Background: The majority of variants of unknown clinical significance (VUCS) in the CFTR gene are missense variants. While change on the CFTR protein structure or function is often suspected, impact on splicing may be neglected. Such undetected splicing default of variants may complicate the interpretation of genetic analyses and the use of an appropriate pharmacotherapy. Methods: We selected 15 variants suspected to impact CFTR splicing after in silico predictions on 319 missense variants (214 VUCS), reported in the CFTR -France database. Six specialized laboratories assessed the impact of nucleotide substitutions on splicing (minigenes), mRNA expression levels (quantitative PCR), synthesis and maturation (western blot), cellular localization (immunofluorescence) and channel function (patch clamp) of the CFTR protein. We also studied maturation and function of the truncated protein, consecutive to in-frame aberrant splicing, on additional plasmid constructs. Results: Six of the 15 variants had a major impact on CFTR splicing by in-frame ( n = 3) or out-of-frame ( n = 3) exon skipping. We reclassified variants into: splicing variants; variants causing a splicing defect and the impairment of CFTR folding and/or function related to the amino acid substitution; deleterious missense variants that impair CFTR folding and/or function; and variants with no consequence on the different processes tested. Conclusion: The 15 variants have been reclassified by our comprehensive approach of in vitro experiments that should be used to properly interpret very rare exonic variants of the CFTR gene. Targeted therapies may thus be adapted to the molecular defects regarding the results of laboratory experiments. (c) 2022 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

BACKGROUND:The development of whole-exome sequencing (WES) and whole-genome sequencing (WGS) for clinical purposes now allows the identification of multiple pathogenic variants in patients with a rare disease. This occurs even when a single causative gene was initially suspected. We report the case of an 8-year-old patient with global developmental delays and dysmorphic features, with a possibly pathogenic variant in three distinct genes.METHODS:Trio-based exome sequencing was performed by IntegraGen SA (Evry, France), on an Illumina HiSeq4000 (Illumina, San Diego, CA, USA). Sanger sequencing was performed to confirm the variants that were found.RESULTS:WES showed the presence of three possibly deleterious variants: KMT2A: c.9068delA;p.Gln3023Argfs*3 de novo, PAX3: c.530C>G;p.Ala177Gly de novo and DLG3: c.127delG;p.Asp43Metfs*22 hemizygous inherited from the mother. KMT2A pathogenic variants are involved in Wiedemann-Steiner syndrome, and PAX3 is the gene responsible for Waardenburg syndrome. DLG3 variants have been described in a non-syndromic X-related intellectual disability.CONCLUSIONS:Considering the dysmorphic features and intellectual disability presented by this patient, these three variants were imputed as pathogenic and their association was considered responsible for his phenotype. Dual molecular diagnoses have already been found by WES in several cohorts with an average of diagnostic yield of 7%. This case demonstrates and reminds us of the importance of analyzing exomes rigorously and exhaustively because, in some cases (< 10%), it can explain superimposed traits or blended phenotypes.

Results:The patient was diagnosed with CF at the age of 3 years (bronchitis and moderately pancreatic insufficiency from the first weeks of life, 74.2 mmol/l sweat chloride concentration).In the future, up to the age of 30, she did not carry out any basic therapy, against the background of a stable state and not a clearly aggravated cough in the autumn-winter period!In 2017 fluorography changes were detected and M.intracellulare was diagnosed.In 2018 was performed sweat chloride retest -94 mmol/l (≪Nanoduct≫); Pseudomonas aeruginosa was detected in the respiratory tract.Spirometry: FEV1 73%, FVC 86%.No pathological changes have been identified on ultrasonography of the abdominal cavity.There are single bronchiectasis in CT of the lungs.A molecular-genetic analysis revealed two rare (not previously described) CFTR variants: c.3815_3816insTTG ( p.Val1272_Ser1273insTrp) and CFTRdele23.Conclusion: Thus, a phenotypically stable patient with no pronounced damage of target organs is presented.The pancreas is clinically intact.It is highly probable that the mutation c.3815_3816insTTG has mild features.

