Diagnostic application of kidney allograft-derived absolute cell-free DNA levels during transplant dysfunction.

Graft-derived cell-free DNA (donor-derived cell-free DNA) is an emerging marker of kidney allograft injury. Studies examining the clinical validity of this biomarker have previously used the graft fraction, or proportion of total cell-free DNA that is graft-derived. The present study evaluated the diagnostic validity of absolute measurements of graft-derived cell-free DNA, as well as calculated graft fraction, for the diagnosis of graft dysfunction. Plasma graft-derived cell-free DNA, total cell-free DNA, and graft fraction were correlated with biopsy diagnosis as well as individual Banff scores. Sixty-one samples were included in the analysis. For the diagnosis of antibody mediated rejection, the receiver-operator characteristic area under the curves of graft-derived cell-free DNA and graft fraction were 0.91 (95% CI 0.82-0.98) and 0.89 (95% CI 0.79-0.98), respectively. Both measures did not diagnose borderline or type 1A cellular mediated rejection. Graft fraction was associated with a broader range of Banff lesions, including lesions associated with cellular mediated rejection, while graft-derived cell-free DNA appeared more specific for antibody mediated rejection. Limitations of this study include a small sample size and lack of a validation cohort. The capacity for absolute quantification, and lower barriers to implementation of this methodology recommend it for further study.

Significantly Elevated FMR1 mRNA and Mosaicism for Methylated Premutation and Full Mutation Alleles in Two Brothers with Autism Features Referred for Fragile X Testing.

Although fragile X syndrome (FXS) is caused by a hypermethylated full mutation (FM) expansion with ≥200 cytosine-guanine-guanine (CGG) repeats, and a decrease in FMR1 mRNA and its protein (FMRP), incomplete silencing has been associated with more severe autism features in FXS males. This study reports on brothers (B1 and B2), aged 5 and 2 years, with autistic features and language delay, but a higher non-verbal IQ in comparison to typical FXS. CGG sizing using AmplideX PCR only identified premutation (PM: 55-199 CGGs) alleles in blood. Similarly, follow-up in B1 only revealed PM alleles in saliva and skin fibroblasts; whereas, an FM expansion was detected in both saliva and buccal DNA of B2. While Southern blot analysis of blood detected an unmethylated FM, methylation analysis with a more sensitive methodology showed that B1 had partially methylated PM alleles in blood and fibroblasts, which were completely unmethylated in buccal and saliva cells. In contrast, B2 was partially methylated in all tested tissues. Moreover, both brothers had FMR1 mRNA ~5 fold higher values than those of controls, FXS and PM cohorts. In conclusion, the presence of unmethylated FM and/or PM in both brothers may lead to an overexpression of toxic expanded mRNA in some cells, which may contribute to neurodevelopmental problems, including elevated autism features.

ADGRV1 is implicated in myoclonic epilepsy.

OBJECTIVE: To investigate the significance of variation in ADGRV1 (also known as GPR98, MASS1, and VLGR1), MEF2C, and other genes at the 5q14.3 chromosomal locus in myoclonic epilepsy. METHODS: We studied the epilepsy phenotypes of 4 individuals with 5q14.3 deletion and found that all had myoclonic seizures. We then screened 6 contiguous genes at 5q14.3, MEF2C, CETN3, MBLAC2, POLR3G, LYSMD3, and ADGRV1, in a 95-patient cohort with epilepsy and myoclonic seizures. Of these genes, point mutations in MEF2C cause a phenotype involving seizures and intellectual disability. A role for ADGRV1 in epilepsy has been proposed previously, based on a recessive mutation in the Frings mouse model of audiogenic seizures, as well as a shared homologous region with another epilepsy gene, LGI1. RESULTS: Six patients from the myoclonic epilepsy cohort had likely pathogenic ultra-rare ADGRV1 variants, and statistical analysis showed that ultra-rare variants were significantly overrepresented when compared to healthy population data from the Genome Aggregation Database. Of the remaining genes, no definite pathogenic variants were identified. SIGNIFICANCE: Our data suggest that the ADGRV1 variation contributes to epilepsy with myoclonic seizures, although the inheritance pattern may be complex in many cases. In patients with 5q14.3 deletion and epilepsy, ADGRV1 haploinsufficiency likely contributes to seizure development. The latter is a shift from current thinking, as MEF2C haploinsufficiency has been considered the main cause of epilepsy in 5q14.3 deletion syndrome. In cases of 5q14.3 deletion and epilepsy, seizures likely occur due to haploinsufficiency of one or both of ADGRV1 and MEF2C.

