Accuracy of saliva and nasopharyngeal sampling for detection of SARS-CoV-2 in community screening: a multicentric cohort study.

Nasopharyngeal sampling for nucleic acid amplification testing (NAAT) is the standard diagnostic test of coronavirus disease 2019. Our objectives were to assess, in real-life conditions, the diagnostic accuracy of a nasopharyngeal point-of-care antigen (Ag) test and of saliva NAAT for detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in ambulatory care. This was a prospective cohort study from 19 October through 18 December 2020 in two community COVID-19 screening centers in Paris, France. Two nasopharyngeal swabs and one saliva sample were simultaneously collected. Diagnostic accuracies of nasopharyngeal Ag testing and of three saliva NAAT methods were assessed as compared to nasopharyngeal NAAT. A total of 1452 ambulatory children and adults were included. Overall, 129/1443 (9%) participants tested positive on nasopharyngeal NAAT (102/564 [18%] in symptomatic and 27/879 [3%] in asymptomatic participants). Sensitivity was 94%, 23%, 96%, and 94% for the three different protocols of saliva NAAT and for the nasopharyngeal Ag test, respectively. Estimates of specificity were above 95% for all methods. Diagnostic accuracy was similar in symptomatic and asymptomatic individuals. Diagnostic accuracy of nasopharyngeal Ag testing and of saliva NAAT is similar to that of nasopharyngeal NAAT, subject to compliance with specific protocols for saliva. Registration number: NCT04578509.

Loss-of-function mutations in SOX10 cause Kallmann syndrome with deafness.

Transcription factor SOX10 plays a role in the maintenance of progenitor cell multipotency, lineage specification, and cell differentiation and is a major actor in the development of the neural crest. It has been implicated in Waardenburg syndrome (WS), a rare disorder characterized by the association between pigmentation abnormalities and deafness, but SOX10 mutations cause a variable phenotype that spreads over the initial limits of the syndrome definition. On the basis of recent findings of olfactory-bulb agenesis in WS individuals, we suspected SOX10 was also involved in Kallmann syndrome (KS). KS is defined by the association between anosmia and hypogonadotropic hypogonadism due to incomplete migration of neuroendocrine gonadotropin-releasing hormone (GnRH) cells along the olfactory, vomeronasal, and terminal nerves. Mutations in any of the nine genes identified to date account for only 30% of the KS cases. KS can be either isolated or associated with a variety of other symptoms, including deafness. This study reports SOX10 loss-of-function mutations in approximately one-third of KS individuals with deafness, indicating a substantial involvement in this clinical condition. Study of SOX10-null mutant mice revealed a developmental role of SOX10 in a subpopulation of glial cells called olfactory ensheathing cells. These mice indeed showed an almost complete absence of these cells along the olfactory nerve pathway, as well as defasciculation and misrouting of the nerve fibers, impaired migration of GnRH cells, and disorganization of the olfactory nerve layer of the olfactory bulbs.

SEMA3A, a gene involved in axonal pathfinding, is mutated in patients with Kallmann syndrome.

Kallmann syndrome (KS) associates congenital hypogonadism due to gonadotropin-releasing hormone (GnRH) deficiency and anosmia. The genetics of KS involves various modes of transmission, including oligogenic inheritance. Here, we report that Nrp1(sema/sema) mutant mice that lack a functional semaphorin-binding domain in neuropilin-1, an obligatory coreceptor of semaphorin-3A, have a KS-like phenotype. Pathohistological analysis of these mice indeed showed abnormal development of the peripheral olfactory system and defective embryonic migration of the neuroendocrine GnRH cells to the basal forebrain, which results in increased mortality of newborn mice and reduced fertility in adults. We thus screened 386 KS patients for the presence of mutations in SEMA3A (by Sanger sequencing of all 17 coding exons and flanking splice sites) and identified nonsynonymous mutations in 24 patients, specifically, a frameshifting small deletion (D538fsX31) and seven different missense mutations (R66W, N153S, I400V, V435I, T688A, R730Q, R733H). All the mutations were found in heterozygous state. Seven mutations resulted in impaired secretion of semaphorin-3A by transfected COS-7 cells (D538fsX31, R66W, V435I) or reduced signaling activity of the secreted protein in the GN11 cell line derived from embryonic GnRH cells (N153S, I400V, T688A, R733H), which strongly suggests that these mutations have a pathogenic effect. Notably, mutations in other KS genes had already been identified, in heterozygous state, in five of these patients. Our findings indicate that semaphorin-3A signaling insufficiency contributes to the pathogenesis of KS and further substantiate the oligogenic pattern of inheritance in this developmental disorder.

