Comprehensive Analysis of PKD1 and PKD2 by Long-Read Sequencing in Autosomal Dominant Polycystic Kidney Disease.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is mainly caused by heterogeneous variants in the PKD1 and PKD2 genes. Genetic analysis of PKD1 has been challenging due to homology with 6 PKD1 pseudogenes and high GC content. METHODS: A single-tube multiplex long-range-PCR and long-read sequencing-based assay termed "comprehensive analysis of ADPKD" (CAPKD) was developed and evaluated in 170 unrelated patients by comparing to control methods including next-generation sequencing (NGS) and multiplex ligation-dependent probe amplification. RESULTS: CAPKD achieved highly specific analysis of PKD1 with a residual noise ratio of 0.05% for the 6 pseudogenes combined. CAPKD identified PKD1 and PKD2 variants (ranging from variants of uncertain significance to pathogenic) in 160 out of the 170 patients, including 151 single-nucleotide variants (SNVs) and insertion-deletion variants (indels), 6 large deletions, and one large duplication. Compared to NGS, CAPKD additionally identified 2 PKD1 variants (c.78_96dup and c.10729_10732dup). Overall, CAPKD increased the rate of variant detection from 92.9% (158/170) to 94.1% (160/170), and the rate of diagnosis with pathogenic or likely pathogenic variants from 82.4% (140/170) to 83.5% (142/170). CAPKD also directly determined the cis-/trans-configurations in 11 samples with 2 or 3 SNVs/indels, and the breakpoints of 6 large deletions and one large duplication, including 2 breakpoints in the intron 21 AG-repeat of PKD1, which could only be correctly characterized by aligning to T2T-CHM13. CONCLUSIONS: CAPKD represents a comprehensive and specific assay toward full characterization of PKD1 and PKD2 variants, and improves the genetic diagnosis for ADPKD.

Third-generation sequencing identified two rare α-chain variants leading to hemoglobin variants in Chinese population.

BACKGROUND: Rare and novel variants of HBA1/2 and HBB genes resulting in thalassemia and hemoglobin (Hb) variants have been increasingly identified. Our goal was to identify two rare Hb variants in Chinese population using third-generation sequencing (TGS) technology. METHODS: Enrolled in this study were two Chinese families from Fujian Province. Hematological screening was conducted using routine blood analysis and Hb capillary electrophoresis analysis. Routine thalassemia gene testing was carried out to detect the common mutations of α- and ß-thalassemia in Chinese population. Rare or novel α- and ß-globin gene variants were further investigated by TGS. RESULTS: The proband of family 1 was a female aged 32, with decreased levels of mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), Hb A2, and abnormal Hb bands in zone 5 and zone 12. No common thalassemia mutations were detected by routine thalassemia analysis, while a rare α-globin gene variant Hb Jilin [α139(HC1)Lys>Gln (AAA>CAA); HBA2:c.418A>C] was identified by TGS. Subsequent pedigree analysis showed that the proband's son also harbored the Hb Jilin variant with slightly low levels of MCH, Hb A2, and abnormal Hb bands. The proband of family 2 was a male at 41 years of age, exhibiting normal MCV and MCH, but a low level of Hb A2 and an abnormal Hb band in zone 12 without any common α- and ß-thalassemia mutations. The subsequent TGS detection demonstrated a rare Hb Beijing [α16(A14)Lys>Asn (AAG>AAT); HBA2:c.51G>T] variant in HBA2 gene. CONCLUSION: In this study, for the first time, we present two rare Hb variants of Hb Jilin and Hb Beijing in Fujian Province, Southeast China, using TGS technology.

A high-fidelity long-read sequencing-based approach enables accurate and effective genetic diagnosis of spinal muscular atrophy.

