OBJECTIVE:To explore the clinical manifestations and genetic etiology of a boy with congenital variant Rett syndrome (RTT). METHODS:A boy presented at the Medical Genetics Center of Northwest Women's and Children's Hospital in June 2023 due to "mental retardation" was selected as the study subject. Clinical data including history of birth, growth and development, and clinical manifestations were collected. Peripheral blood samples were collected from the boy and his parents. Following extraction of genomic DNA, whole-exome sequencing (WES) was carried out. Candidate variant was verified by Sanger sequencing. Pathogenicity of the variant was rated based on the guidelines from the American College of Medical Genetics and Genomics (ACMG). Previous literature on male RTT caused by FOXG1 gene variants was retrieved from databases including China National Knowledge Infrastructure, Wanfang Data Knowledge Service Platform, PubMed, and the phenotypes of the included cases were summarized. This study was approved by the Ethics Committee of the hospital (Ethics No.: 21 -036). RESULTS:The patient, a 6-year-old male, presented with microcephaly, mental retardation, and so forth. WES revealed that he has harbored a heterozygous c.761A>G (p.Tyr254Cys) variant of the FOXG1 gene. The variant was unreported previously. Sanger sequencing verified that the variant was de novo in origin. Based on the ACMG guidelines, the variant was classified as pathogenic (PM1+PS2_Moderate+PP2+PP3_Strong+PM2_Supporting). In total seven relevant articles were retrieved. Together with our case, a total of 10 male RTT patients were included for the analysis of clinical manifestations and genetic etiology. CONCLUSION:The heterozygous c.761A>G (p.Tyr254Cys) variant of the FOXG1 gene probably underlay the pathogenesis of male RTT in this patient. Above finding has enriched the mutational spectrum of the FOXG1 gene and facilitated understanding of the genotype-phenotype correlation of congenital variant RTT.

BackgroundLung cancer remains the leading cause of cancer-related mortality worldwide, with non-small cell lung cancer (NSCLC) being the most common type. SHROOM4, a protein integral to cytoskeletal organization and cellular signaling, has not been extensively studied in NSCLC.MethodsThrough bioinformatics analysis of public databases, we investigated the expression of SHROOM4 and its relationship with clinical outcomes and potential mechanism. And we validated the mRNA and protein expression of SHROOM4 in lung squamous cell carcinoma (LUSC) tissues and corresponding normal tissues.ResultsOur analysis demonstrated a notable downregulation of SHROOM4 mRNA and protein expression, along with its high diagnosis capability in lung cancer, especially pronounced in LUSC, Additionally, higher levels of SHROOM4 were linked to worse clinical outcomes in lung cancer, characterized by reduced survival and more advanced disease stages. Single-cell RNA-seq data and differential analysis show SHROOM4’s high expression in stromal cells and its association with angiogenesis and Wnt/Beta-Catenin pathways possibly through ANGPTL7/SFTPC. Meanwhile, SHROOM4 was found to co-express with PTPN13/CACNA1C impacting the tumor microenvironment (TME) and to participate in critical signaling pathways like cell circle and WNT. Moreover, positive correlations were discovered between SHROOM4 expression and immune infiltration scores in NSCLC.ConclusionThese results underscore the potential of SHROOM4, an anticancer role, as both a diagnostic and therapeutic target, particularly in LUSC. And SHROOM4 may modulate NSCLC progression by affecting the TME in many ways. Further studies are essential to elucidate SHROOM4’s role in lung cancer progression and to validate its clinical utility.

Objectives The aim of this study was to develop and evaluate a new bioinformatics pipeline that improves fetal DNA fraction (FF), determines the origin of chromosomal abnormalities, and better detects sex chromosomal aneuploidies (SCAs) than routine NIPT.Methods We used a bioinformatic strategy that filters out longer DNA fragments, thereby significantly improving the FF. By combining the filtered data with the original data, we determined the origin of detected abnormalities.Results We analyzed samples from 204 pregnancies. Using routing NIPT methods, 36 samples were negative, 37 exhibited low FF, and 131 were detected as positive. Applying the method of this study, all the low FF samples were qualified for analysis. Furthermore, 56 samples initially classified as positive were identified to be false positives, predominantly caused by maternal abnormalities. No false negative results were observed with this method.Conclusion We developed a pipeline for NIPT that significantly improves FF, which helps deduce the origin of abnormalities and detect more karyotypes of SCAs. The strategy has the potential to improve the specificity of NIPT.

