[Prognostic significance of chromosome analysis in childhood acute lymphoblastic leukemia. Children's Cancer and Leukemia Study Group (CCLSG)].

We analyzed the prognostic significance of chromosomal findings in children with acute lymphoblastic leukemia (ALL), treated according to the Children's Cancer and Leukemia Study Group protocols between 1987 and 1993. Patients were classified into 5 groups according to chromosome number. The patients with a hyperdiploid(> 50) karyotype(13%) had the best prognosis [4-year event-free survival (EFS): 83 +/- 6%], while those with a pseudodiploid karyotype (24%) had the worst prognosis(4-year EFS: 52 +/- 6%) (log-rank, p = 0.03). However, multivariate analysis revealed that the ploidy classification had no prognostic significance in terms of EFS. When patients were classified according to chromosome abnormalities, those with any type of translocation had a worse outcome (4-year EFS: 33 +/- 9%) than those with hyperdiploidy(> 50), normal diploidy, and other abnormalities(log-rank, p < 0.0001). Multivariate analysis revealed that chromosome abnormalities were an independent prognostic factor (relative risk 3.98; p < 0.0001). Patients with t(1; 19) had an EFS similar to that of patients with chromosome abnormalities other than translocations or normal diploidy. We conclude that chromosomal findings have prognostic significance, although some chromosome abnormalities lost their statistical significance after modern intensified chemotherapy. Childhood ALL should be further stratified according to chromosome classification.

Agreement between predicted risk and prevalence of Down syndrome in second-trimester triple-marker screening in Japan.

Based on 9350 pregnant Japanese women who were screened by serum triple-marker determination, accuracy of predicted risk for Down syndrome was examined using 24 Down syndrome cases detected either prenatally or postnatally. The correlation is statistically very high (r = 0.98) between the predicted risks and the prevalence of Down syndrome cases. Here we emphasize that this could be accomplished only by an extensive follow-up study, implemented in our prospective intervention programme.

Partially mismatched pediatric transplants with allogeneic CD34(+) blood cells from a related donor.

This was a phase I, multi-center study of 13 pediatric patients (median age, 11 years) to evaluate toxicity, hematopoietic recovery, and graft-versus-host disease (GVHD) after allogeneic transplantation of enriched blood CD34(+) cells obtained from genotypically haploidentical but partially HLA-mismatched related donors (8 parents and 5 siblings). With regard to rejection, donor HLA disparity was 1 (5), 2 (6), or 3 loci (2). With regard to GVHD, recipient HLA disparity was 0 (1), 1 (3), 2 (8), or 3 (1). The patients suffered from acute myelogenous leukemia (6), chronic myelogenous leukemia (4), acute lymphoblastic leukemia (2), or hemolytic anemia plus immunodeficiency disorder (1). To reduce the risk of graft failure through the infusion of a large amount of stem cells, peripheral blood cells (PBC) were mobilized by recombinant granulocyte colony-stimulating factor (G-CSF; lenograstim, 10 microgram/kg/d for 5 days) and collected by 2 to 5 aphereses. To both enhance engraftment and reduce GVHD, CD34(+) cells were enriched using immunomagnetic procedures with the Baxter ISOLEX 300 system (Baxter Healthcare Corp, Irvine, CA) and cryopreserved. After variable cytoreductive regimens, a median of 7.7 (range, 2.2 to 14) x 10(6)/kg of CD34(+) cells and 1.03 (0.05 to 2.09) x 10(5)/kg CD3(+) cells were infused. Using Center-specific posttransplant supportive care and immunosuppressive GVHD prophylaxis, two patients experienced early death; one from veno-occlusive disease at day 17 and one from sepsis at day 18. Nine of 11 patients showed signs of engraftment; however, subsequent rejection was seen in 4 patients, 2 of whom had autologous recovery. Eight patients were evaluated in the early phase of marrow recovery. The median number of days to achieve an absolute granulocyte count of 0.5 x 10(9)/L was 14 (range, 9 to 20) and that to achieve a platelet count of 20 x 10(9)/L was 17.5 (range, 12 to 23). Donor chimerism persisted in five patients until death or current survival. All of the surviving patients with functioning-donor-type hematopoiesis were given total body irradiation. De novo acute GVHD (grades II and IV) was observed in two of the eight evaluated patients. Scheduled donor lymphocyte infusion (DLI), using the CD34(-) fraction, was administered to four patients, free of de novo acute GVHD, beginning between 28 to 43 days after transplant. Three of these patients developed acute GVHD (grades I, II, and IV). Cytomegalovirus infection was a major infectious complication but was successfully managed with gamma-globulin and gancyclovir treatment with or without additional DLI. Five patients are currently surviving, free of disease, with a follow-up ranging from 476 to 937 days. Each survivor has functioning hematopoiesis, three of donor origin and two of autologous origin. In conclusion, our results show that enriched blood CD34(+) cells from a mismatched haploidentical donor are a feasible alternative source of stem cells, but do not appear to ensure engraftment. Because none of the patients who were administered DLI survived, the therapeutic efficacy and safety of periodic DLI, as an integrated part of such transplants, needs to be clarified in further studies.

