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Despite the clinical significance of balanced chromosomal abnormalities (BCAs), their characterization has largely been restricted to cytogenetic resolution. We explored the landscape of BCAs at nucleotide resolution in 273 subjects with a spectrum of congenital anomalies. Whole-genome sequencing revised 93% of karyotypes and demonstrated complexity that was cryptic to karyotyping in 21% of BCAs, highlighting the limitations of conventional cytogenetic approaches. At least 33.9% of BCAs resulted in gene disruption that likely contributed to the developmental phenotype, 5.2% were associated with pathogenic genomic imbalances, and 7.3% disrupted topologically associated domains (TADs) encompassing known syndromic loci. Remarkably, BCA breakpoints in eight subjects altered a single TAD encompassing MEF2C, a known driver of 5q14.3 microdeletion syndrome, resulting in decreased MEF2C expression. We propose that sequence-level resolution dramatically improves prediction of clinical outcomes for balanced rearrangements and provides insight into new pathogenic mechanisms, such as altered regulation due to changes in chromosome topology., Competing Interests: The authors have none to declare.

In this exciting era of "next-gen cytogenetics," integrating genomic sequencing into the prenatal diagnostic setting is possible within an actionable time frame and can provide precise delineation of balanced chromosomal rearrangements at the nucleotide level. Given the increased risk of congenital abnormalities in newborns with de novo balanced chromosomal rearrangements, comprehensive interpretation of breakpoints could substantially improve prediction of phenotypic outcomes and support perinatal medical care. Herein, we present and evaluate sequencing results of balanced chromosomal rearrangements in ten prenatal subjects with respect to the location of regulatory chromatin domains (topologically associated domains [TADs]). The genomic material from all subjects was interpreted to be "normal" by microarray analyses, and their rearrangements would not have been detected by cell-free DNA (cfDNA) screening. The findings of our systematic approach correlate with phenotypes of both pregnancies with untoward outcomes (5/10) and with healthy newborns (3/10). Two pregnancies, one with a chromosomal aberration predicted to be of unknown clinical significance and another one predicted to be likely benign, were terminated prior to phenotype-genotype correlation (2/10). We demonstrate that the clinical interpretation of structural rearrangements should not be limited to interruption, deletion, or duplication of specific genes and should also incorporate regulatory domains of the human genome with critical ramifications for the control of gene expression. As detailed in this study, our molecular approach to both detecting and interpreting the breakpoints of structural rearrangements yields unparalleled information in comparison to other commonly used first-tier diagnostic methods, such as non-invasive cfDNA screening and microarray analysis, to provide improved genetic counseling for phenotypic outcome in the prenatal setting., (Copyright © 2016 American Society of Human Genetics. All rights reserved.)

A 2 month old male infant was found to have mild growth retardation, prominent forehead, low set ears, low nasal bridge, rounded facies, cleft palate, webbed neck, shawl scrotum, and absent right kidney. The propositus, a product of a consanguineous marriage, had extremely rare abnormal cytogenetic findings. His karyotype contained three derivative chromosomes that originated from a familial translocation, t(16;18)(p13.3;p11.2) carried by both parents. Based on parental studies, the infant's unbalanced karyotype was defined as: [46,XY,t(16;18)(p13.3;p11.2), der(18)t(16;18).ish t(16;18)(16ptel-,16qtel+,18ptel+,wcp16+,wcp18+;16ptel+,18ptel-,wcp16+,wcp18+), der(18)t(16;18)(16ptel+,18ptel-,wcp16+,wcp18+)]. We describe this child at 2 months of age with a follow up at 4 1/2 years, exhibiting a mixed clinical picture with features of both 18p- and partial trisomy 16p13.3.

A three year-old boy was evaluated because of growth and developmental delay, hypotonia and dysmorphic features. G-banding analysis revealed a small interstitial deletion of the long arm of chromosome four described as 46,XY,del (4)(q21.1q21.3). This patient's findings on physical exam included relative macrocephaly, frontal bossing, short fingers with clinodactyly and were consistent with the phenotypes of previously reported deletions involving the 4q21--> 4q22 band region (Am. J. Med. Genet. 68 (1997) 400-405). To date there are 10 reported live-born cases with such deletions and similar features. The case reported here delimits a minimal critical region for this phenotype to chromosomal region 4q21. Our patient was also found to have cysts in both his kidneys. The gene for type II polycystic kidney disease (PKD2) has been mapped to chromosomal region 4q21--> 4q23. FISH analysis, with a probe including the PKD2 gene, demonstrated hemizygosity at this locus. Thus the absence of one of the PKD2 alleles in the case reported here is associated with early bilateral cyst development. Kidney ultrasound/autopsy studies were reported in seven of the patients with the characteristic phenotype, and were positive for cysts in four cases including the one presented here (Clin. Genet. 31 (1987) 199-205; Am. J. Med. Genet. 68 (1997) 400-405; Am. J. Med. Genet. 40 (1991) 77-790. Our report supports the presence of a distinct phenotype associated with a deleted chromosomal region within 4q21. Hemizygosity for the PKD2 gene is likely in such deletions and may lead to renal cyst formation.

