Long-range PCR Research Articles

Cytochrome P450 2D6 (CYP2D6) and glucose-6-phosphate dehydrogenase (G6PD) genetic variations in Thai vivax malaria patients: Implications for 8-aminoquinoline radical cure.

Primaquine and tafenoquine are the only licensed drugs that effectively kill the hypnozoite stage and are used to prevent Plasmodium vivax malaria relapse. However, both primaquine and tafenoquine can cause acute hemolysis in glucose-6-phosphate dehydrogenase (G6PD)-deficient people with varying degrees of severity depending on G6PD variants. Additionally, primaquine efficacy against malaria parasites was decreased in individuals with impaired cytochrome P450 2D6 (CYP2D6) activity due to genetic polymorphisms. This study aimed to characterize G6PD and CYP2D6 genetic variations in vivax malaria patients from Yala province, a malaria-endemic area along the Thai-Malaysian border, and determine the biochemical properties of identified G6PD variants. Multiplexed high-resolution melting assay and DNA sequencing detected five G6PD variants, including G6PD Kaiping, G6PD Vanua Lava, G6PD Coimbra, G6PD Mahidol, and G6PD Kerala-Kalyan. Biochemical and structural characterization revealed that G6PD Coimbra markedly reduced catalytic activity and structural stability, indicating a high susceptibility to drug-induced hemolysis. While Kerala-Kalyan had minor effects, it is possible to develop mild adverse effects when receiving radical treatment. CYP2D6 genotyping was performed using long-range PCR and DNA sequencing, and the phenotypes were predicted using the combination of allelic variants. Decreased and no-function alleles were detected at frequencies of 53.4% and 14.2%, respectively. The most common alleles were CYP2D6*36+*10 (25.6%), *10 (23.9%), and *1 (22.2%). Additionally, 51.1% of the intermediate metabolizers showed CYP2D6*10/*36+*10 as the predominant genotype (15.9%). Our findings provide insights about genetic variations of G6PD and CYP2D6 in 88 vivax malaria patients from Yala, which may influence the safety and effectiveness of radical treatment. Optimization of 8-aminoquinoline administration may be required for safe and effective treatment in the studied population, which could be a significant challenge in achieving the goal of eliminating malaria.

Impact of Salmonella genome rearrangement on gene expression.

In addition to nucleotide variation, many bacteria also undergo changes at a much larger scale via rearrangement of their genome structure (GS) around long repeat sequences. These rearrangements result in genome fragments shifting position and/or orientation in the genome without necessarily affecting the underlying nucleotide sequence. To date, scalable techniques have not been applied to GS identification, so it remains unclear how extensive this variation is and the extent of its impact upon gene expression. However, the emergence of multiplexed, long-read sequencing overcomes the scale problem, as reads of several thousand bases are routinely produced that can span long repeat sequences to identify the flanking chromosomal DNA, allowing GS identification. Genome rearrangements were generated in Salmonella enterica serovar Typhi through long-term culture at ambient temperature. Colonies with rearrangements were identified via long-range PCR and subjected to long-read nanopore sequencing to confirm genome variation. Four rearrangements were investigated for differential gene expression using transcriptomics. All isolates with changes in genome arrangement relative to the parent strain were accompanied by changes in gene expression. Rearrangements with similar fragment movements demonstrated similar changes in gene expression. The most extreme rearrangement caused a large imbalance between the origin and terminus of replication and was associated with differential gene expression as a factor of distance moved toward or away from the origin of replication. Genome structure variation may provide a mechanism through which bacteria can quickly adapt to new environments and warrants routine assessment alongside traditional nucleotide-level measures of variation.

The Influence of Structural Variants of the CES1 Gene on the Pharmacokinetics of Enalapril, Presumably Due to Linkage Disequilibrium with the Intronic rs2244613.

