A 2.09 Mb fragment translocation on chromosome 6 causes abnormalities during meiosis and leads to less seed watermelon

Seedlessness is a valuable agronomic trait in watermelon (Citrullus lanatus) breeding. Conventional less seed watermelons are mainly triploid, which has many disadvantages due to unbalanced genome content. Less seed watermelon can be achieved at the diploid level when certain reproductive genes are mutated or by chromosome translocation, which leads to defects during meiosis. However, the formation mechanism of diploid less seed watermelons remains largely unknown. Here, we identified a spontaneous mutant line, watermelon line “148”, which can set seeds normally when self-pollinated. A total of 148 × JM F1 hybrid plants exhibited seed number reductions to 50.3% and 47.3% of those of the two parental lines, respectively, which are considered to be less seed. Examination of pollen viability and hybridization experiments revealed that F1 hybrids produce semisterile pollen and ovules. Further cytological observations indicated that semisterility was a result of a reciprocal translocation of chromosomes, which exhibited one quadrivalent ring of four chromosomes at prometaphase I during meiosis. RT-qPCR analysis indirectly confirmed that the semisterile phenotype is caused by chromosome translocation rather than disruption of specific meiotic gene expression. F2 population genetic analysis indicated that the “148” watermelon line is a homozygous translocation and that the less seed phenotype of the F1 hybrid is prompted by one chromosome fragment translocation. The translocated fragment was further fine mapped to a 2.09 Mb region on chromosome 6 by whole-genome resequencing and genetic map cloning procedures. Our work revealed that a 2.09 Mb chromosome fragment translocation on chromosome 6, causing meiotic defects at metaphase I during meiosis, leads to diploid less seed watermelon. Our findings provide a new promising method for less seed watermelon breeding at the diploid level, as well as a fragment size reference for breeding less seed watermelon through artificially induced chromosome translocation.

Marfan Syndrome Caused by Disruption of the FBN1 Gene due to A Reciprocal Chromosome Translocation

Marfan syndrome (MFS) is a hereditary connective tissue disease caused by heterozygous mutations in the fibrillin-1 gene (FBN1) located on chromosome 15q21.1. A complex chromosomal rearrangement leading to MFS has only been reported in one case so far. We report on a mother and daughter with marfanoid habitus and no pathogenic variant in the FBN1 gene after next generation sequencing (NGS) analysis, both showing a cytogenetically reciprocal balanced translocation between chromosomes 2 and 15. By means of fluorescence in situ hybridization of Bacterial artificial chromosome (BAC) clones from the breakpoint area on chromosome 15 the breakpoint was narrowed down to a region of approximately 110 kb in FBN1. With the help of optical genome mapping (OGM), the translocation breakpoints were further refined on chromosomes 2 and 15. Sequencing of the regions affected by the translocation identified the breakpoint of chromosome 2 as well as the breakpoint of chromosome 15 in the FBN1 gene leading to its disruption. To our knowledge, this is the first report of patients with typical clinical features of MFS showing a cytogenetically reciprocal translocation involving the FBN1 gene. Our case highlights the importance of structural genome variants as an underlying cause of monogenic diseases and the useful clinical application of OGM in the elucidation of structural variants.

Highly precise breakpoint detection of chromosome balanced translocation in a Chronic Myelogenous Leukemia patient

Chronic Myelogenous Leukemia (CML) has a special phenomenon of chromosome translocation, which is called Philadelphia chromosome translocation. However, the detailed connection of this structure is troublesome and expensive to be identified. Low-coverage whole genome sequencing (LCWGS) could not only detect the chromosomal translocation which does not be known in advance, but also provide the breakpoint candidate small region (with an accuracy of ±200 bases). Importantly, the sequencing cost of LCWGS is about US$300. Then, with the Sanger DNA sequencing, the precise breakpoint can be determined at a single base level. In our project, with LCWGS, BCR and ABL1 are successfully identified and were disrupted at chr22:23,632,356 and chr9:133,590,450, respectively. Due to the reconnection after chromosome breakage, classical fusion gene (BCR-ABL1) was found in bone marrow and peripheral blood. The precise breakpoints were helpful to study the pathogenic mechanism of CML and could better guide the classification of CML subtypes. This LCWGS method is universal and can be used to detect all diseases related to chromosome variation, such as solid tumors, liquid tumors and birth defects.

