[Analysis of the results for genetic disease screening among 1 000 newborns from Huzhou].

OBJECTIVE: To analyze the types and distribution of pathogenic variants for neonatal genetic diseases in Huzhou, Zhejiang Province. METHODS: One thousand neonates (48 ~ 42 h after birth) born to Huzhou region were selected as the study subjects. Dry blood spot samples were collected from the newborns, and targeted capture high-throughput sequencing was carried out for pathogenic genes underlying 542 inherited diseases. Candidate variants were verified by Sanger sequencing. RESULTS: Among the 1 000 newborns, the male to female ratio was 1.02 : 1.00. No pathogenic variants were detected in 253 cases, whilst 747 cases were found to carry at least one pathogenic variant, which yielded a carrier rate of 74.7%. The most frequently involved pathogenic gene was FLG, followed by GJB2, UGT1A1, USH2A and DUOX2. The variants were classified as homozygous, compound heterozygous, and hemizygous variants. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), 213 neonates were verified to have carried pathogenic and/or likely pathogenic variants, with a positive rate of 21.3%. The most commonly involved genes had included UGT1A1, FLG, GJB2, MEFV and G6PD. CONCLUSION: Newborn screening based on high-throughput sequencing technology can expand the scope of screening and improve the positive predictive value. Genetic counseling based on the results can improve the patients' medical care and reduce neonatal mortality and childhood morbidity, while provide assistance to family members' health management and reproductive decisions.

[Genetic analysis of a child with XYY syndrome in conjunct with 3-methylglutaenedioic aciduria type I].

OBJECTIVE: To explore the genetic basis for a child with mental retardation. METHODS: The child was subjected to chromosomal microarray analysis (CMA) and targeted capture next-generation sequencing for the exons of genes related to genetic and metabolic diseases. Candidate variants were verified by Sanger sequencing of the child and his parents. RESULTS: CMA suggested that the child has a 47,XYY karyotype. Next-generation sequencing revealed that the child has harbored compound heterozygous variants of the AUH gene, including c.677G>A (p.R226H) and c.373C>T (p.R125W), which were respectively inherited from his parents. Based on the American college of Medical Genetics and Genomics (ACMG) standards and guidelines, the c.677G>A (P.r226h) variant was predicted as variant of uncertain significance (PM2+PP4+PP3), whilst the c.373C>T (P.R125W) variant was predicted as likely pathogenic (PM1+PM2+PP3+PP4). CONCLUSION: The child had XYY syndrome in conjunct with 3-methylglutaenedioic aciduria type I due to biallelic pathogenic variants of the AUH gene.

[Genetic analysis of a rare case of Pitt-Hopkins syndrome due to partial deletion of TCF4 gene].

OBJECTIVE: To explore the genetic basis for a child featuring delayed intellectual development. METHODS: The child and his parents were subjected to conventional G-banding karyotyping and single nucleotide polymorphism array (SNP-array) analysis. Suspected copy number variations (CNVs) were verified in both parents. RESULTS: No karyotypic abnormality was found with the child and his parents. SNP-array results for both parents were normal. The child was found to harbor a de novo 172 kb deletion at 18q21.2 with a physical position of 52 957 042-53 129 237. The deletion only involved one OMIM gene, namely TCF4, resulting in removal of its exons 6 to 8. CONCLUSION: The SNP-array assay has facilitated with the diagnosis of this child. Deletion of 18q21.2 region probably accounts for the Pitt-Hopkins syndrome (PTHS) in this patient.

[Identification of a c.1A>G initial codon variation of ARX gene in a child with severe mental retardation].

OBJECTIVE: To explore the genetic basis for a child featuring severe mental retardation. METHODS: The child was subjected to target region capture and next generation sequencing. Suspected variants were verified by Sanger sequencing. RESULTS: The child was found to harbor a hemizygous c.1A>G (pMet1?) variation of the ARX gene, for which his mother was a heterozygous carrier. The mutation was unreported previously and was predicted to be "probably pathogenic" by bioinformatic analysis. CONCLUSION: The c.1A>G (pMet1?) variant of the ARX gene may underlie the occurrence of severe mental retardation in this child.

[Application value of chromosome microarray analysis for prenatal diagnosis of fetus with ultrasonic abnormalities].

