Familial transmission of chromoanagenesis leads to unpredictable unbalanced rearrangements through meiotic recombination.

Chromoanagenesis is a cellular mechanism that leads to complex chromosomal rearrangements (CCR) during a single catastrophic event. It may result in loss and/or gain of genetic material and may be responsible for various phenotypes. These rearrangements are usually sporadic. However, some familial cases have been reported. Here, we studied six families in whom an asymptomatic or paucisymptomatic parent transmitted a CCR to its offspring in an unbalanced manner. The rearrangements were characterized by karyotyping, fluorescent in situ hybridization, chromosomal microarray (CMA) and/or whole genome sequencing (WGS) in the carrier parents and offspring. We then hypothesized meiosis-pairing figures between normal and abnormal parental chromosomes that may have led to the formation of new unbalanced rearrangements through meiotic recombination. Our work indicates that chromoanagenesis might be associated with a normal phenotype and normal fertility, even in males, and that WGS may be the only way to identify these events when there is no imbalance. Subsequently, the CCR can be transmitted to the next generation in an unbalanced and unpredictable manner following meiotic recombination. Thereby, prenatal diagnosis using CMA should be proposed to these families to detect any pathogenic imbalances in the offspring.

Disruption and deletion of the proximal part of TCF4 are associated with mild intellectual disability: About three new patients.

TCF4 gene (18q21.1) encodes for a transcription factor with multiple isoforms playing a critical role during neurodevelopment. Molecular alterations of this gene are associated with Pitt-Hopkins syndrome, a severe condition characterized by intellectual disability, specific facial features and autonomic nervous system dysfunction. We report here three patients presenting with structural variations of the proximal part of TCF4 associated with a mild phenotype. The first patient is a six-years-old girl carrier of a pericentric inversion of chromosome 18, 46,XX,inv(18)(p11.2q21.1). Whole genome sequencing (WGS) characterized the breakpoint at the base-pair level at chr18:1262334_1262336 and chr18:53254747_53254751 (hg19). This latter breakpoint disrupted the proximal promotor region of TCF4 in the first intron of the gene. The second and third patients are a son and his mother, carrier of a 46 kb deletion characterized by high-resolution chromosomal micro-array and WGS (chr:18:53243454_53287927, hg19) encompassing the first three exon of TCF4 gene and including the proximal promotor region. Expression studies on blood lymphocytes in these patients showed a marked decrease of mRNA level for long isoforms of TCF4 and an increased level for shorter isoforms. The patients described here, together with previously reported patients with proximal structural alterations of TCF4, help to delineate a phenotype of mild ID with non-specific facial dysmorphism without characteristic features of PTHS. It also suggests a gradient of phenotypic severity inversely correlated with the number of intact TCF4 promotor regions, with expression of short isoforms compensating in part the loss of longer isoforms.

Complete characterisation of two new large Xq28 duplications involving F8 using whole genome sequencing in patients without haemophilia A.

INTRODUCTION: Depending on the location of insertion of the gained region, F8 duplications can have variable clinical impacts from benign impact to severe haemophilia A phenotype. AIM: To characterize two large Xq28 duplications involving F8 incidentally detected by chromosome microarray analysis (CMA) in two patients presenting severe intellectual disability but no history of bleeding disorder. METHODS: Whole genome sequencing (WGS) was performed in order to characterize the two large Xq28 duplications at nucleotide level. RESULTS: In patient 1, a 60-73 kb gained region encompassing the exons 23-26 of F8 and SMIM9 was inserted at the int22h-2 locus following a non-homologous recombination between int22h-1 and int22h-2. We hypothesized that two independent events, micro-homology-mediated break-induced replication (MMBIR) and break-induced replication (BIR), could be involved in this rearrangement. In patient 2, the CMA found duplication from 101 to 116-kb long encompassing the exons 16-26 of F8 and SMIM9. The WGS analysis identified a more complex rearrangement with the presence of three genomic junctions. Due to the multiple micro-homologies observed at breakpoints, a replication-based mechanism such as fork stalling and template switching (FoSTeS) was greatly suspected. In both cases, these complex rearrangements preserved an intact copy of the F8. CONCLUSION: This study highlights the value of WGS to characterize the genomic junction at the nucleotide level and ultimately better describe the molecular mechanisms involved in Xq28 structural variations. It also emphasizes the importance of specifying the structure of the genomic gain in order to improve genotype-phenotype correlation and genetic counselling.

A 14q distal chromoanagenesis elucidated by whole genome sequencing.

Chromoanagenesis represents an extreme form of genomic rearrangements involving multiple breaks occurring on a single or multiple chromosomes. It has been recently described in both acquired and rare constitutional genetic disorders. Constitutional chromoanagenesis events could lead to abnormal phenotypes including developmental delay and congenital anomalies, and have also been implicated in some specific syndromic disorders. We report the case of a girl presenting with growth retardation, hypotonia, microcephaly, dysmorphic features, coloboma, and hypoplastic corpus callosum. Karyotype showed a de novo structurally abnormal chromosome 14q31qter region. Molecular characterization using SNP-array revealed a complex unbalanced rearrangement in 14q31.1-q32.2, on the paternal chromosome 14, including thirteen interstitial deletions ranging from 33 kb to 1.56 Mb in size, with a total of 4.1 Mb in size, thus suggesting that a single event like chromoanagenesis occurred. To our knowledge, this is one of the first case of 14q distal deletion due to a germline chromoanagenesis. Genome sequencing allowed the characterization of 50 breakpoints, leading to interruption of 10 genes including YY1 which fit with the patient's phenotype. This precise genotyping of breaking junction allowed better definition of genotype-phenotype correlations.

