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Abstract By examining 19 human cell lines derived from brain tumors for altered expression of expressed sequence tags (ESTs) in chromosomal band 4q21-22, we detected loss of expression, in 10 cell lines, of two sequences, WI6336 and WI7913. Both corresponded to the c-Jun NH[sub 2]-terminal kinase (JNK) 3. In the present study, genomic cloning revealed that the JNK3 gene consists of 14 exons interrupted by 13 introns; its transcription-initiation site is within exon 3 and the termination codon lies in exon 14. Fluorescence in situ hybridization (FISH) and radiation-hybrid mapping confirmed the gene to 4q21-22. Together with prior evidence that, in JNK3-deficient mice, the JNK3 signaling pathway mediates apoptosis in central nervous tissue, our results suggest that loss of expression of the JNK3 gene may play an important role in the development of brain tumors in humans. [ABSTRACT FROM AUTHOR]

Three types of chromosomal translocations, t(4;14)(p16;q32), t(14;16)(q32;q23), and t(11;14)(q13;q32), are associated with prognosis and the decision making of therapeutic strategy for multiple myeloma (MM). In this study, we developed a new diagnostic modality of the multiplex FISH in immunophenotyped cells in suspension (Immunophenotyped-Suspension-Multiplex (ISM)-FISH). For the ISM-FISH, we first subject cells in suspension to the immunostaining by anti-CD138 antibody and, then, to the hybridization with four different FISH probes for genes of IGH, FGFR3, MAF, and CCND1 tagged by different fluorescence in suspension. Then, cells are analyzed by the imaging flow cytometry MI-1000 combined with the FISH spot counting tool. By this system of the ISM-FISH, we can simultaneously examine the three chromosomal translocations, i.e, t(4;14), t(14;16), and t(11;14), in CD138-positive tumor cells in more than 2.5 × 10 4 nucleated cells with the sensitivity at least up to 1%, possibly up to 0.1%. The experiments on bone marrow nucleated cells (BMNCs) from 70 patients with MM or monoclonal gammopathy of undetermined significance demonstrated the promising qualitative diagnostic ability in detecting t(11;14), t(4;14), and t(14;16) of our ISM-FISH, which was more sensitive compared with standard double-color (DC) FISH examining 200 interphase cells with its best sensitivity up to 1.0%. Moreover, the ISM-FISH showed a positive concordance of 96.6% and negative concordance of 98.8% with standard DC-FISH examining 1000 interphase cells. In conclusion, the ISM-FISH is a rapid and reliable diagnostic tool for the simultaneous examination of three critically important IGH translocations, which may promote risk-adapted individualized therapy in MM., (© 2023. The Author(s).)

MicroRNA (miRNA; miR) is a functionally small non-coding RNA and can negatively regulate gene expression by directly binding to the target gene. Some miRNAs are closely involved in the development and progression of cancer and are abnormally expressed in many cancer types. Therefore, control of the expression of cancer-associated miRNAs is expected as a next-generation drug modality to treat advanced types of cancers with high unmet medical needs. Indeed, miRNA therapeutics, which are based on the functional inhibition of oncogenic miRNA (OncomiR) using antisense oligonucleotides (anti-miR) and the replacement via the introduction of a synthetic miRNA mimic for tumor suppressive miRNA (TS-miR), have been developed. In this review, we summarize cancer-associated miRNAs related to various cancer pathologies and their clinical application to miRNA therapeutics for cancer., (© 2021. The Author(s), under exclusive licence to The Japan Society of Human Genetics.)

