Identification of a novel epigenetic marker for typical and mosaic presentations of Fragile X syndrome.

BACKGROUND: Fragile X syndrome (FXS) is primarily due to CGG repeat expansions in the FMR1 gene. FMR1 alleles are classified as normal (N), intermediate (I), premutation (PM), and full mutation (FM). FXS patients often carry an FM, but size mosaicism can occur. Additionally, loss of methylation boundary upstream of repeats results in de novo methylation spreading to FMR1 promoter in FXS patients. RESEARCH DESIGN AND METHODS: This pilot study investigated the methylation boundary and adjacent regions in 66 males with typical and atypical FXS aged 1 to 30 years (10.86 ± 6.48 years). AmplideX FMR1 mPCR kit was used to discriminate allele profiles and methylation levels. CpG sites were assessed by pyrosequencing. RESULTS: 40 out of 66 FXS patients (60.6%) showed an exclusive FM (n = 40), whereas the remaining (n = 26) exhibited size mosaicism [10 PM_FM (15.15%); 10 N_FM (15.15%); 2 N_PM_FM (3%)]. Four patients (6.1%) had deletions near repeats. Noteworthy, a CpG within FMR1 intron 2 displayed hypomethylation in FXS patients and hypermethylation in controls, demonstrating remarkable specificity, sensitivity, and accuracy when a methylation threshold of 69.5% was applied. CONCLUSIONS: Since intragenic methylation is pivotal in gene regulation, the intronic CpG might be a novel epigenetic biomarker for FXS diagnosis.

Understanding the Landscape of X-linked Variants Causing Intellectual Disability in Females Through Extreme X Chromosome Inactivation Skewing.

Intellectual disability (ID) affects 30% more males than females. This sex bias can be attributed to the enrichment of genes on the X chromosome playing essential roles in the central nervous system and their hemizygous state on males. Moreover, as a result of X chromosome inactivation (XCI), most genes on one of the X chromosomes in female somatic cells are epigenetically silenced, so that females carrying X-linked variants are not expected to be so severely affected as males. Consequently, the knowledge about X-linked ID (XLID) in females is still scarce. Herein, we used extreme XCI skewing (≥ 90%) to predict X-linked variants in females with idiopathic ID. XCI profiles from 53 probands were estimated from blood and buccal mucosa through a methylation-sensitive AR/RP2 assay. DNA samples with extreme XCI skewing were then submitted to array-comparative genomic hybridization and whole-exome sequencing. Seven females (13.2%) exhibited extreme XCI skewing, a percentage significantly higher than expected for healthy females in our population. XLID-potentially related variants were identified in five patients with extreme XCI skewing, including one pathogenic rstructural rearrangement [der(X) chromosome from a t(X;2)] and four single nucleotide variants in NLGN4X, HDAC8, TAF1, and USP9X genes, two of which affecting XCI escape genes. XCI skewing showed to be an outstanding approach for the characterization of molecular mechanisms underlying XLID in females. Beyond expanding the spectrum of variants/phenotypes associated with ID, our results pointed to compensatory biological pathways underlying XCI and uncover new insights into the involvement of escape genes on XLID, impacting genetic counseling.

Maternal 5mCpG Imprints at the PARD6G-AS1 and GCSAML Differentially Methylated Regions Are Decoupled From Parent-of-Origin Expression Effects in Multiple Human Tissues.

