Long-range PCR Research Articles

Multiple paralogues and recombination mechanisms contribute to the high incidence of 22q11.2 Deletion Syndrome

The 22q11.2 deletion syndrome (22q11.2DS) is the most common microdeletion disorder. Why the incidence of 22q11.2DS is much greater than that of other genomic disorders remains unknown. Short read sequencing cannot resolve the complex segmental duplicon structure to provide direct confirmation of the hypothesis that the rearrangements are caused by nonallelic homologous recombination between the low copy repeats on Chromosome 22 (LCR22s). To enable haplotype-specific assembly and rearrangement mapping in LCR22 regions, we combined fiber-FISH optical mapping with whole genome (ultra-)long read sequencing or rearrangement-specific long-range PCR on 24 duos (22q11.2DS patient and parent-of-origin) comprising several different LCR22-mediated rearrangements. Unexpectedly, we demonstrate that not only different paralogous segmental duplicon but also palindromic AT-rich repeats (PATRR) are driving 22q11.2 rearrangements. In addition, we show the existence of two different inversion polymorphisms preceding rearrangement, and somatic mosaicism. The existence of different recombination sites and mechanisms in paralogues and PATRRs which are copy number expanding in the human population are a likely explanation for the high 22q11.2DS incidence.

Identification and Characterization of Novel FSHR Copy Number Variations Causing Premature Ovarian Insufficiency.

Follicle stimulating hormone (FSH) is a key pituitary gonadotropic hormone implicated in human fertility and is crucial for folliculogenesis and recruitment of new antral follicles. Variations in its receptor, FSHR, can lead to diverse reproductive phenotypes including ovarian hyperstimulation syndrome (OHSS) and premature ovarian insufficiency (POI). This study reports a novel case of FSHR-related ovarian insufficiency in a patient with primary amenorrhea, subnormal AMH levels, and delayed puberty. Genetic exploration revealed two compound heterozygous intragenic deletions of FSHR. Specifically, the patient inherited a maternally derived deletion spanning exons 5-10 and a paternally derived deletion involving exons 3-6. Through chromosomal microarray analysis (CMA), exome sequencing, long-range PCR, and Sanger sequencing, we characterized the breakpoints and confirmed the compound heterozygous deletions. The findings reveal a complete loss of function of both FSHR alleles, contributing to the patient's POI phenotype. This case emphasizes the complexity of genotype-phenotype correlations in FSHR-related disorders and the role of CNVs in POI phenotypes. Although these events are rare, our results advocate for the inclusion of CNV detection in the diagnostic workup of POI to ensure accurate diagnosis and better patient management.

Amplicon-based long-read sequencing is useful for confirming zygosity of DSG2 variants associated with Japanese ARVC

Abstract Background Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiomyopathy mainly caused by desmosomal gene variants. Previously, we reported that DSG2 variants were the most common in 153 Japanese ARVC probands (DSG2 multiple, n=62 and single, n=4; PKP2, n=28; others, n=7). Among the multiple DSG2 variant carriers, 22 probands were compound heterozygous carriers, and 25 were homozygous, which were confirmed by genetic testing for family members (Figure A). On the other hand, 88% of their family members with single DSG2 variant carriers were not diagnosed with ARVC, which meant that recessive DSG2 variants were the major cause of ARVC in Japan. However, zygosities in remaining 15 probands with multiple DSG2 variants were unknown due to the lack of genome data from their parents. Purpose This study aimed to clarify the zygosities of multiple DSG2 variants using amplicon-based long-read sequencing. Methods Genomic DNA was extracted from whole blood using standard protocols. The range containing multiple variants was amplified by long-range PCR. Sequencing libraries were prepared using ONT Ligation Sequencing Kit. Each library was loaded onto a flow cell for sequencing on ONT GridION Mk1 running MinKNOW control software. FASTQ files were aligned to hg38 using minimap2. Variants were called using Clair3 and phased using WhatsHap. Integrative Genomics Viewer (IGV) was used for visualization of the phased variants. Results We successfully phased all the multiple DSG2 variants found in the 15 proband. A typical result of amplicon-based LRS was shown in Figure B. It was IGV screen shot of reads at DSG2 containing c.874C>T (p.R292C) and c.1481A>C (p.D494A). The reads colored in red belonged to one haplotype, while the ones in blue belonged to the other. Each of the two variants was on different color reads, indicating they were compound heterozygous variants. Using LRS, we confirmed all the 15 probands had compound heterozygous variants in DSG2. Overall, the 62 probands (40.5%) with DSG2 variants were recessive variant carriers (Figure A). Conclusions Japanese ARVC was mainly caused by autosomal recessive variants in DSG2. Clarifying the zygosity of causative variants is crucial for the diagnosis in recessive diseases including Japanese ARVC. Amplicon-based LRS was helpful for phasing the multiple DSG2 variants directly without genetic testing of the parents.Figure AFigure B

