Akira Kuwano

Evidence that poor metabolizers of (S)-mephenytoin could be identified by haplotypes of CYP2C19 in Japanese.

(S)-Mephenytoin is metabolized by CYP2C19. The purpose of this study was to examine availability of phenotyping of poor metabolizers (PMs) of (S)-mephenytoin by polymerase chain reaction (PCR)/restriction enzyme genotyping of CYP2C19 in a Japanese population. We genotyped 217 unrelated healthy Japanese for functionally defective alleles, CYP2C19m1 and CYP2C19m2. The frequencies of the wild type(wm1) and CYP2C19m1 were 0.726 and 0.274, and the wild type(wm2) and CYP2C19m2 were 0.892 and 0.108 respectively. Although the observed numbers of three genotypes were very similar to those estimated according to the Hardy-Weinberg equilibrium for each defect, CYP2C19m2 was not detected in m1 homozygotes, and CYP2C19m1 was not detected in m2 homozygotes. Two defects were inherited separately in four families indicating CYP2C19m1 and m2 segregate independently at the same gene locus. Based on these data, we calculated the haplotype frequencies of wm1-wm2, CYP2C19m1-wm2 and wm1-CYP2C19m2 to be 0.618, 0.274 and 0.108 respectively. Frequencies of homozygotes for CYP2C19m1 and CYP2C19m2 and compound heterozygotes associated with the PM phenotype, were calculated to be 7.5, 1.2 and 5.9% respectively. In total, 14.6% of Japanese are estimated to be PMs. No significant difference was observed between the frequencies of PMs calculated from our results and that identified by urinary S/R ratio (18%) (p > 0.05, chi 2 = 0.545, fd = 1). Our data indicate that Japanese PMs of (S)-mephenytoin could be identified by PCR-based genotyping of CYP2C19.

Synergistic effect of aphidicolin and ethanol on the induction of common fragile sites.

SummaryThe effect of ethanol on the frequency of aphidicolin-induced common fragile sites was studied using lymphocyte cultures from two normal women. Aphidicolin was added to the cultures at a final concentration of 0.2 μM and ethanol at 0.02%, 0.1%, 0.2%, 0.5%, and 1%, both during the last 26 h of culture. The frequency of common fragile sites increased from 296% in subject 1 and 201% in subject 2 with aphidicolin plus 0.02% ethanol, to 765% and 823%, respectively, with aphidicolin plus 1% ethanol. Ethanol alone added to cultures did not induce common fragile sites. The gaps and breaks induced by aphidicolin plus ethanol were highly nonrandom. Altogether, 35 common fragile sites were identified. The addition of 1% ethanol to aphidicolin increased both random and nonrandom gaps and breaks as compared with that of 0.02% ethanol. Dimethyl sulfoxide added to culture at final concentrations of 0.02% to 1% did not change the frequency of aphidicolin-induced fragile sites. The frequency of fluorodeoxyuridine-induced fragile sites was not affected by the addition of 0.02% to 1% ethanol. It was thus concluded that ethanol enhances the aphidicolin-induced fragile sites, possibly inhibiting the repair mechanism of gaps and breaks induced by aphidicolin.

Fibroblast-specific common fragile sites induced by aphidicolin

SummaryThe distribution and frequency of aphidicolin-induced common fragile sites were studied in chromosomes of cultured skin fibroblasts and PHA-stimulated lymphocytes from five normal individuals; 0.2 μM aphidicolin was added for the last 26 h of culture. Skin fibroblasts from five fra(X)-positive patients were also studied in the same manner. Fragile sites most frequently found in fibroblasts from normal individuals were 3q26.2, 7q11.23, 16q23, 1p31, 10q11.2, 12q23 and 7q31, whereas those in lymphocytes from the same individuals were 3p14, 16q23, Xp22, 7q32 and 14q24. The distribution of fragile sites in fibroblasts from fra(X)-positive patients was essentially identical with that in normal individuals. The average number of gaps and breaks in 100 metaphases was 36.8 in fibroblasts from normal individuals, 113.8 in those from fra(X)-positive patients, and 279 in lymphocytes from normal individuals. Their rates of chromosome-type breaks and gaps were 7.9%, 29.7% and 54.5%, respectively. Thus, the distribution and frequency of aphidicolin-induced fragile sites were different between skin fibroblasts and lymphocytes, possibly reflecting differences in their DNA replication sequence or gene activity.