Evaluation of a saliva molecular point of care for the detection of SARS-CoV-2 in ambulatory care.

Rapid identification of SARS-CoV-2-infected individuals is a cornerstone for the control of virus spread. The sensitivity of SARS-CoV-2 RNA detection by RT-PCR is similar in saliva and nasopharyngeal swabs. Rapid molecular point-of-care tests in saliva could facilitate, broaden and speed up the diagnosis. We conducted a prospective study in two community COVID-19 screening centers to evaluate the performances of a CE-marked RT-LAMP assay (EasyCoV) designed for the detection of SARS-CoV2 RNA from fresh saliva samples, compared to nasopharyngeal RT-PCR, to saliva RT-PCR and to nasopharyngeal antigen testing. Overall, 117 of the 1718 participants (7%) tested positive with nasopharyngeal RT-PCR. Compared to nasopharyngeal RT-PCR, the sensitivity and specificity of the RT-LAMP assay in saliva were 34% and 97%, respectively. The Ct values of nasopharyngeal RT-PCR were significantly lower in the 40 true positive subjects with saliva RT-LAMP (Ct 25.9) than in the 48 false negative subjects with saliva RT-LAMP (Ct 28.4) (p = 0.028). Considering six alternate criteria for reference tests, including saliva RT-PCR and nasopharyngeal antigen, the sensitivity of saliva RT-LAMP ranged between 27 and 44%. The detection of SARS-CoV-2 in crude saliva samples with an RT-LAMP assay had a lower sensitivity than nasopharyngeal RT-PCR, saliva RT-PCR and nasopharyngeal antigen testing.Registration number: NCT04578509.

Biallelic mutations in the prokineticin-2 gene in two sporadic cases of Kallmann syndrome.

Kallmann syndrome is a developmental disease that combines hypogonadotropic hypogonadism and anosmia. Putative loss-of-function mutations in PROKR2 or PROK2, encoding prokineticin receptor-2 (a G protein-coupled receptor), and one of its ligands, prokineticin-2, respectively, have recently been reported in approximately 10% of Kallmann syndrome affected individuals. Notably, given PROKR2 mutations were found in the heterozygous, homozygous, or compound heterozygous state in patients, thus raising the question of a possible digenic inheritance of the disease in heterozygous patients. Indeed, one of these patients was also carrying a missense mutation in KAL1, the gene responsible for the X chromosome-linked form of Kallmann syndrome. Mutations in PROK2, however, have so far been found only in the heterozygous state. Here, we report on the identification of PROK2 biallelic mutations, that is, a missense mutation, p.R73C, and a frameshift mutation, c.163delA, in two out of 273 patients presenting as sporadic cases. We conclude that PROK2 mutations in the homozygous state account for a few cases of Kallmann syndrome. Moreover, since the same R73C mutation was previously reported in the heterozygous state, and because Prok2 knockout mice exhibit an abnormal phenotype only in the homozygous condition, we predict that patients carrying monoallelic mutations in PROK2 have another disease-causing mutation, presumably in still undiscovered Kallmann syndrome genes.

ENaCbeta and gamma genes as modifier genes in cystic fibrosis.

BACKGROUND: Clinical phenotype varies among cystic fibrosis (CF) patients with identical CF transmembrane conductance regulator (CFTR)genotype, suggesting that genetic modifiers exist. Transgenic mice that overexpress SCNN1beta present CF-like lung disease symptoms. Mutations or variants in SCNN1beta may therefore potentially modulate the clinical phenotype in CF patients. METHODS: We analysed by DHPLC SCNN1beta and SCNN1gamma genes in 56 patients with classical CF. Patients were classified into two groups according to their CFTR genotype and their severity: 38 patients with severe genotype and an unexpectedly mild lung phenotype, and 18 patients with mild genotype and a severe lung phenotype. RESULTS: We found 3 patients out of 56 carrying at least one missense mutation. Two were novel (p.Thr313Met in SCNN1beta, p.Leu481Gln in SCNN1gamma) and two were previously described (p.Gly589Ser in SCNN1beta and p.Val546Ileu in SCNNgamma). p.Thr313Met has been identified in a CF patient with mild genotype and severe lung phenotype suggesting that it could act in increasing ENaC activity. The three other variants have been identified in CF patients with severe genotype and mild lung phenotype suggesting that they might decrease ENaC activity. However, the function of ENaC in the nasal epithelia of these patients, evaluated by nasal potential difference measurements, did not support the fact that these variants were functional, at least in nasal epithelium. CONCLUSION: Our results suggest that genetic variants in ENaCbeta and gamma genes do not modulate disease severity in the majority of CF patients.