Identification of Males with Cryptic Fragile X Alleles by Methylation-Specific Quantitative Melt Analysis.

BACKGROUND: FMR1 full mutations (FMs) (CGG expansion >200) in males mosaic for a normal (<45 CGG) or gray-zone (GZ) (45-54 CGG) allele can be missed with the standard 2-step fragile X syndrome (FXS) testing protocols, largely because the first-line PCR tests showing a normal or GZ allele are not reflexed to the second-line test that can detect FM. METHODS: We used methylation-specific quantitative melt analysis (MS-QMA) to determine the prevalence of cryptic FM alleles in 2 independent cohorts of male patients (994 from Chile and 2392 from Australia) referred for FXS testing from 2006 to 2013. All MS-QMA-positive cases were retested with commercial triplet primed PCR, methylation-sensitive Southern blot, and a methylation-specific EpiTYPER-based test. RESULTS: All 38 FMs detected with the standard 2-step protocol were detected with MS-QMA. However, MS-QMA identified methylation mosaicism in an additional 15% and 11% of patients in the Chilean and Australian cohorts, respectively, suggesting the presence of a cryptic FM. Of these additional patients, 57% were confirmed to carry cryptic expanded alleles in blood, buccal mucosa, or saliva samples. Further confirmation was provided by identifying premutation (CGG 55-199) alleles in mothers of probands with methylation-sensitive Southern blot. Neurocognitive assessments showed that low-level mosaicism for cryptic FM alleles was associated with cognitive impairment or autism. CONCLUSIONS: A substantial number of mosaic FM males who have cognitive impairment or autism are not diagnosed with the currently recommended 2-step testing protocol and can be identified with MS-QMA as a first-line test.

Clinical audit of genetic testing and referral patterns for fragile X and associated conditions.

An audit was conducted of laboratory/clinical databases of genetic tests performed between January 2003 and December 2009, and for 2014, as well as referrals to the clinical service and a specialist multidisciplinary clinic, to determine genetic testing request patterns for fragile X syndrome and associated conditions and referrals for genetic counseling/multidisciplinary management in Victoria, Australia. An expanded allele (full mutation, premutation or intermediate) was found in 3.7% of tests. Pediatricians requested ∼70% of test samples, although fewer general practitioners and more obstetricians/gynecologists ordered tests in 2014. Median age at testing for individuals with a full mutation seeking a diagnosis without a fragile X family history was 4.3 years (males) and 9.4 years (females); these ages were lower when pediatricians ordered the tests (2.1 years and 6.1 years, respectively). Individuals with a premutation were generally tested at a later age (median age: males, 33.2 years; females, 36.4 years). Logistic regression showed that a family history of ID (OR 3.28 P = 0.005, CI 1.77-5.98) was the only indication to independently increase the likelihood of a test-positive (FM or PM) result. Following testing, ∼25% of full mutation or premutation individuals may not have attended clinical services providing genetic counseling or multidisciplinary management for these families. The apparent delay in fragile X syndrome diagnosis and lack of appropriate referrals for some may result in less than optimal management for these families. These findings suggest continued need for awareness and education of health professionals around diagnosis and familial implications of fragile X syndrome and associated conditions. © 2016 Wiley Periodicals, Inc.

Partially methylated alleles, microdeletion, and tissue mosaicism in a fragile X male with tremor and ataxia at 30 years of age: A case report.

