Competing Endogenous RNAs Crosstalk in Hippocampus: A Potential Mechanism for Neuronal Developing Defects in Down Syndrome.

Down syndrome (DS) is the most example of aneuploidy, resulting from an additional copy of all or part of chromosome 21. Competing endogenous RNAs (ceRNAs) play important roles in neuronal development and neurological defects. This study aimed to identify hub genes and synergistic crosstalk among ceRNAs in the DS fetal hippocampus as potential targets for the treatment of DS-related neurodegenerative diseases. We profiled differentially expressed long non-coding RNAs (DElncRNAs), differentially expressed circular RNAs (DEcircRNAs), differentially expressed microRNAs (DEmiRNAs), and differentially expressed messenger RNAs (DEmRNAs) in hippocampal samples from patients with or without DS. Functional enrichment analysis and gene set enrichment analysis were performed, and chromosome 21-related ceRNA and protein-protein interaction networks were constructed. Additionally, the correlations between lncRNA-mRNA and miRNA-mRNA expression in the samples and HEK293T cells were validated. Our finding of changes in the expression of some key genes and ncRNAs on chromosome 21 in DS might not fully conform to the gene dosage hypothesis. Moreover, we found that four lncRNAs (MIR99AHG, PLCB4, SNHG14, GIGYF2) and one circRNA (hsa_circ_0061697) may competitively bind with three miRNAs (hsa-miR-548b-5p, miR-730-5p, and hsa-miR-548i) and subsequently regulate five mRNAs (beta-1,3-galactosyltransferase 5 [B3GALT5], helicase lymphoid-specific [HELLS], thrombospondin-2 [THBS2], glycinamide ribonucleotide transformylase [GART], clathrin heavy chain like 1 [CLTCL1]). These RNAs, whether located on chromosome 21 or not, interact with each other and might activate the PI3K/Akt/mTOR and Wnt signaling pathways, which are involved in autophagosome formation and tau hyperphosphorylation, possibly leading to adverse consequences of trisomy 21. These findings provide researchers with a better understanding of the fundamental molecular mechanisms underlying DS-related progressive defects in neuronal development.

[Application of whole exome sequencing for the inferential analysis of recessive genetic disease carrier status for couples with a child died of Primary immunodeficiency].

OBJECTIVE: To explore the value of whole exome sequencing for the inferential analysis of recessive genetic disease carrier status for couples with a child died of Primary immunodeficiency (PID). METHODS: Clinical data was collected from four couples with a childbearing history of PID who had sought genetic counseling and undergone genetic testing at Henan Provincial People's Hospital from February 2017 to December 2021. Whole exome sequencing (WES) was performed on both partners of each couple, and candidate variants were validated by Sanger sequencing and fluorescent quantitative PCR. Prenatal diagnosis was conducted on fetuses of these couples after confirming the variants. RESULTS: A total of six variants were detected in four genes including IL2RG, BTK, CYBB, and DUOX2. Among these, the c.1265G>A and c.3329G>A variants of the DUOX2 gene and the c.676C>T variant of the IL2RG gene were previously known as pathogenic variants. On the other hand, the Exon5_8del variant of the IL2RG gene, the c.184_185delAC variant of the BTK gene, and the c.472A>T variant of the CYBB gene were unreported previously. Based on the guidelines from the American College of Medical Genetics and Genomics, the IL2RG: Exon5_8del, BTK: c.184_185delAC and CYBB: c.472A>T variants were classified as likely pathogenic (PVS1+PM2_Supporting+PP4).Prenatal diagnosis was conducted for three couples during their subsequent pregnancies, and the results revealed that the fetuses had the wild-type genotypes at the c.184_185 position of the BTK gene, the c.472 position of the CYBB gene, and the c.676 position of the IL2RG gene. Follow-up examinations one year after birth has found no abnormality in the infants. CONCLUSION: WES is an important tool to infer and analyze the carrier status for couples who had given births to children died of PID and improve the positive detection rate.

Functional analysis of a novel nonsense variant c.91A>T of the TRAPPC2 gene in a Chinese family with X-linked recessive autosomal spondyloepiphyseal dysplasia tarda.

