Immunophenotypic and gene expression analysis of monoclonal B-cell lymphocytosis shows biologic characteristics associated with good prognosis CLL.

Monoclonal B-cell lymphocytosis (MBL) is a hematologic condition wherein small B-cell clones can be detected in the blood of asymptomatic individuals. Most MBL have an immunophenotype similar to chronic lymphocytic leukemia (CLL), and 'CLL-like' MBL is a precursor to CLL. We used flow cytometry to identify MBL from unaffected members of CLL kindreds. We identified 101 MBL cases from 622 study subjects; of these, 82 individuals with MBL were further characterized. In all, 91 unique MBL clones were detected: 73 CLL-like MBL (CD5(+)CD20(dim)sIg(dim)), 11 atypical MBL (CD5(+)CD20(+)sIg(+)) and 7 CD5(neg) MBL (CD5(neg)CD20(+)sIg(neg)). Extended immunophenotypic characterization of these MBL subtypes was performed, and significant differences in cell surface expression of CD23, CD49d, CD79b and FMC-7 were observed among the groups. Markers of risk in CLL such as CD38, ZAP70 and CD49d were infrequently expressed in CLL-like MBL, but were expressed in the majority of atypical MBL. Interphase cytogenetics was performed in 35 MBL cases, and del 13q14 was most common (22/30 CLL-like MBL cases). Gene expression analysis using oligonucleotide arrays was performed on seven CLL-like MBL, and showed activation of B-cell receptor associated pathways. Our findings underscore the diversity of MBL subtypes and further clarify the relationship between MBL and other lymphoproliferative disorders.

Single-cell analysis reveals oligoclonality among 'low-count' monoclonal B-cell lymphocytosis.

Monoclonal B-cell lymphocytosis (MBL) is a preclinical hematologic syndrome characterized by small accumulations of CD5(+) B lymphocytes. Most MBL share phenotypic characteristics with chronic lymphocytic leukemia (CLL). Although some MBL progress to CLL, most MBL have apparently limited potential for progression to CLL, particularly those MBL with normal absolute B-cell counts ('low-count' MBL). Most CLL are monoclonal and it is not known whether MBL are monoclonal or oligoclonal; this is important because it is unclear whether MBL represent indolent CLL or represent a distinct premalignant precursor before the development of CLL. We used flow cytometry analysis and sorting to determine immunophenotypic characteristics, clonality and molecular features of MBL from familial CLL kindreds. Single-cell analysis indicated four of six low-count MBL consisted of two or more unrelated clones; the other two MBL were monoclonal. 87% of low-count MBL clones had mutated immunoglobulin genes, and no immunoglobulin heavy-chain rearrangements of V(H) family 1 were observed. Some MBL were diversified, clonally related populations with evidence of antigen drive. We conclude that although low-count MBL share many phenotypic characteristics with CLL, many MBL are oligoclonal. This supports a model for step-wise development of MBL into CLL.

Loss of chromosome 13 in a case of soft tissue perineurioma.

Soft tissue perineuriomas are rare mesenchymal tumors that are derived from perineurial cells of the peripheral nerve sheath. Although the histological and immunohistochemical features of soft tissue perineuriomas are well described, little is known regarding the cytogenetic abnormalities in these tumors. Herein, we describe a case of a large (12.2 cm) soft tissue perineurioma that arose in the thigh of a 26-year-old Caucasian female. Histologically, the tumor was composed of a diffuse to fascicular arrangement of spindle cells with bland, elongated nuclei with long, thin, tapering cytoplasmic processes. The immunohistochemical profile was consistent with a perineurial cell origin with expression of epithelial membrane antigen, vimentin, and collagen type IV. Cytogenetic evaluation revealed loss of chromosome 13 as the sole abnormality in the majority of examined cells. In contrast to previous reports, we were unable to demonstrate deletion or structural abnormalities of chromosome 22 by either fluorescence in situ hybridization (FISH) or metaphase cytogenetics. This is the first report of loss of chromosome 13 in soft tissue perineurioma. Although never described in this group of neoplasms, loss of chromosome 13 has been identified in a large number of other soft tissue tumors, particularly sarcomas and malignant peripheral nerve sheath tumors. Herein, we discuss this case and provide a review of the literature.