CGG repeat expansion >200 within FMR1, termed full mutation (FM), has been associated with promoter methylation, consequent silencing of gene expression and fragile X syndrome (FXS)-a common cause of intellectual disability and co-morbid autism. Unmethylated premutation (55-199 repeats) and FM alleles have been associated with fragile X related tremor/ataxia syndrome (FXTAS), a late onset neurodegenerative disorder. Here we present a 33-year-old male with FXS, with white matter changes and progressive deterioration in gait with cerebellar signs consistent with probable FXTAS; there was no evidence of any other cerebellar pathology. We show that he has tissue mosaicism in blood, saliva, and buccal samples for the size and methylation of his expanded alleles and a de novo, unmethylated microdeletion. This microdeletion involves a ∼80 bp sequence in the FMR1 promoter as well as complete loss of the CGG repeat in a proportion of cells. Despite FMR1 mRNA levels in blood within the normal range, the methylation and CGG sizing results are consistent with the diagnosis of concurrent FXS and probable FXTAS. The demonstrated presence of unmethylated FM alleles would explain the manifestation of milder than expected cognitive and behavioral impairments and early onset of cerebellar ataxia. Our case suggests that individuals with FXS, who manifest symptoms of FXTAS, may benefit from more detailed laboratory testing. © 2016 Wiley Periodicals, Inc.

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.

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.

Use of copy number deletion polymorphisms to assess DNA chimerism.

BACKGROUND: We describe a novel approach that harnesses the ubiquity of copy number deletion polymorphisms in human genomes to definitively detect and quantify chimeric DNA in clinical samples. Unlike other molecular approaches to chimerism analysis, the copy number deletion (CND) method targets genomic loci (>50 base pairs in length) that are wholly absent from wild-type (i.e., self) background DNA sequences in a sex-independent manner. METHODS: Bespoke quantitative PCR (qPCR) CND assays were developed and validated using a series of DNA standards and chimeric plasma DNA samples collected from 2 allogeneic kidney transplant recipients and 12 pregnant women. Assay performance and informativeness were assessed using appropriate statistical methods. RESULTS: The CND qPCR assays showed high sensitivity, precision, and reliability for linear quantification of DNA chimerism down to 16 genomic equivalents (i.e., 106 pg). Fetal fraction (%) in 12 singleton male pregnancies was calculated using the CND qPCR approach, which showed closer agreement with single-nucleotide polymorphism-based massively parallel sequencing than the SRY (sex determining region Y) (Y chromosome) qPCR assay. The latter consistently underestimated the fetal fraction relative to the other methods. We also were able to measure biological changes in plasma nonself DNA concentrations in 2 renal transplant recipients. CONCLUSIONS: The CND qPCR technique is suitable for measurement of chimerism for monitoring of rejection in allogeneic organ transplantation and quantification of the cell-free fetal DNA fraction in maternal plasma samples used for noninvasive prenatal genetic testing.

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.

Characterization of core clinical phenotypes associated with recurrent proximal 15q25.2 microdeletions.

A recurrent proximal microdeletion at 15q25.2 with an approximate 1.5 megabase smallest region of overlap has recently been reported in seven patients and is proposed to be associated with congenital diaphragmatic hernia (CDH), mild to moderate cognitive deficit, and/or features consistent with Diamond-Blackfan anemia. We report on four further patients and define the core phenotypic features of individuals with this microdeletion to include mild to moderate developmental delay or intellectual disability, postnatal short stature, anemia, and cryptorchidism in males. CDH and structural organ malformations appear to be less frequent associations, as is venous thrombosis. There is no consistent facial dysmorphism. Features novel to our patient group include dextrocardia, obstructive sleep apnea, and cleft lip.

White matter changes in patients with Parkinson's disease carrying small CGG expansion FMR1 alleles: a pilot study.

