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Acetylated histones are associated with FMR1 in normal but not fragile X-syndrome cells.

Mutation of FMR1 results in fragile X mental retardation. The most common FMR1 mutation is expansion of a CGG repeat tract at the 5´ end of FMR1 (refs 2, 3, 4), which leads to cytosine methylation and transcriptional silencing. Both DNA methylation and histone deacetylation have been associated with transcriptional inactivity. The finding that the methyl cytosine-binding protein MeCP2 binds to histone deacetylases and represses transcription in vivo supports a model in which MeCP2 recruits histone deacetylases to methylated DNA, resulting in histone deacetylation, chromatin condensation and transcriptional silencing. Here we demonstrate that the 5´ end of FMR1 is associated with acetylated histones H3 and H4 in cells from normal individuals, but acetylation is reduced in cells from fragile X patients. Treatment of fragile X cells with 5-aza-2´-deoxycytidine (5-aza-dC) resulted in reassociation of acetylated histones H3 and H4 with FMR1 and transcriptional reactivation, whereas treatment with trichostatin A (TSA) led to almost complete acetylated histone H4 and little acetylated histone H3 reassociation with FMR1, as well as no detectable transcription. Our results represent the first description of loss of histone acetylation at a specific locus in human disease, and advance understanding of the mechanism of FMR1 transcriptional silencing.

Incidence of Fragile X Syndrome by Newborn Screening for Methylated FMR1 DNA

Fragile X syndrome (FXS) results from a CGG-repeat expansion that triggers hypermethylation and silencing of the FMR1 gene. FXS is referred to as the most common form of inherited intellectual disability, yet its true incidence has never been measured directly by large population screening. Here, we developed an inexpensive and high-throughput assay to quantitatively assess FMR1 methylation in DNA isolated from the dried blood spots of 36,124 deidentified newborn males. This assay displays 100% specificity and 100% sensitivity for detecting FMR1 methylation, successfully distinguishing normal males from males with full-mutation FXS. Furthermore, the assay can detect excess FMR1 methylation in 82% of females with full mutations, although the methylation did not correlate with intellectual disability. With amelogenin PCR used for detecting the presence of a Y chromosome, this assay can also detect males with Klinefelter syndrome (KS) (47, XXY). We identified 64 males with FMR1 methylation and, after confirmatory testing, found seven to have full-mutation FXS and 57 to have KS. Because the precise incidence of KS is known, we used our observed KS incidence as a sentinel to assess ascertainment quality and showed that our KS incidence of 1 in 633 newborn males was not significantly different from the literature incidence of 1 in 576 (p = 0.79). The seven FXS males revealed an FXS incidence in males of 1 in 5161 (95% confidence interval of 1 in 10,653-1 in 2500), consistent with some earlier indirect estimates. Given the trials now underway for possible FXS treatments, this method could be used in newborn or infant screening as a way of ensuring early interventions for FXS.

Identification of Novel FMR1 Variants by Massively Parallel Sequencing in Developmentally Delayed Males

Fragile X syndrome (FXS), the most common inherited form of developmental delay, is typically caused by CGG‐repeat expansion in FMR1. However, little attention has been paid to sequence variants in FMR1. Through the use of pooled‐template massively parallel sequencing, we identified 130 novel FMR1 sequence variants in a population of 963 developmentally delayed males without CGG‐repeat expansion mutations. Among these, we identified a novel missense change, p.R138Q, which alters a conserved residue in the nuclear localization signal of FMRP. We have also identified three promoter mutations in this population, all of which significantly reduce in vitro levels of FMR1 transcription. Additionally, we identified 10 noncoding variants of possible functional significance in the introns and 3′‐untranslated region of FMR1, including two predicted splice site mutations. These findings greatly expand the catalog of known FMR1 sequence variants and suggest that FMR1 sequence variants may represent an important cause of developmental delay.

Mosaic FMR1 Deletion Causes Fragile X Syndrome and Can Lead to Molecular Misdiagnosis : A Case Report and Review of the Literature

The most common cause of fragile X syndrome is expansion of a CGG trinucleotide repeat in the 5′UTR of FMR1. This expansion leads to transcriptional silencing of the gene. However, other mutational mechanisms, such as deletions of FMR1, also cause fragile X syndrome. The result is the same for both the expansion mediated silencing and deletion, absence of the gene product, FMRP. We report here on an 11‐year‐old boy with a cognitive and behavioral profile with features compatible with, but not specific to, fragile X syndrome. A mosaic deletion of 1,013,395 bp was found using high‐density X chromosome microarray analysis followed by sequencing of the deletion breakpoints. We review the literature of FMR1 deletions and present this case in the context of other FMR1 deletions having mental retardation that may or may not have the classic fragile X phenotype.

