Mapping the locus of the H-Y gene on the human Y chromosome.

The H-Y locus is on the short arm of the human Y chromosome in most individuals but on the long arm in at least one of 17 individuals with structural abnormalities of the Y.

Deletion of chromosome 1 predicts prognosis in pancreatic endocrine tumors.

Endocrine tumors, such as parathyroid adenomas and pheochromocytomas, frequently have deletions of chromosome 1, suggesting that inactivation of a tumor suppressor gene from chromosome 1 is important in their tumorigenesis. We hypothesized that deletion of chromosome 1 may contribute to pancreatic endocrine tumor formation. Twenty-nine sporadic and MEN1 pancreatic endocrine tumors were studied for loss of heterozygosity (LOH) with 12 chromosome 1 microsatellite markers. LOH on chromosome 1 was identified in 10 of 29 (34%) tumors studied. Allele loss occurred more frequently in tumors with hepatic metastases (7 of 8) than tumors without metastases (3 of 21) (P = 0.004). Tumors in patients with lymph node involvement and patients with multiple endocrine neoplasia type 1 did not demonstrate LOH for chromosome 1 markers. These data suggest that loss of chromosome 1 is associated specifically with the development of hepatic metastases in patients with sporadic pancreatic endocrine tumors.

Confined placental chimerism: prenatal and postnatal cytogenetic and molecular analysis, and pregnancy outcome.

The presence of two cell lines in chorionic villi sampling (CVS) represents a significant complication in CVS analysis, interpretation, and counseling. We report on the cytogenetic and molecular analysis of a pregnancy that was conceived on clomiphene citrate. Two cell lines (46,XX and 47,XY,+9) were discovered in CVS analysis done for maternal age; 94% of the cells in the culture were 46,XX and 6% were 47,XY, +9 (the direct preparation was 46,XX). As neither line could have derived from the other, chimerism and not mosaicism was suspected, with the 47,XY,+9 cells deriving from a co-twin whose demise was the result of the autosomal trisomy. At a subsequent amniocentesis, only normal female cells were observed and a normal female infant was delivered at term. Cytogenetic analysis done on the infant's peripheral blood and on a sample of an umbilical vessel showed only 46,XX cells, while amnion and a fibrotic area of the placenta contained 2 cell lines, 46,XX and 47,XY,+9. Molecular analysis of 3 different tissues was done by the polymerase chain reaction (PCR) and Southern blotting, using Y specific primers and probes, respectively. The presence of Y specific DNA was detected in the placenta and amnion, but not in the umbilical blood vessel. These data excluded true chimerism in the fetal tissues at the level of about 1 in 10(5) cells and have defined for the first time probable confined placental chimerism (CPC), the result most likely of a "vanishing twin." Whenever two cell lines are found in CVS, especially in the setting of pharmacologically stimulated ovulation, the possibility of CPC should be considered. The effects of CPC on placental function and fetal outcome merit further study.

Mosaic trisomy 16 ascertained through amniocentesis: evaluation of 11 new cases.

Trisomy 16, once thought to result uniformly in early pregnancy loss, has been detected in chorionic villus samples (CVS) from on-going pregnancies and was initially ascribed to a second, nonviable pregnancy. Prenatally detected trisomy 16 in CVS and its resolution to disomy has led to the reexamination of the viability of trisomy 16. This study evaluates 11 cases of mosaic trisomy 16 detected through second trimester amniocentesis. In 9 of the 11 cases, amniocenteses were performed in women under the age of 35 because of abnormal levels of maternal serum alpha-fetoprotein (MSAFP) or maternal serum human chorionic gonadotropin (MShCG). The other two amniocenteses were performed for advanced maternal age. Five of the 11 pregnancies resulted in liveborn infants, and six pregnancies were electively terminated. The liveborn infants all had some combination of intrauterine growth retardation (IUGR), congenital heart defects (CHD), or minor anomalies. Two of them died neonatally because of complications of severe congenital heart defects. The three surviving children have variable growth retardation, developmental delay, congenital anomalies, and/or minor anomalies. In the terminated pregnancies, the four fetuses evaluated by ultrasound or autopsy demonstrated various congenital anomalies and/or IUGR. Cytogenetic and fluorescent in situ hybridization studies identified true mosaicism in 5 of 10 cases examined, although the abnormal cell line was never seen in more than 1% of cultured lymphocytes. Placental mosaicism was seen in all placentas examined and was associated with IUGR in four of seven cases. Maternal uniparental disomy was identified in three cases. Mosaic trisomy 16 detected through amniocentesis is not a benign finding but associated with a high risk of abnormal outcome, most commonly IUGR, CHD, developmental delay, and minor anomalies. The various outcomes may reflect the diversity of mechanisms involved in the resolution of this abnormality. As 80% of these patients were ascertained because of the presence of abnormal levels of MSAFP or MShCG, the increased use of maternal serum screening should bring more such cases to clinical attention.