BACKGROUND/AIMS: Alleles of the FMR1 gene containing small expansions of the CGG-trinucleotide repeat comprise premutation and grey-zone alleles. Premutation alleles may cause late-onset Fragile X-associated tremor/ataxia syndrome attributed to the neurotoxic effect of elevated FMR1 transcripts. Our earlier data suggested that both grey-zone and low-end premutation alleles might also play a significant role in the acquisition of the parkinsonian phenotype due to mitochondrial dysfunction caused by elevated FMR1 mRNA toxicity. These data were obtained through clinical and molecular comparisons between carriers of grey-zone/low-end premutation alleles and group-matched non-carrier controls from patients with idiopathic Parkinson's disease (iPD). We aimed to explore the relationship between grey-zone alleles, parkinsonism and white matter changes. METHODS: This study compared the extent and severity of white matter hyperintensity (WMH) on magnetic resonance imaging, using a semi-quantitative method, between 11 grey-zone/low-end premutation carriers and 20 non-carrier controls with iPD from our earlier study. Relationships between WMH scores, and cognitive and motor test scores were assessed for carriers and non-carriers. RESULTS: Supratentorial WMH scores, and tremor and ataxia motor scores were significantly higher in carriers compared with disease controls. Moreover, some associations between cognitive decline and WMH scores were specific for each respective carrier status category. CONCLUSIONS: The results support our earlier claim that grey-zone alleles contribute to the severity of parkinsonism and white matter changes.

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.

Application of a new molecular technique for the genetic evaluation of products of conception.

OBJECTIVES: Karyotyping is a well-established method of investigating the genetic content of product of conceptions (POCs). Because of the high rate of culture failure and maternal cell contamination, failed results or 46,XX findings are often obtained. Different molecular approaches that are not culture dependent have been proposed to circumvent these limits. On the basis of the robust experience previously obtained with bacterial artificial chromosomes (BACs)-on-Beads™ (BoBs™), we evaluated the same technology that we had used for the analysis of prenatal samples on POCs. METHOD: KaryoLite™ BoBs™ includes 91 beads, each of which is conjugated with a composite of multiple neighboring BACs according to the hg19 assembly. It quantifies proximal and terminal regions of each chromosome arm. The study included 376 samples. RESULTS: The failure rate was 2%, and reproducibility >99%; false-positive and false-negative rates were <1% for non-mosaic aneuploidies and imbalances effecting all three BACs in a contig. Detection rate for partial terminal imbalances was 65.5%. The mosaic detection threshold was 50%, and the success rate in macerated samples was 87.8%. The aneuploidy detection rate in samples with cell growth failure was 27.8%, and maternal cell contamination was suspected in 23.1% of 46,XX cultured cells. CONCLUSION: KaryoLite™ BoBs™ as a 'first-tier' test in combination with other approaches showed beneficial, cost-effective and clearly enhanced POC testing.

High resolution chromosomal microarray in undiagnosed neurological disorders.

AIM: Despite advances in medical investigation, many children with neurological conditions remain without a diagnosis, although a genetic aetiology is often suspected. Chromosomal microarray (CMA) screens for copy number variants (CNVs) and long continuous stretches of homozygosity (LCSH) and may further enhance diagnostic yield. Although recent studies have identified pathogenic CNVs in intellectual disability, autism and epilepsy, the utility of CMA testing in a broader cohort of children with neurologic disorders has not been reported. METHODS: Two hundred fifteen patients with neurological conditions of unknown aetiology were seen over a 6-month period and were prospectively tested by CMA using high-resolution single nucleotide polymorphism (SNP) microarrays (Illumina HumanCytoSNP-12 v2.1 or Affymetrix 2.7M). RESULTS: Thirty of 215 (14%) patients tested had an abnormal CMA. Twenty-nine had CNVs (13%) and one (0.5%) a clinically significant stretch of homozygosity. Twenty (9.3%) had a CMA finding considered to be pathogenic or involved in susceptibility to the condition of interest, and 10 (4.7%) had findings of unknown significance. Their phenotypes included infantile spasms and other epilepsies, neuromuscular conditions, ataxia, movement disorders, microcephaly and malformations of cortical development. At least one third of patients did not meet national funding criteria for CMA at the time of presentation. CONCLUSIONS: CMA detected clinically significant abnormalities in a broad range of neurologic phenotypes of unknown aetiology. This test should be considered a first-tier investigation of children with neurologic disorders in whom the initial clinical assessment does not indicate a likely aetiology, especially those with severe epilepsies and neurologically abnormal neonates.

Molecular and clinical characterization of 25 individuals with exonic deletions of NRXN1 and comprehensive review of the literature.

