Reevaluating the splice-altering variant in TECTA as a cause of nonsyndromic hearing loss DFNA8/12 by functional analysis of RNA.

PURPOSE: The aim of this study was to determine the genetic cause of early onset autosomal dominant hearing loss segregating in five-generation kindred of Chinese descent and provide preimplantation genetic testing (PGT)for them. METHODS: Clinical examination, pedigree analysis and exome sequencing were carried out on the family. Minigene-based splicing analysis, in vivo RNA analysis and protein structure prediction by molecular modeling were conducted on the candidate variant. PGT for the causative variation and chromosome aneuploidis based on SNP analysis has been used for avoidance of hearing loss in this family. RESULTS: All the affected individuals presented with moderate down-sloping hearing loss and whole-exome sequencing identified a novel splice-site variant c.5383+6T>A in the tested subjects within the TECTA locus. Genotyping of all the 32 family members confirmed segregation of this variant and the hearing loss phenotype in the extended family. Functional analysis of RNA and molecular modeling indicates that c.5383+6T>A is a pathogenic splice-site variant and should be considered as genetic cause of the hearing loss. Furthermore, a successful singleton pregnancy with no variation in TECTA c.5383+6 was established and a healthy male child was born by PGT. CONCLUSION: We have identified a novel variant c.5383+6T>A in TECTA ZA-ZP inter-domain, which could be attributable to the early-onset autosomal dominant hearing loss. The implications of our study are valuable in elucidating the disrupted RNA splicing and uncovering the genetic cause of hearing loss with TECTA pathogenic variants, as well as providing reproductive approaches to healthy offspring.

Evaluating ClinGen variant curation expert panels' application of PVS1 code.

BACKGROUND: The 2015 American College of Medical Genetics and Genomics (ACMG) and Association for Molecular Pathology (AMP) guidelines articulates that the effects of certain types of variants on gene function can often be seen as a complete absence of the gene product by leading to a lack of transcription or nonsense-mediated decay(NMD). However, detailed information considering different types of loss of function(LOF) variants, refined steps assimilating details concerning location of variant, changes in strength levels, NMD boundary, or any additional information pointing to a true null effect, were all left to expert judgement. As part of its Clinical Genome Resource (ClinGen) initiative, Variant Curation Expert Panels (VCEPs) are designated to make gene/disease-centric specifications in accordance with the ACMG/AMP guidelines, including a more detailed definition of what constitutes an appropriate LOF evidence. Our goal was to evaluate the current LOF guidelines developed by the VCEPs and analyse the prior curated variants concerning the PVS1 criteria, bringing people occupied in genetic data analysis a comprehensive understanding of this code. METHODS: Our study evaluated 7 VCEPs for their LOF criteria (PVS1). Subsequently, we assessed the predictive criteria by considering the underlying disease mechanism, protein transcript, and variant types delineated. Then, we meticulously curated the LOF evidence referenced by each VCEP in their preliminary variant classification, thereby scrutinizing the recommendations put forth by VCEPs and their application in the interpretation of the aforementioned predictive criteria. Based on these, an extensive curation of evidence summary considering PVS1 applied by VCEPs according to their classification of pilot variants for the purpose of analyzing VCEP criteria specifications and their use in the understanding of LOF was conducted. RESULTS: We observed in this article that the VCEPs discussed followed the majority of Sequence Variant Interpretation (SVI) recommendations concerning the application of this LOF criteria, except for some disease/gene specific considerations. We highlighted the wide range of PVS1 strength levels approved by VCEP, reflecting the diversity of evidence for each variants type. In addition, we observed substantial differences in the approach used to determine relative strengths for different types of null variants and in the attitude towards these principles concerning variant location, NMD and influence to protein function between VCEPs. CONCLUSIONS: It is difficult to understand the intricacies of the predictive data(PVS1), which often requires expert-level knowledge of disease/gene. The VCEP criteria specifications for the predictive evidence play an important role in making it more accessible for the curators to apply the predictive data by providing details concerning this complex criteria. Despite this, we believe there is a need for more guidance on standardizing this process and ensuring consistency in the application of this predictive evidence.

Identification of five novel SCN1A variants.