Rubinstein-Taybi syndrome (RSTS; OMIM 180849) is an autosomal dominant developmental disorder characterized by facial dysmorphism, broad thumbs and halluces associated with intellectual disability. RSTS is caused by alterations in CREBBP (about 60%) and EP300 genes (8%). RSTS is often diagnosed at birth or during early childhood but generally not suspected during antenatal period. We report nine cases of well-documented fetal RSTS. Two cases were examined after death in utero at 18 and 35 weeks of gestation and seven cases after identification of ultrasound abnormalities and termination of pregnancy. On prenatal sonography, a large gallbladder was detected in two cases, and brain malformations were noted in four cases, especially cerebellar hypoplasia. However, the diagnosis of RSTS has not been suggested during pregnancy. Fetal autopsy showed that all fetuses had large thumbs and/or suggestive facial dysmorphism. A CREBBP gene anomaly was identified in all cases. Alterations were similar to those found in typical RSTS children. This report will contribute to a better knowledge of the fetal phenotype to consider the hypothesis of RSTS during pregnancy. Genotyping allows reassuring genetic counseling.

Aquagenic palmoplantar keratoderma (APK) is a rare skin disorder characterized by skin wrinkling with oedema and whitish papules on the palms and/or soles, pruritus, burning, and pain after contact with water. 1 Most cases are sporadic, but familial cases also have been reported, and APK pathogenesis is poorly understood. In 1974, Elliot first described APK in patients with cystic fibrosis (CF). This article is protected by copyright. All rights reserved.

Objectives: Congenital bilateral aplasia of vas deferens (CBAVD) syndrome is common genetic cause of male infertility with obstructive azoospermia.About 80% CBAVD patients have mutations and/or 5T allele of CFTR gene, but the frequency and spectrum of individual CFTR gene variants vary relative to specific populations.Methods: We analyzed 30 CFTR mutations (CFTRdele2,3(Del21kb), F508del, I507del, 1677delTA, 2143delT, 2184insA, 394delTT, 3821DelT, L138ins, G542X, W1282X, N1303K, R334W, 3849 + 10kbС>Т, 604insA, 3944delGT, S1196X, 621+1g>t, E92K, 3272-26A>G, 4015delA, 4022insT, W1282R, 2785 + 5G>A, 3272-16T>A, S466X, 1898 + 1G>A, 3120 + 1G>A, R347P, S945L) and IVS8Tn polymorphism in 72 Russian men with CBAVD syndrome.Insertions and deletions were detected by AFLP-analysis; point mutations were tested by MLPA.Results: CFTR mutations were found in 65.3% CBAVD patients in the heterozygous (n = 46) and the compound heterozygote (n = 1).In 66% analyzed alleles the following CFTR gene variants were detected: F508del (18.1%),CFTRdele2,3(21kb) (4.2%), W1282X (2.8%), that comprised 54.2%, 12.5%, and 8.2% of detected mutations, respectively.F508del mutation was commonly presented in a combination with 5T/9T (61.5%) or 7T/9T (30.8%) genotypes.IVS8-T5 variant was found in 54.2% patients, including homozygous (5.6%) and heterozygous (48.6%).As many as 44.4% CBAVD patients had two CFTR gene variants, including 5T allele.Compoundheterozygous genotypes with CFTR mutation and 5T allele were detected in 37.5% patients.In total, pathogenic CFTR gene variants and/or 5T allele were found in 81.9% Russian CBAVD patients. Conclusion:The results of presented study demonstrated high frequency of the CFTR gene mutations and 5T allele in Russian CBAVD patients.The frequency and spectrum of CFTR mutations in CBAVD are similar to Russian patients with Cystic Fibrosis.The most common CFTR gene variants were F508del and IVS8-T5 allele, which were detected in 38% CBAVD patients.

Objective— Dominant mutations of the X-linked filamin A ( FLNA ) gene are responsible for filaminopathies A, which are rare disorders including brain periventricular nodular heterotopia, congenital intestinal pseudo-obstruction, cardiac valves or skeleton malformations, and often macrothrombocytopenia. Approach and Results— We studied a male patient with periventricular nodular heterotopia and congenital intestinal pseudo-obstruction, his unique X-linked FLNA allele carrying a stop codon mutation resulting in a 100–amino acid–long FLNa C-terminal extension (NP_001447.2: p.Ter2648SerextTer101 ). Platelet counts were normal, with few enlarged platelets. FLNa was detectable in all platelets but at 30% of control levels. Surprisingly, all platelet functions were significantly upregulated, including platelet aggregation and secretion, as induced by ADP, collagen, or von Willebrand factor in the presence of ristocetin, as well as thrombus formation in blood flow on a collagen or on a von Willebrand factor matrix. Most importantly, patient platelets stimulated with ADP exhibited a marked increase in α IIb β 3 integrin activation and a parallel increase in talin recruitment to β 3 , contrasting with normal Rap1 activation. These results are consistent with the mutant FLNa affecting the last step of α IIb β 3 activation. Overexpression of mutant FLNa in the HEL megakaryocytic cell line correlated with an increase (compared with wild-type FLNa) in PMA-induced fibrinogen binding to and in talin and kindlin-3 recruitment by α IIb β 3 . Conclusions— Altogether, our results are consistent with a less binding of mutant FLNa to β 3 and the facilitated recruitment of talin by β 3 on platelet stimulation, explaining the increased α IIb β 3 activation and the ensuing gain-of-platelet functions.

