Detection of Genetic Rearrangements in the Regulators of Complement Activation RCA Cluster by High-Throughput Sequencing and MLPA

García-Fernández, Jesús; Vilches-Arroyo, Susana; Olavarrieta, Leticia; Pérez-Pérez, Julián; Córdoba, Santiago Rodrı́guez de

doi:10.1007/978-1-0716-1016-9_16

Cited by 9 publications

(6 citation statements)

References 36 publications

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“…Computational and Mathematical Methods in Medicine detection, although gene sequencing is recognized as an accurate and sensitive detection method, its experimental requirements are high and costly, and the long cycle time is not suitable for clinical studies, especially epidemiological investigations [17]. The isothermal signal amplification technique is a newly developed molecular biology technique based on the amplification mechanism of circular DNA molecules in nature, which is based on the fact that rolling loops can only be replicated in a single-stranded closed state with the presence of corresponding primers in a homothermal rolling [18]. The isothermal signal amplification technique is highly specific and requires low sample purity, while its branched chain amplification effect can increase the sensitivity of detecting mutant molecules to about 10 copies, making it extremely sensitive [19].…”

Section: Discussionmentioning

confidence: 99%

Application of Isothermal Signal Amplification Technique in the Etiological Diagnosis of Gonorrhea and Drug Resistance Gene Detection

Chen

Zhang

et al. 2022

Computational and Mathematical Methods in Medicine

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Background: Isothermal signal amplification technique is developed based on the rolling ring amplification mechanism of cyclic DNA molecules in nature. This technique plays an extremely beneficial role in gonorrhea pathogen identification and drug resistance gene detection. Aims: This study analyzes the isothermal signal amplification techniques in the etiological diagnosis of gonorrhea and drug resistance gene detection. Materials and Methods: Urethral, cervical secretion, or prostatic fluid samples from 322 cases of gonorrhea collected from January 2018 to December 2021 at the STD clinic of our hospital dermatology department were selected for direct smear examination and gonococcal culture examination; DNA was extracted from urethral, cervical secretion, or prostatic fluid samples and then used for pathogen identification by SAT assay and rolling loop nucleic acid amplification technique, smear examination and pathogen culture examination methods, SAT assay, and isothermal signal amplification technique for comparative sensitivity and specificity analysis. Results: The highest rate of gonorrhea positivity was for the urine rolling loop nucleic acid amplification technique, followed by the swab rolling loop nucleic acid amplification technique, and the lowest rate of gonorrhea positivity was for the urine SAT test. The difference in the positivity rate between the two urine testing methods was statistically significant ( P < 0.05 ). The highest sensitivity of the urine rolling loop nucleic acid amplification technique method for the detection of gonorrhea pathogens and the lowest sensitivity of the urine SAT method were statistically significant ( P < 0.01 ). The differences in sensitivity and specificity between the swab rolling loop nucleic acid amplification technique and the swab SAT method were not statistically significant ( P > 0.05 ). ROC curves were plotted based on sensitivity and specificity, with swab SAT assay ( A U C = 0.998 ) > rolling loop nucleic acid amplification technique ( A U C = 0.981 ). Comparing the negative rates of urine and swab rolling loop nucleic acid amplification technique and urine SAT assay, the differences were not statistically significant ( P > 0.05 ). Conclusion: The isothermal signal amplification technique improves the shortcomings of gonorrhea pathogen identification means and drug resistance gene detection methods, with good detection sensitivity and specificity, simple operation, low price, and easy promotion, which has obvious advantages in clinical applications and epidemiological studies.

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Section: Discussionmentioning

confidence: 99%

Application of Isothermal Signal Amplification Technique in the Etiological Diagnosis of Gonorrhea and Drug Resistance Gene Detection

Chen

Zhang

et al. 2022

Computational and Mathematical Methods in Medicine

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“…C4BPA and ZP3r/C4BPAP1 are members of the RCA cluster, the genes in this locus are largely conserved across even distantly related species, with sequence variation between species being driven by positive selection, indels, and intragenic domain duplications and losses (Garcia-Fernandez, Vilches-Arroyo, Olavarrieta, Perez-Perez, & Rodriguez de Cordoba, 2021; Heinen et al, 2006; Sanchez-Corral, Pardo-Manuel de Villena, Rey-Campos, & Rodriguez de Cordoba, 1993; Wu, Li, & Zhang, 2012). However, some variation in RCA cluster gene content driven by clade-specific duplication or loss events have also been observed (Pardo-Manuel de Villena, 1995; Sanchez-Corral et al, 1993; Wu et al, 2012).…”

