An efficient molecular genetic testing strategy for incontinentia pigmenti based on single-tube long fragment read sequencing.

Incontinentia pigmenti (IP) is a rare X-linked dominant neuroectodermal dysplasia that primarily affects females. The only known causative gene is IKBKG, and the most common genetic cause is the recurrent IKBKG△4-10 deletion resulting from recombination between two MER67B repeats. Detection of variants in IKBKG is challenging due to the presence of a highly homologous non-pathogenic pseudogene IKBKGP1. In this study, we successfully identified four pathogenic variants in four IP patients using a strategy based on single-tube long fragment read (stLFR) sequencing with a specialized analysis pipeline. Three frameshift variants (c.519-3_519dupCAGG, c.1167dupC, and c.700dupT) were identified and subsequently validated by Sanger sequencing. Notably, c.519-3_519dupCAGG was found in both IKBKG and IKBKGP1, whereas the other two variants were only detected in the functional gene. The IKBKG△4-10 deletion was identified and confirmed in one patient. These results demonstrate that the proposed strategy can identify potential pathogenic variants and distinguish whether they are derived from IKBKG or its pseudogene. Thus, this strategy can be an efficient genetic testing method for IKBKG. By providing a comprehensive understanding of the whole genome, it may also enable the exploration of other genes potentially associated with IP. Furthermore, the strategy may also provide insights into other diseases with detection challenges due to pseudogenes.

Efficient removal of tetracycline hydrochloride by high entropy oxides in visible photo-Fenton catalytic process.

A novel type of oxide material, high entropy oxide (Mn0.2Fe0.2Co0.2Ni0.2Cu0.2)3O4 (MFO) composites with spinel structure were successfully synthesized by a simple solution combustion in this paper, and it was first applied to the degradation of antibiotic organic pollutants in water by photo-Fenton. SEM and BET characterization showed that the composite was porous and had a large specific surface area. XPS results showed that Fe, Mn, Cu, Co and Ni all participated in the redox reaction of the catalytic process. The redox pairs of Mn2+/Mn3+, Cu+/Cu2+, Co2+/Co3+, Ni2+/Ni3+ can accelerate the Fe2+/Fe3+ redox cycling in MFO to activate H2O2 and produce more reactive oxygen species. The catalytic performance of MFO composite was investigated using tetracycline hydrochloride (TC-HCl) as a model pollutant. The results displayed that the degradation rate of TC-HCl by MFO was 92.9% when the initial pH was 4, the dose of H2O2 was 50 mM, and the irradiation time was 60 min. The high entropy oxide MFO composites could build up an internal electric field, which restrains electron-hole recombination, improves the transfer of photogenerated charge carriers and maximize photocatalytic property. In addition, the free radical capture experiment determined that the main active species of the degradation reaction were e-, •O2- and •OH. The synergistic effect of the five components in the high entropy oxide strengthens lattice distortion and defects, increases oxygen vacancies, and thus has enhanced catalytic effect for TC-HCl degradation. This work shows that high entropy oxides have excellent catalytic performance for tetracycline organic pollutants, and it is speculated that high entropy oxides have good application prospects in the field of advanced oxidation technology for the degradation of organic pollutants.

Case report: A compound heterozygous mutations in ASNS broadens the spectrum of asparagine synthetase deficiency in the prenatal diagnosis.

Asparagine synthetase deficiency (ASNSD) is a rare congenital disorder characterized by severe progressive microcephaly, global developmental delay, spastic quadriplegia, and refractory seizures. ASNSD is caused by variations of the ASNS gene. The present study showed a Chinese family with a fetus suffering microcephaly. Whole-exome sequencing and Sanger sequencing were used to identify the disease-associated genetic variants. Compound heterozygous variants c.97C>T p. (R33C) and c.1031-2_1033del were identified in the ASNS gene and the variants were inherited from the parents. The mutation site c.97C>T was highly conserved across a wide range of species and predicted to alter the local electrostatic potential. The variant c.1031-2_1033del was classified pathogenic. However, there is no case report of prenatal diagnosis of ASNSD. This is the first description of fetal compound mutations in the ASNS gene leading to ASNSD, which expanded the spectrum of ASNSD.

