Identification of a novel CFAP61 homozygous splicing variant associated with multiple morphological abnormalities of the flagella.

In this study, we investigated the role of a newly identified homozygous variant (c.1245 + 6T > C) in the CFAP61 gene in the development of multiple morphologically abnormal flagella (MMAF) in an infertile patient. Using exome sequencing, we identified this variant, which led to exon 12 skipping and the production of a truncated CFAP61 protein. Transmission electron microscopy analysis of the patient's spermatozoa revealed various flagellar abnormalities, including defective nuclear chromatin condensation, axoneme disorganization, and mitochondria embedded in residual cytoplasmic droplets. Despite a fertilization rate of 83.3% through ICSI, there was no successful pregnancy due to poor embryo quality.Our findings suggest a link between the identified CFAP61 variant and MMAF, indicating potential disruption in radial spokes' assembly or function crucial for normal ciliary motility. Furthermore, nearly half of the observed sperm heads displayed chromatin condensation defects, possibly contributing to the low blastulation rate. This case underscores the significance of genetic counseling and testing, particularly for couples dealing with infertility and MMAF. Early identification of such genetic variants can guide appropriate interventions and improve reproductive outcomes.

Recurrent "outsider" intronic variation in the SLC5A6 gene causes severe mixed axonal and demyelinating neuropathy, cyclic vomiting and optic atrophy in 3 families from Maghreb.

Sodium dependent multivitamin transporter (SMVT) deficiency is a very rare autosomal recessive disorder characterized by multisystemic clinical manifestations due to combined biotin, panthotenic acid and lipoic acid deficiency. About 10 families have been described so far. Accurate diagnosis is crucial because of the possibility of a supplementation treatment with proven efficacy. Here we describe 4 new patients (3 additional families) originating from the same world region (Algeria, Maghreb). All patients, born form consanguineous parents, were homozygous carriers of the same intronic variation, outside of canonical sites, in the SLC5A6 gene encoding SMVT. RNA study in one family allowed confirming the pathogenic effect of the variation and re-classifying this variant of uncertain significance as pathogenic, opening the possibility of genetic counseling and treatment. The identification of the same variation in three distinct and apparently unrelated families is suggestive of a founder effect. The phenotype of all patients was very similar, with systematic optic atrophy (initially considered as a very rare sign), severe cyclic vomiting, and rapidly progressive mixed axonal and demyelinating sensory motor neuropathy.

PLS3 missense variants affecting the actin-binding domains cause X-linked congenital diaphragmatic hernia and body-wall defects.

Congenital diaphragmatic hernia (CDH) is a relatively common and genetically heterogeneous structural birth defect associated with high mortality and morbidity. We describe eight unrelated families with an X-linked condition characterized by diaphragm defects, variable anterior body-wall anomalies, and/or facial dysmorphism. Using linkage analysis and exome or genome sequencing, we found that missense variants in plastin 3 (PLS3), a gene encoding an actin bundling protein, co-segregate with disease in all families. Loss-of-function variants in PLS3 have been previously associated with X-linked osteoporosis (MIM: 300910), so we used in silico protein modeling and a mouse model to address these seemingly disparate clinical phenotypes. The missense variants in individuals with CDH are located within the actin-binding domains of the protein but are not predicted to affect protein structure, whereas the variants in individuals with osteoporosis are predicted to result in loss of function. A mouse knockin model of a variant identified in one of the CDH-affected families, c.1497G>C (p.Trp499Cys), shows partial perinatal lethality and recapitulates the key findings of the human phenotype, including diaphragm and abdominal-wall defects. Both the mouse model and one adult human male with a CDH-associated PLS3 variant were observed to have increased rather than decreased bone mineral density. Together, these clinical and functional data in humans and mice reveal that specific missense variants affecting the actin-binding domains of PLS3 might have a gain-of-function effect and cause a Mendelian congenital disorder.

Neurotrophic tropomyosin receptor kinase (NTRK) fusion positive tumors: a historical cohort analysis.