Section: Resultsmentioning

confidence: 99%

Recurrent Independent Pseudogenization Events of the Sperm Fertilization Gene ZP3r in Apes and Monkeys

Carlisle

Gurbuz

Swanson

2021

Preprint

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In mice, ZP3r/sp56 is a binding partner to the egg coat protein ZP3 and may mediate induction of the acrosome reaction. ZP3r, as a member of the RCA cluster, is surrounded by paralogs, some of which have been shown to be evolving under positive selection. Sequence divergence paired with paralogous relationships with neighboring genes, has complicated the accurate identification of the human ZP3r ortholog. Here, we phylogenetically and syntenically resolve that the human ortholog of ZP3r is the pseudogene C4BPAP1. We investigate the evolution of this gene within primates. We observe independent pseudogenization events of ZP3r in all Apes with the exception of Orangutans, and many monkey species. ZP3r in both primates that retain ZP3r and rodents contains positively selected sites. We hypothesize that redundant mechanisms mediate ZP3 recognition in mammals and ZP3rs relative importance to ZP recognition varies across species.

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“…Genetic analysis using next-generation sequencing makes it possible to search for nucleotide variants in the whole complement gene set quickly and inexpensively. Nevertheless, some particularities of complement genes should be considered, including the high sequence homology between CFH and CFHRs genes ( 9 ), and additional techniques, such as MLPA, are often required to correctly identify the generation of structural variations in the Regulators of Complement Activation (RCA) gene cluster ( 27 ). Among the variants identified through genetic analyses in aHUS patients, only a subgroup can be defined as pathogenic with certainty, based on either published functional studies or a clear-cut effect on transcription/translation ( 9 , 28 ).…”

Section: Discussionmentioning

confidence: 99%

An ex vivo test to investigate genetic factors conferring susceptibility to atypical haemolytic uremic syndrome

Gastoldi

Aiello²,

Galbusera³

et al. 2023

Front. Immunol.

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IntroductionComprehensive genetic analysis is essential to clinical care of patients with atypical haemolytic uremic syndrome (aHUS) to reinforce diagnosis, and to guide treatment. However, the characterization of complement gene variants remains challenging owing to the complexity of functional studies with mutant proteins. This study was designed: 1) To identify a tool for rapid functional determination of complement gene variants; 2) To uncover inherited complement dysregulation in aHUS patients who do not carry identified gene variants.MethodsTo address the above goals, we employed an ex-vivo assay of serum-induced C5b-9 formation on ADP-activated endothelial cells in 223 subjects from 60 aHUS pedigrees (66 patients and 157 unaffected relatives).ResultsSera taken from all aHUS patients in remission induced more C5b-9 deposition than control sera, independently from the presence of complement gene abnormalities. To avoid the possible confounding effects of chronic complement dysregulation related to aHUS status, and considering the incomplete penetrance for all aHUS-associated genes, we used serum from unaffected relatives. In control studies, 92.7% of unaffected relatives with known pathogenic variants exhibited positive serum-induced C5b-9 formation test, documenting a high sensitivity of the assay to identify functional variants. The test was also specific, indeed it was negative in all non-carrier relatives and in relatives with variants non-segregating with aHUS. All but one variants in aHUS-associated genes predicted in-silico as likely pathogenic or of uncertain significance (VUS) or likely benign resulted as pathogenic in the C5b-9 assay. At variance, variants in putative candidate genes did not exhibit a functional effect, with the exception of a CFHR5 variant. The C5b-9 assay in relatives was helpful in defining the relative functional effect of rare variants in 6 pedigrees in which the proband carried more than one genetic abnormality. Finally, for 12 patients without identified rare variants, the C5b-9 test in parents unmasked a genetic liability inherited from an unaffected parent.DiscussionIn conclusion, the serum-induced C5b-9 formation test in unaffected relatives of aHUS patients may be a tool for rapid functional evaluation of rare complement gene variants. When combined with exome sequencing the assay might be of help in variant selection, to identify new aHUS-associated genetic factors.

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Detection of Genetic Rearrangements in the Regulators of Complement Activation RCA Cluster by High-Throughput Sequencing and MLPA

Cited by 9 publications

References 36 publications

Application of Isothermal Signal Amplification Technique in the Etiological Diagnosis of Gonorrhea and Drug Resistance Gene Detection

Application of Isothermal Signal Amplification Technique in the Etiological Diagnosis of Gonorrhea and Drug Resistance Gene Detection

Recurrent Independent Pseudogenization Events of the Sperm Fertilization Gene ZP3r in Apes and Monkeys

An ex vivo test to investigate genetic factors conferring susceptibility to atypical haemolytic uremic syndrome

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