Cryptic Rearrangements Research Articles

3rd International Meeting on Cryptic Chromosomal Rearrangements in Mental Retardation and Autism

2nd International Meeting on Cryptic Chromosomal Rearrangements in Mental Retardation and Autism

Cryptic duplication of 12q24.33 → qter in a child with Angelman syndrome—simultaneous occurrence of two unrelated cytogenetic events

Simultaneous occurrence of two unrelated cytogenetic events is rare. We present a case of Angelman Syndrome (AS) deletion and 12q duplication in a child with a history of developmental delay, microcephaly, cerebral palsy, and seizures. Traditional cytogenetic studies showed a normal 46,XY karyotype. Fluorescence in situ hybridization (FISH) using probe D15S10 (AS region/15q11.2) revealed a deletion. In addition, we serendipitously detected 12q24.3 duplication by FISH with 12q subtelomere probe. He inherited this duplication from the mother who presented with a balanced translocation karyotype 46,XX,add(12)(q24.3).ish t(12;13)(q24.3;p11.2)(12qtel-;12qtel+,D13Z1/D21Z1+,RB1+). Array comparative genomic hybridization (array-CGH) revealed a duplication of three bacterial artificial chromosome (BAC) clones (RP11-46H11, RP11-386I8, and RP11-309H3) covering about 423 Kb of DNA sequence. The published 12q terminal duplication cases had a detectable segment by classical banded cytogenetics techniques. To our knowledge, this is the smallest 12q cryptic rearrangement characterized by array-CGH and confirmed by BAC-clone FISH analysis. Based on these findings, we attempted to separate the clinical features associated with AS deletion and those features that are probably due to partial 12q duplication. We then reviewed the genes mapped in the duplicated region using the human genome database to understand the clinical significance. A subsequent pregnancy in the mother revealed an apparently balanced t(12;13) karyotype. We compare our case with the published cases, and discuss the implications of our findings and its relevance in addressing genetic counseling issues.

The impact of translocations and gene fusions on cancer causation

Chromosome aberrations, in particular translocations and their corresponding gene fusions, have an important role in the initial steps of tumorigenesis; at present, 358 gene fusions involving 337 different genes have been identified. An increasing number of gene fusions are being recognized as important diagnostic and prognostic parameters in malignant haematological disorders and childhood sarcomas. The biological and clinical impact of gene fusions in the more common solid tumour types has been less appreciated. However, an analysis of available data shows that gene fusions occur in all malignancies, and that they account for 20% of human cancer morbidity. With the advent of new and powerful investigative tools that enable the detection of cytogenetically cryptic rearrangements, this proportion is likely to increase substantially.

MLPA as first screening method for the detection of microduplications and microdeletions in patients with X-linked mental retardation

PurposeRoutine protocols for the study of mental retardation include karyotype, analysis for fragile X syndrome, and subtelomeric rearrangements. Nevertheless, detection of cryptic rearrangements requires more sensitive techniques. Mutation screening in all known genes responsible for X-linked mental retardation is not feasible, and linkage analysis is sometimes limited. Multiplex ligation probe amplification is a recently developed technique based on the amplification of specific probes that allows relative quantification of 40 to 46 different target DNA sequences in a single reaction. MethodsIn the present study, we assessed multiplex ligation probe amplification for the detection of microduplications/microdeletions in 80 male patients with suspicion of X-linked mental retardation. ResultsWe detected four copy number aberrations (5%): three duplications (GDI1, RPS6KA3, and ARHGEF6) and one deletion (OPHN1). All these changes were confirmed by other molecular techniques, and patients were clinically re-evaluated. ConclusionsWe strongly recommend the use of multiplex ligation probe amplification as a first screening method for the detection of copy number aberrations in patients with mental retardation because of its cost-effectiveness.

INHERITED DELETION OF THE 17P TELOMERE: A CLINICALLY SIGNIFICANT FINDING OR FAMILIAL VARIANT?

