Development of a low-cost and accurate carrier screening method for spinal muscular atrophy in developing countries.

Heterozygous carriers of the survival of motor neuron 1 (SMN1) gene deletion in parents account for approximately 95% of neonatal spinal muscular atrophy cases. Given the severity of the disease, professional organizations have recommended periconceptional spinal muscular atrophy carrier screening to all couples, regardless of race or ethnicity. However, the prevalence of screening activities in mainland China remains suboptimal, mainly attributed to the limitations of the existing carrier screening methods. Herein, we aimed to develop a low-cost, accessible, and accurate carrier screening method based on duplex droplet digital PCR (ddPCR), to cover a wider population in developing countries, including China. The receiver operating characteristic curve was used to determine the cut-off value of SMN1 copy numbers. Performance validation was conducted for linearity, precision, and accuracy. In total, 482 cases were considered to validate the concordance between the developed ddPCR assay and multiplex ligation-dependent probe amplification. Linear correlations were excellent between the expected concentration of the reference gene and the observed values (R2 > 0.99). Both the intra- and inter-assay precision of our ddPCR assays were less than 6.0%. The multiplex ligation-dependent probe amplification and ddPCR results were consistent in 480 of the 482 cases (99.6%). Two cases with multiplex ligation-dependent probe amplification, suggestive of two copies of SMN1 exon 7, were classified into three copies by ddPCR analysis. The overall correct classification of the samples included in our ddPCR assay was 100%. This study demonstrates that an appropriate cut-off value is an important prerequisite for establishing a semi-quantitative method to determine the SMN1 copy numbers. Compared to conventional methods, our ddPCR assay is low-cost, highly accurate, and has full potential for application in population spinal muscular atrophy carriers screening.

Comparison of the accuracy of multiplex digital PCR versus multiplex ligation-dependent probe amplification in quantification of the survival of motor neuron genes copy numbers.

For over two decades, multiplex ligation-dependent probe amplification (MLPA) has served as the gold standard for genetic testing of spinal muscular atrophy. However, there is emerging evidence questioning the reliability of MLPA in determining the copy numbers (CNs) of the survival of motor neuron (SMN) gene in certain cases. Recently, digital polymerase chain reaction (dPCR) has shown potential for better performance in copy number variant detection. This study aimed to compare MLPA and dPCR in quantifying SMN1 and SMN2 CNs, identify reasons for observed discrepancies, and explore the clinical implications of false results. A total of 733 DNA samples, previously subjected to MLPA analysis, were tested using multiplex droplet dPCR assays. Samples exhibiting inconsistent results between the two methods underwent repeated dPCR assays. When inconsistencies persisted, a third method was employed for verification. Digital PCR yielded results consistent with those of MLPA in 94.4% (692/733) of samples. Forty-one cases exhibited quantitative disparities in SMN1 and/or SMN2 CNs between the two methods. Confirmatory tests revealed that 37 inaccurate results were produced by the MLPA analysis, whereas four were attributed to the dPCR method. The dPCR technique exhibits better accuracy than MLPA and is qualified for SMA genetic testing across various clinical scenarios.

