Legume nodulation and nitrogen fixation require interaction of DnaJ-like protein and lipid transfer protein.

The lipid transport protein (LTP) product of the AsE246 gene of Chinese milk vetch (Astragalus sinicus) contributes to the transport of plant-synthesized lipids to the symbiosome membranes (SMs) that are required for nodule organogenesis in this legume. However, the mechanisms used by nodule-specific LTPs remain unknown. In this study, a functional protein in the DnaJ-like family, designated AsDJL1, was identified and shown to interact with AsE246. Immunofluorescence showed that AsDJL1 was expressed in infection threads (ITs) and in nodule cells and that it co-localized with rhizobium, and an immunoelectron microscopy assay localized the protein to SMs. Via co-transformation into Nicotiana benthamiana cells, AsDJL1 and AsE246 displayed subcellular co-localization in the cells of this heterologous host. Co-immunoprecipitation assays confirmed that AsDJL1 interacted with AsE246 in nodules. The essential interacting region of AsDJL1 was determined to be the zinc finger domain at its C-terminus. Chinese milk vetch plants transfected with AsDJL1-RNAi had significantly decreased numbers of ITs, nodule primordia and nodules as well as reduced (by 83%) nodule nitrogenase activity compared with the controls. By contrast, AsDJL1 overexpression led to increased nodule fresh weight and nitrogenase activity. RNAi-AsDJL1 also significantly affected the abundance of lipids, especially digalactosyldiacylglycerol, in early-infected roots and transgenic nodules. Taken together, the results of this study provide insights into the symbiotic functions of AsDJL1, which may participate in lipid transport to SMs and play an essential role in rhizobial infection and nodule organogenesis.

A novel MED12 pathogenic variant in a female fetus with facial cleft and cardiac defects identified in the first trimester.

Trio exome sequencing was performed on a female fetus with an increased nuchal translucency, along with nasal bone hypoplasia, suspected cleft palate and abnormal outflow tract of the heart. A de novo heterozygous variant c.5500_5507del, p.(Tyr1834Argfs × 58) in the MED12 gene was detected. Loss-of-function variants in MED12 in females are associated with Hardikar syndrome (HS). A follow-up ultrasound at 15+5 weeks of gestation identified multiple fetal anomalies including bilateral cleft lip and palate, diaphragmatic hernia, atrioventricular septal defect, persistent truncus arteriosus, and bilateral renal pelvis dilation. Fetal autopsy confirmed the prenatal sonographic findings, and the MED12 variant was discussed by our multidisciplinary team to be the cause of fetal anomalies. Our case is the first prenatal one in which HS was diagnosed due to first trimester structural malformations. This case report presents another example of early identification of a major anomaly which allows earlier genetic diagnosis and more time for clinical management.

Phenotypic and genotypic analysis of 11 fetal cases with Bardet-Biedl syndrome.

OBJECTIVE: To present the prenatal sonographic features and genomic spectrum of pregnancies with fetal Bardet-Biedl syndrome (BBS). METHODS: This was a retrospective study of 11 cases with BBS diagnosed by prenatal ultrasound and confirmed by genetic testing. Clinical and laboratory data were collected and reviewed for these cases, including maternal demographics, prenatal sonographic findings, molecular testing sequencing results, and pregnancy outcomes. RESULTS: All cases had unremarkable first-trimester ultrasound scans without reporting limb malformations. All had second-trimester abnormal ultrasounds: postaxial polydactyly in nine cases (9/11), renal abnormalities in seven (7/11), reduced amniotic fluid volume in two (2/11), central nervous system anomalies in two (2/11), and ascites in three (3/11). Ten fetuses presented with at least two-system anomalies, and one (Case 11) presented with only postaxial polydactyly. Variants were detected in five genes, including BBS2, ARL6/BBS3, BBS7, CEP290/BBS14 and IFT74/BBS22. Ten pregnancies were terminated in the second trimester, while one continued to term. CONCLUSION: Enlarged hyperechogenic kidneys and postaxial polydactyly are the two most common sonographic features of fetal BBS. Prenatal diagnosis of BBS can be done with ultrasound and genetic testing although the diagnosis may be made in the second trimester.