Use of conditioned media in cell culture can mask cytogenetic abnormalities in acute leukemia.

Conditioned media (CM) from a human lung adenocarcinoma cell line expressing interleukins 1 and 6 (IL-1, IL-6), granulocyte (G), macrophage (M), and GM colony-stimulating factors (G, M, GM-CSF) and transforming growth factor beta (TGF beta) were used to stimulate growth of bone marrow (BM) cells from 18 persons with leukemia, myelodysplastic syndrome, or lymphoma. The objective was to increase numbers of analyzable metaphases and to enhance the likelihood of detecting cytogenetic abnormalities. Although more mitotic cells were observed with CM, the detection rate of cytogenetic abnormalities decreased in 12 of 18 cases. These data indicate that use of CM for cytogenetic analyses may favor growth of normal versus leukemia cells and mask cytogenetic abnormalities.

Isolation of human chromosome 13-specific DNA sequences cloned from flow sorted chromosomes and potentially linked to the retinoblastoma locus.

A recombinant DNA library has been constructed using flow sorted chromosome #13 DNA and the phage vector, Charon 21A. Roughly 90% of the phage inserts in the library hybridize to human repetitive DNA. Phage containing human nonrepetitive inserts have been screened for chromosome #13 specificity by Southern blot analysis using the genomic DNA of human-rodent cell hybrids containing different regions of the human #13 autosome. Of 18 phage inserts characterized, 13 have been assigned to the 13q12----q22 subregion, three appear to be localized in the 13pter----q12 region, and two are not #13-specific. By Southern blot analysis of the DNA of a retinoblastoma patient exhibiting a deletion of band 13q14 and of karyotypically normal individuals, two phage inserts have been putatively assigned to band 13q14, the currently accepted locus for a genetic determinant for retinoblastoma. These two DNA probes show quantitative differences in hybridization band intensity in the genomic DNA of the 13q--patient relative to that of the normals. In situ hybridization data support these conclusions. A recombinant phage library that shows an approximate 90% enrichment for human chromosome #13-specific DNA fragments should prove useful not only in studies related to retinoblastoma, but also in the molecular analysis of the structure and function of chromosome #13.

Translocation (3;21;8)(q21;q22;q22) in a patient with acute myeloid leukemia. A case report and review of prognostic indicators.

We report a patient with acute myeloid leukemia (AML) and t(3;21;8)(q21;q22;q22). This translocation has not been previously described in de novo or relapsed AML. The patient is a 25-year-old woman who presented with WBC 6.2 x 10(9)/L, Hgb 10.2 g/dL, Hct 28.4%, and platelets 67 x 10(9)/L. A bone marrow biopsy revealed a 70% hematopoietic cellularity with 65% blasts. Immunophenotyping showed aberrant expression of lymphoid-associated marker CD19. Cytogenetic analysis on a 72-hour culture of bone marrow cells supplemented with conditioned media was evaluated by G-banding at about the 400-band level. The patient's age, cytogenetics, WBC, and immunophenotype at diagnosis would seem to suggest a favorable prognosis, according to previous studies of prognostic indicators. She was treated with induction and consolidation chemotherapy, followed by myeloablative conditioning and autologous peripheral blood stem cell transplant (PBSCT). Despite multiple favorable prognostic factors, the patient relapsed 7 months after PBSCT. Translocation of chromosomes 8 and 21 is common in AML and is generally considered a good prognostic factor. We suspect that the effect of the 3q21 translocation in an otherwise favorable translocation of chromosomes 8 and 21 may be responsible for this patient's early relapse.

Burkitt translocation (8;22)(q24;q11) in a patient with multiple myeloma.