Mutation within the TP53 tumor suppressor gene is a frequent occurrence in human cancers, resulting in a poor prognosis, response to therapy, and overall survival time. Mutations have been primarily detected in advanced prostate cancer; however, the involvement of the gene through loss of heterozygosity (LOH) in primary prostate cancers has not been investigated due to lack of identifiable polymorphisms within this gene. Using the nonisotopic RNAse cleavage assay (NIRCA), we screened for point mutations and identified an ApaI restriction site polymorphism located in intron 7 within the TP53 gene. This polymorphism allowed us to detect LOH in informative samples in a population of patients that underwent prostate biopsies and a population that underwent radical prostatectomies. Within the combined study population, 31 of 80 patients (38.75%) were informative for the polymorphism. Loss of heterozygosity was detected in 10 of the 31 samples (32.3%). Point mutations were identified in two samples. The identification of LOH in these patients suggests that the TP53 tumor suppressor gene may play a more active role in prostate cancer than was previously believed.

Approximately 5% of patients with chronic myelogenous leukemia (CML) do not reveal the Philadelphia (Ph) chromosome cytogenetically and are termed Ph-negative CML cases. We report one such case, which appeared normal by routine banding techniques. The BCR/ABL rearrangement was detected by reverse transcriptase-polymerase chain reaction and Southern blotting analysis, which suggested a b3-a2 splice junction. Dual color fluorescence in situ hybridization (FISH) with BCR and ABL DNA probes showed that the chimeric fusion gene was localized on chromosome 9q34, rather than at the typical location on chromosome 22q11. The BCR/ABL rearrangement was detected in 75% of the patient's bone-marrow population, whereas the remaining 25% of the cells appeared normal. The use of dual color FISH in the diagnosis of CML is extremely valuable not only in identifying cases of Ph-negative CML, but also in quantifying the proportion of transformed cell populations. This information ultimately results in an enhancement of our ability to monitor therapy, follow disease progression, and determine transplant eligibility.

Denaturation of chromosomal DNA for fluorescence in situ hybridization (FISH) is an essential step in a procedure associated with a number of variables. In our experience, shorter denaturation time in 70% formamide/2 x SSC at 72 C provides sufficient denaturation, where the hydrogen bonds are broken between the purines and pyrimidines of the double helix. This shortened exposure improves retention of morphology of human chromosomes from lymphocytes, aminocytes, fibroblasts and bone marrow, and allows the same metaphases to be denatured repeatedly and rehybridized with different probes. This approach is useful in investigations where sample volume is limited.

A newborn infant was referred for evaluation because of ambiguous genitalia. Examination of the genitalia revealed a hypospadiac phallus measuring 1.5 cm in length with chordee. Subtle phenotypic features consistent with Turner syndrome were present including hypertelorism, anti-mongoloid slant to the eyes, mild widening of the neck, but no definitive webbing, shield like chest and positive cubitus valgus. A pelvic and renal sonogram confirmed the presence of a uterus and normal-appearing kidneys. There was incomplete fusion of the scrotum. No gonads were palpable within the scrotal sac. The patient was assigned a female gender on the basis of the presence of a uterus, the phenotypic appearance of the genitalia and the malignant potential of the gonads. The cytogenetic findings with QFQ-banding revealed an abnormal karyotype, i.e., mos 46,X,idic(Y) (p11.2)[77]/45,X[29]/46,X,idic(Y) (p11?) [2]/ 47,XY,idic(Y)(p11.2)[2]/47,X,idic(Y)(p11.2), + idic(Y)(p11.2)[1]/46,XY[1]. The presence of an abnormal isodicentric Y-chromosome was evaluated by FISH-technique to ensure a finer characterization than routine methods. The genotype-phenotype correlation could not be established since mosaicisms of highly variable nature can exhibit an unpredictable outcome.

A patient was referred with a high leukocyte count and diagnosed with chronic myelogenous leukemia (CML). Although practically asymptomatic since the time of diagnosis, he had a variable and inconsistent response to treatment. All of his bone marrow cells had a complex, three-way translocation, involving chromosomes 4, 9 and 22. Translocation of chromosome 4 to chromosome 9 was undetectable by routine cytogenetic techniques; however, by the fluorescence in situ hybridization technique, a three-way translocation was identified, 46,XY,t(4;9;22)(p16;q34;q11). Although, other chromosomes are frequently involved in complex or variant translocations with chromosome 9 and 22, participation of chromosome 4 is a very rare event. So far, two previous cases have been described in the literature with translocations involving chromosome 4p16. We present a third case of CML having similar break points whose clinical presentation is unusual.

