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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

Cytogenetic characterization of renal cell carcinoma in von Hippel-Lindau syndrome.

We report the case of a 26-year-old man with von Hippel-Lindau syndrome (VHL) and two renal cell carcinomas (RCC), one of which was studied cytogenetically. Chromosomal analysis of the RCC showed a translocation that involved chromosomes 3 and 8 with subsequent loss of the derivative chromosome 8. The patient's peripheral lymphocytes showed a normal karyotype that indicated that there was not a constitutional chromosomal translocation. This is the third reported case of RCC in a patient with VHL in which loss of a portion of the short arm of chromosome 3 (3p) has occurred. Similar chromosomal changes that involve 3p have been reported in both familial and sporadic cases of RCC and have led to speculation that a tumor suppressor gene may be located in this region. Cytogenetic characterization of renal tumors could assume increasing significance in the diagnosis and classification of RCC and potentially may guide therapy. These studies may also lead to a better understanding of the biologic behavior of RCC and result in more informed patient evaluation and counseling.

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.

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.

Differential amplification, assembly, and relocation of multiple DNA sequences in human neuroblastomas and neuroblastoma cell lines.

DNA amplification, manifested by homogeneously staining regions in chromosomes and by extrachromosomal, double minute bodies, is characteristic of many neuroblastoma cell lines. Sequences recruited from a specific domain on the short arm of chromosome 2 (2p) are amplified in advanced-stage primary neuroblastomas, whereas sequences from distinctly different regions of 2p are amplified in the neuroblastoma cell line IMR-32. Five different DNA segments, which include the oncogene N-myc, three other fragments derived from the homogeneously staining region of the neuroblastoma cell line IMR-32, and a fifth fragment, derived from the neuroblastoma cell line NB-9, showed differential and variable amplification in 24 advanced-stage neuroblastoma tumors out of 112 tested specimens. All five fragments were mapped within the chromosomal region 2p23-2p25 by three different approaches. However, eight other fragments cloned from the homogeneously staining region of IMR-32 cells, which were not amplified in the tumor tissues examined, were mapped to two more proximal domains of 2p, thousands of kilobases apart from each other and from the chromosomal domain that is amplified in the tumors. These results establish the amplification, to different degrees, of a variable-sized segment of one domain near the terminus of 2p in advanced neuroblastomas. These tumors might ultimately be distinguished according to the pattern of amplification of DNA segments within this domain. The data presented also indicate the existence of a new and complex amplification mechanism in at least one neuroblastoma cell line (IMR-32), which involves not only relocation of DNA from specific genomic domains but also the formation of novel units by splicing together very distant DNA segments.

Identification of inverted duplicated #15 chromosomes using bivariate flow cytometric analysis.

A dual laser FACS IV cell sorter has been used to obtain bivariate flow histograms of human metaphase chromosomes stained with the DNA-specific dyes, 33258 Hoechst and chromomycin A3. Approximately twenty distinct chromosomal fluorescence populations can be resolved using this double staining technique and the flow cytometer which has been modified only by the substitution of a specially designed air-spaced achromat for the standard focusing lens. Metaphase chromosomes from two different cell lines bearing inverted duplicated #15 autosomes have been subjected to bivariate chromosome analysis. In both cases, the inverted duplicated #15 chromosomes have been identified in the bivariate flow histogram. This identification was supported by experiments in which doubly stained chromosomes were counterstained with either netropsin or distamycin A, resulting in a relative increase in the 33258 Hoechst fluorescence intensity of the structurally abnormal #15 chromosomes, compared with the other chromosomes, as predicted by cytological studies. The possibility of identifying and separating small abnormal autosomes using commercially available instrumentation should facilitate the use of recombinant DNA techniques for the construction of libraries which are highly enriched for DNA sequences from limited autosomal subregions important in the study of chromosomal abnormalities such as deletions, translocations and inversion duplications.

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.

Molecular genetic approaches to human diseases involving mental retardation.

Recombinant DNA techniques provide new approaches to the diagnosis and analysis of inherited human diseases associated with mental retardation. Examples of such diseases include the Lesch-Nyhan syndrome, phenylketonuria, the Fragile X syndrome, Down syndrome, and those associated with deletions or duplications of subchromosomal regions, e.g., the proximal short arm of human chromosome #15. For a limited but increasing number of diseases, the DNA sequences responsible for the phenotype (e.g., sequences coding for abnormal proteins) can be isolated directly. In many other cases, DNA segments mapping near genes responsible for diseases of interest can be isolated, e.g., from recombinant phage libraries enriched for specific regions of the genome by metaphase chromosome flow-sorting and then used in molecular linkage studies to "track" the abnormal gene in a pedigree. Both the necessary technology and the methods for its application continue to improve, and the impact of recombinant DNA studies in the field of mental retardation should increase markedly in the very near future.