The prevalence of chromosomal abnormalities in multiple myeloma (MM) has been difficult to detect by karyotyping primarily because of the low proliferative rate of malignant plasma cells. The reported incidences of abnormal karyotypes range from 24% to 63% in bone marrows obtained from MM patients, with the higher rates being seen in aggressive disease [1-8]. Detection of abnormal karyotypes in MM has been associated with a poor prognosis. We report a MM patient with an 8;22 Burkitt translocation, the first such reported case.

Differences in murine procarcinogen activation enzymes are not accompanied by parallel differences in procarcinogen-induced sister-chromatid exchange.

C57Bl/6 and DBA/2 mice, strains in which there is marked induction of hepatic monooxygenase activity by phenobarbital, were tested for in vivo sister-chromatid exchange (SCE) formation in response to cyclophosphamide, an agent metabolized by this inducible enzyme system. Baseline SCE frequencies were between 4 and 6 SCEs/cell in regenerating liver and bone marrow of both strains of mice. Administration of cyclophosphamide (5mg/kg) led to an increase of nearly 8 SCEs/cell in both tissues of C57Bl/6 mice and an increase of more than 10 SCEs/cell in DBA/2 mice. Prior exposure to phenobarbital induced p-chloromethylaniline demethylase activity in regenerating liver of both mouse strains approx. 6-fold, but the changes in measured SCE frequencies were not significantly different from those obtained in the absence of enzyme induction. These results, together with our previous observation that induction by 3-methylcholanthrene of benzo[a]pyrene hydroxylase activity in the same mouse strains was not accompanied by a comparable change in benzo[a]pyrene-induced SCE formation, reinforce the impression that simple assays of differences in mixed function oxidase activities may not necessarily be good predictors of hereditary differences in the response to genetic damage by procarcinogens which are presumed to be metabolized by these enzymes.

In vivo induction of sister-chromatid exchanges in liver and marrow cells by drugs requiring metabolic activation.

A highly sensitive method for the detection of in vivo induction of sister-chromatid exchange (SCE) has been developed in mice subjected to partial hepatectomy. SCE induction by either acetylaminofluorene (AAF) or cyclophosphamide, drugs requiring metabolic activation, is significantly greater in both regenerating liver and bone-marrow cells of partial hepatectomized animals than in marrow cells of unhepatectomized mice. These experiments have confirmed the ability of AAF, a well known mutagen-carcinogen, to induce SCE formation, even though the cytogenic effects of this drug on non-hepatectomized mice is very small. The in vivo system described has demonstrated the influence of the liver on drug-induced damage to extra-hepatic tissues. The procedures developed should facilitate the detection of drug-induced cytogenic damage and permit the comparison of inter-tissue differences in SCE induction with tissue-specific differences in drug-activation pathways.

Chromosomal mosaicism in chorionic villus sampling.

The observation of multiple, chromosomally distinct cell lines in chorionic villus samples is not an unusual finding and occurs in 1 per 100 samples. This frequency is ten times greater than the level of mosaicism observed in newborn surveys and, thus, must reflect phenomenon other than true fetal mosaicism. Indeed, only 23% of mosaicism detected at CVS is confirmed in the fetus (2.3 per 1,000 CVS), which is much closer to the newborn rate (1 per 1,000). This indicates that most mosaicism encountered in CVS is unrelated to the fetal karyotype and as such is an inaccurate prediction of the fetal genotype, the purpose of prenatal diagnosis. Most of the mosaicism detected in CVS is due to confined placental mosaicism. Either as a result of error-prone cell division generating an excess of abnormal cells in extraembryonic tissues or reduced selection against aneuploid cells in these tissues allowing their persistence, chorionic villi and placenta appear to show much higher levels of mosaicism than seen in fetuses. This explains the more frequent finding of multiple cell lines in CVS than in amniocentesis or liveborn individuals. The discrepancy between levels of mosaicism present in chorionic villi and fetal tissues means that most instances of mosaicism detected in CVS are not associated with a fetal abnormality and should be evaluated by further prenatal testing, i.e., amniocentesis or fetal blood sampling. Because of the frequency of chromosomal mosaicism in CVS and its attendant need for further testing, a discussion of mosaicism should be included in counseling prior to CVS. The higher frequency of discrepant results in direct CVS preparation emphasizes the prudence of delaying decision making until the results of the CVS culture have been obtained. Although the observation of mosaicism clearly complicates genetic counseling and decision making, it does not appear to be associated with an adverse fetal outcome. Whereas most of the mosaicism observed in CVS is the result of confined placental mosaicism, other types of discrepancies also occur. Maternal cell contamination occurs in about 1% of cases, but is easily evaluated by examining the direct preparation and analyzing chromosome polymorphism. The incidence of pseudomosaicism in CVS cultures is unclear but probably low. Interestingly, CVS analysis has suggested that twinning may be a more common phenomenon at conception than reported at birth and that some discrepancies may reflect the nonviability of twins with abnormal karyotypes. Chorionic villi sampling remains a viable alternative to amniocentesis for early prenatal diagnosis. An understanding of the origins of mosaicism in CVS is necessary for