Variants in the CES1 gene encoding carboxylesterase 1 may affect the metabolism of enalapril to the active metabolite enalaprilat. It was shown that the A allele of rs71647871 and the C allele of rs2244613 led to a decrease in plasma enalaprilat concentrations. This study aimed to estimate the effect of structural haplotypes of CES1 containing the pseudogene CES1P1, or a hybrid of the gene and the pseudogene CES1A2, on the pharmacokinetics of enalapril. We included 286 Caucasian patients with arterial hypertension treated with enalapril. Genotyping was performed using real-time PCR and long-range PCR. Peak and trough plasma enalaprilat concentrations were lower in carriers of CES1A2. The studied haplotypes were in linkage disequilibrium with rs2244613: generally, the A allele was in the haplotype containing the CES1P1, and the C allele was in the haplotype with the CES1A2. Thus, carriers of CES1A2 have reduced CES1 activity against enalapril. Linkage disequilibrium of the haplotype containing the CES1P1 or CES1A2 with rs2244613 should be taken into account when genotyping the CES1 gene.

Mito-SiPE is a sequence-independent and PCR-free mtDNA enrichment method for accurate ultra-deep mitochondrial sequencing

The analysis of somatic variation in the mitochondrial genome requires deep sequencing of mitochondrial DNA. This is ordinarily achieved by selective enrichment methods, such as PCR amplification or probe hybridization. These methods can introduce bias and are prone to contamination by nuclear-mitochondrial sequences (NUMTs), elements that can introduce artefacts into heteroplasmy analysis. We isolated intact mitochondria using differential centrifugation and alkaline lysis and subjected purified mitochondrial DNA to a sequence-independent and PCR-free method to obtain ultra-deep (>80,000X) sequencing coverage of the mitochondrial genome. This methodology avoids false-heteroplasmy calls that occur when long-range PCR amplification is used for mitochondrial DNA enrichment. Previously published methods employing mitochondrial DNA purification did not measure mitochondrial DNA enrichment or utilise high coverage short-read sequencing. Here, we describe a protocol that yields mitochondrial DNA and have quantified the increased level of mitochondrial DNA post-enrichment in 7 different mouse tissues. This method will enable researchers to identify changes in low frequency heteroplasmy without introducing PCR biases or NUMT contamination that are incorrectly identified as heteroplasmy when long-range PCR is used.

PMON115 Long term follow-up of patient with gigantism harboring an exon 2 deletion in the AIP gene

Abstract Introduction Pituitary gigantism is a rare endocrinopathy that has been linked to inactivating germline mutations in AIP gene with a prevalence of 30%. AIP is a tumor suppressor gene associated with high predisposition to pituitary adenoma. Thus, the genetic screening of AIP mutations has been recommended to cases with sporadic young-onset pituitary adenoma. Even with a phenotype suggesting gigantism the diagnosis could be delayed, making the treatment more complex, due the large pituitary tumor. Here we describe a giant patient with deletion of exon 2 of the AIP gene detected by Multiplex Ligation-dependent Probe Amplification assay (MLPA) 33 years after the diagnosis. Material and Methods In 1988, a 13-year-old afro-descendent boy with no history of familial pituitary adenoma, presented with seizures and tall stature. On exam he showed signs of gigantism and hypopituitarism: height 2.05 m (SD: + 7), target height was 1.65 (SD: -1), weight 75 kg (SD: + 3.2), no signs of puberty and impaired vision field. Basal GH was > 40 ng/mL, IGF-1: > 1200 ng/mL (ULN: +4). MRI revealed an invasive macroadenoma (Knosp IV). He underwent pituitary surgery twice with an adenoma GH/PRL positive with no remission. He did not respond to the treatment with somatostatin receptor analogue presenting remission only after radiotherapy, developing permanent deficiency of ACTH/TSH/LH/FSH/GH and diabetes insipidus. Peripheral blood was collected to genetic analysis in 2021. Results The genetic analysis supported by Sanger sequencing resulted negative to pathogenic germline variants in intron-exon frontiers and coding areas of the AIP and MEN1 genes. Thus, MLPA was performed using the SALSA MLPA kit P244 designed to detect large deletions or amplifications in AIP, CDKN1B and MEN1 genes (MRC-Holland, Amsterdam, The Netherlands). Once the exon 2 heterozygous deletion from the AIP gene was found via MLPA the confirmation was carried out through long-range PCR covering full open reading frame of the AIP gene. By agarose gel electrophoresis, the presence of two bands of different sizes confirmed this deletion. Discussion More than 130 AIP mutations have been described over the last decade and most of them, 70% generate truncated proteins (including insertions/deletions). However, gross AIP deletions were exceptionally reported so far (13 cases only: 11 had gigantism or acromegaly). Despite rarity, the present case reinforces the importance of investigate large AIP deletions, especially in gigantism. Cases harboring AIP mutations are associated with more aggressive tumors, lesser response to somatostatin receptor analogues needing, frequently, of an extensive multidisciplinary approach. It was also suggested that cases harboring truncated AIP mutations may develop pituitary adenoma in younger age than patients with non-truncated mutations. Thus, the characterization of genotype could help to refine making-decision on management of this complex disorder in an earlier stage avoiding multimodal treatment. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