Ku70 is Associated with the Frequency of Chromosome Translocation in Human Lymphocytes After Radiation and T-Cell Acute Lymphoblastic Leukemia

Background: Chromosome translocation is one of the most common chromosomal causes to T-cell acute lymphoblastic leukemia (T-ALL). Ku70 is one of the key factors of error-prone DNA repair that may end in translocation. So far, the direct correlation between Ku70 and translocation has not been assessed. Our study aimed to investigate the association of Ku70 and translocation in human lymphocytes after radiation and T-ALL. Methods: Peripheral blood lymphocytes (PBLs) from volunteers and human lymphocyte cell line AHH-1were irradiated with X-rays to form chromosome translocations. The frequency of translocation was detected by fluorescence in situ hybridization (FISH), meanwhile, the expression of Ku70 was also detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot. Furthermore, Ku70 interference, overexpression and chemical inhibition were used in AHH-1 cell lines to confirm the correlation. Finally, we detected the expression of Ku70 in T-ALL samples with or without translocation. Results: Expression of Ku70 and frequencies of translocation were both significantly increased in PBLs after irradiated by X-rays, and a positive correlation between the expression (both mRNA and protein level) of Ku70 and the frequency of translocation was detected (r = 0.4877, P = 0.004; r = 0.3038, P = 0.0358 respectively). Moreover, Ku70 interference decreased the frequency of translocations while the frequency of translocations was not significantly affected after Ku70 overexpression. The expression of Ku70 and frequencies of translocation were both significantly increased in cells after irradiated combined with chemical inhibition (P <0.01). The protein level and mRNA level of Ku70 in T-ALL with translocation were significantly higher than T-ALL with normal karyotype (P = 0.009, P = 0.049 respectively). Conclusions: Ku70 is closely associated with the frequency of chromosome translocation in human lymphocytes after radiation and T-ALL. Ku70 might be a radiation damage biomarker and a potential tumor therapy target.

Highly Conservative Pattern of Sex Chromosome Synapsis and Recombination in Neognathae Birds

We analyzed the synapsis and recombination between Z and W chromosomes in the oocytes of nine neognath species: domestic chicken Gallus gallus domesticus, grey goose Anser anser, black tern Chlidonias niger, common tern Sterna hirundo, pale martin Riparia diluta, barn swallow Hirundo rustica, European pied flycatcher Ficedula hypoleuca, great tit Parus major and white wagtail Motacilla alba using immunolocalization of SYCP3, the main protein of the lateral elements of the synaptonemal complex, and MLH1, the mismatch repair protein marking mature recombination nodules. In all species examined, homologous synapsis occurs in a short region of variable size at the ends of Z and W chromosomes, where a single recombination nodule is located. The remaining parts of the sex chromosomes undergo synaptic adjustment and synapse non-homologously. In 25% of ZW bivalents of white wagtail, synapsis and recombination also occur at the secondary pairing region, which probably resulted from autosome−sex chromosome translocation. Using FISH with a paint probe specific to the germline-restricted chromosome (GRC) of the pale martin on the oocytes of the pale martin, barn swallow and great tit, we showed that both maternally inherited songbird chromosomes (GRC and W) share common sequences.

Hyperdiploid Multiple Myeloma with Novel Complex Structural Chromosome Abnormalities Associated with Poor Prognosis : A Rare Case Report

Hyperdiploid multiple myeloma (MM) is associated with better prognosis and non-hyperdiploid subtype is associated with variable to adverse prognosis based on the nature of karyotype abnormality. Rarely exceptions to this hyperdiploid and non-hyperdiploid divisions do exist in a minority. We report an adult male MM patient who showed hyperdiploid karyotype with few novel complex abnormalities and who showed poor clinical outcome. Conventional cytogenetic analysis carried out in 22 GTG banded metaphases showed 53,Y,der(X)t(X;22)(q27;q11.2),+3,+5,+6,+9,+11,+15,der(17)ins(17;1;3)(q11.2;?;?),der(17)ins(17;1;3)(q11.2;?;?),+19,-22,+mar karyotype pattern in 15 metaphases whereas 7 metaphases showed 46,XY karyotype pattern. Interphase FISH revealed biallelic del(13q14) and del(17p13) but no translocations involving the 14q32 region. Through Spectral karyotyping FISH, the origin of complex abnormalities involving der(17) chromosome, translocation t(X;22), and marker chromosome could be clearly delineated. Although the present case showed hyperdiploid karyotype, he showed an adverse prognosis probably due to the co-existence of high risk and complex abnormalities and expired 5 months after initial diagnosis despite standard treatment given.

Novel TNC-PDGFD fusion in fibrosarcomatous dermatofibrosarcoma protuberans: a case report