BACKGROUND: We aimed to develop a high-fidelity long-read sequencing (LRS)-based approach to detect SMN gene variants in one step. It is challenging for conventional step-wise methods to simultaneously detect all kinds of variations between homologous SMN1 and SMN2. METHODS: In this study, LRS was developed to analyze copy numbers (CNs), full sequences, and structure of SMN1 and SMN2. The results were compared with those from the step-wise methods in 202 samples from 67 families. RESULTS: LRS achieved 100% (202/202) and 99.5% (201/202) accuracy for SMN1 and SMN2 CNs, respectively. It corrected SMN1 CNs from MLPA, which was caused by SNVs/indels that located in probe-binding region. LRS identified 23 SNVs/indels distributing throughout SMN1, including c.22dup and c.884A > T in trans-configuration, and a de novo variant c.41_42delinsC for the first time. LRS also identified a SMN2 variant c.346A > G. Moreover, it successfully determined Alu-mediated 8978-bp deletion encompassing exon 2a-5 and 1415-bp deletion disrupting exon 1, and the exact breakpoints of large deletions. Through haplotype-based pedigree trio analysis, LRS identified SMN1 2 + 0 carriers, and determined the distribution of SMN1 and SMN2 on two chromosomes. CONCLUSIONS: LRS represents a more comprehensive and accurate diagnosis approach that is beneficial to early treatment and effective management of SMA.

An Effective and Universal Long-Read Sequencing-Based Approach for SMN1 2 + 0 Carrier Screening through Family Trio Analysis.

BACKGROUND: Population-wide carrier screening for spinal muscular atrophy (SMA) is recommended by professional organizations to facilitate informed reproductive options. However, genetic screening for SMN1 2 + 0 carriers, accounting for 3%-8% of all SMA carriers, has been challenging due to the large gene size and long distance between the 2 SMN genes. METHODS: Here we repurposed a previously developed long-read sequencing-based approach, termed comprehensive analysis of SMA (CASMA), to identify SMN1 2 + 0 carriers through haplotype analysis in family trios (CASMA-trio). Bioinformatics pipelines were developed for accurate haplotype analysis and SMN1 2 + 0 deduction. Seventy-nine subjects from 24 families composed of, at the minimum, 3 were enrolled, and CASMA-trio was employed to determine whether an index subject with 2 SMN1 copies was a 2 + 0 carrier in these families. For the proof-of-principle, SMN2 2 + 0 was also analyzed. RESULTS: Among the 16 subjects with 2 SMN1 copies, CASMA-trio identified 5 subjects from 4 families as SMN1 2 + 0 carriers, which was consistent with pedigree analysis involving an affected proband. CASMA-trio also identified SMN2 2 + 0 in six out of 43 subjects with 2 SMN2 copies. Additionally, CASMA-trio successfully determined the distribution pattern of SMN1 and SMN2 genes on 2 alleles in all 79 subjects. CONCLUSIONS: CASMA-trio represents an effective and universal approach for SMN1 2 + 0 carriers screening, as it does not reply on the presence of an affected proband, certain single-nucleotide polymorphisms, ethnicity-specific haplotypes, or complicated single-nucleotide polymorphism analysis across 3 generations. Incorporating CASMA-trio into existing SMA carrier screening programs will greatly reduce residual risk ratio.

Molecular spectrum and prevalence of thalassemia investigated by third-generation sequencing in the Dongguan region of Guangdong Province, Southern China.

BACKGROUND: PCR, Sanger sequencing and NGS are often employed for carrier screening of thalassemia but all of these methods have limitations. In this study, we evaluated a new third-generation sequencing-based approach termed comprehensive analysis of thalassemia alleles (CATSA) to explore the prevalence of thalassemia in the Dongguan region of southern China. METHODS: 19,932 subjects were recruited for thalassemia screening and hemoglobin testing was performed for each of them. Routine PCR was performed for all the hemoglobin testing-positive subjects and CATSA was conducted for randomly selected subjects from hemoglobin testing-positive and negative subjects. RESULTS: In the 2716 subjects tested both by PCR and CATSA, 2569 had the same results and 147 had discordant results between the two methods. Sanger sequencing, specially designed PCR and MLPA confirmed the results of CATSA were all correct. In total, CATSA correctly detected 787 subjects with variants while routine PCR correctly detected 640 subjects with variants. CATSA yielded a 5.42% (147 of 2716) increment compared with routine PCR. In the 447 hemoglobin testing-negative subjects, CATSA identified pathogenic variants in 12 subjects. Moreover, CATSA identified a novel deletion (chr16:171262-202032) in the α-globin gene cluster. As a result, the deduced carrier frequency of α-thalassemia,ß-thalassemia and α-/ß-thalassemia was 5.62%, 3.85% and 0.93%, respectively. CONCLUSIONS: Our study demonstrated CATSA was a more comprehensive and precise approach than the routine PCR in a large scale of samples, which is highly beneficial for carrier screening of thalassemia. It provided a broader molecular spectrum of hemoglobinopathies and a better basis for a control program in Dongguan region.

