Results of a Pilot External Quality Assessment Scheme for Genetic Testing of Newborns with Spinal Muscular Atrophy.

BACKGROUND: This study aims to evaluate the ability of laboratories to perform spinal muscular atrophy (SMA) genetic testing in newborns based on dried blood spot (DBS) samples, and to provide reference data and advance preparation for establishing the pilot external quality assessment (EQA) scheme for SMA genetic testing of newborns in China. METHODS: The pilot EQA scheme contents and evaluation principles of this project were designed by National Center for Clinical Laboratories (NCCL), National Health Commission. Two surveys were carried out in 2022, and 5 batches of blood spots were submitted to the participating laboratory each time. All participating laboratories conducted testing upon receiving samples, and test results were submitted to NCCL within the specified date. RESULTS: The return rates were 75.0% (21/28) and 95.2% (20/21) in the first and second surveys, respectively. The total return rate of the two examinations was 83.7% (41/49). Nineteen laboratories (19/21, 90.5%) had a full score passing on the first survey, while in the second survey twenty laboratories (20/20, 100%) scored full. CONCLUSIONS: This pilot EQA survey provides a preliminary understanding of the capability of SMA genetic testing for newborns across laboratories in China. A few laboratories had technical or operational problems in testing. It is, therefore, of importance to strengthen laboratory management and to improve testing capacity for the establishment of a national EQA scheme for newborn SMA genetic testing.

All-Optical Reconfigurable Excitonic Charge States in Monolayer MoS2.

Excitons are quasi-particles composed of electron-hole pairs through Coulomb interaction. Due to the atomic-thin thickness, they are tightly bound in monolayer transition metal dichalcogenides (TMDs) and dominate their optical properties. The capability to manipulate the excitonic behavior can significantly influence the photon emission or carrier transport performance of TMD-based devices. However, on-demand and region-selective manipulation of the excitonic states in a reversible manner remains challenging so far. Herein, harnessing the coordinated effect of femtosecond-laser-driven atomic defect generation, interfacial electron transfer, and surface molecular desorption/adsorption, we develop an all-optical approach to manipulate the charge states of excitons in monolayer molybdenum disulfide (MoS2). Through steering the laser beam, we demonstrate reconfigurable optical encoding of the excitonic charge states (between neutral and negative states) on a single MoS2 flake. Our technique can be extended to other TMDs materials, which will guide the design of all-optical and reconfigurable TMD-based optoelectronic and nanophotonic devices.

Quantum Emitters with Narrow Band and High Debye-Waller Factor in Aluminum Nitride Written by Femtosecond Laser.

Solid-state quantum emitters (QEs) are central components for photonic-based quantum information processing. Recently, bright QEs in III-nitride semiconductors, such as aluminum nitride (AlN), have attracted increasing interest because of the mature commercial application of the nitrides. However, the reported QEs in AlN suffer from broad phonon side bands (PSBs) and low Debye-Waller factors. Meanwhile, there is also a need for more reliable fabrication methods of AlN QEs for integrated quantum photonics. Here, we demonstrate that laser-induced QEs in AlN exhibit robust emission with a strong zero phonon line, narrow line width, and weak PSB. The creation yield of a single QE could be more than 50%. More importantly, they have a high Debye-Waller factor (>65%) at room temperature, which is the highest result among reported AlN QEs. Our results illustrate the potential of laser writing to create high-quality QEs for quantum technologies and provide further insight into laser writing defects in relevant materials.

Reversible Three-Color Fluorescence Switching of an Organic Molecule in the Solid State via "Pump-Trigger" Optical Manipulation.

In photoswitches that undergo fluorescence switching upon ultraviolet irradiation, photoluminescence and photoisomerization often occur simultaneously, leading to unstable fluorescence properties. Here, we successfully demonstrated reversible solid-state triple fluorescence switching through "Pump-Trigger" multiphoton manipulation. A novel fluorescence photoswitch, BOSA-SP, achieved green, yellow, and red fluorescence under excitation by pump light and isomerization induced by trigger light. The energy ranges of photoexcitation and photoisomerization did not overlap, enabling appropriate selection of the multiphoton light for "pump" and "trigger" photoswitching, respectively. Additionally, the large free volume of the spiropyran (SP) moiety in the solid state promoted reversible photoisomerization. Switching between "pump" and "trigger" light is useful for three-color tunable switching cell imaging, which can be exploited in programmable fluorescence switching. Furthermore, we exploited reversible dual-fluorescence switching in a single molecular system to successfully achieve two-color super-resolution imaging.