Mapping basigin (BSG), a member of the immunoglobulin superfamily, to 19p13.3

Basigin is a novel member of the immunoglobulin superfamily ubiquitously expressed in various tissues. We mapped the basigin gene at 19p13.3, using a 1.6-kb cDNA fragment of the gene as a probe and sequential fluorescence in situ hybridization and G-banding on human metaphase chromosomes.

Possible mapping of the gene for transient myeloproliferative syndrome at 21q11.2

SummaryThe parental origin of the extra chromosome 21 was studied in 20 patients with trisomy 21-associated transient myeloproliferative syndrome (TMS) using chromosomal heteromorphisms as markers; this was combined with a study of DNA polymorphisms in 5 patients. Of these, 10 were shown to result from duplication of a parental chromosome 21, viz., maternal in 8 and paternal in 2. A patient with Down syndrome-associated TMS had a paracentric inversion in two of his three chromosomes 21 [47,XY,-21, +inv(21)(q11.2q22.13)mat, +inv(21)(q11.2 q22.13)mat). These findings support our hypothesis of “disomic homozygosity” of a mutant gene on chromosome 21 in 21-trisomic cells as being a mechanism responsible for the occurrence of TMS. The finding also suggests that the putative TMS gene locus is at either 21q11.2 or 21q22.13, assuming that the gene is interrupted at either site because of the inversion. The study of 5 TMS patients using DNA polymorphic markers detected a cross-over site on the duplicated chromosomes 21 between 21q11.2 (or q21.2) and 21q21.3 in one patient, and a site between 21q21.3 and q22.3 in another patient, evidence that confined the gene locus to the 21cen-q21.3 segment. These findings suggest that the putative TMS gene is located at 21q11.2. The extra chromosome 21 in the latter two TMS patients probably resulted from maternal second meiotic non-disjunction, in view of the presence of recombinant heterozygous segments on their duplicated chromosomes 21.

Cell type-dependent difference in the distribution and frequency of aphidicolin-induced fragile sites: T and B lymphocytes and bone marrow cells.

SummaryThe distribution and frequency of aphidicolin-induced common fragile sites were studied in Epstein-Barr virus-transformed B lymphocytes from eight normal individuals, and in bone marrow cells from six children in remission from malignant blood diseases. PHA-stimulated helper T lymphocytes from the same individuals were also studied. These cells were cultured in MEM, and treated with 0.2μM aphidicolin for 26 h. The results, together with those of our previous study on cultured skin fibroblasts, indicated that the distribution and frequency of aphidicolin-induced fragile sites are different among different types of cells.

Mosaicism for del(17) (p11.2p11.2) underlying the Smith-Magenis syndrome

Smith-Magenis syndrome (SMS) is a multiple congenital anomalies/mental retardation syndrome associated with deletion of band p11.2 of chromosome 17. The deletion is typically detected by high-resolution cytogenetic analysis of chromosomes from peripheral lymphocytes. Fluorescence in situ hybridization (FISH) has been previously used to rule out apparent mosaicism for del(17)(p11.2p11.2) indicated by routine cytogenetics. We now report mosaicism for del(17)(p11.2p11.2) in a child with SMS. The mosaicism had gone undetected during previous routine cytogenetic analysis. FISH analysis of peripheral lymphocytes as well as immortalized lymphoblasts using markers from 17p11.2 revealed that approximately 60% of cells carried the deletion. To our knowledge, this is the first case of SMS associated with mosaicism for del(17)(p11.2p11.2).