Duane syndrome associated with features of the cat-eye syndrome and mosaicism for a supernumerary chromosome probably derived from number 22.

A 12-year-old boy with a supernumerary chromosome, probably derived from number 22, had typical features of Duane syndrome with limitation of abduction and retraction of the globe upon adduction. Additionally, the patient had antimongoloid slant of the eyes, epicanthal folds, preauricular sinuses, cardiac malformations, skeletal malformations, and mental retardation suggestive of the cat-eye syndrome. The cat-eye syndrome has been often associated with a supernumerary chromosome derived from number 22. Our patient's karyotype was 46,XY/47,XY, + mar, with the supernumerary chromosome probably derived from number 22. These findings supplement previous findings of chromosome 22 abnormality associated with an ocular motility disorder.

Karyotyping.

Relevant portions of the new International System for Human Cytogenetic Nomenclature (ISCN 1995) have been reproduced in this appendix (with permission from Karger, the original publisher). The new rules supersede all previous rules and include guidelines for cancer cytogenetics as well as new recommendations for nomenclature when in situ hybridization techniques are used in the analysis of chromosomes.

Chromosome banding techniques.

Chromosome banding techniques produce a series of consistent landmarks along the length of metaphase chromosomes that allow for both recognition of individual chromosomes within a genome and identification of specific segments of individual chromosomes. These landmarks facilitate assessment of chromosome normalcy, identification of sites of chromosome breaks and alterations, and location of specific genes. This unit covers these basic banding techniques (Q-banding, G-banding, and R-banding), which produce virtually identical patterns of bands along the length of human chromosomes, although the bands and polymorphic regions highlighted may differ with each technique. These techniques highlight reproducible landmarks along the length of the chromosome and specialized staining techniques can be used to highlight particular regions of chromosomes, such as heterochromatic and repeated-sequence segments. These specialized techniques, nucleolar organizer region (NOR) staining, centromeric heterochromatin staining (C-banding), methylated satellite DNA staining (distamycin-DAPI banding), and replication banding are also presented in this unit.

Comparison of benzo(a)pyrene metabolism and sister chromatid exchange induction in mice.

Genetic differences in the inducible arylhydrocarbon hydroxylase (EC 1.14.14.2) (AHH) system, which is involved in the multi-step metabolism of hydrocarbons, are known to exist in both humans and mice. However, the predictive value of AHH activity in human or murine tissues, assayed as benzo(a)pyrene hydroxylation, as an index of individual susceptibility to mutagens and carcinogens, remains unclear because of apparent inconsistencies between results obtained from different in vitro and in vivo systems. This situation may in part reflect the complexity of the pathways involved in drug metabolism, which combine both activation and detoxification. To determine the relationship of metabolic potential to an easily quantified, short-term in vivo end point of genetic damage, we compared the ability of AHH inducible and uninducible mice to metabolize a procarcinogen, benzo(a)pyrene (BP), with the in vivo induction by BP of sister chromatid exchanges (SCEs). SCE induction has been shown to correlate with mutagenesis. We report here that although BP did cause an increase in SCEs in test animals, the extent of this increase did not differ between the inducible C57BL/6 mice and the uninducible DBA/2 mice. Moreover, prior exposure to an AHH inducer, 3-methyl-cholanthrene (3-MC), did not increase the number of BP-induced SCEs in C57BL/6 mice. This lack of correlation between benzo(a)pyrene hydroxylase (BP-OH) inducibility and SCE response reinforces the idea that other metabolic steps, such as detoxification or DNA repair, may influence the overall genetic impact of a drug.

ISCN standard idiograms.