We report a case of human T-cell lymphotropic virus type 1 (HTLV-1)-infected adult T-cell lymphoma that has multiple chromosomal abnormalities, including the presence of an additional 7q22-36, which contains the locus of the T-cell receptor (TCR) beta chain gene. Specific TCR J beta 1/J beta 2 gene rearrangements were detected in both marrow and peripheral blood DNA, with evidence of further evolution of the transformed clonal population within the peripheral lymphocytes. To our knowledge, this is the first case in which gene rearrangements have been associated with additional TCR loci. Consequently, it is advised that every effort should be made to correlate chromosomal abnormalities with gene rearrangement by molecular methods.

Unlabelled: Trisomy 4p syndrome is a distinct clinical entity which was noted almost a quarter century ago by Wilson et al. [71] and later was delineated by Gonzalez and colleagues [29]. The variation in the length of duplicated segment usually associated with monosomy of other genetic material which has resulted in confusion and as a result a so-called 4p syndrome could not be recognized without cytogenetic analysis. We wish to draw the attention of clinicians to this subject by presenting the description of over 75 cases including one from our clinic and stress the point that molecular approaches are imperative to characterize this anomaly. After extensive review, it appears that patients retaining at least the distal two-thirds to the entire short arm share an overlapping phenotypic expression that constitutes pure trisomy 4p syndrome which includes prominent glabella, bulbous nose with flat or depressed nasal bridge, retrognathia, pointed chin, short neck with low hairline, enlarged ears with abnormal helix and antihelix, rocker-bottom feet with prominent heel. Arachnodactyly and camptodactyly. Molecular characterization of 4p is imperative. We have also included an extensive bibliography for clinicians who may find it useful as a single reference source for evaluating their future cases., Conclusion: The 4p-syndrome is a distinct entity but without cytogenetic evaluation, the syndrome can not be recognized.

We report on a new case of AML [M2] where chromosome 11 was rearranged in a highly complex manner involving band 11q23. Routine G banding failed to identify the nature of this aberration which later was accomplished using whole chromosome 11-specific painting probe by the FISH technique. Chromosome 11 band q23 is frequently involved in reciprocal translocations with various chromosomes, but to have sole morbidity of such a complex nature is an atypical finding in AML [M2]. The band q23 of chromosome 11 involves the mixed lineage leukemia gene and rearrangement of this chromosomal region has been found in a variety of neoplasias. Henceforth, its precise identification has become imperative for classification of hematological malignancies and in turn to devise therapeutic strategies.

We report on a so-called rare variant where a G-positive band was sandwiched within the secondary constriction (qh) region of chromosome 9 and is apparently different from previous cases when characterized by the fluorescence in situ hybridization technique. The major differences included duplication of beta-satellite and satellite III DNA sequences and bands 9q13-->q21.1, without duplication or inversion of the alphoid sequences. Based on the reported cases, at least four types of variations can be accounted for. A variety of mechanisms have been proposed to describe the origin of a G-positive band within the 9qh region, which appears to be similar when studied by routine cytogenetic techniques but differs by molecular methods. It is hypothesized that the clinical consequences depend upon the size of the G-positive band(s) duplicated, and a genetic inactivation mechanism might have some sort of influence during the so-called heterochromatinization process. It appears that heterochromatin, once thought to be composed of junk DNA, may have some role after all in suppression of gene(s) and/or spreading of inactivation, if genes are embedded within the heterochromatic region. Apparently, the mixture of different types of DNA creating patches of genetic debris have become a fundamental hidden treasure, where genetically active chromatin could be inactivated without dire consequences. The variable nature of heterochromatin has resulted in cytogenetic heteromorphisms of a number of human chromosomes. Their characterization by molecular techniques is becoming imperative, because fetal wastage have occurred in many situations where variant chromosomes were wrongly identified as chromosomal abnormalities.

A 12 1/2-month-old girl was referred because of short stature, short neck, large internipple distance and simian crease on her right hand. By routine cytogenetic techniques the presence of an unidentifiable marker chromosome and loss of one X chromosome was noted (i.e. 45, X/46, X, mar/47, X, mar, +mar). By fluorescence in situ hybridization (FISH) technique, the marker chromosome was identified as an isodicentric non-fluorescent Y chromosome ((45, X/46, X, idic (Ynf)/47, X, idic (Ynf), +idic (Ynf)). Although the clinical significance of this finding cannot be assessed at present, possible development of gonadoblastoma in such cases is a major concern and warrants follow-up evaluations.