To the Editors: Several syndromes are associated with a higher risk of nephroblastoma, such as Wilm’s tumour, aniridia, genitourinary abnormalities, and mental retardation (WAGR) and Denys-Drash syndromes, caused by WT1 mutations. Somatic genetic abnormalities have been described in nephroblastoma, such as mutations in WT1, WTX, CTNNB1, and TP53. However in many cases, the underlying genetic alterations are unknown. We report the case of a 4-year-old Caucasian male, who has been diagnosed at the age of 1 year with a right epithelial type nephroblastoma. He was born with a posterior cleft palate, and evolved with a mild psychomotor delay. He had a mild facial dysmorphism, with a bulging forehead, a broad nasal root, a long philtrum with a thin upper lip, and a left single palmar crease. Array-CGH (8*60K, Agilent Technologies) identified a de novo 6p22.3 deletion of 389 kb, encompassing a unique coding gene, SOX4 (Figure 1(a)). No mutations in SOX4 were identified by Sanger sequencing on tumor DNA, which does not support the 2-hit Knudson hypothesis. No mutations or deletions in SOX4 were identified in 3 other patients presenting with a familial form of nephroblastoma. By RT-qPCR on RNA samples in the nephroblastoma and in nontumoral kidney from proband, we found a 12-fold increased expression of SOX4 in the tumor compared to non-tumoral kidney (n = 5) (Figure 1(b)). In 2 other patients, RT-qPCR showed 4 and 6-fold increased expressions of SOX4 on RNA samples from nephroblastomas compared to non-tumoral kidney (n = 3). We observed that the upregulation of SOX4 expression was 2 times more increased in the proband, who has SOX4 deletion, than in the 2 other patients without any genomic deletion of SOX4 (Figure 1(c)). Further information concerning material and methods is available upon request. SOX4 codes for a transcriptional factor which has a critical role during the embryogenesis, especially in neurogenesis. Previous studies showed that SOX4 is required for normal nephrogenesis, and is a target of WT1. SOX4 is also involved in tumorigenesis of many human malignancies. In our patient, the paradoxical increased tumor/normal kidney SOX4 expression ratio could be the consequence of late events in the tumorogenesis process. Nephroblastoma is the result of an excessive proliferation of pluripotent renal precursors in the embryonic kidney. The deleterious effect of a SOX4 deletion could take place during this antenatal period, due to haploinsufficiency, even if, because of an additional mechanism that occurred later, SOX4 is found to be upregulated, as seen in many cancers. Several studies had shown a similar ambiguous role for WT1, which can be an oncogene or a tumor suppressor gene, depending on the context. Four patients with 6p22.3 microdeletion encompassing SOX4 have been described, with 3 of them having an autosomal dominant mesomelic dysplasia Savarirayan type. The authors supposed that the physiopathologic mechanism was the deletion of a topologically associated domain (TAD), with ectopic interactions between enhancers and the ID4 gene. In our patient, this deletion only encompasses the boundary between 2 TADs (Figure 1(a)), and it could therefore impact the regulation of the expression of E2F3, a well-known oncogene, implicated in the p53 pathway. To conclude, we suspect the deletion of SOX4 and the potential dysregulation of E2F3 to be part of a tumorogenesis process, leading to the occurrence of a nephroblastoma in our patient. Other clinical reports of patients carrying the same heterozygous deletion could help to assert this link.

