Mre11-Sae2 and RPA Collaborate to Prevent Palindromic Gene Amplification.

Foldback priming at DNA double-stranded breaks is one mechanism proposed to initiate palindromic gene amplification, a common feature of cancer cells. Here, we show that small (5-9 bp) inverted repeats drive the formation of large palindromic duplications, the major class of chromosomal rearrangements recovered from yeast cells lacking Sae2 or the Mre11 nuclease. RPA dysfunction increased the frequency of palindromic duplications in Sae2 or Mre11 nuclease-deficient cells by ∼ 1,000-fold, consistent with intra-strand annealing to create a hairpin-capped chromosome that is subsequently replicated to form a dicentric isochromosome. The palindromic duplications were frequently associated with duplication of a second chromosome region bounded by a repeated sequence and a telomere, suggesting the dicentric chromosome breaks and repairs by recombination between dispersed repeats to acquire a telomere. We propose secondary structures within single-stranded DNA are potent instigators of genome instability, and RPA and Mre11-Sae2 play important roles in preventing their formation and propagation, respectively.

Myelodysplastic/myeloproliferative neoplasms-unclassifiable with isolated isochromosome 17q represents a distinct clinico-biologic subset: a multi-institutional collaborative study from the Bone Marrow Pathology Group.

Classification of myeloid neoplasms with isolated isochromosome i(17q) [17p deletion with inherent monoallelic TP53 loss plus 17q duplication] is controversial. Most cases fall within the WHO unclassifiable myelodysplastic/myeloproliferative neoplasms (MDS/MPN-U) category. The uniformly dismal outcomes warrant better understanding of this entity. We undertook a multi-institutional retrospective study of 92 adult MDS/MPN-U cases from eight institutions. Twenty-nine (32%) patients had isolated i(17q) [MDS/MPN-i(17q)]. Compared to MDS/MPN without i(17q), MDS/MPN-i(17q) patients were significantly younger, had lower platelet and absolute neutrophil counts, and higher frequency of splenomegaly and circulating blasts. MDS/MPN-i(17q) cases showed frequent bilobed neutrophils (75% vs. 23%; P = 0.03), hypolobated megakaryocytes (62% vs. 20%; P = 0.06), and a higher frequency of SETBP1 (69% vs. 5%; P = 0.002) and SRSF2 (63% vs. 5%; P = 0.006) mutations that were frequently co-existent (44% vs. 0%; P = 0.01). TP53 mutations were rare. The mutation profile of MDS/MPN-U-i(17q) was similar to other myeloid neoplasms with i(17q) including atypical chronic myeloid leukemia, chronic myelomonocytic leukemia, myelodysplastic/myeloproliferative neoplasm with ring sideroblasts and thrombocytosis, myelodysplastic syndrome and acute myeloid leukemia, with frequent concomitant SETBP1/SRSF2 mutations observed across all the diagnostic entities. Over a median follow-up of 52 months, patients with MDS/MPN-i(17q) showed a shorter median overall survival (11 vs. 28 months; P < 0.001). The presence of i(17q) retained independent poor prognostic value in multivariable Cox-regression analysis [HR 3.686 (1.17-11.6); P = 0.026] along with splenomegaly. We suggest that MDS/MPN-i(17q) warrants recognition as a distinct subtype within the MDS/MPN-U category based on its unique clinico-biologic features and uniformly poor prognosis.

Molecular Cytogenetic Classification of Down Syndrome and Screening of Somatic Aneuploidy in Mothers.

Down Syndrome (DS) caused by trisomy 21 results in various congenital and developmental complications in children. It is crucial to cytogenetically diagnose the DS cases early for their proper health management and to reduce the risk of further DS childbirths in mothers. In this study, we performed a cytogenetic analysis of 436 suspected DS cases using karyotyping and fluorescent in situ hybridization. We detected free trisomies (95.3%), robertsonian translocations (2.4%), isochromosomes (0.6%), and mosaics (1.2%). We observed a slightly higher incidence of DS childbirth in younger mothers compared to mothers with advanced age. We compared the somatic aneuploidy in peripheral blood of mothers having DS children (MDS) and control mothers (CM) to identify biomarkers for predicting the risk for DS childbirths. No significant difference was observed. After induced demethylation in peripheral blood cells, we did not observe a significant difference in the frequency of aneuploidy between MDS and CM. In conclusion, free trisomy 21 is the most common type of chromosomal abnormality in DS. A small number of DS cases have translocations and mosaicism of chromosome 21. Additionally, somatic aneuploidy in the peripheral blood from the mother is not an effective marker to predict DS childbirths.