SlCarE054 in Spodoptera litura (Lepidoptera: Noctuidae) showed direct metabolic activity to ß-cypermethrin with stereoselectivity.

Carboxylesterases (CarEs) is an important detoxification enzyme system in phase Ⅰ participating in insecticides resistance. In our previous study, SlCarE054, a CarEs gene from lepidoptera class, was screened out to be upregulated in a pyrethroids and organophosphates resistant population. Its overexpression was verified in two field-collected populations of Spodoptera litura (Lepidoptera: Noctuidae) resistant to pyrethroids and organophosphates by qRT-PCR. Spatiotemporal expression results showed that SlCarE054 was highly expressed in the pupae stage and the digestive tissue midgut. To further explore its role in pyrethroids and organophosphates resistance, its metabolism activity to insecticides was determined by UPLC. Its recombinant protein showed significant metabolism activity to cyhalothrin and fenvalerate, but not to phoxim or chlorpyrifos. The metabolic activity of SlCarE054 to ß-cypermethrin showed stereoselectivity, with higher metabolic activity to θ-cypermethrin than the enantiomer α-cypermethrin. The metabolite of ß-cypermethrin was identified as 3-phenoxybenzaldehyde. Further modelling and docking analysis indicated that ß-cypermethrin, cyhalothrin and fenvalerate could bind with the catalytic triad of the 3D structure of SlCarE054. The interaction of ß-cypermethrin with SlCarE054 also showed the lowest binding energy. Our work provides evidence that SlCarE054 play roles in ß-cypermethrin resistance in S. litura.

Fetal Phenotype of CHARGE Syndrome with a Molecular Confirmation: A Series of 13 Cases.

INTRODUCTION: CHARGE syndrome is an autosomal dominant genetic disorder with known pattern of features. The aim of the study was to present the fetal features of CHARGE syndrome to gain awareness that the antenatal characteristics can be very nonspecific. CASE PRESENTATION: This was a retrospective study of 13 cases with CHARGE syndrome diagnosed by prenatal or postnatal genetic testing and physical examination. Two (15.4%; 2/13) had normal ultrasound scans during pregnancy. One (7.7%; 1/13) with first-trimester cystic hygroma presented intrauterine fetal demise at 16 weeks gestation. The remaining 10 (76.9%; 10/13) cases had abnormal ultrasound features in utero; among these, 1 had an increased nuchal translucency in the first trimester, 5 had second-trimester abnormal ultrasounds including micrognathia, cardiac defects, and facial defects, and 4 third-trimester abnormal ultrasounds including micrognathia, isolated fetal growth restriction, and polyhydramnios. Among the 11 cases with abnormal prenatal ultrasound scans, no fetus could reach the diagnostic criteria of CHARGE syndrome if only based on the results of ultrasound. However, the diagnosis was made in all cases when CHD7 defects were detected. DISCUSSION/CONCLUSION: The CHARGE syndrome presents non-specific abnormal ultrasound markers in utero. Exome sequencing in the genetic workup will aid in prenatal diagnosis of this syndrome.

Early Onset of Severe Anemia Caused by Hb Calgary (HBB: C.194G > T): Another Case Report.

Unstable hemoglobin (Hb) variants are a rare cause of congenital hemolytic anemia. We describe a Chinese girl who presented with transfusion-dependent anemia in early infancy. Her diagnosis of Hb Calgary [ß64(E8)Gly > Val; HBB:c.194G > T] was not made until molecular testing was performed at the age of 5 years. Our case highlights the importance of early genetic testing in order to make the diagnosis, which may not only be useful for patient management and family counseling, but also for avoiding further unnecessary investigative attempts.

A 6-Year Follow-up of a Chinese Child with Homozygous ß0-Thalaasemia and a Heterozygous KLF1 Mutation.

