Small nuclear ribonucleoprotein polypeptide N (SNRPN), an expressed gene in the Prader-Willi syndrome critical region.

Prader-Willi syndrome (PWS) is associated with paternally derived chromosomal deletions in region 15q11-13 or with maternal disomy for chromosome 15. Therefore, loss of the expressed paternal alleles of maternally imprinted genes must be responsible for the PWS phenotype. We have mapped the gene encoding the small nuclear RNA associated polypeptide SmN (SNRPN) to human chromosome 15q12 and a processed pseudogene SNRPNP1 to chromosome region 6pter-p21. Furthermore, SNRPN was mapped to the minimal deletion interval that is critical for PWS. The fact that the mouse Snrpn gene is maternally imprinted in brain suggests that loss of the paternally derived SNRPN allele may be involved in the PWS phenotype.

Giemsa-11 staining of chromosome 1: a newly described heteromorphism.

Sequential Giemsa-11 and C-band staining of the heterochromatic region of chromosome 1 from 30 unrelated individuals revealed a high degree of variability within this region, more than was identifiable with either stain alone. The Giemsa-11 stained material usually appeared as a single band of only slightly varying size within the heterochromatic region. The position of this band ranged from a location immediately adjacent to the centromere, to one farther along the long arm or at the junction of the C-band heterochromatin and euchromatin. Two individuals had a chromosome 1 with no detectable Giemsa-11 band but an average-size C-band. Two others with a large heterochromatic segment by C-banding had two Giemsa-11 positive bands. Additional studies of five members of one family were consistent with transmission of these hetermorphisms in codominant Mendelian fashion.

Localization of the gene encoding the human interleukin-2 receptor on chromosome 10.

The human interleukin-2 receptor is an inducible growth factor receptor present on the surface of activated T lymphocytes. The receptor is required for a normal T-cell immune response. High-resolution fluorescence-activated chromosome sorting and DNA spot-blot analysis with complementary DNA's for the interleukin-2 receptor indicated that the receptor gene was located on chromosome 9, 10, 11, or 12. In situ hybridization studies showed that the interleukin-2 receptor gene is on the short arm of chromosome 10, p14----15.

Human von Willebrand factor (vWF): isolation of complementary DNA (cDNA) clones and chromosomal localization.

Human factor VIII--von Willebrand factor (vWF) is a large, multimeric glycoprotein that plays a central role in the blood coagulation system, serving both as a carrier for factor VIIIC (antihemophilic factor) and as a major mediator of platelet-vessel wall interaction. Diminished or abnormal vWF activity results in von Willebrand's disease (vWD), a common and complex hereditary bleeding disorder. Overlapping vWF cDNA clones that span 8.2 kilobases of the vWF messenger RNA have been obtained. vWF accounts for approximately 0.3 percent of endothelial cell messenger RNA and was undetectable in several other tissues examined. A large single copy gene for vWF is located on the short arm of chromosome 12 (12p12----12pter). No gross gene rearrangement or deletion was detected in the DNA of two patients with severe vWD.

Detection of bcr-abl fusion in chronic myelogeneous leukemia by in situ hybridization.

Chronic myelogeneous leukemia (CML) is genetically characterized by fusion of the bcr and abl genes on chromosomes 22 and 9, respectively. In most cases, the fusion involves a reciprocal translocation t(9;22)(q34;q11), which produces the cytogenetically distinctive Philadelphia chromosome (Ph1). Fusion can be detected by Southern (DNA) analysis or by in vitro amplification of the messenger RNA from the fusion gene with polymerase chain reaction (PCR). These techniques are sensitive but cannot be applied to single cells. Two-color fluorescence in situ hybridization (FISH) was used with probes from portions of the bcr and abl genes to detect the bcr-abl fusion in individual blood and bone marrow cells from six patients. The fusion event was detected in all samples analyzed, of which three were cytogenetically Ph1-negative. One of the Ph1-negative samples was also PCR-negative. This approach is fast and sensitive, and provides potential for determining the frequency of the abnormality in different cell lineages.