The detection of isochromosome i(12p) in malignant germ cell tumours and tumours with somatic malignant transformation by the use of quantitative real-time polymerase chain reaction.

AIMS: Malignant germ cell tumours (GCTs) of the testis are rare neoplasms, but the most common solid malignancies in young men. World Health Organization guidelines divide GCTs into five types, for which numerous immunohistochemical markers allow exact histological subtyping in the majority of cases. In contrast, a germ cell origin is often hard to prove in metastatic GCTs that have developed so-called somatic malignant transformation. A high percentage, up to 89%, of GCTs are characterised by the appearance of isochromosome 12p [i(12p)]. Fluorescence in-situ hybridisation has been the most common diagnostic method for the detection of i(12p) so far, but has the disadvantages of being time-consuming, demanding, and not being a stand-alone method. The aim of the present study was to establish a quantitative real-time polymerase chain reaction assay as an independent method for detecting i(12p) and regional amplifications of the short arm of chromosome 12 by using DNA extracted from formalin-fixed paraffin-embedded tissue. METHODS AND RESULTS: A cut-off value to distinguish between the presence and absence of i(12p) was established in a control set consisting of 36 tumour-free samples. In a training set of 149 GCT samples, i(12p) was detectable in 133 tumours (89%), but not in 16 tumours (11%). In a test set containing 27 primary and metastatic GCTs, all 16 tumours with metastatic spread and/or somatic malignant transformation were successfully identified by the detection of i(12p). CONCLUSION: In summary, the qPCR assay presented here can help to identify, further characterise and assign a large proportion of histologically inconclusive malignancies to a GCT origin.

Isochromosome 11q is Associated with Unique Characteristics and Poor Prognosis in Patients with Acute Myeloid Leukemia.

BACKGROUND: Isochromosome 11q in patients with acute myeloid leukemia is rarely reported, and little is known about its main features. METHODS: The presence of isochromosome 11q was identified in four patients (three adults and one child) from screening 441 patients with an acute myeloid leukemia diagnosis between 2009 and 2018 by using R-banding and fluorescence in situ hybridization. RESULTS: The child, patient 1 with unreported isochromosome (partial 11q isochromosome), accompanied with t(1;11) translocation, initially achieved remission after receiving chemotherapy. However, 4 months later this patient experienced a relapse. While multiple treatments were tried, it had no effect and the patient survived for 16 months. The remaining patients with isochromosome 11q exhibited numerical/structural chromosomal abnormal-ities involving myelodysplastic syndrome-related chromosomes 5, 7, 8, and 20. In patients 2 and 3, we found a derivative chromosome 21. Patient 3 was newly diagnosed with acute myeloid leukemia and was treated with many chemotherapy protocols, unfortunately with no effect. The patient then received traditional Chinese medicine and survived for 10 months, although she still has not achieved complete remission. Patients 2 and 4 received chemotherapy but experienced rapid disease progression and died within 2 months. CONCLUSIONS: In summary, patients with isochromosome 11q/partial 11q isochromosome have a poorer prognosis, especially for isochromosome 11q. Furthermore, these chromosome aberrations may be risk factors for the presence of isochromosome 11q or myelodysplastic syndrome-related genes, both of them may be associated with a failure to respond to treatment and poor outcomes. Hence, these discoveries may lay a foundation to study mechanisms and explore treatments.

Low-Level Trisomy 14 Mosaicism: A Carrier of an Isochromosome 14 and a Supernumerary Marker Chromosome 14.