Patients with the genotype of ß0/ß0 for ß-thalassemia (ß-thal) usually behave as ß-thal major (ß-TM) phenotype which is transfusion-dependent. The pathophysiology of ß-thal is the imbalance between α/ß-globin chains. The degree of α/ß-globin imbalance can be reduced by the more effective synthesis of γ-globin chains, and increased Hb F levels, modifying clinical severity of ß-TM. We report a Chinese child who had homozygous ß0-thal and a heterozygous KLF1 mutation. The patient had a moderate anemia since 6 months old, keeping a baseline Hb value of 8.0-9.0 g/dL. She had normal development except for a short stature (3rd percentile) until 6 years old, when splenomegaly and facial bone deformities occurred. Although genetic alteration of KLF1 expression in ß0/ß0 patients can result in some degree of disease alleviation, our case shows that it is insufficient to ameliorate satisfactorily the presentation. This point should be borne in mind for physicians who provide the genetic counseling and prenatal diagnosis to at-risk families.

Prenatal Diagnosis of Oral Teratoma by Ultrasound.

A pregnant woman had a normal second-trimester anatomic survey at 22 weeks gestation. She was revealed to have a fetal oral mass with polyhydramnios and invisible stomach bubble by ultrasound at 28 weeks. A 50 mm × 36 mm × 42 mm, solid mass was found in the fetal mouth, filling the entire oral cavity. Fetal magnetic resonance imaging showed a homogeneous solid mass in the oral cavity compressing the hypopharynx. At 33 weeks, preterm labor occurred because of the continuation of increased amniotic fluid volume, and a female infant was vaginally delivered. The infant died shortly after tracheal intubation attempt failed. Autopsy confirmed the prenatal sonographic finding. The final pathologic diagnosis was oral immature teratoma. Our study indicates that although oral teratomas are rare, they are readily apparent at prenatal sonographic examinations. Respiratory compromise is the frequent complication of oral teratomas, which is associated with high perinatal mortality.

A Lipopolysaccharide O-Antigen Synthesis Gene in Mesorhizobium huakuii Plays Differentiated Roles in Root Nodule Symbiotic Compatibility with Astragalus sinicus.

Lipopolysaccharide (LPS) is a ubiquitous microbial-associated molecular pattern. Plants can sense the three components of LPS, including core polysaccharide, lipid A, and O-antigen. LPS biosynthesis is an essential factor for the successful establishment of symbiosis in the rhizobium-legume plant system. The MCHK_1752 gene (Mesorhizobium huakuii 7653R gene) encodes O-antigen polymerase and affects the synthesis of O-antigen. Here, we investigated the symbiotic phenotypes of six Astragalus sinicus accessions inoculated with the MCHK_1752 deletion mutant strain. The results revealed that the MCHK_1752 deletion mutant strain had a suppressing effect on the symbiotic nitrogen fixation of two A. sinicus accessions, a promoting effect in three A. sinicus accessions, and no significant effect in one A. sinicus accessions. In addition, the effect of MCHK_1752 on the phenotype was confirmed by its complementary strains and LPS exogenous application. Deletion of MCHK_1752 showed no effect on the growth of a strain, but affected biofilm formation and led to higher susceptibility to stress in a strain. At the early symbiotic stage, Xinzi formed more infection threads and nodule primordia than Shengzhong under inoculation with the mutant, which might be an important reason for the final symbiotic phenotype. A comparison of early transcriptome data between Xinzi and Shengzhong also confirmed the phenotype at the early symbiotic stage. Our results suggest that O-antigen synthesis genes influence symbiotic compatibility during symbiotic nitrogen fixation. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

High accuracy of single-molecule real-time sequencing in detecting a rare α-globin fusion gene in carrier screening population.

