Biomedical subjects
Publications and source records attributed to N Niikawa.
We report on 2 girls with a terminal deletion of the short arm of chromosome X. They had microphthalmia, cloudy corneae, mild linear skin lesions, and agenesis of corpus callosum. A comparison of clinical and cytogenetic findings in similar cases suggested that the critical genes for the Goltz and Aicardi syndromes might be contiguous in the region Xp22.31.
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We describe a chromosome-band-specific painting method that involves (1) microdissection of the chromosome, chromosomal region or band, (2) amplification of a variety of chromosome/region/band-specific DNA fragments with the polymerase chain reaction (PCR), and (3) chromosome in situ suppression hybridization (CISS) with the direct use of the PCR products as a probe pool. With this method, it was possible 1) to paint an entire X or Y chromosome, a distal one-fourth of 2q, and only a band at 8q24.1, 2) to identify the origin of a minute marker chromosome in a mentally retarded patient, 3) to detect an X;Y translocation in another patient, and 4) to identify one human chromosome 2 in a human-mouse hybrid cell line. This method allows us to identify not only structural chromosome abnormalities at the band level, but also the origin of cytogenetically unidentifiable marker chromosomes. It will also be useful in studies of evolutionary cytogenetics.
We previously constructed a chromosome 2q-specific genomic library and isolated a number of microclones. In the present study, we first analyzed with Southern hybridization whether any of the microclones represent restriction fragment length polymorphisms (RFLPs), and then tried to map RFLP markers physically, using the recently developed chromosome microdissection/enzymatic amplification method. Of 13 clones analyzed, two were RFLP markers; a clone, pM2C83, showed a four-allele MspI RFLP, and the other, pM2C8, a two-allele RsaI RFLP. In order to assign the two polymorphic markers, two chromosomal segments, 2q32-q35 and 2q35-qter, on the chromosome 2 from a karyotypically normal person were microdissected, and the DNA from each segment was amplified with the polymerase chain reaction (PCR) using marker sequence-specific primers. With this method, both of the clones were assigned to 2q35. These two RFLP markers must be useful for linkage analysis of genetic diseases whose loci are at around 2q35.
Human chromosomal regions 8q23.3-q24.11 and 2q33-qter were microdissected, DNAs from the regions were amplified with the primer-linker method of polymerase chain reaction (PCR), and their DNA libraries were constructed by cloning into pUC19. The primer-linker PCR involved Sau3AI digestion of microdissected chromosomal DNAs, ligation of the digests to a 10mer DNA linker and 24mer primer, filling the recessed 3' ends, and PCR amplification using the 24mer DNA as a primer. A total of 3.5 x 10(4) pUC19 recombinants (8q library) from the 8q region and 5.0 x 10(4) pUC clones (2q library) from the 2q region were obtained. From the 8q library, 60 pUC clones were selected, while 88 pUC-clones were selected from the 2q library. These clones were Southern blot analyzed on hybrid cell panels with or without human chromosome 8 or 2. Twelve (20%) of the 60 8q-derived clones were unique DNA sequences, and 9 were subjected to deletion analysis in the genomic DNA of two patients, one with trichorhino-phalangeal syndrome (TRPS) type I and the other with TRPS type II, both with del(8) (q23.3q24.13). Five of the 9 pUC clones tested showed a one-copy density in both patients, an indication that the clones map to the region deleted in both patients. Screening a genomic DNA library constructed in the phage revealed a clone with a 9.4-kb insert and a one-copy density in both patients. From the 2q library, 15 (17%) of the 88 pUC clones obtained were unique sequences. When a phage library was screened, 8 clones were obtained: 4 were identical and 2 were overlapping sequences.(ABSTRACT TRUNCATED AT 250 WORDS)
We described cloning and characterization of an inversion breakpoint of chromosome 2 inv(2)(q35q37.3) observed in a patient with Waardenburg syndrome type I (WSI). Genomic cosmid clones containing the HuP2 gene, which was considered as a candidate for WSI, were isolated from a library constructed from the patient DNA. One of the clones contained the inversion breakpoint and revealed signals at both 2q35 and 2q37 by fluorescent in situ hybridization (FISH), indicating disruption of the HuP2 gene by the inversion. Our result further supports that the HuP2 gene is a candidate for Waardenburg syndrome type I and is located at q35.
Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct mental retardation disorders associated with deletions of proximal 15q (q11-q13) of different parental origin. Yeast artificial chromosome (YAC) clones were isolated for 9 previously mapped DNA probes from this region, and for one newly derived marker, LS6-1 (D15S113). A YAC contig of 1-1.5 Mb encompassing four markers (ML34, IR4-3R, PW71, and TD189-1) was constructed. Multi-color fluorescence in situ hybridization (FISH) analysis of interphase nuclei was combined with YAC contig information to provide the following order of markers: cen-IR39-ML34-IR4-3R-PW71-TD189-1-LS6++ +-1-TD3-21-GABRB3-IR10-1-CMW1-tel. FISH analysis was performed on 8 cases of PWS and 3 cases of AS, including 5 patients with normal karyotypes. All eleven patients were deleted for YACs in the interval from IR4-3R to GABRB3. On the proximal side of the deletion interval, 10/10 breakpoints fell within a single ML34 YAC of 370 kb. On the distal side, 8/9 breakpoints fell within a single IR10-1 YAC of 200 kb. These results indicate a striking consistency in the location of the proximal and distal breakpoints in PWS and AS patients. FISH analysis on a previously reported case of familial AS confirmed a submicroscopic deletion including YACs corresponding to LS6-1, TD3-21 and GABRB3 and supports the separation of the PWS and AS critical regions. Since these three YACs do not overlap each other, the minimum size of the AS critical region is > or = 650 kb.
An alternative method for amplification of DNA with unknown sequences was developed. This involves the direct ligation of a primer oligodeoxyribonucleotide itself to restricted DNA fragments with unknown sequences to be amplified by PCR. The oligonucleotide need not be phosphorylated and need not be annealed with its complementary oligonucleotide in advance for ligation. The ligation reaction seems to be independent of the concentration of unknown DNA, proceeds in short time, and is efficient. The ligation efficiency was more than 30% at a low concentration, 10 fg/microliters, of DNA. This method was applied to a microdissection/microcloning of the short arm of human chromosome 2. Of 65 clones screened for the highly repetitive sequences with total human genomic DNA, eleven (17%) were positive. Their inserts ranged in size from 150 to 1,200 bp (average, 460 bp). In Southern blot analysis, thirty consecutive clones all detected signals common to both total human genomic DNA and mouse-human hybrid cell DNA containing only chromosome 2 of human origin. Among them, 24 (80%) were unique sequences, and 6 (20%) were multi-copy (or intermediate-repeat) sequences. Thus, this method is simple and efficient, and provides an alternative way to amplify unknown DNA.
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We describe a girl, one of monozygotic (MZ) twins, with endocrine dysfunction with precocious puberty, café-au-lait nevi and polyostotic fibrous dysplasia (PFD), McCune-Albright syndrome (MAS). After treatment with cyproterone acetate for 7 years the precocious puberty and excess growth improved but the bone-age still remain advanced. The co-twin had an advanced bone-age and a small café-au-lait spot, but showed neither endocrinopathy nor fibrous dysplasia of bone. On the basis of the findings in these twins, together with those in previously reported familial cases of MAS, including two pairs of MZ twins, a 2-hit mutation hypothesis is proposed: a dominant mutation may be inherited and leads to PFD in offspring as the primary defect of MAS; the second mutation may occur in somatic cell leading to mosaicism and thus resulting in MAS. This concept explains not only sporadic cases of MAS but also reported familial cases. If we assume that the second mutation occurred in an early somatic division, it would explain the discrepancy of clinical manifestation between MZ twins.
We report clinical and cytogenetic findings of 4 children (2 boys and 2 girls) with the Smith-Magenis syndrome. All 4 patients had an interstitial deletion of 17p: del(17) (p11.2p11.2). Their clinical manifestations included brachycephaly, midface hypoplasia, prognathism, upper lip eversion, short and broad hands with short fingers, clinodactyly of the fifth fingers, fingertip pads, moderate mental retardation, and behavior problems. Analysis of the metacarpophalangeal pattern profiles in patient 2 showed progressive shortness from the metacarpals to the proximal, middle, and the distal phalanges. The fingerpads observed in all 4 patients have hitherto been noted in only one of 26 previously reported patients with the syndrome. These findings serve as a useful clue to the diagnosis of the syndrome.