Chromosome banding is used mainly to identify both normal and rearranged chromosomes, to define chromosome breakpoints, and to describe the specific location of DNA sequences on chromosomes. A nomenclature has been developed to standardize the identification of chromosomes and the naming of chromosome bands. The system currently in use is An International System for Human Cytogenetic Nomenclature, referred to as "ISCN 1995." It is the report of the standing committee on human cytogenetic nomenclature edited by Felix Mitelman. The report includes a chromosome band nomenclature, as well as standard idiograms, which are "diagrammatic representations of a karyotype, which may be based on measurements of the chromosomes" (ISCN 1995). The idiograms presented here, with the permission of S. Karger and Cytogenetics and Cell Genetics, are drawings of G-banded chromosomes with band numbers indicated. Heterochromatic regions, which contain classes of repetitive DNA and can show individual differences in size, are indicated by patterned areas.

Tissue specificity of chromosomal rearrangements in ataxia-telangiectasia.

Cytogenetic studies of lymphocytes and fibroblasts from individuals with ataxia-telangiectasia (AT) demonstrate spontaneous chromosomal breakage. In the AT lymphocytes, this damage results in a high frequency of balanced rearrangements involving chromosome bands 7p14, 7q35, 14q12, and 14q32. The T-cell receptor alpha, beta, and gamma chain gene complexes and the immunoglobulin heavy chain gene complex, all of which may be functional in lymphocytes, have been localized to these bands. To assess the relationship between genes at these breakpoints and the entirety of the AT phenotype, we undertook a detailed cytogenetic analysis of fibroblasts and lymphocytes from seven AT homozygotes. Our findings indicate that the rearrangements present in the lymphocytes are not commonly observed in the fibroblasts, despite the increased instability of chromosomes from the cells relative to lymphocytes. Furthermore, the changes in the fibroblasts are neither consistent within nor between patients, suggesting that chromosome rearrangement occurs more randomly in this tissue. Therefore, differential site-specific damage in separate tissue may generate the distinct features of the disease in those tissues and may account for the pleiotrophic effects of the AT gene.

Tetrasomy 12p--unusual presentation in CVS.

CVS direct preparations usually achieve limited resolution and are better at detecting numerical rather than structural abnormalities. A CVS direct preparation analyzed using G-banding revealed a 47,XY,+G karyotype in 5 of 11 cells and was reported as mosaic for trisomy 21. Subsequent analysis of the CVS culture found only normal male cells. Amniocentesis revealed both normal male cells and cells with an extra F-group chromosome. Fluorescence in situ hybridization (FISH) identified this chromosome to be an isochromosome from the short arm of chromosome 12 [i(12)(p10)]. The amniocyte karyotype was reported as 47,XY,+i(12)(p10)[12]/46,XY[8].ish i(12)(p10)(wcp12+), which is associated with Pallister-Killian syndrome. Reexamination of the CVS direct preparation by FISH with a chromosome 12 centromere probe confirmed the karyotype of this tissue to be 47,XY,+mar[5]/46,XY[6].nuc ish 12cen(D12Z3 x 3)/12cen(D12Z3 x 2). Thus, multiple studies, including amniocentesis and fluorescence in situ hybridization, may be required to fully and accurately evaluate abnormalities detected by CVS. This case also indicates that mosaicism for supernumerary isochromosomes may have a complex origin.

Preferential derivation of abnormal human G-group-like chromosomes from chromosome 15.

The marked binding of antibodies specific for 5-methylcytidine to the short arm of chromosome 15 distinguishes this chromosome from the other human acrocentrics. This method has been used to study over 60 individuals including 12 who did not have Down's syndrome, but who did have an extra G-group sized acrocentric chromosome. In six cases the extra chromosome did not show intensive binding of anti-5-methylcytidine. In the other six cases, the extra chromosome contained a 5-methylcytidine rich band at each end indicating that both ends were derived from chromosome 15 and contained centromeric heterochromatin normally present on the short arm of chromosome 15. The duplication of short arm material in the abnormal chromosomes was confirmed in all cases by quinacrine staining, nucleolar organizer (Ag-AS) staining or C-banding. In three cases, the abnormal chromosome appeared to arise from two different chromosomes 15. Several possible mechanisms for the production of the abnormal chromosome are discussed. The individuals with this abnormal chromosome all showed some degree of mental retardation, but few common physical findings.