Spondyloepiphyseal dysplasia tarda (SEDT) is a condition involving late-onset, X-linked recessive skeletal dysplasia caused by mutations in the TRAPPC2 gene. In this paper, we identified a novel nonsense variant in a SEDT pedigree and analyzed the function of the variant in an attempt to explain the new pathogenesis of the TRAPPC2 protein in SEDT. Briefly, DNA and RNA samples from the peripheral blood of SEDT individuals were prepared. The causative variant in the Chinese SEDT family was identified by clinic whole-exome sequencing analysis. Then, we observed the mRNA expression of TRAPPC2 in patients and the mutant TRAPPC2 level in vitro and analyzed the protein stability and subcellular distribution by cell fluorescence and Western blotting. We also investigated the effect of TRAPPC2 knockdown on the expression and secretion of COL2A1 in SW1353 cells or primary human chondrocytes. Herein, we found a nonsense variant, c.91A>T, of the TRAPPC2 gene in the pedigree. TRAPPC2 mRNA expression levels were significantly decreased in the available peripheral blood cell samples of two affected patients. An in vitro study showed that the mutant plasmid exhibited significantly lower mRNA and protein of TRAPPC2, and the mutant protein changed its membrane distribution. TRAPPC2 knockdown resulted in decreased COL2A1 expression and collagen II secretions. Our data indicate that the novel nonsense variant, c.91A>T, of the TRAPPC2 gene is the cause of SEDT in this pedigree. The variant results in a lowered expression of TRAPPC2 and then affects the COL2A1 expression and collagen II secretions, which may explain the mechanism of loss of function of the variant.

[Expert consensus on the genetic diagnosis for Dystrophinopathies].

Dystrophinopathies, including Duchenne muscular dystrophy, Becker muscular dystrophy and dilated cardiomyopathy, are X-linked recessive genetic disorders due to variants of the dystrophin gene, which can seriously affect quality of life and health. Genetic diagnosis plays a crucial role in their diagnosis, treatment, and prevention. How to rationally select and standardize the use of various genetic techniques is a skill that clinicians must acquire. By compiling expertise of experts from the relevant areas and guidelines published home and abroad, this consensus has provided a guidance from the perspective of genetic diagnosis for the selection of genetic techniques, testing strategies, and detection process for dystrophinopathies.

[Genetic analysis of a Chinese pedigree with Cohen syndrome due to compound heterozygous variants of VPS13B gene].

OBJECTIVE: To investigate the clinical phenotype and genetic characteristics of a Chinese pedigree affected with Cohen syndrome. METHODS: A proband who was admitted to Zhengzhou People's Hospital on June 2, 2021 due to intellectual disability and developmental delay, in addition with her younger sister and other family members, were selected as the study subjects. Clinical data of the proband and her younger sister were collected. Genomic DNA was extracted from peripheral venous blood and chorionic villi samples. Chromosomal abnormalities were detected with chromosomal microarray analysis (CMA). Whole exome sequencing (WES) and Sanger sequencing were carried out to detect candidate variants in the proband. With RNA extracted from the peripheral blood samples, VPS13B gene transcripts and expression were analyzed by PCR and real-time quantitative PCR. Prenatal diagnosis was carried out at 12 weeks' gestation. RESULTS: The proband was a 10-year-old female with clinical manifestations including development delay, obesity, severe myopia and peculiar facial features. Her sister was 3 years old with a similar phenotype. CMA revealed no chromosomal abnormality in the proband, while WES results revealed that the proband and her sister had both harbored compound heterozygous variants of the VPS13B gene, namely c.10076_10077delCA (p.T3359fs*29) and c.6940+1G>T, which were respectively inherited from their mother and father. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), both variants were classified as pathogenic (PVS1+PS4+PM4+PP1; PVS1+PM2_Supporting+PM3+PP1). In vivo splicing assay confirmed that the c.6940+1G>T variant has produced a frameshift transcript with skipping of exon 38. Compared with the control group, the expression of RNA in the peripheral blood of the proband's parents has decreased to 65% ~ 70% (P < 0.01), whilst that in the proband and her sister has decreased to 40% (P < 0.001). Prenatal diagnosis at 12 weeks of gestation has found that the fetus only harbored the heterozygous c.10076_ 10077delCA variant. CONCLUSION: The c.10076_10077delCA (p.T3359fs*29) frameshift variant and c.6940+1G>T splicing variant probably underlay the Cohen syndrome in this pedigree. Genetic testing has facilitated the diagnosis of this disease.

