Illumina Infinium array. RESULTS: From the DEPTH analysis, we identified 14 regions associated with prostate cancer risk that had been reported previously, five of which would not have been identified by conventional logistic regression. We also identified 112 novel putative susceptibility regions. CONCLUSIONS: DEPTH can reveal new risk-associated regions that would not have been identified using a conventional logistic regression analysis of individual SNPs. IMPACT: This study demonstrates that the DEPTH algorithm could identify additional genetic susceptibility regions that merit further investigation. Cancer Epidemiol Biomarkers Prev; 25(12); 1619-24. ©2016 AACR.

Detection of skewed X-chromosome inactivation in Fragile X syndrome and X chromosome aneuploidy using quantitative melt analysis.

Methylation of the fragile X mental retardation 1 (FMR1) exon 1/intron 1 boundary positioned fragile X related epigenetic element 2 (FREE2), reveals skewed X-chromosome inactivation (XCI) in fragile X syndrome full mutation (FM: CGG > 200) females. XCI skewing has been also linked to abnormal X-linked gene expression with the broader clinical impact for sex chromosome aneuploidies (SCAs). In this study, 10 FREE2 CpG sites were targeted using methylation specific quantitative melt analysis (MS-QMA), including 3 sites that could not be analysed with previously used EpiTYPER system. The method was applied for detection of skewed XCI in FM females and in different types of SCA. We tested venous blood and saliva DNA collected from 107 controls (CGG < 40), and 148 FM and 90 SCA individuals. MS-QMA identified: (i) most SCAs if combined with a Y chromosome test; (ii) locus-specific XCI skewing towards the hypomethylated state in FM females; and (iii) skewed XCI towards the hypermethylated state in SCA with 3 or more X chromosomes, and in 5% of the 47,XXY individuals. MS-QMA output also showed significant correlation with the EpiTYPER reference method in FM males and females (P < 0.0001) and SCAs (P < 0.05). In conclusion, we demonstrate use of MS-QMA to quantify skewed XCI in two applications with diagnostic utility.

Evidence linking FMR1 mRNA and attentional demands of stepping and postural control in women with the premutation.

Recent studies in young adult females with the fragile X mental retardation 1 (FMR1) gene premutation (PM) have shown subtle but significant impairments in executive control and postural stability. Less is known about the influence of age and FMR1 gene expression on executive control and postural stability in females with the PM. Here, we examined the attentional demands of reactive stepping using a well-validated measure of choice stepping reaction time under dual-task interference. We explored the interrelationships between step initiation times during a concurrent verbal fluency task and specific impairments in executive control previously reported in females with the PM. Our results showed increased dual-task interference on step initiation times and variability in female PM compared with control subjects. In addition, we observed greater choice stepping reaction time dual-task costs above the breakpoint of 81 CGG repeats relative to below this CGG range. Dual-task interference on both reaction time and movement time were significantly predicted by low working memory capacity in female PM carriers. Importantly, we revealed that FMR1 messenger RNA level is the most significant predictor accounting for dual-task stepping variability in both reaction time and movement time in PM females. These findings for the first time provide evidence linking elevated FMR1 messenger RNA levels that have been previously associated with FMR1 RNA toxicity and deficits in cerebellar motor and cognitive networks in a subgroup of at-risk PM women.

Early detection of fragile X syndrome: applications of a novel approach for improved quantitative methylation analysis in venous blood and newborn blood spots.