Cryptic chromosome rearrangements have been detected in developmentally delayed and/or dysmorphic individuals with normal karyotypes using conventional cytogenetic techniques. We report on a 10-month-old boy with bilateral cleft lip, cleft palate, strabismus, moderate bilateral hydroureteronephrosis, unilateral cryptorchidism, reduced growth, delayed development, and dysmorphic features, including upslanting palpebral fissures, bilateral epicanthic folds, a broad nasal bridge, fifth finger clinodactyly, and hypoplastic fifth fingernails. High-resolution G-banded chromosome analysis showed a 46,XY karyotype. However, FISH analysis with subtelomeric probes revealed a deletion of the 17p telomere without evidence of additional chromosome imbalance. Subtelomeric probe analysis in the parents showed a 17p telomere deletion in his phenotypically normal mother. Array comparative genomic hybridization analysis on the patient and his mother is pending. Telomere deletions have been described in phenotypically normal individuals, emphasizing the importance of differentiating pathogenic telomeric imbalance from benign familial variants or copy number variants in the population. In particular, paternally inherited deletions of the 17p telomere up to 600 kb in size have been described in individuals with no identifiable phenotype (Martin et al. J Med Genet 2002;39:734). The variability between the patient9s and his mother9s phenotype may result from unmasking of a recessive allele(s), modifier genes, environmental influences, or a difference in the size of their respective deletions. Our case provides additional evidence that the distal 17p13 telomeric region may represent an area of the genome in which reduced gene dosage can still be associated with a normal phenotype.

Subtelomeric rearrangements and 22q11.2 deletion syndrome in anomalous growth‐restricted fetuses with normal or balanced G‐banded karyotype

To determine the frequencies of cryptic subtelomeric rearrangements and 22q11.2 deletion in anomalous growth-restricted fetuses with normal or balanced G-banded karyotypes. This was a study of 27 consecutive fetuses at a median gestational age of 26 (range, 19-33) weeks, that had intrauterine growth restriction (IUGR) as well as at least one major structural anomaly, and a normal or balanced G-banded karyotype. The median maternal age was 29 (range, 17-39) years. Fluorescence z in-situ hybridization (FISH) diagnosis of the cultured amniocytes with the probe TUPLE 1, and then the Chromoprobe Multiprobe-T system were used, respectively, to screen for the frequency of 22q11.2 deletion syndrome and subtelomeric rearrangements involving the 41 unique chromosome ends (i.e. excluding the five short arms of acrocentric autosomes (no. 13, 14, 15, 21 and 22)). Those that had suspected deleted subtelomeres were reanalyzed with a specific subtelomeric probe, TelVysion. Of the 27 fetuses, three (11%) were affected with 22q11.2 deletion syndrome and two (7.4%) had subtelomeric deletions (one monosomy 21q22.3, one monosomy 1p36.3). Of the 11 fetuses with congenital heart defects, three (27.3%) had 22q11.2 deletion syndrome and one (9.1%) had monosomy 1p36.3. In the remaining 16 fetuses without congenital heart defects, none had 22q11.2 deletion syndrome. However, one (6.3%) had cryptic rearrangement involving subtelomeres. Prenatal subtelomeric FISH screening is technically feasible using cultured amniocytes. We propose that 22q11.2 deletion syndrome and cryptic subtelomere rearrangements may be important etiologies of fetuses with IUGR and at least one structural anomaly, along with a normal karyotype or one that is balanced by traditional G-banding. Fetuses with congenital heart defects and IUGR should undergo FISH to exclude 22q11.2 deletion syndrome. In fetuses with IUGR and at least one major structural anomaly but without congenital heart defects, screening of subtelomeric rearrangements may contribute to further elucidation of the underlying etiology.

Trisomy 22 as the Sole Abnormality Is an Important Marker for Inversion 16 in Acute Myeloid Leukemia.

Inv(16) has been reported in 10%~12% of acute myeloid leukemia (AML), mostly being associated with the M4Eo subtype, and is associated with a relatively favorable outcome. However, it is a cryptic rearrangement and often difficult to recognize in conventional cytogenetics (CC). Trisomy 22 is an uncommon karyotypic aberration in AML and is often associated with inv(16)(p13q22). In order to explore the value of trisomy 22 in the diagnosis of AML with inv(16), dual-color interphase fluorescence in situ hybridization (FISH) was performed in 19 AML cases with trisomy 22 abnormality. The probe was two-color break apart probe for CBFb with SpectrumRed on the centromeric side and SpectrumGreen on the telomeric side. And the results were compared with that of R-banding CC. CC did not reveal inv(16) in any of the 19 AML with trisomy 22, but FISH analysis showed inv(16) in 11 cases and del(16)(q22) in one case. Among 11 cases with inv(16), 9 were trisomy 22 as the sole abnormality, one was complicated with trisomy 8, and one was del(16)(q22). Four AML patients with trisomy 22 and inv(16) were analyzed by multiplex FISH (M-FISH) which revealed trisomy 22 only. This study further confirmed that trisomy 22 as the sole abnormality can be regarded as an important marker for the inv(16) in AML.

