Filipe Brum Machado

High-resolution combined linkage physical map of short tandem repeat loci on human chromosome band Xq28 for indirect haemophilia A carrier detection

Summary.  Haemophilia A, the most common severe hereditary bleeding disorder in humans, is chiefly caused by mutations in the coagulation factor VIII F8 gene, which maps on chromosome band Xq28. Linkage analysis with F8 intragenic and/or closely located extragenic short tandem repeat (STR) elements is an effective method for indirect tracing of F8 pathogenic allelic variants in at‐risk families. STR profiling is currently limited to 14 markers, 11 of which are dinucleotide elements. The aim of this study was to define novel polymorphic STR loci for haemophilia A carrier screening. The combined linkage physical map was restricted to a 2.4‐Mb region on Xq28 that hosts 81 annotated genes, F8 inclusive. The inventory was in silico through comparative analyses with three X chromosome reference sequences, using microsatellite mining and validation computer software. Genetic distances for unmapped markers were interpolated on the Rutgers map of the human genome. The effort yielded 94 STR loci: 53 extragenic and 41 intragenic (14 STR elements map on the F8 gene; the other 27 on 19 further genes). The distribution per repeat period size was 61.7% di‐, 5.3% tri‐, 26.6% tetra‐ and 6.4% pentanucleotide loci. The success rate of validation of polymorphism for the new STR loci was 56.3%. For STR elements 0.78 Mb equidistant of the F8 gene, the interpolated downstream genetic length is 3.27 times the upstream genetic length. The inventory represents a 5.7‐fold increase in polymorphic STR loci useful in carrier detection. Genotyping with the upstream extragenic tetra‐ and pentanucleotide markers is thus apprized.

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.

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.

Improved criterion‐referenced assessment in indirect tracking of haemophilia A using a 0.23 cM‐resolution dense polymorphic marker set

Summary.  In haemophilia A, linkage analysis with coagulation factor VIII (F8) intragenic and/or neighbouring extragenic short tandem repeats (STRs) enables indirect tracking F8 pathogenic allelic variant‐carriers. Even where DNA sequencing is available, linkage analysis still has a role if no causative or candidate mutation is unveiled. The cumulative heterozygosity rate of the available multiplexed STRs haplotyping assays rarely reaches 100%. This means that in a proportion of women these loci are uninformative. The norm‐referenced assessment is based on at least one informative marker criterion. We reasoned that by typing a dense market set, spanning a small fraction of recombination, we should be able to improve assessment. The aim of this study was to improve criterion‐referenced assessment in polymorphism segregation analyses using a low‐recombination fraction and dense informative STRs set. The multiplex quantitative fluorescence PCR assay comprises four novel tetranucleotide and pentanucleotide STRs distant ≤0.15 cM from the F8 gene, and three F8 intragenic dinucleotide STRs, mapped to a 0.23 cM interval spanning the F8 on human chromosome band Xq28. We determined heterozygosity rates and allele frequencies from 100 unrelated healthy females. To investigate about segregation stability, we typed 50 true trios (mother, daughter and father) and 50 true mother‐and‐son duos from the general population. The heterozygosity rates for the extragenic markers ranged 0.49–0.76. The 0.23 cM‐resolution heptaplex rendered a cumulative heterozygosity of 0.89 for a minimum of two informative markers, with at least one F8 intragenic. The heptaplex assay enabled improving the criterion‐referenced assessment in indirect carrier‐detection.

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.

Informativeness of a novel multiallelic marker‐set comprising an F8 intron 21 and three tightly linked loci for haemophilia A carriership analysis

Summary.  The extraordinary heterogeneous nature of the deleterious mutations in the F8 gene that lead to functional deficiency of clotting factor VIII in haemophilia A makes routine direct mutation profiling difficult. When direct mutation analysis cannot be performed or a causative/candidate mutation is not found, a second‐line approach to track the defective F8 gene within at‐risk families is linkage genetic analysis with, tried‐and‐tested, F8‐intragenic and/or extragenic non‐recombining multiallelic short tandem repeats (STR). Although several typing STR loci within and around F8 have been described, there is need for improving assessment, because the combined informativeness of available assays rarely reaches 100%. Here, we characterized a newly identified 0.28 cM‐resolution marker‐set, consisting of a dinucleotide STR located on F8 intron 21 (F8Int21; [AC]n) and three extragenic tetranucleotide STR located on GAB3 intron 1 (GAB3Int1; [TAAA]n) and TMLHE intron 1 (TMLHEInt1.1; [GAAA]n and TMLHEInt1.3; [ATTC]n). Heterozygosity rates determined in 100 unrelated females ranged from 0.25 (GAB3Int1) to 0.63 (F8Int21). The set rendered a combined informativeness of 0.91 for at least one marker and 0.60 for a minimum of two loci, with at least one F8‐intragenic. Multiallelic interlocus non‐random association analysis revealed that GAB3Int1 is not in significant gametic disequilibrium (GD) with F8Int21, F8Int9.2, TMLHEInt1.3 or TMLHEInt1.1. Gametic disequilibrium breakdown attests historical recombination between GAB3Int1 and the F8 gene. Through computational analysis of reference assembly sequence data, we note in the GD breakdown region and in the F8 gene a higher than average density of the 13‐mer CCNCCNTNNCCNC consensus motif, commonly associated with recombination hotspots.