Single-cell landscape analysis reveals systematic senescence in mammalian Down syndrome.

BACKGROUND: Down syndrome (DS), which is characterized by various malfunctions, is the most common chromosomal disorder. As the DS population continues to grow and most of those with DS live beyond puberty, early-onset health problems have become apparent. However, the cellular landscape and molecular alterations have not been thoroughly studied. METHODS: This study utilized single-cell resolution techniques to examine DS in humans and mice, spanning seven distinct organs. A total of 71 934 mouse and 98 207 human cells were analyzed to uncover the molecular alterations occurring in different cell types and organs related to DS, specifically starting from the fetal stage. Additionally, SA-ß-Gal staining, western blot, and histological study were employed to verify the alterations. RESULTS: In this study, we firstly established the transcriptomic profile of the mammalian DS, deciphering the cellular map and molecular mechanism. Our analysis indicated that DS cells across various types and organs experienced senescence stresses from as early as the fetal stage. This was marked by elevated SA-ß-Gal activity, overexpression of cell cycle inhibitors, augmented inflammatory responses, and a loss of cellular identity. Furthermore, we found evidence of mitochondrial disturbance, an increase in ribosomal protein transcription, and heightened apoptosis in fetal DS cells. This investigation also unearthed a regulatory network driven by an HSA21 gene, which leads to genome-wide expression changes. CONCLUSION: The findings from this study offer significant insights into the molecular alterations that occur in DS, shedding light on the pathological processes underlying this disorder. These results can potentially guide future research and treatment development for DS.

A novel F8 variant in a Chinese hemophilia A family and involvement of X-chromosome inactivation: A case report.

RATIONALE: Hemophilia A (HA) is an X-linked recessive bleeding disorder, which shows factor VIII (FVIII) deficiency caused by genetic variant in F8 gene. PATIENT CONCERNS: Males with F8 variants are affected, whereas female carriers with a wide range of FVIII levels are usually asymptomatic, it is possible that different X-chromosome inactivation (XCI) may effect the FVIII activity. DIAGNOSES: We identified a novel variant F8: c.6193T > G in a Chinese HA proband, it was inherited from the mother and grandmother with different FVIII levels. INTERVENTIONS: We performed Androgen receptor gene (AR) assays and RT-PCR. OUTCOMES: AR assays revealed that the X chromosome with the F8 variant was severely skewed inactivated in the grandmother with higher FVIII levels, but not in the mother with lower FVIII levels. Further, RT-PCR of mRNA confirmed that only the wild allele of F8 was expressed in the grandmother, with lower expression in the wild allele of the mother. LESSONS: Our findings suggest that F8: c.6193T > G could be the cause of HA and that XCI affected the FVIII plasma levels in female carriers.

Validation and depth evaluation of low-pass genome sequencing in prenatal diagnosis using 387 amniotic fluid samples.

BACKGROUND: Low-pass genome sequencing (LP GS) is an alternative to chromosomal microarray analysis (CMA). However, validations of LP GS as a prenatal diagnostic test for amniotic fluid are rare. Moreover, sequencing depth of LP GS in prenatal diagnosis has not been evaluated. OBJECTIVE: The diagnostic performance of LP GS was compared with CMA using 375 amniotic fluid samples. Then, sequencing depth was evaluated by downsampling. RESULTS: CMA and LP GS had the same diagnostic yield (8.3%, 31/375). LP GS showed all copy number variations (CNVs) detected by CMA and six additional variant of uncertain significance CNVs (>100 kb) in samples with negative CMA results; CNV size influenced LP GS detection sensitivity. CNV detection was greatly influenced by sequencing depth when the CNV size was small or the CNV was located in the azoospermia factor c (AZFc) region of the Y chromosome. Large CNVs were less affected by sequencing depth and more stably detected. There were 155 CNVs detected by LP GS with at least a 50% reciprocal overlap with CNVs detected by CMA. With 25 M uniquely aligned high-quality reads (UAHRs), the detection sensitivity for the 155 CNVs was 99.14%. LP GS using samples with 25 M UAHRs showed the same performance as LP GS using total UAHRs. Considering the detection sensitivity, cost and interpretation workload, 25 M UAHRs are optimal for detecting most aneuploidies and microdeletions/microduplications. CONCLUSION: LP GS is a promising, robust alternative to CMA in clinical settings. A total of 25 M UAHRs are sufficient for detecting aneuploidies and most microdeletions/microduplications.

