Grands réarrangements géniques des gènes CFTR et NF2 (utilisation de la PCR quantitative en temps réel comme technique de confirmation de la MLPA)

LILLE2-BU Santé-Recherche (593502101) / SudocSudocFranceF

Applicability and Efficiency of NGS in Routine Diagnosis: In-Depth Performance Analysis of a Complete Workflow for CFTR Mutation Analysis.

BACKGROUND:Actually, about 2000 sequence variations have been documented in the CFTR gene requiring extensive and multi-step genetic testing in the diagnosis of cystic fibrosis and CFTR-related disorders. We present a two phases study, with validation and performance monitoring, of a single experiment methodology based on multiplex PCR and high throughput sequencing that allows detection of all variants, including large rearrangements, affecting the coding regions plus three deep intronic loci. METHODS:A total of 340 samples, including 257 patients and 83 previously characterized control samples, were sequenced in 17 MiSeq runs and analyzed with two bioinformatic pipelines in routine diagnostic conditions. We obtained 100% coverage for all the target regions in every tested sample. RESULTS:We correctly identified all the 87 known variants in the control samples and successfully confirmed the 62 variants identified among the patients without observing false positive results. Large rearrangements were identified in 18/18 control samples. Only 17 patient samples showed false positive signals (6.6%), 12 of which showed a borderline result for a single amplicon. We also demonstrated the ability of the assay to detect allele specific dropout of amplicons when a sequence variation occurs at a primer binding site thus limiting the risk for false negative results. CONCLUSIONS:We described here the first NGS workflow for CFTR routine analysis that demonstrated equivalent diagnostic performances compared to Sanger sequencing and multiplex ligation-dependent probe amplification. This study illustrates the advantages of NGS in term of scalability, workload reduction and cost-effectiveness in combination with an improvement of the overall data quality due to the simultaneous detection of SNVs and large rearrangements

Axonemal Dynein Intermediate-Chain Gene (DNAI1) Mutations Result in Situs Inversus and Primary Ciliary Dyskinesia (Kartagener Syndrome)

Kartagener syndrome (KS) is a trilogy of symptoms (nasal polyps, bronchiectasis, and situs inversus totalis) that is associated with ultrastructural anomalies of cilia of epithelial cells covering the upper and lower respiratory tracts and spermatozoa flagellae. The axonemal dynein intermediate-chain gene 1 (DNAI1), which has been demonstrated to be responsible for a case of primary ciliary dyskinesia (PCD) without situs inversus, was screened for mutation in a series of 34 patients with KS. We identified compound heterozygous DNAI1 gene defects in three independent patients and in two of their siblings who presented with PCD and situs solitus (i.e., normal position of inner organs). Strikingly, these five patients share one mutant allele (splice defect), which is identical to one of the mutant DNAI1 alleles found in the patient with PCD, reported elsewhere. Finally, this study demonstrates a link between ciliary function and situs determination, since compound mutation heterozygosity in DNAI1 results in PCD with situs solitus or situs inversus (KS)

Novel ADGRG2 truncating variants in patients with X‐linked congenital absence of vas deferens

