Ko-ichiro Yoshiura

Detection of cell free placental DNA in maternal plasma: direct evidence from three cases of confined placental mosaicism

Fetal cells are consistently found in the maternal circulation, and polymerase chain reaction based studies have led to the identification of cell free fetal DNA (fetal DNA) in maternal blood. Approximately 1.2 nucleated fetal cells/ml of whole blood from women carrying a male fetus were detectable,1 and relative enrichment of fetal DNA was detected in the maternal plasma and serum.2 The amount of fetal DNA in the maternal blood increases with progression of pregnancy, and 3.4–6.2% of the total maternal plasma DNA during pregnancy was of fetal origin.3 Therefore, cell free fetal DNA in pregnant women’s plasma is useful for non-invasive prenatal diagnosis, especially for detection of fetal sex,3,4 RhD blood type,5–7 and gene mutations of paternal origin.8–10 Previous studies indicated that pregnant women with pre-eclampsia,11 placenta previa12 and fetal chromosome abnormalities13 tend to have elevated levels of fetal DNA in their plasma. Since functional or structural abnormalities of the placenta and destruction of the trophoblast may be associated with these diseases,14 it is suggested that cell free fetal DNA is of placental origin. This implies that quantitative analysis of fetal DNA may be valuable to screen for placental dysfunction. Ng et al15 recently reported that placental mRNA is present in the maternal circulation, and suggested that the same might occur for placental DNA,16 However, no direct evidence has been given for placenta derived cell free fetal DNA in the maternal blood, although its clinical use is growing.17 Confined placental mosaicism, which is defined by the presence of abnormal karyotypes only in the placenta while the fetus itself is usually diploid,18 may occur through a loss of the extra chromosome in a trisomic zygote during an early mitotic cell division in only the …

Complex low-copy repeats associated with a common polymorphic inversion at human chromosome 8p23

To characterize a submicroscopic, common 8p23 polymorphic inversion, we constructed a complete BAC/PAC-based physical map covering the entire 4.7-Mb inversion and its flanking regions. Two low-copy repeats (LCRs), REPD (approximately 1.3 Mb) and REPP (approximately 0.4 Mb), were identified at each of the inversion breakpoints. Comparison of the REPD and REPP sequences revealed that REPD showed high homology to REPP, with complex direct and inverted orientations. REPD and REPP contain six and five olfactory receptor gene-related sequences, respectively. LCRs at 8p23 showed multiple FISH signals from an Old World monkey to the human. Thus, multiplication of the LCR may have occurred at least 21-25 million years ago. We also investigated the frequency of the 4.7-Mb inversion in the general Japanese population and found that the allele frequency for the 8p23 inversion was estimated to be 27%.

BAC array CGH reveals genomic aberrations in idiopathic mental retardation

Array using 2,173 BAC clones covering the whole human genome has been constructed. All clones spotted were confirmed to show a unique signal at the predicted chromosomal location by FISH analysis in our laboratory. A total of 30 individuals with idiopathic mental retardation (MR) were analyzed by comparative genomic hybridization using this array. Three deletions, one duplication, and one unbalanced translocation could be detected in five patients, which are likely to contribute to MR. The constructed array was shown to be an efficient tool for the detection of pathogenic genomic rearrangements in MR patients as well as copy number polymorphisms (CPNs).

Molecular characterization of del(8)(p23.1p23.1) in a case of congenital diaphragmatic hernia

A 36‐week‐old fetus was referred to the medical center because of his cystic mass and fluid in left thoracic cavity, and was delivered by cesarean section to manage neonatal problems at 37 weeks of gestation. Emergent surgical repair of the left diaphragmatic hernia was performed, but severe hypoxia persisted, and he expired on the following day. Chromosome analysis of cultured amniotic fluid cells indicated 46,XY,del(8)(p23.1p23.1). This is the fourth case of 8p23.1 deletion associated with diaphragmatic hernia. Microarray comparative genomic hybridization analysis using DNA of cultured amniotic fluid cells showed that six clones were deleted, which were mapped to the region between two low copy repeats (LCRs) at 8p23.1 previously described. Microsatellite analysis revealed that the deletion was of paternal origin, and his parents did not carry 8p23.1 polymorphic inversion. These data strongly suggested that the 8p23.1 interstitial deletion should have arisen through a different mechanism from that of inv dup del(8p) whose structural abnormality is always of maternal origin and accompanies heterozygous 8p23.1 polymorphic inversion in mother.