OR12-6 Rocking Around the Cortex and Medulla

Abstract Introduction Pituitary gigantism is a rare endocrinopathy that has been linked to inactivating germline mutations in AIP gene with a prevalence of 30%. AIP is a tumor suppressor gene associated with high predisposition to pituitary adenoma. Thus, the genetic screening of AIP mutations has been recommended to cases with sporadic young-onset pituitary adenoma. Even with a phenotype suggesting gigantism the diagnosis could be delayed, making the treatment more complex, due the large pituitary tumor. Here we describe a giant patient with deletion of exon 2 of the AIP gene detected by Multiplex Ligation-dependent Probe Amplification assay (MLPA) 33 years after the diagnosis. Material and Methods In 1988, a 13-year-old afro-descendent boy with no history of familial pituitary adenoma, presented with seizures and tall stature. On exam he showed signs of gigantism and hypopituitarism: height 2. 05 m (SD: + 7), target height was 1.65 (SD: -1), weight 75 kg (SD: + 3.2), no signs of puberty and impaired vision field. Basal GH was > 40 ng/mL, IGF-1: > 1200 ng/mL (ULN: +4). MRI revealed an invasive macroadenoma (Knosp IV). He underwent pituitary surgery twice with an adenoma GH/PRL positive with no remission. He did not respond to the treatment with somatostatin receptor analogue presenting remission only after radiotherapy, developing permanent deficiency of ACTH/TSH/LH/FSH/GH and diabetes insipidus. Peripheral blood was collected to genetic analysis in 2021. Results The genetic analysis supported by Sanger sequencing resulted negative to pathogenic germline variants in intron-exon frontiers and coding areas of the AIP and MEN1 genes. Thus, MLPA was performed using the SALSA MLPA kit P244 designed to detect large deletions or amplifications in AIP, CDKN1B and MEN1 genes (MRC-Holland, Amsterdam, The Netherlands). Once the exon 2 heterozygous deletion from the AIP gene was found via MLPA the confirmation was carried out through long-range PCR covering full open reading frame of the AIP gene. By agarose gel electrophoresis, the presence of two bands of different sizes confirmed this deletion. Discussion More than 130 AIP mutations have been described over the last decade and most of them, 70% generate truncated proteins (including insertions/deletions). However, gross AIP deletions were exceptionally reported so far (13 cases only: 11 had gigantism or acromegaly). Despite rarity, the present case reinforces the importance of investigate large AIP deletions, especially in gigantism. Cases harboring AIP mutations are associated with more aggressive tumors, lesser response to somatostatin receptor analogues needing, frequently, of an extensive multidisciplinary approach. It was also suggested that cases harboring truncated AIP mutations may develop pituitary adenoma in younger age than patients with non-truncated mutations. Thus, the characterization of genotype could help to refine making-decision on management of this complex disorder in an earlier stage avoiding multimodal treatment. Presentation: Sunday, June 12, 2022 11:00 a.m. - 12:30 p.m.