4 Valuable collaboration between a molecular CFTR database and a national CF registry: the French experience C. Bareil, L. Lemonnier, C. Dehillotte, V. Colomb-Jung, C. Theze, M.-P. Audrezet, C. Ferec, T. Bienvenu, E. Girodon, P. Fanen, C. Mekki, E. Bieth, V. Gaston, P. Fergelot, M.-P. Reboul, A. Kitzis, G. Lalau, A. Pagin, M.-C. Malinge, C. Raynal, M. Claustres. CHRU de Montpellier, Laboratoire de Genetique Moleculaire, Montpellier, France; Association Vaincre La Mucoviscidose, Paris, France; CHRU Brest, Laboratoire de Genetique Moleculaire, Brest, France; Hopital Cochin, APHP, Service de Genetique et Biologie Moleculaires, Paris, France; GH Henri Mondor, Departement de Genetique, Creteil, France; CHRU de Toulouse, Service de Genetique Medicale, Toulouse, France; Hopital Pellegrin – CHU de Bordeaux, Laboratoire de Genetique Moleculaire, Bordeaux, France; CHU de Poitiers, Service de Genetique, Poitiers, France; CHRU de Lille, Service de Toxicologie et Genopathies, Lille, France; CHU Angers, UF de Genetique Moleculaire – Departement de Biochimie Genetique, Angers, France In 2013 the French CF Registry and CFTR-France database managers started collaborating to address the limitations of each base, due to the lack of genetics and clinical data cross comparison. The aim was to check and update clinical data in CFTR-France and genetic data in the Registry. Records from the two databases were compared using name, date of birth, gender, sex and mutations. Matched and unmatched records were identified. Discrepancies were checked and confirmed by the laboratories and then sent to the CF centres. Comparison of 8807 patients in the Registry and 4617 in CFTR-France identified 1924 full match and 321 partially matched records. Molecular laboratories confirmations added 216 full matches. The remaining 105 patients still need confirmation by laboratories and CF centres. Among the 321 patients, personal data and/or genotype discrepancies were identified for respectively 70 and 129 patients. This database crossing allowed 110 genotypes to be corrected/completed in the Registry, which is of major interest. Indeed, if those incorrect data are also present in the CF centre medical files, they can impact familial genetic counselling and impair patients’ access to optimal mutation targeted therapies. Clinical data provided by the Registry are currently being analysed to complete the CFTR-France phenotypic status of patients. In-depth analysis of these data allows patients to be re-classified in different phenotypic groups (CF, CBAVD, isolated bronchiectasis or pancreatitis) and genotype/phenotype relationships to be refined, in order to better manage rare variants interpretation, medical care strategy and genetic counselling to families.

Otopalatodigital spectrum disorders (OPDSD) include OPD syndromes types 1 and type 2 (OPD1, OPD2), Melnick-Needles syndrome (MNS), and frontometaphyseal dysplasia (FMD). These conditions are clinically characterized by variable skeletal dysplasia associated in males, with extra-skeletal features including brain malformations, cleft palate, cardiac anomalies, omphalocele and obstructive uropathy. Mutations in the FLNA gene have been reported in most FMD and OPD2 cases and in all instances of typical OPD1 and MNS. Here, we report a series of 10 fetuses and a neonatally deceased newborn displaying a multiple congenital anomalies syndrome suggestive of OPDSD and in whom we performed FLNA analysis. We found a global mutation rate of 44%. This series allows expanding the clinical and FLNA mutational spectrum in OPDSD. However, we emphasize difficulties to correctly discriminate OPDSD based on clinical criteria in fetuses due to the major overlap between these conditions. Molecular analyses may help pathologists to refine clinical diagnosis according to the type and the location of FLNA mutations. Discriminating the type of OPDSD is of importance in order to improve the genetic counseling to provide to families.

Clear cell renal cell carcinomas (ccRCCs) represent 70% of renal cancers, and several clinical and histolopathological factors are implicated in their prognosis. We recently demonstrated that the overexpression of PAR-3 protein encoded by the PARD3 gene could be implicated in renal oncogenesis. The object of this work was to study the association of intratumoral PAR-3 expression with known prognostic parameters and clinical outcome. In this aim, PAR-3 expression was assessed by immunohistochemistry in ccRCC tumors of 101 patients from 2003 to 2005. The immunostaining of PAR-3 was scored either as membranous (mPAR-3) or as both membranous and cytoplasmic (cPAR-3). Cytoplasmic PAR-3 was significantly associated with worse histopathological and clinical prognostic factors: Fuhrman grades 3 and 4, tumor necrosis, sarcomatoid component, adrenal invasion, renal and hilar fat invasion, eosinophilic component, a noninactivated VHL gene, higher tumor grade, lymph node involvement, metastasis, and worse clinical Eastern Cooperative Oncology Group and S classification scores. After multivariate analysis, 2 parameters were independently associated with cPAR-3: necrosis and eosinophilic components. In addition, cPAR-3 patients had shorter overall and progression-free survivals independently from strong prognostic validated factors like metastases. A cytoplasmic expression of PAR-3 is therefore implicated in worse clinical and pathological cancer features in ccRCC and could be useful to identify patients with high-risk tumors.

