Next-generation sequencing based newborn screening and comparative analysis with MS/MS.

BACKGROUND: Newborn screening (NBS), such as tandem mass spectrometry (MS/MS), may yield false positive/negative results. Next-generation sequencing (NGS) has the potential to provide increased data output, efficiencies, and applications. This study aimed to analyze the types and distribution of pathogenic gene mutations in newborns in Huzhou, Zhejiang province, China and explore the applicability of NGS and MS/MS in NBS. METHODS: Blood spot samples from 1263 newborns were collected. NGS was employed to screen for pathogenic variants in 542 disease-causing genes, and detected variants were validated using Sanger sequencing. Simultaneously, 26 inherited metabolic diseases (IMD) were screened using MS/MS. Positive or suspicious samples identified through MS/MS were cross-referenced with the results of NGS. RESULTS: Among all newborns, 328 had no gene mutations detected. NGS revealed at least one gene mutation in 935 newborns, with a mutation rate of 74.0%. The top 5 genes were FLG, GJB2, UGT1A1, USH2A, and DUOX2. According to American College of Medical Genetics guidelines, gene mutations in 260 cases were classified as pathogenic or likely pathogenic mutation, with a positive rate of 20.6%. The top 5 genes were UGT1A1, FLG, GJB2, MEFV, and G6PD. MS/MS identified 18 positive or suspicious samples for IMD and 1245 negative samples. Verification of these cases by NGS results showed no pathogenic mutations, resulting in a false positive rate of 1.4% (18/1263). CONCLUSION: NBS using NGS technology broadened the range of diseases screened, and enhanced the accuracy of diagnoses in comparison to MS/MS for screening IMD. Combining NGS and biochemical screening would improve the efficiency of current NBS.

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.

Targeted exome sequencing strategy (NeoEXOME) for Chinese newborns using a pilot study with 3423 neonates.

BACKGROUND: Newborn screening (NBS) aims to detect congenital anomalies, and next-generation sequencing (NGS) has shown promise in this aspect. However, the NBS strategy for monogenic inherited diseases in China remains insufficient. METHODS: We developed a NeoEXOME panel comprising 601 genes that are relevant to the Chinese population found through extensive research on available databases. An interpretation system to grade the results into positive (high-risk, moderate-risk, and low-risk genotypes), negative, and carrier according to the American College of Medical Genetics (ACMG) guidelines was also developed. We validated the panel to evaluate its efficacy by using data from the "1000 Genomes Project" and conducted a pilot multicenter study involving 3423 neonates. RESULTS: The NGS positive rate in the 1000 Genomes Project was 7.6% (23/301), whereas the rate was 12.0% in the multicenter study, including 3249 recruited neonates. Notably, in 200 neonates, positive per conventional NBS, 58.5% (69/118) showed results consistent with NGS. In the remaining 3049 neonates showing negative results in conventional NBS, 271 (8.9%) were positive per NGS, and nine of them were clinically diagnosed with diseases in the follow-up. CONCLUSION: We successfully designed a NeoEXOME panel for targeted sequencing of monogenic inherited diseases in NBS. The panel demonstrated high performance in the Chinese population, particularly for the early detection of diseases with no biochemical markers.

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.

[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.

Asymmetric synthesis of planar-chiral macrocycles via organocatalyzed enantioselective macrocyclization.

A novel enantioselective macrocyclization method has been developed for the asymmetric synthesis of planar-chiral macrocycles through chiral phosphoric acid-catalyzed intramolecular addition of the hydroxy group with the allenamide moiety. A series of planar-chiral macrocycles bearing various ring sizes (18-member to 22-member) and various functional groups were generated with good to high enantioselectivities.

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.

Prenatal diagnosis of recurrent moderate skeletal dysplasias in lamin B receptors.