HPV-16 E2 contributes to induction of HPV-16 late gene expression by inhibiting early polyadenylation.

We provide evidence that the human papillomavirus (HPV) E2 protein regulates HPV late gene expression. High levels of E2 caused a read-through at the early polyadenylation signal pAE into the late region of the HPV genome, thereby inducing expression of L1 and L2 mRNAs. This is a conserved property of E2 of both mucosal and cutaneous HPV types. Induction could be reversed by high levels of HPV-16 E1 protein, or by the polyadenylation factor CPSF30. HPV-16 E2 inhibited polyadenylation in vitro by preventing the assembly of the CPSF complex. Both the N-terminal and hinge domains of E2 were required for induction of HPV late gene expression in transfected cells as well as for inhibition of polyadenylation in vitro. Finally, overexpression of HPV-16 E2 induced late gene expression from a full-length genomic clone of HPV-16. We speculate that the accumulation of high levels of E2 during the viral life cycle, not only turns off the expression of the pro-mitotic viral E6 and E7 genes, but also induces the expression of the late HPV genes L1 and L2.

Suppression of HPV-16 late L1 5'-splice site SD3632 by binding of hnRNP D proteins and hnRNP A2/B1 to upstream AUAGUA RNA motifs.

Human papillomavirus type 16 (HPV-16) 5'-splice site SD3632 is used exclusively to produce late L1 mRNAs. We identified a 34-nt splicing inhibitory element located immediately upstream of HPV-16 late 5'-splice site SD3632. Two AUAGUA motifs located in these 34 nt inhibited SD3632. Two nucleotide substitutions in each of the HPV-16 specific AUAGUA motifs alleviated splicing inhibition and induced late L1 mRNA production from episomal forms of the HPV-16 genome in primary human keratinocytes. The AUAGUA motifs bind specifically not only to the heterogeneous nuclear RNP (hnRNP) D family of RNA-binding proteins including hnRNP D/AUF, hnRNP DL and hnRNP AB but also to hnRNP A2/B1. Knock-down of these proteins induced HPV-16 late L1 mRNA expression, and overexpression of hnRNP A2/B1, hnRNP AB, hnRNP DL and the two hnRNP D isoforms hnRNP D37 and hnRNP D40 further suppressed L1 mRNA expression. This inhibition may allow HPV-16 to hide from the immune system and establish long-term persistent infections with enhanced risk at progressing to cancer. There is an inverse correlation between expression of hnRNP D proteins and hnRNP A2/B1 and HPV-16 L1 production in the cervical epithelium, as well as in cervical cancer, supporting the conclusion that hnRNP D proteins and A2/B1 inhibit HPV-16 L1 mRNA production.

Novel JAG1 variants leading to Alagille syndrome in two Chinese cases.

Alagille Syndrome (ALGS) is a complex genetic disorder characterized by cholestasis, congenital cardiac anomalies, and butterfly vertebrae. The variable phenotypic expression of ALGS can lead to challenges in accurately diagnosing affected infants, potentially resulting in misdiagnoses or underdiagnoses. This study highlights novel JAG1 gene mutations in two cases of ALGS. The first case with a novel p.Pro325Leufs*87 variant was diagnosed at 2 months of age and exhibited a favorable prognosis and an unexpected manifestation of congenital hypothyroidism. Before the age of 2, the second patient was incorrectly diagnosed with liver structural abnormalities, necessitating extensive treatment. In addition, he exhibited delays in language acquisition that may have been a result of SNAP25 haploinsufficiency. The identification of ALGS remains challenging, highlighting the importance of early detection and genetic testing for effective patient management. The variant p.Pro325Leufs*87 is distinct from reported variants linked to congenital hypothyroidism in ALGS patients, thereby further confirming the clinical and genetic complexity of ALGS. This emphasizes the critical need for individualized and innovative approaches to diagnosis and medical interventions, uniquely intended to address the complexity of this syndrome.

Super-Resolution Exciton Imaging of Nanobubbles in 2D Semiconductors with Near-Field Nanophotoluminescence Microscopy.