We found an abnormal 47,XX,+mar karyotype in a patient with developmental delay, hypotonia, microcephaly, failure to thrive, and cognitive delay. When metaphases were hybridized with Prader-Willi and Angelman loci-specific probes by the FISH technique, two sites were noted at opposite positions on the marker chromosome. The alphoid satellite DNA probe documented the isodicentric nature while retention of the p arms on both sides of the marker chromosome was demonstrated by beta satellite probe. The patient does not exhibit manifestations of either syndrome despite the presence of these loci in tetrasomic dose. The present investigation suggests that other marker chromosomes be reevaluated, as their clinical manifestations are quite variable.

Human T-lymphotropic virus type I (HTLV-1) integration has been associated with the development of adult T-cell leukemia/lymphoma (ATL). Recently, a correlation between T-cell receptor (TCR) gene rearrangements and chromosomal aberrations has been implicated in this leukemia. We present a case of HTLV-1 infected adult T-cell lymphoma that initially presented with a normal karyotype and germline J beta I/J beta II loci. As the disease evolved to the aggressive stage, a complex chromosomal rearrangement with a duplication of chromosome 6p23-->pter translocated to a derivative chromosome 16, was identified by molecular cytogenetic techniques. The nature of this complex abnormality would have been difficult to determine if only conventional banding techniques were performed. Rearrangement involving one J beta allele was also detected at that time. After initiation of chemotherapy, no germline J beta loci were detected, suggesting a possible second rearrangement involving this locus that was homologous. Although no known immune response genes are located at the breakpoints involved in this abnormality, the chromosomal aberration concurred with the initial J beta rearrangement.

A case with typical features of chronic myelogenous leukemia (CML) with two complex aberrations in addition to the standard t(9;22) is reported. Cytogenetic evaluation of the patient's bone marrow cells (BMC) showed 46,XX,t(6;19)(q16;p13.3),t(9;22)(q34;q11) in 60% of the mitotic cells and 46,XX,idem, t(6;15)(p25;q22) in the remaining 40% dividing cells. The patient's peripheral blood smear exhibited the usual differential observed in chronic-phase CML and was clinically indistinguishable from patients with the t(9;22) as the only translocation. We performed Southern blotting on BglII-digested DNA with the Trans-Probe (OSI) and in addition to the 4.8-, 2.3-, and 1.1-kilobase (kb) germline fragments, we detected an additional fragment at 7 kb. This probe spans the entire 5.8-kb M-breakpoint cluster region (BCR), and a single breakpoint in this region will appear as either one or two additional fragments. Because only one additional fragment was observed, both cell lines apparently share the same breakpoint in the ABL/BCR gene. Apparently the second aberrant cell line with the additional t(6;15) represents clonal evolution of the original abnormal clone.

In a small percentage of cases of chronic myelogenous leukemia (CML), where the Ph chromosome is masked because of highly complex translocations and sub-microscopic rearrangements, precise identification of chromosomal aberrations by routine banding techniques has been difficult. We report on a new case of CML in which a single copy of a masked Ph chromosome was duplicated during blast crisis, i.e., the karyotype was 47,XY,dir ins(22;9)(q11;q34.1q34.2),t(1;22) (q21;q11), + der (22)t(1;22)(q21;q11). The chromosome in situ suppression hybridization (CISS) technique with whole chromosome 1 and 22 specific painting probes demonstrated that 22q11-qter had been translocated to 1q21, whereas 22q11 was the recipient of 1q21-qter. Furthermore, a cosmid probe identified the location of the ABL gene on only one chromosome 9 (band q34). The other ABL gene could be detected on both derivative chromosomes 22 at band q11 which was flanked by the translocated part of the long arm of chromosome 1, thus providing direct visualization of the ABL insertion in a double masked Ph chromosome. A breakpoint within the 5.8 kb major breakpoint cluster [M-BCR] region was shown by Southern blotting.

Heterochromatin confined to pericentromeric (c) and secondary constriction (qh) regions plays a major role in morphological variation of chromosome 9, because of its size and affinity for pericentric inversion. Consequently, pairing at pachytene may lead to some disturbances between homologous chromosomes having such extreme variations and may result in abnormalities involving bands adjacent to the qh region. We encountered such a case, where a G-positive band has originated de novo, suggesting a maternal origin from the chromosome 9 that has had a complete pericentric inversion. In previously reported cases, the presence of an extra G-positive band within the 9qh region has been familial, and in the majority of those cases it was not associated with any clinical consequences. Therefore, this anomaly has been referred to as a "rare" variant. The qh region consists of a mixture of various tandemly repeated DNA sequences, and routine banding techniques have failed to characterize the origin of this extra genetic material. By the chromosome in situ suppression hybridization technique using whole chromosome paint, the probe annealed with the extra G-band, suggesting a euchromatic origin from chromosome 9, presumably band p12. By the fluorescence in situ hybridization technique using alpha- and beta-satellite probes, the dicentric nature was further revealed, supporting the concept of unequal crossing-over during maternal meiosis I, which could account for a duplication of the h region. The G-positive band most likely became genetically inert when it was sandwiched between two blocks of heterochromatin, resulting in a phenotypically normal child. Therefore, an earlier hypothesis, suggesting its origin from heterochromatin through so-called euchromatinization, is refuted here.(ABSTRACT TRUNCATED AT 250 WORDS)