Targeted long-read sequencing identifies missing pathogenic variant in unsolved 11β-hydroxylase deficiency

Background11β-hydroxylase deficiency (11β-OHD), caused by homozygosity or compound heterozygosity CYP11B1 variants, is the second most common cause of congenital adrenal hyperplasia (CAH). Due to the high degree of sequence identity between CYP11B1 and CYP11B2, chimeric genes, and complex structural variants (SVs), the conventional approach to gene testing for 11β-OHD is facing challenges. The study aimed to clarify the underlying genetic causes of two siblings of a Chinese family with 11β-OHD.MethodsPeripheral blood samples and clinical information were collected from subjects and their family members. Sex steroid concentrations were measured using LC-MS/MS. Long-range PCR-based next-generation sequencing (NGS), PCR assay and target long-read sequencing were used to detect the pathogenic variants.ResultsEarly onset hypertension, increased serum levels of adrenocorticotropin (ACTH), progesterone, testosterone, and decreased cortisol and potassium were detected in both affected siblings. Long-range PCR-based NGS identified a heterozygous missense variant (NM_000497.4:c.281 C > T, p.P94> L) in CYP11B1 gene in the two siblings. PCR detected no chimeric CYP11B2/CYP11B1 gene. We finally identified a second pathogenic variant in CYP11B1 gene via target long-read sequencing (T-LRS). This novel variant was a deletion-insertion variant and located chr8:143957269–143,957,579 (hg19) with the insertion of ‘ACAG’ (NM_000497.4:c.954 + 78_980delinsACAG), which was in trans with CYP11B1: c.281 C > T.ConclusionsOur study suggests that the integrated long-range PCR-based NGS and T-LRS seem to be the most reliable and accurate method for 11β-OHD genetic diagnosis and carrier sequencing.

Clinical characteristics and genetics functional analysis of two children with Spinal muscular atrophy

To explore the characteristics of SMN1 gene variants and carry out functional verification for two children with Spinal muscular atrophy (SMA). Two male children with complicated SMA diagnosed at the Children's Hospital Affiliated to Capital Institute of Pediatrics respectively in July 2021 and April 2022 due to delayed or retrograde motor development were selected as the study subjects. Clinical data of the children were collected. Primary culture of skin fibroblasts was carried out, and peripheral blood samples were collected from both children and their parents. Multiplex ligation-dependent probe amplification, combined long-range PCR and nested PCR, and Sanger sequencing were carried out to detect the copy number and variants of the SMN1 gene. Absolute quantitative real-time PCR, Western blotting and immunofluorescence were used to determine the transcriptional level of the SMN gene, expression of the SMN protein, and the number of functional SMN protein complexes (gems body), respectively. This study was approved by the Children's Hospital Affiliated to Capital Institute of Pediatrics (Ethics No. SHERLLM2021009). Child 1, a 1-year-old boy, was clinically diagnosed with type 1 SMA. Child 2, a 2-and-a-half-year-old boy, was clinically diagnosed with type 3 SMA. Both children were found to harbor a paternally derived SMN1 deletion and a maternally derived SMN1 gene variant, namely c.824G>T (p.Gly275Val) and c.884A>T (p.*295Leu). Compared with the normal controls and carriers, the levels of full-length SMN1 transcripts in their peripheral blood and skin fibroblast cell lines were significantly decreased (P < 0.05), and the levels of SMN protein normalized to that of β-actin, and the numbers of gems bodies in the primary fibroblast cells were also significantly lower (P < 0.05). Based on the guidelines from the American College of Medical Genetics and Genomics, both variants were classified as likely pathogenic (PS3+PM3+PM5+PP3; PS3+PM3+PM4+PP3). Following the diagnosis, both children had received nusinersen treatment. Although their motor function was improved, child 1 still died at the age of 2 due to severe pulmonary infection. The walking ability of child 2 was significantly improved, and his prognosis appeared to be good. Two cases of clinically complicated SMA have been confirmed by genetic testing and experimental studies, which has provided a reference for their accurate treatment.