Molecular and cytogenetic characterization of a novel rearrangement involving chromosomes 9, 12, and 17 resulting in ETV6 (TEL) and ABL fusion.

We performed chromosome analysis on the bone marrow of a patient with BCR/ABL negative chronic myelogenous leukemia (CML). By interphase fluorescence in situ hybridization (FISH), an extra ABL signal was present in interphase nuclei and appeared to be located at 17p in the metaphase cells. Chromosome analysis showed a subtle abnormality at 17p13 and 12p13 but no visible rearrangement at 9q34 (ABL). Additional FISH experiments disclosed a rearrangement between the short arms of chromosomes 12 and 17 at approximately bands 12p13 and 17p13, respectively. In addition, subtelomeric FISH analysis confirmed the presence of terminal 12p at 17p13 and showed terminal 9q34 to be intact on each chromosome 9. Taken together, these results indicated a rearrangement involving chromosomes 9, 12, and 17 that suggested the possibility of juxtaposition of part of the ETV6 (also known as TEL) locus (12p13) with a portion of ABL (9q34) together at 17p13. The ETV6/ABL fusion was confirmed by RT-PCR, which showed that the first 5 exons of ETV6 were fused in frame with ABL at exon 2. Wild-type ETV6 and ABL were also expressed, in accordance with the FISH results that showed no loss of the second ETV6 or ABL allele.

A case of infantile acute lymphoblastic leukemia presenting with rearrangement of MLL at 11q23 and apparent insertion or translocation at 10p12.

We report the case of an 11-month-old patient with a clinical diagnosis of infantile acute lymphoblastic leukemia and an MLL (11q23) rearrangement in 69% of nuclei, revealed with interphase fluorescence in situ hybridization (FISH). Routine chromosome analysis of the bone marrow showed a very subtle rearrangement involving the short arm of chromosome 10 and the long arm of chromosome 11 in the abnormal cells. To clarify the nature of this rearrangement, we hybridized the MLL break-apart probe to previously G-banded slides. The rearrangement was interpreted as a small inversion within the band 11q23, separating the 5' MLL from the 3' MLL region. This segment on the long arm of chromosome 11 containing the rearranged MLL locus was either inserted in or translocated to the short arm of chromosome 10 at approximately band 10p12. The inversion affecting MLL may have followed insertion or preceded it. Molecular characterization of this rearrangement was not possible, due to limited sample material. There have been previous reports of rearrangements of MLL with the MLLT10 (alias AF10) gene locus at 10p12, including an interstitial inverted insertion of 11q13q23 in one case and insertion of 11q14q23 at 10p12 in another. These both resulted in a large derivative chromosome 10 and transcription of an MLL/MLLT10 fusion product. To our knowledge, the novel and cryptic rearrangement detected in our patient has not been described previously. A follow-up study of the patient's bone marrow at the end of induction therapy showed no morphologic evidence of residual leukemia and both FISH and chromosome analyses were normal.

Cytogenetic and molecular analysis of an unusual case of acute promyelocytic leukemia with a t(15;17;17)(q22;q23;q21).

We present a 52-year-old female with a clinical history of acute myelocytic leukemia, probable acute promyelocytic leukemia (APL). Flow cytometry results were somewhat unusual. Specifically, the promyelocytic population showed partial positivity for antigens not usually expressed in APL (HLA-DR and CD117). The interpretation of these results was that the abnormal population contained a proportion of very early promyeolocytes that had not completely lost all their "precursor" antigens. Cytogenetic analysis of a bone marrow aspirate showed a t(15:17;17)(q22;q23;q21) in all cells analyzed. Fluorescence in situ hybridization (FISH) analysis using the PML-RARA DNA probe showed a positive signal pattern (fusion) in 100% of 200 total interphase and metaphase cells examined, confirming the presence of the PML-RARA rearrangement. Multicolor FISH, which produces 24 colors to differentiate all chromosomes in a single hybridization, was applied. This study confirmed the cytogenetic interpretation of the rearrangement. No material from any other chromosome was detected on the second smaller derivative chromosome 17. Additional studies using the RARA(17q21) break-apart DNA FISH probe showed that 17q21 (RARA) was not rearranged on the derivative chromosome 17 that received the q22-->qter segment from chromosome 15. The RARA locus on the smaller derivative 17 was the allele involved in the fusion in this three-way rearrangement. The signal pattern was consistent in 100% of interphase and metaphase cells scored. This unusual t(15;17;17) prompted us to investigate further using reverse-transcription polymerase chain reaction with primers from the 3' and 5' regions of both the RARA and PML loci. These studies showed that the PML-RARA fusion was present, but the complementary fusion RARA-PML, which is usually detectable, was absent. The patient is responding well to standard treatment protocols.