Spectrum of sperm mtDNA deletions in men exposed to industrial air pollution.

Sperm mtDNA status can serve as a molecular marker of oxidative stress and environmental exposure. High levels of air pollution may be associated with increased mitochondrial DNA (mtDNA) deletion rates in sperm. We compared the length spectra of sperm mtDNA deletions in semen samples collected from city policemen exposed to traffic and industrial air pollution in two seasons with different levels of air pollution. We used long-range PCR to amplify a fragment of mtDNA (8066bp) frequently affected by deletions, visualized the PCR products by gel electrophoresis, and analysed aberrant bands corresponding to deleted mtDNA, using gel documentation software. The predominance of undeleted sperm mtDNA was accompanied by a variety of shorter PCR product lengths in the vast majority of sperm samples, in both seasons. Sperm mtDNA molecules and bands corresponding to long deletions were more frequently detected than shorter deletions, in both seasons. We did not detect any difference in the total number of electrophoretic bands corresponding to deleted sperm mtDNA and in the number of deleted sperm mtDNA molecules between the two seasons. In our study, air pollution during sperm maturation did not induce formation of large mtDNA deletions detectable by long PCR and gel electrophoresis (>1kb) in maturing sperm mtDNA.

Comprehensive Analysis of Fragile X Syndrome: Full Characterization of the FMR1 Locus by Long-Read Sequencing.

Fragile X syndrome (FXS) is the most frequent cause of inherited X-linked intellectual disability. Conventional FXS genetic testing methods mainly focus on FMR1 CGG expansions and fail to identify AGG interruptions, rare intragenic variants, and large gene deletions. A long-range PCR and long-read sequencing-based assay termed comprehensive analysis of FXS (CAFXS) was developed and evaluated in Coriell and clinical samples by comparing to Southern blot analysis and triplet repeat-primed PCR (TP-PCR). CAFXS accurately detected the number of CGG repeats in the range of 93 to at least 940 with mass fraction of 0.5% to 1% in the background of normal alleles, which was 2-4-fold analytically more sensitive than TP-PCR. All categories of mutations detected by control methods, including full mutations in 30 samples, were identified by CAFXS for all 62 clinical samples. CAFXS accurately determined AGG interruptions in all 133 alleles identified, even in mosaic alleles. CAFXS successfully identified 2 rare intragenic variants including the c.879A > C variant in exon 9 and a 697-bp microdeletion flanking upstream of CGG repeats, which disrupted primer annealing in TP-PCR assay. In addition, CAFXS directly determined the breakpoints of a 237.1-kb deletion and a 774.0-kb deletion encompassing the entire FMR1 gene in 2 samples. Long-read sequencing-based CAFXS represents a comprehensive assay for identifying FMR1 CGG expansions, AGG interruptions, rare intragenic variants, and large gene deletions, which greatly improves the genetic screening and diagnosis for FXS.

Runx2 regulates chromatin accessibility to direct the osteoblast program at neonatal stages.

SUMMARYThe transcriptional regulator Runx2 (runt-related transcription factor 2) has essential but distinct roles in osteoblasts and chondrocytes in skeletal development. However, Runx2-mediated regulatory mechanisms underlying the distinctive programming of osteoblasts and chondrocytes are not well understood. Here, we perform an integrative analysis to investigate Runx2-DNA binding and chromatin accessibility ex vivo using neonatal osteoblasts and chondrocytes. We find that Runx2 engages with cell-type-distinct chromatin-accessible regions, potentially interacting with different combinations of transcriptional regulators, forming cell-type-specific hotspots, and potentiating chromatin accessibility. Genetic analysis and direct cellular reprogramming studies suggest that Runx2 is essential for establishment of chromatin accessibility in osteoblasts. Functional enhancer studies identify an Sp7 distal enhancer driven by Runx2-dependent binding and osteoblast-specific chromatin accessibility, contributing to normal osteoblast differentiation. Our findings provide a framework for understanding the regulatory landscape encompassing Runx2-mediated and cell-type-distinct enhancer networks that underlie the specification of osteoblasts.