A newborn infant was referred because of low-set ears, mild downward slant of the palpebral fissures, micrognathia with high-arched palate, a flat midface, small mouth, and thin upper lip with cupid bow configuration. To some extent her cry resembled that associated with cri du chat syndrome. Cytogenetic findings with G- and Q-banding alone failed to characterize precisely the complex translocations. By the chromosome in situ suppression (CISS) hybridization technique using whole chromosome specific probes, a complex 4 breakpoint rearrangement involving both arms of a single chromosome 1 with the long arms of chromosomes 5 and 11 was disclosed, i.e., 46,XX, der(1),t(1;5) t(1;11) (5qter-->5q31::1p31.3-->1q44::11q23-->11 qter;5pter-->5q31::1p31.3-->1pter;11pter-- >11q 23::1q44-->1qter). Gene deregulation and position effect may explain the multiple anomalies in individuals with apparently balanced translocations. The molecular characterization of such cytogenetically balanced translocations may shed some light towards unveiling the clinical consequences associated with aberrations which are presumably balanced.

A genomic clone encoding prothymosin alpha (gene symbol: PTMA), a nuclear-targeted protein associated with cell proliferation, was isolated and the 5'-regulatory region subcloned and sequenced. Because of previously reported discrepancies between several cDNA clones and a genomic clone for prothymosin alpha, we determined the sequence of the first exon and of a 1.7-kb region 5' to the first exon. The sequence of the genomic clone reported here corresponds to the published cDNA sequences, suggesting that the previously noted discrepancies may be due to genetic polymorphism in this region. In addition, our sequence data extend the known 5'-upstream sequence by an additional 1.5 kb allowing the identification of numerous, potential cis-acting regulatory sites. This 5'-flanking cloned probe permitted us to localize the prothymosin gene to chromosome 2 in humans.

A patient with the typical features of the stable phase of chronic myeloid leukemia (CML) displayed two karyotypically related subclones. In addition to the t(9;22), cells from one clone contained a deletion of the short arm of chromosome 7, del(7)(p12), [46,XY,del(7)(p12),t(9;22)(q34;q11)]; the other contained only the standard translocation [46,XY,t(9;22)(q34;q11)]. Cells with a deletion of the short arm of chromosome 7 at band p12 as the only additional abnormality have not been observed previously in CML. Conventional chemotherapy with hydroxyurea and then with recombinant interferon-alpha (rIFN-alpha) did not reduce the population of either subclone. However, after treatment with a combination of rIFN-alpha and low-dose cytosine arabinoside (LoDac) continuously infused subcutaneously (s.c.), cells from the clone with the deleted chromosome 7 disappeared and normal metaphases were demonstrable.

A number of selective staining techniques have been utilized to decipher the variability of pericentromeric heterochromatin. One such technique is called DA/DAPI and it is believed to be stain specific. However, we demonstrate otherwise and suggest that pericentromeric regions of all human chromosomes stain positive by DA/DAPI-technique. It must be emphasized that the incidence of DA/DAPI positive stained chromosomes, other than 1, 9, 15, 16 and Y, is a rare occurrence and only a small portion of the pericentromeric region is DA/DAPI positive, as reported here using 50 normal individuals.

The presence of human T-cell leukemia virus (HTLV-1) in patients with adult T-cell leukemia (ATL) was investigated by Southern blotting and in situ hybridization. In all seven patients, HTLV-1 provirus was detected. A large and variable number of labeled restriction fragments were observed, indicating multiple integrations. Two of the patients analyzed by in situ hybridization had two, while the third patient had three, sites of viral integration on six different chromosomes, suggesting random integration. A single site of integration was shared by two patients, which was on chromosome 10 at bands p11-->p15. One of these sites was on an apparently normal chromosome 10 and the other was on a derivative chromosome 10,t(10;14)(p12;q32). The interleukin 2 receptor (IL2R) has previously been localized to this region (10p14-->p15). The alpha-chain of the IL2R is continuously expressed on affected T-cells in this disease. Southern blotting with pIL2R showed the presence of a novel 3.5 kb fragment in five out of the seven patients. This novel fragment has not been previously reported. No direct correlation was found between the novel 3.5 kb fragment, present in patients both cytogenetically normal and abnormal, and viral integration in the 10p11-->p15 region in two patients. Therefore, it is suggested that the presence of the 3.5 kb fragment and the numerous chromosomal breaks associated with this disease may not be direct results of viral integration.