Evaluation of strategies for identification of infants with pathogenic glucose-6-phosphate dehydrogenase variants in China.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, which is caused by pathogenic variants of G6PD that result in decreased G6PD activity, is an X-linked inherited inborn error of metabolism that occurs worldwide. Individuals with G6PD deficiency and heterozygous females with normal G6PD activity (i.e., all individuals with pathogenic G6PD variants) are at risk of developing hemolytic anemia under increased oxidative challenge. However, this risk can be minimized by timely diagnosis. Currently, two assays are used to diagnose G6PD deficiency in China: evaluation of enzymatic activity and targeted genotyping. In terms of identification of all individuals with pathogenic G6PD variants, the performance and cost of different diagnostic strategies (isolated or combined evaluation of G6PD activity and G6PD genotyping) can vary, and these factors should be comprehensively evaluated. In this study, we examined 555 infants (437 males and 118 females) who were positive for the newborn screening of G6PD deficiency. We first evaluated the diagnostic performances of enzymatic testing and targeted genotyping. Both assays attained 100% specificities and positive predictive values for both male and female infants. In contrast, the sensitivities and negative predictive values (NPVs) of the diagnostic tests were different for male and female infants. For male infants, the sensitivities were 99.8 and 98.3%, and the NPVs were 94.1% and 69.6%, for enzymatic testing and targeted genotyping, respectively. For female infants, the sensitivities were 62.5% and 97.9%, and the NPVs were 37.9% and 91.7%, for enzymatic testing and targeted genotyping, respectively. We also evaluated the cost of the five different diagnostic strategies. The combination of G6PD activity testing of all infants, followed by genotyping of female infants with normal G6PD activity, attained high diagnostic sensitivity (99.8%) at a low cost (8.60 USD per diagnosed case). In the future, simultaneous examination of G6PD activity and whole-exon or whole-gene G6PD sequencing could become a standard clinical practice. Our data provide references for clinical practice on the standardization of current and future interventions for G6PD deficiency in China.

Genome-wide identification and genetic characterization of the CaMYB family and its response to five types of heavy metal stress in hot pepper (Capsicum annuum cv. CM334).

MYB proteins play a crucial role in plant growth and development and stress responses. In this study, 160 members of the MYB gene family from the pepper genome database were used to analyze gene structures, chromosome localization, collinearity, genetic affinity and expression in response to heavy metals. The results identified R2R3-MYB members and further phylogenetically classified them into 35 subgroups based on highly conserved gene structures and motifs. Collinearity analysis showed that segmental duplication events played a crucial role in the functional expansion of the CaMYB gene family by intraspecific collinearity, and at least 12 pairs of CaMYB genes existed between species prior to the differentiation between monocots and dicots. Moreover, the upstream CaMYB genes were mainly localized to the phytohormone elements ABRE and transcription factor elements MYB and MYC. Further analysis revealed that MYB transcription factors were closely associated with a variety of abiotic stress-related proteins (e.g., MAC-complex and SKIP). Under the stress of five metal ions, Cd2+, Cu2+, Pb2+, Zn2+, and Fe3+, the expression levels of some CaMYB family genes were upregulated. Of these genes, pairing homologous 1 (PH-1), PH-13, and PH-15 in the roots of Capsicum annuum were upregulated to the greatest extent, indicating that these three MYB family members are particularly sensitive to these five metals. This study provides a theoretical reference for the analysis of the molecular regulatory mechanism of MYB family genes in mediating the response to heavy metals in plants. This study reveals the mode of interaction between MYB and a variety of abiotic stress proteins and clarifies the biological functions of CaMYB family members in the regulation of heavy metal stress.

Newborn Screening for G6PD Deficiency in Xiamen, China: Prevalence, Variant Spectrum, and Genotype-Phenotype Correlations.

Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzymatic defect. The purpose of this study was to evaluate the profile of G6PD deficiency and investigate the factors associated with the accuracy of newborn screening (NBS) in Xiamen, China. Methods: A total of 99,546 newborns were screened by modified fluorescent spot test at the Women and Children's Hospital, Xiamen University. High-risk neonates were recalled for diagnosis by either a measurement of G6PD activity or genetic testing for the presence of pathogenic G6PD variants using a quantitative G6PD enzymatic assay or the MeltPro® G6PD assay, respectively. Results: In the first-tier screening, 1,256 newborns were categorized as high risk. Of these, 1,051 were diagnosed with G6PD deficiency, indicating a prevalence of 1.39% in Xiamen, China. Among the 1,013 neonates who underwent genotyping, 851 carried hemizygous, heterozygous, homozygous, or compound heterozygous variants, for a positive predictive value (PPV) of 84.01%. In total, 12 variants and 32 genotypes were identified, and the six most common variants were c.1376G>T, c.1388G>A, c.95A>G, c.1024C>T, c.871G>A, and c.392G>T, which accounted for approximately 94% of the identified alleles. Different variants showed characteristic enzymatic activities, although high phenotypic heterogeneity was observed for each variant. The use of cold-chain transportation significantly improved the PPV of NBS. Conclusions: We determined the profile of G6PD deficiency in Xiamen, including the prevalence, variant spectrum, and genotype-phenotype correlations and confirmed that maintaining a low temperature during sample transport is essential to ensure the high screening accuracy of NBS. Our data provides epidemiological, genotypic, phenotypic, and clinical practice references to standardize future interventions for G6PD deficiency.