Translocation t(X;21)(q13.3; p11.1) in a girl with Menkes disease

A girl with a 46,X,t(X;21) (q13.3;p11.1) karyotype presented with skin redundancy, especially in the neck, prominent occiput and micrognathia, and later developed hypotonia, hypopigmentation, sparse scalp hair, and profound mental retardation characteristic of Menkes disease. Her serum copper (14 microg/dl) and ceruloplasmin (9 mg/dl) levels were extremely low. Fluorescent in situ hybridization analysis with a 100-kb P1-derived artificial chromosome probe containing the Menkes disease gene demonstrated three twin-signals, one on the normal X chromosome and one each on derivative chromosomes X and 21, indicating that the Xq13.3 breakpoint was located within the gene. Replication pattern analysis showed that the normal X chromosome was late replicating, whereas the derivative X chromosome was selectively early replicating. These results indicated that Menkes disease in our patient resulted from a de novo translocation that disrupts the disease gene.

Inverted insertion (9)(q34.3q22.3q21.2) and its recombination product: Duplication 9q21.2q22.3

To the Editor: We read with interest a recent article in your journal by Dr. Narahara and his colleagues (1986) who described a kindred with two carriers of what they interpreted as an intrachromosomal direct shift, dir ins(9)(q34.3q22.1q31.3), and a now 9-yearold girl with a recombinant chromosome 9. It was deduced that the rec(9) in the girl resulted from crossing over at one of two loops formed during meiosis I in her carrier mother, and thus carried duplication of the 9q22.1---~q31.3 segment. The ABO locus was assigned to 9q31.3-~ qter in view of the fact that the girl was a recombinant for the locus. We studied another kindred in which an apparently identical ins(9) chromosome is segregating through five generations. Our kindred and the maternal grandfather of the proband in Naraharas kindred both live in Tokuyama, a city with a 113,000 population. It is thus likely, but yet to be proven, that the two kindreds are related with each other. Our interpretation of the ins(9) is different from Naraharas (Fig. l, upper row). It involves insertion of an inverted 9@1.2,q22.3 segment into 9q34.3. Pairing of the inv ins(9) chromosome with a normal chromosome 9 during meiosis I in a carrier individual would produce two loops, one involving the inverted q21.2--~ q22.3 segment and the other the @2.3--+q34.3 segment (Fig. l, lower row). Odd numbers of crossing over in the latter loop would produce a recombinant chromosome with duplication of the q21.2--, q22.3 segment and one with deficiency of the same segment (Fig. 2). Family studies in our kindred disclosed three ins(9) carriers and two individuals with dup 9q21.2--,q22.3 (Fig. 3). It was deduced that both l 1-2 and 11-3, and also either I-i or I-2, were carriers of the inv ins(9) chromosome. Thus, the trait was transmitted through at least five generations. V-4, the proband in our kindred, a 2 year 2 month-old girl with dup 9q21.2 ~-, q22.3, weighed 1,874 g at birth. She walked at 15 months but did not speak meaningful words at age 2 2/12 years. She measured 76.6 cm ( 3 . 2 SD) and weighed 8 kg ( 3 SD). She had ocular hypertelorism, a short nose with a depressed nasal tip, short neck, low-set, malformed ears, fifth finger clinodactyly, absence of bilateral palmar triradii C and distally placed axial triradii. Her bone age was correspondent to her chronological age. III-5, a maternal distant relative of the proband, also carries the dup(q) chromosome. She is now 45 years old and mentally retarded, but no other details are known to us. The proband in Naraharas kindred, a 5year-old girl with dup 9q, had a low birth weight, growth deficiency, ocular hypertelorism, and dermatoglyphic abnormalities. Her IQ was 92 and her bone age

Cell type-dependent difference in the distribution and frequency of excess thymidine-induced common fragile sites: T lymphocytes and skin fibroblasts

SummaryCommon fragile sites were induced by excess thymidine in phytohemagglutin-stimulated T lymphocytes from 4 normal individuals, and skin fibroblasts from 4 normal and 5 fra(X) positive individuals. The results indicate that the frequency and distribution of excess thymidine-induced fragile sites are different between these two types of cells. The sites at 1p13 and 2p11.2, induced in both types of cells, have not previously been described, and are thus considered to be excess thymidine-specific fragile sites. These findings extend and support our previous studies on cell type-dependent difference in aphidicolin-induced common fragile sites.