Partial trisomy 7p defined by analysis of a complex chromosome rearrangement using a BAC clone panel.

PURPOSE: To illustrate the use of bacterial artificial chromosome (BAC) clone panels for molecular cytogenetic analysis of complex chromosome rearrangements (CCRs). METHODS: High resolution cytogenetics followed by fluorescence in situ hybridization (FISH) analysis using chromosome band-specific BAC probes, in addition to commercially available probes. RESULTS: High resolution cytogenetics in conjunction with FISH using commercially available probes proved inadequate to resolve problems in characterizing a balanced CCR in the mother of a patient who had inherited an unbalanced form of the CCR. Accurate interpretation of the CCR and the unbalanced rearrangement in the patient as trisomy 7p12.2-->p21.3 was accomplished only through use of the BAC clone panel. CONCLUSION: Use of BAC clone panels can enhance the power of FISH analysis in defining chromosome rearrangements that cannot be resolved by high resolution chromosome analysis.

Delayed membranous ossification of the cranium associated with familial translocation (2;3)(p15;q12).

The relationship of delayed membranous cranial ossification to cranium bifidum and parietal foramina syndromes is unclear. We report on a family with delayed cranial membranous ossification (OMIM 155980) that segregates with an apparently balanced reciprocal translocation between chromosomes 2 and 3. The propositus had apparently low-set ears, proptosis, and a soft skull at birth. A radiographic survey of the skeleton showed markedly decreased ossification of the cranial bones and no other skeletal abnormalities. The mother and maternal grandmother of the propositus have brachycephaly, hypertelorism, and a history of a soft skull at birth. Chromosome analysis of peripheral blood from the propositus showed 46,XY,t(2;3)(p15;q12). The propositus, mother, and grandmother carry the same reciprocal translocation, whereas the mother's two phenotypically normal sibs have a normal karyotype. We used an STS-linked BAC resource to define the translocation breakpoint by identifying flanking BAC clones from both chromosomes 2, 1006D24 (D2S2279) and 1060A5 (D2S2231), and chromosome 3, 3D17 (WI8558) and 3D18 [CITB Human BAC Library, J.R.K.]. This represents the second report of a family with delayed membranous ossification of the cranium and the first report of the phenotype segregating with a chromosome rearrangement.

Molecular cytogenetic evaluation in a patient with a translocation (3;21) associated with blepharophimosis, ptosis, epicanthus inversus syndrome (BPES).

Blepharophimosis, ptosis, epicanthus inversus syndrome type I (BPES; OMIM 110100) is an autosomal dominant disorder affecting craniofacial development and ovarian function. We have identified a patient with BPES who carried a de novo reciprocal translocation [46, XX,t(3;21)(q23;q22.1)]. Fluorescence in situ hybridization analysis at band 3q23 using probes derived from BAC 175G20 (Research Genetics), PACs 108L15 and 169C10 (RPCI1), and cosmids AC174D4, AC68D3, AC44F5, and AC125C5 (Lawrence Livermore National Laboratory) was performed. The patient's breakpoint was found to lie within the overlapping region of the BAC and PACs but centromeric to all the cosmids. However, a 10.5-kb BamHI-digested fragment, common to the BAC and PAC clones, was shown to cross the breakpoint. The results have placed our patient's breakpoint proximal to that of the previously reported patient [46,XY,t(3;4)(q23;p15.2)] and within a 10.5-kb interval. This is the second patient in which a breakpoint was refined by molecular cytogenetics. Our findings emphasize the significance of this region for BPES.

