Cryptic breakpoint identified by whole-genome mate-pair sequencing in a rare paternally inherited complex chromosomal rearrangement

Abstract Background Precise characterization of apparently balanced complex chromosomal rearrangements in non-affected individuals is crucial as they may result in reproductive failure, recurrent miscarriages or affected offspring. Case presentation We present a family, where the non-affected father and daughter were found, using FISH and karyotyping, to be carriers of a three-way complex chromosomal rearrangement [t(6;7;10)(q16.2;q34;q26.1), de novo in the father]. The family suffered from two stillbirths, one miscarriage, and has a son with severe intellectual disability. In the present study, the family was revisited using whole-genome mate-pair sequencing. Interestingly, whole-genome mate-pair sequencing revealed a cryptic breakpoint on derivative (der) chromosome 6 rendering the rearrangement even more complex. FISH using a chromosome (chr) 6 custom-designed probe and a chr10 control probe confirmed that the interstitial chr6 segment, created by the two chr6 breakpoints, was translocated onto der(10). Breakpoints were successfully validated with Sanger sequencing, and small imbalances as well as microhomology were identified. Finally, the complex chromosomal rearrangement breakpoints disrupted the SIM1, GRIK2, CNTNAP2, and PTPRE genes without causing any phenotype development. Conclusions In contrast to the majority of maternally transmitted complex chromosomal rearrangement cases, our study investigated a rare case where a complex chromosomal rearrangement, which most probably resulted from a Type IV hexavalent during the pachytene stage of meiosis I, was stably transmitted from a fertile father to his non-affected daughter. Whole-genome mate-pair sequencing proved highly successful in identifying cryptic complexity, which consequently provided further insight into the meiotic segregation of chromosomes and the increased reproductive risk in individuals carrying the specific complex chromosomal rearrangement. We propose that such complex rearrangements should be characterized in detail using a combination of conventional cytogenetic and NGS-based approaches to aid in better prenatal preimplantation genetic diagnosis and counseling in couples with reproductive problems

Epidemiology and risk factors for resistance to treatment of Kawasaki disease in Cyprus

Kawasaki disease (KD) is one of the most common vasculitides of early childhood. There are no previous studies on KD in Cyprus. The aim of this study was to evaluate the epidemiology of KD in Cyprus, risk factors for resistance to treatment and the development of cardiac complications. This is a retrospective multicenter study of pediatric patients with KD hospitalized between January 2000 and-December 2019. The data were collected from medical records. A total of 136 patients with KD were included in the study. 83% of patients were < 5 years of age and 10% were < 6 months. Thirty patients (22%) developed coronary artery lesions. Serum sodium ≤ 133 mmol/L, albumin ≤ 3.2 g/dl, ALT ≥ 80 U/L and neutrophils percentage ≥ 80% at diagnosis, were identified as risk factors for resistance to IVIG. Clinical and epidemiological characteristics of KD in Cyprus population were similar to those reported in the literature. Although the majority of cases received appropriate treatment in time, cardiac complications still occurred

Εμβολιασμός κατά COVID-19 στον παιδιατρικό πληθυσμό της Κύπρου. Παράγοντες που επηρεάζουν τις αποφάσεις των γονέων

Τα εμβόλια έναντι covid-19 έχουν εγκριθεί για τα παιδιά ≥12 ετών και 5-11 ετών από τον Σεπτέμβριο και Δεκέμβριο 2021, αντίστοιχα. Όμως, αρκετοί γονείς διστάζουν να εμβολιάσουν τα παιδιά τους. Ο σκοπός αυτής της μελέτης είναι η διερεύνηση των απόψεων των γονέων στην Κύπρο για τον εμβολιασμό. Πρόκειται για μια συγχρονική μελέτη, κατά την οποία διαμοιράστηκαν ερωτηματολόγια σε γονείς που ένα τουλάχιστον παιδί τους ήταν 5-18 ετών, και επισκέφθηκαν παιδίατρο της πρωτοβάθμιας φροντίδας υγείας σε όλες τις επαρχίες της Κύπρου. Συνολικά συμπληρώθηκαν 610 έγκυρα ερωτηματολόγια. Το 84% των συμμετεχόντων ήταν γυναίκες, και η πλειοψηφία ήταν 30-49 ετών (91%). Το 30% των γονέων εμβολίασε ήδη τα παιδιά του, το 9% σκόπευε να τα εμβολιάσει αλλά δεν το είχε πράξει ακόμα, το 24% δεν είχε αποφασίσει και το 37% αρνήθηκε τον εμβολιασμό. Ο κύριος λόγος της απροθυμίας των γονέων να εμβολιάσουν τα παιδιά τους ήταν η ανησυχία για τις ανεπιθύμητες ενέργειες. Παρόλο που ο εμβολιασμός αποτελεί σημαντικό εργαλείο για την προστασία των παιδιών, μόνο το ένα τρίτο των γονέων εμβολίασε τα παιδιά του σύμφωνα με τα αποτελέσματά μας.Covid-19 vaccines have been approved for children ≥12 and 5-11 years since September 2021 and December 2021, respectively. However, many parents still hesitate to vaccinate their children. The aim of this study is to investigate the parents’ attitudes to offer the vaccine to their children. This is a cross-sectional study, in which questionnaires were distributed to parents whose at least one child was 5 to 18 years, and who visited a primary care paediatrician’s office across all regions of Cyprus. A total of 610 questionnaires were completed. 84% of participants were female, and the majority were 30-49 years (91%). 30% of parents have already vaccinated their children, 9% intended to do so, 24% have not decided yet and 37% refused to offer the vaccine. The main reason for which parents were not willing to vaccinate their children, were concerns regarding related adverse events. Even though vaccination in childhood is a very important tool which protects children, only one third of parents in Cyprus vaccinated their children, according to our findings