Two-dimensional (2D) semiconductors, such as transition metal dichalcogenides, have emerged as important candidate materials for next-generation chip-scale optoelectronic devices with the development of large-scale production techniques, such as chemical vapor deposition (CVD). However, 2D materials need to be transferred to other target substrates after growth, during which various micro- and nanoscale defects, such as nanobubbles, are inevitably generated. These nanodefects not only influence the uniformity of 2D semiconductors but also may significantly alter the local optoelectronic properties of the composed devices. Hence, super-resolution discrimination and characterization of nanodefects are highly demanded. Here, we report a near-field nanophotoluminescence (nano-PL) microscope that can quickly screen nanobubbles and investigate their impact on local excitonic properties of 2D semiconductors by directly visualize the PL emission distribution with a very high spatial resolution of ∼10 nm, far below the optical diffraction limit, and a high speed of 10 ms/point under ambient conditions. By using nano-PL microscopy to map the exciton and trion emission intensity distributions in transferred CVD-grown monolayer tungsten disulfide (1L-WS2) flakes, it is found that the PL intensity decreases by 13.4% as the height of the nanobubble increases by every nanometer, which is mainly caused by the suppression of trion emission due to the strong doping effect from the substrate. In addition to the nanobubbles, other types of nanodefects, such as cracks, stacks, and grain boundaries, can also be characterized. The nano-PL method is proven to be a powerful tool for the nondestructive quality inspection of nanodefects as well as the super-resolution exploration of local optoelectronic properties of 2D materials.

Novel HPD mutation p.A244V compound with p.T219M causing tyrosinemia type III in a Chinese girl and review of the genotype-phenotype spectrum.

BACKGROUND: Hereditary tyrosinemia type III (HT III) is an extremely rare form of tyrosinemia, characterized by autosomal recessive inheritance and biallelic mutations in the HPD gene. The clinical presentation of HT III is variable and poorly understood, with symptoms ranging from developmental delay and intellectual impairment to seizures and intermittent ataxia. This study aimed to provide further insights into the clinical and genetic characteristics of HT III. METHODS: A 3-year-old girl, identified through newborn screening, was diagnosed with HT III using targeted next-generation sequencing. A comprehensive literature review was conducted, and the clinical, biochemical, and genetic findings of previously reported HT III patients were summarized and analyzed. RESULTS: The genetic analysis of the proband revealed compound heterozygous mutations in the HPD gene such as c.731C>T (p.A244V) and c.656C>T (p.T219M). Notably, the HPD p.A244V mutation had not been previously documented in public databases or the scientific literature. Bioinformatics analysis classified both variants as pathogenic variants. The patient exhibited persistent tyrosinemia, elevated levels of related metabolite derivatives, confirming the diagnosis of HT III. The review of previously published cases contributed to a better understanding of the clinical and genetic characteristics associated with HT III. CONCLUSION: Early diagnosis and prompt treatment in infancy are crucial for managing HT III effectively. Dietary therapy, particularly during childhood, plays a significant role in disease management. The findings from this study enhance our understanding of the genotype-phenotype associations in HT III and emphasize the importance of early intervention for improved patient outcomes.

Optofluidic crystallithography for directed growth of single-crystalline halide perovskites.

Crystallization is a fundamental phenomenon which describes how the atomic building blocks such as atoms and molecules are arranged into ordered or quasi-ordered structure and form solid-state materials. While numerous studies have focused on the nucleation behavior, the precise and spatiotemporal control of growth kinetics, which dictates the defect density, the micromorphology, as well as the properties of the grown materials, remains elusive so far. Herein, we propose an optical strategy, termed optofluidic crystallithography (OCL), to solve this fundamental problem. Taking halide perovskites as an example, we use a laser beam to manipulate the molecular motion in the native precursor environment and create inhomogeneous spatial distribution of the molecular species. Harnessing the coordinated effect of laser-controlled local supersaturation and interfacial energy, we precisely steer the ionic reaction at the growth interface and directly print arbitrary single crystals of halide perovskites of high surface quality, crystallinity, and uniformity at a high printing speed of 102 µm s-1. The OCL technique can be potentially extended to the fabrication of single-crystal structures beyond halide perovskites, once crystallization can be triggered under the laser-directed local supersaturation.