OBJECTIVE: To assess the application value of chromosomal microarray analysis (CMA) for prenatal diagnosis of fetus with ultrasound abnormalities. METHODS: For 293 fetuses with ultrasound abnormalities (including 168 with structural abnormalities and 125 with non-structured abnormalities) but no common chromosomal abnormalities, CMA assay was performed. RESULTS: Sixteen pathogenic copy number variants (pCNVs) were detected by CMA with a detection rate of 5.46%. The detection rates were 5.95% (10/168) for those with structural abnormalities and 4.80% (6/125) for those with non-structural abnormalities. CONCLUSION: Compared with conventional karyotyping analysis, CMA can improve the detection of fetal chromosomal abnormality and provide an effective means for prenatal diagnosis.

[Genetic analysis and prenatal diagnosis of a pregnant woman with Sheldon-Hall syndrome].

OBJECTIVE: To provide genetic testing and prenatal diagnosis for a woman with Sheldon-Hall syndrome. METHODS: The woman was subjected to targeted capture and next-generation sequencing for variant of genes associated with skeletal disorders. And the result was verified in her parents and fetus. RESULTS: The woman was found to harbor a c.188G>A variant of the TNNT3 gene, which was also found in her affected mother and the fetus. Her grandmother and grandmother's brother had similar manifestations, which was in line with an autosomal dominant inheritance. The same variant was not found in her father. CONCLUSION: The c.188G>A variant of the TNNT3 gene probably underlay the distal joint contracture in this pedigree, based on which prenatal diagnosis was attained.

[Prenatal diagnosis of three fetuses with small supernumerary marker chromosomes].

OBJECTIVE: To explore the origin and mechanism of small supernumerary marker chromosomes (sSMC) in three fetuses. METHODS: The three fetuses were predicted to have carried chromosomal abnormalities by non-invasive prenatal testing (NIPT). G-banding chromosomal karyotyping analysis were carried out on amniotic fluid samples of the fetuses and peripheral blood samples from their parents. Single nucleotide polymorphism array (SNP-array) was used to determine the origin, size and genetic effect of sSMCs. RESULTS: In fetus 1, SNP array has detected two microduplications respectively at 4p16.3p15.2 (24.7 Mb) and 18p11.32q11.2 (20.5 Mb) which, as verified by fluorescence in situ hybridization (FISH), have derived from a balanced 46,XY,t(4;18)(p15.2q11.2) translocation carried by its father. Fetus 2 has carried a de novo microduplication of 15q11.2-q13.3 (9.7 Mb). The sequence of SMC in fetus 3 has derived from 21q11.2-q21.1 (8.3 Mb), which was inherited from its mother. CONCLUSION: Both NIPT and SNP-array are highly accurate for the detection of sSMC. SNP-array can delineate the origin and size of abnormal chromosomes, which in turn can help with clarification of sSMC-related genotype-phenotype correlation and facilitate prenatal diagnosis and genetic counseling for the family.

TIGAR knockdown enhanced the anticancer effect of aescin via regulating autophagy and apoptosis in colorectal cancer cells.

Our previous study showed that TP53-induced glycolysis and apoptosis regulator (TIGAR) regulated ROS, autophagy, and apoptosis in response to hypoxia and chemotherapeutic drugs. Aescin, a triterpene saponin, exerts anticancer effects and increases ROS levels. The ROS is a key upstream signaling to activate autophagy. Whether there is a crosstalk between TIGAR and aescin in regulating ROS, autophagy, and apoptosis is unknown. In this study, we found that aescin inhibited cell viability and colony formation, and induced DNA damage, cell cycle arrest, and apoptosis in cancer cell lines HCT-116 and HCT-8 cells. Concurrently, aescin increased the expression of TIGAR, ROS levels, and autophagy activation. Knockdown of TIGAR enhanced the anticancer effects of aescin in vitro and in vivo, whereas overexpression of TIGAR or replenishing TIGAR downstream products, NADPH and ribose, attenuated aescin-induced apoptosis. Furthermore, aescin-induced ROS elevation and autophagy activation were further strengthened by TIGAR knockdown in HCT-116 cells. However, autophagy inhibition by knockdown of autophagy-related gene ATG5 or 3-methyladenine (3-MA) exaggerated aescin-induced apoptosis when TIGAR was knocked down. In conclusion, TIGAR plays a dual role in determining cancer cell fate via inhibiting both apoptosis and autophagy in response to aescin, which indicated that inhibition of TIGAR and/or autophagy may be a junctional therapeutic target in treatment of cancers with aescin.