Production of a mixture of antimicrobial organic acids from lactose by co-culture of Bifidobacterium longum and Propionibacterium freudenreichii.

The antimicrobial activities of standard solutions of three organic acids (lactic, acetic, and propionic acids) were compared using Micrococcus luteus, Pseudomonas sp. and Staphylococcus aureus as test microorganisms. At the same concentrations of the undissociated form, the antimicrobial activities of acetic and propionic acids were higher than that of lactic acid, irrespective of test microorganisms. In a single cultivation of Bifidobacterium longum, a mixture of lactic (17 g/l) and acetic (20 g/l) acids was produced from 50 g/l lactose and its antimicrobial activities against M. luteus, Pseudomonas sp., and S. aureus correspond to that of 32, 19, and 25 g/l of acetic acid, respectively. To increase the total antimicrobial activity, a co-culture of B. longum and Propionibacterium freudenreichii, in which lactic acid produced once from lactose by B. longum was converted to acetic and propionic acids by P. freudenreichii, was done using TPY medium containing commercially available peptones as a nitrogen source. By the sequential conversion of lactose using the two microorganisms, the culture supernatant containing a mixture of acetic (27 g/l) and propionic (13 g/l) acids without lactic acid was produced. The antimicrobial activities of the mixture against M. luteus, Pseudomonas sp., and S. aureus were 35, 30, and 26 g/l as a concentration of acetic acid, respectively, higher than that obtained in the cultivation of B. longum alone. When the medium containing an enzymatic hydrolyzate of whey proteins with a protease was used in the co-culture of B. longum and P. freudenreichii, the culture supernatant containing the mixture of organic acids was also obtained in the same manner as the co-culture using TPY medium and the activities were 43, 29, and 29 g/l as a concentration of acetic acid for M. luteus, Pseudomonas sp. and S. aureus, respectively.

Transient neurotoxicity associated with FK506: MR findings.

We report a patient presenting with transient neurotoxicity who developed edema in the cerebral and cerebellar cortex following bone marrow transplant and administration of FK506, a widely used immunosuppresive agent.

Genetic detection of colorectal cancer cells in circulation and lymph nodes.

PURPOSE: This study was undertaken to investigate the clinical implications of detection of genetic alterations in blood and lymph nodes in colorectal cancer patients. METHODS: The reverse transcriptase polymerase chain reaction product of the cytokeratin gene in blood was examined as a cancer cell-specific expression in 35 colorectal cancer patients. The K-ras or p53 gene mutations in the lymph nodes histopathologically negative for metastasis were studied by the mutant-allele-specific amplification method in 26 colorectal cancer patients. RESULTS: The reverse transcriptase polymerase chain reaction assay was able to detect a cytokeratin reverse transcriptase polymerase chain reaction product at a concentration from a single to ten colon cancer cells per 10(6) normal peripheral blood mononuclear cells. Cytokeratin reverse transcriptase polymerase chain reaction products were detected in nine patients' blood samples, although none of the samples were cytologically detectable. The blood's cytokeratin positivity correlated with the invasive mode of the tumor (P < 0.05) and the presence of distant metastasis (P < 0.01). Two (50 percent) of four patients whose blood was positive for cytokeratin had recurrences. Of 17 patients with the K-ras or p53 gene mutation in primary tumors, 9 (53 percent) had the corresponding mutations in lymph nodes. Mutation positivity in lymph nodes correlated with the presence of lymphatic invasion of the primary tumor (P < 0.05). All patients with mutation-negative lymph nodes remained disease-free for more than two years after surgery. CONCLUSION: Detection of cytokeratin reverse transcriptase polymerase chain reaction products in the blood and K-ras or p53 gene mutations in the lymph nodes histologically negative for metastasis may be applicable for clinical use, despite some limitations, and may serve as a useful clinical factor for stratifying patients who are at high or low risk for recurrence after surgery.