Trisomy 14 (T14) mosaicism is a rare chromosomal condition characterised by various clinical features, including developmental delay, growth impairment, and dysmorphism. Here, we report on a 12-year-old female referred for cytogenetic analysis due to short stature. Standard GTG-banding analysis on the patient's peripheral blood revealed mosaic Τ14 in the form of an i(14)(q10) in 3% of cells. Furthermore, a small supernumerary marker chromosome (sSMC) had been detected in the first trimester of pregnancy in chorionic villus sampling. A skin biopsy in the patient revealed the presence of a metacentric sSMC in 100% of cells. Cytogenetic and FISH studies showed that it was a de novo metacentric bisatellited sSMC derived from chromosomes 14 or 22. Oligonucleotide array-CGH using skin cells revealed no copy number variations. Studies for uniparental disomy 14 by microsatellite analysis confirmed biparental inheritance. To the best of our knowledge, this is the second report of a patient with 2 abnormal cell lines involving chromosome 14 in different tissues, one with mosaic T14 in the form of i(14)(q10) and one with an sSMC derived from chromosome 14, present in blood and skin, respectively. A rare mechanism of trisomy rescue events is proposed to explain the presence of the different cell lines in the tissues examined. This case highlights the importance of providing the cytogenetics laboratory with adequate clinical data to test for low mosaicism and analyse different tissues if necessary, thus contributing to the suitable clinical management of the patient.

Misdivision of Telocentrics and Isochromosomes in Wheat.

For normal transition through meiosis, chromosomes rely on pairing with their homologues. Chromosomes which fail to pair, univalents, behave irregularly and may undergo various types of breakage across their centromeres. Here, we analyzed the meiotic behavior of misdivision products themselves: isochromosomes and telocentrics in wheat. Both types of chromosomes behaved in the same fashion as standard 2-armed chromosomes. The 2 most frequent scenarios were separation of sister chromatids in anaphase I or monopolar/bipolar attachment of the univalent to the spindle apparatus with unseparated chromatids. Misdivision was rare, and its frequency appeared directly related to the size of the centromere. The previously deduced relationship between misdivision frequency and chromosome size was likely erroneous and can be explained by a general relationship between chromosome length and the size of its centromere. Pairing of identical arms in isochromosomes did not protect them from misdivision. It is not chiasmate pairing that protects from misdivision but mechanistic issues that arise through that pairing.

Clinicopathologic Features and Chromosome 12p Status of Pediatric Sacrococcygeal Teratomas: A Multi-institutional Analysis.

Chromosome 12p gains are typically present in postpubertal male patients with testicular malignant germ cell tumors, including most teratomas, and absent in pure ovarian teratomas, both mature and immature. We sought to evaluate the clinicopathologic features and chromosome 12p status of pediatric patients with sacrococcygeal teratomas (SCTs) using the institutional databases of 2 tertiary medical centers. Seven mature teratomas (3 pure, 2 with yolk sac tumor, 1 with medulloepithelioma, and 1 with ependymoma) and 3 immature teratomas (2 pure: grade 2 and grade 3 and 1 mixed: grade 3 with yolk sac tumor) were identified. All patients underwent surgery and 2 received adjuvant chemotherapy. Fluorescence in situ hybridization analysis was performed to elucidate chromosome 12p gains, including isochromosome 12p. All 10 tumors analyzed lacked 12p gains regardless of the components. No patient had evidence of disease at their most recent interval follow-up (mean: 30, range: 7-91 months), irrespective of margin status or of receiving chemotherapy. Overall, our study suggests an absence of chromosome 12p abnormalities in clinically nonaggressive SCTs. Additional data are required to confirm these findings before definitive patient care recommendations can be made.

A rare case in literature: Isochromosome Xq in Klinefelter syndrome.

