SVInterpreter: A Comprehensive Topologically Associated Domain-Based Clinical Outcome Prediction Tool for Balanced and Unbalanced Structural Variants

With the advent of genomic sequencing, a number of balanced and unbalanced structural variants (SVs) can be detected per individual. Mainly due to incompleteness and the scattered nature of the available annotation data of the human genome, manual interpretation of the SV’s clinical significance is laborious and cumbersome. Since bioinformatic tools developed for this task are limited, a comprehensive tool to assist clinical outcome prediction of SVs is warranted. Herein, we present SVInterpreter, a free Web application, which analyzes both balanced and unbalanced SVs using topologically associated domains (TADs) as genome units. Among others, gene-associated data (as function and dosage sensitivity), phenotype similarity scores, and copy number variants (CNVs) scoring metrics are retrieved for an informed SV interpretation. For evaluation, we retrospectively applied SVInterpreter to 97 balanced (translocations and inversions) and 125 unbalanced (deletions, duplications, and insertions) previously published SVs, and 145 SVs identified from 20 clinical samples. Our results showed the ability of SVInterpreter to support the evaluation of SVs by (1) confirming more than half of the predictions of the original studies, (2) decreasing 40% of the variants of uncertain significance, and (3) indicating several potential position effect events. To our knowledge, SVInterpreter is the most comprehensive TAD-based tool to identify the possible disease-causing candidate genes and to assist prediction of the clinical outcome of SVs. SVInterpreter is available at http://dgrctools-insa.min-saude.pt/cgi-bin/SVInterpreter.py.

Rare t(X;14)(q28;q32) translocation reveals link between MTCP1 and chronic lymphocytic leukemia

Rare, recurrent balanced translocations occur in a variety of cancers but are often not functionally interrogated. Balanced translocations with the immunoglobulin heavy chain locus (IGH; 14q32) in chronic lymphocytic leukemia (CLL) are infrequent but have led to the discovery of pathogenic genes including CCND1, BCL2, and BCL3. Following identification of a t(X;14)(q28;q32) translocation that placed the mature T cell proliferation 1 gene (MTCP1) adjacent to the immunoglobulin locus in a CLL patient, we hypothesized that this gene may have previously unrecognized importance. Indeed, here we report overexpression of human MTCP1 restricted to the B cell compartment in mice produces a clonal CD5+/CD19+ leukemia recapitulating the major characteristics of human CLL and demonstrates favorable response to therapeutic intervention with ibrutinib. We reinforce the importance of genetic interrogation of rare, recurrent balanced translocations to identify cancer driving genes via the story of MTCP1 as a contributor to CLL pathogenesis.

Genomic imbalance in a patient with two balanced translocations and abnormal phenotype

Представлены клинические и молекулярно-генетические результаты обследования пациента с задержкой психомоторного развития, аномалиями фенотипа и множественными врожденными пороками систем и органов. При стандартном цитогенетическом исследовании определены две реципрокные транслокации между хромосомами 2 и 6 и хромосомами 7 и 11, подтвержденные FISH-методом. Хромосомный микроматричный анализ позволил выявить делецию 6q14.1 в точке разрыва на длинном плече хромосомы 6. Делеция включает несколько десятков генов, в том числе гены PHIP, FILIP1, MYO6, HTR1B, IMPG1, EVOLV4, TENT5A, которые вероятнее всего ассоциированы с клиническими проявлениями у пациента. The results of clinical and molecular genetic study of the patient with psychomotor delay, phenotype abnormalities and multiple congenital malformations of systems and organs are presented. A standard cytogenetic study determined a double translocation between chromosomes 2 and 6 and chromosomes 7 and 11, confirmed by the FISH method. Chromosomal microarray analysis revealed a deletion of 6q14.1 region at the break point on the long arm of chromosome 6. The deletion involves several dozen genes, including PHIP, FILIP1, MYO6, HTR1B, IMPG1, EVOLV4, TENT5A genes, which are most likely associated with clinical symptoms in the patient.

Analysis of parental abnormal chromosomal karyotype and subsequent live births in Chinese couples with recurrent pregnancy loss

The frequency and distribution of chromosomal abnormalities and the impact of parental chromosomal aberration on the pregnancy outcomes of couples with recurrent pregnancy loss remains controversial. 3235 RPL couples who experienced two or more miscarriages before 20 weeks were diagnosed in our tertiary referral hospital during 2008–2018 and included in the single-center retrospective cohort study covering a 10-year period. Chromosome aberration was detected in 121 (3.74%) among 3235 RPL couples which included 75 female and 46 male cases at an individual level. 101 cases were structural aberrations including balanced translocations in 46(38.0%) cases, Robertsonian translocations in 13(10.7%) cases, inversions in 42(34.7%) cases and 20(16.5%) cases were numerical aberrations. 121 carriers and 428 non-carriers were followed up for two years, 55 carriers and 229 non-carriers were subsequent pregnant after diagnosis by natural conception or intrauterine insemination. The frequency of carriers to have a health newborn was not significantly different with non-carriers (72.7% vs. 71.2%, adjusted P = 0.968). This study described the majority of carriers were balanced translocations and chromosome aberrations had a limited influence on live birth rate from the present data. The results of the study also remind us that natural conception may be also a good alternative rather than PGD (Pre-implantation Genetic Diagnosis) which is common in many other reproductive centers for such patients.

