HIROKI KURAHASHI

Human trisomy 21, responsible for Down syndrome, is the most prevalent genetic cause of cognitive impairment and remains a key focus for prenatal and preimplantation diagnosis. However, research directed toward eliminating supernumerary chromosomes from trisomic cells is limited. The present study demonstrates that allele-specific multiple chromosome cleavage by clustered regularly interspaced palindromic repeats Cas9 can achieve trisomy rescue by eliminating the target chromosome from human trisomy 21 induced pluripotent stem cells and fibroblasts. Unlike previously reported allele-nonspecific strategies, we have developed a comprehensive allele-specific (AS) Cas9 target sequence extraction method that efficiently removes the target chromosome. The temporary knockdown of DNA damage response genes increases the chromosome loss rate, while chromosomal rescue reversibly restores gene signatures and ameliorates cellular phenotypes. Additionally, this strategy proves effective in differentiated, nondividing cells. We anticipate that an AS approach will lay the groundwork for more sophisticated medical interventions targeting trisomy 21.

UBA1 is an E1 ubiquitin-activating enzyme that initiates the ubiquitylation of target proteins and is thus a key component of the ubiquitin signaling pathway. Three disorders are associated with pathogenic variants of the UBA1 gene: vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS) syndrome, lung cancer in never smokers (LCINS), and X-linked spinal muscular atrophy (XL-SMA, SMAX2). We here report a case of infantile respiratory distress syndrome followed by continuing neuromuscular symptoms. We identified a de novo hemizygous mutation, c.1660 C > T (p.Pro554Ser), in exon 15 of the UBA1 gene in this baby. This missense mutation was located with the AAD (active adenylation domain) of the protein, a known hotspot of SMAX2 mutations. This case lends support to the genotype-phenotype correlation regarding the UBA1 mutation and its related diseases.

＜文献概要＞▽網羅的染色体解析による検査手法の確立により,着床前診断(preimplantation genetic testing:PGT)の診断精度は大きく向上した.▽PGTにまつわる倫理的な議論,特にPGT-M(重篤な遺伝性疾患を対象とした着床前遺伝学的検査)に関連した内容は継続して行われており,検査適応については症例ごとの詳細な検討が必要となる.▽PGTに関連した技術は現在も進歩を続けており,検査の応用範囲は今後さらに拡大していく可能性が高い.

Mixed gonadal dysgenesis (MGD) is a disorder of sex development caused by mosaicism of the Y chromosome, represented by 45,X/46,XY. Prophylactic gonadectomy is recommended as soon as possible after its diagnosis, owing to a high risk of malignancy. In the present case, a 21-year-old woman presented with primary amenorrhea. Although the patient's external genitalia were female, the patient exhibited a hypoplastic uterus, wherein the ovaries were difficult to identify. The patient's height was 146 cm; they had cubitus valgus and webbing of the neck, leading to the consideration of a disorder of sex development. Chromosomal examination revealed 45,X/46,XY mosaicism. Thus, the patient was diagnosed with MGD. After thorough counseling, laparoscopic bilateral gonadectomy was performed. Pathological examination revealed a gonadoblastoma of the left gonad. Postoperatively, the patient had no recurrence and continued on Kaufmann therapy. In conclusion, prophylactic gonadectomy is recommended immediately following a diagnosis of MGD; however, the timing of the surgery should be carefully considered and adequate counseling should be conducted by a multidisciplinary team.

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.