Background Dermatofibrosarcoma protuberans (DFSP) is a superficial fibroblastic tumor characterized by high rate of local recurrence and low metastatic potential. Fibrosarcomatous transformation can rarely arise in DFSP either de novo or as recurrent, which represents a form of tumor progression and carries an increased risk of metastasis over classic DFSP. Cytogenetically, DFSP is characterized by a recurrent unbalanced chromosome translocation t (17;22)(q22;q13), leading to the formation of COL1A1-PDGFB fusion transcript that is present in more than 90% of cases. Alternative fusions involving the PDGFD with partners of COL6A3 or EMILIN2 have recently been documented in less than 2% of cases. Herein, we report a DFSP with fibrosarcomtous morphology harboring a novel TNC-PDGFD fusion. Case presentation A 54-year-old female presented with a slowly growing mass in the right thigh. Excision demonstrated a 2-cm ovoid, well-circumscribed, gray-white, mass. Microscopic examination revealed a partially encapsulated subcutaneous nodule without dermal connection. The neoplasm was composed of cellular and fairly uniform spindle cells with brisk mitoses, arranged in elongated fascicles and herringbone patterns, with focal collagenized stroma. The neoplastic cells were positive for CD34 and smooth muscle actin. Fluorescence in-situ hybridization analyses showed negative for COL1A1-PDGFB fusion as well as NTRK1/2/3 rearrangements. A subsequent RNA sequencing detected an in-frame fusion between exon 15 of TNC and exon 6 of PDGFD. This fusion was further confirmed by nested reverse transcription polymerase chain reaction amplification followed by Sanger sequencing. A diagnosis of fibrosarcomatous DFSP was rendered and the patient was in good status at a follow-up of 12 months after the operation. Conclusions We report a fibrosarcomatous DFSP with novel TNC-PDGFD fusion, which adds to the pathologic and genetic spectrum of PDGFD-rearranged DFSP.

P–540 A feasible diagnostic approach for the cryptic subtelomeric traslocations in early recurrent miscarriage patients by preimplantation genetic testing (PGT)

Study question Could cryptic subtelomeric traslocations in early recurrent miscarriage patients be diagnosed by preimplantation genetic testing? Summary answer PGT is a powerful tool to detect subtelomeric cryptic traslocations identifying the cause of early recurrent miscarriage and allowing subsequent genetic counselling. What is known already: Chromosome translocations are frequently associated with birth defects, spontaneous early pregnancy losses and infertility. However, submicroscopic traslocations (so-called cryptic traslocations) are too small to be detected by conventional karyotyping.. Due to balanced status, high resolution molecular techniques as arrayCGH are not able to detect it. Thus, cryptic traslocations detection is challenging. PGT is able to detect CNVs at higher resolution than routine karyotyping. Therefore, the recurrent diagnosis of CNV at embryo level could suggest a subchromosomal parental traslocation. The aim of this study is to investigate the feasibility of using PGT as an indicator of parental balanced cryptic traslocations. Study design, size, duration We included three couples who underwent PGT for unexplained repeated pregnancy loss (RPL) in our clinic from February 2020 to November 2020. Common established causes of RPL (uterine anomalies, antiphospholipid syndrome, immunological, hormonal and metabolic disorders) were previously rouled-out. Even couple karyotypes were normal. Twenty-three embryos from those couples were biopsied at blastocyst and analysed for CNVs detection using low coverage whole genome NGS. Participants/materials, setting, methods PGT by NGS was performed by Veriseq-NGS (Illumina), with previous whole genome amplification. Fluorescence in situ hybridization (FISH) using parental blood samples were performed to validate the origin of subchromosomal number variation. Commercially available subtelomeric specific probes were selected according to the CNV identified and the procedures were performed according to the manufacturer’s protocols. Main results and the role of chance Overall, CNVs of terminal duplication and deletion that imply unbalanced traslocation derivatives were detected in the 43.5% of biopsied embryos. For couple 1, 4 out of 5 embryos (80%) carried deletion of telomeric region on chromosomes 5 and 21. Three out of 6 biopsed embyos (50%) were diagnosed with subchromosomal copy variants at telomeric region on chromosomes 6 and 16 for couple 2. In the case of couple 3, three out of 12 embryos (25%) were carriers of CNV at subtelomeric region on chromosomes 2 and 6. The size of CNVs detected ranges from 8Mb to 20Mb. Accurate diagnosis with the parental study was made by FISH. The combination of probes to detect the structural chromosome alteration were: Tel5qter-LSI21q, Tel6pter-CEP16 and Tel6pter-CEP6 for each couple respectively. The FISH studies reveal that CNVs were inherited from one parent carrying the balanced cryptic traslocation. Ultimately, the abnormal karyotype from the carrier parent were 46,XY,t(5;21)(q33.2;q21.2) for couple 1, 46,XY,t(6;16)(p22.3;q22.1) for couple 2 and 46,XY,t(2;6)(p25.1;p24.2) for couple 3. Finally, each couple performed a cryotransfer of a single normal balanced embryo. Two pregnancies are ongoing. Limitations, reasons for caution The main limitation of this approach is the NGS- PGT resolution. CNVs smaller than 5Mb could not be detected. Wider implications of the findings: This study shows the value of PGT for unexplained RPL, followed by parental FISH to better characterize CNVs and identify couples in whom one partner carries a cryptic translocation. Accurate diagnosis of parental chromosome translocation can achieve with FISH only, but FISH would not be performed unless PGT showed CNVs. Trial registration number Not applicable