7030 Molecular Analysis Of Congenital Adrenal Hyperplasia-X Syndrome And Development Of A Pilot Panel For Analyzing Structural Variations With Long-Read Sequencing

Abstract Disclosure: J. Kim: None. S. Park: None. J. Yoon: None. D. Kim: None. S. Hwang: None. G. Kim: None. J. Kim: None. J. Choi: None. H. Yoo: None. Purpose: Genomic recombination events between the CYP21A2 and CYP21A1P genes are frequent due to their high sequence homology. Deletions in both the CYP21A2 and TNXB genes result in a chimeric TNXA/TNXB gene, leading to congenital adrenal hyperplasia (CAH)-X syndrome, characterized by a hypermobile form of Ehlers-Danlos syndrome. The aim of study was to determine the frequency of CAH-X syndrome and to develop a pilot panel for structural variation analysis using long-read sequencing technology. Methods: Among the 165 unrelated families with confirmed genetic mutations in the CYP21A2 gene, 44 probands had large deletions in one or both alleles. Long-range PCR was performed in 12 patients out of 44 probands using a forward primer for the 5’-UTR of the CYP21A2 and CYP21A1P and a reverse primer specific for exon 32 of TNXB. The amplified PCR products were then subjected to Sanger sequencing. We constructed a custom panel of 140 kb containing the RCCX modules arranged as STK19-C4A-CYP21A1P-TNXA-STK19B-C4B-CYP21A2-TNXB. In 4 patients with deletions in both the CYP21A2 and TNXB genes, long-read sequencing was performed using the custom panel on the PacBio Sequel II platform. Results: Out of 12 probands, eight harbored TNXA/TNXB chimeras. One patient was homozygous for CAH-X CH1 with a 120 bp deletion in exon 35 and had a Beighton score of 6 for joint hypermobility and multiple diverticula in the ascending colon. Two patients were heterozygous for CAH-X CH1, while three were heterozygous for the CAH-X CH2 with an intact exon 35 and the c.P4058W in exon 40. Two patients carried heterozygous mutations p.S4175N in exon 43, which is an unclassified type. Using long-read sequencing with a custom panel, missense mutations and TNXA/TNXB chimeras were detected in four patients previously identified by conventional direct sequencing and MLPA. Conclusions: This study identified 8 patients with CAH-X syndrome carrying four different TNXA/TNXB chimeras by long-range PCR. Long-read sequencing emerged as a comprehensive and efficient method for the molecular analysis of CAH, especially for the identification of structural variations. Biallelic deletion of TNXB was associated with severe clinical manifestations. Therefore, screening for CAH-X is essential for clinical management and prevention of the long-term consequences of CAH-X syndrome. Presentation: 6/1/2024

9266 Molecular Analysis Of Congenital Adrenal Hyperplasia-X Syndrome And Development Of A Pilot Panel For Analyzing Structural Variations With Long-Read Sequencing

Abstract Disclosure: J. Kim: None. S. Park: None. J. Yoon: None. D. Kim: None. S. Hwang: None. G. Kim: None. J. Kim: None. J. Choi: None. H. Yoo: None. Purpose: Genomic recombination events between the CYP21A2 and CYP21A1P genes are frequent due to their high sequence homology. Deletions in both the CYP21A2 and TNXB genes result in a chimeric TNXA/TNXB gene, leading to congenital adrenal hyperplasia (CAH)-X syndrome, characterized by a hypermobile form of Ehlers-Danlos syndrome. The aim of study was to determine the frequency of CAH-X syndrome and to develop a pilot panel for structural variation analysis using long-read sequencing technology. Methods: Among the 165 unrelated families with confirmed genetic mutations in the CYP21A2 gene, 44 probands had large deletions in one or both alleles. Long-range PCR was performed in 12 patients out of 44 probands using a forward primer for the 5’-UTR of the CYP21A2 and CYP21A1P and a reverse primer specific for exon 32 of TNXB. The amplified PCR products were then subjected to Sanger sequencing. We constructed a custom panel of 140 kb containing the RCCX modules arranged as STK19-C4A-CYP21A1P-TNXA-STK19B-C4B-CYP21A2-TNXB. In 4 patients with deletions in both the CYP21A2 and TNXB genes, long-read sequencing was performed using the custom panel on the PacBio Sequel II platform. Results: Out of 12 probands, eight harbored TNXA/TNXB chimeras. One patient was homozygous for CAH-X CH1 with a 120 bp deletion in exon 35 and had a Beighton score of 6 for joint hypermobility and multiple diverticula in the ascending colon. Two patients were heterozygous for CAH-X CH1, while three were heterozygous for the CAH-X CH2 with an intact exon 35 and the c.P4058W in exon 40. Two patients carried heterozygous mutations p.S4175N in exon 43, which is an unclassified type. Using long-read sequencing with a custom panel, missense mutations and TNXA/TNXB chimeras were detected in four patients previously identified by conventional direct sequencing and MLPA. Conclusions: This study identified 8 patients with CAH-X syndrome carrying four different TNXA/TNXB chimeras by long-range PCR. Long-read sequencing emerged as a comprehensive and efficient method for the molecular analysis of CAH, especially for the identification of structural variations. Biallelic deletion of TNXB was associated with severe clinical manifestations. Therefore, screening for CAH-X is essential for clinical management and prevention of the long-term consequences of CAH-X syndrome. Presentation: 6/1/2024