Background: Epilepsy is characterized by recurrent unprovoked seizures. Mutations in the voltage-gated sodium channel alpha subunit 1 (SCN1A) gene are the main monogenic cause of epilepsy. Type and location of variants make a huge difference in the severity of SCN1A disorder, ranging from the mild phenotype (genetic epilepsy with febrile seizures plus, GEFS+) to the severe phenotype (developmental and epileptic encephalopathies, DEEs). Dravet Syndrome (DS) is an infantile-onset DEE, characterized by drug-resistant epilepsy and temperature sensitivity or febrile seizures. Genetic test results reveal SCN1A variants are positive in 80% DS patients and DS is mainly caused by de novo variants. Methods: Trio-whole exome sequencing (WES) was used to detect variants which were associated with clinical phenotype of five probands with epilepsy or twitching. Then, Sanger sequencing was performed to validate the five novel SCN1A variants and segregation analysis. After analyzing the location of five SCN1A variants, the pathogenic potential was assessed. Results: In this study, we identified five novel SCN1A variants (c.4224G > C, c.3744_3752del, c.209del, c.5727_5734delTTTAAAACinsCTTAAAAAG and c.5776delT) as the causative variants. In the five novel SCN1A variants, four were de novo and the remaining one was inherited. All novel variants would be classified as "pathogenic" or "likely pathogenic." Conclusion: The five novel SCN1A variants will enrich the SCN1A mutations database and provide the corresponding reference data for the further genetic counseling.

Analysis of chromosomal structural variations in patients with recurrent spontaneous abortion using optical genome mapping.

Background and aims: Certain chromosomal structural variations (SVs) in biological parents can lead to recurrent spontaneous abortions (RSAs). Unequal crossing over during meiosis can result in the unbalanced rearrangement of gamete chromosomes such as duplication or deletion. Unfortunately, routine techniques such as karyotyping, fluorescence in situ hybridization (FISH), chromosomal microarray analysis (CMA), and copy number variation sequencing (CNV-seq) cannot detect all types of SVs. In this study, we show that optical genome mapping (OGM) quickly and accurately detects SVs for RSA patients with a high resolution and provides more information about the breakpoint regions at gene level. Methods: Seven couples who had suffered RSA with unbalanced chromosomal rearrangements of aborted embryos were recruited, and ultra-high molecular weight (UHMW) DNA was isolated from their peripheral blood. The consensus genome map was created by de novo assembly on the Bionano Solve data analysis software. SVs and breakpoints were identified via alignments of the reference genome GRCh38/hg38. The exact breakpoint sequences were verified using either Oxford Nanopore sequencing or Sanger sequencing. Results: Various SVs in the recruited couples were successfully detected by OGM. Also, additional complex chromosomal rearrangement (CCRs) and four cryptic balanced reciprocal translocations (BRTs) were revealed, further refining the underlying genetic causes of RSA. Two of the disrupted genes identified in this study, FOXK2 [46,XY,t(7; 17)(q31.3; q25)] and PLXDC2 [46,XX,t(10; 16)(p12.31; q23.1)], had been previously shown to be associated with male fertility and embryo transit. Conclusion: OGM accurately detects chromosomal SVs, especially cryptic BRTs and CCRs. It is a useful complement to routine human genetic diagnostics, such as karyotyping, and detects cryptic BRTs and CCRs more accurately than routine genetic diagnostics.

Screening and mutation analysis of phenylalanine hydroxylase deficiency in newborns from Jiangxi province.

Background: Phenylalanine hydroxylase deficiency (PAHD) is an autosomal recessive disorder of amino acid metabolism and caused by mutations in the phenylalanine hydroxylase (PAH) gene. Without timely and appropriate dietary management, the disturbance of amino acid metabolism may impair cognitive development and neurophysiological function. Newborn screening (NBS) can aid the early diagnosis of PAHD, which can give accurate therapy to PAHD patients in time. In China, the PAHD incidence and PAH mutation spectrum vary enormously across the provinces. A total of 5,541,627 newborns from Jiangxi province were screened by NBS between 1997 and 2021. Method: One seventy one newborns from Jiangxi province were diagnosed with PAHD. By Sanger sequencing and the multiplex ligation-dependent probe amplification (MLPA) analysis, mutation analysis was performed in 123 PAHD patients. Using an arbitrary values (AV)-based model, we compared the observed phenotype with the predicted phenotype based on the genotype. Results: In this study, we speculated the PAHD incidence of Jiangxi province was about 30.9 per 1,000,000 live births (171/5,541,627). We summarized the PAH mutation spectrum in Jiangxi province for the first time. Two novel variants (c.433G > C, c.706 + 2T > A) were found. The most prevalent variant was c.728G > A (14.1%). The overall prediction rate of the genotype-phenotype was 77.4%. Conclusion: This mutation spectrum is very meaningful to improve the diagnostic rate of PAHD and to increase the accuracy genetic counseling. This study offers data for the genotype-phenotype prediction suitable for Chinese population.