A hallmark of imprinted genes in mammals is the occurrence of parent-of-origin-dependent asymmetry of DNA cytosine methylation (5mC) of alleles at CpG islands (CGIs) in their promoter regions. This 5mCpG asymmetry between the parental alleles creates allele-specific imprinted differentially methylated regions (iDMRs). iDMRs are often coupled to the transcriptional repression of the methylated allele and the activation of the unmethylated allele in a tissue-specific, developmental-stage-specific and/or isoform-specific fashion. iDMRs function as regulatory platforms, built through the recruitment of chemical modifications to histones to achieve differential, parent-of-origin-dependent chromatin segmentation states. Here, we used a comparative computational data mining approach to identify 125 novel constitutive candidate iDMRs that integrate the maximal number of allele-specific methylation region records overlapping CGIs in human methylomes. Twenty-nine candidate iDMRs display gametic 5mCpG asymmetry, and another 96 are candidate secondary iDMRs. We established the maternal origin of the 5mCpG imprints of one gametic (PARD6G-AS1) and one secondary (GCSAML) iDMRs. We also found a constitutively hemimethylated, nonimprinted domain at the PWWP2AP1 promoter CGI with oocyte-derived methylation asymmetry. Given that the 5mCpG level at the iDMRs is not a sufficient criterion to predict active or silent locus states and that iDMRs can regulate genes from a distance of more than 1 Mb, we used RNA-Seq experiments from the Genotype-Tissue Expression project and public archives to assess the transcriptional expression profiles of SNPs across 4.6 Mb spans around the novel maternal iDMRs. We showed that PARD6G-AS1 and GCSAML are expressed biallelically in multiple tissues. We found evidence of tissue-specific monoallelic expression of ZNF124 and OR2L13, located 363 kb upstream and 419 kb downstream, respectively, of the GCSAML iDMR. We hypothesize that the GCSAML iDMR regulates the tissue-specific, monoallelic expression of ZNF124 but not of OR2L13. We annotated the non-coding epigenomic marks in the two maternal iDMRs using data from the Roadmap Epigenomics project and showed that the PARD6G-AS1 and GCSAML iDMRs achieve contrasting activation and repression chromatin segmentations. Lastly, we found that the maternal 5mCpG imprints are perturbed in several hematopoietic cancers. We conclude that the maternal 5mCpG imprints at PARD6G-AS1 and GCSAML iDMRs are decoupled from parent-of-origin transcriptional expression effects in multiple tissues.

Population genetic structure of Guiana dolphin (Sotalia guianensis) from the southwestern Atlantic coast of Brazil.

Sotalia guianensis is a small dolphin that is vulnerable to anthropogenic impacts. Along the Brazilian Atlantic coast, this species is threatened with extinction. A prioritized action plan for conservation strategies relies on increased knowledge of the population. The scarcity of studies about genetic diversity and assessments of population structure for this animal have precluded effective action in the region. Here, we assessed, for the first time, the genetic differentiation at 14 microsatellite loci in 90 S. guianensis specimens stranded on the southeastern Atlantic coast of the State of Espírito Santo, Brazil. We estimated population parameters and structure, measured the significance of global gametic disequilibrium and the intensity of non-random multiallelic interallelic associations and constructed a provisional synteny map using Bos taurus, the closest terrestrial mammal with a reference genome available. All microsatellite loci were polymorphic, with at least three and a maximum of ten alleles each. Allele frequencies ranged from 0.01 to 0.97. Observed heterozygosity ranged from 0.061 to 0.701. The mean inbreeding coefficient was 0.103. Three loci were in Hardy-Weinberg disequilibrium even when missing genotypes were inferred. Although 77 of the 91 possible two-locus associations were in global gametic equilibrium, we unveiled 13 statistically significant, sign-based, non-random multiallelic interallelic associations in 10 two-locus combinations with either coupling (D' values ranging from 0.782 to 0.353) or repulsion (D' values -0.517 to -1.000) forces. Most of the interallelic associations did not involve the major alleles. Thus, for either physically or non-physically linked loci, measuring the intensity of non-random interallelic associations is important for defining the evolutionary forces at equilibrium. We uncovered a small degree of genetic differentiation (FST = 0.010; P-value = 0.463) with a hierarchical clustering into one segment containing members from the southern and northern coastal regions. The data thus support the scenario of little genetic structure in the population of S. guianensis in this geographic area.

Erratum to: Familial chromosomal translocation X; 22 associated with infertility and recurrent X mosaicism.

[This corrects the article DOI: 10.1186/s13039-016-0249-5.].

Familial chromosomal translocation X; 22 associated with infertility and recurrent X mosaicism.