Single-Molecule Sequencing of the C9orf72 Repeat Expansion in Patient iPSCs.

A hexanucleotide GGGGCC repeat expansion in the C9orf72 gene is the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD). C9orf72 repeat expansions are currently identified with long-range PCR or Southern blot for clinical and research purposes, but these methods lack accuracy and sensitivity. The GC-rich and repetitive content of the region cannot be amplified by PCR, which leads traditional sequencing approaches to fail. We turned instead to PacBio single-molecule sequencing to detect and size the C9orf72 repeat expansion without amplification. We isolated high molecular weight genomic DNA from patient-derived iPSCs of varying repeat lengths and then excised the region containing the C9orf72 repeat expansion from naked DNA with a CRISPR/Cas9 system. We added adapters to the cut ends, capturing the target region for sequencing on PacBio's Sequel, Sequel II, or Sequel IIe. This approach enriches the C9orf72 repeat region without amplification and allows the repeat expansion to be consistently and accurately sized, even for repeats in the thousands. Key features • This protocol is adapted from PacBio's previous "no-amp targeted sequencing utilizing the CRISPR-Cas9 system." • Optimized for sizing C9orf72 repeat expansions in patient-derived iPSCs and applicable to DNA from any cell type, blood, or tissue. • Requires high molecular weight naked DNA. • Compatible with Sequel I and II but not Revio.

Identification and characterisation of pathogenic and non-pathogenic FGF14 repeat expansions

Repeat expansions in FGF14 cause autosomal dominant late-onset cerebellar ataxia (SCA27B) with estimated pathogenic thresholds of 250 (incomplete penetrance) and 300 AAG repeats (full penetrance), but the sequence of pathogenic and non-pathogenic expansions remains unexplored. Here, we demonstrate that STRling and ExpansionHunter accurately detect FGF14 expansions from short-read genome data using outlier approaches. By combining long-range PCR and nanopore sequencing in 169 patients with cerebellar ataxia and 802 controls, we compare FGF14 expansion alleles, including interruptions and flanking regions. Uninterrupted AAG expansions are significantly enriched in patients with ataxia from a lower threshold (180–200 repeats) than previously reported based on expansion size alone. Conversely, AAGGAG hexameric expansions are equally frequent in patients and controls. Distinct 5’ flanking regions, interruptions and pre-repeat sequences correlate with repeat size. Furthermore, pure AAG (pathogenic) and AAGGAG (non-pathogenic) repeats form different secondary structures. Regardless of expansion size, SCA27B is a recognizable clinical entity characterized by frequent episodic ataxia and downbeat nystagmus, similar to the presentation observed in a family with a previously unreported nonsense variant (SCA27A). Overall, this study suggests that SCA27B is a major overlooked cause of adult-onset ataxia, accounting for 23–31% of unsolved patients. We strongly recommend re-evaluating pathogenic thresholds and integrating expansion sequencing into the molecular diagnostic process.

