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Biomedical subjects

N Niikawa

Publications and source records attributed to N Niikawa.

At least 163 records · Page 9Linked to original sources
PubMed1994

A comparison of GC content and the proportion of Alu/KpnI-repetitive sequences in a single dark- and light-band region from a human chromosome.

To obtain direct evidence for a molecular basis of differentiation between regular Giemsa dark (G)- and light (R)-band regions of human chromosomes, two regions of chromosome 11, q14-q22 (G-band) and q23-q25 (R-band), were microdissected. The DNA fragments were amplified by the linker-primer polymerase chain reaction and cloned into pUC19. Microclones from each library were then compared by colony hybridization with repetitive DNA sequences, by Southern blot hybridization of each microclone to total human genomic DNA and mouse-human hybrid cell DNA containing only human chromosome 11, and by sequencing of unique and Alu-repetitive clones. Among the G-band-derived microclones analyzed, 43.0% were single-copy (unique) sequences and 23.2% contained highly repetitive sequence elements; in the R-band-derived library, 54.2% were unique clones and 20.3% had highly repetitive elements. The G-band library was significantly richer in clones positive for KpnI sequences (4.1% in the G-band library vs 2.8% in the R-band library). No significant difference was found in the proportion of Alu repetitive clones present in the two libraries, but Class IV Alu sequence derivatives were more frequently observed in the R-library than in the G-library. Sequence analysis revealed no significant difference in GC content or in the ratio of CpG sequence to GpC dinucleotide between 30 microclones derived from G-bands and 29 microclones derived from R-bands.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Composition↗
PubMed1994

Isochromosome 15q of maternal origin in two Prader-Willi syndrome patients previously diagnosed erroneously as cytogenetic deletions.

Since our previous report on two Prader-Willi syndrome (PWS) patients with t(15q;15q) (Niikawa and Ishikiriyama; Hum Genet 69:22-27, 1985) was erroneous, we report here new data and a corrected interpretation. Reexamination of the parental origin of their t(15q;15q) using polymorphic DNA markers that are mapped to various regions of 15q documented no molecular deletions at the 15q11-q13 region in either patient. Both patients were homozygous at all loci examined and their haplotypes on 15q coincided with one of those in their respective mothers. These results indicate that the presumed t(15q;15q) in each patient was actually an isochromosome 15q producing maternal uniparental disomy, consistent with genomic imprinting at the PWS locus.

Aneuploidy↗
PubMed1994

Excess functional copy of allele at chromosomal region 11p15 may cause Wiedemann-Beckwith (EMG) syndrome.

Wiedemann-Beckwith syndrome (WBS) is a genetic disorder with overgrowth and predisposition to Wilms' tumor. The putative locus of the gene responsible for this syndrome is assigned to chromosome region 11p15.5, and genomic imprinting in this region has been proposed: the paternally derived gene(s) at 11p15.5 is selectively expressed, while the maternally transmitted gene(s) is inactive. We examined 18 patients for the parental origin of their 11p15 regions. DNA polymorphism analyses using 6 loci on chromosome 11 showed that 2 patients with duplications of 11p15 regions from their respective fathers and one from the mother, indicating the transmission of an excessive paternal gene at 11p15 to each patient. Our results, together with the previous findings in karyotypically normal or abnormal patients and in overgrowth mouse experiments, are consistent with imprinting hypothesis that overexpression of paternally derived gene(s) at 11p15.5, probably the human insulin-like growth factor II (IGF-II) gene, may cause the phenotype. Total constitutional uniparental paternal disomy (UPD) or segmental UPD for the 6 loci examined of chromosome 11 was not observed in our 12 sporadic patients. In order to explain completely the inheritance of this syndrome in patients with various chromosomal constitutions, we propose an alternative imprinting mechanism involving the other locus that may be paternally imprinted and may suppress the expression of this gene.

Alleles↗
PubMed1994

Case of 46,XX/47,XY, +21 chimerism in a newborn infant with ambiguous genitalia.

We describe whole-body chimerism in a newborn infant with small phallus, pseudo-vaginal perineal hypospadias, and a bifid scrotum containing gonads. The human testis determining factor gene (SRY) was detected by PCR amplification. GTG-banding chromosome analysis in peripheral blood lymphocytes and cultured fibroblasts derived from right cubital skin showed a 46,XX/47,XY, +21 karyotype. Their ratios in each cell line were 294:5 and 178:7, respectively. QFQ-banding chromosome analysis documented 3 heteromorphic satellites on trisomic chromosomes 21 in the 47,XY, +21 cell line and a homozygous satellite pattern in the 46,XX cell line. Heteromorphic patterns of chromosomes 4, 13, 14, and 22 were also different between the two cell lines. To our knowledge, such disomy/trisomy chimeras have not been described previously.