Early-onset developmental and epileptic encephalopathy (DEE) is a group of devastating disorders that appear during the neonatal and infantile periods. Despite great progress in the discovery of genes leading to early-onset DEE, many cases with unexplained etiology remain. Furthermore, to date, the association of copy number variations (CNVs) with early-onset DEE has seldom been addressed. Here, we investigated the contribution of CNVs to epilepsy in a cohort of Japanese children with a variety of early-onset DEEs. Single nucleotide polymorphism (SNP) array analysis was performed for 83 cases that were previously negative for pathogenic single nucleotide variants (SNVs) in 109 genes known or suspected to cause epileptic seizures. Rare CNVs were detected in a total of 12 cases (14.4%), of which three cases (3.6%) involved clearly pathogenic CNVs and nine cases (10.8%) were CNVs of uncertain significance. The three pathogenic CNVs included two de novo heterozygous deletions involving known epileptic encephalopathy genes, such as GABRG2 and PCDH19, and one maternally inherited duplication encompassing MECP2. Our findings indicate rare CNVs are also relevant for the diagnosis of early-onset DEEs, highlighting the importance of not relying only on the investigation of SNVs/small indels at the risk of missing large deletions and duplications.

Intellectual disability (ID) is a heterogeneous condition affecting 2-3% of the population, often associated with multiple congenital anomalies (MCA). The genetic cause remains largely unexplained for most cases. To investigate the causes of ID/MCA of unknown etiology in the Japanese population, 645 subjects have been recruited for the screening of pathogenic copy-number variants (CNVs). Two screenings using bacterial artificial chromosome (BAC) arrays were previously performed, which identified pathogenic CNVs in 133 cases (20.6%; Hayashi et al., J. Hum. Genet., 2011). Here, we present the findings of the third screening using a single-nucleotide polymorphism (SNP) array, performed in 450 negative cases from our previous report. Pathogenic CNVs were found in 22 subjects (4.9%), in which 19 CNVs were located in regions where clinical significance had been previously established. Among the 22 cases, we identified PPFIA2 as a novel candidate gene for ID. Analysis of copy-neutral loss of heterozygosity (CNLOH) detected one case in which the CNLOH regions seem to be significant. The SNP array detected a modest fraction of small causative CNVs, which is explained by the fact that the majority of causative CNVs have larger sizes, and those had been mostly identified in the two previous screenings.

By screening patients with undiagnosed multiple congenital anomalies and intellectual disability using array-comparative genomic hybridization, we identified an 884 kb heterozygous microdeletion at 14q13.3 in two siblings presenting with oligodontia, hypothyroidism and persistent pulmonary hypertension of the newborn, resulting from their parental gonosomal mosaicism. Among the six genes included in the deletion, haploinsufficiency of PAX9 and NKX2-1 was probably associated with their phenotypes. These results highlighted a possibility of recurrence of pathogenic copy-number variants associated with parental mosaicism, which requires careful genetic counseling.

Approximately 3% of the live-born infants have major dysmorphic features, and about two-thirds of which are observed in the maxillofacial region; however, in many cases, the etiology of the dysmorphic features remains uncertain. Recently, the genome-wide screening of large patient cohorts with congenital disorders has made it possible to discover genomic aberrations corresponding to the pathogenesis. In our analyses of more than 536 cases of clinically undiagnosed multiple congenital anomalies and mental retardation (MR) by microarray-based comparative genomic hybridization, we detected two non-consanguineous unrelated patients with microdeletions at 10p11.23-p12.1, which overlapped for 957 kb, including four protein-coding genes: ARMC4, MPP7, WAC and BAMBI. As the two patients had similar phenotypes; for example, MR and multiple maxillofacial abnormalities including midface retrusion, wide mouth and large tongue, we assessed the phenotypes in detail to define the common features, using quantitative evaluations of the maxillofacial dysmorphism. The concordance of the genetic and phenotypic alterations is a good evidence of a new syndrome. Although an interstitial deletion of 10p is rare, the current study is the first trial to examine precisely the craniofacial characteristics of patients with a heterozygous deletion at 10p11.23-p12.1, and presents good evidence to diagnose potential patients with the same genetic cause.

Investigations of chromosomal rearrangements in patients with mental retardation (MR) are particularly informative in the search for genes involved in MR. Here we report a family with concomitant duplications of methyl CpG binding protein 2 (MECP2) at Xq28 and ATRX (the causative gene for X-linked alpha thalassemia/mental retardation) at Xq21.1 detected by array-comparative genomic hybridization. The alterations were observed in a 25-year-old man who inherited them from his mother, who showed a normal phenotype and completely skewed X-chromosome inactivation, and also in his cousin, a 32-year-old man. The proband and his cousin showed severe MR, muscular hypotonia, recurrent respiratory infections and various other features characteristic of MECP2 duplication syndrome. However, the proband also had cerebellar atrophy never reported before in MECP2 duplication syndrome, suggesting that his phenotypes were modified through the ATRX duplication in an additive or epistatic manner.