Array-CGH Analysis of T-ALL Patients and Cell Lines.

BackgroundMolecular analysis of T-cell acute lymphoblastic leukemia (T-ALL) has provided evidence that a stepwise alteration of at least four specific pathways is required during transformation of thymocytes to leukemic T-cells. Genetic alterations in hematopoietic precursor cells lead to loss of cell cycle control, impaired differentiation, proliferation and survival advantages, and unlimited self-renewal capacity. These defects include inactivation of CDKN2A (P16) present in 96 % of the patients, deregulated expression of transcription factors, and mutation of NOTCH1 in 56% of patients. However, the molecular lesions leading to the proliferative and survival advantages of T-ALL cells are less well characterized, remaining unknown in 80 % of the T-ALLs.AimsOur aim was to set up a genome-wide analysis of T-ALL in order to detect cryptic deletions and amplifications, with a special focus on the 90 protein tyrosine kinase genes present in the human genome.MethodsWe used the array-CGH (micro-array comparative genomic hybridization) technology with slides containing genomic BAC probes spaced every 1 Mb over the human genome. An additional 480 probes were added covering the genomic locations of each of the 90 protein tyrosine kinases genes. A total of 27 T-ALL cases and 12 cell lines were included in the study.ResultsAn interstitial deletion on chromosome 9p24 directly upstream of JAK2 was identified in one patient. The deletion was confirmed by FISH. Quantitative PCR (qPCR) analyses indicated that the deletion was 700 kb in size including exons 1–4 of JAK2. In two cell lines, deletions affecting ALK and ERBB4 were detected. Molecular analyses to characterize the possible presence of fusion transcripts involving tyrosine kinases are in progress. We did not detected other rearrangements involving tyrosine kinase genes in neither of the 26 other T-ALL cases nor in the 10 cell lines, indicating that cryptic deletions or amplifications involving tyrosine kinase genes are relatively rare in T-ALL.The most frequent aberration was the deletion of CDKN2A (16/27 cases and 9/12 cell lines) ranging from only one clone to almost the complete 9p arm. MYB duplication was found in two cases and 4 cell lines, and was confirmed by qPCR and FISH analysis. Two of the 4 cell lines had overexpression of MYB detected by qPCR, which can interfere with apoptosis enhancing the survival of the cells, as has been previously described. PTEN deletion was present in one case and one cell line. Other unbalanced aberrations of various size were detected: del (2p) in 5/12 cell lines, del (5q) in 1/27 samples and 4/12 cell lines, del(6q) in 3/27 samples and 2/12 cell lines, or del(9p) in 5/27 samples and 4/12 cell lines. Some of these rearrangements were not observed by standard cytogenetics. Strikingly, cell lines had significantly more chromosomal abnormalities than primary T-ALL cases.ConclusionsWe detected a novel cryptic rearrangement of JAK2 in one T-ALL case, and duplication of MYB in two T-ALL cases (2/27; 7.5%) and four cell lines (4/12; 33%). Our results suggest that cryptic deletions or amplifications involving tyrosine kinase genes are relatively rare in T-ALL.

Prenatal detection of subtelomeric rearrangements by multi‐subtelomere FISH in a cohort of fetuses with major malformations

Cryptic unbalanced subtelomeric rearrangements have been identified as an important contributor ( approximately 6%) to the etiology of mental retardation and dysmorphism. Our objective was to study the role of these rearrangements in the development of fetal malformations. Multi-subtelomere FISH was performed on cells from 48 fetuses with major malformations diagnosed by prenatal ultrasound with a normal karyotype at a minimal 400 band resolution. We developed a method of performing multi-subtelomere FISH on a single slide of amniocyte metaphase spreads. We identified five subtelomeric abnormalities: two derivative chromosomes inherited from a parent carrying a balanced translocation, two known polymorphisms, and one novel familial variant. These results show a similar frequency (4%) of clinically significant subtelomeric rearrangements to that found in children with multiple malformations. This study adds to a growing number of reports of cryptic subtelomeric rearrangements associated with congenital malformations and highlights the relevance and technical feasibility of multi-subtelomere FISH screening of prenatal samples.