Targeted comparative genomic hybridization array for the detection of single- and multiexon gene deletions and duplications

Purpose: To develop a high resolution microarray based method to detect single- and multiexons gene deletions and duplications.Methods: We have developed a high-resolution comparative genomic hybridization array to detect single- and multiexon deletions and duplications in a large set of genes on a single microarray, using the NimbleGen 385K array with an exon-centric design.Results: We have successfully developed, validated, and implemented a targeted gene comparative genomic hybridization arrays for detecting single- and multiexon deletions and duplication in autosomal and X-linked disease-associated genes.Conclusion: The comparative genomic hybridization arrays can be adopted readily by clinical molecular diagnostic laboratories as a rapid, cost-effective, highly sensitive, and accurate approach for the detection of single- and multiexon deletions or duplications, particularly in cases where direct sequencing fails to identify a mutation.

Genetic Screening for OPA1 and OPA3 Mutations in Patients with Suspected Inherited Optic Neuropathies

PURPOSE Autosomal-dominant optic atrophy (DOA) is one of the most common inherited optic neuropathies, and it is genetically heterogeneous, with mutations in both OPA1 and OPA3 known to cause disease. Approximately 60% of cases harbor OPA1 mutations, whereas OPA3 mutations have been reported in only 2 pedigrees with DOA and premature cataracts. The aim of this study was to determine the yield of OPA1 and OPA3 screening in a cohort of presumed DOA cases referred to a tertiary diagnostic laboratory. DESIGN Retrospective case series. PARTICIPANTS One hundred eighty-eight probands with bilateral optic atrophy referred for molecular genetic investigations at a tertiary diagnostic facility: 38 patients with an autosomal-dominant pattern of inheritance and 150 sporadic cases. METHODS OPA1 and OPA3 genetic testing was initially performed using polymerase chain reaction-based sequencing methods. The presence of large-scale OPA1 and OPA3 genomic rearrangements was assessed further with a targeted comparative genomic hybridization microarray platform. The 3 primary Leber hereditary optic neuropathy (LHON) mutations, m.3460G→>A, m.11778G→A, and m.14484T→C, also were screened in all patients. MAIN OUTCOME MEASURES The proportion of patients with OPA1 and OPA3 pathogenic mutations. The clinical profile observed in molecularly confirmed DOA cases. RESULTS Twenty-one different OPA1 mutations were found in 27 (14.4%) of the 188 probands screened. The mutations included 6 novel pathogenic variants and the first reported OPA1 initiation codon mutation at c.1A→T. An OPA1 missense mutation, c.239A→G (p.Y80C), was identified in an 11-year-old black girl with optic atrophy and peripheral sensorimotor neuropathy in her lower limbs. The OPA1 detection rate was significantly higher among individuals with a positive family history of visual failure (50.0%) compared with sporadic cases (5.3%). The primary LHON screen was negative in the patient cohort, and additional molecular investigations did not reveal any large-scale OPA1 rearrangements or OPA3 genetic defects. The mean baseline visual acuity for the OPA1-positive group was 0.48 logarithm of the minimum angle of resolution (units mean Snellen equivalent, 20/61; range, 20/20-20/400; 95% confidence interval, 20/52-20/71), and visual deterioration occurred in 54.2% of patients during follow-up. CONCLUSIONS OPA1 mutations are the most common genetic defects identified in patients with suspected DOA, whereas OPA3 mutations are very rare in isolated optic atrophy cases.