Clinical validation and application of targeted long-range polymerase chain reaction and long-read sequencing–based analysis for hemophilia: experience from a hemophilia treatment center in China

BackgroundTargeted long-read sequencing (LRS) is expected to comprehensively analyze diverse complex variants in hemophilia A (HA) and hemophilia B (HB) caused by the F8 and F9 genes, respectively. However, its clinical applicability still requires extensive validation. ObjectivesTo evaluate the clinical applicability of targeted LRS-based analysis compared with routine polymerase chain reaction (PCR)–based methods. MethodsGene variants of retrieved subjects were retrospectively and prospectively analyzed. Whole-genome sequencing was performed to further analyze undiagnosed cases. Breakpoints of novel genomic rearrangements were mapped and validated using long-distance PCR and long-range PCR combined with sequencing. ResultsIn total, 122 subjects were retrieved. In retrospective analysis of the 90 HA cases, HA-LRS assay showed consistent results in 84 cases compared with routine methods and characterized 6 large deletions with their exact breakpoints confirmed by further validation in 6 cases (routine methods only presented failure in amplifying the involved exons). In prospective analysis of the 21 HA subjects, 20 variants of F8 were identified in 20 cases. For the remaining HA patient, no duplication/deletion or single-nucleotide variant (SNV)/insertion and deletion (InDel) was found, but a potential recombination involving exons 14 and 21 of F8 was observed by LRS. Whole-genome sequencing analysis and further verification defined a 30 478 base pairs (bp) tandem repeat involving exons 14 to 21 of F8. Among the 11 HB patients, HB-LRS analysis detected 11 SNVs/InDels in F9, consistent with routine methods. ConclusionTargeted LRS-based analysis was efficient and comprehensive in identifying SNVs/InDels and genomic rearrangements of hemophilia genes, especially when we first expanded the panel to include F9. However, further investigation for complex gross rearrangement is still essential.

A novel dual CRISPR-Cas assay for detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in penaeid shrimp without false positives from its endogenous viral elements (EVEs)

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a single-stranded DNA virus posing significant economic challenges in shrimp aquaculture. Depending on the shrimp species, IHHNV infections can lead to mass mortalities, deformities, or growth retardation, all of which impact production output. Timely identification of IHHNV, particularly using nucleic acid detection methods, is paramount for effective prevention. However, PCR and methods relying on nucleic acid probes face challenges due to the presence of chromosome-integrated, endogenous viral elements (EVEs) of IHHNV sharing identical sequences to their infectious counterparts. While long-range PCR assays, such as LongAmp PCR (LA-PCR), have shown promise in minimizing false positive results, their applicability in the field is limited by their long turnaround times and reliance on costly equipment. To address these limitations, we have developed a rapid and potentially field-deployable platform termed Cas9-RPA-Cas12a (“CRPAC”) capable of differentiating the infectious form of IHHNV from EVEs, capitalizing on the fact that the genome of IHHNV is single-stranded, while EVEs are double-stranded DNA. This method employs Cas9 endonuclease to deplete EVEs, thereby enriching for single-stranded genomic DNA. This enriched DNA is then subjected to recombinase polymerase amplification (RPA) coupled with the Cas12a diagnostic assay. Here, we show that CRPAC gives positive results exclusively for infectious IHHNV, appearing as strong fluorescence or a distinct pattern on lateral flow strips that facilitates naked-eye evaluation. In field sample testing, CRPAC demonstrated excellent agreement with LA-PCR, identifying 8 out of 16 samples containing infectious IHHNV, while the WOAH-recommended PCR assays gave false positive results to all samples containing EVEs. Collectively, our findings underscore the utility of CRPAC as a robust and field-adaptable tool for distinguishing between infectious IHHNV and EVEs, irrespective of their nucleotide sequence similarities.

Implementation of long-read sequencing for routine molecular diagnosis of familial mediterranean fever

BackgroundLong-read sequencing technology, widely used in research, is proving useful in clinical diagnosis, especially for infectious diseases. Despite recent advances, it hasn't been routinely applied to constitutional human diseases. Long-read sequencing detects intronic variants and phases variants, crucial for identifying recessive diseases. MethodsWe integrated long-read sequencing into the clinical diagnostic workflow for the MEFV gene, responsible for familial Mediterranean fever (FMF), using a Nanopore-based workflow. This involved long-range PCR amplification, native barcoding kit library preparation, GridION sequencing, and in-house bioinformatics. We compared this new workflow against our validated method using 39 patient samples and 3 samples from an external quality assessment scheme to ensure compliance with ISO15189 standards. ResultsOur evaluation demonstrated excellent performance, meeting ISO15189 requirements for reproducibility, repeatability, sensitivity, and specificity. Since October 2022, 150 patient samples were successfully analyzed with no failures. Among these samples, we identified 13 heterozygous carriers of likely pathogenic (LP) or pathogenic (P) variants, 1 patient with a homozygous LP/P variant in MEFV, and 4 patients with compound heterozygous variants. ConclusionThis study represents the first integration of long-read sequencing for FMF clinical diagnosis, achieving 100 % sensitivity and specificity. Our findings highlight its potential to identify pathogenic variants without parental segregation analysis, offering faster, cost-effective, and accurate clinical diagnosis. This successful implementation lays the groundwork for future applications in other constitutional human diseases, advancing precision medicine.