The gene for enhancer binding proteins E12/E47 lies at the t(1;19) breakpoint in acute leukemias.

The gene (E2A) that codes for proteins with the properties of immunoglobulin enhancer binding factors E12/E47 was mapped to chromosome region 19p13.2-p13.3, a site associated with nonrandom translocations in acute lymphoblastic leukemias. The majority of t(1;19)(q23;p13)-carrying leukemias and cell lines studied contained rearrangements of E2A as determined by DNA blot analyses. The rearrangements altered the E2A transcriptional unit, resulting in the synthesis of a transcript larger than the normal-sized E2A mRNAs in one of the cell lines with this translocation. These observations indicate that the gene for a transcription factor is located at the breakpoint of a consistently recurring chromosomal translocation in many acute leukemias and suggest a direct role for alteration of such factors in the pathogenesis of some malignancies.

Importance of bone marrow cytogenetic evaluation before autologous bone marrow transplantation for Hodgkin's disease.

Alkylating agents used either with or without radiation therapy have been associated with the development of myelodysplastic syndrome (MDS) and acute nonlymphoblastic leukemia (ANLL) after treatment of both malignant and nonmalignant disorders. This report describes seven patients with recurrent Hodgkin's disease (HD) evaluated for bone marrow transplantation (BMT) who developed chromosomal abnormalities, and emphasizes the importance of bone marrow cytogenetic studies before bone marrow harvest. Three patients with histologically normal bone marrow underwent autologous BMT and subsequently developed an MDS or ANLL. Four patients had the clonal abnormality detected before bone marrow harvest and did not proceed to BMT.

Expression of bcr-abl fusion transcripts following bone marrow transplantation for Philadelphia chromosome-positive leukemia.

A modified polymerase chain reaction (PCR) procedure was used to study the expression of bcr-abl fusion transcripts following allogeneic bone marrow transplantation (BMT) for Philadelphia chromosome (Ph1) positive acute and chronic leukemias. The technique was applied to RNA preparations of peripheral blood and bone marrow cells from 10 patients with chronic myelogenous leukemia (CML) and one patient with acute lymphoblastic leukemia (ALL), all of whom had undergone allogenic BMT and were in clinical and cytogenetic remission. Pre-BMT samples available for eight of 11 patients contained detectable bcr-abl fusion products serving as a baseline for comparison to post-BMT studies. Six patients showed no PCR-detectable bcr-abl transcripts in each of several serial analyses post-BMT (1-36 months post-BMT). The remaining five patients demonstrated various patterns of bcr-abl transcript expression after transplantation. In three patients, bcr-abl transcripts persisted for up to 3 months post-BMT but subsequently were undetectable. Molecular relapse was observed 3 and 6 months post-BMT in the remaining two patients whose earlier post-BMT samples showed no bcr-abl fusion transcripts. No bcr-abl transcripts were detected in subsequent samples from both of these patients 6 months and 1 year post-BMT, respectively. These data confirm that Ph1 carrying cells expressing the bcr-abl fusion mRNA may persist or recur for several months following BMT in the absence of clinical and cytogenetic relapse. The significance of these observations is discussed with respect to results reported recently by others using similar techniques.

Effects of recombinant human granulocyte colony stimulating factor and granulocyte-monocyte colony stimulating factor on in vitro hemopoiesis in the myelodysplastic syndromes.