[Analysis of a child with mental retardation due to a de novo variant of the KAT6A gene].

OBJECTIVE: To explore the genetic etiology for a child featuring mental retardation and speech delay. METHODS: Clinical data of the child was collected. DNA was extracted from peripheral blood samples of the child and members of his pedigree. Whole exome sequencing was carried out for the child, and candidate variants were verified by Sanger sequencing. Prenatal diagnosis was provided for his mother upon her subsequent pregnancy. RESULTS: The child has mainly featured mental retardation, speech delay, ptosis, strabismus, photophobia, hyperactivity, and irritability. Whole exome sequencing revealed that he has harbored a pathogenic heterozygous variant of the KAT6A gene, namely c.5314dupA (p.Ser1772fs*20), which was not detected in either of his parents. The child was diagnosed with Arboleda-Tham syndrome. The child was also found to harbor a hemizygous c.56T>G (p.Leu19Trp) variant of the AIFM1 gene, for which his mother was heterozygous and his phenotypically normal maternal grandfather was hemizygous. Pathogenicity was excluded. Prenatal diagnosis has excluded the c.5314dupA variant of the KAT6A gene in the fetus. CONCLUSION: The heterozygous c.5314dupA (p.Ser1772fs*20) variant of the KAT6A gene probably underlay the Arboleda-Tham syndrome in this child. Above finding has enabled genetic counseling and prenatal diagnosis for this pedigree.

[Expert consensus on the application of prenatal exome sequencing for fetal structural anomalies].

Prospective research have shown that whole exome sequencing (WES) may be considered when a diagnosis cannot be obtained using routine prenatal methods, e.g., chromosomal karyotyping and copy number variation sequencing, for fetuses with significant structural anomalies. WES can increase the diagnostic rate of genetic disorders in such fetuses by 8% - 10%. Prenatal WES has been gaining wide acceptance. However, due to the limitations of fetal phenotypic evaluation and complexity of ethical issues in prenatal diagnosis, to justify and standardize the application of prenatal WES and maximize its clinical utility has become an urgent need. In view of this, a consensus has been formed by referring to the latest guidelines, expert consensus and authoritative literature. This consensus has put forward suggestions on the suitable objects of prenatal WES, pre-test consultation, sampling and laboratory testing, results report, post-test consultation, pregnancy outcome follow-up, multidisciplinary consultation of difficult cases, preservation of prenatal WES samples and data information.

[Application of genomic copy number variation detection technology in prenatal diagnosis of 7617 pregnant women with serological screening abnormalities during the second trimester of pregnancy].

OBJECTIVE: To analyze the genomic variation characteristics of fetal with abnormal serological screening, and to further explore the value of copy number variation (CNV) detection technology in prenatal diagnosis of fetal with abnormal serological screening. METHODS: 7617 singleton pregnant women who underwent amniocentesis for prenatal diagnosis solely due to abnormal Down's serological screening were selected. According to the results of serological screening, the patients were divided into high risk group, borderline risk group and single abnormal multiple of median (MOM) group. CMA and CNV-Seq were used to detect the copy number variation of amniotic fluid cell genomic DNA and combined with amniotic fluid cell karyotype analysis for prenatal diagnosis. Outpatient revisit combined with telephone inquiry was used for postnatal follow-up. RESULTS: Among 7617 amniotic fluid samples, aneuploidy was detected in 138cases (1.81%) by CMA and CNV-Seq, 9 cases of aneuploid chimerism were detected by amniotic fluid cell karyotype analysis, and 203 cases of fetus carrying pathogenic and likely pathogenic CNV (P/LP CNV) were detected, the variant of uncertain significance (VUS) was detected in 437 cases (5.7%), the overall abnormal detection rate was 10.33%. The detection rate of aneuploidy by CMA and CNV-Seq in three group were 123 cases (2.9%), 13 cases (1.3%) and 2 cases (0.4%), respectively,and showing no significant difference (χ 2=7.469, P=0.024). The detection rate of pathogenic and likely pathogenic CNV in three group were 163cases (2.6%); 24 cases (2.6%) and 16 cases (3.3%), respectively, and showing no significant difference (χ 2=0.764, P=0.682). The CMA reported 2.9% (108/3729)P/LP CNV, and CNV-seq reported 2.4% (95/3888)P/LP CNV, both tests showed similar detective capabilities (χ 2=1.504, P=0.22).The most popular P/LP CNV in this cohort were Xp22.31 microdeletion, 16p13.11 microduplication /microdeletion, 22q11.21 microduplication /microdeletion. In fetuses with P/LP CNV CNV, 59 fetuses were terminated pregnancy, and 32 of 112 fetuses born had abnormal clinical manifestations. Non-medically necessary termination of pregnancy occurred in 11 fetuses carrying VUS CNV, 322 fetuses carrying VUS CNV were born, 4 of them presented abnormal clinical manifestations. CONCLUSION: Compared with the traditional chromosome karyotype, CMA and CNV-Seq can improve the detection rate of pathogenic and likely pathogenic CNV. CMA and CNV-seq can be used for first tier diagnosis of pregnant women in the general population with abnormal Down's serological screening.