Identification of two novel and one rare mutation in DYRK1A and prenatal diagnoses in three Chinese families with intellectual Disability-7.

Background and purpose: Intellectual disability-7 (MRD7) is a subtype disorder of intellectual disability (MRD) involving feeding difficulties, hypoactivity, and febrile seizures at an age of early onset, then progressive intellectual and physical development deterioration. We purposed to identify the underlying causative genetic factors of three individuals in each Chinese family who presented with symptoms of intellectual disability and facial dysmorphic features. We provided prenatal diagnosis for the three families and genetic counseling for the prevention of this disease. Methods: We collected retrospective clinical diagnostic evidence for the three probands in our study, which included magnetic resonance imaging (MRI), computerized tomography (CT), electroencephalogram (EEG), and intelligence tests for the three probands in our study. Genetic investigation of the probands and their next of kin was performed by Trio-whole exome sequencing (WES). Sanger sequencing or quantitative PCR technologies were then used as the next step to verify the variants confirmed with Trio-WES for the three families. Moreover, we performed amniocentesis to explore the state of the three pathogenic variants in the fetuses by prenatal molecular genetic diagnosis at an appropriate gestational period for the three families. Results: The three probands and one fetus were clinically diagnosed with microcephaly and exhibited intellectual developmental disability, postnatal feeding difficulties, and facial dysmorphic features. Combining probands' clinical manifestations, Trio-WES uncovered the three heterozygous variants in DYRK1A: a novel variant exon3_exon4del p.(Gly4_Asn109del), a novel variant c.1159C>T p.(Gln387*), and a previously presented but rare pathogenic variant c.1309C>T p.(Arg437*) (NM_001396.5) in three families, respectively. In light of the updated American College of Medical Genetic and Genomics (ACMG) criterion, the variant of exon3_exon4del and c.1159C>T were both classified as likely pathogenic (PSV1+PM6), while c1309C>T was identified as pathogenic (PVS1+PS2_Moderate+PM2). Considering clinical features and molecular testimony, the three probands were confirmed diagnosed with MRD7. These three discovered variants were considered as the three causal mutations for MRD7. Prenatal diagnosis detected the heterozygous dominant variant of c.1159C>T p.(Gln387*) in one of the fetuses, indicating a significant probability of MRD7, subsequently the gestation was intervened by the parents' determination and professional obstetrical operation. On the other side, prenatal molecular genetic testing revealed wild-type alleles in the other two fetuses, and their parents both decided to sustain the gestation. Conclusion: We identified two novel and one rare mutation in DYRK1A which has broadened the spectrum of DYRK1A and provided evidence for the diagnosis of MRD7 at the molecular level. Besides, this study has supported the three families with MRD7 to determine the causative genetic factors efficiently and provide concise genetic counseling for the three families by using Trio-WES technology.

Mutation spectrum of PTS gene in patients with tetrahydrobiopterin deficiency from jiangxi province.