International audienceBACKGROUND:Congenital absence of vas deferens (CAVD) represents a major cause of obstructive azoospermia and is mainly related to biallelic alteration of the CFTR gene, also involved in cystic fibrosis. Using whole exome sequencing, we recently identified hemizygous loss-of-function mutations in the Adhesion G Protein-coupled Receptor G2 gene (ADGRG2) as responsible of isolated CAVD in the absence of associated unilateral renal agenesis.OBJECTIVES:The objective of this study was to retrospectively perform ADGRG2 sequencing on a large cohort of patients with CAVD, and 0 or only 1 CFTR defective allele identified after comprehensive testing in order to (a) define more precisely the spectrum and the frequency of ADGRG2 mutations within Caucasian population (b) explore the possibility of co-occurrence of CFTR and ADGRG2 mutations.MATERIALS AND METHODS:We collected 53 DNA samples from CAVD patients with 0 (n = 23) or 1 (n = 30) alteration identified after comprehensive CFTR testing in order to perform ADGRG2 sequencing. Twenty patients had normal ultrasonographic renal examination, and renal status was not documented for 33 patients.RESULTS:We identified six new truncating ADGRG2 mutations in 8 patients including two twin brothers: c.251C > G (p.Ser84*), c.1013delC (p.Pro338Hisfs*4), c.1460delG (p.Gly487Alafs*9), c.2096dupT (p.Phe700Ilefs*29), c.2473C > T (p.Arg825*), and c.1731_1839 + 373del (p.Asn578Thrfs*12), which is a 596 base pair deletion affecting the last five bases of exon 21 and the whole exon 22. Five of the eight patients also harbored an heterozygous CFTR mutation which we consider as incidental regarding the high penetrance expected for ADGRG2 truncating variants. The frequency of ADGRG2 truncating mutation was 26% (5/19 unrelated patients) when presence of both kidneys was attested by ultrasonography and 6.1% (2/33) among patients with unknown renal status.DISCUSSION & CONCLUSION:Our results confirm the interest of ADGRG2 sequencing in patients with CAVD not formerly related to CFTR dysfunction, especially in the absence of associated unilateral renal agenesis

Impact of primer trimming on variant calling.

<p>(A) Overestimation of WT allele at variant A locus due to the presence of the primers sequence in the sequencing reads of Amplicon 2 as observed for c.1000C>T (the same principle applies to c.2583del and c.2589_2599del, which are located under the R2 primer of Amplicon 1). When no allele drop-out occurred, variant A is present in 3/12 reads in R1 direction (3/6 from Amplicon1 and 0/6 from Amplicon2) and 3/6 reads in R2 direction for Amplicon1 for a total VAF of 6/18 = 33%. There is a risk of false negative for variant A if the depth of coverage of Amplicon2 is superior to Amplicon1, resulting in a VAF under the detection threshold. In the case of an allele drop-out of Amplicon 2 due to the presence of variant A, then variant A is present in 3/9 reads in R1 direction (3/6 from Amplicon1 and 0/3 from Amplicon2) and 3/6 reads in R2 direction for Amplicon1 for a total VAF of 6/15 = 40%. In this case there is also a risk of false negative if a second variant is associated in cis with variant A (as illustrated by variant B). (B) Impact of primer trimming to limit the risk of false negative results. When primer sequences are removed from the reads, the VAF for variant A is 50% independently of the risk of allele drop-out. In the case of a complex allele involving variant A and B, the risk of false negative result for variant B due to allele drop-out of Amplicon 2 is still present and can be addressed by concomitant CNVs detection followed by Sanger sequencing with a different primer set.</p

CNVs analysis of Phase 2 samples.

<p>RPC_CNV for (1) every amplicon of 3 negative samples verified by MLPA, (2) deleted amplicons for CFTRdele2_3 (n = 14) and CFTRdele1_24 (n = 1) positive controls, (3) apparently deleted amplicons in false positive samples (n = 6 samples for 15 amplicons), (4) duplicated amplicons for CFTRdup4_10 controls (n = 2) and (5) apparently duplicated amplicons in false positive samples (n = 14 samples for 20 amplicons). Three samples showed false positive signals for both deletion and amplification. Dot lines indicate thresholds for deletion (0.7) and duplication (1.3).</p

Depth of coverage analysis for phase 2 samples.

<p>The coverage analysis is based on the number of R1 reads counted for each amplicon with MiSeq Reporter. Values were calculated for the 275 samples tested in phase 2 (CNVs positive controls were excluded). (A) Box plot diagram of the RPC_COV. Box plots show mean (horizontal grey line), 95^th percentile values (box outline) and minimal/maximal values (whiskers). (B) Mean (solid line) and median (dotted line) depth of coverage values by amplicon.</p

Genotypes tested in validation phase (phase 1).

<p>Genotypes tested in validation phase (phase 1).</p

Variants identified in the performance monitoring phase (Phase 2).

<p>Variants identified in the performance monitoring phase (Phase 2).</p

Advanced measurements for in-situ THermo-mechanical monitORing of large sample uNder thermal grAdient (ATHORNA)

International audienc