BACKGROUND: Standard fragile X syndrome (FXS) diagnostic tests that target methylation of the fragile X mental retardation 1 (FMR1) CpG island 5' of the CGG expansion can be used to predict severity of the disease in males from birth, but not in females. METHODS: We describe methylation specific-quantitative melt analysis (MS-QMA) that targets 10 CpG sites, with 9 within FMR1 intron 1, to screen for FXS from birth in both sexes. The novel method combines the qualitative strengths of high-resolution melt and the high-throughput, quantitative real-time PCR standard curve to provide accurate quantification of DNA methylation in a single assay. Its performance was assessed in 312 control (CGG <40), 143 premutation (PM) (CGG 56-170), 197 full mutation (FM) (CGG 200-2000), and 33 CGG size and methylation mosaic samples. RESULTS: In male and female newborn blood spots, MS-QMA differentiated FM from control alleles, with sensitivity, specificity, and positive and negative predictive values between 92% and 100%. In venous blood of FM females between 6 and 35 years of age, MS-QMA correlated most strongly with verbal IQ impairment (P = 0.002). In the larger cohort of males and females, MS-QMA correlated with reference methods Southern blot and MALDI-TOF mass spectrometry (P < 0.05), but was not significantly correlated with age. Unmethylated alleles in high-functioning FM and PM males determined by both reference methods were also unmethylated by MS-QMA. CONCLUSIONS: MS-QMA has an immediate application in FXS diagnostics, with a potential use of its quantitative methylation output for prognosis in both sexes.

Relationships between age and epi-genotype of the FMR1 exon 1/intron 1 boundary are consistent with non-random X-chromosome inactivation in FM individuals, with the selection for the unmethylated state being most significant between birth and puberty.

Methylation of the fragile X-related epigenetic element 2 (FREE2) located on the exon 1/intron 1 boundary of the FMR1 gene is related to FMRP expression and cognitive impairment in full mutation (FM; CGG>200) individuals. We examined the relationship between age, the size of the FMR1 CGG expansion and the methylation output ratio (MOR) at 12 CpG sites proximal to the exon 1/intron 1 boundary using FREE2 MALDI-TOF MS. The patient cohort included 119 males and 368 females, i.e. 121 healthy controls (CGG<40), 176 premutation (CGG 55-170) and 190 FM (CGG 213-2000). For all CpG units examined, FM males showed a significantly elevated MOR compared with that in hypermethylated FM females. In FM males the MOR for most CpG units significantly positively correlated with both age and CGG size (P< 0.05). In FM females the skewing towards the unmethylated state was significant for half of the units between birth and puberty (P < 0.05). The methylation status of intron 1 CpG10-12 that was most significantly related to cognitive impairment in our earlier study, did not change significantly with age in FM females. These results challenge the concept of fragile X syndrome (FXS)-related methylation being static over time, and suggest that due to the preference for the unmethylated state in FM females, X-inactivation at this locus is not random. The findings also highlight that the prognostic value of FXS methylation testing is not uniform between all CpG sites, and thus may need to be evaluated on a site-by-site basis.

Fragile X-related element 2 methylation analysis may provide a suitable option for inclusion of fragile X syndrome and/or sex chromosome aneuploidy into newborn screening: a technical validation study.

PURPOSE: We show that a novel fragile X-related epigenetic element 2 FMR1 methylation test can be used along with a test for sex-determining region Y (SRY) to provide the option of combined fragile X syndrome and sex chromosome aneuploidy newborn screening. METHODS: Fragile X-related epigenetic element 2, SRY, and FMR1 CGG repeat analyses were performed on blood and saliva DNA, and in adult and newborn blood spots. The cohort consisted of 159 controls (CGG <40), 187 premutation (CGG 56-170), and 242 full-mutation (CGG ~200-2,000) males and females, 106 sex chromosome aneuploidy individuals, and 151 cytogenetically normal controls. RESULTS: At the 0.435 threshold, fragile X-related epigenetic element 2 analysis in males was robust on both blood DNA and newborn blood spots, with specificity and sensitivity of ~100% for full-mutation genotype. In females, the specificity was 99%, whereas half of full-mutation females were above the 0.435 threshold in both blood DNA and newborn blood spots. Furthermore, at this threshold, the test could not differentiate individuals with Klinefelter syndrome from female controls without using the SRY marker. When combined with SRY analysis, the test was consistent with most results for sex chromosome aneuploidies from karyotyping. CONCLUSION: Setting specific thresholds for fragile X-related epigenetic element 2 analysis and including the SRY marker provides the option to either include or exclude detection of sex chromosome aneuploidies as part of fragile X syndrome newborn screening.