BACKGROUND: Individuals with apparently balanced translocations, often, show no clinical findings. However, in meiosis, translocations tend to cause errors on chromosome disjunction and the ones involving sex chromosomes have particular implications for the phenotype. Male carriers of balanced X-autosome translocations are almost invariably infertile due to interruption of the spermatogenesis, but the mechanism is not fully understood. CASE PRESENTATION: In this case report, we performed a combination of classical cytogenetics (G-banding), molecular cytogenetics (fluorescence in situ hybridization and X-chromosome inactivation study), and cytogenomics (microarray-based comparative genomic hybridization) techniques for characterization of an inherited (X;22) translocation in a family originally referred for infertility investigation. Both proband and his sister are infertile and present the maternally inherited translocation. Interestingly, the maternal grandmother was mosaic for X chromosome monosomy suggesting that the t(X;22) in the proband's mother arose by errors at oogenesis. The presence of the same mosaicism of the X chromosome in the proband's aunt is consistent with this consideration. Array- CGH analysis showed no constitutional pathogenic gains or losses in the translocation carriers. The X-chromosome inactivation studies revealed that the translocated X;22 was active in 99.3% of cells in the mother and in 88% of cells in the daughter. We suggest that incomplete skewing of X inactivation (>97 %) of the daughter could justify the infertility. This study is the first description of recurrent mosaicism of the X chromosome associated with a familial X-autosome translocation. CONCLUSIONS: The phenotype of infertility was probably caused by disruption of spermatogenesis due to gametogenesis specific errors resulted from meiotic pairing and segregation anomalies on the translocated chromosomes.

Trisomy 21 Alters DNA Methylation in Parent-of-Origin-Dependent and -Independent Manners.

The supernumerary chromosome 21 in Down syndrome differentially affects the methylation statuses at CpG dinucleotide sites and creates genome-wide transcriptional dysregulation of parental alleles, ultimately causing diverse pathologies. At present, it is unknown whether those effects are dependent or independent of the parental origin of the nondisjoined chromosome 21. Linkage analysis is a standard method for the determination of the parental origin of this aneuploidy, although it is inadequate in cases with deficiency of samples from the progenitors. Here, we assessed the reliability of the epigenetic 5mCpG imprints resulting in the maternally (oocyte)-derived allele methylation at a differentially methylated region (DMR) of the candidate imprinted WRB gene for asserting the parental origin of chromosome 21. We developed a methylation-sensitive restriction enzyme-specific PCR assay, based on the WRB DMR, across single nucleotide polymorphisms (SNPs) to examine the methylation statuses in the parental alleles. In genomic DNA from blood cells of either disomic or trisomic subjects, the maternal alleles were consistently methylated, while the paternal alleles were unmethylated. However, the supernumerary chromosome 21 did alter the methylation patterns at the RUNX1 (chromosome 21) and TMEM131 (chromosome 2) CpG sites in a parent-of-origin-independent manner. To evaluate the 5mCpG imprints, we conducted a computational comparative epigenomic analysis of transcriptome RNA sequencing (RNA-Seq) and histone modification expression patterns. We found allele fractions consistent with the transcriptional biallelic expression of WRB and ten neighboring genes, despite the similarities in the confluence of both a 17-histone modification activation backbone module and a 5-histone modification repressive module between the WRB DMR and the DMRs of six imprinted genes. We concluded that the maternally inherited 5mCpG imprints at the WRB DMR are uncoupled from the parental allele expression of WRB and ten neighboring genes in several tissues and that trisomy 21 alters DNA methylation in parent-of-origin-dependent and -independent manners.

5meCpG epigenetic marks neighboring a primate-conserved core promoter short tandem repeat indicate X-chromosome inactivation.

X-chromosome inactivation (XCI) is the epigenetic transcriptional silencing of an X-chromosome during the early stages of embryonic development in female eutherian mammals. XCI assures monoallelic expression in each cell and compensation for dosage-sensitive X-linked genes between females (XX) and males (XY). DNA methylation at the carbon-5 position of the cytosine pyrimidine ring in the context of a CpG dinucleotide sequence (5meCpG) in promoter regions is a key epigenetic marker for transcriptional gene silencing. Using computational analysis, we revealed an extragenic tandem GAAA repeat 230-bp from the landmark CpG island of the human X-linked retinitis pigmentosa 2 RP2 promoter whose 5meCpG status correlates with XCI. We used this RP2 onshore tandem GAAA repeat to develop an allele-specific 5meCpG-based PCR assay that is highly concordant with the human androgen receptor (AR) exonic tandem CAG repeat-based standard HUMARA assay in discriminating active (Xa) from inactive (Xi) X-chromosomes. The RP2 onshore tandem GAAA repeat contains neutral features that are lacking in the AR disease-linked tandem CAG repeat, is highly polymorphic (heterozygosity rates approximately 0.8) and shows minimal variation in the Xa/Xi ratio. The combined informativeness of RP2/AR is approximately 0.97, and this assay excels at determining the 5meCpG status of alleles at the Xp (RP2) and Xq (AR) chromosome arms in a single reaction. These findings are relevant and directly translatable to nonhuman primate models of XCI in which the AR CAG-repeat is monomorphic. We conducted the RP2 onshore tandem GAAA repeat assay in the naturally occurring chimeric New World monkey marmoset (Callitrichidae) and found it to be informative. The RP2 onshore tandem GAAA repeat will facilitate studies on the variable phenotypic expression of dominant and recessive X-linked diseases, epigenetic changes in twins, the physiology of aging hematopoiesis, the pathogenesis of age-related hematopoietic malignancies and the clonality of cancers in human and nonhuman primates.