Abstract Fusobacterium nucleatum (F.n.) infection is closely related to poor prognosis and high mortality in colorectal cancer (CRC) patients, promotes the proliferation and migration of CRC cells, induces inflammation, and creates an immunosuppressive tumor microenvironment. Compared with CRC cells not infected with F.n., F.n.-infected CRC cells exhibit greater glycolytic activity, considered as a beneficial metabolic change for establishing a symbiotic relationship between CRC cells and F.n. In this study, the classical glycolysis inhibitors 2-deoxy-d-glucose (2-DG) and tanshinone IIA (TAN IIA) were coloaded into zeolitic imidazolate framework-8 nanoparticles (DTZP NPs) to suppress F.n.-infected CRC growth. TAN IIA not only eliminated intratumoral F.n. but also acted as a chemotherapeutic drug against F.n.-infected CRC cells. DTZP NPs strongly inhibited both the F.n.-infected MC38 tumor model and the azoxymethane (AOM)/dextran sulfate sodium salt (DSS)-induced spontaneous CRC model by downregulating the NF-κB/HIF-1α/glycolysis pathway. DTZP NPs’ treatment improved the immunosuppressive tumor microenvironment, decreased the inflammatory levels in both serum and tumor tissue, and modulated the gut microbiota and its metabolites. In summary, we proposed combining anti-intratumoral F.n. with adjusting the tumor metabolism strategy, which could provide new insights into the treatment of F.n.-infected CRC in the future.

IntroductionMeier-Gorlin syndrome 7 (MGORS7) is a rare autosomal recessive disorder characterized by primordial dwarfism, craniosynostosis, and patellar aplasia, caused by pathogenic variants of CDC45. Here, we report a Chinese patient presenting with classic hallmarks of MGORS7 alongside atypical clinical features, including hearing and visual impairments.MethodsClinical and radiological data were collected. Whole-genome sequencing and Sanger sequencing were performed to identify and validate the causative variants. Their functional effects were investigated using an exon-trapping assay, and a literature review of previously reported MGORS7 cases was conducted.ResultsGenetic analysis identified two compound heterozygous CDC45 variants: c.1416C>T (p.H472=) and c.1559+2T>A, which are a recurrent variant in the East Asian population and a novel variant, respectively. Our exon-trapping assay indicated that c.1559+2T>A induced aberrant splicing, generating transcripts predicted to undergo nonsense-mediated mRNA decay. Additionally, growth hormone therapy was initiated in our patient, with a noted improvement in growth parameters in the initial assessment and without immediate complications. The literature review identified a total of 32 CDC45 variants in 29 patients with MGORS7, who showed high heterogeneity in clinical phenotypes.DiscussionOur study further expanded the mutational spectrum of CDC45 and provided a preliminary clinical observation suggesting that growth hormone therapy may be beneficial for growth retardation in patients with MGORS7.

Genetically encoded fluorescent biosensors (GEFBs) are invaluable tools for spatiotemporal metabolite monitoring in cellular metabolism, yet their development for many key metabolites is hampered by a lack of specific biorecognition elements. Here, we report a versatile strategy to engineer metabolite-responsive GEFBs by leveraging the allosteric properties of regulatory domains from allosteric enzymes. Using regulatory domains from chorismate mutase, 2-acetolactate synthase, and D-citramalate synthase as biorecognition elements, we construct three biosensors for specific L-phenylalanine, L-valine, and L-isoleucine detection. We further demonstrate that multi-ligand-binding regulatory domains can be exploited to derive diverse specific biosensors, and apply this strategy to develop two S-adenosyl-L-methionine biosensors and an S-methyl-5'-thioadenosine biosensor. We also showcase the utility of these biosensors for real-time, in situ tracking of target metabolites in living cells, as well as bioprocess monitoring and clinical diagnostics. Overall, this study establishes a flexible strategy that provides insights to construct GEFBs targeting other metabolites.