Typical pantothenate kinase-associated neurodegeneration caused by compound heterozygous mutations in PANK2 gene in a Chinese patient: a case report and literature review.

Pantothenate kinase-associated neurodegeneration (PKAN) is a rare genetic neurodegenerative disorder with brain iron accumulation characterized as dysarthria, spasticity, cognitive impairment, parkinsonism, and retinopathy. PKAN is caused by biallelic mutations in the mitochondrial pantothenate kinase 2 (PANK2) gene. Herein, we report a 4-year-old patient with PKAN from a Han Chinese family, who presented with developmental regression, progressive inability to walk, and limb tremors. Neuroimaging demonstrated "eye-of-the-tiger" sign. Whole exome sequencing (WES) identified compound heterozygous mutations of c.1213T>G (p.Tyr405Asp) and c.1502T>A (p.Ile501Asn) in PANK2 gene. In addition, a review of all known PANK2 variants observed in reported PKAN patients was conducted, to improve understanding of the genotype-phenotype associations that occur in PKAN patients.

A GATA3 gene mutation that causes incorrect splicing and HDR syndrome: a case study and literature review.

Hypoparathyroidism, deafness, and renal dysplasia (HDR) syndrome is an infrequent autosomal dominant genetic disorder caused by haploinsufficiency of the GATA binding protein 3 (GATA3) gene. In this report, we present a case study of a 6-year-old female patient manifesting seizures, tetany, hypoparathyroidism, and sensorineural hearing loss. A heterozygous variant, c.1050 + 2T>C, in the GATA3 gene was discovered by genetic testing. Moreover, a minigene splicing experiment revealed that the aforementioned variation causes incorrect splicing and premature cessation of protein synthesis. The clinical profile of the patient closely resembles the well-known phenomenology of HDR syndrome, supporting the association between the condition and the GATA3 variant. The challenges in early diagnosis highlight the importance of employing next-generation sequencing for timely detection of rare diseases. Additionally, this research contributes to a deeper understanding of the genotype-phenotype correlations in HDR syndrome, underscoring the critical need for improved diagnostic and therapeutic strategies.

A multicenter prospective study of next-generation sequencing-based newborn screening for monogenic genetic diseases in China.

BACKGROUND: Newborn screening (NBS) is an important and successful public health program that helps improve the long-term clinical outcomes of newborns by providing early diagnosis and treatment of certain inborn diseases. The development of next-generation sequencing (NGS) technology provides new opportunities to expand current newborn screening methodologies. METHODS: We designed a a newborn genetic screening (NBGS) panel targeting 135 genes associated with 75 inborn disorders by multiplex PCR combined with NGS. With this panel, a large-scale, multicenter, prospective multidisease analysis was conducted on dried blood spot (DBS) profiles from 21,442 neonates nationwide. RESULTS: We presented the positive detection rate and carrier frequency of diseases and related variants in different regions; and 168 (0.78%) positive cases were detected. Glucose-6-Phosphate Dehydrogenase deficiency (G6PDD) and phenylketonuria (PKU) had higher prevalence rates, which were significantly different in different regions. The positive detection of G6PD variants was quite common in south China, whereas PAH variants were most commonly identified in north China. In addition, NBGS identified 3 cases with DUOX2 variants and one with SLC25A13 variants, which were normal in conventional NBS, but were confirmed later as abnormal in repeated biochemical testing after recall. Eighty percent of high-frequency gene carriers and 60% of high-frequency variant carriers had obvious regional differences. On the premise that there was no significant difference in birth weight and gestational age, the biochemical indicators of SLC22A5 c.1400C > G and ACADSB c.1165A > G carriers were significantly different from those of non-carriers. CONCLUSIONS: We demonstrated that NBGS is an effective strategy to identify neonates affected with treatable diseases as a supplement to current NBS methods. Our data also showed that the prevalence of diseases has significant regional characteristics, which provides a theoretical basis for screening diseases in different regions.

Combining Z-Score and Maternal Copy Number Variation Analysis Increases the Positive Rate and Accuracy in Non-Invasive Prenatal Testing.