The heterogeneity in femoral neck structure and strength.

Most measures of femoral neck strength derived using dual-energy X-ray absorptiometry or computed tomography (CT) assume the femoral neck is a cylinder with a single cortical thickness. We hypothesized that these simplifications introduce errors in estimating strength and that detailed analyses will identify new parameters that more accurately predict femoral neck strength. High-resolution CT data were used to evaluate 457 cross-sectional slices along the femoral neck of 12 postmortem specimens. Cortical morphology was measured in each cross-section. The distribution of cortical thicknesses was evaluated to determine whether the mean or median better estimated central tendency. Finite-element models were used to calculate the stresses in each cross-section resulting from the peak hip joint forces created during a sideways fall. The relationship between cortical morphology and peak bone stress along the femoral neck was analyzed using multivariate regression analysis. In all cross-sections, cortical thicknesses were non-normally distributed and skewed toward smaller thicknesses (p < 0.0001). The central tendency of cortical thickness was best estimated by the median, not the mean. Stress increased as the median cortical thickness decreased along the femoral neck. The median, not mean, cortical thickness combined with anterior-posterior diameter best predicted peak bone stress generated during a sideways fall (R(2) = 0.66, p < 0.001). Heterogeneity in the structure of the femoral neck determines the diversity of its strength. The median cortical thickness best predicted peak femoral neck stress and is likely to be a relevant predictor of femoral neck fragility.

Does eczema in infancy cause hay fever, asthma, or both in childhood? Insights from a novel regression model of sibling data.

BACKGROUND: The atopic march hypothesis proposes that eczema precedes the development of asthma and allergic rhinitis. OBJECTIVE: We sought to assess the evidence for a causal effect of infantile eczema on childhood hay fever, asthma, or both. METHODS: We used parental reports on infantile eczema and childhood asthma and hay fever for 3778 pairs of 7-year-olds matched to their sibling closest in age within 2 years from the Tasmanian Longitudinal Health Study. We analyzed paired sibling data using a logistic regression model that allowed inference about a causal effect of a familial predictor on a child's outcome by examining the change in association with their cosibling's predictor after adjusting for their own predictor status. RESULTS: Siblings were concordant for infantile eczema (tetrachoric correlation, 0.40). For having both hay fever and asthma by age 7 years, the association with cosibling's eczema was an odds ratio (OR) of 1.98 (95% CI, 1.37-2.86), which reduced after adjusting for own eczema to an OR of 1.65 (95% CI, 1.17-2.34). For having hay fever only, the association with cosibling's eczema was an OR of 1.68 (95% CI, 1.22-2.31) before and an OR of 1.59 (95% CI, 1.19-2.14) after adjusting for own eczema. There was no association between having asthma only and cosibling's eczema (OR, 1.00; 95% CI, 0.77-1.30). CONCLUSIONS: Eczema in infancy might have a causal effect on hay fever in children with and perhaps without asthma. The association of infantile eczema on asthma in children without hay fever, which might be early transient wheeze, is unlikely to be causal or familial. These findings have implications for hay fever prevention.

A meta-analysis of genome-wide association studies of breast cancer identifies two novel susceptibility loci at 6q14 and 20q11.