BACKGROUND: NTRK gene fusions have been identified in various tumors; some requiring aggressive therapy and sometimes new TRK inhibitors (TRKi). We aimed to describe a national, unselected, retrospective, multicenter cohort. RESEARCH DESIGN AND METHODS: Patients were identified through the French sarcoma diagnostic laboratory at Institut Curie through samples analyzed by RT-qPCR or whole-transcriptome sequencing. RESULTS: From 2001 to 2019, 65 NTRK fusion tumors were identified within 2120 analyses (3.1%): 58 by RNA sequencing (including 20 after RT-qPCR analysis) and 7 exclusively by RT-qPCR. Of the 61 patients identified, 37 patients had infantile soft tissue or kidney fibrosarcomas (IFS), 15 other mesenchymal (Other-MT) and nine central nervous system (CNS) tumors. They encompassed 14 different tumor types with variable behaviors. Overall, 53 patients had surgery (3 mutilating), 38 chemotherapy (20 alkylating agents/anthracycline), 11 radiotherapy, two 'observation strategy' and 13 received TRKi. After a median follow-up of 61.0 months [range, 2.5-226.0], 10 patients died. Five-year overall survival is, respectively, 91.9% [95%CI, 83.5-100.0], 61.1% [95%CI, 34.2-100.0] and 64.8% [95%CI, 39.3-100.0] for IFS, Other-MT, and CNS groups. CONCLUSIONS: NTRK-fusion positive tumors are rare but detection is improved through RNA sequencing. TRKi could be considered at diagnosis for CNS NTRK-fusion positive tumors, some IFS, and Other-MT. TRIAL REGISTRATION: Not adapted.

Monoallelic and biallelic variants in LEF1 are associated with a new syndrome combining ectodermal dysplasia and limb malformations caused by altered WNT signaling.

PURPOSE: LEF1 encodes a transcription factor acting downstream of the WNT-ß-catenin signaling pathway. It was recently suspected as a candidate for ectodermal dysplasia in 2 individuals carrying 4q35 microdeletions. We report on 12 individuals harboring LEF1 variants. METHODS: High-throughput sequencing was employed to delineate the genetic underpinnings of the disease. Cellular consequences were characterized by immunofluorescence, immunoblotting, pulldown assays, and/or RNA sequencing. RESULTS: Monoallelic variants in LEF1 were detected in 11 affected individuals from 4 unrelated families, and a biallelic variant was detected in an affected individual from a consanguineous family. The phenotypic spectrum includes various limb malformations, such as radial ray defects, polydactyly or split hand/foot, and ectodermal dysplasia. Depending on the type and location of LEF1 variants, the inheritance of this novel Mendelian condition can be either autosomal dominant or recessive. Our functional data indicate that 2 molecular mechanisms are at play: haploinsufficiency or loss of DNA binding are responsible for a mild to moderate phenotype, whereas loss of ß-catenin binding caused by biallelic variants is associated with a severe phenotype. Transcriptomic studies reveal an alteration of WNT signaling. CONCLUSION: Our findings establish mono- and biallelic variants in LEF1 as a cause for a novel syndrome comprising limb malformations and ectodermal dysplasia.

Identification of limb-specific Lmx1b auto-regulatory modules with Nail-patella syndrome pathogenicity.

LMX1B haploinsufficiency causes Nail-patella syndrome (NPS; MIM 161200), characterized by nail dysplasia, absent/hypoplastic patellae, chronic kidney disease, and glaucoma. Accordingly in mice, Lmx1b has been shown to play crucial roles in the development of the limb, kidney and eye. Although one functional allele of Lmx1b appears adequate for development, Lmx1b null mice display ventral-ventral distal limbs with abnormal kidney, eye and cerebellar development, more disruptive, but fully concordant with NPS. In Lmx1b functional knockouts (KOs), Lmx1b transcription in the limb is decreased nearly 6-fold, indicating autoregulation. Herein, we report on two conserved Lmx1b-associated cis-regulatory modules (LARM1 and LARM2) that are bound by Lmx1b, amplify Lmx1b expression with unique spatial modularity in the limb, and are necessary for Lmx1b-mediated limb dorsalization. These enhancers, being conserved across vertebrates (including coelacanth, but not other fish species), and required for normal locomotion, provide a unique opportunity to study the role of dorsalization in the fin to limb transition. We also report on two NPS patient families with normal LMX1B coding sequence, but with loss-of-function variations in the LARM1/2 region, stressing the role of regulatory modules in disease pathogenesis.

TAR syndrome: Clinical and molecular characterization of a cohort of 26 patients and description of novel noncoding variants of RBM8A.

Thrombocytopenia-absent radius (TAR) syndrome is characterized by radial defect and neonatal thrombocytopenia. It is caused by biallelic variants of RBM8A gene (1q21.1) with the association of a null allele and a hypomorphic noncoding variant. RBM8A encodes Y14, a core protein of the exon junction complex involved in messenger RNA maturation. To date, only two hypomorphic variants have been identified. We report on a cohort of 26 patients affected with TAR syndrome and carrying biallelic variants in RBM8A. Half patients carried a 1q21.1 deletion and one of the two known hypomorphic variants. Four novel noncoding variants of RBM8A were identified in the remaining patients. We developed experimental models enabling their functional characterization in vitro. Two variants, located respectively in the 5'-untranslated region (5'-UTR) and 3'-UTR regions, are responsible for a diminished expression whereas two intronic variants alter splicing. Our results bring new insights into the molecular knowledge of TAR syndrome and enabled us to propose genetic counseling for patients' families.