Characterization of an unusual deletion of the galactose-1-phosphate uridyl transferase ( GALT ) gene

Purpose: We previously reported a deletion of the Galactose-1-Phosphate Uridyl Transferase (GALT) gene. This deletion can cause apparent homozygosity for variants located on the opposite allele, potentially resulting in a discrepancy between the biochemical phenotype and the apparent genotype in an individual. The purpose of this study was to determine the deletion breakpoints, allowing the development of a rapid and reliable molecular test for the mutation.Methods: A Polymerase Chain Reaction walking strategy was used to map the 5′ and 3′ breakpoints. The junction fragment was amplified and sequenced to precisely characterize the deletion breakpoints.Results: The deletion has a bipartite structure involving two large segments of the GALT gene, while retaining a short internal segment of the gene. Molecular characterization allowed the development of a deletion specific Polymerase Chain Reaction-based assay. In 25 individuals who had a biochemical carrier galactosemia phenotype, but tested negative for 8 common GALT gene variants, 3 carried this deletion.Conclusion: This deletion occurs at an appreciable frequency and should be considered when there is a discrepancy between the genotype and biochemical phenotype. Many of the individuals carrying the allele were of Ashkenazi Jewish ancestry suggesting that the deletion may be a common cause of galactosemia in that population.

Origins, distribution and expression of the Duarte-2 (D2) allele of galactose-1-phosphate uridylyltransferase

Duarte galactosemia is a mild to asymptomatic condition that results from partial impairment of galactose-1-phosphate uridylyltransferase (GALT). Patients with Duarte galactosemia demonstrate reduced GALT activity and carry one profoundly impaired GALT allele (G) along with a second, partially impaired GALT allele (Duarte-2, D2). Molecular studies reveal at least five sequence changes on D2 alleles: a p.N314D missense substitution, three intronic base changes and a 4 bp deletion in the 5′ proximal sequence. The four non-coding sequence changes are unique to D2. The p.N314D substitution, however, is not; it is found together with a silent polymorphism, p.L218(TTA), on functionally normal Duarte-1 alleles (D1, also called Los Angeles or LA alleles). The HapMap database reveals that p.N314D is a common human variant, and cross-species comparisons implicate D314 as the ancestral allele. The p.N314D substitution is also functionally neutral in mammalian cell and yeast expression studies. In contrast, the 4 bp 5′ deletion characteristic of D2 alleles appears to be functionally impaired in reporter gene transfection studies. Here we present allele-specific qRT–PCR evidence that D2 alleles express less mRNA in vivo than their wild-type counterparts; the difference is small but statistically significant. Furthermore, we characterize the prevalence of the 4 bp deletion in GG, NN and DG populations; the deletion appears exclusive to D2 alleles. Combined, these data strongly implicate the 4 bp 5′ deletion as a causal mutation in Duarte galactosemia and suggest that direct tests for this deletion, as proposed here, could enhance or supplant current tests, which define D2 alleles on the basis of the presence and absence of linked coding sequence polymorphisms.

Molecular diagnostic testing for congenital disorders of glycosylation (CDG): detection rate for single gene testing and next generation sequencing panel testing.

Congenital disorders of glycosylation (CDG) are comprised of over 60 disorders with the majority of defects residing within the N-glycosylation pathway. Approximately 20% of patients do not survive beyond five years of age due to widespread organ dysfunction. A diagnosis of CDG is based on abnormal glycosylation of transferrin but this method cannot identify the specific gene defect. For many individuals diagnosed with CDG the gene defect remains unknown. To improve the molecular diagnosis of CDG we developed molecular testing for 25 CDG genes including single gene testing and next generation sequencing (NGS) panel testing. From March 2010 through November 2012, a total of 94 samples were referred for single gene testing and 68 samples were referred for NGS panel testing. Disease causing mutations were identified in 24 patients resulting in a molecular diagnosis rate of 14.8%. Coverage of the 24 CDG genes using panel testing and whole exome sequencing (WES) was compared and it was determined that many exons of these genes were not adequately covered using a WES approach and a panel approach may be the preferred first option for CDG patients. A collaborative effort between physicians, researchers and diagnostic laboratories will be very important as NGS testing using panels and exome becomes more widespread. This technology will ultimately improve the molecular diagnosis of patients with CDG in hard to solve cases.