Subtelomere specific microarray based comparative genomic hybridisation: a rapid detection system for cryptic rearrangements in idiopathic mental retardation

Mental retardation (MR) occurs in 2–3% of the general population, and more than half of MR patients are categorised as idiopathic—that is, the cause is unknown.1,2 Patients with idiopathic MR are presumed to be affected with certain genetic disorders or undetectable chromosomal abnormalities. MR may also be caused by environmental factors independently or by their interaction with genetic factors. Subtelomeric rearrangements comprise about half of segmental aneusomies,3 and are one of the major causes of MR.4,5 A recent review showed that subtelomeric rearrangements were detected in 131 (5.1%) of 2585 children with MR.1,4–6 Conventional cytogenetic analysis can detect many, but not all, rearrangements, depending on its powers of resolution.4 Other methods, such as fluorescent in situ hybridisation (FISH) using a complete set of subtelomeric probes, multicolour FISH (M-FISH), comparative genomic hybridisation (CGH), spectrum karyotyping, multiple amplifiable probe hybridisation, primed in situ labelling, and genotyping have been designed to detect subtelomeric rearrangements, but none of them is ideal in terms of sensitivity and/or efficiency.4,6 Microarray based CGH is a promising, high throughput method of detecting subtelomeric rearrangements.4 Veltman et al recently reported a microarray CGH system using crude bacterial/plasmid derived artificial chromosome (BAC/PAC) DNA for the analysis of subtelomeric aberrations, and suggested that degenerate oligonucleotide primed (DOP)-PCR products could also be used instead of crude clone DNA, although the performance of DOP-PCR products might be less sensitive.7 We have developed a microarray CGH system to identify rearrangements involving a subtelomeric region, using DOP-PCR that amplifies subtelomeric BAC/PAC DNA. Here we describe details of the method and the results of microarray CGH analyses of five cases of Wolf-Hirschhorn syndrome (WHS) associated with terminal 4p deletions as positive controls, and of 69 patients with idiopathic MR with or without multiple …

Role of DNA methylation and histone H3 lysine 27 methylation in tissue-specific imprinting of mouse Grb10.

ABSTRACT Mouse Grb10 is a tissue-specific imprinted gene with promoter-specific expression. In most tissues, Grb10 is expressed exclusively from the major-type promoter of the maternal allele, whereas in the brain, it is expressed predominantly from the brain type promoter of the paternal allele. Such reciprocally imprinted expression in the brain and other tissues is thought to be regulated by DNA methylation and the Polycomb group (PcG) protein Eed. To investigate how DNA methylation and chromatin remodeling by PcG proteins coordinate tissue-specific imprinting of Grb10, we analyzed epigenetic modifications associated with Grb10 expression in cultured brain cells. Reverse transcriptase PCR analysis revealed that the imprinted paternal expression of Grb10 in the brain implied neuron-specific and developmental stage-specific expression from the paternal brain type promoter, whereas in glial cells and fibroblasts, Grb10 was reciprocally expressed from the maternal major-type promoter. The cell-specific imprinted expression was not directly related to allele-specific DNA methylation in the promoters because the major-type promoter remained biallelically hypomethylated regardless of its activity, whereas gametic DNA methylation in the brain type promoter was maintained during differentiation. Histone modification analysis showed that allelic methylation of histone H3 lysine 4 and H3 lysine 9 were associated with gametic DNA methylation in the brain type promoter, whereas that of H3 lysine 27 regulated by the Eed PcG complex was detected in the paternal major-type promoter, corresponding to its allele-specific silencing. Here, we propose a molecular model that gametic DNA methylation and chromatin remodeling by PcG proteins during cell differentiation cause tissue-specific imprinting in embryonic tissues.