The lamin B receptor (LBR) gene is located in chromosome 1q42.12 and encodes the lamin B receptor, an intracellular protein that binds to lamin B. LBR mutations are associated with a broad phenotypic spectrum ranging from non-lethal to lethal skeletal dysplasias. The typical phenotypes include the Pelger-Huet anomaly (PHA) and embryonic lethal Greenberg dysplasia (GRBGD). With the further study of this gene, other phenotypes have been found in different individuals. This retrospective study analyzed recurrent prenatal moderate skeletal dysplasias in Chinese fetuses. Nothing malformed was detected in the fetal karyotype and microarray, while the whole-exome sequencing identified a homozygous variant (NM_002296.4:c.1757G>A, NP_002287.2:p.Arg586His) in exon 14 of the LBR gene in both fetuses. Mutation analysis in the parents confirmed that the c.1757G>A variation is heterozygous by Sanger sequencing. Intensive analysis on bioinformatics and familial co-segregation suggest that the homozygous variation in the LBR gene is responsible for this recurrent prenatal moderate skeletal dysplasia. Moreover, moderate skeletal dysplasias differ from typical GRBGD phenotypes. Our findings are based on the DNA base test and the prenatal diagnosis of skeletal dysplasia, which can be helpful in proper phenotyping and contribute to a better understanding of the correlation between the phenotype and genotype.

[Analysis of pathogenic variants of USH2A gene in a child with Usher syndrome type II].

OBJECTIVE: To detect pathogenic variant in a child featuring Usher syndrome type II. METHODS: Peripheral blood samples of the child and his parents were collected for the analysis of variants of hearing impairment-related genes. The findings were verified in 100 individuals with normal hearing. RESULTS: The child was found to harbor compound heterozygous variants of the USH2A gene, namely c.8224-1G>C in intron 41 and c.5678C>G(p.Ser1893X) in exon 28, which were inherited respectively from his mother and father. Based on the American College of Medical Genetics and Genomics standards and guidelines, both c.8224-1G>C and c.5678C>G(p.Ser1893X) variants of USH2A gene were predicted to be pathogenic(PVS1+PM2+PM3). CONCLUSION: The compound heterozygous variants c.8224-1G>C and c.5678C>G of the USH2A gene probably underlay the disease in this child. Above finding has enriched the spectrum of USH2A gene variants.

[Prenatal diagnosis of a fetus with chromosome 18p deletion and duplication].

OBJECTIVE: To assess the value of chromosomal microarray analysis (CMA) to verify a fetus with partial 18p deletion signaled by non-invasive prenatal testing. METHODS: G-banding chromosomal karyotyping analysis was carried out on amniotic fluid sample of the fetus and peripheral blood samples from the parents. Amniotic DNA was also subjected to CMA analysis. The fetus was also subjected to systematic ultrasound scan. RESULTS: The fetus was found to have a karyotype of 46,XX,18p+. CMA has revealed a 5 Mb deletion at 18p11.32-p11.31, a 2.9 Mb duplication at 18p11.31-p11.23, and a 2.5 Mb duplication at 18p11.23-p11.22. No chromosomal aberration or microdeletion/microduplication was detected in either parent. CONCLUSION: Non-invasive prenatal testing and CMA are both sensitive for the detection of chromosomal microdeletions and microduplications. CMA can help with clarification of genotype-phenotype correlation and facilitate prenatal diagnosis and genetic counseling for the family.

Prevalence and genotype distribution of human papillomavirus infection in Huzhou City, eastern China, 2018-2019.

BACKGROUND: Human papillomavirus (HPV) infection is involved in cervical cancer development, and hence understanding its prevalence and genotype distribution is important. However, there are few reports on the prevalence and genotype distribution of HPV in the city of Huzhou in China. METHODS: In this retrospective cross-sectional study, 11,506 women who visited Huzhou Maternity & Child Health Care Hospital between January 2018 and October 2019 were enrolled. The results of HPV genotyping and cytology tests were analyzed. RESULTS: The overall prevalence of HPV infection was 15.5%. The rate of high-risk (HR) HPV infection (13.5%) was higher than that of single low-risk (LR) HPV infection (2.0%) (p<0.05). The five most common HPV genotypes were HPV52 (3.3%), 16 (1.9%), 58 (1.7%), 53 (1.5%), and 81 (1.2%). The infection rate of HPV peaked in women aged 16-24 and women aged ≥55. The infection rate of HPV58 or HPV81 appeared as a single peak in women aged ≥55. The rates of HR-HPV and LR-HPV infection were higher in subjects with abnormal cytology (p<0.05). CONCLUSIONS: HPV infection is high in Huzhou, and HPV53 and HPV81 are the prevalent genotypes. HPV infection rate is associated with age and cytology. Regional HPV surveillance is essential to optimize current HPV prevention and vaccine development.