The application value of whole exome sequencing technology in diagnosis of hereditary renal cysts

The data of 57 renal cyst patients who visited the First Affiliated Hospital of Zhengzhou University from January 2023 to March 2024 were retrospectively analyzed. The age of patients ranged from three months to 60 years old, with 31 males and 26 females. The whole exome sequencing (WES) detected pathogenic or suspected pathogenic (P/LP) variants in 48 renal cystic probands, with a detection rate of 84.2% (48/57), including PKD1, PKD2, PKHD1, LRP5, COL4A4 and ALG8 gene variants as well as copy number variations (CNV). In addition, four PKD1 gene variants of uncertain significance (VUS) were detected. In five WES negative families, one PKD1 nonsense variation was detected through long-range PCR (LR-PCR)+Oxford nanopore technologies, and one heterozygous deletion in exon 22 of PKD1 gene was detected through multiplex ligation-dependent probe amplification (MLPA). In summary, WES can detect multiple types of variations, which is helpful for early diagnosis and prognosis prediction of renal cyst patients. However, there is still a risk of failing to detect PKD1 gene by WES, therefore, healthcare practitioners should beware of the negative results of WES.

Detection of hemophilia A genetic variants using third-generation long-read sequencing

BackgroundHemophilia A (HA) is an X-linked recessive genetic disorder caused by pathogenic variations of the factor VIII −encoding gene, F8 gene. Due to the large size and diverse types of variations in the F8 gene, causative mutations in F8 cannot be simultaneously detected in one step by traditional molecular analysis, and genetic molecular diagnosis and prenatal screening of HA still face significant difficulties and challenges in clinical practice. Therefore, we aimed to develop and validate an efficient, accurate, and time-saving method for the genetic detection of HA. MethodsA comprehensive analysis of hemophilia A (CAHEA) method based on long-range PCR and long-read sequencing (LRS) was used to detect F8 gene mutations in 14 clinical HA samples. The LRS results were compared with those of the conventional methods to evaluate the accuracy and sensitivity of the proposed approach. ResultsThe CAHEA method successfully identified 14 F8 variants in all probands, including 3 small insertion deletions, 4 single nucleotide variants, and 7 intron 22 inversions in a “one-step” manner, of which 2 small deletions have not been reported previously. Moreover, this method provided an opportunity to analyze the mechanism of rearrangement and the pathogenicity of F8 variants. The LRS results were validated and found to be in 100% agreement with those obtained using the conventional method. ConclusionOur proposed LRS-based F8 gene detection method is an accurate and reproducible genetic screening and diagnostic method with significant clinical value. It provides efficient, comprehensive, and accurate genetic screening and diagnostic services for individuals at high risk of HA as well as for premarital and prenatal populations.

Evaluation of pathogenic variants detected in high homology regions of the PMS2 gene. How effective is long-range PCR?

Lynch syndrome (LS) is an inherited cancer predisposition syndrome characterized by a high risk of colorectal and extracolonic tumors. Germline pathogenic variants (GPV) in the PMS2 gene are associated with <15% of all cases. The PMS2CL pseudogene presents high homology with PMS2, challenging molecular diagnosis by next-generation sequencing (NGS). Due to the high methodological complexity required to distinguish variants between PMS2 and PMS2CL, most laboratories do not clearly report the origin of this molecular finding. The aim of this study was to confirm the GPVs detected by NGS in regions of high homology segments of the PMS2 gene in a Brazilian sample. An orthogonal and gold standard long-range PCR (LR-PCR) methodology to separate variants detected in the PMS2 gene from those detected in the pseudogene. A total of 74 samples with a PMS2 GPV detected by NGS in exons with high homology with PMS2CL pseudogene were evaluated. The most common was NM_000535.6:c.2182_2184delinsG, which was previously described as deleterious mutation in a study of African-American patients with LS and has been widely reported by laboratories as a pathogenic variant associated with the LS phenotype. Of all GPVs identified, only 6.8% were confirmed by LR-PCR. Conversely, more than 90% of GPV were not confirmed after LR-PCR, and the diagnosis of LS was ruled out by molecular mechanisms associated with PMS2. In conclusion, the use of LR-PCR was demonstrated to be a reliable approach for accurate molecular analysis of PMS2 variants in segments with high homology with PMS2CL. We highlight that our laboratory is a pioneer in routine diagnostic complementation of the PMS2 gene in Brazil, directly contributing to a more assertive molecular diagnosis and adequate genetic counseling for these patients and their families.