We evaluated the effects of recombinant human granulocyte colony stimulating factor (rhG-CSF) and granulocyte-monocyte colony stimulating factor (rhGM-CSF) on the in vitro proliferative, differentiative, and regenerative responsiveness of marrow cells from myelodysplastic syndrome patients (MDS) in comparison to those from normal individuals. Our studies showed decreased primary clonogenicity of myeloid (CFU-GM) and erythroid (BFU-E) hemopoietic progenitor cells from the MDS patients. rhGM-CSF had more potent stimulatory effects than rhG-CSF for MDS marrow CFU-GM growth; no enhanced cellular proliferation in the MDS patients was observed in liquid culture with either rhGM-CSF or rhG-CSF. Decreased myeloid clonal cell self-generation and/or recruitment occurred in the MDS patients upon exposure to either rhG-CSF or rhGM-CSF. rhG-CSF demonstrated more potent granulocytic differentiation effects than rhGM-CSF both for normals and MDS patients using marrow enriched for immature myeloid cells with lesser differentiation noted for MDS. Cytogenetic abnormalities, present with or without additional normal karyotypes in native marrow of four MDS patients, persisted after culture with rhG-CSF, indicating induced differentiation of both normal and abnormal clones. Although proliferative and differentiative effects were seen with both factors these data show MDS marrow cells in vitro to have predominantly differentiative responsiveness to rhG-CSF and proliferative responsiveness to rhGM-CSF.

Effects of granulocyte colony-stimulating factor therapy on in vitro hemopoiesis in myelodysplastic syndromes.

We evaluated the effects of 2 months of G-CSF treatment on in vitro hematopoiesis in 17 patients with myelodysplastic syndromes (MDS). Although in vitro marrow myeloid progenitor cell (CFU-GM) growth stimulated by G-CSF generally remained subnormal, in the majority of neutrophil responders significantly augmented incremental change (termed AIC) of CFU-GM numbers occurred after treatment, as did neutrophilic differentiation. The neutrophil non-responders had less prominent in vitro myeloid responses and lower basal neutrophil levels (p < 0.05). Following G-CSF treatment, the initially subnormal erythroid burst-forming unit (BFU-E) values underwent AIC in five of 11 patients along with increased reticulocyte responses in vivo, whereas four of the five patients who lacked AIC of BFU-E did not. Three patients with persisting cytogenetic abnormalities and increased neutrophilic differentiation in vitro also responded in vivo, suggesting that G-CSF induced in vivo cellular differentiation from the abnormal clone. Two of the three patients who developed blastic responses in vivo had increased CFU-GM growth pre- and post-therapy. These results indicate in vivo-in vitro correlations for myeloid and erythroid responses of MDS marrow cells which related to treatment with G-CSF.

Characterization of spontaneous factor-independent cell lines derived from the human leukemic cell line TF-1: a dominant event.

Uncontrolled proliferation of acute myeloid leukemia (AML) cells is an important step during leukemogenesis. However, little is known about the mechanisms leading to growth autonomy. Studies using immortalized murine hematopoietic cell lines have suggested that autocrine production of growth factors, or the constitutive activation of molecules in growth factor signalling pathways, are involved. We have established six spontaneous factor-independent cell lines from the human growth factor-dependent TF-1 cell line. The factor-independent cells showed no detectable growth factor activity. Immunoblotting analyses of tyrosine phosphorylation, Raf-1 and extracellular signal-regulated kinase 2 (ERK-2) showed a similar pattern in all the cell lines including TF-1 cells. Furthermore, somatic-cell hybrids between TF-1 and the factor-independent cells grew in absence of growth factor. Taken together this data demonstrates that the factor independence in this system is dominant and suggests that the molecular event is located either downstream of the Raf-1 and MAP kinases pathway or on an alternative pathway. Finally, the karyotype analysis of one factor-independent cell line TF-1i1 and TF-1H- (G418 resistant, HAT sensitive TF-1 cells) and their hybrids demonstrated an unstable derivative chromosome [der(19) t(19;?) (q13.1;?)] which seemed to correlate with the factor-independence capacity. This model may help in our understanding of autonomous proliferation by human myeloid leukemias.

Association of methylenetetrahydrofolate reductase polymorphism C677T and dietary folate with the risk of cervical dysplasia.