Preferential Paternal Origin of Microdeletions Caused by Prezygotic Chromosome or Chromatid Rearrangements in Sotos Syndrome

Sotos syndrome (SoS) is characterized by pre- and postnatal overgrowth with advanced bone age; a dysmorphic face with macrocephaly and pointed chin; large hands and feet; mental retardation; and possible susceptibility to tumors. It has been shown that the major cause of SoS is haploinsufficiency of the NSD1 gene at 5q35, because the majority of patients had either a common microdeletion including NSD1 or a truncated type of point mutation in NSD1. In the present study, we traced the parental origin of the microdeletions in 26 patients with SoS by the use of 16 microsatellite markers at or flanking the commonly deleted region. Deletions in 18 of the 20 informative cases occurred in the paternally derived chromosome 5, whereas those in the maternally derived chromosome were found in only two cases. Haplotyping analysis of the marker loci revealed that the paternal deletion in five of seven informative cases and the maternal deletion in one case arose through an intrachromosomal rearrangement, and two other cases of the paternal deletion involved an interchromosomal event, suggesting that the common microdeletion observed in SoS did not occur through a uniform mechanism but preferentially arose prezygotically.

Four novel NIPBL mutations in Japanese patients with Cornelia de Lange syndrome

Cornelia de Lange syndrome (CdLS, OMIM #122470) is a multiple congenital anomaly syndrome characterized by dysmorphic facial features, hirsutism, severe growth and developmental delay, and malformed upper limbs [Ireland et al., 1993; Jackson et al., 1993]. The prevalence is estimated to be 1/10,000 [Opitz, 1985]. Recently, two independent groups proved that CdLS is caused by NIPBL mutations [Krantz et al., 2004; Tonkin et al., 2004]. NIPBL consists of 47 exons and encodes delangin, a 2,804 amino-acid protein, from exon 2 to 47. We analyzed 15 Japanese sporadic patients (CdL 1–15) with typical CdLS features (Table I) and their parents after obtaining written informed consent. All protocols in this study were approved by the Committee for the Ethical Issues on Human Genome and Gene analysis, Nagasaki University. Clinical geneticists diagnosed these patients based on mental and growth retardation, and characteristic facial features. Genomic DNA was extracted using a standard protocol. Fourty-six coding exons (from exon 2 to 47) of NIPBL were amplified by PCR as described previously [Krantz et al., 2004] except for exons 4, 33, 37, and 41, of which primers were originally designed (available on request). Sequence analysis was performed as described previously [Kurotaki et al., 2003]. We identified three novel nonsense mutations and one missense mutation in NIPBL among the 15 Japanese patients examined: 1885C>T (R629X) (CdL 4) and 1921G>T (E641X) (CdL 2) in exon 10, 3346G>T (E1116X) (CdL 15) in exon 12, and 5483G>A (R1828Q) (CdL 10) in exon 29. All the four mutations were not found in any of 97 normal Japanese controls or in the JSNP database (http://snp.ims.u-tokyo.ac.jp/). The altered amino acid (R1828Q) was de novo and located in the evolutionally conserved sequences at least in the human, rat, mouse, and fly homologs, thus the change is likely to be pathological. The C-terminal half 1500 amino acids of delangin is well conserved among homologs of flies, worms, plants, and fungi, and is expected to be biologically important [Tonkin et al., 2004], though it was not found to contain any obvious functional domains by analysis using PROSITE (http://kr. expasy.org/cgi-bin/prosite/PSScan.cgi). Three protein truncation mutations at amino acid positions 629, 641, and 1116 and a missense mutation at amino acid position 1828 could lose or impair the C-terminal half function. The Drosophila homolog of NIPBL, Nipped-B, is involved in activating the Ubx and Cut homeobox genes. Ubx suppresses the limb formation by repressing Dll that requires for the distal limb development, and Cut mutations cause leg and wing abnormalities [Tonkin et al., 2004]. Thus, it is plausible that reduced expression of human NIPBL may lead to limb anomalies in CdLS. Interestingly, limb abnormalities (oligodactyly and ulner deficiency) were observed in three of our four patients with a mutation, but only one of seven patients without any mutation whose clinical information was available did show some limb abnormality (oligodactyly), though Gillis et al. [2004] reported that severity of limb defects was not statistically different between mutation-positive and mutation-negative patients. Additionally, three single nucleotide polymorphisms (SNPs), 1151A>G (N384S) in exon 9, 2021A>G (N674S) in exon 10 and 5874T>C (S1958S) in exon 33, were identified, as they were found among normal controls and the second substitution (2021A>G) was previously reported as a SNP [Gillis et al., 2004]. Allele frequencies of the three SNPs in normal Japanese controls are 3.2% (6/186), 13.0% (25/192), and 64.5% (129/200), respectively. To exclude a submicroscopic deletion around NIPBL and its franking regions, fluorescence in situ hybridization (FISH) analysis was performed in 10 of 15 cases on their metaphase chromosomes using two BAC clones covering the NIPBL gene (Table I), RP11-14I21, and RP11-7M4, selected from the UCSC genome browser, 2003 July version (http://genome.ucsc.edu/ cgi-bin/hgGateway). FISH and subsequent photomicroscopy were performed as described previously [Miyake et al., 2004]. However, none of them showed any deletion. We also investigated core promoter regions in 11 affected individuals not having detectable point mutations in the coding regions. Two core promoter regions were identified, ranging 800 to 500 bp (CPR-A) and 400 to þ 200 bp (CPRB) from the beginning of NIPBL cDNA (NM_015384.3) using four different promoter prediction programs: neural network promoter prediction program (http://www.fruitfly.org/seq_ tools/promoter.html), human core-promoter finder (http:// rulai.cshl.org/tools/genefinder/CPROMOTER/human.htm), promoter 2.0 prediction server (http://www.cbs.dtu.dk/services/ promoter/), bioinformatics & molecular analysis section (http:// bimas.dcrt.nih.gov/molbio/proscan/). No nucleotide changes were detected among the 11 patients in the two core promoter regions except for a part of CPR-B sequence ( 60 þ 60), which was hardly determined due to high GC ratio (75.83%), suggesting that promoter mutations in NIBPL is less likely. In conclusion, we identified four novelNIPBLmutations and three SNPs. It is important to describe a full spectrum of phenotype in more patients with positive mutations and establish comprehensive diagnostic criteria. *Correspondence to: Dr. Naomichi Matsumoto, Department of Human Genetics, Yokohama City University Graduate School of Medicine, Fukuura 3-9, Kanazawa-ku, Yokohama 236-0004, Japan. E-mail: naomat@yokohama-cu.ac.jp