BACKGROUND: Klinefelter syndrome(KS), affecting 1 in 500-1,000 newborn males, is the most common sex chromosome aneuploidy among males with primary hypogonadism. Isochromosome Xq on the other hand is a rare variant of Klinefelter syndrome, accounting approximately 0.3% of all KS and associated with normal height and androgenisation compared to classical KS. Here, we present a case of isochromosome Xq variant of KS with similar clinical and cytogenetic findings with the few cases reported before. MATERIALS AND METHODS: A 25-year-old male patient referred to our clinic with complaint of infertility. He is the son of a consanguineous couple who are first cousins and there was no family history of reproductive difficulty. In physical examination synophrys, prominent ear and small testicles noted. The patient's spermiogram showed azoospermia and scrotal USG revealed testicular atrophy. RESULTS: Karyotype analysis using G-banding resulted as 47,X,i(X)(q10),Y, and STR analysis showed no deletion in AZF and SRY loci of interest. CONCLUSION: Although several isochromosome Xq variant of KS cases can be found in literature, it is our duty to emphasise the importance of karyotyping for patients with reproductive difficulty who may not have all features of classical Klinefelter syndrome.

Cytogenetic analysis of patients with primary amenorrhea in Eastern India.

Amenorrhea is the absence of menstruation in women of reproductive age. Previous reports suggest that chromosomal abnormality is the second most common cause of amenorrhoea. Early referral for cytogenetic evaluation is recommended for the identification of underlying chromosomal aberrations in amenorrhoea patients. This was an observational study which was conducted in Vivekananda Institute of Medical sciences, Kolkata, during January 2013-December 2015. This study aims to estimate the frequency and types of chromosomal abnormalities in primary amenorrhoea (PA) patients in Eastern India and correlate their hormonal profile with chromosomal reports. Clinical features of 150 patients were recorded with clinical expertise. Peripheral venous blood was taken following informed consent, followed by karyotyping for chromosomal analysis. Results revealed 76.1% of PA with normal female karyotype (46, XX) and 23.9% with different abnormal karyotypes. Among the abnormal karyotype constituents, 50% numerical abnormalities, most frequent being Turner syndrome, pure (n = 12, 8%) and mosaic (n = 5, 3.3%). Three cases (2%) showed male (XY) karyotype. The other cases showed X structural abnormalities. This study emphasises the need for cytogenetic analysis as integral part of the diagnostic protocol in case of PA for precise identification of chromosomal abnormalities and for appropriate management and counselling of these patients.

The Turner syndrome life course project: Karyotype-phenotype analyses across the lifespan.

INTRODUCTION: Turner syndrome (TS) is associated with a variety of morbidities affecting nearly every body system, some of which increase in prevalence in adult life. The severity of clinical features in TS is roughly in parallel with the magnitude of the deficit of X-chromosome material. The aim of this study was to extend the established karyotype-phenotype relationships using data from a large adult cohort. MATERIALS AND METHODS: Karyotypes were available in 656 women with TS. 611 of whom could be classified into five major groups within the cohort: 45,X; 45,X mosaicism (45,X/46,XX); isochromosome X (isochromosome Xq); mosaicism 45,X/46,XY and ring X. Continuous variables such as blood pressure and biochemical markers from clinic data were binarised allocating those in the upper quartile to represent at-risk individuals. With the exception of bone mineral density T-score for which the lower quartile was allocated as at risk. For comorbidities, initiation of formal treatment was recorded. RESULTS: 45,X/46,XX had considerably lower frequency of comorbidities compared to 45,X. The isochromosome group experienced similar outcomes to 45,X. Novel associations were found between the XY mosaic karyotype group and a decreased prevalence of thyroid disease and severe hearing loss. A previously unreported increased incidence of metabolic syndrome was noted within the ring chromosome subgroup. CONCLUSIONS: Karyotype may play an important factor against stratifying risk of comorbidity in TS and should be taken into consideration when managing adults with TS. Further investigations of the isochromosome (Xq) and ring groups are necessary to further clarify their associations with comorbidities.

Evidence of a dual histogenetic pathway of sacrococcygeal teratomas.