INTRODUCTION: The α-globin fusion gene between the HBA2 and HBAP1 genes becomes clinically important in thalassemia screening because this fusion gene can cause severe hemoglobin (Hb) H disease when combining with α0 -thalassemia (α0 -thal). Due to its uncommon rearrangement in the α gene cluster without dosage changes, this fusion gene is undetectable by common molecular testing approaches used for α-thal diagnosis. METHODS: In this study, we used the single-molecule real-time (SMRT) sequencing technique to detect this fusion gene in 23 carriers identified by next-generation sequencing (NGS) among 16,504 screened individuals. Five primers for α and ß thalassemia were utilized. RESULTS: According to the NGS results, the 23 carriers include 14 pure heterozygotes, eight compound heterozygotes with common α-thal alleles, and one homozygote. By using SMRT, the fusion mutant was successfully detected in all 23 carriers. Furthermore, SMRT corrected the diagnosis in two "pure" heterozygotes: one was compound heterozygote with anti-3.7 triplication, and the other was homozygote. CONCLUSION: Our results indicate that SMRT is a superior method compared to NGS in detecting the α fusion gene, attributing to its efficient, accurate, and one-step properties.

Application of real-time PCR-based multicolor melting curve with automatic analysis system in pregestational and prenatal thalassemia diagnoses.

BACKGROUND: To evaluate the value of the real-time PCR-based multicolor melting curve analysis (MMCA) with an automatic analysis system used in a mass thalassemia screening and prenatal diagnosis program. METHODS: A total of 18,912 peripheral blood samples from 9456 couples and 1150 prenatal samples were detected by MMCA assay. All prenatal samples were also tested by a conventional method. Samples with unknown melting peaks, unusual peak height ratios between a wild allele and a mutant allele, or a discordant phenotype-genotype match were further studied by using multiplex ligation-dependent probe amplification (MLPA) or Sanger sequencing. All MMCA results were automatically analyzed and manually checked. The consistency between MMCA assay and conventional methods among prenatal samples was investigated. RESULTS: Except for initiation codon (T > G) (HBB:c.2T > G), all genotypes of thalassemia inside the scope of conventional methods were detected by MMCA assay. Additionally, 27 carriers with 10 rare HBB variants, 13 with α fusion gene, 1 with a rare deletion in α globin gene, and 1 with rare HBA variant were detected by using MMCA assay. CONCLUSION: MMCA can be an alternative approach used in routine thalassemia carrier screening and prenatal diagnosis for its high throughput, sufficient stability, low cost, and easy operation.

Whole exome sequencing improves genetic diagnosis of fetal clubfoot.

OBJECTIVE: This retrospective study aimed to investigate the value of whole exome sequencing (WES) for clubfoot (CF) fetuses with or without other structural abnormalities and to further explore the genetic causes of fetal CF. METHODS: this study included 83 singleton pregnancies diagnosed with fetal CF referred to our center between January 2016 and March 2022; cases were divided into two groups: isolated CF and non-isolated CF. After excluding cases with positive karyotyping and chromosomal microarray analysis results, WES was performed for the eligible fetuses and parents. Monogenic variants detected by WES and perinatal outcomes were recorded and evaluated at postnatal follow-up. RESULTS: overall, clinically significant variations were identified in 12.0% (10/83) of fetuses, and the detection rate was significantly higher in the non-isolated than in the isolated CF group (8/36, 22.2% vs. 2/47, 4.3%, p = 0.031). We additionally detected eight (9.6%) fetuses harboring variants of unknown significance. We identified 11 clinically significant variations correlating with clinical phenotypes in nine genes from ten fetuses, with KLHL40 being the most frequent (n = 2). Furthermore, we observed a significant difference in termination and survival rates between isolated and non-isolated CF cases (27.6 vs. 77.8% and 59.6 vs. 19.4%, p < 0.001 for both). CONCLUSION: our data indicate that WES has a high additional diagnostic yield for the molecular diagnosis of fetal CF, markedly enhancing existing prenatal diagnostic capabilities and expanding our understanding of intrauterine genetic disorders, thus assisting us to better interpret fetal phenotype in the future.

Prenatal diagnosis in the fetal hyperechogenic kidneys: assessment using chromosomal microarray analysis and exome sequencing.