Cells, Cultured↗
PubMed1994

cDNA cloning and chromosomal mapping of human N-acetylglucosaminyltransferase V+.

Human N-acetylglucosaminyltransferase V (GnT-V, EC 2.4.1.155) cDNA was isolated from a human fetal liver cDNA library. Oligonucleotide primers for polymerase chain reaction were designed according to the amino acid sequence of human GnT-V. Screening for the cDNA was carried out by plaque hybridization using PCR products of about 500 bp. Human GnT-V has 741 amino acids and six putative N-glycosylation sites. The homology to rat GnT-V is 88% at the nucleotide level and is 97% at the amino acid level, and there is one amino acid insertion. Using the cDNA clones as probe, five overlapping genomic clones have been isolated from a human phagemid DNA library. The GnT-V gene has been mapped to chromosome 2q21 using fluorescence in situ hybridization.

Amino Acid Sequence↗
PubMed1994

Mapping of the 8q23 translocation breakpoint of t(8;13) observed in a patient with multiple exostoses.

A detailed cytogenetic map was constructed around the chromosomal breakpoint of t(8;13) observed in a patient with multiple exostoses. The order of seven loci defined by cosmid clones mapped to 8q23 was determined by means of two-color fluorescence in situ hybridization (FISH) on elongated prophase chromosomes, and localizations of these markers relative to the breakpoint were examined. The results indicated that loci defined by cC18-553 and cC18-1512 flank the breakpoint. By pulsed-field gel electrophoresis of DNA digested with BssHII and Southern hybridization with cC18-1512, DNA from the patient showed a band which was not observed in DNA isolated from either parent. As the normal size of this BssHII fragment is 600 kb, the chromosomal breakpoint probably lies less than 600 kb away from cC18-1512.

Cell Line↗
PubMed1994

Isolation of two isoforms of the PAX3 gene transcripts and their tissue-specific alternative expression in human adult tissues.

We have isolated two isoforms of cDNA clones from the human PAX3 gene, a candidate gene responsible for Waardenburg syndrome type I (WSI) as well as a gene associated with development of alveolar rhabdomyosarcoma. The gene product is considered to be one of transcription factors, and the two cDNA clones isolated, termed PAX3A and PAX3B, were generated by alternative splicing. The transcripts coded 215 and 206 amino acids, respectively, and shared 196 amino acids at the NH2 end. The amino acid sequence in the common region (residues 1-196) showed a 100% identity with that of exons 1-4 of the mouse Pax-3 gene. However, both of the PAX3 cDNAs lacked the DNA sequence corresponding to the paired-type homeodomain of the mouse Pax-3 gene. Analysis of gene expression in human adult tissues by reverse transcriptase polymerase chain reaction (RT-PCR) revealed tissue-specific expression of this gene. PAX3B was expressed in most of the tissues examined, but the PAX3A type of transcript was detected only in the cerebellum, esophagus, and skeletal muscle.

Adult↗
PubMed1994

Molecular analysis of a patient with Beckwith-Wiedemann syndrome, rhabdomyosarcoma and renal cell carcinoma.

We described a patient with Beckwith-Wiedemann syndrome associated with rhabdomyosarcoma (RMS), and renal cell carcinoma (RCC). Karyotypes of peripheral lymphocytes and RMS cells were normal. DNA analyses showed maternal loss of heterozygosity (LOH) at 11p15 region in RMS but not in RCC. The insulin-like growth factor II gene (IGF2) was found to be expressed at a moderate level in RMS but not in RCC by in situ hybridization. Each of parental allele-derived IGF2 transcript was detected in RCC, while maternal allele-derived transcript was weak in RMS because of maternal LOH. These results suggest that (1) loss of imprinting (LOI) of IGF2 might be responsible for BWS, (2) on the other hand, LOI itself might not induce tumor occurrence in tissues where the control of tissue-specific expression of IGF2 is maintained, (3) increased expression of IGF2 due to maternal loss of a putative controller gene for IGF2 at 11p15 might predispose to sustaining tumorigenic mutations and tumor progression, (4) loss of a putative onco-suppressor gene at 11p15 might induce RMS occurrence. The cause of RCC was thought to be different from that of RMS.