[Prenatal diagnosis of a case with Branchi-oto-renal syndrome].

OBJECTIVE: To carry out prenatal diagnosis for a women with Branchio-oto-renal syndrome by using chromosomal microarray analysis (CMA). METHODS: Peripheral blood chromosomal karyotyping and CMA were used to analyze the gravida with an abnormal phenotype. Pathological copy number variants (CNVs) were validated in other members of the family members and her fetus. RESULTS: The gravida and her daughter both had Branchio-oto-renal syndrome and a 8q13.3 microdeletion encompassing the EYA1 gene. The same microdeletion was also found in the fetus. No phenotypic or genotypic anomaly was found with other members of the family. CONCLUSION: Mutation of the EYA1 gene probably underlies the Branchio-oto-renal syndrome in this family, which is consistent with an autosomal dominant inheritance.

[Analysis of SYNE1 gene variant in an infant featuring epilepsy and developmental disorders].

OBJECTIVE: To explore the clinical features and molecular basis for a child featuring infantile epilepsy and developmental disorders. METHODS: Clinical data and peripheral blood samples of the child and his parents were collected. The coding regions of genes associated with nervous system development were subjected to target region capture sequencing. RESULTS: The child developed generalized spasm at 3 months and was diagnosed with epilepsy at 6 months of age. He was treated with Depakin but was diagnosed with mental retardation and developmental retardation at 3 years of age. A novel heterozygous c.3842T to G variant of the SYNE1 gene was detected. His father was found to carry the same variant and had a history of convulsions in infancy but with no mental or developmental anomalies. CONCLUSION: A novel variant of SYNE1 gene was identified in this child, and the prognosis may be poor.

[Genetic analysis and prenatal diagnosis for a pedigree affected with X-linked Norrie disease].

OBJECTIVE: To detect mutation of NDP gene in a pedigree affected with Norrie disease. METHODS: Sanger sequencing was used to analyze the NDP gene at Xp11.3. Prenatal diagnosis was performed on amniotic fluid sample after the causative gene was detected. RESULTS: Sanger sequencing has revealed a c.2T>C (p.M1T) missense mutation of the NDP gene in the proband and the fetus. The same variation was not found in ClinVar and HGMD database. CONCLUSION: The c.2T>C mutation of the NDP gene probably underlies the Norrie disease in this pedigree.

Aescin-induced reactive oxygen species play a pro-survival role in human cancer cells via ATM/AMPK/ULK1-mediated autophagy.

Aescin, a natural mixture of triterpene saponins, has been reported to exert anticancer effect. Recent studies show that aescin increases intracellular reactive oxygen species (ROS) levels. However, whether the increased ROS play a role in the anticancer action of aescin remains to be explored. In this study, we demonstrated that aescin (20-80 µg/mL) dose-dependently induced apoptosis and activated mammalian target of rapamycin (mTOR)-independent autophagy in human hepatocellular carcinoma HepG2 cells and colon carcinoma HCT 116 cells. The activation of autophagy favored cancer cell survival in response to aescin, as suppression of autophagy with ATG5 siRNAs or 3-methyladenine (3-MA), a selective inhibitor of autophagy, promoted aescin-induced apoptosis in vitro, and significantly enhanced the anticancer effect of aescin in vivo. Meanwhile, aescin dose-dependently elevated intracellular ROS levels and activated Ataxia-telangiectasia mutated kinase/AMP-activated protein kinase/UNC-51-like kinase-1 (ATM/AMPK/ULK1) pathway. The ROS and ATM/AMPK/ULK1 pathway were upstream modulators of the aescin-induced autophagy, as N-acetyl-L-cysteine (NAC) or ATM kinase inhibitor (KU-55933) remarkably suppressed aescin-induced autophagy and consequently promoted aescin-induced apoptosis, whereas overexpression of ATG5 partly attenuated NAC-induced enhancement in aescin-induced apoptosis. In conclusion, this study provides new insights into the roles of aescin-mediated oxidative stress and autophagy in cancer cell survival. Our results suggest that combined administration of the antioxidants or autophagic inhibitors with aescin might be a potential strategy to enhance the anticancer effect of aescin.

Human serum albumin templated MnO2 nanosheets are oxidase mimics for colorimetric determination of hydrogen peroxide and for enzymatic determination of glucose.

