TALEN-mediated single-base-pair editing identification of an intergenic mutation upstream of BUB1B as causative of PCS (MVA) syndrome.

Cancer-prone syndrome of premature chromatid separation with mosaic variegated aneuploidy [PCS (MVA) syndrome] is a rare autosomal recessive disorder characterized by constitutional aneuploidy and a high risk of childhood cancer. We previously reported monoallelic mutations in the BUB1B gene (encoding BUBR1) in seven Japanese families with the syndrome. No second mutation was found in the opposite allele of any of the families studied, although a conserved BUB1B haplotype and a decreased transcript were identified. To clarify the molecular pathology of the second allele, we extended our mutational search to a candidate region surrounding BUB1B. A unique single nucleotide substitution, G > A at ss802470619, was identified in an intergenic region 44 kb upstream of a BUB1B transcription start site, which cosegregated with the disorder. To examine whether this is the causal mutation, we designed a transcription activator-like effector nuclease-mediated two-step single-base pair editing strategy and biallelically introduced this substitution into cultured human cells. The cell clones showed reduced BUB1B transcripts, increased PCS frequency, and MVA, which are the hallmarks of the syndrome. We also encountered a case of a Japanese infant with PCS (MVA) syndrome carrying a homozygous single nucleotide substitution at ss802470619. These results suggested that the nucleotide substitution identified was the causal mutation of PCS (MVA) syndrome.

Meiosis error and subsequent genetic and epigenetic alterations invoke the malignant transformation of germ cell tumor.

Germ cell tumors (GCTs) are thought to arise from primordial germ cells (PGCs) that undergo epigenetic reprogramming. To explore the mechanisms of GCT formation, we analyzed single-nucleotide polymorphism array comparative genomic hybridization patterns and the methylation status of 15 tumor suppressor genes (TSGs) and differentially methylated regions (DMRs) of two imprinted genes, H19 and SNRPN, in 28 children with GCTs. Three GCTs with 25-26 segmental uniparental disomies (UPDs), heterozygous centromeric regions, and a highly methylated SNRPN DMR may have occurred through meiosis I error. Three other GCTs with whole UPD and homozygous centromeric regions of all chromosomes may have occurred through endoreduplication of a haploid set in an ovum or testis. The other 22 GCTs had heterozygous centromeric regions of all chromosomes and no or a small number of segmental or whole UPDs and may have developed from premeiotic PGCs before imprint erasure or a reestablishment of imprinting. Gain and amplification of 3p24-p22 and 20q13-q13, and loss and UPD of 1p36-p35, 4q21-q21, 5q11-q13, and 6q26-qter were found in five or more tumors. 1p36-p35 loss was frequent, and found in 19 tumors; RUNX3 residing at 1p36 was methylated in the promoter regions of 16 tumors. Two yolk sac tumors with many segmental UPDs or whole UPD of all chromosomes had gain of 20q13-q13 and loss of 1p36-p35, and seven or eight methylated TSGs. These genetic and epigenetic alterations may have caused malignant transformation because they were rarely found in teratomas with segmental or whole UPDs.

Insufficiency of BUBR1, a mitotic spindle checkpoint regulator, causes impaired ciliogenesis in vertebrates.

Budding uninhibited by benzimidazole-related 1 (BUBR1) is a central molecule of the spindle assembly checkpoint. Germline mutations in the budding uninhibited by benzimidazoles 1 homolog beta gene encoding BUBR1 cause premature chromatid separation (mosaic variegated aneuploidy) [PCS (MVA)] syndrome, which is characterized by constitutional aneuploidy and a high risk of childhood cancer. Patients with the syndrome often develop Dandy-Walker complex and polycystic kidneys; implying a critical role of BUBR1 in morphogenesis. However, little is known about the function of BUBR1 other than mitotic control. Here, we report that BUBR1 is essential for the primary cilium formation, and that the PCS (MVA) syndrome is thus a novel ciliopathy. Morpholino knockdown of bubr1 in medaka fish also caused ciliary dysfunction characterized by defects in cerebellar development and perturbed left-right asymmetry of the embryo. Biochemical analyses demonstrated that BUBR1 is required for ubiquitin-mediated proteasomal degradation of cell division cycle protein 20 in the G0 phase and maintains anaphase-promoting complex/cyclosome-CDC20 homolog 1 activity that regulates the optimal level of dishevelled for ciliogenesis.