Analytical validation of the amplification refractory mutation system polymerase chain reaction-capillary electrophoresis assay to diagnose spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by homozygous deletion and compound heterozygous mutations in survival motor neuron 1 (SMN1), with severity tied to the copy number of survival motor neuron 2 (SMN2). This study aimed to develop a rapid and comprehensive method for the diagnosis of SMA. A total of 292 children with clinically suspected SMA and 394 family members were detected by the amplification refractory mutation system polymerase chain reaction-capillary electrophoresis (ARMS-PCR-CE) method, which targeted 19 reported mutations, and the results were compared with those in multiplex ligation-dependent probeamplification (MLPA). Individuals with identified point mutations were further confirmed by SMN1 long-range PCR and Sanger sequencing. A total of 202 children with SMA, 272 carriers, and 212 normal individuals were identified in this study. Nodifference was found in the R-value distribution of exons 7 and 8 in SMN1 and SMN2 among these cohorts, with coefficients of variation consistently below 0.08. To detect exon 7 and 8 copy numbers in SMN1 and SMN2, the ARMS-PCR-CE results were concordant with those of MLPA. Approximately 4.95 % (10/202) of the study patients had compound heterozygous mutations. The ARMS-PCR-CE assay is a comprehensive, rapid, and accurate diagnostic method for SMA that simultaneously detects copy numbers of exons 7 and 8 in SMN1/SMN2, as well as 19 point mutations in SMN1 and 2 enhancers in SMN2. This approach can effectively reduce thetime frame for diagnosis, facilitating early intervention and preventing birth defects.

RFC1 Repeat Distribution in the Cypriot Population: Study of a Large Cohort of Patients With Undiagnosed Ataxia and Non-Disease Controls.

The intronic biallelic AAGGG expansion in the replication factor C subunit 1 (RFC1) gene was recently associated with a phenotype combining cerebellar ataxia, neuropathy, and vestibular areflexia syndrome, as well as with late-onset ataxia. Following this discovery, studies in multiple populations extended the phenotypic and genotypic spectrum of this locus. Multiple benign and additional pathogenic configurations are currently known. Our main objectives were to study the prevalence of the pathogenic AAGGG expansion in the Cypriot population, to further characterize the RFC1 repeat locus allele distribution, and to search for possible novel repeat configurations. Cypriot undiagnosed patients, in the majority presenting at least with cerebellar ataxia and non-neurologic disease controls, were included in this study. A combination of conventional methods was used, including standard PCR flanking the repeat region, repeat-primed PCR, long-range PCR, and Sanger sequencing. Bioinformatics analysis of already available in-house short-read whole-genome sequencing data was also performed. A large group of undiagnosed patients (n = 194), mainly presenting with pure ataxia or with ataxia accompanied by neuropathy or additional symptoms, as well as a group of non-disease controls (n = 100), were investigated in the current study. Our findings include the diagnosis of 10 patients homozygous for the pathogenic AAGGG expansion and a high percentage of heterozygous AAGGG carriers in both groups. The benign AAAAGn, AAAGGn, and AAGAGn configurations were also identified in our cohorts. We also report and discuss the identification of 2 recently reported novel and possibly benign repeat configurations, AAAGGGn and AAGACn, thus confirming their existence in another distinct population, and we highlight an increased frequency of the AAAGGGn in the patient group, including a single case of homozygosity. Our findings indicate the existence of genetic heterogeneity regarding the RFC1 repeat configurations and that the AAGGG pathogenic expansion is a frequent cause of ataxia in the Cypriot population.

SPAST Intragenic CNVs Lead to Hereditary Spastic Paraplegia via a Haploinsufficiency Mechanism.