A de novo frameshift variant of ANKRD11 (c.1366_1367dup) in a Chinese patient with KBG syndrome.

BACKGROUND: KBG syndrome is a rare autosomal dominant genetic disease mainly caused by pathogenic variants of ankyrin repeat domain-containing protein 11 (ANKRD11) or deletions involving ANKRD11. Herein, we report a novel de novo heterozygous frameshift ANKRD11 variant via whole exome sequencing in a Chinese girl with KBG syndrome. CASE PRESENTATION: A 2-year-2-month-old girl presented with a short stature and developmental delay. Comprehensive physical examinations, endocrine laboratory tests and imaging examination were performed. Whole-exome sequencing and Sanger sequencing were used to detect and confirm the variant associated with KBG in this patient, respectively. The pathogenicity of the variant was further predicted by several in silico prediction tools. The patient was diagnosed as KBG syndrome with a short stature and developmental delay, as well as characteristic craniofacial abnormalities, including a triangular face, long philtrum, wide eyebrows, a broad nasal bridge, prominent and protruding ears, macrodontia of the upper central incisors, dental crowding, and binocular refractive error. Her skeletal anomalies included brachydactyly, fifth finger clinodactyly, and left-skewed caudal vertebrae. Electroencephalographic results generally showed normal background activity with sporadic spikes and slow wave complexes, as well as multiple spikes and slow wave complexes in the bilateral parietal, occipital, and posterior temporal regions during non-rapid-eye-movement sleep. Brain MRI showed a distended change in the bilateral ventricles and third ventricle, as well as malformation of the sixth ventricle. Whole exome sequencing revealed a novel heterozygous frameshift variant in the patient, ANKRD11 c.1366_1367dup, which was predicted to be pathogenic through in silico analysis. The patient had received physical therapy since 4 months of age, and improvement of gross motor dysfunction was evident. CONCLUSIONS: The results of this study expand the spectrum of ANKRD11 variants in KBG patients and provide clinical phenotypic data for KBG syndrome at an early age. Our study also demonstrates that whole exome sequencing is an effective method for the diagnosis of rare genetic disorders.

Clinical Utility of a High-Resolution Melting Test for Screening Numerical Chromosomal Abnormalities in Recurrent Pregnancy Loss.

Recurrent pregnancy loss (RPL) occurs in approximately 5% of clinically identified pregnancies. Determining the cause of RPL is essential. Genetic testing, accompanied by an evidence-based workup, is the well-accepted process for evaluating RPL; however, current genetic tests have limitations in clinical practice. We, thus, developed a high-resolution melting analysis-based test (HRM test) to screen for the most common numerical chromosomal abnormalities present in the products of conception. We examined 765 products-of-conception samples with known karyotypes retrospectively using the HRM test, which showed high technical sensitivity (96.1%) and specificity (96.3%) as well as a high positive predictive value (95.9%) for the screening of chromosomal abnormalities. The cost-effectiveness of four RPL evaluation strategies that employ different genetic tests, karyotyping, chromosomal microarray/next-generation sequencing, the HRM test, and a combination of the HRM test and chromosomal microarray/next-generation sequencing, was then compared. The costs of diagnosing an explained RPL using karyotyping or the HRM test alone were similar. Performance of the HRM screening test before chromosomal microarray/next-generation sequencing analysis improved cost-effectiveness by approximately 30%. Cost-effectiveness was more prominent in the advanced maternal age group. Thus, the HRM test could be used as an initial screening tool, followed by other diagnostic methods to improve the cost-effectiveness of RPL evaluation, or as an alternative genetic test when other methods are unavailable or unaffordable.