Background: Hyperphenylalaninemia (HPA) is the most common inborn error in amino acid metabolism. It can be primarily classified into phenylalanine hydroxylase (PAH) deficiency and tetrahydrobiopterin (BH4) deficiency. BH4 deficiency (BH4D) is caused by genetic defects in enzymes involved in the biosynthesis and regeneration of BH4. 6-pyruvoyl-tetrahydropterin synthase (PTPS/PTS), which is encoded by the PTS gene, participates in the biosynthesis of BH4. PTPS deficiency (PTPSD) is the major cause of BH4D. In this study, we investigated that the prevalence of BH4D in Jiangxi province was approximately 12.5 per 1,000,000 live births (69/5,541,627). Furthermore, the frequency of BH4D was estimated to be 28.8% (69/240) in the HPA population of Jiangxi. In this study, we aimed to characterize the mutational spectrum of the PTS gene in patients with PTPSD from Jiangxi province. Method: Newborn screening data of Jiangxi province from 1997 to 2021 were analyzed and 53 families with PTPSD were enrolled for the analysis of the PTS gene variants by Sanger sequencing. Results: 106 variants were identified in 106 alleles of 53 patients with PTPSD, including 13 types of variants reported previously, and two novel variants (c.164-36A>G and c.146_147insTG). The predominant variant was c.259C>T (47.2%), followed by c.84-291A>G (19.8%), c.155A>G (8.5%), c.286G>A (6.6%) and c.379C>T (4.7%). Conclusion: The results of this study can not only provide guidance for the molecular diagnosis and genetic counseling in cases of PTPS deficiency but also enrich the PTS mutation database.

In Vitro Inhibitory Effects of Maqian Essential Oil against Ectopic Endometrial Stromal Cells and LPS-Induced Endometrial Epithelial Cells.

Previous studies revealed that MQEO (Maqian fruits essential oil), which is extracted from the fruit of Maqian (Zanthoxylum myriacanthum var. Pubescens), had a good anti-inflammatory effect, but the effect on endometriosis in vitro remains unknown. In the present study, the inhibitory effects of MQEO against the EESCs (ectopic endometrial stromal cells) were investigated. Cells were treated with a concentration gradient (from 0.025 % to 0.15 %) of MQEO for 24 h and cell viability was detected by CCK-8. In addition, apoptotic rates were investigated using flow cytometry. The effect of MQEO on cell migration was determined by wound-healing and transwell assay. The expression of apoptosis-associated and cell adhesion-related proteins was assessed by western blotting. The transcriptional levels of IL-1, IL-6 and TNF-α were determined by Real-time qPCR. RNA-seq was used to identify the DEGs (differentially expressed genes) in MQEO-pretreated EESCs. We found that the MQEO condition dosage-dependently reduced the cell viability of EESCs. Based on flow cytometry results, the number of apoptotic cells increased significantly with dosage. The wound-healing and transwell results showed that MQEO group exhibited a significantly decreased cell motility and migration ability in comparison with the normal group. Western blotting results showed that MQEO down-regulated the expression of Bcl-2, ICAM-1 (intercellular adhesion molecule 1) and CD44, but up-regulated the cleaved caspase-3 expression in EESCs. What's more, MQEO also inhibited the LPS-induced inflammation in human EECs (endometrial epithelial cells). RNA-seq revealed that 221 DEGs were up-regulated genes and 284 DEGs were down-regulated in MQEO-pretreated EESCs. Our data uncovered the beneficial effects of MQEO in endometriosis and provided new insights into the mechanism of the effect of MQEO on EESCs, suggesting MQEO could be a promising new therapeutic agent for endometriosis.

Concurrent newborn hearing and genetic screening of common hearing loss variants with bloodspot-based targeted next generation sequencing in Jiangxi province.