Noninvasive Prenatal Screening for Common Fetal Aneuploidies Using Single-Molecule Sequencing.

Amplification biases caused by next-generation sequencing (NGS) for noninvasive prenatal screening (NIPS) may be reduced using single-molecule sequencing (SMS), during which PCR is omitted. Therefore, the performance of SMS-based NIPS was evaluated. We used SMS-based NIPS to screen for common fetal aneuploidies in 477 pregnant women. The sensitivity, specificity, positive predictive value, and negative predictive value were estimated. The GC-induced bias was compared between the SMS- and NGS-based NIPS methods. Notably, a sensitivity of 100% was achieved for fetal trisomy 13 (T13), trisomy 18 (T18), and trisomy 21 (T21). The positive predictive value was 46.15% for T13, 96.77% for T18, and 99.07% for T21. The overall specificity was 100% (334/334). Compared with NGS, SMS (without PCR) had less GC bias, a better distinction between T21 or T18 and euploidies, and better diagnostic performance. Overall, our results suggest that SMS improves the performance of NIPS for common fetal aneuploidies by reducing the GC bias introduced during library preparation and sequencing.

Case Report: De novo DDX3X mutation caused intellectual disability in a female with skewed X-chromosome inactivation on the mutant allele.

Skewed XCI plays an important role in the phenotypic heterogeneities of many X-linked disorders, even involving in diseases caused by XCI-escaping genes. DDX3X-related intellectual disability is more common in females and less common in males, who usually inherit from unaffected heterozygous mothers. As an X inactivation (XCI) escaping gene, the role of skewed XCI in the phenotype of DDX3X mutant female is unknown. Here we reported a DDX3X: c.694_711dup18 de novo heterozygous mutation in a female with intellectual disability on the maternal X chromosome on the basis of SNPs detected by PCR-sanger sequencing. AR assay revealed that the maternal mutant X chromosome was extremely inactivated in the proband. Using RNA sequencing and whole-exome sequencing, we quantified allelic read counts and allele-specific expression, and confirmed that the mutant X chromosome was inactive. Further, we verified that the mutant DDX3X allele had a lower expression level by RNA sequencing and RT-PCR, and the normal and mutated DDX3X expression accounted for respectively 70% and 30% of total. In conclusion, we found a symptomatic female with extreme skewing XCI in the DDX3X mutant allele. It was discovered that XCI in the mutant allele was insufficient to reverse the phenotype of DDX3X-related neurodevelopmental disorder. It contributed to a better understanding of the role of skewed XCI in phenotypic differences, which can aid in the genetic counseling and prenatal diagnosis of disorders in females with DDX3X defects.

Prenatal diagnosis of trisomy 8 mosaicism, initially identified by cffDNA screening.

BACKGROUND: So called cell-free fetal DNA (cffDNA) in the maternal plasma, which is derived from placenta, is widely used to screen fetal aneuploidies, including trisomy 21, 18, 13 and sex chromosomes. Here we reported a case of trisomy 8 mosaicism (T8M), which was initially identified via cffDNA screening in noninvasive prenatal testing (NIPT). METHODS: A 35-year-old woman received cffDNA screening at 17th week of gestation. Amniocentesis was performed subsequently, and karyotyping, single-nucleotide polymorphism array (SNP-array) and BACs-on-Beads™ (BoBs™) were used to determine fetal chromosome content. Interphase fluorescence in situ hybridization (FISH) was applied to determine the copy number of chromosome 8. RESULTS: An enhanced risk for fetal trisomy 8 was identified by cffDNA screening in the studied pregnant woman. After amniocentesis trisomy 8 was found in 1 of 73 metaphases. SNP-array on DNA derived from cultured amniocytes and neonatal cord blood cells suggested the presence of T8M. Interphase FISH on native neonatal cord blood cells confirmed T8M with a percentage of 10%. The Bobs™ fluorescence data also suggested that 8q23-8q24 was amplified. CONCLUSIONS: The current study shows that NIPT is suited to provide hints on rare autosomal trisomies, which have to be further validated and confirmed by other approaches.