AIMS: Sacrococcygeal teratomas are rare tumours that occur most frequently in neonates, although adult cases also occur. The molecular pathogenesis of these tumours and their long-term prognosis is uncertain. We investigated the i(12p) status of a large number of primary sacrococcygeal teratomas in both children and adults, including cases with malignant germ cell tumour elements. METHODS AND RESULTS: Fifty-four sacrococcygeal teratoma specimens from 52 patients were identified, and available follow-up information was obtained. Fluorescence in-situ hybridization analysis was performed to identify isochromosome 12p [i(12p)] abnormalities on paraffin blocks of the tumours. Among the 48 paediatric patients, there were 44 teratomas and four tumours with combined teratoma and yolk sac tumour (one of whom also had primitive neuroectodermal tumour). The teratomas included 37 mature teratomas and 11 immature teratomas (four grade 1, two grade 2, and five grade 3). The 44 teratomas lacking a yolk sac tumour component were all negative for i(12p). The four tumours with a yolk sac tumour component were all positive for i(12p). The four adult cases all lacked non-teratomatous germ cell tumour components, immature elements, and i(12p). Follow-up information was available for 32 patients. Two patients with teratoma had recurrence, but were alive with no evidence of disease after long-term follow-up. One patient with combined teratoma and yolk sac tumour had recurrence 7 months after resection. The other patients were alive with no evidence of disease at last follow-up. CONCLUSIONS: Our data suggest that paediatric sacrococcygeal teratomas should be considered as two distinct groups with divergent histogenetic pathways. The prognosis of these tumours is excellent, despite rare recurrence.

Prader-Willi syndrome and Tay-Sachs disease in association with mixed maternal uniparental isodisomy and heterodisomy 15 in a girl who also had isochromosome Xq.

Malsegregation of chromosomes during reproduction can result in uniparental disomy when associated with trisomy rescue, monosomy rescue or gamete complementation. Pathogenicity stemming from uniparental disomy in liveborns results from imprinting disorders or autozygosity for autosomal recessive disorders. We report on a girl with Prader-Willi syndrome and Tay-Sachs disease resulting from maternal uniparental disomy of chromosome 15. The child also had an isochromosome Xq. To further characterize the etiology of the aberrant chromosome 15 and the isochromosome Xq, SNP loci from both chromosomes were assessed in the proband and parents, and genome-wide DNA methylation analysis was performed. SNP and DNA methylation analysis confirmed maternal uniparental heterodisomy around the Prader-Willi locus, while the region around the HEXA locus showed maternal uniparental isodisomy. This result is consistent with trisomy rescue of a maternal meiosis l error in a chromosome 15 with two meiotic recombinations. SNP analysis of the X chromosomes is consistent with a maternal origin for the isochromosome.

Supernumerary isochromosome 1, idic(1)(p12), leading to tetrasomy 1q in Burkitt lymphoma.

Burkitt lymphoma (BL) is an aggressive mature B-cell neoplasm. The cytogenetic hallmark are MYC-involving translocations, most frequently as t(8;14)(q24;q32). Additional cytogenetic abnormalities are seen in the majority of cases. The most frequent additional aberration involves the long arm of chromosome 1, either as partial or complete trisomy 1q. A very rare additional aberration is a supernumerary isochromosome 1q, i(1)(q10), resulting in tetrasomy 1q. The biological significance of this aberration is unclear. We present a highly aggressive case of BL in a child with immature B-cell immunophenotype (IP) and supernumerary i(1)(q10). Diagnostic karyotyping showed 47,XY,+i(1)(q10),t(8;14)(q24;q32)[2]/47,idem,del(15)(q24)[21]/46,XY[2]. aCGH analysis detected a gain of 1p12qter and a loss of 15q22q25. FISH analysis confirmed the isodicentric chromosome 1, which has not previously been reported in BL. In the literature, supernumerary i(1)(q10) was found in 11 cases of which >80% presented with immature B-cell IP and >60% relapsed or died. Tetrasomy 1q resulting from supernumerary idic(1)(p12) or i(1)(q10) is a rare genetic event in BL and probably associated with immature B-cell IP. We propose that high amplification of genes on chromosome 1p12qter may contribute to the BL IP and disease progression.

Molecular features of hepatosplenic T-cell lymphoma unravels potential novel therapeutic targets.