The most common form of hereditary spastic paraplegia (HSP), SPG4 is caused by single nucleotide variants and microrearrangements in the SPAST gene. The high percentage of multi-exonic deletions or duplications observed in SPG4 patients is predisposed by the presence of a high frequency of Alu sequences in the gene sequence. In the present study, we analyzed DNA and RNA samples collected from patients with different microrearrangements in SPAST to map gene breakpoints and evaluate the mutation mechanism. The study group consisted of 69 individuals, including 50 SPG4 patients and 19 healthy relatives from 18 families. Affected family members from 17 families carried varying ranges of microrearrangements in the SPAST gene, while one individual had a single nucleotide variant in the 5'UTR of SPAST. To detect the breakpoints of the SPAST gene, long-range PCR followed by sequencing was performed. The breakpoint sequence was detected for five different intragenic SPAST deletions and one duplication, revealing Alu-mediated microhomology at breakpoint junctions resulting from non-allelic homologous recombination in these patients. Furthermore, SPAST gene expression analysis was performed using patient RNA samples extracted from whole blood. Quantitative real-time PCR tests performed in 14 patients suggest no expression of transcripts with microrearrangements in 5 of them. The obtained data indicate that nonsense-mediated decay degradation is not the only mechanism of hereditary spastic paraplegia in patients with SPAST microrearrangements.

Whole genome analysis of Human Mastadenovirus D causing Keratoconjunctivitis in India - A multicentre study.

Human mastadenovirus (HAdV) types 8, 37, 64 have been considered the major contributors in Epidemic keratoconjunctivitis (EKC) epidemics, but recent surveillance data have shown the involvement of emerging recombinants, including HAdV-53, HAdV-54, and HAdV-56. In our initial work, positive samples for adenovirus revealed that our strains were closer to HAdV-54 than HAdV-8. Hence, the current study aimed to use whole genome technology to identify the HAdV strain correctly. Oxford Nanopore technique was used, wherein a Targeted sequencing approach using long-range PCR amplification was performed. Primers were designed using HAdV-54 (AB448770.2) and HAdV-8 (AB897885.1) as reference sequences. Amplicons were sequenced on the GridION sequencer. Sequences were annotated using Gatu software, and similarities with standard reference sequence was calculated using Bioedit software. The phylogenetic tree was built after alignment in MEGA v7.0 using Neighbour joining method for each of the genes: Penton, Hexon, and Fiber. The effect of novel amino acid changes was evaluated using the PROVEAN tool. The Recombination Detection Program (RDP) package Beta 4.1 was used to identify recombinant sequences. Of the five samples sequenced, OL450401, OL540403, and OL540406 showed nucleotide similarity to HAdV-54 in the penton region. Additionally, OL450401 showed a statistically significant recombination event with HAdV-54 as minor and HAdV-8 as major parents. This was further supported by phylogenetic analysis as well. In the present study, we have found evidence of a shift from HAdV-8 towards HAdV-54, thus stressing the need for surveillance of HAdVs and to stay updated on the rise of new recombinants.

An Amplicon-Based Application for the Whole-Genome Sequencing of GI-19 Lineage Infectious Bronchitis Virus Directly from Clinical Samples.

Infectious bronchitis virus (IBV) causes a highly contagious respiratory disease in chickens, leading to significant economic losses in the poultry industry worldwide. IBV exhibits a high mutation rate, resulting in the continuous emergence of new variants and strains. A complete genome analysis of IBV is crucial for understanding its characteristics. However, it is challenging to obtain whole-genome sequences from IBV-infected clinical samples due to the low abundance of IBV relative to the host genome. Here, we present a novel approach employing next-generation sequencing (NGS) to directly sequence the complete genome of IBV. Through in silico analysis, six primer pairs were designed to match various genotypes, including the GI-19 lineage of IBV. The primer sets successfully amplified six overlapping fragments by long-range PCR and the size of the amplicons ranged from 3.7 to 6.4 kb, resulting in full coverage of the IBV genome. Furthermore, utilizing Illumina sequencing, we obtained the complete genome sequences of two strains belonging to the GI-19 lineage (QX genotype) from clinical samples, with 100% coverage rates, over 1000 × mean depth coverage, and a high percentage of mapped reads to the reference genomes (96.63% and 97.66%). The reported method significantly improves the whole-genome sequencing of IBVs from clinical samples; thus, it can improve understanding of the epidemiology and evolution of IBVs.