Human myosin light chain-2 (MYL2) is an important protein involved in the regulation of myosin ATPase activity in smooth muscle. In cardiac muscle, the precise role of MYL2 is not well understood; however, an increase in ventricular MYL2 is observed during myocardial hypertrophy in cardiac patients with valve stenosis. The chromosomal location of the gene coding for MYL2 was identified using a cloned cDNA for human MYL2. Southern blot analysis of DNA from a human/rodent somatic cell hybrid mapping panel showed that the BamHI fragment that hybridized with this cDNA probe was concordant with chromosome 12. The 768-bp cDNA was hybridized to human metaphase chromosomes. The results revealed a significant clustering of silver grains over chromosome 12 bands q23-q24.3, indicating that the gene coding for MYL2 is located in this region.

A case of acute myelogenous leukemia (AML-M2) with an unusual chromosomal finding is presented. In addition to the most frequently observed translocation in this neoplasia, involving the long arms of chromosomes 8 and 21, there was a partial deletion of the short arm of chromosome 2 band (p23), i.e., 46,XX,del(2)(p23),t(8;21)(q22;q22). Deletion of the short arm of chromosome 2 has been described in association with other chromosome abnormalities in two other cases of AML and as the sole abnormality in three cases of AML, indicating that this abnormality is nonrandom and may be associated with leukemic transformation of hematopoietic cells. Therefore, we propose that the del(2)(p23),t(8;21)(q22;q22) abnormality be accorded status III and possibly considered a subset of AML (M2).

A new reciprocal, apparently balanced translocation between chromosomes 7 and 22, i.e., t(7;22)(p22;q13), in association with inv(16)(p13q22) in a child with acute nonlymphocytic leukemia (M4) is reported. Five percent of her bone marrow cells contained both of these aberrations while 90% of her cells have the pericentric inversion of chromosome 16 as the sole abnormality. A clonal evolution of the unusual cell line may be associated with atypical clinical presentation. The presence of inversion 16 in adults has a better prognosis as compared with children. A concise review on the cytogenetic findings in children with ANLL(M4) is also provided.

We report a new case of refractory anemia with excess blasts (RAEB) having complex chromosomal abnormalities. The 5q- associated with RAEB and other preleukemic syndromes was present in 100% of the cells; however, 60% of the cells had a highly unusual derivative chromosome involving the short arm of chromosome 1 and the long arm of chromosome 5, i.e., t(1;5)(p36;q14). Although the patient presented with highly complex chromosomal abnormalities, his initial clinical presentation was that of typical refractory anemia with excess blasts. Shortly after diagnosis (7 months), the patient developed acute leukemia.

Structural abnormalities involving heterochromatic regions of the human genome are difficult to characterize because these segments are G-band negative by GTG technique, a routinely used procedure in clinical cytogenetic laboratories. Chromosome abnormalities of such cases have gone undetected or were incorrectly characterized because these regions are so-called heteromorphisms or variants. Consequently, much anxiety has been aroused by the confusion between a chromosome abnormality and a normal heteromorphic variant. We report the first documented case with a so-called highly unusual h region of chromosome 9 which is not a variation but a structural rearrangement involving a paracentric inversion and a duplication. The major clinical features were psychomotor retardation, microcephaly, narrow palpebral fissures, renal and genital anomalies, vertebral anomalies, protruding tongue, and learning and behavioral problems. A concise review of variable duplicated segments of 9q is also provided.

The most recent addition to the various selective staining methods is the technique using restriction endonucleases to digest the metaphase chromosomes and subsequent staining by Giemsa (endonuclease/Giemsa technique). One of the endonucleases, AluI, which induces a characteristic modified C-band pattern is evaluated for its application in cancer cytogenetics using malignant lymphoid cells. The pattern obtained by AluI/Giemsa has been routinely useful in identifying some of the unusual markers that are difficult by routine banding. The centromeric regions are found to be far more heterogeneous by AluI resulting in frequent heteromorphic markers on several chromosomes. This has provided additional information to detect the donor cells in grafts after bone marrow transplantation.