Gametic phase disequilibrium between the syntenic multiallelic HTG4 and HMS3 markers widely used for parentage testing in Thoroughbred horses.

Validation of parentage and horse breed registries through DNA typing relies on estimates of random match probabilities with DNA profiles generated from multiple polymorphic loci. Of the twenty-seven microsatellite loci recommended by the International Society for Animal Genetics for parentage testing in Thoroughbred horses, eleven are located on five chromosomes. An important aspect in determining combined exclusion probabilities is the ascertainment of the genetic linkage status of syntenic markers, which may affect reliable use of the product rule in estimating random match probabilities. In principle, linked markers can be in gametic phase disequilibrium (GD). We aimed at determining the extent, by frequency and strength, of GD between the HTG4 and HMS3 multiallelic loci, syntenic on chromosome 9. We typed the qualified offspring (n (1) = 27; n (2) = 14) of two Quarter Bred stallions (registered by the Brazilian Association of Quarter Horse Breeders) and 121 unrelated horses from the same breed. In the 41 informative meioses analyzed, the frequency of recombination between the HTG4 and HMS3 loci was 0.27. Consistent with genetic map distances, this recombination rate does not fit to the theoretical distribution for independently segregated markers. We estimated sign-based D' coefficients as a measure of GD, and showed that the HTG4 and HMS3 loci are in significant, yet partial and weak, disequilibrium, with two allele pairs involved (HTG4 M/HMS3 P, D'(+) = 0.6274; and HTG4 K/HMS3 P, D'(-) = -0.6096). These results warn against the inadequate inclusion of genetically linked markers in the calculation of combined power of discrimination for Thoroughbred parentage validation.

Genetic data on 15 STR autosomal loci for a sample population of the Northern Region of the State of Rio de Janeiro, Brazil.

Allele frequencies data, paternity and forensic parameters for 15 autosomal short tandem repeat (STR) autosomal markers (D8S1179, D21S11, D7S820, CSF1PO, D5S818, D3S1358, TH01, D13S317, D16S539, D2S1338, TPOX, D19S433, vWA, D18S51, FGA) were determined for a sample of 494 unrelated individuals undergoing kinship analysis and molecular cytogenetic testing from the population of the city of Campos dos Goytacazes, Northern State of Rio de Janeiro, Brazil. The loci with the highest polymorphism information content were D18S51 (0.874), D2S1338 (0.853), FGA (0.852), D21S11 (0.838). The combined power of discrimination and the combined power of exclusion were 0.999999999999999 and 0.999526684, respectively. At the available common loci CSF1PO, TH01, TPOX, vWA, D16S539, D7S820 and D13S317, allele frequencies were compared with population databases from State of Alagoas, State of Amazonas, State of São Paulo (Brazilian mulattoes, descendants of Europeans, Africans or Asians), State of Mato Grosso do Sul and State of Rio de Janeiro. No significant distances were observed. The interpopulation genetic distance (Fst coefficients) to the present database, ranged 0.0022 (p=0.446) (Northern State Rio de Janeiro-State of São Paulo European-descendants) to 0.0138 (p=0.993) (Northern State Rio de Janeiro-State of São Paulo Asian-descendants). The Asian-descendants Brazilians are the least admixed. All other groups are admixed as one unique population.