Fetal hyperechogenic kidneys (HEK) is etiologically a heterogeneous disorder. The aim of this study was to identify the genetic causes of HEK using prenatal chromosomal microarray analysis (CMA) and exome sequencing (ES). From June 2014 to September 2022, we identified 92 HEK fetuses detected by ultrasound. We reviewed and documented other ultrasound anomalies, microscopic and submicroscopic chromosomal abnormalities, and single gene disorders. We also analyzed the diagnostic yield of CMA and ES and the clinical impact the diagnosis had on pregnancy management. In our cohort, CMA detected 27 pathogenic copy number variations (CNVs) in 25 (25/92, 27.2%) fetuses, with the most common CNV being 17q12 microdeletion syndrome. Among the 26 fetuses who underwent further ES testing, we identified 7 pathogenic/likely pathogenic variants and 8 variants of uncertain significance in 9 genes in 12 fetuses. Four novel variants were first reported herein, expanding the mutational spectra for HEK-related genes. Following counseling, 52 families chose to continue the pregnancy, and in 23 of them, postnatal ultrasound showed no detectable renal abnormalities. Of these 23 cases, 15 had isolated HEK on prenatal ultrasound. Taken together, our study showed a high rate of detectable genetic etiologies in cases with fetal HEK at the levels of chromosomal (aneuploidy), sub-chromosomal (microdeletions/microduplications), and single gene (point mutations). Therefore, we speculate that combined CMA and ES testing for fetal HEK is feasible and has good clinical utility. When no genetic abnormalities are identified, the findings can be transient, especially in the isolated HEK group.

Prenatal diagnosis of autosomal recessive Noonan syndrome associated with biallelic LZTR1 variants presented with thick nuchal translucency and cardiac abnormalities.

Noonan syndrome (NS) is a common clinical variable disease characterized by a number of features, mainly including congenital heart defects, short stature, and a variable degree of developmental delay. This disorder is transmitted mostly in an autosomal dominant manner and is genetically heterogeneous. We report three prenatal cases of LZTR1-related recessive NS. One case had a recurrent cystic hygroma at 13 weeks gestation and the pregnancy was terminated. Two cases had an increased nuchal translucency at 12 weeks' gestation, but a normal second trimester ultrasound; both presented with hypertrophic cardiomyopathy in the third trimester. The two infants were diagnosed with NS after birth. All of the three cases had invasive genetic investigations during pregnancy, and trio exome sequencing revealed biallelic likely pathogenic or pathogenic LZTR1 variants in the fetuses. All parents were LZTR1 variant carriers. Our report further strengthens the association of LZTR1 with an autosomal recessive form of NS. The affected fetuses are more likely to have cardiac anomalies. Clarification of molecular diagnosis has important implications in these families because they carry a 25% recurrence risk.

Fetal Hemoglobin H Hydrops Fetalis: Another Three Case Reports.

We report three cases of fetalis hydrops associated with nondeletional α-thalassemia. Two cases were caused by hemoglobin (Hb) H-Quong Sz disease, and one caused by homozygous Hb Constant Spring. Fetal hydrops occurred in the late second trimester in all three cases. Our study indicates that for pregnancies at risk for fetal nondeletional Hb H disease, strict ultrasound follow-up is particularly important. Even without techniques of intrauterine transfusion treatment, early prenatal diagnosis can enable parents to make timely decisions.

ß-Thalassemia Trait Caused by SUPT5H Defects: Another Case Report.

We identified a novel mutation in the SUPT5H gene in a Chinese female who presented with a ß-thalassemia trait. The substitution of c.193C > T (p.Arg65*) leads to a premature stop codon on residue 65 and could be associated with haploinsufficiency. This variant was inherited from the mother who also had the asymptomatic phenotype of ß-thalassemia trait. Our case further supports the role of SUPT5H as a potential ß-globin chain production-modulating gene.

Fetal Rhabdomyoma Leads to Family Diagnosis of Tuberous Sclerosis Complex.