Cryptic and complex chromosomal rearrangements and the deletion of TP53 gene in a patient with leukemic mantle cell lymphoma

We present a case of leukemic mantle cell lymphoma with cryptic and complex chromosomal rearrangements, including multiple-way translocations involving chromosomes 8q24, 14q11/q32, 17p13.3, 17p13.1, 21q22, and 21q22; a deletion of the long arm of chromosome 10 [del(10)(q24)]; and a deletion of the TP53 gene in addition to t(11;14). We speculate that this series of chromosomal changes may disrupt the IgH gene, activate the c-MYC oncogene, inactivate the p53 tumor suppressor gene, and disrupt other cancer-related genes either within or flanking the chromosomal breakpoints. This combinational effect causes the progression of mantle cell lymphoma.

Cryptic terminal chromosome rearrangements in colorectal carcinoma cell lines detected by subtelomeric FISH analysis

Epithelial tumour karyotypes are often difficult to study by standard cytogenetic methods because of poor chromosome preparation quality and the high complexity of their genomic rearrangements. Subtelomeric fluorescence in situ hybridisation (FISH) has proved to be a useful method for detecting cryptic constitutional chromosomal rearrangements but little is known about its usefulness for tumour cytogenetic analysis. Using a combination of chromosome banding, multicolour karyotyping and subtelomeric FISH, five colorectal cancer cell lines were characterised. The resulting data were compared to results from previous studies by comparative genomic hybridisation and spectral karyotyping or multicolour FISH. Subtelomeric FISH made it possible to resolve several highly complex chromosome rearrangements, many of which had not been detected or were incompletely characterised by the other methods. In particular, previously undetected terminal imbalances were found in the two cell lines not showing microsatellite instability.

Array-based comparative genomic hybridisation identifies high frequency of cryptic chromosomal rearrangements in patients with syndromic autism spectrum disorders

Background: Autism spectrum disorders (ASD) refer to a broader group of neurobiological conditions, pervasive developmental disorders. They are characterised by a symptomatic triad associated with qualitative changes in social interactions,...

Screening of subtelomeric rearrangements in autistic disorder: Identification of a partial trisomy of 13q34 in a patient bearing a 13q;21p translocation

Within the framework of a FISH screening protocol to detect cryptic subtelomeric rearrangements in autistic disorder (AD), a patient bearing three copies of the subtelomeric portion of the q arm of chromosome 13 has been identified. Beside AD, the patient also has severe mental retardation and displays several dysmorphic features. Further FISH analyses revealed that the trisomy was caused by the translocation of a 13q subtelomeric fragment to the acrocentric tip of one chromosome 21 [46,XY.ish der(21) t(13;21) (q34;p13)(D13S1825+)]. Gene dosage experiments carried out with three multiallelic polymorphisms of the subtelomeric region of chromosome 13q showed that the putative length of the triplicate region does not exceed 300 kb about, that is, the distance from telomere to the first normally inherited marker. In addition, gene dosage analysis performed on the derivative chromosome 21, did not reveal loss of the most telomeric protein-encoding genes on 21p. The potential relationship between a postulated increased expression of genes on 13q34 and the complex phenotype in this trisomic patient is discussed.

Temporary blast reduction after immunoglobulin administration for congenital cytomegalovirus infection masking infant leukemia with cryptic MLL rearrangement

Differentiation between reactive bone marrow suppression due to viral infection and early stages of leukemia can be difficult particularly in young infants. We report on a 2-month-old girl presenting with pancytopenia and positive markers for congenital cytomegalovirus (CMV) infection. Definitive diagnosis of coexisting pro-B cell infant leukemia with cryptic MLL rearrangement was delayed by the transient regeneration of normal hematopoiesis and reduction of abnormal blastoid cells in the bone marrow following immunoglobulin administration. Molecular diagnosis could only be established using interphase fluorescence in situ hybridization (FISH) analysis which may be considered as a valuable additional diagnostic tool in similar cases.