Costello syndrome (CS) is a congenital disease that is characterized by a distinctive facial appearance, failure to thrive, mental retardation and cardiomyopathy. In 2005, we discovered that heterozygous germline mutations in HRAS caused CS. Several studies have shown that CS-associated HRAS mutations are clustered in codons 12 and 13, and mutations in other codons have also been identified. However, a comprehensive comparison of the substitutions identified in patients with CS has not been conducted. In the current study, we identified four mutations (p.G12S, p.G12A, p.G12C and p.G12D) in 21 patients and analyzed the associated clinical manifestations of CS in these individuals. To examine functional differences among the identified mutations, we characterized a total of nine HRAS mutants, including seven distinct substitutions in codons 12 and 13, p.K117R and p.A146T. The p.A146T mutant demonstrated the weakest Raf-binding activity, and the p.K117R and p.A146T mutants had weaker effects on downstream c-Jun N-terminal kinase signaling than did codon 12 or 13 mutants. We demonstrated that these mutant HRAS proteins induced senescence when overexpressed in human fibroblasts. Oncogene-induced senescence is a cellular reaction that controls cell proliferation in response to oncogenic mutation and it has been considered one of the tumor suppression mechanisms in vivo. Our findings suggest that the HRAS mutations identified in CS are sufficient to cause oncogene-induced senescence and that cellular senescence might therefore contribute to the pathogenesis of CS.

Recent advances in the analysis of patients with congenital abnormalities using array-based comparative genome hybridization (aCGH) have uncovered two types of genomic copy-number variants (CNVs); pathogenic CNVs (pCNVs) relevant to congenital disorders and benign CNVs observed also in healthy populations, complicating the screening of disease-associated alterations by aCGH. To apply the aCGH technique to the diagnosis as well as investigation of multiple congenital anomalies and mental retardation (MCA/MR), we constructed a consortium with 23 medical institutes and hospitals in Japan, and recruited 536 patients with clinically uncharacterized MCA/MR, whose karyotypes were normal according to conventional cytogenetics, for two-stage screening using two types of bacterial artificial chromosome-based microarray. The first screening using a targeted array detected pCNV in 54 of 536 cases (10.1%), whereas the second screening of the 349 cases negative in the first screening using a genome-wide high-density array at intervals of approximately 0.7 Mb detected pCNVs in 48 cases (13.8%), including pCNVs relevant to recently established microdeletion or microduplication syndromes, CNVs containing pathogenic genes and recurrent CNVs containing the same region among different patients. The results show the efficient application of aCGH in the clinical setting.

X-linked mental retardation (XLMR) is a common, clinically complex and genetically heterogeneous disease arising from many mutations along the X chromosome. Although research during the past decade has identified >90 XLMR genes, many more remain uncharacterized. In this study, copy-number variations (CNVs) were screened in individuals with MR from 144 families by array-based comparative genomic hybridization (aCGH) using a bacterial artificial chromosome-based X-tiling array. Candidate pathogenic CNVs (pCNVs) were detected in 10 families (6.9%). Five of the families had pCNVs involving known XLMR genes, duplication of Xq28 containing MECP2 in three families, duplication of Xp11.22-p11.23 containing FTSJ1 and PQBP1 in one family, and deletion of Xp11.22 bearing SHROOM4 in one family. New candidate pCNVs were detected in five families as follows: identical complex pCNVs involved in dup(X)(p22.2) and dup(X)(p21.3) containing part of REPS2, NHS and IL1RAPL1 in two unrelated families, duplication of Xp22.2 including part of FRMPD4, duplication of Xq21.1 including HDX and deletion of Xq24 noncoding region in one family, respectively. Both parents and only mother samples were available in six and three families, respectively, and pCNVs were inherited from each of their mothers in those families other than a family of the proband with deletion of SHROOM4. This study should help to identify the novel XLMR genes and mechanisms leading to MR and reveal the clinical conditions and genomic background of XLMR.