Exploring the Genetic Causality of Discordant Phenotypes in Familial Apparently Balanced Translocation Cases Using Whole Exome Sequencing

Familial apparently balanced translocations (ABTs) are usually not associated with a phenotype; however, rarely, ABTs segregate with discordant phenotypes in family members carrying identical rearrangements. The current study was a follow-up investigation of four familial ABTs, where whole exome sequencing (WES) was implemented as a diagnostic tool to identify the underlying genetic aetiology of the patients’ phenotypes. Data were analysed using an in-house bioinformatics pipeline alongside VarSome Clinical. WES findings were validated with Sanger sequencing, while the impact of splicing and missense variants was assessed by reverse-transcription PCR and in silico tools, respectively. Novel candidate variants were identified in three families. In family 1, it was shown that the de novo pathogenic STXBP1 variant (NM_003165.6:c.1110+2T>G) affected splicing and segregated with the patient’s phenotype. In family 2, a likely pathogenic TUBA1A variant (NM_006009.4:c.875C>T, NP_006000.2:p.(Thr292Ile)) could explain the patient’s symptoms. In family 3, an SCN1A variant of uncertain significance (NM_006920.6:c.5060A>G, NP_008851.3:p.(Glu1687Gly)) required additional evidence to sufficiently support causality. This first report of WES application in familial ABT carriers with discordant phenotypes supported our previous findings describing such rearrangements as coincidental. Thus, WES can be recommended as a complementary test to find the monogenic cause of aberrant phenotypes in familial ABT carriers

Position effect, cryptic complexity, and direct gene disruption as disease mechanisms in de novo apparently balanced translocation cases.

The majority of apparently balanced translocation (ABT) carriers are phenotypically normal. However, several mechanisms were proposed to underlie phenotypes in affected ABT cases. In the current study, whole-genome mate-pair sequencing (WG-MPS) followed by Sanger sequencing was applied to further characterize de novo ABTs in three affected individuals. WG-MPS precisely mapped all ABT breakpoints and revealed three possible underlying molecular mechanisms. Firstly, in a t(X;1) carrier with hearing loss, a highly skewed X-inactivation pattern was observed and the der(X) breakpoint mapped ~87kb upstream an X-linked deafness gene namely POU3F4, thus suggesting an underlying long-range position effect mechanism. Secondly, cryptic complexity and a chromothripsis rearrangement was identified in a t(6;7;8;12) carrier with intellectual disability. Two translocations and a heterozygous deletion disrupted SOX5; a dominant nervous system development gene previously reported in similar patients. Finally, a direct gene disruption mechanism was proposed in a t(4;9) carrier with dysmorphic facial features and speech delay. In this case, the der(9) breakpoint directly disrupted NFIB, a gene involved in lung maturation and development of the pons with important functions in main speech processes. To conclude, in contrast to familial ABT cases with identical rearrangements and discordant phenotypes, where translocations are considered coincidental, translocations seem to be associated with phenotype presentation in affected de novo ABT cases. In addition, this study highlights the importance of investigating both coding and non-coding regions to decipher the underlying pathogenic mechanisms in these patients, and supports the potential introduction of low coverage WG-MPS in the clinical investigation of de novo ABTs

Accurate Breakpoint Mapping in Apparently Balanced Translocation Families with Discordant Phenotypes Using Whole Genome Mate-Pair Sequencing