Epidemiological studies have been inconsistent regarding a role for folate in the etiology of cervical dysplasia. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the synthesis of 5-methyltetrahydrofolate, which is involved in the methylation of homocysteine to methionine. A common variant of this enzyme, resulting from a 677C-->T (Ala-->Val) substitution in the gene, has been shown to have reduced activity and is associated with mild hyperhomocysteinemia. A multiethnic case-control study was used to examine the association of dietary folate and MTHFR genotype with the odds ratios (ORs) for cervical dysplasia among women identified from several clinics on Oahu, Hawaii, between 1992 and 1996. We collected blood samples for DNA extraction, cervical smears for cytological diagnosis, exfoliated cervical cells for human papillomavirus (HPV) DNA testing, and personal interviews from 150 women with squamous intraepithelial lesions (SILs) and from 179 women with cytologically normal (Pap) smears. We found a positive, monotonic trend (P = 0.02) in the ORs for cervical SILs associated with the number of variant MTHFR T alleles, after multivariate adjustment. Women with the heterozygous CT genotype had twice the risk of cervical SILs [OR, 2.0; 95% confidence interval (CI), 1.1-3.7], and women with the homozygous TT genotype had almost three times the risk of SILs (OR, 2.9; 95% CI, 1.0-8.8) compared to women with the homozygous MTHFR CC genotype. The dietary intakes of folate, vitamin B(6), and vitamin B(12) were inversely related to the ORs for cervical SILs, after adjustment for HPV DNA and other confounders. The OR among women in the highest quartile compared with women in the lowest quartile of folate intake was 0.3 (95% CI, 0.1-0.7; P for trend = 0.002). Women with the variant T allele and folate intakes below the median were at significantly elevated risk of cervical SILs (OR, 5.0; 95% CI, 2.0-12.2) compared to women with CC alleles and folate intakes above the median. HPV infection was a strong risk factor for cervical dysplasia, particularly among women with the variant T allele (OR, 46.6; 95% CI, 15.9-136.2). All associations of MTHFR genotype with the ORs for cervical SILs were independent of other risk factors under study. These findings suggest that the MTHFR T allele and reduced dietary folate may increase the risk for cervical SILs.

Relationships of TP53 codon 72 and HRAS1 polymorphisms with lung cancer risk in an ethnically diverse population.

Tobacco smoking is a strong cause of lung cancer. However, because only a small proportion of smokers develop the disease, other factors, including genetic susceptibility, may be important in determining lung cancer risk. Polymorphisms in the TP53 tumor suppressor gene and HRAS1 proto-oncogene have been associated in some studies with this cancer; we sought to replicate these associations in an ethnically diverse population in Hawaii. We conducted a population-based case-control study among 334 incident lung cancer cases and 446 controls of Caucasian, Japanese, or Native Hawaiian origin. In-person interviews collected detailed information on lifestyle risk factors. DNA was extracted from peripheral blood leukocytes, and genotyping was performed using a PCR-based assay for the TP53 codon 72 polymorphism and Southern blot analysis and PCR for allelic polymorphisms in the HRAS1 minisatellite. Logistic regression analyses were used to compute odds ratios (ORs) and 95% confidence intervals (CIs) adjusting for smoking and other risk factors. The presence of two rare HRAS1 alleles was associated with a 2.2-fold (95% CI, 1.0-5.0) increased lung cancer risk for all ethnic groups combined. The association was present in Native Hawaiians (OR, 5.2; 95% CI, 1.1-24.4) and was suggested for Japanese (OR, 2.8; 95% CI, 0.6-12.5); no association was observed in Caucasians (OR, 0.8; 95% CI, 0.2-3.6). This association was also observed for each lung cancer cell type. The presence of only one rare allele did not increase risk for any ethnic group or cell type. No significant association was found between the TP53 codon 72 polymorphism and lung cancer [OR, 1.4 (95% CI, 0.8-2.4) for the Pro/Pro genotype compared with the Arg/Arg genotype]. This study suggests that the presence of two rare HRAS1 alleles confers an increased lung cancer risk in Native Hawaiians and Japanese but possibly not in Caucasians. The amino acid replacement of arginine by proline at codon 72 of TP53 appears not to be important in determining lung cancer risk in this population.