The clear cell subtype of renal carcinoma (CCRCC) is highly vascularized and despite a slow progression rate, it is potentially a highly aggressive tumor. Although a doubling of median progression-free survival in CCRCC patients treated by targeted therapies has been observed, the fact that tumors escape after anti-VEGF treatment suggests alternative pathways. The chick chorioallantoic membrane (CAM) is a well-established model, which allows in vivo studies of tumor angiogenesis and the testing of anti-angiogenic molecules. However, only a few data exist on CCRCC grafted onto CAM. We aimed to validate herein the CAM as a suitable model for studying the development of CCRCC and the interactions with the surrounding stroma. Our study uses both CCRCC cell lines and fresh tumor samples after surgical resection. We demonstrate that in both cases CCRCC can be grafted onto the CAM, to survive and to induce an angiogenic process. We further provide insights into the transcriptional regulation of the model by performing a differential analysis of tumor-derived and stroma-derived transcripts.

Clear cell renal cell carcinoma (ccRCC) is the most common histological subtype of kidney cancer and is often characterized by mutations or deletions of the Von Hippel Lindau (VHL) tumour suppressor gene. Aurora gene family members are implicated in proper mitotic progression and spindle checkpoint function and play a crucial role in cancer progression. In the present study, we assessed the expression of Aurora-A in a cohort of 30 ccRCC with fully characterized VHL status (wt/wt or mut/del) and Fuhrman grade. Aurora-A transcript and protein levels were significantly increased in high Fuhrman grade tumours and in VHLwt/wt tumours. These results suggest that Aurora-A and VHL interact in the ccRCC. We demonstrated that the two proteins interact in vivo and identified the Ser72 on the sequence of VHL as the unique site phosphorylated by Aurora-A.

Angiogenesis in clear cell renal cell carcinoma has received recent focus with the development of antiangiogenic therapies. Although tumor progression is known to be correlated with intratumoral and plasma levels of vascular endothelial growth factor-A, the role of tumor induced-angiogenesis remains unclear in these tumors. We analyzed the vascular network in a cohort of 73 clear cell renal cell carcinoma cases using endothelial immunostaining. We studied protein expression of vascular endothelial growth factor, Von Hippel Lindau, and carbonic anhydrase IX by immunohistochemistry, Von Hippel Lindau gene alteration by sequencing, deletion- and methylation-specific Multiplex Ligation-dependent Probe Amplification, and gene expression by pangenomic microarray and quantitative polymerase chain reaction in a subcohort of 39 clear cell renal cell carcinoma cases. We described 2 distinct angiogenic phenotypes in comparison with the normal kidney vasculature: low and high angiogenic phenotypes. The low angiogenic phenotype was associated with more aggressive prognostic factors such as T3 to T4 (62% versus 31%, P = .002), N+ (29% versus 3% P = .004), M+ (53% versus 21%, P = .004) stages, Fuhrman grade (grade 3-4: 91% versus 36%, P < .001), and intratumoral vascular endothelial growth factor expression (74% versus 28%, P < .001); was less associated with Von Hippel Lindau inactivation (56% versus 80%, P = .03); and was a predictor of poor prognosis in terms of progression-free, cancer-specific, and overall survival (log-rank test, P = .002, P = .011, and P = .035, respectively). The low angiogenic phenotype was also associated with a relative down-regulation of gene expression (platelet-derived growth factor D, N-acetyl transferase 8, and N-acetyl transferase 8 B). In conclusion, the histologic and molecular distinction between these 2 angiogenic phenotypes could help to better understand the biologic behavior of clear cell renal cell carcinoma angiogenesis and could be analyzed in a prospective study of the effects of antiangiogenic drugs.