This paper reports on a colorimetric assay for H2O2 and glucose. It is based on the use of human serum albumin-templated MnO2 nanosheets that possess oxidase-like activity. They are capable of oxidizing 3,3',5,5'-tetramethylbenzidine (TMB) with oxygen to give a blue product (oxTMB) with an absorbance maximum at 652 nm. When H2O2 is introduced, the MnO2 nanosheets are reduced to Mn(II) ions, and this inhibits the formation of oxTMB. Based on these findings, a colorimetric assay was established for H2O2 that has a 0.56 µM detection limit. If glucose is oxidized by glucose oxidase under formation of H2O2, the nanosheets can be used to quantify H2O2 and thereby to sense glucose. Response is linear in the 0.5 µM to 50 µM glucose concentration range, and the detection limit is 0.32 µM. The method was applied to the determination of glucose in spiked serum samples and gave satisficatory results. Graphical abstract Human serum albumin (HSA) is used as a template for the synthesis of MnO2 nanosheet. These possess oxidase mimicking activity. H2O2 can reduce the nanosheets. The effect is exploited in colorimetric assays for H2O2 and glucose using tetramethylbenzidine (TMB) as a chromogenic substrate.

[Genetic analysis of two cases with Dandy-Walker deformed fetus].

OBJECTIVE: To explore the genetic etiology of two fetuses with Dandy-Walker malformation using single nucleotide polymorphism microarray (SNP-array). METHODS: The fetuses and their parents were subjected to G banding karyotype analysis. The fetuses were also subjected to SNP-array analysis. RESULTS: The parents of both fetuses showed a normal karyotype. One fetus has a 46,X,?i(X)(q10), while for another conventional cell culture has failed. SNP-array showed that one fetus carried a 6p25.3p25.2 microdeletion, and another carried a Xp22.33p22.2 deletion and a Yq11.221q11 duplication. The abnormal fragments have involved FOXC1, SHOX and STS genes, which are associated with Dandy-Walker malformation. CONCLUSION: Alteration of 6p25.3p25.2, Xp22.33p22.2 copy numbers probably underlies the Dandy-Walker syndrome in the fetuses. The disorder may be attributed to abnormal expression of FOXC1, SHOX, and STS genes. SNP-array can provide an important supplement for prenatal diagnosis.

[Prenatal genetic analysis of a fetus with Wolf-Hirschhorn syndrome and Edward syndrome].

OBJECTIVE: To screen for genomic copy number variants (CNVs) in a fetus with cardiac abnormalities and intrauterine growth retardation through single nucleotide polymorphism microarray (SNP array) and karyotyping analysis. METHODS: The fetus and its parents were subjected to conventional G banding and SNP-array analysis. The results were confirmed with fluorescence in situ hybridization (FISH). RESULTS: G-banding analysis showed that the fetus has a karyotype of 47,XX,+mar. The father has a karyotype of 46,XY,t(4;18) (p15.2q11.2), while the mother showed a normal karyotype. SNP-array detected two microduplications at 18p11.32q11.2 (20.5 Mb) and 4p16.3p15.2 (24.7 Mb) in the fetus. The supernumerary marker chromosome carried by the fetus has derived from the balanced translocation carried by its father. The result was confirmed by FISH. CONCLUSION: Based on the two microduplications, the fetus was diagnosed as Wolf-Hirschhorn syndrome in conjunction with Edward syndrome. Verification of the origin of the supernumerary marker chromosome by SNP-array has provided a basis for prenatal genetic diagnosis.

[Prenatal diagnosis of a fetus with 5p15.33 microdeletion].

OBJECTIVE: To screen for genomic copy number variants (CNVs) in a fetus with one sibling affected with Prader-Willi syndrome using single nucleotide polymorphism (SNP) array. METHODS: The fetus and its parents were subjected to chromosomal karyotyping and SNP array analysis. RESULTS: A 5p15.33 microdeletions was identified in the fetus and its phenotypically normal mother with a size of 344 kb (113 576 to 457 213). The father was normal for both testing. Analysis of literature and CNVs database indicated the above CNV to be variant of unclear significance. The couple decided to continue with the pregnancy and gave birth to a healthy boy at full-term. No abnormalities were found during the follow-up. CONCLUSION: This study may provide further data for the phenotype-genotype correlation of 5p15.33 microdeletion, which differs from Cri du Chat syndrome.