Molecular cytogenetic characterization of human papillomavirus16-transformed foreskin keratinocyte cell line 16-MT

Anogenital cancers are closely associated with human papillomavirus (HPV), and HPV-infected individuals, particularly those with high-grade dysplasias, are at increased risk for cervical and anal cancers. Although genomic instability has been documented in HPV-infected keratinocytes, the full spectrum of genetic changes in HPV-associated lesions has not been fully defined. To address this, we examined an HPV16-transformed foreskin keratinocyte cell line, 16-MT, by GTG-banding, spectral karyotyping (SKY), and array comparative genomic hybridization (array CGH); these analyses revealed multiple numerical, complex, and cryptic chromosome rearrangements. Based on GTG-banding, the 16-MT karyotype was interpreted as 78∼83,XXY,+add(1)(p36.3),+3,+4,+5,+5,+7,+8,+i(8)(q10)×2,+10,?der(12),der(13;14)(q10;q10),+15,+16,add(19)(q13.3),+21,+21,−22[cp20]. Multicolor analysis by SKY confirmed and further characterized the anomalies identified by GTG banding. The add(1) was identified as a der(1)(1qter→1q25::1p36.1→1qter), the add(19) as a dup(19), and the der(12) interpreted as a der(11) involving a duplication of chromosome 11 material and rearrangement with chromosome 19. In addition, previously unidentified der(9)t(9;22), der(3)t(3;19), and der(4)t(4;9) were noted. The 16-MT cell line showed losses and gains of DNA due to unbalanced translocations and complex rearrangements of regions containing known tumor suppressor genes. Chromosomal changes in these regions might explain the increased risk of cancer associated with HPV. Also, array CGH detected copy-number gains or amplifications of chromosomes 2, 8, 10, and 11 and deletions of chromosomes 3, 4, 11, and 15. These results provide the basis for the identification of candidate oncogenes responsible for cervical and anal cancer in amplified regions, and for putative tumor suppressor genes in commonly deleted regions like 11q22∼23. Furthermore, these data represent the first full characterization of the HPV-positive cell line 16-MT.

Molecular cytogenetic studies of Xq critical regions in premature ovarian failure patients

Premature ovarian failure (POF) is defined as amenorrhoea for more than 6 months, occurring before the age of 40, with an FSH serum level higher than 40 mIU/ml. Cytogenetically visible rearrangements of the X chromosome are associated with POF. Our hypothesis was that cryptic Xq chromosomal rearrangements could be an important etiological contributor of POF. Ninety POF women were recruited and compared to 20 control women. Peripheral blood samples were collected and metaphase chromosomes were prepared using standard cytogenetic methods. To detect Xq chromosomal micro-rearrangements, fluorescence in situ hybridization (FISH) analysis was performed using a selection of 30 bacterial artificial chromosome (BAC) and P1 artificial chromosome clones, spanning Xq13-q27. We further localized the translocation breakpoints by FISH with additional BAC clones. Chromosomal abnormalities were identified in 8.8% of our 90 patients [one triple X, three large Xq deletions 46,X,del(X)(q22.3), 46,X,del(X)(q21.2) and 46,X,del(X)(q21.32), two balanced X;autosome translocations 46,X,t(X;1) (q21.1;q32) and 46,X,t(X;9)(q21.31;q21.2) and two Robertsonian translocations 45,XX,der(15;22)(q10;q10) and 45,XX,der(14;21)(q10;q10)]. The two Xq translocation breakpoints were among a cluster of repetitive elements without any known genes. FISH analysis did not reveal any Xq chromosomal micro-rearrangement. Karyotyping is definitely helpful in the evaluation of POF patients. No submicroscopic chromosomal rearrangements affecting Xq region were identified. Further analysis using DNA microarrays should help delineate Xq regions involved in POF.