Long-read sequencing: An effective method for genetic analysis of CYP21A2 variation in congenital adrenal hyperplasia.

BACKGROUND: The sequence similarity between CYP21A2 gene and its inactive pseudogene CYP21A1P, and copy number variation (CNV) caused by unequal crossover, make it challenging to characterize the CYP21A2 gene through traditional methods. This study aimed to evaluate the clinical utility of the long-read sequencing (LRS) method in carrier screening and genetic diagnosis of congenital adrenal hyperplasia (CAH) by comparing the efficiency of the LRS method with the conventional multiplex ligation-dependent probe amplification (MLPA) plus Sanger sequencing approaches in CYP21A2 analysis. METHODS: In a retrospective study, full sequence analysis of the CYP21A2 and CYP21A1P was performed for three pedigrees through long-range locus-specific PCR followed by LRS based on the Pacific Biosciences (PacBio, California, USA) single-molecule real-time (SMRT) platform, and the results were compared with those obtained from next-generation sequencing (NGS)-based whole exome sequencing (WES) and the traditional methods of MLPA plus Sanger sequencing. RESULTS: The LRS method successfully identified seven CYP21A2 variants, including three single nucleotide variants (NM_000500.9:c.1451G > C p.(Arg484Pro), c.293-13A/C > G (IVS2-13A/C > G), c.518 T > A p.(Ile173Asn)), one 111-bp polynucleotide insertion, one set of 3'URT variants (NM_000500.9:c.*368 T > C, c.*390A > G, c.*440C > T, c.*443 T > C) and two types of chimeric genes and straightforwardly depicted the inheritance patterns of these variants within families. Moreover, the LRS method enabled us to determine the cis-trans configuration of multiple variants in one assay, without the need to analyze additional family samples. Compared with traditional methods, this LRS method can achieve a precise, comprehensive and intuitive result in the genetic diagnosis of 21-hydroxylase deficiency (21-OHD). CONCLUSION: The LRS method is comprehensive in CYP21A2 analysis and intuitive in result presentation, which holds substantial promise in clinical application as a crucial tool for carrier screening and genetic diagnosis of CAH.

Comprehensive Analysis of Hemophilia A (CAHEA): Towards Full Characterization of the F8 Gene Variants by Long-Read Sequencing.

BACKGROUND: Hemophilia A (HA) is the most frequently occurring X-linked bleeding disorder caused by heterogeneous variants in the F8 gene, one of the largest genes known. Conventional molecular analysis of F8 requires a combination of assays, usually including long-range polymerase chain reaction (LR-PCR) or inverse-PCR for inversions, Sanger sequencing or next-generation sequencing for single-nucleotide variants (SNVs) and indels, and multiplex ligation-dependent probe amplification for large deletions or duplications. MATERIALS AND METHODS: This study aimed to develop a LR-PCR and long-read sequencing-based assay termed comprehensive analysis of hemophilia A (CAHEA) for full characterization of F8 variants. The performance of CAHEA was evaluated in 272 samples from 131 HA pedigrees with a wide spectrum of F8 variants by comparing to conventional molecular assays. RESULTS: CAHEA identified F8 variants in all the 131 pedigrees, including 35 intron 22-related gene rearrangements, 3 intron 1 inversion (Inv1), 85 SNVs and indels, 1 large insertion, and 7 large deletions. The accuracy of CAHEA was also confirmed in another set of 14 HA pedigrees. Compared with the conventional methods combined altogether, CAHEA assay demonstrated 100% sensitivity and specificity for identifying various types of F8 variants and had the advantages of directly determining the break regions/points of large inversions, insertions, and deletions, which enabled analyzing the mechanisms of recombination at the junction sites and pathogenicity of the variants. CONCLUSION: CAHEA represents a comprehensive assay toward full characterization of F8 variants including intron 22 and intron 1 inversions, SNVs/indels, and large insertions and deletions, greatly improving the genetic screening and diagnosis for HA.

