倉橋 浩樹

BACKGROUND: Intrachromosomal insertion is a rare form of structural chromosomal rearrangement that often cannot be accurately delineated by conventional G-banding, making it difficult to predict reproductive outcomes. In clinical practice, such insertions are often misinterpreted as inversions or remain undetected, leading to recurrent segmental imbalances in offspring. We aimed to characterize an unresolved structural rearrangement identified in a family and to clarify its reproductive implications through advanced cytogenetic and molecular analyses. METHODS: Cytogenetic and molecular studies were conducted in a family where the proband exhibited a 17.8 Mb duplication at 9q21.31-q22.33. Although G-banding suggested a parental structural abnormality, its configuration could not be precisely defined. Subsequent preimplantation genetic testing for structural rearrangements (PGT-SR) using shallow whole-genome sequencing was performed on embryos, and further structural characterization was achieved through fluorescence in situ hybridization (FISH) and nanopore long-read sequencing. RESULTS: PGT-SR identified recurrent segmental imbalances involving the same region as in the proband, including four duplications and one deletion among 13 embryos. FISH and long-read sequencing demonstrated that the paternal rearrangement represented an intrachromosomal inverted insertion, described as ins(9)(q34.13q22.33q21.31). The father was phenotypically normal but transmitted unbalanced gametes generated by recombination between the insertion and original sites, leading to recurrent chromosomal abnormalities. CONCLUSIONS: This case highlights the potential of intrachromosomal insertions, although balanced in carriers, to cause recurrent segmental duplications or deletions in offspring. Comprehensive analysis using FISH and long-read sequencing is essential for accurate diagnosis, appropriate genetic counseling, and informed reproductive decision-making.

BACKGROUND: LHX8 gene encodes a germ cell specific transcription factor that is required for oocyte development. We evaluated two unrelated women with primary infertility who showed reproducible oocyte abnormalities across in vitro fertilization cycles, and we performed genomic and functional assays to clarify the role of LHX8. RESULTS: Whole exome sequencing identified heterozygous loss-of-function variants in LHX8 (NM_001001933.1) in both patients: c.778 C > T (p.Gln260Ter) in family 1 and c.581-1G > A in family 2. Both variants met the American College of Medical Genetics and Genomics criteria for likely pathogenicity. The two patients had high proportions of degenerated or immature oocytes and showed consistent morphologic features, including multiple cytoplasmic vacuoles, impaired zona pellucida function with accumulation of sperm in the perivitelline space, and poor embryo development. The splice site variant was inherited from a fertile mother, which indicates incomplete penetrance. A minigene assay confirmed the use of a cryptic acceptor site that produced a one nucleotide deletion and a frameshift, consistent with loss of function. CONCLUSIONS: These findings expand the phenotypic spectrum of LHX8 related infertility and provide mechanistic evidence that partial reduction of LHX8 activity compromises oocyte quality. Recognition of the characteristic morphology may guide genetic testing and counseling in cases of unexplained infertility. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13048-026-01978-2.

OBJECTIVE: Myotonic dystrophy type 1 (DM1) is an autosomal dominant neurodevelopmental disorder caused by CTG repeat expansion in the DMPK gene. Although the clinical classification of DM1 is determined by the CTG repeat length in DMPK, conventional sizing relies on Southern blotting, which is a suboptimal method in prenatal and PGD contexts as it requires large amounts of genomic DNA. We here evaluated the utility of nanopore long read sequencing (LRS) for DM1 diagnosis in these contexts. METHOD: LRS was performed with adaptive sampling or CRISPR/Cas9-mediated enrichment targeting DMPK. The use of whole genome amplified DNA (WGA-DNA) prepared with RepliG was also assessed. RESULTS: Adaptive sampling and Cas9-based LRS enabled detection of both the normal and expanded alleles. Further, LRS with CRISPR/Cas9-mediated enrichment improved efficiency and enabled accurate sizing of expanded CTG repeats exceeding 1000 units. In contrast, the use of whole genome amplified DNA prepared with RepliG did not permit reliable CTG repeat sizing, even when combined with adaptive sampling or CRISPR/Cas9. CONCLUSION: Nanopore sequencing can potentially replace Southern blotting for prenatal DM1 diagnosis, including repeat sizing. However, further improvement is needed for PGD using WGA-DNA.

RESEARCH QUESTION: What is the prevalence of tubulin beta 8 class VIII (TUBB8) variants in Japanese women with oocyte/zygote/embryo maturation arrest (OZEMA), and what are the phenotype-genotype correlations in this population? DESIGN: This multicentre retrospective study analysed 39 Japanese women with primary infertility characterized by OZEMA. Whole-exome sequencing was performed to identify TUBB8 variants, followed by Sanger sequencing, in-silico analysis, and structural modelling. Clinical data were collected across multiple treatment cycles, including oocyte maturation, fertilization, and embryonic development. Time-lapse imaging observed abnormal fertilization processes. RESULTS: Six TUBB8 variants were identified in 15.4% (6/39) of participants, including one novel homozygous nonsense variant (p.Thr136*) and two novel heterozygous missense variants (p.Phe294Leu and p.Trp344Arg). All six patients exhibited degrees of oocyte maturation arrest, predominantly at metaphase I. The novel homozygous nonsense variant was uniquely associated with multipronuclear (MPN) zygote formation. Both novel heterozygous missense variants, one confirmed de novo, caused complete metaphase I arrest. Time-lapse imaging in the patient with the homozygous nonsense variant demonstrated impaired polar body extrusion characterized by unusually broad cytoplasmic protrusions. These protrusions were reabsorbed into the oocyte, resulting in MPN zygotes. CONCLUSIONS: In this first study of TUBB8 in Japanese patients with OZEMA, three novel variants with unique phenotype-genotype correlations were identified. The phenotypes observed with the homozygous p.Thr136* variant suggest that complete TUBB8 loss disrupts not only meiotic progression but also post-fertilization events, including second polar body extrusion and pronuclear formation. These findings expand the spectrum of TUBB8-related reproductive phenotypes, and suggest that genetic testing can help avoid repeated unsuccessful treatments for primary infertility.

INTRODUCTION: Alternative RNA splicing adds diverse variations to gene function, and its abnormalities are occasionally associated with the etiology of disease. We examined this possibility in pre-eclampsia. METHODS: We performed transcriptome analysis of placentas from pre-eclamptic and normotensive pregnancies and screened for disease-specific aberrant splicing. RESULTS: We identified aberrant splicing at exon 14 in the ZC3H4 gene. This in-frame exon is generally skipped in placentas from normal pregnancies but often observed in those from pre-eclampsia patients. The level of exon inclusion did not correlate with disease severity, such as blood pressure or fetal weight, but showed an association with the decrease in placental weight. Significantly, placental blood flow resistance measured by Doppler ultrasound correlated with the level of ZC3H4 exon 14 inclusion, suggesting that this retention leads to the onset and/or symptoms of pre-eclampsia. ZC3H4 is known to act on transcriptional regulation via suppression of lncRNA expression. Moreover, the SOD1 gene, encoding superoxide dismutase that eliminates toxic free superoxide radicals, was identified in the downstream gene group for ZC3H4. Indeed, the expression of SOD1 was found in this current study to be decreased in the pre-eclamptic placenta in correlation with the levels of ZC3H4 exon 14 retention. DISCUSSION: Aberrant splicing of ZC3H4 gene may induce excessive oxidative stress in the placenta via the downregulation of downstream SOD1 expression thereby leading to the onset and development of pre-eclampsia.