Background:Clear cell renal cell carcinoma (ccRCC), is highly metastatic with unfavorable oncologic outcomes. The metastatic dissemination and underlying mechanisms of ccRCC remain insufficiently understood. The expression of fucosyltransferases (FUTs) has been explored in multiple cancer types, which affect survival of tumor cells and oncology progress. However, the role of fucosyltransferase 10 (FUT10), a member of the FUT family, is still unclear in ccRCC. We aimed to investigate the effects of FUT10 on the prognosis and immune infiltration of ccRCC via The Cancer Genome Atlas (TCGA) database. Methods:The relationship between FUT10 expression and clinical-pathologic features was evaluated by Welch's t-test, Wilcoxon signed-rank test, Dunn's test, and logistic regression based on TCGA datasets. The FUT10 expression level was converted into a categorical variable by receiver operating characteristic (ROC) and the area under the curve (AUC). The factors associated with the prognosis were determined by Kaplan-Meier method. The function of FUT10 was identified by functional enrichment analysis, gene set enrichment analysis (GSEA), gene correlation analysis, and immune infiltration analysis. At last, we verified the FUT10 messenger RNA (mRNA) expression in ccRCC and adjacent kidney tissues by quantitative real-time polymerase chain reaction (qRT-PCR). Results:Downregulated FUT10 expression in ccRCC was associated with the clinical stage (P<0.001), T stage (P<0.001), M stage (P<0.001), and overall survival (OS) event (P<0.001). The ROC curve suggested that FUT10 had a certain accuracy in the diagnostic ability in ccRCC (AUC =0.787). It was shown that patient survival was prolonged in the FUT10 high-expression group. Meanwhile, multivariate analysis displayed that FUT10 was an independent risk factor for ccRCC patients (P=0.003). Moreover, we uncovered that FUT10 was involved in the phenotype of the immune response, oxidative phosphorylation (OXPHOS), arachidonic acid (AA) metabolism, and primary immunodeficiency (PID) by function enrichment analysis and GSEA. In addition, in the high FUT10 expression group, natural killer (NK) CD56bright cells exhibited lower enrichment scores, and central memory T cells exhibited higher enrichment scores. Especially, ARL8B, a key factor in NK-mediated cytotoxicity, had a certain correlation with FUT10 (r=0.590, P<0.001). Compared to the normal kidney tissues, the FUT10 mRNA expression in the ccRCC was decreased (P=0.004). Conclusions:FUT10 might be a promising immune therapy target and prognostic biomarker in ccRCC.

Comprehensive genetic characterization and screening for congenital adrenal hyperplasia (CAH) have not yet been achieved at the population level because of the complexity of the CYP21A2 locus. This prospective study incorporated long-read sequencing (LRS) into the current first-tier biochemical newborn screening (NBS) to comprehensively characterize the variant spectrum of CYP21A2, fully investigate the carrier frequency and expected incidence of classic and non-classic CAH (NCCAH), and evaluate the clinical feasibility of genetic NBS for CAH. A total of 21,239 newborns were consecutively recruited from 11 centers across China between June 2023 and May 2024. All the participants underwent biochemical and genetic NBS. In vitro enzymatic activity and minigene assays were performed to determine the pathogenicity of novel variants. A 30.8-kb long amplicon, followed by LRS, was performed to determine the phasing of duplication chimera and single-nucleotide variations (SNVs) and indels in CYP21A2. Eligible genetic screening results were obtained for 21,234 (99.98

Newborn screening (NBS) through disease biomarkers has significantly reduced severe outcomes of congenital disorders. Moreover, exploratory newborn genetic screening programs are increasingly being implemented. This consensus, developed by multidisciplinary experts, aims to standardize the combined screening of genes and biomarkers for neonatal diseases in China, balancing ethical, technical, and clinical considerations. This consensus synthesizes evidence from peer-reviewed literature (PubMed, CNKI, etc.) up to 2024 and integrates clinical experiences from multidisciplinary experts in neonatology, genetics, and laboratory medicine, focusing on disease biomarker-based NBS, newborn genetic screening, and the clinical utility of combined screening. The consensus defines principles for combined screening: (1) disease/gene selection: 154 disease-causing genes covering 67 inherited metabolic disorders (e.g., amino acid metabolism disorders, organic acid metabolism disorders), prioritized by treatability, onset age (< 5 years), and cost-effectiveness; (2) methodology: integrating dried blood spot biomarker analysis with next-generation sequencing-based targeted capture (coverage > 300 ×), validated by MLPA/Sanger and long-range sequencing for complex variants (e.g., CYP21A2, SLC25A13); and (3) operational workflow: standardized workflows for informed consent, sample collection/delivery, and result interpretation, with dual reporting of marker and genetic findings within 15 days. Positive cases require family verification and/or other genetic sequencing techniques. This consensus establishes a practical framework for integrating marker and genetic screening, aiming to improve diagnostic accuracy and achieve rapid and effective interventions, thereby saving lives and reducing the occurrence of severe complications. Implementation requires interdisciplinary collaboration and ongoing quality control to maximize clinical utility.