Molecular characterization of a novel 83.9-kb deletion of the α-globin upstream regulatory elements by long-read sequencing.

Inherited deletions of upstream regulatory elements of α-globin genes give rise to α-thalassemia, which is an autosomal recessive monogenic disease. However, conventional thalassemia target diagnosis often fails to identify these rare deletions. Here we reported a family with two previous pregnancies of Hb Bart's hydrops fetalis and was seeking for prenatal diagnosis during the third pregnancy. Both parents had low level of Hemoglobin A2 indicating α-thalassemia. Conventional Gap-PCR and PCR-reverse dot blot showed the father carried -SEA deletion but did not identify any variants in the mother. Multiplex ligation-dependent probe amplification identified a deletion containing two HS-40 probes but could not determine the exact region. Finally, a long-read sequencing (LRS)-based approach directly identified that the exact deletion region was chr16: 48,642-132,584, which was located in the α-globin upstream regulatory elements and named (αα)JM after the Jiangmen city. Gap-PCR and Sanger sequencing confirmed the breakpoint. Both the mother and fetus from the third pregnancy carried heterozygous (αα)JM, and the fetus was normally delivered at gestational age of 39 weeks. This study demonstrated that LRS technology had great advantages over conventional target diagnosis methods for identifying rare thalassemia variants and assisted better carrier screening and prenatal diagnosis of thalassemia.

Case report: Long-read sequencing identified a novel 14.9-kb deletion of the α-globin gene locus in a family with α-thalassemia in China.

Background: Thalassemia is a hereditary blood disease resulting from globin chain synthesis impairment because of α- and/or ß-globin gene variants. α-thalassemia is characterized by non-deletional and deletional variants in the HBA gene locus, of which rare deletional variants are difficult to detect by conventional polymerase chain reaction (PCR)-based methods. Case report: We report the case of a one-month-old boy, who and his mother had abnormal hematological parameters, while his father had normal hematology. Conventional PCR-reverse dot blot (RDB) was performed for all family members to analyze the 23 most common thalassemia variants in China, but did not identify any pathologic variants. Single-molecule real-time (SMRT) long-read sequencing (LRS) technology was then performed and identified an unreported 14.9-kb large deletion (hg38 chr16:168,803-183,737) of the α-globin gene locus, which disrupted both HBA1 and HBA2 genes in the proband and his mother. The exact breakpoints of the deletion were confirmed by gap-PCR and Sanger sequencing. Conclusion: We have detected a novel large deletion in α-globin gene locus in China, which not only enriches the variant spectrum of thalassemia, but also demonstrates the accuracy and efficiency of LRS in detecting rare and novel deletions.

Evaluating the Clinical Utility of a Long-Read Sequencing-Based Approach in Prenatal Diagnosis of Thalassemia.

BACKGROUND: The aim is to evaluate the clinical utility of a long-read sequencing-based approach termed comprehensive analysis of thalassemia alleles (CATSA) in prenatal diagnosis of thalassemia. METHODS: A total of 278 fetuses from at-risk pregnancies identified in thalassemia carrier screening by PCR-based methods were recruited from 9 hospitals, and PCR-based methods were employed for prenatal diagnosis. CATSA was performed retrospectively and blindly for all 278 fetuses. RESULTS: Among the 278 fetuses, 263 (94.6%) had concordant results and 15 (5.4%) had discordant results between the 2 methods. Of the 15 fetuses, 4 had discordant thalassemia variants within the PCR detection range and 11 had additional variants identified by CATSA. Independent PCR and Sanger sequencing confirmed the CATSA results. In total, CATSA and PCR-based methods correctly detected 206 and 191 fetuses with variants, respectively. Thus, CATSA yielded a 7.9% (15 of 191) increment as compared with PCR-based methods. CATSA also corrected the predicted phenotype in 8 fetuses. Specifically, a PCR-based method showed one fetus had homozygous HBB c.52A > T variants, while CATSA determined the variant was heterozygous, which corrected the predicted phenotype from ß-thalassemia major to trait, potentially impacting the pregnancy outcome. CATSA additionally identified α-globin triplicates in 2 fetuses with the heterozygous HBB c.316-197C > T variant, which corrected the predicted phenotype from ß-thalassemia trait to intermedia and changed the disease prognosis. CONCLUSIONS: CATSA represents a more comprehensive and accurate approach that potentially enables more informed genetic counseling and improved clinical outcomes compared to PCR-based methods.