Background and aims: Concurrent hearing and genetic screening of newborns have been widely adopted as an effective strategy in early diagnosis and intervention for hearing loss in many cities in China. Here, we aimed to firstly explore the efficacy of combining conventional hearing screening with genetic screening among the large-scale newborns in Jiangxi Province. Methods: A total of 24,349 newborns from Jiangxi Maternal and Child Health Hospital were enrolled in our study from April 2021 to June 2022. Newborn hearing screening was conducted using otoacoustic emission (OAE) and automated auditory brainstem response (AABR). Meanwhile, newborn dried blood spots were collected and twenty common variants in four genes, including GJB2, SLC26A4, MT-RNR1(12SrRNA), and GJB3, were screened using a BGISEQ-500 next generation sequencing platform. Whole coding regions sequencing of GJB2 and SLC26A4 were performed by Sanger sequencing and NGS, respectively. Following up of hearing for the newborns was undertaken by phone interviews. Results: Among the 24,349 newborns, 7.00% (1,704/24,349) were bilaterally or unilaterally referred in their initial hearing screening, whereas 1.30% (316/24,349) exhibited bilateral or unilateral hearing loss in the repeated screening. Genetic screening revealed that 4.813% (1,172/24,349) of the screened newborns were positive for at least one mutant allele (heterozygote, homozygote, or compound heterozygote in one gene, mtDNA homoplasmy or heteroplasmy and combined variants in different genes). A total of 1,146 individuals were identified with mutant allele in one gene, including 525 of GJB2, 371 of SLC26A4, 189 as homoplasmic or heteroplasmic of MT-RNR1, and 61 of GJB3, indicating that GJB2 and SLC26A4 are the most common endemic deafness-associated genes among newborns in Jiangxi Province. Nineteen newborns were detected with combined heterozygous variants in different genes, with "c.235delC heterozygous and c.919-2A > G heterozygous" as the most prevalent genotype. Additionally, seven newborns were screened as homozygotes or compound heterozygotes responsible for congenital or late-onset prelingual hearing loss, including three cases with GJB2 c.235delC homozygous and one with SLC26A4 c.919-2A > G homozygous variant, one case with compound heterozygous variants for GJB2 and two with compound heterozygous variants for SLC26A4. Coding regions sequencing of GJB2 or SLC26A4 for overall 265 infants revealed that 14 individuals were identified as compound heterozygote with a second pathogenic variant not screened by our genetic panel. Conclusions: Herein our study firstly investigated the efficacy of concurrent hearing screening and genetic screening of common hearing impairment variants among large-scale newborns in Jiangxi Province. Concurrent screening provides a more comprehensive approach for management of congenital or delayed onset prelingual hearing loss and prevention of drug-induced hearing impairment for newborns at risk as well as their maternal relatives. An insight into the molecular epidemiology for hearing loss genes among Jiangxi population will also be beneficial to the genetic counseling and birth defect prevention.

Molecular prevalence of HBB-associated hemoglobinopathy among reproductive-age adults and the prenatal diagnosis in Jiangxi Province, southern central China.

Background and aims: Hemoglobinopathy associated with the HBB gene, with its two general subtypes as thalassemia and abnormal hemoglobin (Hb) variants, is one of the most prevalent hereditary Hb disorders worldwide. Herein we aimed to elucidate the prevalence of ß-thalassemia and abnormal hemoglobin variants and the prenatal diagnosis of the HBB gene in Jiangxi Province, southern central China. Methods: Hematological indices and capillary Hb electrophoresis were conducted for 136,149 subjects who were admitted to Jiangxi Maternal and Child Health Hospital and requested for hemoglobinopathy investigation. Routine α- and ß-globin genotyping were performed by gap-polymerase chain reaction (Gap-PCR) and reverse dot-blot (RDB) hybridization for the 11,549 individuals suspected to be thalassemia carriers. For participants whose genotypes could not explain their hematological indices, further Sanger sequencing and Gap-PCR were conducted for the detection of rare or novel variants in related globin genes. Prenatal diagnosis was performed for 77 pregnant couples both carrying ß-thalassemia trait at appropriate gestational ages. Results: Among the 11,549 subjects, 2,548 individuals were identified with HBB-associated hemoglobinopathy based on molecular analysis. A total of 2,358 subjects were identified as ß-thalassemia heterozygous carriers and nine cases were diagnosed as compound heterozygous ß-thalassemia. Additionally, 125 cases were detected with composite α- and ß-thalassemia and the remaining 56 individuals with abnormal Hb variants in the HBB. A total of 35 types of variants were identified in the HBB gene, including 26 types of ß-thalassemia and nine types of abnormal Hb variants. Four novel variants were firstly reported, including one variant in HBA2 and three variants in HBB. Overall, 77 prenatal samples underwent ß-thalassemia molecular diagnosis; 20 fetuses were identified with normal ß-thalassemia genotypes, 30 fetuses as ß-thalassemia heterozygotes, 11 as homozygotes, and 16 as compound heterozygotes in HBB. Conclusion: We have demonstrated a relatively high prevalence rate at 1.872% of ß-hemoglobinopathies including common and rare ß-thalassemia as well as abnormal Hb variants among large child-bearing population in the Jiangxi area of southern central China for the first time. Our data presents that prenatal diagnosis is an effective way to prevent and control birth defects of ß-thalassemia.

Identification of a Novel Nonsense Mutation in PLA2G6 and Prenatal Diagnosis in a Chinese Family With Infantile Neuroaxonal Dystrophy.