Transmembrane protein 53 (TMEM53) is an outer nuclear membrane protein that plays a crucial role in maintaining skeletal homeostasis. Pathogenic variants in TMEM53 have been identified as the genetic cause of craniotubular dysplasia, Ikegawa type (CTDI), a rare form of sclerosing bone dysplasia characterized by skull hyperostosis, cranial deformities, and increased bone density. To date, the causal association of bi-allelic pathogenic variants of TMEM53 in CTDI has been identified in 14 patients from eight unrelated families. Mechanistically, TMEM53 negatively regulates BMP–SMAD signaling by restricting the nuclear import of phosphorylated SMAD1/5/9, thereby modulating osteoblast differentiation and bone formation. This review summarizes the current understanding of TMEM53 function and the consequences of its deficiency. We aim to clarify genotype-phenotype correlations, outline therapeutic prospects for CTDI, and explore the distinct mechanisms underlying cranial and tubular bone formation.

OBJECTIVE:To investigate the X-chromosome inactivation (XCI) patterns and origin in four children with Rett syndrome (RTT), and to explore the genetic basis of their phenotypic variability. METHODS:Four pediatric RTT cases diagnosed at Northwest Women's and Children's Hospital between August 1, 2022 and October 31, 2024 were enrolled. Clinical data were collected, and whole exome sequencing (WES) and Sanger sequencing were performed on the children and their parents to identify pathogenic variants. XCI analysis and linkage studies were conducted to determine the origin of variants and assess skewed XCI. This study was approved by the Medical Ethics Committee of the Northwest Women's and Children's Hospital (Ethics No. 21-036). RESULTS:WES and Sanger sequencing revealed that the four children carried the following MECP2 (NM_001110792.2) variants. c.916C>T (p.Arg306Cys), c.842delG (p.G281Afs*20), c.763C>T (p.R255X), and c.686C>T (p.Pro229Leu). The c.916C>T variant was maternally inherited, while the other three were de novo. All four variants have been previously reported: c.916C>T, c.842delG, and c.763C>T were classified as pathogenic, whereas c.686C>T was deemed likely pathogenic. XCI analysis demonstrated skewed inactivation in child 2 and 3 and their mothers, with maternal X-chromosome recombination during gametogenesis observed in child 3. All variants were located on the maternal X chromosome. CONCLUSION:Skewed XCI is a common pathogenic mechanism in MECP2-related RTT, and MECP2 variants may exhibit a maternal origin bias. Clinical evaluation should incorporate XCI status for comprehensive genetic analysis.

Craniotubular dysplasia, Ikegawa type (CTDI) is a rare autosomal recessive skeletal dysplasia characterized by hyperostosis of the calvaria and skull base, metadiaphyseal undermodeling of the long tubular bones, and mild shortening and diaphyseal broadening of the short tubular bones. Its causal gene is TMEM53. Six CTDI families have been reported; however, its clinical course and prognosis still remain to be determined. Here, we report two Iranian siblings carrying a novel homozygous missense variant of TMEM53. The affected individuals were referred for progressive severe visual loss of unknown cause. The patient had severe optic atrophy and optic canal narrowing. Radiographic evaluation suggested the diagnosis of CTDI, which was confirmed by the identification of TMEM53 variant (c.704G > T, p.R235L) co-segregating in the consanguineous family. The proband underwent trans-nasal endoscopic optic canal decompression and showed remarkable improvement in visual acuity and daily visual tasks. We recommend early comprehensive clinical and genetic evaluation followed by proper treatment to improve the prognosis of CTDI.