Back-to-Back Comparison of Third-Generation Sequencing and Next-Generation Sequencing in Carrier Screening of Thalassemia.

CONTEXT.­: Recently, new technologies, such as next-generation sequencing and third-generation sequencing, have been used in carrier screening of thalassemia. However, there is no direct comparison between the 2 methods in carrier screening of thalassemia. OBJECTIVE.­: To compare the clinical performance of third-generation sequencing with next-generation sequencing in carrier screening of thalassemia. DESIGN.­: Next-generation sequencing and third-generation sequencing were simultaneously conducted for 1122 individuals in Hainan Province. RESULTS.­: Among 1122 genetic results, 1105 (98.48%) were concordant and 17 (1.52%) were discordant between the 2 methods. Among the 17 discordant results, 4 were common thalassemia variants, 9 were rare thalassemia variants, and 4 were variations with unknown pathogenicity. Sanger sequencing and polymerase chain reaction for discordant samples confirmed all the results of third-generation sequencing. Among the 685 individuals with common and rare thalassemia variants detected by third-generation sequencing, 512 (74.74%) were carriers of α-thalassemia, 110 (16.06%) were carriers of ß-thalassemia, and 63 (9.20%) had coinheritance of α-thalassemia and ß-thalassemia. Three thalassemia variants were reported for the first time in Hainan Province, including -THAI, -α2.4, and ααααanti3.7. Eleven variants with potential pathogenicity were identified in 36 patients with positive hemoglobin test results. Among 52 individuals with negative hemoglobin test results, 17 were identified with thalassemia variants. In total, third-generation sequencing and next-generation sequencing correctly detected 763 and 746 individuals with variants, respectively. Third-generation sequencing yielded a 2.28% (17 of 746) increment compared with next-generation sequencing. CONCLUSIONS.­: Third-generation sequencing was demonstrated to be a more accurate and reliable approach in carrier screening of thalassemia compared with next-generation sequencing.

Evaluating the clinical utility of a long-read sequencing-based approach in genetic testing of fragile-X syndrome.

BACKGROUND: Fragile X syndrome (FXS) arises from the FMR1 CGG expansion. Comprehensive genetic testing for FMR1 CGG expansions, AGG interruptions, and microdeletions is essential to provide genetic counseling for females carrying premutation alleles. However, conventional PCR-based FMR1 assays mainly focus on CGG repeats, and could detect AGG interruption only in males. METHODS: The clinical utility of a long-read sequencing-based assay termed comprehensive analysis of FXS (CAFXS) was evaluated in 238 high-risk samples by comparing to conventional PCR assays. RESULTS: PCR assays identified five premuation and three full mutation categories alleles in all the samples, and CAFXS successfully called all the FMR1 CGG expansion. CAFXS identified 24-bp microdeletions upstream to the trinucleotide region with 30 CGG repeats, which was miscalled by the length-based PCR methods. CAFXS also identified a 187-bp deletion in about 1/7 of the sequencing reads in a male patient with mosaic full mutation alleles. CAFXS allowed for precise constructing the FMR1 CGG repeat and AGG interruption pattern in all the samples, and identified a novel and alternative CGA interruption in one normal female sample. CONCLUSIONS: CAFXS represents a more comprehensive and accurate approach for FXS genetic testing that potentially enables more informed genetic counseling compared to PCR-based methods.

Application of third-generation sequencing for genetic testing of thalassemia in Guizhou Province, Southwest China.