Case report: FOXP1 syndrome caused by a de novo splicing variant (c.1652+5 G>A) of the FOXP1 gene.

FOXP1 syndrome is a rare neurodevelopmental disorder characterized by global developmental delay, intellectual disability, and language delay, with or without autistic features. Several splicing variants have been reported for this condition, but most of them lack functional evidence, and the actual effects of the sequence changes are still unknown. In this study, a de novo splicing variant (c.1652 + 5 G>A) of the FOXP1 gene was identified in a patient with global developmental delay, mild intellectual disability, speech delay, and autistic features. Assessed by TA-cloning, the variant promoted the skipping of exon 18 and a premature stop codon (p.Asn511*), resulting in a predicted truncated protein. This variant, that is lacking the forkhead-box DNA-binding domain and nuclear localization signal 2, may disrupt the protein function and thus cause FOXP1 syndrome-related symptoms. Our study extends the phenotypic and allelic spectra of the FOXP1 syndrome.

Case report: Prenatal diagnosis of Kagami-Ogata syndrome in a Chinese family.

The aim of this work was to explore the genetic cause of the proband (Ⅲ2) presenting with polyhydramnios and gastroschisis. Copy number variation sequencing (CNV-seq), methylation-specific multiplex PCR (MS-PCR), and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) were used to characterize the genetic etiology. CNV-seq revealed a deletion of 732.26 kb at 14q32.2q32.31 in the proband (Ⅲ2) and its mother (Ⅱ2). MS-PCR showed the maternal allele was missing in the proband, while paternal allele was missing in its mother. MS-MLPA showed deletion of the DLK1, MEG3, MIR380, and RTL1 genes of both the proband and its mother. MEG3 imprinting gene methylation increased in the proband, while decreased in its mother. It was indicated that a maternally transmitted deletion was responsible for Kagami-Ogata syndrome in the proband (Ⅲ2), and the de novo paternal deletion resulted in Temple syndrome in the mother (Ⅱ2). Prenatal diagnosis was provided at 17+3 weeks of pregnancy on the mother's fourth pregnancy (Ⅲ4). Fortunately, the karyotype and single-nucleotide polymorphism array (SNP array) results were normal. The current investigation provided the detection methods for imprinted gene diseases, expanded the phenotype spectrum of the disease, and obtained the insight into the diagnosis, prenatal diagnosis, and genetic counseling of the disease.

Case Report: Identification Pathogenic Abnormal Splicing of BBS1 Causing Bardet-Biedl Syndrome Type I (BBS1) due to Missense Mutation.

Conventionally, protein features affected by missense mutation was attributed to destroy an important domain with amino acid alternation, and it was difficult to clearly specify the pathogenicity of a novel missense mutation. Nevertheless, the associations between missense mutations and abnormal splicing are nowadays increasingly reported. Rarely, some missense mutations, locating at the non-canonical splicing sites, are observed to damage the splicing process. In this study, a couple has three adverse pregnancy history that the affected fetus presented typical polydactyly, renal abnormalities, and cerebral ventriculomegaly. To identify its genetic etiology, whole-exome sequencing (WES) was performed and a missense mutation c.1339G > A was identified, which was located at the non-canonical splicing sites of the BBS1 gene. Then, reverse transcription polymerase chain reaction was carried out and demonstrated extra 115bp originating from intron 13 cut into cDNA, which generated a predicted premature termination codon (PTC) in the BBS1 protein. Further expression analysis by using real-time reverse-transcribed PCR confirmed the occurrence of nonsense-mediated decay (NMD). Therefore, the pathogenicity of the missense mutation c.1339G > A was explicit and our study helped to extend the spectrum of pathogenic mutations in Bardet-Biedl syndrome type I.

Corrigendum: Case Report: Prenatal Diagnosis for a Rett Syndrome Family Caused by a Novel MECP2 Deletion With Heteroduplexes of PCR Product.

[This corrects the article DOI: 10.3389/fped.2021.748641.].

Enhanced catalytic performance of Cu-doped MnFe2O4 magnetic ferrites: Tetracycline hydrochloride attacked by superoxide radicals efficiently in a strong alkaline environment.