OBJECTIVE:To analyze the results of prenatal diagnosis for fetuses with a high risk for sex chromosome aneuploidies (SCAs) indicated by non-invasive prenatal testing (NIPT), and to assess the influence of maternal chromosomal factors on the results of NIPT. METHODS:A retrospective analysis was conducted on the clinical data of 454 pregnant women with a high risk for SCAs indicated by NIPT undergoing invasive prenatal diagnosis at the Medical Genetics Center of Northwest Women's and Children's Hospital from January 2022 to September 2024. The data has included prenatal diagnosis indications, results, pregnancy outcomes, and the chromosomal results of pregnant women. RESULTS:Among the 454 women (including 10 with twin pregnancy) with a high risk for SCAs indicated by NIPT, 149 (including 4 twin cases) were diagnosed with SCAs through invasive prenatal diagnosis. These had included 47,XXX (37 cases), 47,XXY (56 cases), 47,XYY (29 cases), 45,X (1 case), 48,XXYY (1 case), mosaicism (20 cases), sex chromosome structural abnormalities (6 cases), and small-scale pathogenic copy number variations (3 cases). 383 pregnant women (including 7 with twin pregnancy) had accepted chromosomal karyotyping analysis. In total 49 cases of SCAs abnormalities were detected. Among them, 41 cases were pregnant women with SCAs but normal fetal chromosomes, which yielded a false positive rate for NIPT caused by maternal factors by 10.7%. In addition, 8 cases (including 1 twin case) had SCAs abnormalities in both the pregnant woman and the fetus. Among the 383 pregnant women, 129 cases (including 3 twin cases) of fetal SCAs were diagnosed, which yielded an overall positive predictive value (PPV) of NIPT for SCAs by 33.7% (129/383). With the 41 false positive cases caused by maternal SCAs abnormalities excluded, the PPV of NIPT for SCAs will be increased to 37.7% (129/342). Among the 454 pregnant women, twin pregnancies have accounted for 2.2% (10/454). Among the confirmed cases of SCAs abnormalities, twin cases accounted for 2.7% (4/149). Among the 383 pregnant women undergoing chromosomal karyotyping, twin cases accounted for 1.8% (7/383). Among the detected cases of chromosomal abnormalities, twin cases accounted for 2.0% (1/49). By calculation, the proportion of singleton pregnant women with a high risk for SCAs indicated by NIPT was approximately 32.1%, and the proportion of twin pregnant women was approximately 38.6%, indicating that twin pregnancies could increase the positive rate of NIPT. CONCLUSION:NIPT can improve the screening efficiency for SCAs, but its PPV is limited. Therefore, pregnant women with a high risk for SCAs indicated by NIPT need to undergo invasive prenatal diagnosis for a definite diagnosis, and twin pregnancies can increase the positive rate of NIPT. The study confirmed that chromosomal abnormalities in pregnant women can significantly affect the accuracy of NIPT in detecting fetal SCAs. Therefore, when NIPT indicates SCAs abnormalities, it is recommended to simultaneously conduct chromosomal testing on the pregnant women. The combined application of chromosomal karyotyping analysis, fluorescence in situ hybridization, and copy number variation detection techniques can significantly improve the diagnostic accuracy for SCAs, especially for the detection of mosaicisms.

Spontaneous preterm birth (sPTB) is a significant global health concern, contributing to adverse outcomes for both pregnant women and newborns. Early identification of women with risk of sPTB is essential for mitigating these negative effects and improving maternal and neonatal health outcomes. The aim of this study is to explore the feasibility of using machine learning to predict sPTB risk and to analyze the contribution of variables. All data were collected retrospectively. Prediction models were developed using eight different machine learning algorithms combined with six variable selection methods. The models’ predictive performance was evaluated using area under the receiver operating characteristic curve (AUROC), area under the precision recall curve (AUPRC), accuracy, sensitivity, F1-score, positive predictive value, and negative predictive value. A total of 1122 pregnant women, of whom 187 had preterm birth and 935 had term birth, were enrolled. The model by combining the categorical boosting algorithm and backward elimination had the best predictive performance with the highest AUROC (0.8762) and AUPRC (0.7061), and the Brier score was 0.12 on the test set. The top 5 variables for predicting sPTB risk in this study were free triiodothyronine, albumin/globulin, thyroglobulin antibody, total thyroxine, red cell volume distribution width. The machine learning model may help identify pregnant women at high risk of sPTB, and individual risk factor analysis could provide reference for clinical decision. However, as some key variables are not part of routine laboratory tests during pregnancy worldwide, the model’s generalizability and clinical applicability require further study.