OBJECTIVES: To explore the application of third-generation sequencing (TGS) for genetic diagnosis and prenatal genetic screening of thalassemia genes. METHODS: Two groups of subjects were enrolled in this study. The first group included 176 subjects with positive hematological phenotypes for thalassemia. Thalassemia-associated genes were detected simultaneously in each sample using both the PacBio TGS platform based on single-molecule real-time (SMRT) technology and the conventional PCR-reverse dot blot (PCR-RDB). Sanger sequencing was used for validation when results were discordant between the two methods. The second group included 53 couples with at least one partner having a positive thalassemia hematological phenotype, and they were screened for homotypic thalassemia variants by TGS, and the risk of pregnancies with babies presenting with severe thalassemia, was assessed. RESULTS: Of the 176 subjects, 175 had concordant genotypes between the two methods, including 63 normal subjects and 112 α- and/or ß-thalassemia gene carriers, with a concordance rate of 99.43%. TGS detected a rare ß-thalassemia gene variant -50 (G > A) that was not detected by conventional PCR-RDB. TGS identified seven of the 53 couples as homotypic thalassemia gene carriers, five of whom were at risk of pregnancies with severe thalassemia. CONCLUSION: TGS could effectively detect common and rare thalassemia variants with high accuracy and efficiency. This approach would be suitable for prenatal thalassemia genetic screening in areas with high incidence of thalassemia.

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

BACKGROUND: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

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.

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.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Detection of rare thalassemia mutations using long-read single-molecule real-time sequencing.

Gap- polymerase chain reaction (PCR), reverse dot-blot assay (RDB), real-time PCR based multicolor melting curve analysis (MMCA assay), multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing are conventional methods to diagnose thalassemia but all of them have limitations. In this study, we applied single-molecule real-time (SMRT) sequencing following multiplex long-range PCR to uncover rare mutations in nine patients and their family members. The patients with different results between Gap-PCR and MMCA assay or with phenotype not matching genotype were included. Using SMRT sequencing, we first identified the carriers with αααanti3.7/HKαα, -α762bpα/αα (chr16:172,648-173,409), ααfusion/αQSα (in a trans configuration), two cases with novel gene rearrangements and another case with a novel 341 bp insertion in α-globin gene cluster, respectively. One carrier with --SEA/αααanti4.2, and two carriers with the coexistence of globin variant and an α-globin gene duplication were also found. Most importantly, we could determine two defects in α-globin gene cluster being a cis or trans configuration in a single test. Our results showed that SMRT has great advantages in detection of α-globin gene triplications, rare deletions and determination of a cis or trans configuration. SMRT is a comprehensive and one-step method for thalassemia screening and diagnosis, especially for detection of rare thalassemia mutations.

Third-generation sequencing: A novel tool detects complex variants in the α-thalassemia gene.

OBJECTIVE: Thalassemia is a monogenic disorder with a high carrier rate in the southern region of China. Most laboratories currently follow the protocol of testing hematologic indicators in individuals with positive hematologic indicators and then using the hot-spot mutation test kit. A novel thalassemia gene test is performed if there is a mismatch between the hematology and hot-spot mutation test results. However, due to the large population in southern China, some individuals carry complex α-globin gene cluster (CAGC) variants in NG_000006.1, which are difficult to detect using conventional thalassemia genetic analysis protocols, leading to missed or false genetic test results for individuals carrying these complex α-globin gene cluster variants. When an individual carries a complex α-thalassemia gene variant, and an individual carries a ß- thalassemia gene variant, there may be clinical symptoms that might complicate clinical consultation and prenatal diagnosis if not accurately detected. Third-generation sequencing (TGS) enables long-read single-molecule sequencing with high detection accuracy, and long-length DNA chain reads in high-fidelity reads mode. TGS can be used to analyze high homology and rich GC DNA sequences. RESULTS: Four samples that showed abnormalities in the thalassemia genetic test were studied using TGS, revealing that they carried genotypes with complex α-globin gene cluster variants, one of which was a complex variant αα anti3.7 α anti3.7 α 17.2. CONCLUSIONS: TGS detects complex α-globin gene cluster variants. This study may provide a reference protocol for the use of TGS for the detection of complex α-globin gene cluster variants. TGS can reveal individuals with complex α-thalassemia genotypes in the population and improve the accuracy of genetic counseling and prenatal diagnosis.