Fryns type mesomelic dysplasia of the upper limbs caused by inverted duplications of the HOXD gene cluster.

The HoxD cluster is critical for vertebrate limb development. Enhancers located in both the telomeric and centromeric gene deserts flanking the cluster regulate the transcription of HoxD genes. In rare patients, duplications, balanced translocations or inversions misregulating HOXD genes are responsible for mesomelic dysplasia of the upper and lower limbs. By aCGH, whole-genome mate-pair sequencing, long-range PCR and fiber fluorescent in situ hybridization, we studied patients from two families displaying mesomelic dysplasia limited to the upper limbs. We identified microduplications including the HOXD cluster and showed that microduplications were in an inverted orientation and inserted between the HOXD cluster and the telomeric enhancers. Our results highlight the existence of an autosomal dominant condition consisting of isolated ulnar dysplasia caused by microduplications inserted between the HOXD cluster and the telomeric enhancers. The duplications likely disconnect the HOXD9 to HOXD11 genes from their regulatory sequences. This presumptive loss-of-function may have contributed to the phenotype. In both cases, however, these rearrangements brought HOXD13 closer to telomeric enhancers, suggesting that the alterations derive from the dominant-negative effect of this digit-specific protein when ectopically expressed during the early development of forearms, through the disruption of topologically associating domain structure at the HOXD locus.

Supravalvular Aortic Stenosis Caused by a Familial Chromosome 7 Inversion Disrupting the ELN Gene Uncovered by Whole-Genome Sequencing.

Apparently, balanced chromosomal rearrangements usually have no phenotypic consequences for the carrier. However, in some cases, they may be associated with an abnormal phenotype. We report herein the case of a 4-year-old boy presenting with clinically isolated supravalvular aortic stenosis (SVAS). No chromosomal imbalance was detected by array CGH. The karyotype showed a balanced paracentric chromosome 7 inversion. Breakpoint characterization using paired-end whole-genome sequencing (WGS) revealed an ELN gene disruption in intron 1, accounting for the phenotype. Family study showed that the inversion was inherited, with incomplete penetrance. To our knowledge, this is the first case of a disruption of the ELN gene characterized by WGS. It contributes to refine the genotype-phenotype correlation in ELN disruption. Although this disruption is a rare etiology of SVAS, it cannot be detected by the diagnostic tests usually performed, such as array CGH or sequencing methods (Sanger, panel, or exome sequencing). With the future perspective of WGS as a diagnostic tool, it will be important to include a structural variation analysis in order to detect balanced rearrangements and gene disruption.

Identification of mobile retrocopies during genetic testing: Consequences for routine diagnosis.

Human retrocopies, that is messenger RNA transcripts benefitting from the long interspersed element 1 machinery for retrotransposition, may have specific consequences for genomic testing. Next genetration sequencing (NGS) techniques allow the detection of such mobile elements but they may be misinterpreted as genomic duplications or be totally overlooked. We report eight observations of retrocopies detected during diagnostic NGS analyses of targeted gene panels, exome, or genome sequencing. For seven cases, while an exons-only copy number gain was called, read alignment inspection revealed a depth of coverage shift at every exon-intron junction where indels were also systematically called. Moreover, aberrant chimeric read pairs spanned entire introns or were paired with another locus for terminal exons. The 8th retrocopy was present in the reference genome and thus showed a normal NGS profile. We emphasize the existence of retrocopies and strategies to accurately detect them at a glance during genetic testing and discuss pitfalls for genetic testing.

Whole genome paired-end sequencing elucidates functional and phenotypic consequences of balanced chromosomal rearrangement in patients with developmental disorders.

BACKGROUND: Balanced chromosomal rearrangements associated with abnormal phenotype are rare events, but may be challenging for genetic counselling, since molecular characterisation of breakpoints is not performed routinely. We used next-generation sequencing to characterise breakpoints of balanced chromosomal rearrangements at the molecular level in patients with intellectual disability and/or congenital anomalies. METHODS: Breakpoints were characterised by a paired-end low depth whole genome sequencing (WGS) strategy and validated by Sanger sequencing. Expression study of disrupted and neighbouring genes was performed by RT-qPCR from blood or lymphoblastoid cell line RNA. RESULTS: Among the 55 patients included (41 reciprocal translocations, 4 inversions, 2 insertions and 8 complex chromosomal rearrangements), we were able to detect 89% of chromosomal rearrangements (49/55). Molecular signatures at the breakpoints suggested that DNA breaks arose randomly and that there was no major influence of repeated elements. Non-homologous end-joining appeared as the main mechanism of repair (55% of rearrangements). A diagnosis could be established in 22/49 patients (44.8%), 15 by gene disruption (KANSL1, FOXP1, SPRED1, TLK2, MBD5, DMD, AUTS2, MEIS2, MEF2C, NRXN1, NFIX, SYNGAP1, GHR, ZMIZ1) and 7 by position effect (DLX5, MEF2C, BCL11B, SATB2, ZMIZ1). In addition, 16 new candidate genes were identified. Systematic gene expression studies further supported these results. We also showed the contribution of topologically associated domain maps to WGS data interpretation. CONCLUSION: Paired-end WGS is a valid strategy and may be used for structural variation characterisation in a clinical setting.