[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.

[Genetic analysis of a child with global developmental delay and neurofibromatosis type 1].

OBJECTIVE: To explore the genetic basis for a child with global developmental delay and neurofibromatosis type 1 (NF1). METHODS: The patient underwent clinical examination. Whole exome sequencing (WES) was carried out to detect pathogenic genetic variants. RESULTS: The child had cafe au lait spots all over her body, pigmentation in the back, and global developmental delay as assessed by Gese II. Cranial MRI revealed globular abnormal density in the lower hemisphere of left posterior cranial fossa. WES detected a novel variant of the NF1 gene, c.6513-6515del (p.Tyr2171), which was strongly correlated with her clinical phenotype. The same variant was not found in either parent and was unreported previously. CONCLUSION: The c.3842T>G variant of the NF1 gene probably underlay the NF1 and global developmental delay in this child, for whom prompt symptomatic treatment and regular follow-up were recommended.

[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.

[Identification of LINS1 gene variant in a patient with severe mental retardation].

OBJECTIVE: To explore the genetic basis of a child with idiopathic mental retardation. METHODS: Clinical data and peripheral blood sample of the child were collected. Genomic DNA was extracted and subjected to copy number analysis using single nucleotide polymrophism array comparative genome hybridization (SNP-aCGH) and targeted capture and next generation sequencing (NGS). RESULTS: No microdeletion/microduplication were detected by SNP-aCGH. NGS has detected homozygous c.722delA (p.Asp241fs) variant of the LISN1 gene, which is known to underlie autosomal recessive mental retardation-27 (MRT 27). Both parents are carriers of the variant, conforming to the autosomal recessive inheritance. CONCLUSION: A novel pathogenic variant of the LINS1 gene has been identified, which probably underlies the MRT 27 in the patient.

[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.

Iron sulfur clusters in protein nanocages for photocatalytic hydrogen generation in acidic aqueous solutions.

We took advantage of the iron binding affinity of apoferritin to immobilize iron-sulfur clusters into apoferritin up to 312 moieties per protein, with a loading rate as high as 25 wt%. The photocatalytic hydrogen generation activity in acidic aqueous solutions was achieved with TONs up to 31 (based on a single catalyst moiety) or 8.3 × 103 (based on a single protein) upon 3 h of visible light irradiation. The present study provides a versatile strategy to construct uniform protein/photocatalyst supramolecular systems with FeFe-H2ase activity.

[Pre-implantation genetic screening of discarded embryos through whole genome amplification and next-generation sequencing].

OBJECTIVE: To assess the value of whole-genome amplification (WGA) and next generation sequencing (NGS) for the pre-implantation screening of discarded embryos. METHODS: In total 476 discarded embryos were collected. After continued culture, 23 high-quality blastocysts were obtained. Blastocysts graded as 4BC or above based on Gardner classification were subjected for blastula biopsy. Five to ten nourish ectoderm cells were hatched with a biopsy needle. Following WGA and NGS, deletion and/or duplication of chromosomal fragments and numerical chromosomal aberrations were analyzed. RESULTS: In total 148 trophoblast cells were obtained from the 23 blastocysts. Following WGA, 60 amplification products were selected for NGS. The results showed that there were 39 abnormal chromosomes derived from 14 blastocysts, which gave an abnormal rate of blastocyst of 60.87% (14/23). CONCLUSION: WGA combined with NGS can enable pre-implantation genetic screening for discarded embryos, which may improve the efficacy of in vitro fertilization as well as reduce the risk for birth defects.

[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.