Phenotypic Variability in a Family with Acrodysostosis Type 2 Caused by a Novel PDE4D Mutation Affecting the Serine Target of Protein Kinase-A Phosphorylation.

Acrodysostosis is a very rare congenital multisystem condition characterized by skeletal dysplasia with severe brachydactyly, midfacial hypoplasia, and short stature, varying degrees of intellectual disability, and possible resistance to multiple G protein-coupled receptor signalling hormones. Two distinct subtypes are differentiated: acrodysostosis type 1 resulting from defects in protein kinase type 1-α regulatory subunit and acrodysostosis type 2 caused by mutations in phosphodiesterase 4D (PDE4D). Most cases are sporadic. We report on a rare multigenerational familial case of acrodysostosis type 2 due to a novel autosomal dominantly inherited PDE4D mutation. A 3.5-year-old boy presented with short stature, midfacial hypoplasia, severe brachydactyly, developmental delay, and behavioural problems. Laboratory investigations revealed mild thyrotropin resistance. His mother shared some characteristic features, such as midfacial hypoplasia and severe brachydactyly, but did not show short stature, intellectual disability or hormonal resistance. Genetic analysis identified the identical, novel heterozygous missense mutation of the PDE4D gene c.569C>T (p.Ser190Phe) in both patients. This case illustrates the significant phenotypic variability of acrodysostosis even within one family with identical mutations. Hence, a specific clinical diagnosis of acrodysostosis remains challenging because of great interindividual variability and a substantial overlap of the two subtypes as well as with other related Gsα-cAMP-signalling-linked disorders.

A frequent large rearrangement in the CFTR gene in cystic fibrosis patients from Reunion Island.

Reunion Island is a French province, 800 km east of Madagascar and 200 km west of Mauritius. On Reunion Island, the birth prevalence of cystic fibrosis (CF) is particularly high in the population of European origin, approximately 1:1000. In a previous study, we demonstrated that the screening of the 27 exons of the CF transmembrane conductance regulator (CFTR) gene by denaturing high-pressure liquid chromatography (DHPLC) in 114 CF families allowed the detection of about 93% of the molecular defects present on Reunion Island. Unidentified CF mutations may lie in introns or in regulatory regions that are not routinely investigated, or may correspond to gene rearrangements such as large, heterozygous deletions that escape detection using current PCR-based techniques. Using a combination of different methods (such as multiplex ligation-dependent probe amplification), 6 of the 13 unidentified CF alleles (46%) were found to harbor a deletion of 5288 bp, spanning from exon 17a to 18. Identification and examination of the breakpoint sequences showed that this deletion is different from the 3120+1kbdel8.6Kb previously found in the Palestinian Arabs. The chromosomes bearing IVS16+3316_IVS18+644del5288 did not have a common extragenic haplotype. Clinical evaluation of homozygotes (2 unrelated patients) and compound heterozygotes indicated that this deletion represents a severe mutation associated with positive sweat chloride test, pancreatic insufficiency, and early age at diagnosis.

Neurofibromatosis type 1 molecular diagnosis: what can NGS do for you when you have a large gene with loss of function mutations?

Molecular diagnosis of neurofibromatosis type 1 (NF1) is challenging owing to the large size of the tumour suppressor gene NF1, and the lack of mutation hotspots. A somatic alteration of the wild-type NF1 allele is observed in NF1-associated tumours. Genetic heterogeneity in NF1 was confirmed in patients with SPRED1 mutations. Here, we present a targeted next-generation sequencing (NGS) of NF1 and SPRED1 using a multiplex PCR approach (230 amplicons of ∼150 bp) on a PGM sequencer. The chip capacity allowed mixing 48 bar-coded samples in a 4-day workflow. We validated the NGS approach by retrospectively testing 30 NF1-mutated samples, and then prospectively analysed 279 patients in routine diagnosis. On average, 98.5% of all targeted bases were covered by at least 20X and 96% by at least 100X. An NF1 or SPRED1 alteration was found in 246/279 (88%) and 10/279 (4%) patients, respectively. Genotyping throughput was increased over 10 times, as compared with Sanger, with ∼90[euro ] for consumables per sample. Interestingly, our targeted NGS approach also provided quantitative information based on sequencing depth allowing identification of multiexons deletion or duplication. We then addressed the NF1 somatic mutation detection sensitivity in mosaic NF1 patients and tumours.

The prevalence of CHD7 missense versus truncating mutations is higher in patients with Kallmann syndrome than in typical CHARGE patients.