Heterozygous TGFBR2 mutations in Marfan syndrome.

Marfan syndrome is an extracellular matrix disorder with cardinal manifestations in the eye, skeleton and cardiovascular systems associated with defects in the gene encoding fibrillin (FBN1) at 15q21.1 (ref. 1). A second type of the disorder (Marfan syndrome type 2; OMIM 154705) is associated with a second locus, MFS2, at 3p25-p24.2 in a large French family (family MS1). Identification of a 3p24.1 chromosomal breakpoint disrupting the gene encoding TGF-beta receptor 2 (TGFBR2) in a Japanese individual with Marfan syndrome led us to consider TGFBR2 as the gene underlying association with Marfan syndrome at the MSF2 locus. The mutation 1524G-->A in TGFBR2 (causing the synonymous amino acid substitution Q508Q) resulted in abnormal splicing and segregated with MFS2 in family MS1. We identified three other missense mutations in four unrelated probands, which led to loss of function of TGF-beta signaling activity on extracellular matrix formation. These results show that heterozygous mutations in TGFBR2, a putative tumor-suppressor gene implicated in several malignancies, are also associated with inherited connective-tissue disorders.

Maternal isodisomy for 14q21-q24 in a man with diabetes mellitus.

We report a 20-year-old man with maternal uniparental disomy for chromosome 14 (UPD14) and maturity-onset diabetes mellitus (DM). He had pre- and postnatal growth retardation, developed DM at age 20 years without any autoimmune antibodies, and had a mosaic 45,XY,der(14;14)(q10;q10)[129]/46,XY,+14,der(14;14)(q10;q10)[1] karyotype. Allelotyping using microsatellite markers covering the entire 14q indicated segmental maternal isodisomy for 14q21-q24 and maternal heterodisomy of the remaining regions of the chromosome. It is thus tempting to speculate that the segmental isodisomy led to reduction to homozygosity for a mutant gene and thus caused his DM, although the possibility of coincidental occurrence of the two events cannot totally be ruled out. Fluorescence in situ hybridization (FISH) analysis using BAC clone probes revealed that the isodisomic segment did not overlap any known IDDM or NIDDM susceptibility loci on chromosome 14, suggesting a novel locus for a subset of DM at the isodisomic segment.

Duplication of 8p23.2: a benign cytogenetic variant?

We describe a duplication of the 8p23.2 band in seven individuals from four families. The duplication was recognizable as an enlarged 8p23.2 band on G-banded chromosomes at the 550 band level. It was transmitted from a parent to offspring in three of the four families in which both parents were karyotyped. Each proband in the four families had the enlarged band and showed various phenotypic abnormalities, but the abnormalities were inconsistent. Chromosomal and interphase fluorescence in situ hybridization (FISH) analysis of the enlarged band region defined a 2.5-Mb duplicated segment common to all seven individuals studied. Interphase FISH analysis of peripheral blood lymphocytes from 50 unrelated normal individuals showed the duplication in three individuals. In view of these findings, it is most likely that the 8p23.2 duplication we described is a normal variant.

Monoallelic BUB1B mutations and defective mitotic-spindle checkpoint in seven families with premature chromatid separation (PCS) syndrome.