Deletion and RFLP studies with 5 cloned DNA markers localized at 15q11.2 were performed in 50 patients with the Prader-Willi syndrome (PWS). A one-copy density (deletion) for at least one of 4 loci, D15S9, D15S11, D15S10, D15S12, was detected in 32 (64%) of the 50 patients; deletions of each of the 4 loci were found in 29, 30, 29, and 28 patients, respectively. Three patients showed 4 or more copy density for D15S12 locus, in addition to deletions. The remaining 18 patients showed two-copy densities for each of the 4 loci. A common site of rearrangements among our 32 patients as well as the reported patients seemed to be confined to a segment between D15S9 and D15S11, suggesting the putative PWS gene locus in this segment. Of 6 patients who have cytologic deletions but did not show any molecular deletions, 3 have normal size of hands and feet, and 4 have normally pigmented skin and hair. The normal pigmentation was also observed in 3 patients who had small molecular deletions in the examined 5-locus segment. These observations may support the conception of contiguous gene syndrome. RFLP analysis demonstrated maternal uniparental isodisomy of chromosomes 15 in both a patient with 45,t(15q;15q) and a karyotypically normal patient. Based on the results of the present study, a new model is proposed to explain the occurrence of PWS with a variety of chromosome abnormalities, including partial monosomy, disomy, trisomy, and/or tetrasomy for 15q11.2. The normal development may require an even or more "number ratio" of paternally derived allele(s) to maternally derived allele(s) of the gene(s) localized at 15q11.2, and a disturbance of the ratio would lead to the PWS phenotype.
DNA deletion studies using 5 DNA markers localized at 15q11-q12 were performed in 14 Angelman syndrome (AS) patients (9 sporadic and 5 familial cases). A one-copy density for one or more of the 5 loci was detected in 8 (57.1%) of the 14 patients. A deletion of only the D15S11 locus was detected in one sporadic patient, that involving only the D15S10 in 3 familial patients (sibs in a family), that spanning 3 loci (D15S11, D15S10, D15S12) in one sporadic patient, and that spanning 4 loci (D15S9, D15S11, D15S10, D15S12) in the other 3 sporadic patients. The deletion common to our patients as well as to the reported patients may be confined to a segment between D15S11 and D15S10, if the 5 loci are ordered as cen-D15S18-(D15S9-D15S11-D15S10)-D15S12-qt er. This site overlaps but is more distal to the common deletion site in Prader-Willi syndrome (PWS) patients. In the family of the 3 sibs, both of the phenotypically normal mother and maternal grandfather also have deletions of the D15S10 locus. These results were consistent with the genomic imprinting hypothesis for the occurrence of AS, i.e., the lack of a maternally derived locus leads to AS, but may not support a model that AS is the alternative phenotype of PWS at the identical locus.
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In a Japanese family, a propositus and his mother had crossed polydactyly type I. A maternal grandaunt also had preaxial polydactyly of the feet. The findings that both of the mother and son had the identical type of polydactyly are consistent with an autosomal dominant inheritance with variable expressivity. Other explanations include X-linked recessive inheritance, polygenic inheritance, and a chance occurrence of the 2 different kinds of polydactyly.
We describe a 6 month-old girl with a 49, XX-XXX chromosome constitution. The patient had a characteristic round face, a low hairline, hypertelorism, epicanthus, a long philtrum, high-arched palate, short and webbed neck, small hands and feet, clinodactyly of the fifth fingers, overlapping toes, and separation between the first and the second toes. She also had atrial septal defect and patent ductus arteriosus complicated by myocarditis which exacerbated the course of her congestive heart failure. Psychomotor development was retarded with opisthotonoid posture, axial hypotonia, and with a borderline abnormal EEG. A densitometric, transmission analysis on X-linked polymorphic DNA-fragments of the Southern blots of the patient and the parents, using P20/MspI and pERT87-1/XmnI as probe/enzyme combinations, showed that the pentasomy X had resulted from 3 successive nondisjunctions at maternal meiosis. Clinical manifestations among 22 previously reported penta X syndrome patients are also reviewed.