This study aimed to elucidate the observed variable phenotypic expressivity associated with NRXN1 (Neurexin 1) haploinsufficiency by analyses of the largest cohort of patients with NRXN1 exonic deletions described to date and by comprehensively reviewing all comparable copy number variants in all disease cohorts that have been published in the peer reviewed literature (30 separate papers in all). Assessment of the clinical details in 25 previously undescribed individuals with NRXN1 exonic deletions demonstrated recurrent phenotypic features consisting of moderate to severe intellectual disability (91%), severe language delay (81%), autism spectrum disorder (65%), seizures (43%), and hypotonia (38%). These showed considerable overlap with previously reported NRXN1-deletion associated phenotypes in terms of both spectrum and frequency. However, we did not find evidence for an association between deletions involving the ß-isoform of neurexin-1 and increased head size, as was recently published in four cases with a deletion involving the C-terminus of NRXN1. We identified additional rare copy number variants in 20% of cases. This study supports a pathogenic role for heterozygous exonic deletions of NRXN1 in neurodevelopmental disorders. The additional rare copy number variants identified may act as possible phenotypic modifiers as suggested in a recent digenic model of neurodevelopmental disorders.

Methylation of novel markers of fragile X alleles is inversely correlated with FMRP expression and FMR1 activation ratio.

The fragile X syndrome (FXS) is caused by silencing of the fragile X mental retardation gene (FMR1) and the absence of its product, fragile X mental retardation protein (FMRP), resulting from CpG island methylation associated with large CGG repeat expansions (more than 200) termed full mutation (FM). We have identified a number of novel epigenetic markers for FXS using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), naming the most informative fragile X-related epigenetic element 1 (FREE1) and 2 (FREE2). Methylation of both regions was correlated with that of the FMR1 CpG island detected using Southern blot (FREE1 R = 0.97; P < 0.00001, n = 23 and FREE2 R = 0.93; P < 0.00001, n = 23) and negatively correlated with lymphocyte expression of FMRP (FREE1 R = -0.62; P = 0.01, n = 15 and FREE2 R = -0.55; P = 0.03, n = 15) in blood of partially methylated 'high functioning' FM males. In blood of FM carrier females, methylation of both markers was inversely correlated with the FMR1 activation ratio (FREE1 R = -0.93; P < 0.0001, n = 12 and FREE2 R = -0.95; P < 0.0001, n = 9). In a sample set of 49 controls, 18 grey zone (GZ 40-54 repeats), 22 premutation (PM 55-170 repeats) and 22 (affected) FXS subjects, the FREE1 methylation pattern was consistent between blood and chorionic villi as a marker of methylated FM alleles and could be used to differentiate FXS males and females from controls, as well as from carriers of GZ/PM alleles, but not between GZ and PM alleles and controls. Considering its high-throughput and specificity for pathogenic FM alleles, low cost and minimal DNA requirements, FREE MALDI-TOF MS offers a unique tool in FXS diagnostics and newborn population screening.

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.

Extending the scope of diagnostic chromosome analysis: detection of single gene defects using high-resolution SNP microarrays.

Microarray analysis has provided significant advances in the diagnosis of conditions resulting from submicroscopic chromosome abnormalities. It has been recommended that array testing should be a "first tier" test in the evaluation of individuals with intellectual disability, developmental delay, congenital anomalies, and autism. The availability of arrays with increasingly high probe coverage and resolution has increased the detection of decreasingly small copy number changes (CNCs) down to the intragenic or even exon level. Importantly, arrays that genotype SNPs also detect extended regions of homozygosity. We describe 14 examples of single gene disorders caused by intragenic changes from a consecutive set of 6,500 tests using high-resolution SNP microarrays. These cases illustrate the increased scope of cytogenetic testing beyond dominant chromosome rearrangements that typically contain many genes. Nine of the cases confirmed the clinical diagnosis, that is, followed a "phenotype to genotype" approach. Five were diagnosed by the laboratory analysis in the absence of a specific clinical diagnosis, that is, followed a "genotype to phenotype" approach. Two were clinically significant, incidental findings. The importance of astute clinical assessment and laboratory-clinician consultation is emphasized to optimize the value of microarrays in the diagnosis of disorders caused by single gene copy number and sequence mutations.

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.