OBJECTIVE:To explore a case of abnormal fetal development due to a rare paternal t(10;14)(p11.2;p11) translocation. METHODS:A fetus undergoing prenatal diagnosis at Northwest Women's and Children's Hospital on June 21,2024 was selected as the study subject. Clinical data were collected. Amniotic fluid sample of the fetus and peripheral venous blood samples of its parents were collected for chromosomal karyotyping and copy number variation (CNV) analysis. This study was approved by the Ethics Committee of the hospital (Ethics No.: 2024-132). RESULTS:Ultrasound scan at 23+4 gestational weeks revealed nasal bone dysplasia. Amniotic fluid analysis revealed that the fetus has a karyotype of 46,X?,der(14)t(10;14)(p11.2;p11)dpat, while its father had a 46,XY,t(10;14)(p11.2;p11) karyotype. No chromosomal abnormality was found in its mother. CNV analysis revealed that the fetus had a 30.46 Mb duplication in the 10p15.3-p11.23 region. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the duplication was classified as pathogenic. CONCLUSION:By combining conventional cytogenetic methods with molecular techniques, the fetus was diagnosed with partial trisomy 10p syndrome caused by a rare paternal t(10;14)(p11.2;p11) translocation. Above finding holds significant clinical value for genetic counseling and prenatal diagnosis for the family.

The purpose of this review is to explore the multifaceted roles of the ERI1 exoribonuclease, particularly in RNA metabolism and bone development, and to address the genotype-phenotype complexity in patients and mice with ERI1 pathogenic variants. The 3’-to-5’ exoribonuclease 1 encoded by the ERI1 gene performs a variety of biologically essential functions, including modulating RNA interference, heterochromatin formation, rRNA maturation, and histone mRNA degradation. Recently, the relationship between ERI1 variants and human skeletal dysplasia has garnered increasing attention. In a phenotypic dichotomy associated with bi-allelic ERI1 variants, patients with at least one missense pathogenic variant exhibited severe spondylo-epi-metaphyseal dysplasia (SEMD), while those with bi-allelic nonsense pathogenic variant only presented mild anomaly in digits. The biological mechanisms underlying the bone dysplasia caused by ERI1 pathogenic variants remain unknown. Although Eri1 knockout (KO) mice showed mild skeletal phenotypes, neither SEMD nor digital anomaly were found, further underscoring a complex genotype-phenotype relationship of ERI1 pathogenic variants. We systematically reviewed the advances in exploring the multiple functions of ERI1 with emphasis on its roles in RNA metabolism and skeletal development. Our review would contribute to the understanding of the phenotypic spectrum caused by ERI1 pathogenic variants and the limitations of existing disease models in revealing the corresponding pathomechanism.

OBJECTIVE:To analyze four patients with a 16q22 fragile site with miscarriage or infertility by using cytogenetic methods. METHODS:Four patients presented at Northwest Women's and Children's Hospital between January 2022 and December 2024 were selected as the study subjects. Peripheral blood samples were collected from the patients and subjected to G-banded chromosomal karyotyping, among whom two were also subjected to copy number variation (CNV) sequencing. This study has been approved by the Ethics Committee of the Hospital (Ethics No. 2020-022). RESULTS:The chromosomal karyotypes of the patients were mos 46,XX,fra(16)(q22)[26]/47,XX,del(16)(q22),+chrb(16)(q22)[4]/46,XX,del(16)(q22)[3]/46,XX[91], mos 46,XY,fra(16)(q22)[21]/46,XY,del(16)(q22)[3]/46,XY[76], mos 46,XX,fra(16)(q22)[21]/ 46,XX,del(16)(q22)[4]/46,XX[75] and mos 46,XX,fra(16)(q22)[16]/46,XX,del(16)(q22)[7]/47,XX,del(16)(q22),+chrb(16)(q22)[6]/47,XX,fra(16)(q22),+chrb(16)(q22)[3]/46,XX[68], respectively. CNV sequencing of patients 2 and 4 revealed no deletion or duplication on chromosome 16. CONCLUSION:Identification of the 16q22 fragile site has facilitated genetic counseling for these patients.