Multi-type RFC1 repeat expansions as the most common cause of hereditary sensory and autonomic neuropathy.

Non-coding repeat expansions within RFC1 and NOTCH2NLC genes have lately been linked to multisystem neurodegenerative diseases, which also shed light on yet undiagnosed patients with inherited peripheral neuropathies. The aim of this study was to identify the genetic basis of patients with hereditary sensory and autonomic neuropathy (HSAN). We collected 79 unrelated DNA samples clinically suspected with HSAN from multiple regions of Japan. Mutation screening was first performed using gene panel sequencing and whole-exome sequencing. Pathogenic/likely pathogenic variants were identified from genes of WNK1/HSN2 (6 cases), SCN9A (3 cases), NTRK1 (3 cases), and DNMT1 (2 cases). Subsequently, long-range flanking PCR and repeat-primed PCR were applied to analyze repeat expansions in RFC1 and NOTCH2NLC. Bi-allelic RFC1 repeat expansions were detected from 20 adult-onset HSAN patients, consisting of [(AAGGG)exp/(AAGGG)exp] (8 cases), [(ACAGG)exp/(ACAGG)exp] (8 cases), and [(AAGGG)exp/(ACAGG)exp] (4 cases). GGC repeat expansion in NOTCH2NLC was found in 1 case. Single-nucleotide variant-based haplotype analysis of patients harboring disease-associated repeat expansions in RFC1 revealed distinguishable haplotypes among subgroups with different repeat genotypes. These findings substantially redefine the genetic spectrum of HSAN, where multi-type RFC1 repeat expansions account for 25.3% of all patients, highlighting the necessity of genetic screening, particularly for adult-onset patients.

Oculocutaneous albinism and bleeding diathesis due to a novel deletion in the HPS3 gene.

Hermansky–Pudlak syndrome (HPS) is a group of rare autosomal recessive disorders characterized by oculocutaneous albinism (OCA) and bleeding diathesis. To date, 11 HPS types have been reported (HPS-1 to HPS-11), each defined by disease-causing variants in specific genes. Variants in the HPS1 gene were found in approximately 15% of HPS patients, most of whom harbor the Puerto Rican founder mutation. In this study, we report six affected individuals from three nonconsanguineous families of Ashkenazi Jewish descent, who presented with OCA and multiple ecchymoses and had normal platelet number and size. Linkage analysis indicated complete segregation to HPS3. Sequencing of the whole coding region and the intron boundaries of HPS3 revealed a heterozygous c.1163+1G>A variant in all six patients. Long-range PCR amplification revealed that all affected individuals also carry a 14,761bp deletion that includes the 5′UTR and exon 1 of HPS3, encompassing regions with long interspersed nuclear elements. The frequency of the c.1163+1G>A splice site variant was found to be 1:200 in the Ashkenazi Jewish population, whereas the large deletion was not detected in 300 Ashkenazi Jewish controls. These results present a novel HPS3 deletion mutation and suggest that the prevalence of HPS-3 in Ashkenazi Jews is more common than previously thought.

Organization and Unconventional Integration of the Mating-Type Loci in Morchella Species.

True morels (Morchella spp.) are a group of delicious fungi in high demand worldwide, and some species of morels have been successfully cultivated in recent years. To better understand the sexual reproductive mechanisms of these fungi, we characterized the structure of the mating-type loci from ten morel species, and seven of them were obtained using long-range PCR amplification. Among the studied species, eight were heterothallic, two were homothallic, and four types of composition were observed in the MAT loci. In three of the five black morel species, the MAT1-1-1, MAT1-1-10, and MAT1-1-11 genes were in the MAT1-1 idiomorph, and only the MAT1-2-1 gene was in the MAT1-2 idiomorph, while an integration event occurred in the other two species and resulted in the importation of the MAT1-1-11 gene into the MAT1-2 idiomorph and survival as a truncated fragment in the MAT1-1 idiomorph. However, the MAT1-1-11 gene was not available in the four yellow morels and one blushing morel species. M. rufobrunnea, a representative species of the earliest diverging branch of true morels, along with another yellow morel Mes-15, were confirmed to be homothallic, and the MAT1-1-1, MAT1-1-10, and MAT1-2-1 genes were arranged in a tandem array. Therefore, we hypothesized that homothallism should be the ancestral reproductive state in Morchella. RT-PCR analyses revealed that four mating genes could be constitutively expressed, while the MAT1-1-10 gene underwent alternative splicing to produce different splice variants.