Inv dup del(4)(:p14 --> p16.3::p16.3 --> qter) with manifestations of partial duplication 4p and Wolf-Hirschhorn syndrome.

An 8‐year‐old girl with a combination of clinical manifestations of partial duplication 4p and the Wolf–Hirschhorn syndrome was studied. Chromosomal G‐banding and FISH analyses showed a 33.2‐Mb segment of inverted duplication at 4p14‐p16.3 and a 2.8‐Mb segment of deletion at 4p16.3‐pter (including the Wolf–Hirschhorn syndrome critical region). The chromosomes of the parents were normal. Her karyotype was thus 46,XX, inv dup del(4)(:p14 → p16.3::p16.3 → qter) de novo. The inverted duplication deletion was assumed to have arisen through chromatid breakage at 4p16.3, U‐type reunion at the breakpoints to produce a dicentric intermediate, breakage of the dicentric to result in a monocentric, and telomere capture/healing of the broken end. Olfactory receptor gene clusters at 4p16.3 were ruled out as an intermediary of the duplication deletion process.

Identification of eight novel NSD1 mutations in Sotos syndrome

In this study, we validated the spectrum of NSD1 intragenicmutations among 30 newly collected SoS patients.MATERIALS AND METHODSThe subjects studied included 13 Japanese and 17 non-Japanese patients with SoS. No patient with Weaver synromewas included in this study. The 17 non-Japanese casescomprised four Canadians including two Hutterite, threeeach of Brazilians, Germans, and Italians, and one each ofIsraeli-Arab, Israeli, Austrian, and Croatian. Three mainfeatures were considered at the clinical diagnosis: (a) typicalcraniofacial dysmorphology including macrocephaly, highanterior hairline, down slanting palpebral fissures, andprominent jaw, (b) developmental delay (intelligence quo-tient or development quotient ,80), and (c) history ofovergrowth (height and weight .+2 SD). Advanced bone agewas not evaluated, because sufficient data were not available.Adequate clinical information was available in 22 patients. Inthe other eight cases, only limited information was providedfor this study. All patients were referred to us aftermicrodeletions were ruled out by fluorescent in situhybridisation analysis using a P1 derived artificial chromo-some probe (RP11-118M12), as described previously.