Commentary on population screening for fragile X syndrome

In this issue of Genetics in Medicine, Hill et al.1 conduct a systematic review of studies investigating population screening for fragile X syndrome (FXS) among two groups: women of reproductive age and newborns. Most of the studies obtained through their review focus on the psychosocial and counseling issues of screening adult women of reproductive age for the FMR1 mutation alleles, with one small pilot study examining newborn screening for FXS. Because of the unique biology of the FMR1 mutation, population screening for FXS raises concerns that are not found in population screening for disorders such as phenylketonuria and cystic fibrosis. For FMR1, two mutation classes are of medical concern: the premutation and the full mutation. Male premutation carriers pass on the premutation to all their daughters and are themselves at risk of developing fragile X-associated tremor/ataxia syndrome (FXTAS) later in life. Female premutation carriers are at risk of having a child with FXS, of having fragile X-associated primary ovarian insufficiency (FXPOI) and of developing FXTAS; although the latter occurs at a much lower rate than in male carriers. Full mutation carrier males almost always have frank intellectual disabilities. The full mutation carried by women, however, is incompletely penetrant and variably expressed. This has led to a concern that identifying full mutation carrier females via population screening programs could lead to stigmatization of clinically unaffected females. For all these reasons, current guidelines recommend that population screening for FXS be limited to well-defined clinical research protocols. In their review, Hill et al.1 find that screening adult women for FMR1 mutations is less controversial than newborn screening for FXS. Among adult women, preconception screening is preferred to screening during pregnancy. As Hill et al.1 note, preconception screening would actually serve two purposes: assessment of a woman’s risk for having a child with FXS and assessment of her risk for FXPOI. Voluntary screening of adult women for FMR1 mutations to assess reproductive risk is generally viewed favorably by women, even among those who chose not to be screened. To date, there has been no evidence that offering voluntary screening to this population has any negative impact. As with any population screening for a genetic condition, family members would be identified as mutation carriers through cascade testing. Unique to FMR1 mutations, this also serves as predictive testing for the premutation lateonset disorders of FXPOI and FXTAS, in addition to the risk for offspring with FXS. This raises concerns of the impact on family members who did not consent to being screened. These concerns, however, are similar to those raised in testing for familial cancer predisposition syndromes, such as BRCA1 and BRCA2 testing. Unlike newborn screening, population screening of adult women of reproductive age would be done with their fully informed consent, thereby reducing the negative impact of screening. Access to education and expert genetic counseling services would be needed to help balance the risk to benefit ratio for each unique family. Turning to population screening for newborns brings up different concerns. The identification of FXS males in the newborn period would offer the opportunity for early educational intervention, prevent the “diagnostic odyssey” of trying to establish a diagnosis in an affected child, and inform couples about their risk of having another child with FXS. However, there are three major concerns regarding screening for FMR1 mutations during the newborn period. First, there is no diagnostic test to determine which full mutation carrier females are destined to be affected with FXS, and identifying all full mutation carrier females raises the concern that these girls will be at risk for the “vulnerable child syndrome,” where the expectation of disease itself causes disease or worsens subtle manifestations of disease. One way to avoid the vulnerable child syndrome is of course to simply not screen infant girls for FXS; however, excluding girls from screening leads to questions of equity in screening, as full mutation carrier females who are affected with FXS could clearly benefit from early detection, as would males. A second concern is that population screening for FMR1 mutations may result in the incidental identification of infants with sex chromosome aneuploidies. For example, Klinefelter syndrome is approximately eight times more prevalent than FXS in males, whereas Turner syndrome, with a prevalence of about one in 4,000, has a slightly higher prevalence than full mutation carrier females. Therefore, for every infant identified with the FMR1 full mutation, approximately five infants will be identified with a sex chromosome aneuploidy. As with the identification of full mutation carrier females, early identification recognition of sex chromosome aneuploidies can be beneficial. However, stigmatization and the vulnerable child syndrome are also concerns for individuals identified with sex chromosome aneuploidies, because they may show no signs or only mild clinical manifestations of the disease in childhood. Finally, the third concern is the identification of premutation carriers, a significant fraction of whom will be destined to develop adult-onset conditions. Predictive screening of infants for adult-onset conditions is clearly not a goal of newborn screening. Sizing CGG repeats would lead to the detection of premutations, although programmatic decisions could be made not to report premutations. Using aberrant FMR1 methylation as a screening tool for FXS circumvents the problem of detecting premutation carriers, as opposed to detection of expanded CGG repeats. Unfortunately, methylation analysis in females cannot accurately predict whether a girl will be affected with FXS. Development of other high-throughput technologies, perhaps, such as quantification of the FMR1 protein, might provide a solution for these issues. In addition, as Hill et al.1 rightly point out, more studies examining the psychosocial impact of newborn screening are needed in general to better understand the risks and benefits of FMR1 screening. From the Department of Human Genetics, Emory University School of Medicine, Atlanta, GA.