P-561 Long-read amplicon guided haplotype imputation enabling comprehensive preimplantation genetic testing in families with de novo pathogenic variants

Abstract Study question Can we use comprehensive genome-wide haplotyping PGT for couples carrying de novo mutations, given the absence of phasing references to establish the disease-associated haplotype? Summary answer Targeted amplicon long-read sequencing represents a valuable approach for PGT workup that leverages comprehensive PGT application in families with de novo mutations. What is known already Preimplantation genetic testing (PGT) aims to select embryos devoid of inherited pathogenic variants. Current comprehensive genome-wide haplotyping PGT methods cannot be applied for couples where one of the partners carries a de novo mutation, as absence of affected close relatives restricts variant phasing to establish the disease-associated haplotype. For such families targeted approaches are currently used instead, thus missing genome-wide embryo analysis. Study design, size, duration To overcome the forementioned limitation, we developed a long-read amplicon guided haplotype imputation method. From January 2017 till June 2021 thirty-two couples were enrolled into the PGT program at the Centre for Human Genetics, UZ Leuven, in the context of a de novo variant causing a known monogenic disorder in one of the two partners. Participants/materials, setting, methods Genomic DNA from the partner carrying the variant (proband) and his/her parents was used for trio analysis via long-range PCR and long-read amplicon sequencing using the PacBio RSII and/or Oxford Nanopore platforms. Targeted haplotype phasing was then performed to impute the disease-associated allele. Main results and the role of chance The parental origin of the mutant allele was identified in 20 patients, resulting in the current 62.5% success rate. Most de novo mutations occurred on the paternal allele. In the remaining families, the region of interest either had no SNPs or had insufficient number of informative SNPs linked to mutation. From the successfully analyzed couples, eight have proceeded to PGT cycle and so far, three disease-free children have been born. Limitations, reasons for caution A costly family-specific work-up is required, allowing nevertheless the implementation of a generic genome-wide method at the embryo analysis stage. The sequencing cost itself is anticipated to decrease in time. To avoid non-informative results, the analysis of multiple amlpicons is recommended. Wider implications of the findings Current practices for the handling of couples with de novo variants are mostly targeted, require the combination of direct and indirect approaches and often require the multiple biopsies. Targeted amplicon long-read sequencing represents a valuable approach for PGT workup that leverages comprehensive PGT application in families with de novo mutations. Trial registration number not applicable

A closed Candidatus Odinarchaeum chromosome exposes Asgard archaeal viruses

Asgard archaea have recently been identified as the closest archaeal relatives of eukaryotes. Their ecology, and particularly their virome, remain enigmatic. We reassembled and closed the chromosome of Candidatus Odinarchaeum yellowstonii LCB_4, through long-range PCR, revealing CRISPR spacers targeting viral contigs. We found related viruses in the genomes of diverse prokaryotes from geothermal environments, including other Asgard archaea. These viruses open research avenues into the ecology and evolution of Asgard archaea.