The pathogenesis of hepatosplenic T-cell lymphoma (HSTL), a rare entity mostly derived from γδ T cells and usually with a fatal outcome, remains largely unknown. In this study, HSTL samples (7γδ and 2αß) and the DERL2 HSTL cell line were subjected to combined gene-expression profiling and array-based comparative genomic hybridization. Compared with other T-cell lymphomas, HSTL had a distinct molecular signature irrespective of TCR cell lineage. Compared with peripheral T-cell lymphoma, not otherwise specified and normal γδ T cells, HSTL overexpressed genes encoding NK-cell-associated molecules, oncogenes (FOS and VAV3), the sphingosine-1-phosphatase receptor 5 involved in cell trafficking, and the tyrosine kinase SYK, whereas the tumor-suppressor gene AIM1 (absent in melanoma 1) was among the most down-expressed. We found highly methylated CpG islands of AIM1 in DERL2 cells, and decitabine treatment induced a significant increase in AIM1 transcripts. Syk was present in HSTL cells and DERL2 cells contained phosphorylated Syk and were sensitive to a Syk inhibitor in vitro. Genomic profiles confirmed recurrent isochromosome 7q (n = 6/9) without alterations at the SYK and AIM1 loci. Our results identify a distinct molecular signature for HSTL and highlight oncogenic pathways that offer rationale for exploring new therapeutic options such as Syk inhibitors and demethylating agents.

Prenatal diagnosis of isochromosome 21p and isochromosome 21q in a fetus with Down syndrome.

The aim of this study was to present the first case with Down syndrome in conjunction with de novo isochromosomes of both short and long arm of the chromosome 21. Cytogenetics, molecular cytogenetics and molecular genetic analysis were performed on chorionic villus sampling at 12 weeks of gestation of a 42-years-old pregnant woman. According to cytogenetics, molecular cytogenetics and molecular genetic analysis the karyotype was designated as: 47,XY,i(21) (qter --> q10::q10 --> qter),+i(21) (pter --> p10::p10 --> 10pter).ish i(21)(qter --> q10::q10 --> qter)(CEP13/21+,WCP21+),+i(21) (pter --> p10::p10 --> pter)(CEP13/21+,WCP21+). Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR) analysis revealed that isochromosome 21q was maternal in origin. After the detailed genetic counseling, the family decided termination of the pregnancy. This is the first report of co-existence of an isochromosome 21p and an isochromosome 21q in a case with Down syndrome. Our case shows the importance of the molecular cytogenetics and molecular genetic analysis in cases with isochromosomes of the acrocentric chromosomes and supernumerary marker chromosomes regarding to highlight of the formation mechanisms of co-existence of these two rearrangements.

FoSTeS, MMBIR and NAHR at the human proximal Xp region and the mechanisms of human Xq isochromosome formation.

The recently described DNA replication-based mechanisms of fork stalling and template switching (FoSTeS) and microhomology-mediated break-induced replication (MMBIR) were previously shown to catalyze complex exonic, genic and genomic rearrangements. By analyzing a large number of isochromosomes of the long arm of chromosome X (i(Xq)), using whole-genome tiling path array comparative genomic hybridization (aCGH), ultra-high resolution targeted aCGH and sequencing, we provide evidence that the FoSTeS and MMBIR mechanisms can generate large-scale gross chromosomal rearrangements leading to the deletion and duplication of entire chromosome arms, thus suggesting an important role for DNA replication-based mechanisms in both the development of genomic disorders and cancer. Furthermore, we elucidate the mechanisms of dicentric i(Xq) (idic(Xq)) formation and show that most idic(Xq) chromosomes result from non-allelic homologous recombination between palindromic low copy repeats and highly homologous palindromic LINE elements. We also show that non-recurrent-breakpoint idic(Xq) chromosomes have microhomology-associated breakpoint junctions and are likely catalyzed by microhomology-mediated replication-dependent recombination mechanisms such as FoSTeS and MMBIR. Finally, we stress the role of the proximal Xp region as a chromosomal rearrangement hotspot.