Childhood multiple sclerosis treated with plasmapheresis.

We report a case of multiple sclerosis in a 7-year-old boy. He experienced three episodes in 8 months and was repeatedly treated with a high dose of methylprednisolone. During the third episode, to avoid the side effects associated with frequent high doses of steroid, we substituted plasmapheresis for methylprednisolone, initially performing it for 3 days and continuing it every 2 to 3 weeks according to the fluctuating values of antinuclear antibody. The patient improved markedly after initiation of plasmapheresis and has been relapse-free for more than 18 months. The effectiveness of plasmapheresis for treatment of multiple sclerosis in adults is variable and has seldom been reported in children. Our case suggests that plasmapheresis as an alternative therapy is useful for steroid-dependent or severe types of multiple sclerosis even in childhood, especially when its chronic course is assessed by antinuclear antibody titers.

An increased NM23H1 copy number may be a poor prognostic factor independent of LOH on 1p in neuroblastomas.

In a study of 154 neuroblastomas, loss of heterozygosity (LOH) was observed on 1p (13%, 19/143), 11q (19%, 11/59), 14q (15%, 15/97), 17p (5%, 5/105) and 17q (17%, 9/52). We also found an increase in NM23H1 copy number in 14% (13/95) of neuroblastomas. All except one tumour with an increased copy number stained positive with anti-NM23H1 monoclonal antibody. Event-free survival (EFS) was significantly shorter in 19 patients with LOH on 1p than in 128 without (41% vs 77% 4 year EFS, P=0.0093), and in 13 patients with increased NM23H1 copy numbers than in 82 with normal copy numbers of the gene (61% vs 84% 4 year EFS, P=0.0103). LOH on 11q, 14q or 17q did not affect EFS. Most tumours with LOH on 1p, increased NM23H1 copy numbers or MYCN amplification occurred in patients aged 12 months or more, those with advanced stage disease, and those who showed near diploidy or pseudodiploidy. However, LOH on 1p was found in only 1 of the 13 tumours with increased NM23H1 copy numbers, and MYCN amplification of four copies occurred in only one other such tumour. These findings suggest that the increased NM23H1 copy number may be a predictor for poor prognosis, independent of LOH on 1p, and probably also of MYCN amplification.

A comparative study on essential oil components of wild and cultivated Atractylodes lancea and A. chinensis.

To clarify the variation in the pharmacologically active components of the essential oil contained in the rhizomes of Atractylodes lancea and A. chinensis growing in China, we transplanted the rhizomes of the wild plants from 18 populations, including A. koreana, in the same experimental field. After two or three years' cultivation, main essential oil components, i.e., the sesquiterpenes: elemol (1), atractylon (2), hinesol (3), beta-eudesmol (4), selina-4(14),7(11)-dien-8-one (5), and the polyacetylene of atractylodin (6) were determined by capillary gas chromatography. The analytical data of 306 cultivated plants were compared with plants collected in their habitat. A. lancea varied significantly in the contents of the components after cultivation; however, the correlation coefficient in the contents of 3, 4, and 6 between the wild and cultivated plants were 0.985, 0.954, and 0.945, respectively (p < 0.001). Compared to this, A. chinensis had constant content values. Three types of A. lancea and two types of A. chinensis, which are distinguished by the characteristics of the components in the wild conditions, were statistically recognized after cultivation. From these results, it was determined that the geographical variation in the components of these species mainly reflects genetic variability.

Triple marker screening in native Japanese women.

Prenatal screening using the maternal serum markers alpha-fetoprotein, human chorionic gonadotropin, and unconjugated oestriol was investigated in a native Japanese population. Comparison with a Caucasian U.S. population revealed differences which led to modification of the generally used equations for risk calculations. Prenatal screening was shown to be clinically useful.

[Usefulness and problems of high dose chemotherapy using CBDCA, VP-16, and MCNU with peripheral blood stem cell transplantation in pediatric malignant brain tumors].