Tuberous sclerosis complex (TSC) is a multisystem genetic disorder characterized by the growth of numerous noncancerous tumors in many parts of the body. It is highly variable in clinical presentations, including a wide range of cognitive, behavioral, and psychiatric manifestations. Of all the possible manifestations, cognitive and behavioral problems are the greatest concern to parents and physicians. In this study, two fetuses were identified to have rhabdomyomas by prenatal ultrasound. Carefully inquired familial medical history revealed other symptoms of TSC such as skin lesions or psychiatric problems in family members in the two families. Both fetuses and family members with positive clinical symptoms were confirmed to carry a familial TSC2 variant. Our study indicates that fetal echocardiography is not just the evaluation of the fetal heart. When fetal cardiac rhabdomyomas are diagnosed, a full family medical history and clinical assessment for TSC in family members should be undertaken.

Megacystis in the First Trimester as an Unreported Sonographic Finding of Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins Confirmed by Whole-exome Sequencing.

A pregnant woman was revealed to have fetal univentricular heart and megacystis by a routine first-trimester ultrasound. Chorionic villus sampling with the use of karyotyping and microarray found no causative etiologies. A further investigation with whole-exome sequencing (WES) demonstrated a FOXF1 variant. Autopsy confirmed the prenatal findings, and a histological study of the lungs showed the characteristic features of alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). This study indicates that although ultrasound itself has no ability of the identification of pulmonary histological malformations associated with ACDMPV, the early markers of univentricular heart and megacystis might alert clinicians to consider this genetic disorder which is facilitated considerably by the increasingly used WES in prenatal diagnosis.

Adducted Thumb in the First Trimester of Pregnancy: An Early Clue to Prenatal Diagnosis of L1 Syndrome.

A pregnant woman was revealed to have an unusual position of the fetal hand by a routine 12-week ultrasound. Bilateral adducted thumbs and a male phenotype were confirmed by another ultrasound at 14 weeks' gestation. A structural survey at 18 weeks revealed fetal hydrocephalus with severe bilateral ventriculomegaly. The pregnancy was terminated, and postnatal examination with trio exome sequencing detected a hemizygous deletion (1,511 bp in size) variant of L1CAM gene in the fetus, inherited from the mother. The fetus was diagnosed as L1 syndrome (X-linked hydrocephalus). A family study found that this was a familial mutant allele. This study demonstrates that fetal hand abnormalities can be identified in the first trimester. Adducted thumbs might indicate the maldevelopment of the fetal brain, and therefore, examination of fetal hands and fingers should be integrated into fetal anomaly scans.

Analysis of Outer Membrane Vesicles Indicates That Glycerophospholipid Metabolism Contributes to Early Symbiosis Between Sinorhizobium fredii HH103 and Soybean.

Gram-negative bacteria can produce outer membrane vesicles (OMVs), and most functional studies of OMVs have been focused on mammalian-bacterial interactions. However, research on the OMVs of rhizobia is still limited. In this work, we isolated and purified OMVs from Sinorhizobium fredii HH103 under free-living conditions that were set as control (C-OMVs) and symbiosis-mimicking conditions that were induced by genistein (G-OMVs). The soybean roots treated with G-OMVs displayed significant deformation of root hairs. G-OMVs significantly induced the expression of nodulation genes related to early symbiosis, while they inhibited that of the defense genes of soybean. Proteomics analysis identified a total of 93 differential proteins between C-OMVs and G-OMVs, which are mainly associated with ribosome synthesis, flagellar assembly, two-component system, ABC transporters, oxidative phosphorylation, nitrogen metabolism, quorum sensing, glycerophospholipid metabolism, and peptidoglycan biosynthesis. A total of 45 differential lipids were identified through lipidomics analysis. Correlation analysis of OMV proteome and lipidome data revealed that glycerophospholipid metabolism is the enriched Kyoto Encyclopedia of Genes and Genomes metabolic pathway, and the expression of phosphatidylserine decarboxylase was significantly up-regulated in G-OMVs. The changes in three lipids related to symbiosis in the glycerophospholipid metabolism pathway were verified by enzyme-linked immunosorbent assay. Our results indicate that glycerophospholipid metabolism contributes to rhizobia-soybean symbiosis via OMVs.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.