[Genetic analysis and prenatal diagnosis of a Chinese pedigree affected with Usher syndrome due to novel compound heterozygous variants of PCDH15 gene].

OBJECTIVE: To analyze the clinical features and genetic variant in a patient with Usher syndrome. METHODS: Whole exome sequencing was carried out for the patient. Suspected variants were validated by Sanger sequencing of her parents and fetus. RESULTS: The proband was found to harbor compound heterozygous variants c.17_18insA (p.Tyr6Ter*) and c.4095_4096insA (p.Arg1366Lys fs*38) of the PCDH15 gene (NM_033056), which were respectively inherited from her father and mother. The same variants were not detected in 100 healthy controls. Based on the guidelines of the American Society of Medical Genetics and Genomics, both variants were predicted to be pathogenic (PVS1+PM2+PP4). By prenatal diagnosis, her fetus was found to carry the c.4095_4096insA variant. After birth, the child has passed neonatal hearing screening test, and no abnormal auditory and visual function was found after the first year. CONCLUSION: The compound heterozygous variants c.17_18insA (p.Tyr6Ter*) and c.4095_4096insA (p.Arg1366Lys fs*38) of the PCDH15 gene probably underlay the Usher syndrome is this proband.

[Analysis of a Chinese pedigree affected with dyschromatosis symmetrica hereditaria due to a novel variant of ADAR gene].

OBJECTIVE: To explore the genetic basis for a Chinese pedigree affected with dyschromatosis symmetrica hereditaria (DSH). METHODS: PCR and Sanger sequencing were carried out for the proband, and suspected variant was validated by Sanger sequencing in the pedigree. RESULTS: The proband was found to harbor a novel variant of c.1352delA (p.N451Mfs*13) of the ADAR (NM_001111) gene. The same variant was found in her affected mother and sister, but not in her unaffected father, uncle, and 100 healthy individual. CONCLUSION: The novel variant of the ADAR gene probably underlay the pathogenesis of DSH in this pedigree.

Functional Analysis of a Compound Heterozygous Mutation in the VPS13B Gene in a Chinese Pedigree with Cohen Syndrome.

Cohen syndrome (CS) is an autosomal recessive congenital disorder characterized by mutation in the vacuolar protein sorting 13 homolog B (VPS13B; formerly COH1) gene. In the current study, a Chinese family has two young sibling cases having a developmental delay, physical obesity, high myopia, and a special face, which suspected to be CS. The purpose of the study was to identify variants and further analyze their pathogenicity for CS. Next-generation sequencing (NGS) revealed a compound heterozygous mutation in VPS13B gene in the proband, which comprises a frameshift mutation in NM_017890.4: c.10076_10077delCA (p.T3359fs*29) and a putative splice site mutation in c.6940 + 1G > T. Both Minigene assay in vitro and splicing assay in vivo confirmed that the splicing mutation in c.6940 + 1G > T generates a frameshift transcript with whole exon 38 skipping. Eventually, quantitative real-time PCR demonstrated that either of the two mutations can lead to degradation of the VPS13B gene at the transcriptional level. Functional studies of variants identified in CS patients are essential for their subsequent genetic counseling and prenatal diagnoses and could also be the start point for new therapeutic approaches, currently based only on symptomatic treatment.