Genome-wide association studies (GWAS) of breast cancer defined by hormone receptor status have revealed loci contributing to susceptibility of estrogen receptor (ER)-negative subtypes. To identify additional genetic variants for ER-negative breast cancer, we conducted the largest meta-analysis of ER-negative disease to date, comprising 4754 ER-negative cases and 31 663 controls from three GWAS: NCI Breast and Prostate Cancer Cohort Consortium (BPC3) (2188 ER-negative cases; 25 519 controls of European ancestry), Triple Negative Breast Cancer Consortium (TNBCC) (1562 triple negative cases; 3399 controls of European ancestry) and African American Breast Cancer Consortium (AABC) (1004 ER-negative cases; 2745 controls). We performed in silico replication of 86 SNPs at P ≤ 1 × 10(-5) in an additional 11 209 breast cancer cases (946 with ER-negative disease) and 16 057 controls of Japanese, Latino and European ancestry. We identified two novel loci for breast cancer at 20q11 and 6q14. SNP rs2284378 at 20q11 was associated with ER-negative breast cancer (combined two-stage OR = 1.16; P = 1.1 × 10(-8)) but showed a weaker association with overall breast cancer (OR = 1.08, P = 1.3 × 10(-6)) based on 17 869 cases and 43 745 controls and no association with ER-positive disease (OR = 1.01, P = 0.67) based on 9965 cases and 22 902 controls. Similarly, rs17530068 at 6q14 was associated with breast cancer (OR = 1.12; P = 1.1 × 10(-9)), and with both ER-positive (OR = 1.09; P = 1.5 × 10(-5)) and ER-negative (OR = 1.16, P = 2.5 × 10(-7)) disease. We also confirmed three known loci associated with ER-negative (19p13) and both ER-negative and ER-positive breast cancer (6q25 and 12p11). Our results highlight the value of large-scale collaborative studies to identify novel breast cancer risk loci.

Fragile X mental retardation 1 (FMR1) intron 1 methylation in blood predicts verbal cognitive impairment in female carriers of expanded FMR1 alleles: evidence from a pilot study.

BACKGROUND: Cognitive status in females with mutations in the FMR1 (fragile X mental retardation 1) gene is highly variable. A biomarker would be of value for predicting which individuals were liable to develop cognitive impairment and could benefit from early intervention. A detailed analysis of CpG sites bridging exon 1 and intron 1 of FMR1, known as fragile X-related epigenetic element 2 (FREE2), suggests that a simple blood test could identify these individuals. METHODS: Study participants included 74 control females (<40 CGG repeats), 62 premutation (PM) females (55-200 CGG repeats), and 18 full-mutation (FM) females assessed with Wechsler intelligence quotient (IQ) tests. We used MALDI-TOF mass spectrometry to determine the methylation status of FREE2 CpG sites that best identified low-functioning (IQ <70) FM females (>200 CGG repeats), compared the results with those for Southern blot FMR1 activation ratios, and related these assessments to the level of production of the FMR1 protein product in blood. RESULTS: A methylation analysis of intron 1 CpG sites 10-12 showed the highest diagnostic sensitivity (100%) and specificity (98%) of all the molecular measures tested for detecting females with a standardized verbal IQ of <70 among the study participants. In the group consisting of only FM females, methylation of these sites was significantly correlated with full-scale IQ, verbal IQ, and performance IQ. Several verbal subtest scores showed strong correlation with the methylation of these sites (P = 1.2 × 10(-5)) after adjustment for multiple measures. CONCLUSIONS: The data suggest that hypermethylation of the FMR1 intron 1 sites in blood is predictive of cognitive impairment in FM females, with implications for improved fragile X syndrome diagnostics in young children and screening of the newborn population.

FMR1 intron 1 methylation predicts FMRP expression in blood of female carriers of expanded FMR1 alleles.