Cancer-prone syndrome of premature chromatid separation (PCS syndrome) with mosaic variegated aneuploidy (MVA) is a rare autosomal recessive disorder characterized by growth retardation, microcephaly, childhood cancer, premature chromatid separation of all chromosomes, and mosaicism for various trisomies and monosomies. Biallelic BUB1B mutations were recently reported in five of eight families with MVA syndrome (probably identical to the PCS syndrome). We here describe molecular analysis of BUB1B (encoding BubR1) in seven Japanese families with the PCS syndrome. Monoallelic BUB1B mutations were found in all seven families studied: a single-base deletion (1833delT) in four families; and a splice site mutation, a nonsense mutation, and a missense mutation in one family each. Transcripts derived from the patients with the 1833delT mutation and the splice site mutation were significantly reduced, probably due to nonsense-mediated mRNA decay. No mutation was found in the second alleles in the seven families studied, but RT-PCR of BUB1B and Western blot analysis of BubR1 indicated a modest decrease of their transcripts. BubR1 in the cells from two patients showed both reduced protein expression and diminished kinetochore localization. Their expression level of p55cdc, a specific activator of anaphase-promoting complex, was normal but its kinetochore association was abolished. Microcell-mediated transfer of chromosome 15 (containing BUB1B) into the cells restored normal BubR1 levels, kinetochore localization of p55cdc, and the normal responses to colcemid treatment. These findings indicate the involvement of BubR1 in p55cdc-mediated mitotic checkpoint signaling, and suggest that >50% decrease in expression (or activity) of BubR1 is involved in the PCS syndrome.

Induction of premature chromatid separation (PCS) in individuals with PCS trait and in normal controls.

Cultured peripheral blood lymphocytes from ten normal individuals, treated with 0.075 M KCl at 37 degrees C for 20 min, showed 0-2% cells in premature chromatid separation (PCS), a configuration with split centromeres and chromatids of most or all chromosomes. When treated for 30 min, they increased to 19% in the average, and at 45 min to 63%. Similar and significant effects of temperature and duration of hypotonic treatment on the frequencies of PCSs were found also in mitotic lymphocytes from patients with homozygous PCS trait, a cancer-prone disorder with >50% lymphocytes in PCS, mosaic variegated aneuploidy, and a variety of clinical manifestations; and from their heterozygous carrier parents. B lymphoblastoid cells from two infants with the homozygous PCS trait did not show PCSs when processed without hypotonic treatment. The frequencies of their PCSs increased with increasing temperature and duration of hypotonic treatment, attaining more than 65% after 20 min treatment and 90% after 45 min at 37 degrees C. PCS is thus likely to be induced largely by hypotonic treatment. Treatment at 37 degrees C for 20 min was found to be most suitable for the count of PCSs, in which the frequency of PCSs becomes almost zero in cells from normal individuals, and the difference in frequency of PCSs was most remarkable between the patients and heterozygous carriers, and between the heterozygous carriers and normal individuals. Chromosomes from the patients with the homozygous PCS trait tended to be long, and their PCSs tended to have a large number of widely separated sister chromatids. Chromosomes from normal individuals tended to be short, and the sister chromatids in their PCSs were set close to each other.

ICF syndrome in a girl with DNA hypomethylation but without detectable DNMT3B mutation.

A 3-year-old girl with phenotypic and cytogenetic manifestations of the ICF syndrome and DNA hypomethylation but without DNMT3B gene mutation is described. At age 3 months, she had an apneic spell that left her with spastic paraplegia and severe mental retardation. At age 8 months, she suffered meningococcal meningitis and sepsis. When seen by us at age 3 years with virilization, she had a cleft plate, macroglossia, and an atrial septal defect. An adenoma was surgically removed from the right adrenal cortex. Her serum immunoglobulin levels were normal except IgA at the low normal border. Her lymphocytes showed paracentromeric stretching of chromosomes 1 and 16 in 7% of metaphases, and multiradial figures involving these chromosomes in 1% of cells. Hypomethylation of classical satellite 2 DNA was observed with BstBI digestion, but in a lesser degree than those in the individuals with proven DNMT3B mutations. No mutation was found in the coding and promoter regions of the gene. Several alternative interpretations were considered to explain the low frequencies of chromosomal instabilities and the lower degree of DNA hypomethylation, and undetected DNA3B mutations. A mutation may be present in the gene but undetected, present in other DNA methyltransferases (DNMT) genes or in a DNMT-associated protein gene.