GREPore-seq: A Robust Workflow to Detect Changes After Gene Editing Through Long-range PCR and Nanopore Sequencing

To achieve the enormous potential of gene-editing technology in clinical therapies, one needs to evaluate both the on-target efficiency and unintended editing consequences comprehensively. However, there is a lack of a pipelined, large-scale, and economical workflow for detecting genome editing outcomes, in particular insertion or deletion of a large fragment. Here, we describe an approach for efficient and accurate detection of multiple genetic changes after CRISPR/Cas9 editing by pooled nanopore sequencing of barcoded long-range PCR products. Recognizing the high error rates of Oxford nanopore sequencing, we developed a novel pipeline to capture the barcoded sequences by grepping reads of nanopore amplicon sequencing (GREPore-seq). GREPore-seq can assess nonhomologous end-joining (NHEJ)-mediated double-stranded oligodeoxynucleotide (dsODN) insertions with comparable accuracy to Illumina next-generation sequencing (NGS). GREPore-seq also reveals a full spectrum of homology-directed repair (HDR)-mediated large gene knock-in, correlating well with the fluorescence-activated cell sorting (FACS) analysis results. Of note, we discovered low-level fragmented and full-length plasmid backbone insertion at the CRISPR cutting site. Therefore, we have established a practical workflow to evaluate various genetic changes, including quantifying insertions of short dsODNs, knock-ins of long pieces, plasmid insertions, and large fragment deletions after CRISPR/Cas9-mediated editing. GREPore-seq is freely available at GitHub (https://github.com/lisiang/GREPore-seq) and the National Genomics Data Center (NGDC) BioCode (https://ngdc.cncb.ac.cn/biocode/tools/BT007293).

Comprehensive Analysis of Congenital Adrenal Hyperplasia Using Long-Read Sequencing.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder that has been included in newborn screening programs. Current approaches to gene testing for CAH are facing challenges because of the complexity of the CYP21A2 locus and genetic heterogeneity of the disease. A comprehensive analysis of CAH (CACAH) combining long-range locus-specific PCR and long-read sequencing (LRS) was developed to perform full sequence analysis of 5 common CAH candidate genes, including CYP21A2, CYP11B1, CYP17A1, HSD3B2, and StAR. In a blind retrospective study, the clinical utility of CACAH was evaluated in 37 samples by comparing to standard CAH testing using multiplex ligation-dependent probe amplification (MLPA) plus Sanger sequencing. Of the 37 clinical samples, a total of 69 pathogenic variants were identified, comprising 65 CYP21A2 variants, 2 HSD3B2 variants, and 2 CYP17A1 variants. For CYP21A2, the most frequent variant was c.518T > A (29.2%), followed by c.293-13C/A > G (21.5%). Compared with the current CAH testing using MLPA plus Sanger sequencing, the CACAH assay showed 100% specificity and 100% sensitivity, and precisely determined the junction sites of deletions/insertions and cis-trans configuration of multiple variants without analyzing family samples. Moreover, CACAH identified a case carrying 2 copies of CYP21A1 with the c.1451_1452delinsC variant on the same chromosome, which was not confirmed by MLPA plus Sanger sequencing. LRS-based CACAH can determine all genotypes of CAH accurately and reliably in one assay, presenting a comprehensive approach for CAH genetic diagnosis and carrier screening.

Analysis of rare thalassemia genetic variants based on third-generation sequencing

Thalassemia is a group of common hereditary anemias that cause significant morbidity and mortality worldwide. However, precisely diagnosing thalassemia, especially rare thalassemia variants, is still challenging. Long-range PCR and long-molecule sequencing on the PacBio Sequel II platform utilized in this study could cover the entire HBA1, HBA2 and HBB genes, enabling the diagnosis of most of the common and rare types of thalassemia variants. In this study, 100 cases of suspected thalassemia were subjected to traditional thalassemia testing and third-generation sequencing for thalassemia genetic diagnosis. Compared with traditional diagnostic methods, an additional 10 cases of rare clinically significant variants, including 3 cases of structure variants and 7 cases of single nucleotide variations (SNVs) were identified, of which a case with − α3.7 subtype III (− α3.7III) was first identified and validated in the Chinese population. Other rare variants of 11.1 kb deletions (− 11.1/αα), triplicate α-globin genes (aaa3.7/αα) and rare SNVs have also been thoroughly detected. The results showed that rare thalassemia variants are not rare but have been misdiagnosed by conventional methods. The results further validated third-generation sequencing as a promising method for rare thalassemia genetic testing.