[Identification of SPAST gene variant in a pedigree affected with hereditary spastic paraplegia type 4].

OBJECTIVE: To explore the genetic basis for a pedigree affected with hereditary spastic paraplegia type 4 (HSP4). METHODS: Peripheral venous blood samples were taken from members of the four-generation pedigree and 50 healthy controls for the extraction of genomic DNA. Genes associated with peripheral neuropathy and hereditary spastic paraplegia were captured and subjected to targeted capture and next-generation sequencing. The results were confirmed by Sanger sequencing. RESULTS: DNA sequencing suggested that the proband has carried a heterozygous c.1196C>G variant in exon 9 of the SPAST gene, which can cause substitution of serine by threonine at position 399 (p.Ser399Trp) and lead to change in the protein function. The same variant was also detected in other patients from the pedigree but not among unaffected individuals or the 50 healthy controls. Based on the ACMG 2015 guidelines, the variant was predicted to be possibly pathogenic. CONCLUSION: The c.1196C>G variant of the SPAST gene probably underlay the HSP4 in this pedigree.

[Genetic analysis of a patient with late infantile metachromatic leukodystrophy].

OBJECTIVE: To detect variants of ARSA gene in a child featuring late infantile metachromatic leukodystrophy (MLD). METHODS: PCR and Sanger sequencing was carried out for the patient and her parents. RESULTS: The patient had typical features of MLD including ARSA deficiency, regression of walking ability, and demyelination. Compound heterozygous variants of the ARSA gene, namely c.960G>A and c.244C>T, were detected in the patient, for which her mother and father were respectively heterozygous carriers. ARSA c.960G>A was known to be pathogenic, while ARSA c.244C>T was a novel variant. The same variants were not detected among 50 healthy controls. CONCLUSION: The compound heterozygous variants c.960G>A and c.244C>T of the ARSA gene probably underlie the MLD in this patient.

[Prenatal diagnosis of a fetus affected with Finnish type congenital nephrotic syndrome].

OBJECTIVE: To explore the genetic basis for a fetus suspected for congenital nephrotic syndrome of Finland (CNF). METHODS: Genomic DNA was extracted from peripheral and umbilical cord blood samples derived from both parents and the fetus. Potential variants were detected by using next-generation sequencing. Suspected variants were confirmed by Sanger sequencing. RESULTS: The fetus was found to carry compound heterozygous variants c.1440+1G>A and c.925G>T of the NPHS1 gene, which were respectively inherited from its mother and father. CONCLUSION: Identification of the compound heterozygous NPHS1 variants has enabled diagnosis of CNF in the fetus and genetic counseling for the affected family.

[Prenatal diagnosis of partial trisomy 3q in a fetus].

OBJECTIVE: To carry out prenatal diagnosis for a fetus with ultrasonographic abnormality. METHODS: Chromosomal karyotyping and array comparative genomic hybridization (array-CGH) analysis were applied for the diagnosis. Peripheral blood samples were also taken from the parents for chromosome karyotyping analysis. RESULTS: The fetal karyotype showed additional material of unknown-origin attached to Yq. Array CGH analysis confirmed that the material was derived from 3q22.1q29. The father was found to carry a balanced translocation 46, X, t(Y;3)(q12;q23) (which was diagnosed as 46,XY,Y≥18 elsewhere), whilst the mother was found to be normal. CONCLUSION: 3q partial trisomy may present as malformation of multiple systems. Combination of chromosome karyotyping and array-CGH can provide reliable diagnosis for fetuses with abnormalities by ultrasonography.

[Genetic analysis of a pedigree affected with Bartter's syndrome].

OBJECTIVE: To explore the genetic basis for a pedigree affected with Bartter's syndrome (BS). METHODS: Panel-based next-generation sequencing (NGS) was carried out to detect mutation in BS-related genes SLC12A1, KCNJ1, BSND and CLCNKB. Sanger sequencing of MAGED2 gene and chromosomal microarray analysis (CMA) were also performed on the patient. Suspected mutation was validated in her family members. RESULTS: No pathogenic mutation was detected by NGS, while a 0.152 Mb microdeletion at Xp11.21 (54 834 585-54 986 301) was found in the male fetus, which removed the entire coding region of the MAGED2 gene. His mother was a heterozygous carrier of the deletion. His father and sister did not carry the same deletion. CONCLUSION: The loss of the MAGED2 gene may underlie the BS in this pedigree.