CONTEXT: Mutations in CHD7, a gene previously implicated in CHARGE (coloboma, heart defect, choanal atresia, retardation of growth and/or development, genital hypoplasia, ear anomalies) syndrome, have been reported in patients presenting with Kallmann syndrome (KS) or congenital hypogonadotropic hypogonadism (CHH). Most mutations causing CHARGE syndrome result in premature stop codons and occur de novo, but the proportion of truncating vs nontruncating mutations in KS and CHH patients is still unknown. OBJECTIVE: The objective of the study was to determine the nature, prevalence, mode of transmission, and clinical spectrum of CHD7 mutations in a large series of patients. DESIGN: We studied 209 KS and 94 CHH patients. These patients had not been diagnosed with CHARGE syndrome according to the current criteria. We searched for mutations in 16 KS and CHH genes including CHD7. RESULTS: We found presumably pathogenic mutations in CHD7 in 24 KS patients but not in CHH patients. Nontruncating mutations (16 missense and a two-codon duplication) were more prevalent than truncating mutations (three nonsense, three frame shift, and a splice site), which contrasts with patients presenting with typical CHARGE syndrome. Thus, the clinical spectrum associated with CHD7 mutations may be partly explained by genotype/phenotype correlations. Eight patients also had congenital deafness and one had a cleft lip/palate, whereas six had both. For 10 patients, the presence of diverse features of the CHARGE spectrum in at least one relative argues against a de novo appearance of the missense mutation, and this was confirmed by genetic analysis in five families. CONCLUSION: Considering the large prevalence and clinical spectrum of CHD7 mutations, it will be particularly relevant to genetic counseling to search for mutations in this gene in KS patients seeking fertility treatment, especially if KS is associated with deafness and cleft lip/palate.

Greater prevalence of PROKR2 mutations in Kallmann syndrome patients from the Maghreb than in European patients.

CONTEXT: Kallmann syndrome (KS) is a genetically heterogeneous developmental disorder that associates hypogonadotropic hypogonadism and anosmia. Various causative genes have been identified, but their respective involvement in different world regions is poorly documented. OBJECTIVE: We aimed to compare the prevalence of mutations in five routinely analyzed KS genes between Maghrebian and European patients. METHODS: Blood samples from 120 presumably unrelated Maghrebian patients were collected for DNA sequencing by the Sanger technique. The prevalence of the non-synonymous mutations in KAL1, FGFR1, FGF8, PROKR2, and PROK2 was determined for each gene, and compared with those previously obtained from the analysis of 712 European patients. RESULTS: Diverse mutations in PROKR2, a gene involved both in monogenic recessive and digenic/oligogenic KS transmission modes, were found in 23.3% of the Maghrebian patients, but only in 5.1% of the European patients (Fisher's exact test, P<0.001), whereas mutations in each of the other four KS genes were present either at similar frequencies in the Maghrebian and European patients (KAL1, PROK2, FGF8, from 6.6 to 0.8%; Fisher's exact test, P>0.4 for all comparisons) or at a lower frequency in Maghrebian patients (FGFR1, 5.0 vs 11.7%; Fisher's exact test, P<0.05). Homozygosity resulting from consanguineous marriages was not sufficient to account for the greater prevalence of PROKR2 mutations in the Maghrebian patients. CONCLUSIONS: The great prevalence of PROKR2 mutations in Maghrebian patients has practical consequences for molecular diagnosis of the disease and genetic counseling in the Maghrebian population.

Custom oligonucleotide array-based CGH: a reliable diagnostic tool for detection of exonic copy-number changes in multiple targeted genes.

The frequency of disease-related large rearrangements (referred to as copy-number mutations, CNMs) varies among genes, and search for these mutations has an important place in diagnostic strategies. In recent years, CGH method using custom-designed high-density oligonucleotide-based arrays allowed the development of a powerful tool for detection of alterations at the level of exons and made it possible to provide flexibility through the possibility of modeling chips. The aim of our study was to test custom-designed oligonucleotide CGH array in a diagnostic laboratory setting that analyses several genes involved in various genetic diseases, and to compare it with conventional strategies. To this end, we designed a 12-plex CGH array (135k; 135 000 probes/subarray) (Roche Nimblegen) with exonic and intronic oligonucleotide probes covering 26 genes routinely analyzed in the laboratory. We tested control samples with known CNMs and patients for whom genetic causes underlying their disorders were unknown. The contribution of this technique is undeniable. Indeed, it appeared reproducible, reliable and sensitive enough to detect heterozygous single-exon deletions or duplications, complex rearrangements and somatic mosaicism. In addition, it improves reliability of CNM detection and allows determination of boundaries precisely enough to direct targeted sequencing of breakpoints. All of these points, associated with the possibility of a simultaneous analysis of several genes and scalability 'homemade' make it a valuable tool as a new diagnostic approach of CNMs.