The aim of this study was to evaluate efficacy of high dose chemotherapy and restoration of hamatopoiesis following peripheral blood stem cell transplantation (PBSCT). Three patients with pediatric malignant brain tumors (two medulloblastomas and one medullomyoblastoma) underwent high dose chemotherapy including CBDCA, VP-16, and MCNU with PBSCT. Postcontrast-MR images revealed no abnormal enhancing lesions after high dose chemotherapy in all patients. One patient with medulloblastoma has remained complete remission one year and seven months after the termination of treatment. Another patient with medullomyoblastoma died of respiratory distress syndrome one month after the second course of high dose chemotherapy. The other patient with medulloblastoma, which received PBSCT and high dose chemotherapy at the time of tumor recurrence after failure of initial treatment, suffered from multiple disseminated lesions five months after the treatment. PBSCT contributed prompt recovery from hematopoietic dysfunction in all patients. These results indicate that PBSCT may play an important adjuvant to chemotherapy and further offer a safer and more effective high dose chemotherapy in pediatric malignant brain tumor patients.

Tumor necrosis factor-alpha stimulates the production of squamous cell carcinoma antigen in normal squamous cells.

Squamous cell carcinoma (SCC) antigen, a tumor marker of squamous cell carcinoma, is also increased in several nonmalignant skin lesions, e.g. pemphigus. The aim of the present investigation was to determine if tumor necrosis factor-alpha (TNF-alpha), one of the important environmental factors, stimulated the production of SCC antigen in the normal squamous cells. The exposure of normal human epidermal keratinocytes to TNF-alpha (100 IU/ml) for 72 h greatly increased the SCC antigen production. The stimulatory effect of TNF-alpha (1,000 IU/ml) on the production of SCC antigen was also observed in the normal squamous epithelium tissue. These results would be helpful for understanding the increase of SCC antigen in several nonmalignant skin disorders.

Detection of neoplastic clone in the hypoplastic and recovery phases preceding acute lymphoblastic leukemia by in vitro amplification of rearranged T-cell receptor delta chain gene.

This study assessed the clonality of hypoplastic and subsequent recovery phases before the development of overt leukemia by molecular genetic analysis. We describe a boy who had transient granulocytopenia and anemia before the development of acute lymphoblastic leukemia (ALL). Initially, his bone marrow was hypocellular with 23.6% of lymphoblastic cells, whereas subsequent marrow after the administration of granulocyte colony-stimulating factor (G-CSF) appeared almost normal without any lymphoblasts. At diagnosis, we found the rearrangement of T cell receptor (TCR) delta gene in the leukemic cell DNA by Southern blot hybridization. The junctional sequence of the V delta 2-D delta 3 recombination of leukemic cells obtained by polymerase chain reaction (PCR) was used for a clonospecific probe. Using the probe, the presence of leukemic clone in the materials before and after diagnosis was examined. We found that the clonospecific probe could detect one leukemic cell in 10,000 normal cells, and we demonstrated the presence of the leukemic clone at the initial hypoplastic and the subsequent recovery phase. The PCR method is very useful to confirm the presence of leukemic clone even in a retrospective analysis using low-quality materials and may be helpful to understand the pathogenesis of a smoldering preleukemic phase.

Induction of autoantibodies in normal mice by injection of nucleobindin and natural occurrence of antibodies against nucleobindin in autoimmune MRL/lpr/lpr mice.

Our previous works have shown that nucleobindin (Nuc) or recombinant (r) Nuc not only augments anti-DNA antibody production in vitro but also accelerates autoimmune response in vivo in MRL/+/+ (MRL/n) mice which are the substrain of autoimmune MRL/lpr/lpr (MRL/l) mice. To investigate whether rNuc can induce autoimmune response similarly in naive mice, we carried out intraperitoneal (i.p.) injection of rNuc (5 micrograms) without adjuvant into 8-week-old female BALB/c mice and continued injection twice a week for 12 weeks. About 5 weeks after the first injection, all the mice began to show IgG hypergammaglobulinemia (HG) followed by elevation of a number of autoantibodies of the IgG class such as anti-double-stranded (ds) DNA, anti-U1 ribonuclear protein (RNP), anti-ssB(La) and anti-Fc antibodies (RF), but not by anti-Sm antibodies. However, the IgG anti-dsDNA antibody response and histopathological changes in the kidney of these BALB/c mice were not so noticeable as those in MRL/n mice induced by rNuc in our previous experiment. In contrast, the IgG anti-rNuc antibody response of normal BALB/c mice induced by rNuc was stronger than that of MRL/n mice induced by rNuc. Since the titers of each autoantibody of BALB/c mice induced by rNuc were not always associated with the level of IgG HG, and either IgG HG or IgG autoantibodies could not be induced by control administration of extracts (5 micrograms) of Escherichia coli with or without harboring plasmid alone, polyclonal B cell activation (PBA) appeared not to be the mechanism of this autoimmunity.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the human gene (TBXAS1) encoding thromboxane synthase.