Case-control study of ovarian cancer and polymorphisms in genes involved in catecholestrogen formation and metabolism.

Steroid hormones, such as estrogens, appear to be associated with ovarian carcinogenesis, but the precise biological mechanisms are unclear. Polymorphisms in genes that regulate the concentration of estrogens and their metabolites may contribute directly to the individual variation in ovarian cancer risk through a mechanism involving oxidative stress or indirectly by influencing ovarian cancer susceptibility associated with ovulation and reproduction. We conducted a population-based, case-control study of primary ovarian cancer between 1993 and 1999 in Hawaii to test several genetic and related hypotheses. A personal interview and blood specimen were obtained in the subjects' homes. In a sample of 129 epithelial ovarian cancer cases and 144 controls, we compared the frequencies of several polymorphisms in genes that regulate steroid hormone metabolism and catecholestrogen formation. Multivariate unconditional logistic regression was used to model the association of each genetic polymorphism separately after adjusting for age, ethnicity, and other covariates. The high-activity Val432 allele of the CYP1B1 gene, which may be linked to oxidative stress through elevated 4-hydroxylated catecholestrogen formation, was associated with an increased risk of ovarian cancer. The Val/Leu genotype for CYP1B1 was associated with an odds ratio of 1.8 (95% confidence interval, 1.0-3.3) and the Val/Val genotype with an odds ratio of 3.8 (95% confidence interval, 1.2-11.4) compared with the Leu/Leu genotype (P = 0.005). Tobacco smokers with at least one CYP1A1 (MspI) m2 allele, one CYP1B1 Val allele, one COMT Met allele, or two CYP1A2 A alleles were at significantly increased risk of ovarian cancer compared to never-smokers with CYP1A1 (MspI) ml/ml, CYP1B1 Leu/Leu, COMT Val/Val, or CYP1A2 A/A genotypes, respectively. We found a positive statistical interaction (P = 0.03) between tobacco smoking and the CYP1A1 (MspI) polymorphism on the risk of ovarian cancer. None of the other gene-environment (pregnancy, oral contraceptive pill use) or gene-gene interactions were statistically significant. Although not significant, there was a suggestion that the effect of the CYP1B1 Val allele was reduced substantially in the presence of the high-activity COMT Met allele. These findings suggest that the CYP1B1-Val allele and perhaps other genetic polymorphisms in combination with environmental or hormonal exposures are susceptibility factors for ovarian cancer.

Combined effects of well-done red meat, smoking, and rapid N-acetyltransferase 2 and CYP1A2 phenotypes in increasing colorectal cancer risk.

Heterocyclic amines (HAAs) are suspected carcinogens that are formed in meat when it is cooked at high temperature for long durations. These compounds require metabolic activation by CYP1A2 and N-acetyltransferase (NAT) 2 or NAT1 before they can bind to DNA. It has been hypothesized that well-done meat increases the risk of colorectal cancer (CRC), especially in individuals with the rapid phenotype for CYP1A2 and NAT2. This association may be particularly strong in smokers because smoking is known to induce CYP1A2. We conducted a population-based case-control study on Oahu, Hawaii to specifically test this hypothesis. An in-person interview assessed the diet and preference for well-done red meat of 349 patients with CRC and 467 population controls. A urine collection after caffeine challenge and a blood collection were used to assess phenotype for CYP1A2 and NAT2 and genotype for NAT2 and NAT1, respectively. No statistically significant main effect association with CRC was found for red meat intake, preference for well-done red meat, the NAT2 rapid genotype, the CYP1A2 rapid phenotype or the NAT1*10 allele. However, in ever-smokers, preference for well-done red meat was associated with an 8.8-fold increased risk of CRC (95% confidence interval, 1.7-44.9) among subjects with the NAT2 and CYP1A2 rapid phenotypes, compared with smokers with low NAT2 and CYP1A2 activities who preferred their red meat rare or medium. No similar association was found in never-smokers, and there was no increased risk for well-done meat among smokers with a rapid phenotype for only one of these enzymes or for smokers with both rapid phenotypes who did not prefer their red meat well-done. These data provide additional support to the hypothesis that exposure to carcinogens (presumably HAAs) through consumption of well-done meat increases the risk of CRC, particularly in individuals who are genetically susceptible (as determined by a rapid phenotype for both NAT2 and CYP1A2) and suggest that smoking, by inducing CYP1A2, facilitates this effect.