Congenital chloride diarrhea (CCD) is an autosomal recessive disease, characterized by watery diarrhea, hypochloremia and metabolic alkalosis. It is associated with defects in solute carrier family 26 member 3 (SLC26A3) which acts as Na+-independent Cl−/HCO3− exchanger. Early diagnosis allows planning of perinatal care and timely treatment to improve the prognosis of CCD. However, only few cases were diagnosed in the fetus period, while most of CCD cases were diagnosed after birth without timely diagnosis and treatment. This study was conducted to verify the disease-causing gene by prenatal genetic and functional tests for assisting prenatal diagnosis of a fetus with suspected CCD and reviewed the mutation spectrum of Chinese CCD patients for the first time. Here, we reported a suspected CCD fetus with signs of polyhydramnios and intestinal dilatation by prenatal ultrasound. Subsequent prenatal Trio-whole-exome sequencing identified a novel homozygous mutation (c.383-5A > G) of SLC26A3, which was classified as uncertain significance (VUS). Mini-Gene Splicing Assay confirmed the effect of VUS variant on abnormal splicing of SLC26A3, increasing pathogenicity evidence, and determining the prenatal diagnosis and subsequent treatment of CCD. Literature review of 18 CCD cases showed that t c.270_271insAA (p.G91Kfs*3) was the most frequent mutation in China. In conclusion, our study found the novel c.383-5A > G mutation of SLC26A3 as the pathogenic cause in the proband, which expanded the mutation spectrum of SLC26A3. There also highlights the importance of integrative genetic and functional tests that provide a reference for prenatal diagnosis of suspected CCD to access postnatal management in a timely manner.

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders predominantly characterized by impaired corticosteroid synthesis. Clinical phenotypes include hypoadrenocorticism, electrolyte disturbances, abnormal gonadal development, and short stature, of which severe hyponadrenocorticism and salt wasting can be life-threatening. Genetic analysis can help in the clinical diagnosis of CAH. However, the 21-OHD-causing gene CYP21A2 is arranged in tandem with the highly homologous CYP21A1P pseudogene, making it difficult to determine the exact genotypes using the traditional method of multiplex ligation-dependent probe amplification (MLPA) plus Sanger sequencing or next-generation sequencing (NGS). We applied a long-read sequencingbased approach termed comprehensive analysis of CAH (CACAH) to 48 newborns with CAH that were diagnosed by clinical features and the traditional MLPA plus Sanger sequencing method for retrospective analysis, to evaluate its efficacy in the clinical diagnosis of neonatal CAH. Compared with the MLPA plus Sanger sequencing method, CACAH showed 100 % consistency in detecting SNV/indel variants located in exons and exon-intron boundary regions of CAH-related genes. It can directly determine the cis-trans relationship without the need to analyze parental genotypes, which reduces the time to diagnosis. Moreover, CACAH was able to distinguish different CYP21A1P/CYP21A2 and TNXA/TNXB chimeras, and detect additional variants (CYP21A2 variants c.121C > T, c.*13G > A, c.*52C > T, c.*440C > T, c.*443 T > C, and TNXB variants c.12463 + 2 T > C, c.12204 + 5G > A). We also identified the TNXB variant c.11435_11524 + 30del alone instead of as a part of the TNXA/ TNXB-CH-1 chimera in two newborns, which might be introduced by gene conversion. All of these characteristics enabled clinicians to better explain the phenotype of subjects and manage them more effectively. CACAH has a great advantage over the traditional MLPA and Sanger sequencing methods, showing substantial potential in the genetic diagnosis and screening of neonatal CAH.

Spondylocostal dysostosis (SCDO) encompasses a group of skeletal disorders characterized by multiple segmentation defects in the vertebrae and ribs. SCDO has a complex genetic etiology. This study aimed to analyze and identify pathogenic variants in a fetus with SCDO. Copy number variant sequencing and whole exome sequencing were performed on a Chinese fetus with SCDO, followed by bioinformatics analyses, in vitro functional assays and a systematic review on the reported SCDO cases with LFNG pathogenic variants. Ultrasound examinations in utero exhibited that the fetus had vertebral malformation, scoliosis and tethered cord, but rib malformation was not evident. We found a novel homozygous variant (c.1078 C > T, p.R360C) within the last exon of LFNG. The variant was predicted to cause loss of function of LFNG by in silico prediction tools, which was confirmed by an in vitro assay of LFNG enzyme activity. The systematic review listed a total of 20 variants of LFNG in SCDO. The mutational spectrum spans across all exons of LFNG except the last one. This study reported the first Chinese case of LFNG-related SCDO, revealing the prenatal phenotypes and expanding the mutational spectrum of the disorder.