稲垣 秀人

Cytogenetic information about the product of conception (POC) is important to determine the presence of recurrent chromosomal abnormalities that are an indication for preimplantation genetic testing for aneuploidy or structural rearrangements. Although microscopic examination by G-staining has long been used for such an evaluation, detection failures are relatively common with this method, due to cell-culture-related issues. The utility of low-coverage whole-genome sequencing (lcWGS) using short-read next-generation sequencing (NGS) has been highlighted recently as an alternative cytogenomic approach for POC analysis. We, here, performed comparative analysis of two NGS-based protocols for this purpose based on different short-read sequencers (the Illumina VeriSeq system using a MiSeq sequencer and the Thermo Fisher ReproSeq system using an Ion S5 sequencer). The cytogenomic diagnosis obtained with each NGS method was equivalent in each of 20 POC samples analyzed. Notably, X chromosome sequence reads were reduced in some female samples with both systems. The possibility of low-level mosaicism for monosomy X as an explanation for this was excluded by FISH analysis. Additional data from samples with various degrees of X chromosome aneuploidy suggested that it was a technical artifact related to X chromosome inactivation. Indeed, subsequent nanopore sequencing indicated that the DNA in the samples showing the artifact was predominantly unmethylated. Our current findings indicate that although X chromosome data must be interpreted with caution, both the systems we tested for NGS-based lcWGS are useful alternatives for the karyotyping of POC samples.

Sonic hedgehog signaling molecule (SHH) is a key molecule in the cilia-mediated signaling pathway and a critical morphogen in embryogenesis. The association between loss-of-function variants of SHH and holoprosencephaly is well established. In mice experiments, reduced or increased signaling of SHH have been shown to be associated with narrowing or excessive expansion of the facial midline, respectively. Herein, we report two unrelated patients with de novo truncating variants of SHH presenting with hypertelorism rather than hypotelorism. The first patient was a 13-year-old girl. Her facial features included hypertelorism, strabismus, telecanthus, malocclusion, frontal bossing, and wide widow's peak. She had borderline developmental delay and agenesis of the corpus callosum. She had a nonsense variant of SHH: Chr7(GRCh38):g.155802987C > T, NM_000193.4:c.1302G > A, p.(Trp434*). The second patient was a 25-year-old girl. Her facial features included hypertelorism and wide widow's peak. She had developmental delay and agenesis of the corpus callosum. She had a frameshift variant of SHH: Chr7(GRCh38):g.155803072_155803074delCGGinsT, NM_000193.4:c.1215_1217delCCGinsA, p.(Asp405Glufs*92). The hypertelorism phenotype contrasts sharply with the prototypical hypotelorism-holoprosencephaly phenotype associated with loss-of-function of SHH. We concluded that a subset of truncating variants of SHH could be associated with hypertelorism rather than hypotelorism.

Constitutional complex chromosomal rearrangements (CCRs) are rare cytogenetic aberrations arising in the germline via an unknown mechanism. Here we analyzed the breakpoint junctions of microscopically three-way or more complex translocations using comprehensive genomic and epigenomic analyses. All of these translocation junctions showed submicroscopic genomic complexity reminiscent of chromothripsis. The breakpoints were clustered within small genomic domains with junctions showing microhomology or microinsertions. Notably, all of the de novo cases were of paternal origin. The breakpoint distributions corresponded specifically to the ATAC-seq (assay for transposase-accessible chromatin with sequencing) read data peak of mature sperm and not to other chromatin markers or tissues. We propose that DNA breaks in CCRs may develop in an accessible region of densely packaged chromatin during post-meiotic spermiogenesis.

OBJECTIVE: Xq chromosome duplication with complex rearrangements is generally acknowledged to be associated with neurodevelopmental disorders such as Pelizaeus-Merzbacher disease (PMD) and MECP2 duplication syndrome. For couples who required a PGT-M (pre-implantation genetic testing for monogenic disease) for these disorders, junction-specific PCR is useful to directly detect pathogenic variants. Therefore, pre-clinical workup for PGT-M requires the identification of the junction of duplicated segments in PMD and MECP2 duplication syndrome, which is generally difficult. METHODS: In this report, we used nanopore long-read sequencing targeting the X chromosome using an adaptive sampling method to identify breakpoint junctions in disease-causing triplications. RESULTS: By long-read sequencing, we successfully identified breakpoint junctions in one PMD case with PLP1 triplication and in another MECP2 triplication case in a single sequencing run. Surprisingly, the duplicated region involving MECP2 was inserted 45 Mb proximal to the original position. This inserted region was confirmed by FISH analysis. With the help of precise mapping of the pathogenic variant, we successfully re-established STR haplotyping for PGT-M and avoided any potential misinterpretation of the pathogenic allele due to recombination. CONCLUSION: Long-read sequencing with adaptive sampling in a PGT-M pre-clinical workup is a beneficial method for identifying junctions of chromosomal complex structural rearrangements. This article is protected by copyright. All rights reserved.