Fragile X syndrome (FXS) is caused by loss of the fragile X mental retardation gene protein product (FMRP) through promoter hypermethylation, which is usually associated with CGG expansion to full mutation size (>200 CGG repeats). Methylation-sensitive Southern blotting is the current gold standard for the molecular diagnosis of FXS. For females, Southern blotting provides the activation ratio (AR), which is the proportion of unmethylated alleles on the active X chromosome. Herein, we examine the relationship of FMRP expression with methylation patterns of two fragile X-related epigenetic elements (FREE) analyzed using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and the AR. We showed that the differential methylation of the FREE2 sequence within fragile X mental retardation gene intron 1 was related to depletion of FMRP expression. We also show that, using the combined cohort of 12 females with premutation (55 to 200 CGG repeats) and 22 females with full mutation alleles, FREE2 methylation analysis was superior to the AR as a predictor of the proportion of FMRP-positive cells in blood. Because matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry is amenable to high-throughput processing and requires minimal DNA, these findings have implications for routine FXS testing and population screening.

Evidence for the toxicity of bidirectional transcripts and mitochondrial dysfunction in blood associated with small CGG expansions in the FMR1 gene in patients with parkinsonism.

PURPOSE: Our previous results showed that both gray zone and lower end premutation range (40-85 repeats) fragile X mental retardation 1 (FMR1) alleles were more common among males with parkinsonism than in the general population. This study aimed to determine whether these alleles have a significant role in the manifestations and pathogenesis of parkinsonian disorders. METHODS: Detailed clinical assessment and genetic testing were performed in 14 male carriers of premutation and gray zone FMR1 alleles and in 24 noncarriers identified in a sample of males with parkinsonism. RESULTS: The premutation + gray zone carriers presented with more severe symptoms than disease controls matched for age, diagnosis, disease duration, and treatment. The Parkinson disease (Unified Parkinson's Disease Rating Scale) motor score and the measures of cognitive decline (Mini-Mental State Examination and/or Addenbrooke's Cognitive Examination Final Revised Version A scores) were significantly correlated with the size of the CGG repeat and the (elevated) levels of antisense FMR1 and Cytochrome C1 mRNAs in blood leukocytes. In addition, the carriers showed a significant depletion of the nicotinamide adenine dinucleotide, reduced dehydrogenase subunit 1 mitochondrial gene in whole blood. CONCLUSION: Small CGG expansion FMR1 alleles (gray zone and lower end premutation) play a significant role in the development of the parkinsonian phenotype, possibly through the cytotoxic effect of elevated sense and/or antisense FMR1 transcripts involving mitochondrial dysfunction and leading to progressive neurodegeneration.

A varying coefficient model to measure the effectiveness of mass media anti-smoking campaigns in generating calls to a Quitline.

BACKGROUND: Anti-smoking advertisements are an effective population-based smoking reduction strategy. The Quitline telephone service provides a first point of contact for adults considering quitting. Because of data complexity, the relationship between anti-smoking advertising placement, intensity, and time trends in total call volume is poorly understood. In this study we use a recently developed semi-varying coefficient model to elucidate this relationship. METHODS: Semi-varying coefficient models comprise parametric and nonparametric components. The model is fitted to the daily number of calls to Quitline in Victoria, Australia to estimate a nonparametric long-term trend and parametric terms for day-of-the-week effects and to clarify the relationship with target audience rating points (TARPs) for the Quit and nicotine replacement advertising campaigns. RESULTS: The number of calls to Quitline increased with the TARP value of both the Quit and other smoking cessation advertisement; the TARP values associated with the Quit program were almost twice as effective. The varying coefficient term was statistically significant for peak periods with little or no advertising. CONCLUSIONS: Semi-varying coefficient models are useful for modeling public health data when there is little or no information on other factors related to the at-risk population. These models are well suited to modeling call volume to Quitline, because the varying coefficient allowed the underlying time trend to depend on fixed covariates that also vary with time, thereby explaining more of the variation in the call model.

Applications of additive semivarying coefficient models: monthly suicide data from Hong Kong.

A semivarying coefficient model for the monthly numbers of suicides in Hong Kong is developed and a new estimation procedure for estimating the parametric component is proposed. The estimators are examined in a small simulation study and fitted to monthly suicide data to estimate a nonparametric long-term trend and parametric seasonal and socioeconomic effects. Fitting the model detected interpretable structure in the data that is consistent with that driving public health policy. While exploratory, the analysis motivates the collection of more detailed data and the development of more sophisticated models to help determine target groups and strategies to reduce the suicide rate in Hong Kong.