Screening and mutation analysis of hyperphenylalaninemia in newborns from Xiamen, China.

In this study, we evaluated the incidence and genetic characteristics of hyperphenylalaninemia (HPA) in Xiamen, China. We analyzed the newborn screening data of HPA, obtained using a fluorometric method and tandem mass spectrometry (MS/MS), from 2013 to 2017. The suspected positive samples were further diagnosed using MassArray technology, multiplex ligation-dependent probe amplification (MLPA), and Sanger sequencing. A total of 418,831 newborns were screened, of whom 19 were diagnosed as HPA patients, with an incidence of 1:22,044. Of these HPA patients, 15 tested positive for phenylketonuria (PKU, 1:27922), and 4 tested positive for tetrahydrobiopterin deficiency (BH4D, 1:104,708). A total of 17 mutations were identified among 38 alleles in the 19 patients, with a detection rate of 94.74%, including 13 PAH and 4 PTS mutations. Among these, the c.721C>T, c.728G>A, c.1197A>T, c.611A>G and c.331C>T mutations, and the c.259C>T and c.155A>G mutations were the most prevalent PAH and PTS mutations in Xiamen, respectively. Therefore, this study systematically demonstrated the incidence and mutation spectrum of HPA in Xiamen. This information would contribute to genetic counseling, prenatal diagnosis, and management of HPA patients. Moreover, combining MS/MS technology with molecular genetic diagnosis is an effective strategy for future newborn HPA screening in Xiamen.

Carrier screening for spinal muscular atrophy with a simple test based on melting analysis.

Carrier screening of spinal muscular atrophy (SMA) can provide reproductive options for carriers and prevent the birth defects. Here, we developed a simple screening test based on melting analysis. The test comprises a duplex PCR with two primer pairs and three probes to simultaneous amplify SMN1, SMN2, and CFTR. By analyzing the melting profiles, we were able to determine the SMN1/SMN2 ratio and SMN1 + SMN2 copy number to subsequently determine the copy number of SMN1. Samples with one copy of SMN1 were considered as "high risk for carrier," while samples with ≥2 copies of SMN1 were considered as "low risk for carrier." We evaluated the clinical performance of this test using 215 clinical samples with various genotypes that had been previously confirmed by multiplex ligation-dependent probe amplification (MLPA). The test showed high sensitivity (100%) and specificity (97.1%) as well as high positive (97.3%) and negative (100%) predictive value, and was in perfect agreement with the gold standard test, MLPA (k = 0.97). Moreover, it is rapid, inexpensive, and easy to perform and automate, with high reproducibility and capacity. Therefore, we expect this test will advance carrier screening for SMA.

Clinical phenotype, in silico and biomedical analyses, and intervention for an East Asian population-specific c.370G>A (p.G124S) COQ4 mutation in a Chinese family with CoQ10 deficiency-associated Leigh syndrome.