Identification of the alpha-aminoadipic semialdehyde synthase gene, which is defective in familial hyperlysinemia.

The first two steps in the mammalian lysine-degradation pathway are catalyzed by lysine-ketoglutarate reductase and saccharopine dehydrogenase, respectively, resulting in the conversion of lysine to alpha-aminoadipic semialdehyde. Defects in one or both of these activities result in familial hyperlysinemia, an autosomal recessive condition characterized by hyperlysinemia, lysinuria, and variable saccharopinuria. In yeast, lysine-ketoglutarate reductase and saccharopine dehydrogenase are encoded by the LYS1 and LYS9 genes, respectively, and we searched the available sequence databases for their human homologues. We identified a single cDNA that encoded an apparently bifunctional protein, with the N-terminal half similar to that of yeast LYS1 and with the C-terminal half similar to that of yeast LYS9. This bifunctional protein has previously been referred to as "alpha-aminoadipic semialdehyde synthase," and we have tentatively designated this gene "AASS." The AASS cDNA contains an open reading frame of 2,781 bp predicted to encode a 927-amino-acid-long protein. The gene has been sequenced and contains 24 exons scattered over 68 kb and maps to chromosome 7q31.3. Northern blot analysis revealed the presence of several transcripts in all tissues examined, with the highest expression occurring in the liver. We sequenced the genomic DNA from a single patient with hyperlysinemia (JJa). The patient is the product of a consanguineous mating and is homozygous for an out-of-frame 9-bp deletion in exon 15, which results in a premature stop codon at position 534 of the protein. On the basis of these and other results, we propose that AASS catalyzes the first two steps of the major lysine-degradation pathway in human cells and that inactivating mutations in the AASS gene are a cause of hyperlysinemia.

Quantitative neurologic assessment of ataxia-telangiectasia.

BACKGROUND: Ataxia telangiectasia (A-T) is a rare disorder with many distinctive neurologic features. Although there is substantial individual variation in the rate of progression of these features, their relationship to one another or to age has not been characterized. METHODS: We formulated and tested multiple elements that assess different neurologic functions known to be affected by A-T. The overall index was applied to 52 patients with A-T, 2 to 29 years of age. RESULTS: Seven elements items proved to be informative, and three elements were added based on face validity. In a linear regression model of individuals under 19 years of age, controlled for correlation within sibships, age accounted for 87% of the variation in the A-T Index. CONCLUSION: Despite substantial individual variability of the phenotypic elements of A-T, scores on this multidimensional index have a very high correlation with age, indicating that there is a characteristic rate of progression of the disease, although functional domains in the brain are differentially affected. The pattern of scores suggests that a severe and a mild form of A-T may be distinguished by this quantitative measure. With further development this index may become useful as an outcome measure for treatment studies and prognosis.

Cryptic subtelomeric translocations in the 22q13 deletion syndrome.

Cryptic subtelomeric rearrangements are suspected to underlie a substantial portion of terminal chromosomal deletions. We have previously described two children, one with an unbalanced subtelomeric rearrangement resulting in deletion of 22q13-->qter and duplication of 1qter, and a second with an apparently simple 22q13-->qter deletion. We have examined two additional patients with deletions of 22q13-->qter. In one of the new patients presented here, clinical findings were suggestive of the 22q13 deletion syndrome and FISH for 22qter was requested. Chromosome studies suggested an abnormality involving the telomere of one 22q (46,XX,?add(22)(q13. 3)). FISH using Oncor D22S39 and Vysis ARSA probes confirmed a terminal deletion. A multi-telomere FISH assay showed a signal from 19qter on the deleted chromosome 22. Results were confirmed with 19qtel and 22qtel specific probes. The patient is therefore trisomic for 19qter and monosomic for 22qter. The patient's mother was found to have a translocation (19;22)(q13.42;q13.31). We also re-examined chromosomes from two patients previously diagnosed with 22q deletions who were not known to have a rearrangement using the multi-telomere assay. One of these patients was found to have a derivative chromosome 22 (der(22)t(6;22)(p25;q13)). No evidence of rearrangement was detected in the other patient. Thus we have found the 22q13 deletion to be associated with a translocation in three of four patients. This report illustrates the usefulness of examining patients with hypotonia, severe language delay, and mild facial dysmorphism for this syndrome and suggests that most of these deletions may be unbalanced subtelomeric rearrangements.

