AIM: To establish cell line FL-POL? - and to study the role of POL?(polymerase kappa) on genetic stability. METHODS: A mammalian expression vector expressing antisense POL? gene fragment pMAMneo -amp -- POL? was constructed by cloning the 1 690-1 918 fragment of POL? gene into the mammalian expression vector pMAMneo-amp - in antisense orientation. FL cells were fransfected with this antisense RNA expressing vector and selected by G418. Based on the shuttle-plasmid pZ189, the mutation assay was made. RESULTS: The spontaneous mutation frequency of supF tRNA gene in the plasmid replicated in the FL-POL? - was 11.2?10 -4 , while it was 4.9?10 -4 and 3.7?10 -4 in the control cells FL and FL-M , respectively. CONCLUSION: POL? playes an important role in maintenance of genetic stability.

OBJECTIVE: To analyze the (CGG)n repeats of FMR1 gene among patients with unexplained mental retardation. METHODS: For 201 patients with unexplained mental retardation, the (CGG)n repeats of the FMR1 gene were analyzed by PCR and FragilEase RESULTS: For the 201 patients with unexplained mental retardation, 15 were identified with full mutations of the FMR1 gene. The prevalence of fragile X syndrome (FXS) in patients with unexplained mental retardation was determined as 7.5% (15/201). Prenatal diagnosis was provided for 6 pregnant women with pre- or full mutations. Analysis revealed that women with mental retardation and full FMR1 mutations exhibited a skewed XCI pattern with primary expression of the X chromosome carrying the mutant allele. CONCLUSION FXS has a high incidence among patients with unexplained mental retardation. Analysis of FMR1 gene (CGG)n repeats in patients with unexplained mental retardation can facilitate genetic counseling and prenatal diagnosis for their families. FMR1 gene (CGG)n repeats screening should be recommended for patients with unexplained mental retardation.
Female ; Fragile X Mental Retardation Protein/genetics* ; Fragile X Syndrome/genetics* ; Humans ; Intellectual Disability/genetics* ; Mutation ; Pregnancy ; Prenatal Diagnosis

Objective:To carry out cytogenetic and molecular genetic analysis for two infertile patients carrying rare small supernumerary marker chromosomes (sSMC).Methods:Two infertile patients who received reproductive and genetic counseling at CITIC Xiangya Reproductive and Genetic Hospital on October 31, 2018 and May 10, 2021, respectively were selected as the study subjects. The origin of sSMCs was determined by conventional G banding, fluorescence in situ hybridization (FISH) and copy number variation sequencing (CNV-seq). Microdissection combined with high-throughput whole genome sequencing (MicroSeq) was carried out to determine the fragment size and genomic information of their sSMCs. Results:For patient 1, G-banded karyotyping and FISH revealed that he has a karyotype of mos47, XY, del(16)(p10p12), + mar[65]/46, XY, del(16)(p10p12)[6]/48, XY, del(16)(p10p12), + 2mar[3].ish mar(Tel 16p-, Tel 16q-, CEP 16-, WCP 16+ ). CNV analysis has yielded a result of arr[GRCh37]16p12.1p11.2(24999364_33597595)×1[0.25]. MicroSeq revealed that his sSMC has contained the region of chromosome 16 between 24979733 and 34023115 (GRCh37). For patient 2, karyotyping and reverse FISH revealed that she has a karyotype of mos 47, XX, + mar[37]/46, XX[23].rev ish CEN5, and CNV analysis has yielded a result of seq[GRCh37]dup(5)(p12q11.2)chr5: g(45120001_56000000)dup[0.8]. MicroSeq results revealed that her sSMC has contained the region of chromosome 5 between 45132364 and 55967870(GRCh37). After genetic counseling, both couples had opted in vitro fertilization (IVF) treatment and preimplantation genetic testing (PGT). Conclusion:For individuals harboring sSMCs, it is vital to delineate the origin and structural characteristics of the sSMCs for their genetic counseling and reproductive guidance. Preimplantation genetic testing after microdissection combined with high-throughput whole genome sequencing (MicroSeq-PGT) can provide an alternative treatment for carrier couples with a high genetic risk.

OBJECTIVE: To summarize the genetic characteristics of 671 Chinese pedigrees affected with Duchenne/Becker muscular dystrophy (DMD/BMD). METHODS: Clinical data of the pedigrees were collected. Multiplex PCR, multiple ligation dependent probe amplification (MLPA), next generation sequencing (NGS), Sanger sequencing and long read sequencing were used to detect the variant of DMD gene in the probands and their mothers, and prenatal diagnosis was provided for high risk pregnant women. RESULTS: Among 178 pedigrees analyzed by multiplex PCR, 44 variants of the DMD gene were detected, with the genetic diagnosis attained in 110 pedigrees. Among 493 pedigrees analyzed by MLPA in combination with NGS or Sanger sequencing, 294 pathogenic/possible pathogenic variants were identified, among which 45 were unreported previously, and the genetic diagnosis attained in 484 pedigrees. Structural variants of the DMD gene were identified in two pedigrees by long-read sequencing. Among 444 probands, 341 have inherited the DMD gene variant from their mothers (76.8%). Among 390 women with a high-risk, 339 have opted to have natural pregnancy and 51 chose preimplantation genetic testing for monogenetic disease (PGT-M). The detection rate of neonatal patients and carriers following natural pregnancy was significantly higher than that for PGT-M. CONCLUSION Combined application of MLPA, NGS, Sanger sequencing and long-read sequencing is an effective strategy to detect DMD/BMD. PGT-M can effectively reduce the risk of fetuses. Above finding has expanded the spectrum of DMD gene variants and provided a basis for reproductive intervention for pregnancies with a high risk for DMD/BMD.
China ; Dystrophin/genetics* ; Exons ; Female ; Genetic Testing ; Humans ; Infant, Newborn ; Multiplex Polymerase Chain Reaction ; Muscular Dystrophy, Duchenne/genetics* ; Mutation ; Pedigree ; Pregnancy ; Prenatal Diagnosis

Primary ciliary dyskinesia (PCD) is a congenital, motile ciliopathy with pleiotropic symptoms. Although nearly 50 causative genes have been identified, they only account for approximately 70% of definitive PCD cases. Dynein axonemal heavy chain 10 (DNAH10) encodes a subunit of the inner arm dynein heavy chain in motile cilia and sperm flagella. Based on the common axoneme structure of motile cilia and sperm flagella, DNAH10 variants are likely to cause PCD. Using exome sequencing, we identified a novel DNAH10 homozygous variant (c.589C > T, p.R197W) in a patient with PCD from a consanguineous family. The patient manifested sinusitis, bronchiectasis, situs inversus, and asthenoteratozoospermia. Immunostaining analysis showed the absence of DNAH10 and DNALI1 in the respiratory cilia, and transmission electron microscopy revealed strikingly disordered axoneme 9+2 architecture and inner dynein arm defects in the respiratory cilia and sperm flagella. Subsequently, animal models of Dnah10-knockin mice harboring missense variants and Dnah10-knockout mice recapitulated the phenotypes of PCD, including chronic respiratory infection, male infertility, and hydrocephalus. To the best of our knowledge, this study is the first to report DNAH10 deficiency related to PCD in human and mouse models, which suggests that DNAH10 recessive mutation is causative of PCD.
Humans ; Male ; Animals ; Mice ; Semen/metabolism* ; Dyneins/metabolism* ; Cilia/metabolism* ; Mutation ; Ciliary Motility Disorders/genetics*