COQ4 mutations have recently been shown to cause a broad spectrum of mitochondrial disorders in association with CoQ10 deficiency. Herein, we report the clinical phenotype, in silico and biochemical analyses, and intervention for a novel c.370 G > A (p.G124S) COQ4 mutation in a Chinese family. This mutation is exclusively present in the East Asian population (allele frequency of ~0.001). The homozygous mutation caused CoQ10 deficiency-associated Leigh syndrome with an onset at 1-2 months of age, presenting as respiratory distress, lactic acidosis, dystonia, seizures, failure to thrive, and detectable lesions in the midbrain and basal ganglia. No renal impairment was involved. The levels of CoQ10 and mitochondrial respiratory chain complex (C) II + III activity were clearly lower in cultured fibroblasts derived from the patient than in those from unaffected carriers; the decreased CII + III activity could be increased by CoQ10 treatment. Follow-up studies suggested that our patient benefitted from the oral supplementation of CoQ10, which allowed her to maintain a relatively stable health status. Based on the genetic testing, preimplantation and prenatal diagnoses were performed, confirming that the next offspring of this family was unaffected. Our cases expand the phenotypic spectrum of COQ4 mutations and the genotypic spectrum of Leigh syndrome.

Rapid screening for Klinefelter syndrome with a simple high-resolution melting assay: a multicenter study.

Klinefelter syndrome (KS) is the set of symptoms that result from the presence of an extra X chromosome in males. Postnatal population-based KS screening will enable timely diagnosis of this common chromosomal disease, providing the opportunity for early intervention and therapy at the time point when they are most effective and may prevent later symptoms or complications. Therefore, through this study, we introduced a simple high-resolution melting (HRM) assay for KS screening and evaluated its clinical sensitivity and specificity in three medical centers using 1373 clinical blood samples. The HRM assay utilized a single primer pair to simultaneously amplify specific regions in zinc finger protein, X-linked (ZFX) and zinc finger protein, Y-linked (ZFY). In cases of KS, the ratios of ZFX/ZFY are altered compared to those in normal males. As a result, the specific melting profiles differ and can be differentiated during data analysis. This HRM assay displayed high analytical specificity over a wide range of template DNA amounts (5 ng-50 ng) and reproducibility, high resolution for detecting KS mosaicism, and high clinical sensitivity (100%) and specificity (98.1%). Moreover, the HRM assay was rapid (2 h per run), inexpensive (0.2 USD per sample), easy to perform and automatic, and compatible with both whole blood samples and dried blood spots. Therefore, this HRM assay is an ideal postnatal population-based KS screening tool that can be used for different age groups.

Evaluation of a Magnetic Cellulose-Based DNA Extraction System to Improve the Performance of HybriBio Human Papillomavirus Genotyping and Screening Tests for Cervical Swab Samples.

BACKGROUND: To ensure the accuracy of clinical human papillomavirus (HPV) testing, the nucleic acid extraction procedure should be thoroughly evaluated for each clinical sample type. Therefore, we evaluated whether the MagCore® Automated Nucleic Acid Extraction system (MagCore system) could improve HybriBio HPV test performance for cervical swab samples. METHODS: We compared the performance of HybriBio HPV genotyping and screening tests using samples prepared with the MagCore system and Cell Lysis Kit, which was provided by the HPV test manufacturer. RESULTS: The MagCore system extracted high quality DNA and outperformed the Cell Lysis Kit in the subsequent analysis. In terms of the HPV genotyping testing, use of the MagCore system-extracted DNA markedly increased the signal intensity compared to that of DNA extracted with the Cell Lysis Kit for low concentrations of HPV DNA. Thus, the analytical sensitivity of testing was increased approximately 10 times by using the MagCore system, and the reproducibility was also increased (97.1% vs. 88.2%). In terms of the HPV screening tests, use of the MagCore system improved the compatibility of the internal control and targets, reducing the risk of false or invalid results. The MagCore system also improved the amplification efficiency and quantification accuracy for HPV16 detection. CONCLUSIONS: We demonstrated that the performance of HybriBio HPV genotyping and screening tests can be improved by using the MagCore system. This study not only provided an alternative, robust DNA extraction method for clinical users but also reemphasized the importance of establishing a reliable DNA extraction procedure for clinical HPV testing.

Whole Exome Sequencing Identifies a c.C2566T Mutation in the Androgen Receptor in a Chinese Family.