A novel informative dinucleotide microsatellite marker located on human factor VIII intron 25.

F. B. MACHADO,* L. P. DUARTE and E. MEDINA-ACOSTA *Hospital Escola Álvaro Alvim, Pelinca, Campos dos Goytacazes, Rio de Janeiro; Núcleo de Diagnóstico e Investigação Molecular, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Parque Califórnia, Campos dos Goytacazes, Rio de Janeiro; and Hemocentro Regional de Campos do Hospital Ferreira Machado, Campos dos Goytacazes, Rio de Janeiro, Brazil

Eyes wide open: the (mis)use of combined power of discrimination for X-linked short tandem repeats

In the field of Genetic Epidemiology it is well established that ignoring interallelic multipoint gametic disequilibrium (GD) introduces false-positive evidence of linkage for sibpair (kinship) analysis, especially in cases of sample deficiencies [1]. In the field of Forensic Genetics this proven concept is, in stark contrast, largely trivialized or ignored. There is an alarming accrual of forensic reports (Online Resource 1) announcing population data on short tandem repeats loci mapping on X chromosome (X-STRs) in which the product rule is applied to syntenic loci (i.e., being located on the same chromosome whether or not there is demonstrable genetic linkage between them), mostly ignoring the second law of Mendelian inheritance, and fundamental notions on genetic linkage status. We believe that the issue has been utterly disregarded, and thus it can no longer go unnoticed without opinion. We center our opinion at the most crucial aspect that underlies meaningful calculation of combined power of discrimination (CPD), as it applies to forensic and medical genetics: accurate knowledge about genetic linkage status of X-STRs loci [2]. X chromosome is endowed with distinctive characteristics mainly derived from how it is inherited by male and female offspring. Contrasting to autosomes, and excluding the pseudoautosomal regions present in both X and Y chromosomes, X chromosome recombination only occurs in females, so every existing X chromosome has evolved two-thirds of its history in them [3]. Syntenic loci may, in principle, be subjected to GD, which is a multifactorial evolutionary dynamic phenomenon that occurs at normal selection, characterized by significant and intensity-variable non-random associations of alleles at different DNA loci, physically mapped on the same (even though not necessarily) chromosomal DNA molecule [4]. GD is affected by, and therefore it is an indicator of, extreme allele frequencies, allele mutation rate, selection, genetic drift, gene flow, recombination, admixture, and the demographic history of populations [5]. A first conundrum with typing syntenic X-STRs loci is whether, and if so how, to compute the genotype evidence (phased our unphased diplotypes and male haplotypes) in favor of a matching hit (i.e., coinciding profiles) or against it (i.e., allelic exclusion). Solving this riddle will help responding the practical question ‘‘what is the evidential value of an X haplotype?’’ This problem is of considerable lesser extent in relation to the field of YSTRs haplotypes [6], from which important lessons ought to be learned. It helps to keep in mind that matching haplotypes can reflect either ‘‘identity by descend’’ (IBD; i.e., related haplotypes with no intervening mutations) or ‘‘identity by Electronic supplementary material The online version of this article (doi:10.1007/s11033-010-0518-z) contains supplementary material, which is available to authorized users.

An IKBKG, and not a G6PD, short tandem repeat marker is used in indirect diagnosis of haemophilia A

Genetic counselling about diagnosis and inheritance of haemophilia A, the most common severe genetic bleeding chromosome X linked recessively inherited disorder in man, are still crucial for comprehensive care of the affected individuals, F8 pathogenic allele variant carriers and close family. In addition to reducing guilt, resentment and blame, ascertainment of the F8 pathogenic allele carrier status is likely to have dramatic impacts in family planning decisions about preimplantation and prenatal genetic diagnosis. Routine direct mutant profiling analysis in haemophilia A families is impractical and costprohibited, particularly in third world countries, due to the heterogeneous nature of the pathogenic mutations, the large size and complexity of the F8 gene. Instead, linkage genetic analysis using multiplelocus F8 intragenic and/or closely located extragenic multiallelic DNA short tandem repeats (STR) is an amenable indirect method of carrier-tracking the defective F8 gene within a family. Two recent studies reported the use of a G6PD (glucose-6-phosphate dehydrogenase) dinucleotide STR element [TG]22 for these purposes [1,2]. In the process of validating in silico the said marker, we found that the reported primer sequences and the STR element refer to the locus IKBKG (inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase gamma), rather than to the G6PD gene, both closely linked at Xq28, approximately 300 kbp downstream of the F8 gene. The expected PCR amplicon comprising this STR element is 188 bp long, as validated in silico using three genome sequences, Fig. 1 Screening of the G6PD gene using Tandem Repeats Finder (http:// tandem.bu.edu/trf/trf.html), however, yielded no suitable STR candidate markers for linkage analysis (not shown). Therefore, we have reassigned the STR marker to the IKBKG locus. The STR element is located within intron 1 of the IKBKG gene and it is also known as UniSTS REN90010 and GBD: 179459. Mutations in the IKBKG gene cause Familial Incontinentia Pigmenti, a X-linked dominant disorder usually lethal prenatally in males [3].

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.