Abdominal Neoplasms↗
PubMed1994

Mosaic and polymorphic imprinting of the WT1 gene in humans.

We have examined the imprinting of the Wilms' tumour suppressor gene (WT1) in human tissues. We confirm that WT1 is biallelically expressed in the kidney, however, in five of nine preterm placentae WT1 was expressed largely or exclusively from the maternal allele. Monoallelic expression of WT1 was also found in two fetal brains. These data demonstrate that WT1 can undergo tissue specific imprinting. Furthermore, because monoallelic expression of WT1 was not found in all placentae examined, WT1 imprinting may be genetically polymorphic within the human population.

Alleles↗
PubMed1994

Isolation and characterization of mutations in the human holocarboxylase synthetase cDNA.

Holocarboxylase synthetase (HCS) plays an essential role in biotin utilization in eukaryotic cells and its deficiency causes biotin-responsive multiple carboxylase deficiency in humans. We have cloned the human HCS cDNA and show that antiserum against the recombinant protein immunoprecipitates human HCS. A one base deletion resulting in a premature termination and a missense mutation (Leu to Pro) were found in cells from siblings with HCS deficiency. Human HCS shows homology to BirA, which acts as both a biotin-[acetyl-CoA-carboxylase] ligase and a biotin repressor in E. coli, suggesting a functional relationship between the two proteins. The human HCS gene maps to chromosome 21q22.1.

Amino Acid Sequence↗
PubMed1994

Assignment of the human PAX4 gene to chromosome band 7q32 by fluorescence in situ hybridization.

Of the nine known members of a human paired box-containing gene family (Pax), only PAX4 has not been precisely localized. We screened a cosmid library of human genomic DNA using polymerase chain reaction products for PAX4 as a probe and isolated three positive cosmid clones. Sequence analysis revealed that at least two of them had exon-like sequences and showed extensive homology to Pax-4 in the mouse. These two cosmid clones were mapped to human chromosome band 7q32 by fluorescence in situ hybridization.

Animals↗
PubMed1994

A 15-year-old girl with pubertal masculinization due to bilateral gonadoblastoma and 45,X/46,X,+mar karyotype.

We report the case of a 15-year-old girl with bilateral gonadoblastoma and 45,X/46,X,+mar karyotype. She was short but had no other stigmata of Turner syndrome. Her genitalia were completely of the female type until she began to show signs of masculinization at age 13 years. She had breasts of Tanner stage 4, primary amenorrhea, clitoromegaly, hirsutism and low voice. Plasma testosterone level was elevated and returned to normal after the successful removal of the bilateral gonadoblastoma. Her karyotype was 45,X/46,X,+mar and it was proved that the marker chromosome was derived from the short arm of the Y chromosome by Southern blot analysis with a Y chromosome specific probe, pDP1007. Even when there are no stigmata of Turner syndrome or signs of intrauterine virilization, the possibility of gonadoblastoma should not be ruled out in girls who show postnatal virilization.

Adolescent↗
PubMed1994

A stable acentric marker chromosome: possible existence of an intercalary ancient centromere at distal 8p.

A centromere is considered to be an essential chromosomal component where microtubule-kinetochore interaction occurs to segregate sister chromatids faithfully and acentric chromosomes are unstable and lost through cell divisions. We report a novel marker chromosome that was acentric but stable through cell divisions. The patient was a 2-year-old girl with mental retardation, patent ductus arteriosus, and mild dysmorphic features. G-banded chromosome analysis revealed that an additional small marker chromosome was observed in all 100 cells examined. By the reverse-chromosome-painting method, the marker was found to originate from the distal region of 8p, and a subsequent two-color FISH analysis with cosmid probes around the region revealed that the marker was an inverted duplication interpreted as 8pter-->p23.1::p23.1-->8pter. No centromeric region was involved in the marker. By FISH, no alpha-satellite sequence was detected on the marker, while a telomere sequence was detected at each end. Anti-kinetochore immunostaining, using a serum from a patient with CREST (calcinosis, Raynaud syndrome, esophageal dismotility, sclerodactyly, and telangiectasia) syndrome, showed a pair of signals on the marker, which indicated that a functional kinetochore was present on the marker. The analysis of this patient might suggest the possibility that an ancient centromere sequence exists at distal 8p (8p23.1-pter) and was activated through the chromosome rearrangement in the patient.