By using an in-house bacterial artificial chromosome-based X-tilling array, we detected a 0.4 Mb novel deletion at Xq24 that included UBE2A in a 4-year-old and 10-month-old boy with mental retardation and various other characteristics inherited from his mother; for example, marked developmental delay, synophrys, ocular hypertelorism, esotropia, low nasal bridge, marked generalized hirsutism and seizure. Although additional nine transcripts around UBE2A were also defective, a phenotypic similarity with a recently reported X-linked familial case involving a novel X-linked mental retardation syndrome and a nonsense mutation of UBE2A indicates a functional defect of UBE2A to be responsible for most of the abnormalities in these cases. Because some characteristics, such as congenital heart disease and proximal placement of the thumb, were not described in the family reported previously, suggesting genes other than UBE2A within the deleted region to be responsible for those abnormalities.

In this study, we report the molecular structure of the breakpoint region in a new chromosomal translocation, t(2;7)(p24.3;p14.2), in a case of acute myeloid leukemia transformed from myelodysplastic syndrome (MDS). An extensive fluorescence in situ hybridization (FISH) analysis showed that NAG (2p24.3) and ELMO1 (7p14.2) were involved at the breakpoints of t(2;7)(p24.3;p14.2). Furthermore, we detected a novel chimeric transcript consisting of NAG and ELMO1. Interestingly, this transcript encoded a truncated molecular form of 3'ELMO1 as the result of a frameshift caused by the chromosomal translocation. Although this study does not provide direct evidence that a defect in NAG-ELMO1 plays a role in the pathogenesis or the leukemic change in MDS, it does suggest that defects in NAG-ELMO1 potentially contributed to the leukemic progression in this case.

The 16q22.1-linked autosomal dominant cerebellar ataxia (16q-ADCA; Online Mendelian Inheritance in Man [OMIN] #117210) is one of the most common ADCAs in Japan. Previously, we had reported that the patients share a common haplotype by founder effect and that a C-to-T substitution (-16C>T) in the puratrophin-1 gene was strongly associated with the disease. However, recently, an exceptional patient without the substitution was reported, indicating that a true pathogenic mutation might be present elsewhere. In this study, we clarified the disease locus more definitely by the haplotype analysis of families showing pure cerebellar ataxia. In addition to microsatellite markers, the single nucleotide polymorphisms (SNPs) that we identified on the disease chromosome were examined to confirm the borders of the disease locus. The analysis of 64 families with the -16C>T substitution in the puratrophin-1 gene revealed one family showing an ancestral recombination event between SNP04 and SNP05 on the disease chromosome. The analysis of 22 families without identifiable genetic mutations revealed another family carrying the common haplotype centromeric to the puratrophin-1 gene, but lacking the -16C>T substitution in this gene. We concluded that the disease locus of 16q-ADCA was definitely confined to a 900-kb genomic region between the SNP04 and the -16C>T substitution in the puratrophin-1 gene in 16q22.1.

The human chromosome X is closely associated with congenital disorders and mental retardation (MR), because it contains a significantly higher number of genes than estimated from the proportion in the human genome. We constructed a high-density and high-resolution human chromosome X array (X-tiling array) for comparative genomic hybridization (CGH). The array contains a total of 1,001 bacterial artificial chromosome (BACs) throughout chromosome X except pseudoautosomal regions and two BACs specific for Y. In four hybridizations using DNA samples from healthy males, the ratio of each spotted DNA was scattered between -3SD and 3SD, corresponding to a log(2) ratio of -0.35 and 0.35, respectively. Using DNA samples from patients with known congenital disorders, our X-tiling array was proven to discriminate one-copy losses and gains together with their physical sizes, and also to estimate the percentage of a mosaicism in a patient with mos 45,X[13]/46,X,r(X)[7]. Furthermore, array-CGH in a patient with atypical Schinzel-Giedion syndrome disclosed a 1.1-Mb duplication at Xq22.3 including a part of the IL1RAPL2 gene as a likely causative aberration. The results indicate our in-house X-tiling array to be useful for the identification of cryptic copy-number aberrations containing novel genes responsible for diseases such as congenital disorders and X-linked MR.