Objective: To evaluate positive rate and accuracy of non-invasive prenatal testing (NIPT) combining Z-score and maternal copy number variation (CNV) analysis. To assess the relationship between Z-score and positive predictive value (PPV). Methods: This prospective study included 61525 pregnancies to determine the correlation between Z-scores and PPV in NIPT, and 3184 pregnancies to perform maternal CNVs analysis. Positive results of NIPT were verified by prenatal diagnosis and/or following-up after birth. Z-score grouping, logistic regression analysis, receiver operating characteristic (ROC) curves, and S-curve trends were applied to correlation analysis of Z-scores and PPV. The maternal CNVs were classified according to the technical standard for the interpretation of ACMG. Through genetic counseling, fetal and maternal phenotypes and family histories were collected. Results: Of the 3184 pregnant women, 22 pregnancies were positive for outlier Z-scores, suggesting fetal aneuploidy. 12 out of 22 pregnancies were true positive (PPV = 54.5%). 17 pregnancies were found maternal pathogenic or likely pathogenic CNVs (> 0.5 Mb) through maternal CNV analysis. Prenatal diagnosis revealed that 7 out of 11 fetuses carried the same CNVs as the mother. Considering the abnormal biochemical indicators during pregnancy and CNV-related clinical phenotypes after birth, two male fetuses without prenatal diagnosis were suspected to carry the maternally-derived CNVs. Further, we identified three CNV-related family histories with variable phenotypes. Statistical analysis of the 61525 pregnancies revealed that Z-scores of chromosomes 21 and 18 were significantly associated with PPV at 3 ≤ Z ≤ 40. Notably, three pregnancies with Z > 40 were both maternal full aneuploidy. At Z < -3, fetuses carried microdeletions instead of monosomies. Sex chromosome trisomy was significantly higher PPV than monosomy. Conclusion: The positive rate of the NIPT screening model combining Z-score and maternal CNV analysis increased from 6.91‰ (22/3184) to 12.25‰ (39/3184) and true positives increased from 12 to 21 pregnancies. We found that this method could improve the positive rate and accuracy of NIPT for aneuploidies and CNVs without increasing testing costs. It provides an early warning for the inheritance of pathogenic CNVs to the next generation.

Clinical evaluation of non-invasive prenatal screening in 32,394 pregnancies from Changzhi maternal and child health care hospital of Shanxi China.

Background: Non-invasive prenatal screening (NIPS) is a highly sensitive and specific screening test to detect fetal chromosomal abnormalities. The primary objective of this study was to evaluate the NIPS as an effective method for prenatal detection of aneuploidies in both high-risk and low-risk pregnancies. Methods: In current study, we performed NIPS in 32,394 pregnancies, out of which results were available in 32,361 (99.9%) of them. Illumina sequencing was performed for NIPS screening. Hypothesis Z test was used to classify fetal autosomal aneuploidy of T21, T18, and T13. Karyotyping was performed to determine the true negative and true positive NIPS results. Results: Among the 32,361 confirmed samples, 164 cases had positive results and 32197 cases had negative results. Of these positive cases, 116 cases were trisomy 21, 34 cases were trisomy 18 and 14 cases were trisomy 13. No false negative results were found in this cohort. The overall sensitivity and specificity were 100% and 99.91%, respectively. There was no significant difference in test performance between the 7,316 high-risk and 25,045 low-risk pregnancies, (sensitivity, 100% vs 100% (P>0.05); specificity, 99.96% vs 99.95% (P > 0.05)). Factors contributing to false-positive results included fetal copy number variants (CNVs), fetal mosaicism and typically producing Z scores between 3 and 4. Moreover, we analyzed NIPS wholegenome sequencing to investigate the Single Nucleotide Polymorphisms (SNPs) associations with drug response or risk of disease. As compare to the 1000g East Asian genome data, the results revealed a significant difference in 7,285,418 SNPs variants of Shanxi pregnant women including 19,293 clinvar recorded variants and 7,266,125 non-clinvar recorded. Conclusions: Our findings showed that NIPS was an effective assay that may be applied as routine screening for fetal trisomies in the prenatal setting. In addition, this study also provides an accurate assessment of significant differences in 7,285,418 SNPs variants in Shanxi pregnant women that were previously unavailable to clinicians in Shanxi population.

Optical Visualization of Photoexcitation Diffusion in All-Inorganic Perovskite at High Temperature.