Five to ten percent of the Ph-positive cases of chronic myelogenous leukemia (CML), termed variant translocations, involve at least one chromosome in addition to 9 and 22 in the abnormality. The involvement of chromosome 9 band q34, where the c-abl oncogene has been localized, is not always cytogenetically detectable in so called variant translocations due to complex rearrangements. We present two cases having the most frequently involved chromosomes (#3 and #17) in such translocations. In one case, both chromosome 9's were cytogenetically normal while in the other, band 9q34 was so called 'masked' or 'hidden'. After molecular evaluation using in situ hybridization and Southern blotting techniques, the involvement of the altered bcr/abl gene was demonstrated and the cytogenetic analysis was revised. Utilization of molecular probes in the evaluation of such cases should become a routine diagnostic procedure in detecting the exchange of bcr and c-abl sequences.

A new case is presented displaying typical features of the stable phase of chronic myelogenous leukemia (CML), with a complex translocation involving chromosomes 8q and 9q. Cytogenetic evaluation revealed an abnormal karyotype, 46,XY,t(8;9)(q22;q34). Both chromosomes 22 were found to be cytogenetically normal. After molecular evaluation the cytogenetic diagnosis was revised to 46,XY,t(8;9;22)(q22;q34;q11). The importance of the chimeric abl/bcr gene fusion product in the pathogenesis of CML is suggested as a characteristic feature, even in some patients with a so-called Philadelphia (Ph) negative CML. Utilization of molecular probes in the evaluation of such cases must become a routine diagnostic procedure. Our patient received the potential benefit of Ph-positive directed therapy because of the present approach.

Hyperdiploidy is common in neoplastic diseases but severe hypodiploidy or near-haploidy is extremely rare. Acute lymphocytic leukemia (ALL) and blast phase of chronic myelocytic leukemia (BC/CML) are the two most common leukemias where metaphases with as low as 23 chromosomes have been reported. Recent studies have indicated that during the course of malignant development, cells undergo numerous changes, however, it is still not known whether malignant transformation proceeds or results from the near-haploid state. Retrospectively, we have examined 100 metaphases with chromosome counts of 23 to 35 in patients with CML who have not yet progressed to the blastic phase, to see whether such metaphases share any common characteristics with published cases. The unusual behavior of chromosomes 8, 17 and the presence of Ph-chromosomes in 85% of the cells are highly unique features in our study. These observations are compatible with those found in BC/CML patients reported earlier. Therefore, it is hypothesized that selective chromosome loss is a gradual phenomenon and one of these near-haploid clones may replace a diploid clone as the dominant component of the population during blast transformation. Several hypotheses are proposed as to the origin of such clones in malignant hematopoietic stem cells.

In an attempt to further substantiate the involvement of the c-abl oncogene in the genesis of chronic myelogenous leukemia (CML) and acute lymphocytic leukemia (ALL), we performed in-situ hybridization with the c-abl probe on metaphase chromosomes of a healthy control, a karyotypically normal non-CML leukemia, three cases of CML with t(9;22), t(13;22) and t(3;9;22), one blast phase CML with t(9;22), Ph, + Ph, and one ALL with t(9;22), Ph, + Ph. The aberrant 9q could be cytogenetically identified in all cases except t(13;22). The molecular data clearly demonstrated the involvement of the c-abl in t(13;22) and the karyotype was revised to t(9;13;22). All patients, except the control, showed the altered c-able/bcr rearrangement that has been demonstrated in the Ph-chromosome from the standard t(9;22) translocation. These findings suggest that genomic diversities have important clinical differences in these two diseases. The amalgamation of information from cytogenetic and molecular data on cases where translocations are not precise or difficult to identify, will lead towards a deeper understanding of leukemias.

Chromosomal studies were performed on six patients with adult T-cell leukemia (ATL). Structural abnormalities of chromosome 3 were the most common. In one case a complete loss of the short arm of chromosome 10 (10 p-) was noted while in another case a balanced translocation involving chromosome 10p and 4q was found. These abnormalities have not been previously reported. After reviewing the literature, it was concluded that chromosomes 3, 6, 10, 13, 14, and X were most frequently involved in abnormalities. Specific and consistent chromosomal abnormalities were noted in each study. Therefore, it is hypothesised that the mutation rate for this virus may be higher than first expected. Furthermore, the relative consistency of heterogeneous findings in different localities may reflect a geographic clustering of specific chromosomal abnormalities which may in turn be related to specific and geographically associated viral mutations. To support these suggestions not only are more cytogenetic data required but a molecular evaluation of these patients must be carried out to establish a relationship, if any, between genetic abnormalities and the epidemiology of ATL.