Molecular cytogenetic analysis and clinical findings in a newborn with prenatally diagnosed rec(7)dup(7q)inv(7)(p22q31.3)pat.

We report prenatal and early postnatal findings in a newborn with a partial trisomy of chromosome 7 (7q31.3-qter), arising from meiotic recombination of a paternal pericentric inversion, inv(7)(p22q31.3). The inversion breakpoints were localized and the regions of duplication and deletion were defined by fluorescence in situ hybridization (FISH) analysis using a series of locus-specific and subtelomeric probes. To our knowledge, only three cases involving a recombinant 7 with duplication of 7q have been reported, two of these being first cousins. The clinical findings in our patient included skeletal abnormalities, facial dysmorphism, dilated cerebral ventricles, microretrognathia and short neck. These findings and some aspects of the neonatal course were consistent with the phenotype previously reported for duplication of distal 7q, without associated monosomy for sequences from another chromosome.

Clinical, cytogenetic, and fluorescence in situ hybridization findings in two cases of "complete ring" syndrome.

The term "ring syndrome" was proposed to describe a phenotype of growth failure without major malformations due to a ring autosome. The growth failure is thought to be caused by instability of the ring chromosome leading to aneusomy and cell death. Most previous studies of ring chromosomes were based on standard cytogenetic banding techniques and were limited to microscopically detectable deletions in the ring chromosomes. We report on two patients with complete ring (4) and ring (9) chromosomes, respectively. The first was a 15-month-old girl and the second was a 16-month-old boy. They both presented with severe, symmetrical growth failure and normal psychomotor development in the absence of malformations. Their parents had a normal phenotype. The first case had a whorled pattern of hyperpigmentation and hypopigmentation on part of the face and chest, and the second case had a patchy hyperpigmented rash on the trunk. Peripheral blood karyotype of the first patient was 46,XX, r(4)(p16.3q35.2) and of the second 45,XY,-9/46,XY,r(9)(p24q34.3). G-band analysis suggested no loss of material in the ring chromosomes. These findings were confirmed by fluorescence in situ hybridization (FISH) analysis using chromosome-specific subtelomeric probes. The common human telomeric sequences were intact in the first patient but absent in the second patient. The cytogenetic and FISH data in our two cases provide further evidence for the existence of a "complete ring" phenotype independent of the autosome involved. Pigmentary skin changes are a useful clinical sign of mosaicism caused by the ring instability.

Hyperornithinaemia-hyperammonaemia-homocitrullinuria syndrome is caused by mutations in a gene encoding a mitochondrial ornithine transporter.

Neurospora crassa ARG13 and Saccharomyces cerevisiae ARG11 encode mitochondrial carrier family (MCF) proteins that transport ornithine across the mitochondrial inner membrane. We used their sequences to identify EST candidates that partially encode orthologous mammalian transporters. We thereby identified such a gene (ORNT1) that maps to 13q14 and whose expression, similar to that of other urea cycle (UC) components, was high in liver and varied with changes in dietary protein. ORNT1 expression restores ornithine metabolism in fibroblasts from patients with hyperammonaemia-hyperornithinaemia-homocitrullinuria (HHH) syndrome. In a survey of 11 HHH probands, we identified 3 ORNT1 mutant alleles that account for 21 of 22 possible mutant ORNT1 genes in our patients: F188delta, which is common in French-Canadian HHH patients and encodes an unstable protein; E180K, which encodes a stable, properly targeted protein that is inactive; and a 13q14 microdeletion. Our results show that ORNT1 encodes the mitochondrial ornithine transporter involved in UC function and is defective in HHH syndrome.