All-inorganic halide perovskites are promising candidates for optoelectronic and photovoltaic devices because of their good thermal stability and remarkable optoelectronic properties. Among those properties, carrier transport properties are critical as they inherently dominate the device performance. The transport properties of perovskites have been widely studied at room and lower temperatures, but their high-temperature (i.e., tens of degrees above room temperature) characteristics are not fully understood. Here, the photoexcitation diffusion is optically visualized by transient photoluminescence microscopy (TPLM), through which the temperature-dependent transport characteristics from room temperature to 80 °C are studied in all-inorganic CsPbBr3 single-crystalline microplates. We reveal the decreasing trend of diffusion coefficient and the almost unchanged trend of diffusion length when heating the sample to high temperature. The phonon scattering in combination with the variation of effective mass is proposed for the explanation of the temperature-dependent diffusion behavior.

3D nanoprinting of semiconductor quantum dots by photoexcitation-induced chemical bonding.

Three-dimensional (3D) laser nanoprinting allows maskless manufacturing of diverse nanostructures with nanoscale resolution. However, 3D manufacturing of inorganic nanostructures typically requires nanomaterial-polymer composites and is limited by a photopolymerization mechanism, resulting in a reduction of material purity and degradation of intrinsic properties. We developed a polymerization-independent, laser direct writing technique called photoexcitation-induced chemical bonding. Without any additives, the holes excited inside semiconductor quantum dots are transferred to the nanocrystal surface and improve their chemical reactivity, leading to interparticle chemical bonding. As a proof of concept, we printed arbitrary 3D quantum dot architectures at a resolution beyond the diffraction limit. Our strategy will enable the manufacturing of free-form quantum dot optoelectronic devices such as light-emitting devices or photodetectors.

Serine/arginine-rich protein 30c activates human papillomavirus type 16 L1 mRNA expression via a bimodal mechanism.

Two splice sites on the human papillomavirus type 16 (HPV-16) genome are used exclusively by the late capsid protein L1 mRNAs: SD3632 and SA5639. These splice sites are suppressed in mitotic cells. This study showed that serine/arginine-rich protein 30c (SRp30c), also named SFRS9, activated both SD3632 and SA5639 and induced production of L1 mRNA. Activation of HPV-16 L1 mRNA splicing by SRp30c required an intact arginine/serine-repeat (RS) domain of SRp30c. In addition to this effect, SRp30c could enhance L1 mRNA production indirectly by inhibiting the early 3'-splice site SA3358, which competed with the late 3'-splice site SA5639. SRp30c bound directly to sequences downstream of SA3358, suggesting that SRp30c inhibited the enhancer at SA3358 and caused a redirection of splicing to the late 3'-splice site SA5639. This inhibitory effect of SRp30c was independent of its RS domain. These results suggest that SRp30c can activate HPV-16 L1 mRNA expression via a bimodal mechanism: directly by stimulating splicing to late splice sites and indirectly by inhibiting competing early splice sites.

Spectrum of PAH gene mutations and genotype-phenotype correlation in patients with phenylalanine hydroxylase deficiency from Shanxi province.

BACKGROUND: Phenylalanine hydroxylase deficiency (PAHD) is an autosomal recessive inborn error that affects phenylalanine (Phe) metabolism. It has a complex phenotype with many variants and genotypes among different populations. Shanxi province is a high-prevalence area of PAHD in China. METHODS: In this study, eighty-nine PAHD patients were subjected to genetic testing using Sanger sequencing, followed by multiplex ligation-dependent probe amplification analysis (MLPA). Allelic and genotypic phenotype values (APV and GPV, respectively) were used for genotype-based phenotypic prediction. RESULTS: Fifty-one types of variants, including three novel forms, were identified. The predominant variant was p.R243Q (22.09%), followed by p.R53H (10.47%), p.EX6-96A > G (9.30%), p.V399V (5.23%) and p.R413P (3.49%). Notably, mild hyperphenylalaninemia (MHP) has a high prevalence in this region (up to 45.76%), and the variant p.R53H was solely observed in patients of MHP. According to the genotype-phenotype prediction, the APV/GPV system was well correlated with the metabolic phenotype of most PAHD patients. CONCLUSION: We have systematically constructed the mutational and phenotypic spectrum of PAH in Shanxi province. Hence, this study will help to further understand the genotype-phenotype associations in PAHD patients, and it may offer more reliable genetic counseling and management.