Background and Purpose: Infantile neuroaxonal dystrophy (INAD) is a subtype of PLA2G6-Associated Neurodegeneration (PLAN) with an age of early onset and severe clinical phenotypes of neurodegeneration. Individuals affected with INAD are characterized by rapid progressive psychomotor deterioration, neuroregression, and hypotonia followed by generalized spasticity, optic atrophy, and dementia. In this case, we aimed to identify the underlying causative genetic factors of a Chinese family with two siblings who presented with walking difficulty and inability to speak. We provided a prenatal diagnosis for the family and information for the prevention of this genetic disease. Methods: Retrospective clinical information and magnetic resonance imaging (MRI) findings of the proband were collected. Trio-whole exome sequencing (WES) including the proband and his parents was performed to explore the genetic causes, while Sanger sequencing was subsequently used to validate the variants identified by Trio-WES in the pedigree. Furthermore, prenatal molecular genetic diagnosis was carried out through amniocentesis to investigate the status of pathogenic mutations in the fetus by Sanger sequencing at an appropriate gestational age. Results: The two siblings were both clinically diagnosed with rapid regression in psychomotor development milestones. Brain MRI showed cerebellar atrophy and typical bilaterally symmetrical T2/FLAIR hyperintense signal changes in periventricular areas, indicating periventricular leukomalacia, and myelin sheath dysplasia. Trio-WES revealed two heterozygous variants in PlA2G6 associated with clinical manifestations in the proband: a novel maternally inherited variant c.217C>T (p.Gln73*) and a previously reported paternally inherited recurrent pathogenic variant c.1894C>T (p.Arg632Trp). These two heterozygous mutations were also detected in the younger brother who had similar clinical features as the proband. The novel variant c.217C>T was classified as "pathogenic (PVS1 + PM2 + PP3)," while the variant c.1894C>T was "pathogenic" (PS1 + PM1 + PM2 + PM3 + PP3) based on the latest American College of Medical Genetics and Genomics (ACMG) guidelines on sequence variants. Combining the molecular evidence and clinical phenotypes, the diagnosis of INAD was established for the two affected siblings. The two variants that were identified were considered the causative mutations for INAD in this family. Prenatal diagnosis suggested compound heterozygous mutations of c.217C>T and c.1894C>T in the fetus, indicating a high risk of INAD, and the parents chose to terminate the pregnancy. Conclusion: We identified a novel pathogenic mutation that broadens the mutation spectrum of PLA2G6 and will provide clues for the molecular diagnosis of INAD. Furthermore, our study has helped to elucidate the causative genetic factors of this Chinese family with INAD effectively and efficiently by using the emerging Trio-WES strategy and providing precise genetic counseling for this family.

Wooden-Tip Electrospray Mass Spectrometry Characterization of Human Hemoglobin in Whole Blood Sample for Thalassemia Screening: A Pilot Study.

Traditional analytical methods for thalassemia screening are needed to process complicated and time-consuming sample pretreatment. In recent decades, ambient mass spectrometry (MS) approaches have been proven to be an effective analytical strategy for direct sample analysis. In this work, we applied ambient MS with wooden-tip electrospray ionization (WT-ESI) for the direct analysis of raw human blood samples that were pre-identified by gene detection. A total of 319 whole blood samples were investigated in this work, including 100 α-thalassemia carriers, 67 ß-thalassemia carriers, and 152 control healthy samples. Only one microliter of raw blood sample was directly loaded onto the surface of the wooden tip, and then five microliters of organic solvent and a high voltage of +3.0 kV were applied onto the wooden tip to generate spray ionization. Multiply charged ions of human hemoglobin (Hb) were directly observed by WT-ESI-MS from raw blood samples. The signal ratios of Hb chains were used to characterize two main types of thalassemia (α and ß types) and healthy control blood samples. Our results suggested that the ratios of charged ions to Hb chains being at +13 would be an indicator for ß-thalassemia screening.

Performance of expanded non-invasive prenatal testing for fetal aneuploidies and copy number variations: A prospective study from a single center in Jiangxi province, China.

