Tingting Liu, Mingtao Huang, Min Dai, Yixuan Liang, Cuiping Zhang, Jianxin Tan, Fengchang Qiao, Yan WangDepartment of Prenatal Diagnosis, Women’s Hospital of Nanjing Medical University, Nanjing Women and Children’s Healthcare Hospital, Nanjing, Jiangsu, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yan Wang, Email wangyan@njmu.edu.cn Fengchang Qiao, Email fengchang-qiao@163.comBackground: Global developmental delay (GDD)/intellectual disability (ID) represent a group of neurodevelopmental disorders (NDDs) with onset in childhood with a global prevalence of 1– 3%. The etiology of GDD/ID is complex, with genetic factors accounting for about half of cases. Exome sequencing (ES) has proven to be an effective diagnostic tool for genetically heterogeneous disorders such as GDD/ID. This study aims to identify the genetic etiologies using ES in a Chinese patient with NDD.Methods: Clinical data were collected through physical examination. Trio-ES was conducted to investigate the potential genetic causes, followed by Sanger sequencing for validation.Results: The proband exhibited facial dysmorphisms, motor and language development delay, ID, autism spectrum disorder, and attention-deficit hyperactivity disorder symptoms. Trio-ES identified a novel maternal inherited hemizygous nonsense variant c.205G>T (p.E69*) in ZNF711, and a novel de novo missense variant c.673C>T (p.R225C) in SRCAP. Both of variants were classified as likely pathogenic. Phenotype dissection revealed ZNF711 dominated neurodevelopmental abnormalities (Intellectual developmental disorder, X-linked 97, XLID97) and SRCAP contributed to multisystemic manifestations (developmental delay, hypotonia, musculoskeletal defects, and behavioral abnormalities, DEHMBA).Conclusion: To our knowledge, this study reports the first case of concurrent ZNF711 and SRCAP variants in China, expanding variant spectrum of both genes. It highlights the critical value of ES for detecting multi-gene variants in probands with complex phenotypes.Keywords: global developmental delay, intellectual disability, exome sequencing, ZNF711, SRCAP, ASD, ADHD
Purpose:To diagnose and perform preimplantation genetic testing for monogenic disorders (PGT-M) in a Chinese family affected by Cornelia de Lange syndrome type 5, resulting from a microdeletion in Xq13.1q13.2 truncating the HDAC8 gene characterized by low-level gonadal mosaicism. Patients and Methods:A de novo copy number variation (CNV) was identified through chromosomal microarray analysis and whole exome sequencing in a family with two unsuccessful pregnancies, indicating germline mosaicism. The CNV was validated via real-time quantitative PCR. Whole-genome low-coverage mate-pair sequencing was conducted on female peripheral blood to exclude chromosomal abnormalities. Long-PCR amplified the deleted fragment, utilizing primers designed nearby breakpoints identified through chromosomal microarray analysis. Oxford Nanopore Technology sequencing pinpointed specific breakpoint positions. Droplet-digital PCR (ddPCR) confirmed germline mosaicism in ovarian samples. Results:A female patient, suspected of harboring a de novo microdeletion at Xq13.1q13.2 exhibiting gonadal mosaicism, along with her husband, participated in this study. The diagnoses of the 172.3 kb microdeletion at Xq13.1q13.2 as low-level gonadal mosaicism was corroborated through nanopore sequencing and ddPCR. We constructed the high-risk haplotype using the affected products of conception as the phasing reference, and performed PGT-M based on SNP haplotype linkage analysis, finally achieving a healthy live birth in February 2023. Conclusion:Our findings underscore the efficacy of PGT-M employing haplotype linkage analysis for CNVs less than 1 Mb, even within cases involving gonadal mosaicism. We present methodologies to address microdeletions associated with gonadal mosaicism utilizing next-generation sequencing, microarray, nanopore sequencing and ddPCR techniques. Our results advocate for an expansion of PGT-M based on haplotype linkage analysis for families with minor pathogenic CNVs.
Lulu Meng,1,* Yan Wang,1,* Junqiang Zhang,2,* Ran Zhou,1 Xingxing Wang,3 Fengchang Qiao,1 Qinxin Zhang,1 Cheng Wan,3 Shujing Jiao,3 Ping Hu,1 Zhengfeng Xu11Department of Prenatal Diagnosis, Women’s Hospital of Nanjing Medical University (Nanjing Women and Children’s Healthcare Hospital), Nanjing, Jiangsu, People’s Republic of China; 2State Key Laboratory of Reproductive Medicine, Department of Reproduction Medicine, Women’s Hospital of Nanjing Medical University (Nanjing Women and Children’s Healthcare Hospital), Nanjing, Jiangsu, People’s Republic of China; 3Department of Research and Development, Yikon Genomics Co. Ltd, Suzhou, Jiangsu, People’s Republic of China*These authors contributed equally to this workCorrespondence: Ping Hu, Department of Prenatal Diagnosis, Women’s Hospital of Nanjing Medical University (Nanjing Women and Children’s Healthcare Hospital), 123 Tianfei Alley, Mochou Road, Nanjing, Jiangsu, 210004, People’s Republic of China, Email njfybjyhuping@163.com Zhengfeng Xu, Department of Prenatal Diagnosis, Women’s Hospital of Nanjing Medical University (Nanjing Women and Children’s Healthcare Hospital), 123 Tianfei Alley, Mochou Road, Nanjing, Jiangsu, 210004, People’s Republic of China, Email zhengfengxu@njmu.edu.cnPurpose: To diagnose and perform preimplantation genetic testing for monogenic disorders (PGT-M) in a Chinese family affected by Cornelia de Lange syndrome type 5, resulting from a microdeletion in Xq13.1q13.2 truncating the HDAC8 gene characterized by low-level gonadal mosaicism.Patients and Methods: A de novo copy number variation (CNV) was identified through chromosomal microarray analysis and whole exome sequencing in a family with two unsuccessful pregnancies, indicating germline mosaicism. The CNV was validated via real-time quantitative PCR. Whole-genome