Inherited duplication Xq27-qter at Xp22.3 in severely affected males: molecular cytogenetic evaluation and clinical description in three unrelated families.

We describe the clinical phenotype in four males from three families with duplication (X)(qter-->q27::p22.3-->qter). This is an unusual duplication of the distal long arm segment, Xq27-qter, onto the distal short arm of the X chromosome at Xp22.3, as shown by fluorescent in situ hybridization analysis with multiple X-specific probes. The patients are young male offspring of three unrelated, phenotypically normal carrier women. The affected males have similar clinical manifestations including severe growth retardation and developmental delay, severe axial hypotonia, and minor anomalies. Such clinical similarity in three unrelated families demonstrates that this chromosome abnormality results in a new and distinct clinical phenotype. Replication studies, performed on two of the mothers, provided evidence that inactivation of the abnormal X chromosome permitted the structural abnormality to persist in these families for a generation or more in females without phenotypic expression.

DNA damage induced via independent generation of the radical resulting from formal hydrogen atom abstraction from the C1'-position of a nucleotide.

BACKGROUND: Deoxyribonucleotide radicals resulting from formal C1'-hydrogen atom abstraction are important reactive intermediates in a variety of DNA-damage processes. The reactivity of these radicals can be affected by the agents that generate them and the environment in which they are produced. As an initial step in determining the factors that control the reactivity of these important radical species, we developed a mild method for their generation at a defined site within a biopolymer. RESULTS: Irradiation of oligonucleotides containing a photolabile nucleotide produced C1'-DNA radicals. In the absence of potential reactants other than O2, approximately 90% of the damage events involve formation of alkaline-labile lesions, with the remainder resulting in direct strand breaks. The ratio of alkaline-labile lesions to direct strand breaks (approximately 9:1) is independent of whether the radical is generated in single-stranded DNA or double-stranded DNA. Strand damage is almost completely quenched under anaerobic conditions in the presence of low thiol concentrations. Competition studies with O2 indicate that the trapping rate of C1'-DNA radicals by beta-mercaptoethanol is approximately 1.1 x 10(7) M-1s-1. CONCLUSIONS: The mild generation of the C1'-DNA radical in the absence of exogenous oxidants makes it possible to examine their intrinsic reactivity. In the absence of other reactants, the formation of direct strand breaks from C1'-radicals is, at most, a minor pathway. Competition studies between beta-mercaptoethanol and O2 indicate that significantly higher thiol concentrations than those in vivo or some means of increasing the effective thiol concentration near DNA are needed for these reagents to prevent the formation of DNA lesions arising from the C1'-radical under aerobic conditions.

Familial tandem duplication of bands q31.1 to q32.3 on chromosome 4 with mild phenotypic effect.

We describe a family carrying a direct duplication of an interstitial segment of the distal long arm of chromosome 4(q31.1q32.3) with a mild clinical phenotype. Affected family members include a 27-year-old woman and 2 of her 4 children, 1 of whom also has Klinefelter syndrome. Although 8 cases of simple duplications or insertions of 4q material have been described, only 3 include bands q31-q32, and these involve additional adjacent material. Affected members of the pedigree reported here have some of the manifestations of previously described cases of duplication 4q, but are less severely affected, suggesting that the genetic material in this region is well tolerated as a partial trisomy.

Deletion of PTEN in a patient with Bannayan-Riley-Ruvalcaba syndrome suggests allelism with Cowden disease.

We report on an 18-month-old boy with an interstitial deletion at 10q23.2-q24.1. This region includes the PTEN gene, mutations of which have been reported to cause Cowden disease. Our patient presented with manifestations of Bannayan-Riley-Ruvalcaba (BRR) syndrome. The BRR syndrome is a rare disorder which presents most commonly in childhood. Cowden disease is a disease of adulthood and is inadequately described in children. Because of the considerable phenotypic overlap between the two disorders, and the cytogenetic and molecular findings in our patient, we suggest that BRR syndrome and Cowden disease are allelic.