<div><p>Familial apparently balanced translocations (ABTs) segregating with discordant phenotypes are extremely challenging for interpretation and counseling due to the scarcity of publications and lack of routine techniques for quick investigation. Recently, next generation sequencing has emerged as an efficacious methodology for precise detection of translocation breakpoints. However, studies so far have mainly focused on <i>de novo</i> translocations. The present study focuses specifically on familial cases in order to shed some light to this diagnostic dilemma. Whole-genome mate-pair sequencing (WG-MPS) was applied to map the breakpoints in nine two-way ABT carriers from four families. Translocation breakpoints and patient-specific structural variants were validated by Sanger sequencing and quantitative Real Time PCR, respectively. Identical sequencing patterns and breakpoints were identified in affected and non-affected members carrying the same translocations. <i>PTCD1</i>, <i>ATP5J2-PTCD1</i>, <i>CADPS2</i>, and <i>STPG1</i> were disrupted by the translocations in three families, rendering them initially as possible disease candidate genes. However, subsequent mutation screening and structural variant analysis did not reveal any pathogenic mutations or unique variants in the affected individuals that could explain the phenotypic differences between carriers of the same translocations. In conclusion, we suggest that NGS-based methods, such as WG-MPS, can be successfully used for detailed mapping of translocation breakpoints, which can also be used in routine clinical investigation of ABT cases. Unlike <i>de novo</i> translocations, no associations were determined here between familial two-way ABTs and the phenotype of the affected members, in which the presence of cryptic imbalances and complex chromosomal rearrangements has been excluded. Future whole-exome or whole-genome sequencing will potentially reveal unidentified mutations in the patients underlying the discordant phenotypes within each family. In addition, larger studies are needed to determine the exact percentage for phenotypic risk in families with ABTs.</p></div

Translocation junction sequences identified in each family.

<p>Translocation junction sequences to the base-pair level as identified by mate-pair sequencing and verified by Sanger sequencing in A) family 1 with t(1;7)(p36.1;q22), B) family 2 with t(7;8)(q32;q24.13), C) family 3 with t(4;10)(q35;q11.2), and D) family 4 with t(1;20)(p35.3;q13.3). Translocation junction sequences (middle line) and matching reference sequences (top and bottom lines) are shown with a different colour depending on the chromosome involved (chr1-orange; chr4-purple; chr7-blue; chr8-red; chr10-yellow; chr20-green). Microhomology observed at the translocation breakpoint sites is highlighted in yellow, deleted sequences around the breakpoints are underlined, duplicated sequences are in bold, capital letters, and inserted sequences not aligning to either chromosome are in bold, lower-case letters.</p

The <i>ZNF423</i> gene and quantitative Real-Time PCR (qRT-PCR) results.

<p>A) Schematic illustration of the <i>ZNF423</i> gene (minus strand). The protein-coding exons and introns of the gene are represented with solid, vertical lines and dotted, horizontal lines, respectively (not to scale). Intron 3 is enlarged to demonstrate the approximate <i>ZNF423</i> deletion position (blue line) as predicted by MPS in the affected sibling in family 2, as well as the amplicon (Z-RT1, Z-RT2, and Z-RT3) positions (red lines) from the qRT-PCR analysis. The actual coordinates are given on the left (GRCh37/hg19). B) qRT-PCR results demonstrating a reduced relative <i>ZNF423</i> copy number in the proband and normal results in the non-affected sibling as compared with a control. Parental genomic material was unavailable and thus not included in the analysis.</p

Breakpoint mapping and sequencing results for each apparently balanced translocation case included in this study.

<p>Breakpoint mapping and sequencing results for each apparently balanced translocation case included in this study.</p

Family 1 results with t(1;7) translocation.

<p>A) Family 1 pedigree showing the proband with intellectual disability, psychomotor delay and epilepsy, as well as the non-affected mother and father. The t(1;7)(p36.1;q22) translocation is maternally inherited. B) Ideograms showing the normal and derivative (der) chromosomes (chr) 1 and 7 (not to scale). Genetic material from chr1 and chr7 is shown with a solid, orange and dotted, blue frame line, respectively. The approximate breakpoint positions on 1p36.1 and 7q22 are indicated by arrows. C) UPD7 analysis results from one of the informative microsatellite markers (D71824) in the affected proband and non-affected parents; by comparing the peak sizes between all family members, normal biparental inheritance was concluded. D) Quantitative Real-Time PCR results demonstrating the paternal inheritance of the chr3 duplication predicted from the structural variant analysis in the affected proband.</p