low-coverage mate-pair sequencing was conducted on female peripheral blood to exclude chromosomal abnormalities. Long-PCR amplified the deleted fragment, utilizing primers designed nearby breakpoints identified through chromosomal microarray analysis. Oxford Nanopore Technology sequencing pinpointed specific breakpoint positions. Droplet-digital PCR (ddPCR) confirmed germline mosaicism in ovarian samples.Results: A female patient, suspected of harboring a de novo microdeletion at Xq13.1q13.2 exhibiting gonadal mosaicism, along with her husband, participated in this study. The diagnoses of the 172.3 kb microdeletion at Xq13.1q13.2 as low-level gonadal mosaicism was corroborated through nanopore sequencing and ddPCR. We constructed the high-risk haplotype using the affected products of conception as the phasing reference, and performed PGT-M based on SNP haplotype linkage analysis, finally achieving a healthy live birth in February 2023.Conclusion: Our findings underscore the efficacy of PGT-M employing haplotype linkage analysis for CNVs less than 1 Mb, even within cases involving gonadal mosaicism. We present methodologies to address microdeletions associated with gonadal mosaicism utilizing next-generation sequencing, microarray, nanopore sequencing and ddPCR techniques. Our results advocate for an expansion of PGT-M based on haplotype linkage analysis for families with minor pathogenic CNVs.Keywords: preimplantation genetic testing for monogenic disease, PGT-M, Cornelia de Lange syndrome, CdLS, low-level gonadal mosaicism, nanopore sequencing, droplet-digital PCR
Purpose:The prevalence of consanguineous marriages remains high in several regions worldwide, particularly the Middle East and South Asia, with a reported rate of 58%. However, the prevalence and genetic characteristics of consanguineous marriages in Mongolia are not well studied. This study aimed to estimate the frequency of consanguineous marriages in selected Mongolian populations, using genealogical and short tandem repeat (STR) analyses. Patients and Methods:This cross-sectional study was conducted at the Institute of Biomedicine at the Mongolian National University of Medical Sciences. A total of 574 individuals from 185 families across 43 soums in three provinces (X1, X2, and X3) were included. Genealogical analysis was used to construct pedigree-based consanguinity profiles, and STR analysis was performed to validate biological relatedness in selected families. Results:Seventeen families were consanguineous, with a mean inbreeding coefficient of 0.089 ± 0.136, whereas 42 families were classified as suspected consanguinity. Among the confirmed cases, seven families with complete biological samples were further analyzed using STR markers, of which five showed molecular evidence of consanguinity (probabilities, 76.18-98.84%). The STR analysis of 29 families classified as suspected or non-consanguineous identified 13 families as non-consanguineous and 10 families showing possible or indeterminate relatedness. Notably, six families initially classified as suspected or non-consanguineous demonstrated molecular evidence of consanguinity (probabilities, 70.75-99.85%). Genetic diversity analysis revealed reduced heterozygosity in offspring from consanguineous families (expected heterozygosity [HE] = 0.7580, observed heterozygosity[HO] = 0.7045) compared with offspring from non-consanguineous families (HE = 0.7858, HO = 0.8110), with a significant reduction in HO (p = 0.0009). Conclusion:This study demonstrates the value of integrating genealogical and STR-based molecular approaches for the assessment of consanguinity in Mongolia. Molecular analyses identified consanguineous relationships that were not evident from pedigree information alone and revealed reduced genetic diversity among offspring from consanguineous families. These findings provide important baseline data for understanding the genetic consequences of consanguinity in the Mongolian population and support the need for large scale epidemiological and genetic investigation.
Purpose:Multiple morphological abnormalities of the sperm flagella (MMAF), uncommonly causing primary infertility, are typical features of aberrant spermatozoa flagellum morphologies, which manifest as shortness, absence, bending, coiling, and irregularity of flagella. CFAP44, an important component of flagella assembly, has attracted significant interest due to its critical role in MMAF pathogenesis. Understanding the variants associated with CFAP44 can provide insights into the molecular mechanisms underlying MMAF. Patients and Methods:A comprehensive clinical evaluation was conducted on an infertile Chinese male patient from a nonconsanguineous family with sever asthenozoospermia (no progressive sperm). By performing whole-exome sequencing (WES), a novel variant of CFAP44 was identified. Sanger sequencing was performed to confirm the variant. To better investigate its pathogenicity, In silico variant analyses, minigene splicing assays and RT-PCR in vivo were performed. Results:As a result, a CFAP44 homozygous deep-intronic variant (NM_001164496.1:c.1890+5G>C) was detected in the proband by WES. Sanger sequencing confirmed this variant in this family. Splice site prediction suggested that this variant may be a disease-causing variant. Then, exon 15 skipping was identified through minigene assays and RT-PCR in vivo, resulting in a 111-bp deletion within the mutated sequence, thereby indicating a disruption in the normal splicing of the CFAP44 transcript. Conclusion:This is the first study to detect a homozygous variant (c.1890+5G>C) within the CFAP44 gene causing MMAF in a Chinese family. Our results confirmed the pathogenicity of this deep-intronic variant and expanded the mutational spectrum of the CFAP44 gene. Consequently, this study may help elucidate the effect of CFAP44 on MMAF and provide a theoretical basis for MMAF.