To evaluate the performance of expanded non-invasive prenatal testing (expanded noninvasive prenatal testing, NIPT-Plus) in screening for fetal chromosomal abnormalities includes aneuploidies and copy number variations, a total of 23,116 pregnant women with a singleton pregnancy were recruited for NIPT-Plus. Screening positive results were verified by karyotype analysis and chromosomal microarray analysis after amniocentesis. A total of 264 pregnancies (1.14%) were positive results as predicted by NIPT-Plus, including 233 aneuploidies and 31 copy number variations. Following genetic counseling, 233 (88.26%) pregnant women underwent invasive prenatal diagnosis and 136 were verified as true positives, comprising 72 common trisomies (T21, T18, T13), 47 sex chromosomal abnormalities two rare autosomal aneuploidies (RATs) and 15 copy number variations The positive predictive value for common trisomies, SCAs, RATs and CNVs were 68.57%, 68.12%, 6.67% and 51.72%, respectively. Pregnant women with screen-positive results for common trisomies have higher rates of invasive prenatal diagnosis and pregnancy termination than those with positive results for SCAs, RATs, and CNVs. NIPT-Plus showed a good performance in detecting common trisomies, SCAs and also contributed to detecting pathogenic CNVs, but higher accuracy was required in the detection of RATs. In summary, this study provides a reference for the clinical application of NIPT-Plus for screening fetal chromosomal abnormalities in this region. Therefore, we suggest that NIPT-Plus could be widely used in clinical screening for fetal chromosomal abnormalities in combination with prenatal diagnosis and genetic counseling.

[Prenatal diagnosis of a fetus with 8q13.3 microdeletion through chromosomal microarray analysis].

OBJECTIVE: To assess the value of chromosomal microarray analysis (CMA) for the prenatal diagnosis of a fetus with structural anomaly detected by ultrasonography. METHODS: The fetus and its parents were subjected to chromosomal karyotyping and CMA analysis. RESULTS: The fetus was found to carry a 46,XN,t(8;11)(q21.2;q13) translocation which was inherited from its mother. CMA has found no copy number variations (CNVs) in both parents but a de novo 2.00 Mb microdeletion in the fetus at 8q13.3. CONCLUSION: CMA is capable of detecting microdeletions and microduplications in fetuses with translocations detected by karyotyping analysis.

[Genetic analysis and prenatal diagnosis for a Chinese pedigree affected with N-acetylglutamate synthase deficiency].

OBJECTIVE: To explore the genetic basis for a Chinese pedigree affected with N-acetylglutamate synthase deficiency. METHODS: Trio whole exome sequencing (WES) was carried out for the pedigree. Pathogenicity of the identified variant was predicted based on the latest recommendation of the American College of Medical Genetics and Genomics (ACMG). Prenatal diagnosis was provided for subsequent pregnancy through Sanger sequencing. RESULTS: Trio WES showed that the proband has carried compound heterozygous c.68delG and c.796G>C variants of NAGS gene, for which the mother and father were respectively heterozygous carriers. Neither variant was reported previously. Based on the ACMG guidelines, the c.68delG variant was classified as "likely pathogenic" (PVS1+PM2), while the c.796G>C variant was classified as with "uncertain significance" (PM2+BP4). Sanger sequencing validated the above findings, and only detected the heterozygous c.796G>C variant in the amniotic fluid sample. The fetus was followed up till 6 month after birth with no obvious abnormality. CONCLUSION: The compound heterozygous c.68delG and c.796G>C variants of the NAGS gene probably underlay the disorder in this pedigree, and the resulth asenabled genetic counseling and prenatal diagnosis for this pedigree.

A Novel ß-Thalassemia Mutation [IVS-I-6 (T>G), HBB: c.92+6T>G] in a Chinese Family.

ß-Thalassemia (ß-thal) is one of the most common inherited hemoglobin (Hb) disorders in southern China. Up to now, the mutation spectrum of ß-thal has been increasingly broadened through various molecular methods. In this study, a 34-year-old female displaying microcytic, hypochromic anemia was first detected with a novel IVS-I-6 (T>G) (HBB: c.92+6T>G) mutation by Sanger sequencing. Pedigree analysis performed on her family showed that her mother and her daughter, who had abnormal hematological indices, also carried this mutation, while her other family members with normal hematological phenotypes, were not detected to carry any mutation. Based on the observed symptoms in this Chinese family, we concluded that this novel mutation was associated with a mild ß-thal phenotype.

A novel TAB2 nonsense mutation (p.S149X) causing autosomal dominant congenital heart defects: a case report of a Chinese family.