It is important to develop a catalyst that can maintain good activity in alkaline environment for Fenton or Fenton-like reactions. In order to achieve stable Fenton catalytic degradation in a wide pH range, this study reports Cu-doped MnFe2O4 heterogeneous catalysts still has excellent effect when the pH is extended to 11 for removing organic pollutants, such as tetracycline hydrochloride (TC-HCl). The synergistic effect among Fe, Mn and Cu ions has been proved to enhanced the catalytic activity in this work. When the molar ratio of Cu/Mn = 4:1, the porous Cu0·8Mn0·2Fe2O4 materials had the highest photo-Fenton catalytic activity compared with pure MnFe2O4, CuFe2O4 and other CuxMn1-xFe2O4. The XPS showed that Cu0·8Mn0·2Fe2O4 formed oxygen vacancies, which exposed more active sites to attract more H2O2 for TC-HCl degradation. Results indicated 94.3% of TC-HCl was efficiently degraded by 0.1 g/L Cu0·8Mn0·2Fe2O4 with 50 mM H2O2 at pH = 11 under 30 min visible light irradiation, and the corresponding apparent rate constant was 0.08286 min-1. With free radicals quenching experiment, O2- was responsible for the high catalytic degradation and OH was participated in the photo-Fenton reaction. To sum up, Cu0·8Mn0·2Fe2O4 exhibited high activity, great stability and easily recyclable, which eliminated the pH limitation of the Fenton reaction and provided practical application performance for water purification.

Novel homozygous silent mutation of ITGB3 gene caused Glanzmann thrombasthenia.

Glanzmann thrombasthenia (GT) is a rare inherited disease characterized by mucocutaneous bleeding due to the abnormalities in quantity or quality of platelet membrane GP IIb (CD41) or GP IIIa (CD61). GP IIb and GP IIIa are encoded by the ITGA2B and ITGB3 genes, respectively. Herein, we described a 7-year-old Chinese boy of the consanguineous couple who was diagnosed with GT based on the typical clinical manifestations, absence of blood clot retraction and the reduced expression of CD41 and CD61 in platelets. A homozygous silent variant c.1431C > T (p. G477=) of the ITGB3 gene was identified by the Whole-exome sequencing and confirmed by Sanger sequencing. The variant was predicted to affect the splicing. RT-PCR and sequencing revealed that the variant caused a deletion of 95 base pairs and frameshift, and subsequently created a premature stop codon in exon 10 of ITGB3 (p. G477Afs*30). It was indicated that the variant c.1431C > T (p. G477=) of ITGB3 was the cause for Glanzmann thrombasthenia. Our findings expanded the mutation spectrum and provided the information for the genetic counseling, prenatal diagnosis and preimplantation genetic testing (PGT).

Case Report: Prenatal Diagnosis for a Rett Syndrome Family Caused by a Novel MECP2 Deletion With Heteroduplexes of PCR Product.

Rett syndrome is an X-linked dominant, postnatal neurological disorder. Approximately 80-90% of classic Rett syndrome patients harbor mutations in the coding region of MECP2. Somatic or germline MECP2 mosaicism is not rare, and paternal germline MECP2 mosaicism occurs in especially high proportions. Here, we report the case of a Chinese girl with Rett syndrome in whom a heterozygous deletion was found in exon 4 of MECP2 using multiplex ligation-dependent probe amplification. To obtain an accurate region of deletion, we narrowed down the deletion region using real-time quantitative PCR, and subsequent long-range PCR was performed to detect the deletion breakpoints. Surprisingly, three DNA bands from long-range PCR products were observed after gel electrophoresis. To exclude somatic mosaicism, we performed T-A cloning and DNA sequencing, the middle DNA band was proved to be a heteroduplex of the PCR product in vitro. Meanwhile, a prenatal diagnosis was performed for the pregnant mother of the patient. Our study showed that the patient was heterozygous for the deletion of 713-base pairs in exon 4 of MECP2 (MECP2: c.441_1153del713), resulting in a frameshift and premature termination of the 487 amino acid protein at the 154th codon. In summary, we reported a novel heterozygous deletion in the MECP2 gene with heteroduplexes of the PCR product in vitro, which can help in the genetic counseling and prenatal diagnosis of disorders of MECP2 defects.