Malignant rhabdoid tumor (MRT) is a highly malignant pediatric cancer, which arises in various sites such as the kidney, brain, and soft tissues. Cytogenetic studies have revealed alterations of 22q11 in MRT. Recently, deletions and mutations of the SNF5/INI1 locus in 22q11.2 have been reported in MRT, suggesting that SNF5/INI1 is a tumor suppressor gene for MRT. Here we report our molecular cytogenetic study for a newly established cell line from extrarenal MRT with t(1;22)(p36;q11.2). Consequently, the reciprocal translocation was associated with the interstitial deletion of a small segment including SNF5/INI1, and another, chromosome 22, showed terminal deletion, the breakpoint of which was located 70-80 kb centromeric to SNF5/INI1, resulting in homozygous deletion of SNF5/INI1 in this cell line.

DP-1 is a heterodimerization partner for members of the E2F family of transcription factors; E2F/DP-1 regulates the expression of various cellular promoters, particularly gene products that are involved in the cell cycle. Our earlier studies identified the DP-1 gene ( TFDP1) as a probable target within a 13q34 amplicon that is frequently detected in hepatocellular carcinomas (HCC) and esophageal squamous-cell carcinomas. The aim of the present study was to investigate the clinicopathological significance of up-regulation of TFDP1 in HCC. We determined expression levels of TFDP1 and E2F1 in 41 primary HCCs by means of quantitative real-time reverse transcription-polymerase chain reactions, and looked for relationships between those data and various clinicopathological parameters. To assess transactivating activity of E2F1/DP-1, we also analyzed expression of ten putative transcriptional targets of this complex in HCCs. Using antisense oligonucleotides, we down-regulated TFDP1 in Hep3B, an HCC cell line that had shown overexpression of the gene, to examine the role of elevated TFDP1 expression in the growth of Hep3B cells. Elevated expression of TFDP1, but not E2F1, was associated significantly with large (> or =5 cm) tumor size (P=0.021). Expression levels of TFDP1 and E2F1 correlated with those of seven transcriptional targets ( TYMS, DHFR, PCNA, RRM1, CCNE1, CDC2, and MYBL2) that play important roles in the G1/S transition, and down-regulation of TFDP1 inhibited growth of Hep3B cells. In conclusion, overexpression of TFDP1 may contribute to progression of some HCCs by promoting growth of the tumor cells.

Comparative genomic hybridization (CGH) analyses have detected gains of copy number on 13q, especially at 13q31-q32, in cell lines and primary cases of various types of lymphoma. Since amplification of chromosomal DNA is one of the mechanisms that can activate tumor-associated genes, and because 13q amplification had been reported in various other types of tumors as well, we attempted to define by fluorescence in situ hybridization (FISH) a common region at 13q31-q32 in which to explore genes that might be targets for the amplification events. Although the commonly amplified region we defined was relatively large (approximately 4 Mb), only one true gene, GPC5, was found there. GPC5 was over-expressed in lymphoma cell lines that had shown amplification, in comparison with those that had not. Our findings suggest that GPC5 is a likely target for amplification, and that over-expression of this gene may contribute to development and/or progression of lymphomas and other tumors.

Using a repetitive sequence of tandemly arrayed pentanucleotides in the human H19 3'-flanking region, we isolated a phage clone (lambda PEN11) which localized to chromosome 11p15.5. The lambda PEN11 phage encodes a 2.3-kb cDNA consisting of seven exons at least. The gene was mainly expressed in brain and pancreas (and less abundantly in testis), and demonstrated differential allele usage, with maternal expression being predominant in placenta, which indicates the gene is an atypical imprinted gene. While the pentamer repeat might contribute to this effect, it is also possible that the differential allele usage might reflect the local chromosomal structure known as the imprinting domain.