Chickens having a trisomic complement for the nucleolar organizer region (NOR) serve as a model for investigations on gene expression, molecular regulation, and physiological alterations resulting from extra genetic material. Of fundamental importance is the question of whether the extra set of ribosomal cistrons in trisomic chickens generates a morphologically and biochemically complete nucleolus. Ultrastructural and cytochemical studies of diploid and trisomic embryonic and adult cells were undertaken. All structures identified in trisomic cells as nucleoli by gross morphology were shown to be RNA rich by acridine orange fluorescence. At the ultrastructural level, the expected granular and fibrillar components were identified in all nucleoli of trisomic cells. Fibrillar centers, indicative of NOR's, were seen in all cases. These and other results from silver staining suggest full gene activity for each of the three chromosomes in the trisomic cell at all stages of development.

Two new variant cases of chronic myelogenous leukemia (CML) are presented. The first case is a 19-year-old male with a 46,XY,t(9;15;22)(q34;q22;q11) karyotype. The second case is a 75-year-old man with a 46,XY,t(6;9;12;22)(p21;q34;q24;q11) karyotype. In both cases, the prognosis was no different from those cases of CML with the standard t(9;22) as the only abnormality. We recommend that all unusual translocations be reported.

A patient with chronic myelogenous leukemia (CML) having the standard [t(9;22), Ph] translocation is presented where the Philadelphia (Ph) chromosome disappeared following bone marrow transplantation (BMT). The Ph chromosome reappeared in host cells after one year of stable hematologic remission. Three additional cell lines, all possessing the Ph chromosome with other abnormalities were consistently present in her marrow cells. Two years after BMT, ninety percent of her dividing bone marrow cells had become leukemic. The patient's clinical status remains unchanged, despite complex cytogenetic findings. The high incidence of multiple aberrant leukemic clones present in this case remains intriguing. Possible mechanisms for this unique transformation after BMT are discussed.

The recent advent of banding techniques has facilitated the identification of human chromosomal abnormalities in neoplasias. Utilization of a single technique for the identification of marker chromosomes has caused ambiguity because staining profiles overlap with many other chromosomal regions in the human genome. Thus, the implication of DA/DAPI technique in clinical cytogenetics has been documented by presenting a variety of cases with neoplastic syndromes.

The incidence of breakpoints in CML patients with variant translocations was investigated. There was no relationship between the length of various chromosomes with breakpoint frequency. However, a significantly higher (p less than 0.05) incidence of breaks were seen on the long arms as compared to the short arms due mainly to the involvement of 9q and 22q in these translocations. Chromosome 17 showed a significantly (p less than 0.005) higher involvement in these translocations, however only when 9q34-qter was not cytogenetically involved. A total of 683 breaks were found in 225 cases. 362 of these were located at c-abl and c-sis, while 110 were at other oncogenetic sites. The prognostic and hematologic features of patients with variant translocations are not significantly different from those of CML cases with the typical 9q;22q translocation. Some of these complex translocation, where the breakpoints are correlated with oncogenetic sites, are further discussed in molecular terms.

It has become routine practice to culture bone marrow and/or unstimulated peripheral blood for cytogenetic analysis due to associations of consistent chromosomal abnormalities within specific subgroups of leukemias. Unfortunately, two leukemias for which numerous chromosomal aberrations have been recorded, acute myelogenous leukemia (AML) and acute lymphocytic leukemia (ALL), frequently present with very low cell counts often resulting in unsuccessful cultures. Furthermore, when cultures are successful, chromosomes are often contracted and metaphase spreads can be of poor quality. In the present investigation, by first treating blood or bone marrow from AML and ALL patients with ammonium chloride (1.5 mM) we were able to obtain chromosomal preparations often similar in quality to those obtained from normal stimulated blood cultures. With this improved culturing method it is also possible to generate high-quality chromosomes and DNA from the same sample.

A new case of ambiguous genitalia and immature tissue in the left gonad is presented. Cytogenetic findings with various techniques demonstrated that the distal two-thirds of the long arm of the Y chromosome is deleted. Q-banding showed a non-fluorescent Y; three positive bands were however noted when the DA/DAPI technique was applied. After a review of the literature, it was concluded that the non-fluorescent Y chromosome (Ynf) when inherited from generation to generation is a heteromorphism in normal males. However, in our case, where the proband's Y is lacking the fluorescent segment, a simple deletion does not appear to adequately explain the DA/DAPI positive bands. Possibly, a deletion followed by a structural rearrangement of the non-fluorescent segment had occurred de novo. The highly Y-specific DNA sequences present in the fluorescent segment are absent in these patients. The abnormal development in these cases is due to the presence of the 45,X cell line. The gene responsible for spermatogenesis has been localized to the non-fluorescent region in the long arm of the Y chromosome. Furthermore, it is concluded that two types of non-fluorescent Y chromosomes can be found in the population; one is a normal inherent heteromorphic variant, while the other appears to be an abnormality, especially in cases with azoospermia. Such distinctions should clearly be established prior to genetic counseling for patients with so called Ynf or del (Yd).