The gene encoding human thromboxane synthase (TBXAS1) was isolated from a human EMBL3 genomic library using human platelet thromboxane synthase cDNA as a probe. Nucleotide sequencing revealed that the human thromboxane synthase gene spans more than 75 kb and consists of 13 exons and 12 introns, of which the splice donor and acceptor sites conform to the GT/AG rule. The exon-intron boundaries of the thromboxane synthase gene were similar to those of the human cytochrome P450 nifedipine oxidase gene (CYP3A4) except for introns 9 and 10, although the primary sequences of these enzymes exhibited 35.8% identity each other. The 1.2-kb of the 5'-flanking region sequence contained potential binding sites for several transcription factors (AP-1, AP-2, GATA-1, CCAAT box, xenobiotic-response element, PEA-3, LF-A1, myb, basic transcription element and cAMP-response element). Primer-extension analysis indicated the multiple transcription-start sites, and the major start site was identified as an adenine residue located 142 bases upstream of the translation-initiation site. However, neither a typical TATA box nor a typical CAAT box is found within the 100-b upstream of the translation-initiation site. Southern-blot analysis revealed the presence of one copy of the thromboxane synthase gene per haploid genome. Furthermore, a fluorescence in situ hybridization study revealed that the human gene for thromboxane synthase is localized to band q33-q34 of the long arm of chromosome 7. A tissue-distribution study demonstrated that thromboxane synthase mRNA is widely expressed in human tissues and is particularly abundant in peripheral blood leukocyte, spleen, lung and liver. The low but significant levels of mRNA were observed in kidney, placenta and thymus.

Isolation and analysis of cDNA encoding a precursor of Canavalia ensiformis asparaginyl endopeptidase (legumain).

Recently, asparaginyl endopeptidase was purified to homogeneity from jack bean (Canavalia ensiformis) seeds, and its NH2-terminal amino acid sequence was determined for 25 residues [Abe, Y. et al. (1993) J. Biol. Chem. 268, 3525-3529]. On the basis of this sequence information, we searched for seed cDNAs encoding this enzyme. Seven clones were obtained and sequenced. By combining four of them, we obtained a cDNA for a precursor of the enzyme containing the reported NH2-terminal sequence. The other three clones seemed to be for precursors of its isozymes. When the deduced amino acid sequences of these enzyme precursors were compared with those in the GeneBank, EMBL, and NBRF databases, only one protein was found with a homologous sequence. It was a precursor of hemoglobinase from a blood fluke (Schistosoma mansoni). Significant homology was observed only in the range of sequence for the mature form. Although hemoglobinase and asparaginyl endopeptidase behave as cysteine proteinases, their protein natures are distinct from either papain-type proteinases or clostripain. Various plant seeds have been reported to contain asparaginyl endopeptidases. This may be the first report, however, that deals with the primary structure of such a proteinase.

Analysis on heterogeneity of squamous cell carcinoma antigen by two-dimensional electrophoresis.

Squamous cell carcinoma (SCC) antigen was separated by two-dimensional electrophoresis combined with immunoblotting into four spots: spot 1 with pI 6.4 and 44.5 kDa, spot 2 with pI 6.3 and 44.5 kDa, spot 3 with pI 6.0 and 44.5 kDa, and spot 4 with pI 5.9 and 45 kDa. In cancer and noncancerous tissues, it was common that spot 1 was the largest spot. In noncancerous tissues, spot 3 was the smallest spot and spot 2 was stained as densely as spot 4. In cancer tissues, however, spot 4 was apparently smaller than spot 2 and 3. Also, spots 2 and 3 in cancer tissues were larger than those in noncancerous tissues. When SCC antigen was treated with alkaline phosphatase prior to isoelectric focusing (IEF), spot 4 disappeared from the immunoblotting pattern. When the SCC antigen was treated with alkaline phosphatase after IEF, spot 4 changed its molecular weight to the same weight as that of the other three spots. These results strongly suggest that spot 4 is phosphorylated SCC antigen.