BACKGROUND: Whole exome sequencing (WES) is one of the most valuable tools for the detection of Mendelian diseases in clinical laboratory. We performed WES for a family of 46,XY disorders of gender development and compared the applicability of public databases for the subsequent phenotype studies of WES-identified mutations. METHODS: DNA samples from the two patients were analyzed by WES. The mutated protein was studied using the HomoloGene database, Polyphen2, and SIFT. The phenotype of the mutation was studied using ClinVar, the androgen receptor gene mutations database, AR database at Leiden Open Variation Database, and PubMed. RESULTS: A c.C2566T (p.R856C) mutation in the androgen receptor gene was detected for the patients. The in silico studies indicated that the p.R856C mutation is deleterious to the function of the androgen receptor. Unlike those of other databases, the variations listed in the androgen receptor gene mutations database were classified as complete androgen insensitivity-, partial androgen insensitivity-, or mild androgen insensitivity-relevant according to their clinical phenotype. In addition, the publications of the collected mutations in the androgen receptor gene mutations database are complete and easily accessible, which facilitates in depth studies of clinically identified mutations. CONCLUSIONS: We identified a c.C2566T (p.R856C) mutation of the AR gene in cases of familial complete androgen insensitivity by WES, and provided genetic counseling to related family members. This is the first study reporting this mutation in Chinese patients. We also compared the applicability of several public databases for phenotype studies of clinically identified Androgen Receptor mutations and suggest that the androgen receptor gene mutations database best satisfies clinical demands.

Rapid detection of G6PD mutations by multicolor melting curve analysis.

The MeltPro G6PD assay is the first commercial genetic test for glucose-6-phosphate dehydrogenase (G6PD) deficiency. This multicolor melting curve analysis-based real-time PCR assay is designed to genotype 16 G6PD mutations prevalent in the Chinese population. We comprehensively evaluated both the analytical and clinical performances of this assay. All 16 mutations were accurately genotyped, and the standard deviation of the measured Tm was <0.3°C. The limit of detection was 1.0ng/µL human genomic DNA. The assay could be run on four mainstream models of real-time PCR machines. The shortest running time (150min) was obtained with LightCycler 480 II. A clinical study using 763 samples collected from three hospitals indicated that, of 433 samples with reduced G6PD activity, the MeltPro assay identified 423 samples as mutant, yielding a clinical sensitivity of 97.7% (423/433). Of the 117 male samples with normal G6PD activity, the MeltPro assay confirmed that 116 samples were wild type, yielding a clinical specificity of 99.1% (116/117). Moreover, the MeltPro assay demonstrated 100% concordance with DNA sequencing for all targeted mutations. We concluded that the MeltPro G6PD assay is useful as a diagnostic or screening tool for G6PD deficiency in clinical settings.

Combination of fluorescence color and melting temperature as a two-dimensional label for homogeneous multiplex PCR detection.

Multiplex analytical systems that allow detection of multiple nucleic acid targets in one assay can provide rapid characterization of a sample while still saving cost and resources. However, few systems have proven to offer a solution for mid-plex (e.g. 10- to 50-plex) analysis that is high throughput and cost effective. Here we describe the combined use of fluorescence color and melting temperature (Tm) as a virtual 2D label that enables homogenous detection of one order of magnitude more targets than current strategies on real-time polymerase chain reaction platform. The target was first hybridized with a pair of ligation oligonucleotides, one of which harbored an artificial sequence that had a unique Tm when hybridized with a reporter fluorogenic probe. The ligated products were then amplified by a universal primer pair and denatured by a melting curve analysis procedure. The targets were identified by their respective Tm values in the corresponding fluorescence detection channels. The proof-of-principle of this approach was validated by genotyping 15 high-risk human papillomaviruses and 48 human single-nucleotide polymorphisms. The robustness of this method was demonstrated by analyzing a large number of clinical samples in both cases. The combined merits of multiplexity, flexibility and simplicity should make this approach suitable for a variety of applications.