P–083 Analysis of chromosomal segregation and interchromosomal effects (ICE) in sperms from balanced translocation carriers using fluorescence in situ hybridization (FISH) after sperm selector separation

Study question Influence of sperm selector separation of sperms on their translocation load, segregation pattern, motility and occurrence of interchromosomal effects Summary answer Sperm selector separation led to reduction of the translocation load, shift in segregation pattern and lower rates of interchromosomal effects within sperm samples What is known already Balanced translocations in men are known to be one of the main causes of reproductive failure. The segregation pattern in sperms is determined by the distribution of the chromosomes during meiosis. Interchromosomal effects can also influence the distribution of chromosomes that are not involved in the translocation. The sperm selector used consists of two concentric chambers, which are overlaid by a U-ring and a cover glass. Motile sperms migrate from the native ejaculate in the medium filled inner chamber by using a capillary bridge created by the U-ring. This avoids potential harmful centrifugation and allows accumulation of motile sperms. Study design, size, duration Twenty-one carriers of balanced translocations participated in the study. In addition, 15 patients were involved as control. All participants signed an informed consent (F–8–15). Samples of three patients did not meet the internal quality criteria and had to be excluded from analysis. The study started in 2015 and is still ongoing. Participants/materials, setting, methods Liquefied native ejaculate was processed with a sperm selector. Native ejaculate, non-migrated sperms from the outer chamber and migrated sperms from the inner chamber were transferred onto glass slides, fixed and underwent a decondensation treatment. For segregation analysis FISH translocation specific FISH probe mixes were used and tested on patient’s blood. Interchromosomal effects were analysed with FISH probes for the chromosomes X, Y, 18 and 13, 21. Evaluation was done manually using fluorescence microscopy. Main results and the role of chance Segregation analysis was done for more than 25,000 sperms from men carrying a balanced translocation (18 patients with reciprocal and 3 patients with Robertsonian translocation). Separation via sperm selector led to a reduction in translocation load (native to separated approach 49,1±11,5% to 34,8±9,4% (P = <0,01), the rate depending on the specific translocation. There was also a shift in the segregation pattern, which seemed to be influenced by the specific translocation and the resulting steric alignment of the corresponding quadrivalent / trivalent. Additionally, more than 90,000 sperms from patients with balanced translocations were analysed for interchromosomal effects. Separation led to reduced maldistribution rate (native to separated approach 7,1±3,5% to 5±3,1%, P = <0,01) whereas the steric alignment of the corresponding quadrivalent / trivalent seems to influence the interchromosomal effect as well. For control, sperms from control patients were analysed regarding the chromosomes X, Y, 18 and 13, 21. In about 90,000 control sperms separation led to reduced maldistribution rate (native to separated approach 5,4±1,5% to 3,8±1,1%; P = <0,01). Limitations, reasons for caution The number of accumulated strandbreak-free sperms depended on the motility and sperm count of the native ejaculate. Examinations are not reproducible, as each sample delivery is influenced by external circumstances Wider implications of the findings: Sperm selector separation can be used before ART to reduce the translocation load and rate of maldistribution in sperms from carriers of balanced translocations. This could have a considerable impact on PGT results after trophectoderm biops. Trial registration number Not applicable

Recurrent spontaneous abortion related to balanced translocation of chromosomes: two case reports

Background Recurrent spontaneous abortion (RSA) is often idiopathic, but structural chromosomal abnormality is an important nosogenesis. Balanced translocations or inversions can lead to unbalanced gametes depending on the specific recombination and segregation patterns during meiosis. An unbalanced karyotype in the conceptus of a couple when one partner has a structural chromosomal abnormality may result in failure to implant, miscarriage, or ongoing pregnancy of a fetus with an unbalanced karyotype. Case presentation We report two rare Han cases of RSA associated with balanced translocation of chromosomes. In case 1, a women who had had four spontaneous abortions, the karyotype was 46, XX, t (4;7) (q31;q22). In case 2, a women who had two spontaneous abortions and one stillborn fetus, the karyotype was 46, XX, t (3;15) (q12;p11.2), inv (5) (P13q13). The abnormal karyotype was not found in other chromosomes. Conclusions It is very important that couples with more than two miscarriages be provided with chromosomal analysis. Referring couples for karyotyping will rule out or confirm possible hereditary etiology and the source of chromosomal abnormalities in recurrent miscarriages.