The complete mitochondrial genome of the White-browed Tit-warbler

The White-browed Tit-warbler (Leptopoecile sophiae) is a tiny and infrequently-seen bird species that is mainly distributed in alpine scrub and forests. This species is poorly understood with respect to its natural history and genetic information. In this paper, we first presented the whole mitochondrial genome of the White-browed Tit-warbler. The whole mitochondrial sequence of this species was determined by long-range PCR and conserved primer walking approaches. The results revealed that the whole mitochondrial genome of L. sophiae was 17,803 bp in length with 54.3% A + T content; the genome has the same gene order as that of other passerine birds, including 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and 1 non-coding control region. The control region (D-loop) of L. sophiae was located between tRNA-Gln and tRNA-Phe with 1155 bp length. Phylogenetic analysis indicated that the genome of Leptopoecile species is more closely related to Aegithalos species than to Phylloscopus species. These data provide value for further studies on molecular evolution and conservation genetics on these poorly-known Leptopoecile species at high altitudes.

Rh Blood Group D Antigen Genotyping Using a Portable Nanopore-based Sequencing Device: Proof of Principle.

Nanopore sequencing is direct sequencing of a single-stranded DNA molecule using biological pores. A portable nanopore-based sequencing device from Oxford Nanopore Technologies (MinION) depends on driving a DNA molecule through nanopores embedded in a membrane using a voltage. Changes in current are then measured by a sensor, thousands of times per second and translated to nucleobases. Genomic DNA (gDNA) samples (n = 13) were tested for Rh blood group D antigen (RHD) gene zygosity using droplet digital PCR. The RHD gene was amplified in 6 overlapping amplicons using long-range PCR. Amplicons were purified, and the sequencing library was prepared following the 1D Native barcoding gDNA protocol. Sequencing was carried out with 1D flow cells R9 version. Data analysis included basecalling, aligning to the RHD reference sequence, and calling variants. Variants detected were compared to the results acquired previously by the Ion Personal Genome Machine (Ion PGM). Up to 500× sequence coverage across the RHD gene allowed accurate variant calling. Exonic changes in the RHD gene allowed RHD allele determination for all samples sequenced except 1 RHD homozygous sample, where 2 heterozygous RHD variant alleles are suspected. There were 3 known variant RHD alleles (RHD*01W.02, RHD*11, and RHD*15) and 6 novel RHD variant alleles, as previously seen in Ion PGM sequencing data for these samples. MinION was effective in blood group genotyping, provided enough sequencing data to achieve high coverage of the RHD gene, and enabled confident calling of variants and RHD allele determination.

Comprehensive Analysis of Spinal Muscular Atrophy: SMN1 Copy Number, Intragenic Mutation, and 2 + 0 Carrier Analysis by Third-Generation Sequencing

Population-wide carrier screening for spinal muscular atrophy (SMA) is recommended by the American College of Medical Genetics and Genomics. However, the methods used currently mainly focus on SMN1 copy number and fail to identify carriers with pathogenic intragenic mutations and silent (2+0) carriers. We developed a method termed comprehensive analysis of SMA (CASMA) based on long-range PCR and third-generation sequencing of full-length and downstream regions of SMN1/2. The sensitivity and specificity of CASMA to detect SMA carriers with one copy of SMN1 were 100% (n=101) and 99.2% (n=236), respectively. CASMA confirmed three SMN1 intragenic mutations and pinpointed an inframe mutation c.661_666del to SMN2, which was misreported to SMN1 by allele-specific long-range nested PCR plus Sanger sequencing. CASMA also correctly predicted 8 of 16 samples (50%) with SMN1 duplication alleles. CASMA was expected to increase the detection rate of SMA carriers from 91% to 98% and decrease the residual risk ratio from 1:415 to 1:1868 after negative results of two SMN1 copies in the Chinese population. CASMA presents a comprehensive approach for identifying SMN1 and SMN2 copy number, intragenic mutations, and potential silent carriers that significantly reduces the residual risk ratio in SMA carrier screening and has great clinical utility.