Transcript levels of the intermediate size or grey zone fragile X mental retardation 1 alleles are raised, and correlate with the number of CGG repeats.

BACKGROUND: Grey zone or intermediate alleles are one of the three recognised classes of the X-linked fragile X mental retardation 1 (FMR1) gene showing intergenerational instability. These classes are defined according to the number of CGG repeats in the FMR1 5'-untranslated region. Although large CGG expansions (>200 repeats) cause a neurodevelopmental anomaly through silencing of the gene, resulting in a deficit of FMR1 specific protein, smaller expansions (approximately 55-200 repeats) are associated with an increased transcription and late-onset specific phenotypes. Those alleles with a CGG repeat number ranging between approximately 41 and 55 are relatively poorly defined with regard to both transcriptional and translational activity, and also potential phenotypic effects. METHODS AND RESULTS: Based on a sample of 33 males carrying FMR1 alleles within the grey zone range, defined here as 41-60 CGGs, we show an increased transcriptional activity relative to that seen in common alleles (5-40 CGGS). This is the first study to report a significant relationship between FMR1 mRNA levels and CGG repeat number within the grey zone range (p<0.001). From a piecewise linear regression model, the threshold for onset of the increase in mRNA levels as a function of CGG repeat size has been determined at approximately 39 repeats (standard error (SE) 3.24), and that for the reduction in the rate of this increase at approximately 54 repeats (SE 4.27). CONCLUSIONS: The ambiguities associated with the definition and transcription dynamics of the FMR1 gene within the grey zone range are dealt with. There may be specific phenotypes associated with the toxic "gain-of-function" effect of raised mRNA.

Autism spectrum phenotype in males and females with fragile X full mutation and premutation.

The behavioural phenotype of autism was assessed in individuals with full mutation and premutation fragile X syndrome (FXS) using the Autism Diagnostic Observation Scale-Generic (ADOS-G) and the Autism Diagnostic Interview (ADI-R). The participants, aged 5-80 years, comprised 33 males and 31 females with full mutation, 7 males and 43 females with premutation, and 38 non-fragile X relatives (29 males, 9 females). In the full mutation group, a total of 67% males and 23% females met either the Autism Disorder (AD) or the Autism Spectrum Disorder (ASD) criteria on at least one of the diagnostic tests. In the premutation group, 14% males and 5% females met the ADOS-G criteria for ASD. The presence of autism manifestations in males and females with full mutation and premutation provide support for a spectrum view.

Molecular and cognitive predictors of the continuum of autistic behaviours in fragile X.

The distributions of scores for autistic behaviours obtained from the Autism Diagnostic Observation Scale-Generic (ADOS-G) were investigated in 147 males and females affected with the full mutation in the fragile X mental retardation 1 (FMR1) gene, in 59 individuals with the premutation, and in 42 non-fragile X relatives, aged 4-70 years. The scores representing communication and social interaction were continuously distributed across the two fragile X groups, and they were significantly elevated compared with the non-fragile X controls. Strong relationships were found between both these scores and FMRP deficits, but they became insignificant for social interaction, and the sum of social interaction and communication scores, when FSIQ was included as another predictor of autism scores. Other significant predictors of these scores in both sexes were those executive skills which related to verbal fluency, and to the regulation and control of motor behaviour. Overall, our data have shown that cognitive impairment, especially of verbal skills, best explains the comorbidity of autism and fragile X. This implies some more fundamental perturbations of specific neural connections which are essential for both specific behaviours and cognition. We also emphasize that FXS offers a unique molecular model for autism since FMRP regulates the translation of many other genes involved in synaptic formation and plasticity which should be natural targets for further exploration.