BACKGROUND: TAB2 is an activator of MAP 3 K7/TAK1, which is required for the IL-1 induced signal pathway. Microdeletions encompassing TAB2 have been detected in various patients with congenital heart defects (CHD), indicating that haploinsufficiency of TAB2 causes CHD. To date, seven variants within TAB2 were reported associated with CHD, only two of them are nonsense mutations. CASE PRESENTATION: Here we describe a three-generation Chinese family that included five CHD patients with heart valvular defects, such as mitral or tricuspid valves prolapse or regurgitation, and aortic valve stenosis or regurgitation. Our proband was a pregnant woman presenting with mitral, tricuspid, and aortic defects; her first child experienced sudden cardiac death at the age of 2 years. Whole-exome sequencing of the proband revealed a novel nonsense variant in TAB2 (c.C446G, p.S149X), which results in the elimination of the majority of C-terminal amino acids of TAB2, including the critical TAK1-binding domain. The variant was identified in five affected patients but not in the eight unaffected family members using Sanger sequencing and was classified as "pathogenic" according to the latest recommendation on sequence variants laid out by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. CONCLUSION: We described a family with CHD caused by a novel TAB2 nonsense mutation. Our study broadens the mutation spectrum of TAB2; to the best of our knowledge, this is the first report of a pathogenic mutation within TAB2 in a Chinese population.

[Study of the phenylalanine hydroxylase gene variants in patients with phenylketonuria from Jiangxi province].

OBJECTIVE: To delineate the variants spectrum of phenytalanine hydroxylase (PAH) gene among 78 unrelated patients with phenylketonuria (PKU) from Jiangxi province. METHODS: The 13 exons and flanking intronic regions of the PAH gene were subjected to PCR amplification and sequencing. RESULTS: A total of 143 variants were detected among the 156 alleles, which included 54 types of variants, which yielded a detection rate of 91.7%. Common variants have included R243Q (26/143, 18.2%), R408Q (10/143, 7.0%), EX6-96A to G(8/143, 5.6%), IVS4-1G to A(7/143, 4.9%), R241C(7/143, 4.9%) and V399V(7/143, 4.9%). In addition, 6 novel variants were detected, which included IVS4-3T to G, Q172H, C284Y, V291L, V329del, and L430R. The variants consisted of missense, splicing, nonsense and deletion variants, which have mainly located in exons 7 (45, 31.5%), 12(17, 11.9%), 11(16, 11.2%) and 6(14, 9.8%). CONCLUSION: Variants of the PAH gene identified in Jiangxi province mainly involve exons 7, 12, 11 and 6, with the most common variants being R243Q and R408Q. Six novel variants were identified.

The mechanism on phosphorylation of Hsp20Ser16 inhibit GA stress and ER stress during OGD/R.

Recent research has demonstrated that small heat shock protein (sHsp) phosphorylation plays a variety of roles in neural cells. While the phosphorylation of serine 16 (Ser16) is blocked, Hsp20 no longer has neuroprotective effects. To further investigate the mechanism underlying this process, oxygen-glucose deprivation and reperfusion (OGD/R) was used with human SH-SY5Y cells and mouse N2a neuroblastoma cells. When SH-SY5Y and N2a cells were transfected with pEGFP-Hsp20(WT), pEGFP-Hsp20(S16A), and pEGFP-Hsp20(S16D) plasmids, the Golgi apparatus (GA) became more swollen and scattered, and many small fragments formed in the MOCK and S16A groups after OGD/R (P < 0.05). Meanwhile, the endoplasmic reticulum (ER) network was reduced, and the lamellar structure increased. However, these changes were not as obvious in the WT and S16D groups. Additionally, after OGD/R, Golgi Stress related protein contents were increased in the WT and S16D groups compared with the MOCK and S16A groups (P < 0.05). However, ER Stress related protein contents were decreased in the WT and S16D groups compared with the MOCK and S16A groups (P < 0.05). Our study demonstrates that Hsp20 phosphorylation on Ser16 protects against not only OGD/R-induced GA fragmentation in SH-SY5Y cells and N2a cells via Golgi stress but also OGD/R-induced ER structural changes in SH-SY5Y cells via ER stress. These findings suggest that Hsp20 is a potential drug target for ischemia stroke treatment.