Deciphering balanced translocations in infertile males by Next Generation Sequencing to identify candidate genes for spermatogenesis disorders

Male infertility affects about 7% of the general male population. Balanced structural chromosomal rearrangements are observed in 0.4 to 1.4% of infertile males and are considered as a well-established cause of infertility. However, underlying pathophysiological mechanisms still need to be clarified. A strategy combining standard and high throughput cytogenetic and molecular technologies was applied in order to identify the candidate genes that might be implicated in the spermatogenesis defect in three male carriers of different balanced translocations. Fluorescence in situ hybridization (FISH) and whole genome paired-end sequencing were used to characterize translocation breakpoints at the molecular level while exome sequencing was performed in order to exclude the presence of any molecular event independent from the chromosomal rearrangement in the patients. All translocation breakpoints were characterized in the three patients. We identified four variants: a position effect on LACTB2 gene in Patient 1, a heterozygous CTDP1 gene disruption in Patient 2, two single nucleotide variations (SNVs) in DNAH5 gene and a heterozygous 17q12 deletion in Patient 3. The variants identified in this study need further validation to assess their roles in male infertility. This study shows that beside the mechanical effect of structural rearrangement on meiosis, breakpoints could result in additional alterations such as gene disruption or position effect. Moreover, additional SNVs or copy number variations may be fortuitously present and could explain the variable impact of chromosomal rearrangements on spermatogenesis. In conclusion, this study confirms the relevance of combining different cytogenetic and molecular techniques to investigate patients with spermatogenesis disorders and structural rearrangements on genomic scale.

A case of deletion 8q22.2q22.3 in a child with de novo balanced translocation t(1;6)

Выяснение этиопатогенеза аномального фенотипа у пациентов со сбалансированными транслокациями является актуальным аспектом в современной клинической цитогенетике. Формирование аномалий развития может быть ассоциировано с наличием скрытого геномного дисбаланса как в точках разрывов, так и на хромосомах, не задействованных в перестройке. Целью данного исследования явилась этиологическая диагностика геномного дисбаланса у пациента со сбалансированной транслокацией и аномалиями развития. Для детекции геномного дисбаланса у пациента со сбалансированной транслокацией использовали хромосомный микроматричный анализ (ХМА) и FISH-исследование. У пациента со сбалансированной транслокацией при ХМА была выявлена делеция на хромосоме 8, не задействованной в транслокации. Таким образом, в статье представлен новый случай делеции 8q22.2q22.3 у пациента со сбалансированной транслокацией t(1;6) и аномалиями развития вследствие делеции. Identification of the etiopathogenesis of the abnormal phenotype in patients with balanced translocations is current trend in cytogenetic laboratories. The formation of developmental anomalies can be associated with the presence of a cryptic genomic imbalance both at breakpoints and on chromosomes not involved in rearrangements.The aim of this study is diagnostics of genomic imbalance in a patient with balanced translocation and abnormal phenotype. The case was characterized by GTG-banding, chromosomal microarray analysis and FISH diagnosis. We present a new case of deletion 8q22.2-q22.3 in child with balanced translocation t(1;6) and developmental delay/congenital defects due to deletion.

Balanced translocations 46, XY, t (8;17)(p23;q11) in brothers with azoospermia

Infertility is one of the most significant human health problems of the reproductive years. The causes of infertility are diverse and numerous-including non-genetic and genetic factors. A review of this case confirmed this. A balanced translocation was found in two siblings diagnosed with azoospermia. After being unable to conceive, the older brother and his wife had two in vitro fertilization (IVF) failed. At the same time, his younger brother and his wife had one IVF. After the cytogenetic analysis was performed in both pairs, it was shown that the cause of their infertility was the same balanced translocation in the brothers. The female showed a regular (46, XX) karyotype, whereas the male was found to carry balanced reciprocal translocation [46, XY, t(8;17)(p23;q11)]. This case support that cytogenetic analysis is still the first and basic diagnostic analysis of patients with azoospermia and other reproductive problems.

Analysis of Parental Abnormal Chromosomal Karyotype and Subsequent Live Births in Chinese Couples With Recurrent Miscarriage

The frequency and distribution of chromosomal abnormalities and the impact of parental chromosomal aberration on the pregnancy outcomes of couples with recurrent miscarriage remains controversial. 3235 RM couples who experienced two or more miscarriages before 20 weeks were diagnosed in our tertiary referral hospital during 2008 to 2018 and included in the single-centre retrospective cohort study covering a 10-year period. Chromosome aberration was detected in 121 (3.74%) among 3235 RM couples which included 75 female and 46 male cases at an individual level. 101 cases were structural aberrations including balanced translocations in 46(38.0%) cases, Robertsonian translocations in 13(10.7%) cases, inversions in 42(34.7%) cases and 20(16.5%) cases were numerical aberrations. 121 carriers and 428 non-carriers were followed up for two years，55 carriers and 229 non-carriers were subsequent pregnant after diagnosis by natural conception or intrauterine insemination. The frequency of carriers to have a health newborn was not significantly different with non-carriers (72.7% vs. 71.2%, adjusted p=0.968). This study described the majority of carriers were balanced translocations and chromosome aberrations had a limited influence on live birth rate from the present data. The results of the study also remind us that natural conception may be also a good alternative rather than PGD (Pre-implantation Genetic Diagnosis) which is common in many other reproductive centers for such patients.