Y-specific DNA sequences in male patients with 46,XX and 47,XXX karyotypes.

Y chromosomal DNA sequences were detected in three of four 46,XX males and in one 47,XXX male. One reiterated Y chromosomal sequence, Y-190, was localized by in situ hybridization to the distal short arm of an X chromosome of the 47,XXX male. This result is compatible with the hypothesis that an aberrant X/Y interchange has occurred, most likely during paternal meiosis, and that this interchange accounts for Y chromosomal material and sex reversal in this 47,XXX individual.

Clinical heterogeneity associated with deletions in the long arm of chromosome 15: report of 3 new cases and their possible genetic significance.

Deletions, duplications, and rearrangements of the long arm of chromosome 15 are frequently associated with the clinical diagnosis of the Prader-Willi syndrome. However, a number of other clinical entities have also been associated with similar, if not identical, cytogenetic defects, arguing for clinical heterogeneity associated with abnormalities in this region of chromosome 15. We present 3 patients who all appear to have deletions in 15q11-15q12, such as described for many patients with Prader-Willi syndrome; however, none of these patients has classical clinical features of the Prader-Willi syndrome. The first patient is a child with Williams syndrome, the second, Angelman (Happy Puppet) syndrome, and the third is a child with hypotonia of infancy, obesity, and developmental delay, but who does not meet specific diagnostic criteria for the Prader-Willi syndrome. It is proposed that different molecular abnormalities involving specific points or segments along the long arm of chromosome 15 might account for the clinical diversity seen among these and other patients.

X-linked recessive torsion dystonia in the Philippines.

The occurrence of an X-linked form of torsion dystonia in the Philippines was demonstrated by the genetic and biochemical analysis of affected males and their relatives. Thirty-six affected males were ascertained in 21 families by clinical neurologic evaluation. The mean age-of-onset of dystonia was 37.9 years with a range from 12 to 52 years. Neurologic symptoms began focally and progressed to either segmental or generalized involvement in all cases. Generalized dystonia developed in 78% of the patients after a mean duration of 6.8 years from the onset of symptoms. A family history of dystonia was elicited in 17 of the 21 kindreds, accounting for a total of 64 males and one possibly affected female, distributed among 224 individuals in 33 sibships. In 18 of the 33 sibships, 2 or more brothers reportedly had dystonia. There were 12 kindreds with a history of multigenerational dystonia. In those, only males of maternal ancestry were affected, and in 7 of these families, maternal grandfathers reportedly had dystonia. There were no instances of male-to-male transmission. Cytogenetic analysis did not show any X chromosome abnormalities in 4 affected propositi. Several secondary causes of torsion dystonia were excluded, including Wilson disease, aminoacidopathies, organic acidurias, oligosaccharidoses, and chronic hexosaminidase A and B deficiency. These findings substantiate the existence of an X-linked recessive form of primary torsion dystonia.

Angelman syndrome in a daughter with del(15) (q11q13) associated with brachycephaly, hearing loss, enlarged foramen magnum, and ataxia in the mother.

We report on a 4-year-old girl with Angelman syndrome who has an apparent de-novo del(15) (q11q13) originating from a maternally derived chromosome. Her mother had severe brachycephaly, sensorineural hearing loss, speech impediment, and mild ataxia. CT brain scans showed an enlarged foramen magnum in the mother and daughter but magnetic resonance imaging (MRI) showed no brainstem abnormality in either. This family demonstrates that some Angelman syndrome cases may be dominantly transmitted with variable expression and associated with abnormal or cytogenetically apparently normal chromosome 15.