Identification of four TTN variants in three families with fetal akinesia deformation sequence.

BACKGROUND: TTN is a complex gene with large genomic size and highly repetitive structure. Pathogenic variants in TTN have been reported to cause a range of skeletal muscle and cardiac disorders. Homozygous or compound heterozygous mutations tend to cause a wide spectrum of phenotypes with congenital or childhood onset. The onset and severity of the features were considered to be correlated with the types and location of the TTN variants. METHODS: Whole-exome sequencing was performed on three unrelated families presenting with fetal akinesia deformation sequence (FADS), mainly characterized by reduced fetal movements and limb contractures. Sanger sequencing was performed to confirm the variants. RT-PCR analysis was performed. RESULTS: TTN c.38,876-2 A > C, a meta transcript-only variant, with a second pathogenic or likely pathogenic variant in trans, was observed in five affected fetuses from the three families. Sanger sequencing showed that all the fetal variants were inherited from the parents. RT-PCR analysis showed two kinds of abnormal splicing, including intron 199 extension and skipping of 8 bases. CONCLUSIONS: Here we report on three unrelated families presenting with FADS caused by four TTN variants. In addition, our study demonstrates that pathogenic meta transcript-only TTN variant can lead to defects which is recognizable prenatally in a recessive manner.

Renal Hypodysplasia/Aplasia 3 Caused by a Rare Variant of GREB1L With Incomplete Penetrance in a Chinese Family.

Renal agenesis represents the most severe form of congenital anomalies of the kidney and urinary tract. Bilateral renal agenesis is almost invariably fatal at birth and has high genetic heterogeneity. Here we report on a Chinese family with two pregnancies affected by a prenatal form of bilateral renal agenesis. Trio-WES was conducted to explore the underlying genetic cause and identified a novel nonsense variant (c .2621G>A: p. Trp874Ter) in the GREB1L gene. Based on previous research, pathogenic mutations in GREB1L can cause renal hypodysplasia/aplasia-3 (RHDA3) with autosomal dominant inheritance. Sanger sequencing performed on the family members revealed that the variant was vertically transmitted from the maternal grandfather through the unaffected mother to the two affected fetuses, fully demonstrating the incomplete dominance of the disease. Our study extends the mutational spectrum associated with RHDA3 and contributes to a more general understanding for the complex genetic inheritance of GREB1L.

A Novel Mutation c.3392G>T of COL2A1 Causes Spondyloepiphyseal Dysplasia Congenital by Affecting Pre-mRNA Splicing.

Spondyloepiphyseal dysplasia congenital (SEDC) is a rare chondrodysplasia caused by dominant pathogenic variants in COL2A1. Here, we detected a novel variant c.3392G > T (NM_001844.4) of COL2A1 in a Chinese family with SEDC by targeted next-generation sequencing. To confirm the pathogenicity of the variant, we generated an appropriate minigene construct based on HeLa and HEK293T cell lines. Splicing assay indicated that the mutated minigene led to aberrant splicing of COL2A1 pre-mRNA and produced an alternatively spliced transcript with a skipping of partial exon 48, which generated a predicted in-frame deletion of 15 amino acids (p. Gly1131_Pro1145del) in the COL2A1 protein. Due to the pathogenicity of the variation, we performed prenatal diagnosis on the proband's wife, which indicated that the fetus carried the same mutation.

Case Report: Prenatal Diagnosis of Postaxial Polydactyly With Bi-Allelic Variants in Smoothened (SMO).

Postaxial polydactyly is a common congenital malformation which involves complex genetic factors. This retrospective study analyzed the cytogenetic and molecular results of a Chinese fetus diagnosed with postaxial polydactyly of all four limbs. Fetal karyotyping and chromosomal microarray analysis (CMA) did not find any abnormality while trio whole-exome sequencing (trio-WES) identified bi-allelic variants in smoothened (SMO) and (NM_005631.5: c.1219C > G, NP_005622.1: p. Pro407Ala, and NM_005631.5: c.1619C > T, NP_005622.1: p. Ala540Val). Sanger sequencing validated these variants. The mutations are highly conserved across multiple species. In-depth bioinformatics analysis and familial co-segregation implied the compound heterozygous variants as the likely cause of postaxial polydactyly in this fetus. Our findings provided the basis for genetic counseling and will contribute to a better understanding of the complex genetic mechanism that underlies postaxial polydactyly.