Abnormalities, Multiple↗
PubMed1993

Microdissection and microcloning of genomic DNA markers from human chromosomal region 11q23.

A human genomic DNA library was constructed by using a microdissection-microcloning procedure with polymerase chain reaction (PCR) techniques on DNA from the chromosome 11q23 region. A total of 450 recombinant pUC clones were isolated from the library. Their insert sizes ranged from 150 to 850 bp with a mean of 320 bp. Fifty pUC clones were randomly selected and analyzed in detail. Southern blot analyses showed that 21 (42%) clones were unique DNA sequences, 20 (40%) clones were repetitive sequences, and 9 (18%) clones had no detectable hybridization. The unique sequences were used further in a secondary screening of a partially digested human genomic DNA library constructed in phage vector, and 4 clones were isolated. The chromosomal locations of these phage clones were confirmed to be in the q23 region of chromosome 11 by fluorescence in situ suppression hybridization. These pUC microclones isolated from the chromosomal region-specific genomic DNA library will be useful in the construction of physical contig maps with yeast artificial chromosome and/or cosmid clones and in the positional cloning of disease-associated genes localized to the q23 region of chromosome 11.

Chromosome Mapping↗
PubMed1993

Isolation of region-specific cosmids by hybridization with microdissection clones from human chromosome 10q11.1-q21.1.

A region-specific plasmid library composed of 20,000 recombinants was constructed by microdissection of human chromosome 10 (10q11.2-q21.1) and subsequent amplification with the primer-linker method of polymerase chain reaction (PCR). Hybridization with total human DNA showed that 32 of 217 microclones studied contained highly repetitive sequences. Further analysis of the remaining 185 microclones proved that 43 microclones, each having an insert longer than 200 bp, contained unique sequences of human chromosome 10 origin. Twenty-five microclones randomly selected from the 43 were used directly as probes to isolate corresponding cosmid clones, resulting in 32 cosmids corresponding to 14 microclones. Of the 25 cosmids that could be mapped by fluorescence in situ hybridization, 24 proved to originate from the microdissected or adjacent region (10p11.2-q22.3) and 1 from a rather distal region (10q24.3-q25.1). In addition, 15 of the 32 cosmids revealed restriction fragment length polymorphisms, including 1 with a variable number of tandem repeats marker. The microdissection library and the obtained cosmids are valuable resources for constructing high-resolution physical and linkage maps of the pericentromeric region of chromosome 10, where the gene predisposing to multiple endocrine neoplasia type 2A (MEN2A) has been mapped.

Base Sequence↗
PubMed1993

The origin of cytologically unidentifiable chromosome abnormalities: six cases ascertained by targeted chromosome-band painting.

De novo chromosome structural abnormalities cannot always be diagnosed by the use of standard cytogenetic techniques. We applied a previously developed chromosome-band-specific painting method to the diagnosis of such rearrangements. The diagnostic procedures consisted of microdissection of an aberrant chromosomal region of a given patient, polymerase chain reaction (PCR) amplification of the dissected chromosomal DNA, and subsequent competitive fluorescence in situ hybridization (FISH) using the PCR products as a probe pool on metaphase chromosomes from the patient and/or a karyotypically normal person. With this strategy, we studied 6 de novo rearrangements (6p+, 6q+, 9p+, 17p+, +mar, and +mar) in 6 patients. These rearrangements had been seen by conventional banding but their origin could not be identified. In all 6 patients, we successfully ascertained the origin. Using an aberrant region-specific probe pool, FISH signals appeared on both the aberrant region and a region of another specific chromosome pair. A reverse probe pool that was generated through the microdissection of normal chromosomes at a candidate region for the origin of the aberration hybridized with both the aberrant and the candidate regions. We thus diagnosed one patient with 17p+ as having trisomy for 14q32-qter, one with 9p+ as having trisomy for 12pter-p12, one with 6q+ as having a tandem duplication (trisomy) of a 6q23-q25 segment, one with 6p+ as having a tandem duplication (trisomy) of a 6p23-q21.3 segment, one with a supernumerary metacentric marker chromosome as having tetrasomy for 18pter-cen, and the last with an additional small marker chromosome as having trisomy for 18p11.1 (or p11.2)-q11.2. The present targeted chromosome-band-painting method provides the simple and rapid preparation of a probe pool for region-specific FISH, and is useful for the diagnosis of chromosome abnormalities of unknown origin.

Base Sequence↗