There may be two tumor suppressor genes on chromosome arm 1p closely associated with biologically distinct subtypes of neuroblastoma.

We studied loss of heterozygosity (LOH) on chromosome arm 1p in 108 neuroblastomas using 14 polymorphic DNA markers. One-hundred and four tumors with one or more informative loci; 21 (20%) of the 104 tumors showed LOH on 1p, and were classified into three groups on the basis of interstitial or terminal allelic loss, and presence or absence of LOH on 1p. Seven of the 21 tumors showed an interstitial deletion which encompassed a small region in 1p36 (group A), and the other 14 showed a terminal deletion which encompassed the region from 1pter to 1p32 (group B). Eighty-three tumors without LOH on 1p were classified as group C. The group A patients were mostly less than 12 months of age (6/7), were frequently found by a mass screening program for infants (5/7), had a tumor of non-adrenal origin, and rarely progressed to stage IV (1/7). Most group B patients were 12 months or older (11/14), were found clinically (11/14), had tumors of adrenal origin, and progressed to stage IV (10/14). Analysis of biologic characteristics in group C tumors suggested that they may comprise group A and B tumors. While all group A tumors were in the triploid range (3n) (4/4), most group B tumors were diploid (2n) or tetraploid (4n) (7/10). MYCN amplification was found in 8 group B tumors, but in none of group A tumors. Event-free survivals of groups A, B, and C patients at 3 years were 86, 49, and 74%, respectively (P = 0.0287). These findings suggest that there may be two tumor suppressor genes on 1p which are closely associated with two biologically distinct subtypes of neuroblastoma.

Characterization of the human gene (PTGS2) encoding prostaglandin-endoperoxide synthase 2.

The human gene (PTGS2) encoding an inducible isozyme of prostaglandin-endoperoxide synthase (prostaglandin-endoperoxide synthase 2) that is distinct from the well-characterized and constitutive isozyme (prostaglandin-endoperoxide synthase 1), was isolated using a polymerase-chain reaction-generated cDNA fragment probe for human prostaglandin-endoperoxide synthase 2. Nucleotide sequence analysis of the entire human prostaglandin-endoperoxide-synthase-2 gene demonstrated that it is more than 8.3 kb in size and consists of ten exons; this gene is very similar to the murine and chicken prostaglandin-endoperoxide-synthase-2 genes. The structures of exons in the human prostaglandin-endoperoxide-synthase-2 gene were also similar to those of the human prostaglandin-endoperoxide-synthase-1 gene (PTGS1). However, the sizes of introns in the human prostaglandin-endoperoxide-synthase-2 gene were generally smaller than those of the human prostaglandin-endoperoxide-synthase-1 gene. Primer-extension analysis indicated that the transcriptional-start site is 134 bases upstream of the translational-initiation site. The sequence of the 1.69-kb region of nucleotides preceding the transcriptional-start site and the first 0.8-kb intron contained a canonical TATA box and various transcriptional-regulatory elements (CArG box, NF-IL6, PEA-1, myb, GATA-1, xenobiotic-response element, cAMP-response element, NF-kappa B, PEA-3, Sp-1 and 12-O-tetradecanoyl-phorbol-13-acetate-response element). The nucleotide sequence of the 5'-flanking region (275 bp) of the human prostaglandin-endoperoxide-synthase-2 gene showed 63% similarity to the sequence of murine prostaglandin-endoperoxide-synthase-2/TIS10 gene, but essentially no homology to the chicken prostaglandin-endoperoxide-synthase-2 gene, and human and murine prostaglandin-endoperoxide-synthase-1 genes. A fluorescence in situ hybridization study showed that the human genes coding for prostaglandin-endoperoxide synthase 1 (PTGS1) and prostaglandin-endoperoxidase synthase 2 (PTGS2) were mapped to distinct chromosomes 9q32-q33.3 and 1q25.2-q25.3, respectively, indicating that these genes are not genetically linked.