[A novel compound heterozygous mutation of GNPTAB gene underlying a case with mucolipidosis type II α/ß].

OBJECTIVE: To analyze the clinical features and genetic mutations in a patient with mucolipidosis type II α/ß by using next generation sequencing. METHODS: Clinical data of the patient was collected. Genomic DNA of the patient and her parents was extracted by a standard method. The patient was subjected to targeted sequencing using an Ion Ampliseq panel, which included genes related to mucolipidosis and mucopolysaccharidosis. Suspected mutations were verified by Sanger sequencing. RESULTS: Compound heterozygous mutations, namely c.1284+1G>T and c.1090C>T (p.Arg364*), were detected in the patient, which were respectively inherited from her mother and father. No other disease-causing mutation was detected in the patient. GNPTAB c.1090C>T was known to be pathogenic, while GNPTAB c.1284+1G>T is a novel mutation. The same mutations were not detected among 50 healthy controls. CONCLUSION: The compound heterozygous mutations c.1284+1G>T and c.1090C>T (p.Arg364*) of GNPTAB gene probably account for the mucolipidosis type II α/ß in the patient. NGS has a great value for the molecular diagnosis and typing of mucolipidosis.

[Identification of two novel Parkin gene mutations in a patient affected with Juvenile Parkinson's syndrome].

OBJECTIVE: To explore the clinical and genetic features of a patient suspected with Juvenile Parkinson's syndrome (JP). METHODS: Clinical features of the patient were analyzed. Genomic DNA of the patient and his parents was extracted from peripheral blood samples and sequenced by exome capture sequencing. The nature and impact of detected mutations were predicted and validated. RESULTS: The patient displayed typical features including resting tremor, bradykinesia, rigidity, but with excellent response to low dose levodopa. DNA sequencing showed that she has carried compound heterozygous mutations of the Parkin gene, namely c.1381dupC and c.619-1G>C, which were respectively inherited from his mother and father. Neither mutation was reported previously. Bioinformatic analysis predicted that both mutations are pathogenic. CONCLUSION: The patient has JP caused by mutations of the Parkin gene. Exome capture sequencing is an accurate and efficient method for genetic diagnosis of such disease.

Targeted next-generation sequencing-based molecular diagnosis of congenital hand malformations in Chinese population.

Congenital hand malformations is rare and characterized by hand deformities. It is highly heterogeneous, both clinically and genetically, which complicates the identification of causative genes and mutations. Recently, targeted next-generation (NGS) sequencing has been successfully used for the detection of heterogeneous diseases, and the use of NGS also has contributed significantly in evaluating the etiology of heterogeneous disease. Here, we employed targeted NGS to screen 248 genes involved in genetic skeletal disorders, including congenital hand malformations. Three pathogenic mutations located in the GJA1, ROR2 and TBX5 genes were detected in three large Chinese families with congenital hand malformations. Two novel mutations were reported, and a known causative mutation was verified in this Chinese population. This is also the first report that the same panel of targeted NGS was employed to perform molecular diagnosis of different subtypes of congenital hand malformations. Our study supported the application of a targeted NGS panel as an effective tool to detect the genetic cause for heterogeneous diseases in clinical diagnosis.

[Analysis of WAS gene mutation in a Chinese family affected with Wiskott-Aldrich syndrome].

OBJECTIVE: To detect potential mutation of the WAS gene in a Chinese family affected with Wiskott-Aldrich syndrome. METHODS: Peripheral blood samples were collected from the proband and his family members. All exons and flanking regions of the WAS gene were subjected to PCR amplification - Sanger sequencing as well as restriction endonuclease analysis. Plasma level of B-cell activating factor (BAFF) was also determined for all family members. RESULTS: A hemizygous mutation (c.257G>A) of the WAS gene was identified in all patients from the family, for which the patient's mother was heterozygous. The same mutation was not found among healthy members of the family. Compared with unaffected members, all patients had a higher level of BAFF. CONCLUSION: The c.257G>A mutation of the WAS gene probably underlies the Wiskott-Aldrich syndrome in this family.