Evaluation of MLPA for the detection of cryptic subtelomeric rearrangements

Chromosomal rearrangements involving the telomeres are implied as a significant cause of idiopathic mental retardation. The most frequently used technique to detect these rearrangements was fluorescent in situ hybridization (FISH), an expensive and labor-intensive technique. One of the most promising alternative techniques is multiplex ligation-dependent probe amplification (MLPA). Here, the authors present the evaluation of a double set of probes (the SALSA P036, P019, and P020 human telomere test kits) on a series of 95 patients and 22 normal controls. Overall, 34 patients had been studied by telomeric FISH and MLPA, which was demonstrated to be a reliable method to detect essentially all subtelomeric rearrangements characterized by FISH. In addition, in these 34 patients, 13 dose imbalances were detected by MLPA, but not by FISH analysis. Overall, 12 alterations were observed only with one of the two sets, and they corresponded to polymorphic variants, as they were inherited from healthy parents or also appear in normal controls. The remaining 61 patients were initially studied with SALSA P036, and any putative dose alteration was confirmed with the two other kits and FISH. In the whole series, the authors found 9 dose imbalances evidenced with 2 MLPA kits and confirmed by FISH, representing 10% of patients with subtelomeric rearrangements. On the other hand, one small duplication at 14q11 may be clinically relevant as it appears de novo in one patient. In conclusion, MLPA can be considered a quick, sensitive, cost-effective, and easy method to screen for subtelomeric rearrangements, but any finding based in the testing of one probe should be confirmed by other sources.

2nd International Meeting on Cryptic Chromosomal Rearrangements in Mental Retardation and Autism

2nd International Meeting on Cryptic Chromosomal Rearrangements in Mental Retardation and Autism

Molecular cytogenetic study of 126 unselected T-ALL cases reveals high incidence of TCRβ locus rearrangements and putative new T-cell oncogenes

Chromosomal aberrations of T-cell receptor (TCR) gene loci often involve the TCRalphadelta (14q11) locus and affect various known T-cell oncogenes. A systematic fluorescent in situ hybridization (FISH) screening for the detection of chromosomal aberrations involving the TCR loci, TCRalphadelta (14q11), TCRbeta (7q34) and TCRgamma (7p14), has not been conducted so far. Therefore, we initiated a screening of 126 T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma cases and 19 T-ALL cell lines using FISH break-apart assays for the different TCR loci. Genomic rearrangements of the TCRbeta locus were detected in 24/126 cases (19%), most of which (58.3%) were not detected upon banding analysis. Breakpoints in the TCRalphadelta locus were detected in 22/126 cases (17.4%), whereas standard cytogenetics only detected 14 of these 22 cases. Cryptic TCRalphadelta/TCRbeta chromosome aberrations were thus observed in 22 of 126 cases (17.4%). Some of these chromosome aberrations target new putative T-cell oncogenes at chromosome 11q24, 20p12 and 6q22. Five patients and one cell line carried chromosomal rearrangements affecting both TCRbeta and TCRalphadelta loci. In conclusion, this study presents the first inventory of chromosomal rearrangements of TCR loci in T-ALL, revealing an unexpected high number of cryptic chromosomal rearrangements of the TCRbeta locus and further broadening the spectrum of genes putatively implicated in T-cell oncogenesis.

Dermatofibrosarcoma protuberans: report of a case with a variant ring chromosome and metastases following pregnancy

The most frequent molecular abnormality observed in dermatofibrosarcoma protuberans (DFSP) is the formation of a supernumerary ring chromosome or translocation resulting in fusion of the gene encoding the alpha-chain of type 1 collagen, COL1A1 from 17q22, to the platelet-derived growth factor beta-chain, PDGFB gene from 22q13. Rare cases documenting variant ring or marker chromosomes involving regions other than 17q22 and 22q13 have been reported. Further analysis in three of these cases demonstrated the presence of the COL1A1 and PDGFB genes. We report a further case of DFSP with a rare variant ring chromosome. The tumor appeared to undergo accelerated growth during pregnancy, then metastasized following pregnancy. We describe the clinical, histological, immunohistochemical, and cytogenetic features. The metastatic tumor showed a variant r(17;?) chromosome. A locus-specific probe was required to demonstrate presence of the PDGFB gene within the ring, indicating cryptic molecular rearrangement between chromosomes 17 and 22, and recombination with an unknown chromosome. Cryptic rearrangement of chromosomes 17 and 22 should be suspected in variant ring chromosomes and translocations. Pregnancy may contribute to accelerated growth of DFSP, and delay in surgical resection should be avoided.