Lack of MECP2 gene transcription on the duplicated alleles of two related asymptomatic females with Xq28 duplications and opposite X-chromosome inactivation skewing.

Xq28 duplication syndrome (MIM# 300815) is a severe neurodevelopmental disorder in males due to MeCP2 overexpression. Most females with MECP2 duplication are asymptomatic carriers, but there are phenotypic heterogeneities. Skewed X-chromosome inactivation (XCI) can protect females from exhibiting clinical phenotypes. Herein we reported two asymptomatic females (mother and grandmother) with interstitial Xq28 duplication. AR and RP2 assays showed that both had extremely skewed XCI, the Xq28 duplicated chromosome was inactivated in the mother, but was surprisingly activated in the grandmother. Interestingly, by combining RNA sequencing and whole-exome sequencing, we confirmed that XIST only expressed in the Xq28 duplication chromosomes of the two females, indicating that the Xq28 duplication chromosomes were inactive. Meanwhile, MECP2 and most XCI genes in the duplicated X-chromosomes were not transcriptionally expressed or upregulated, precluding major clinical phenotypes in the two females, especially the grandmother. We showed that XCI status detected using RNA sequencing was more relevant for establishing the clinical phenotype of MECP2 duplication in females. It suggested that there were other factors maintaining the XCI status in addition to DNA methylation, a possible additional inhibition mechanism occurred at the transcriptional level in the unmethylated X-chromosome, counter balancing the MECP2 duplication's detrimental phenotype effects.

Identification of a likely pathogenic structural variation in the LAMA1 gene by Bionano optical mapping.

Recent advances in Bionano optical mapping (BOM) provide a great insight into the determination of structural variants (SVs), but its utility in identification of clinical likely pathogenic variants needs to be further demonstrated and proved. In a family with two consecutive pregnancies affected with ventriculomegaly, a splicing likely pathogenic variant at the LAMA1 locus (NM_005559: c. 4663 + 1 G > C) inherited from the father was identified in the proband by whole-exome sequencing, and no other pathogenic variant associated with the clinical phenotypes was detected. SV analysis by BOM revealed an ~48 kb duplication at the LAMA1 locus in the maternal sample. Real-time quantitative PCR and Sanger sequencing further confirmed the duplication as c.859-153_4806 + 910dup. Based on these variants, we hypothesize that the fetuses have Poretti-Boltshauser syndrome (PBS) presenting with ventriculomegaly. With the ability to determine single nucleotide variants and SVs, the strategy adopted here might be useful to detect cases missed by current routine screening methods. In addition, our study may broaden the phenotypic spectrum of fetuses with PBS.

The association between the two more common genetic causes of spermatogenic failure: a 7-year retrospective study.

Chromosomal abnormalities and Y chromosome microdeletions are considered to be the two more common genetic causes of spermatogenic failure. However, the relationship between chromosomal aberrations and Y chromosome microdeletions is still unclear. This study was to investigate the incidence and characteristics of chromosomal aberrations and Y chromosome microdeletions in infertile men, and to explore whether there was a correlation between the two genetic defects of spermatogenic failure. A 7-year retrospective study was conducted on 5465 infertile men with nonobstructive azoospermia or oligozoospermia. Karyotype analysis of peripheral blood lymphocytes was performed by standard G-banding techniques. Y chromosome microdeletions were screened by multiplex PCR amplification with six specific sequence-tagged site (STS) markers. Among the 5465 infertile men analyzed, 371 (6.8%) had Y chromosome microdeletions and the prevalence of microdeletions in azoospermia was 10.5% (259/2474) and in severe oligozoospermia was 6.3% (107/1705). A total of 4003 (73.2%) infertile men underwent karyotyping; 370 (9.2%) had chromosomal abnormalities and 222 (5.5%) had chromosomal polymorphisms. Karyotype analysis was performed on 272 (73.3%) patients with Y chromosome microdeletions and 77 (28.3%) had chromosomal aberrations, all of which involved sex chromosomes but not autosomes. There was a significant difference in the frequency of chromosomal abnormalities between men with and without Y chromosome microdeletions (P< 0.05).