Multiplex targeted high-throughput sequencing in a series of 352 patients with congenital limb malformations.

Congenital limb malformations (CLM) comprise many conditions affecting limbs and more than 150 associated genes have been reported. Due to this large heterogeneity, a high proportion of patients remains without a molecular diagnosis. In the last two decades, advances in high throughput sequencing have allowed new methodological strategies in clinical practice. Herein, we report the screening of 52 genes/regulatory sequences by multiplex high-throughput targeted sequencing, in a series of 352 patients affected with various CLM, over a 3-year period of time. Patients underwent a clinical triage by expert geneticists in CLM. A definitive diagnosis was achieved in 35.2% of patients, the yield varying considerably, depending on the phenotype. We identified 112 single nucleotide variants and 26 copy-number variations, of which 52 are novel pathogenic or likely pathogenic variants. In 6% of patients, variants of uncertain significance have been found in good candidate genes. We showed that multiplex targeted high-throughput sequencing works as an efficient and cost-effective tool in clinical practice for molecular diagnosis of congenital limb malformations. Careful clinical evaluation of patients may maximize the yield of CLM panel testing.

Outcome of relapse in children and adolescents with B-cell non-Hodgkin lymphoma and mature acute leukemia: A report from the French LMB study.

INTRODUCTION: In order to describe relapsed B-cell non-Hodgkin lymphoma and mature acute leukemia in children/adolescents treated with the Lymphomes Malins B (LMB) regimen and their outcome in the rituximab era, relapses in the French LMB2001 study were reviewed. METHODS: Between February 2001 and December 2011, 33 patients out of 773 (4.3%) relapsed; 27 had Burkitt lymphoma and six large B-cell histology. Median age at diagnosis was 10.1 years. One patient was initially treated in risk group A, 21 in group B, and 11 in group C. RESULTS: Median time to relapse after diagnosis was 4.5 months (range 2.4-13.6). Thirty-two patients received salvage therapy. Twenty-seven received rituximab mainly in addition to high-dose cytarabine and etoposide (n = 18) and/or ifosfamide, carboplatin, and etoposide (n = 7). First-line salvage chemotherapy response rate was 66% with 47% being complete remission (CR). Twenty-one patients received high-dose chemotherapy (HDC) followed by autologous (n = 13) or allogeneic (n = 8) transplant. With a median follow-up of 6.8 years, the 5-year survival rate after relapse was 36.4% (95% confidence interval [CI] 22-53%). Twelve patients were still alive; all but one (group A) received consolidation treatment. Achieving CR before consolidation was significantly associated with better survival, with a 5-year survival rate of 75% (95% CI 46.8-91.1%) for patients in CR before HDC, 33% (95% CI 9.7-70%) for patients in partial remission, and 0% for nonresponders (P = .033). CONCLUSION: Survival of children/adolescents with mature B-cell lymphoma/leukemia remains poor after relapse with no apparent improvement with rituximab. Response rates to salvage chemo-immunotherapies are insufficient and new drugs are urgently needed to improve disease control.

WNT10B variants in split hand/foot malformation: Report of three novel families and review of the literature.

Split-hand/foot malformation (SHFM) is a genetically heterogeneous congenital limb malformation typically limited to a defect of the central rays of the autopod, presenting as a median cleft of hands and feet. It can be associated with long bone deficiency or included in more complex syndromes. Among the numerous genetic causes, WNT10B homozygous variants have been recently identified in consanguineous families, but remain still rarely described (SHFM6; MIM225300). We report on three novel SHFM families harboring WNT10B variants and review the literature, allowing us to highlight some clinical findings. The feet are more severely affected than the hands and there is a frequent asymmetry without obvious side-bias. Syndactyly of third-fourth fingers was a frequent finding (62%). Polydactyly, which was classically described in SHFM6, was only present in 27% of patients. No genotype-phenotype correlation is delineated but heterozygous individuals might have mild features of SHFM, suggesting a dose-effect of the WNT10B loss-of-function.

Optimization of Next-Generation Sequencing Technologies for von Hippel Lindau (VHL) Mosaic Mutation Detection and Development of Confirmation Methods.

Von Hippel-Lindau disease (VHL) is a monogenic disorder characterized by the development of tumors affecting the central nervous system, kidney, pancreas, or adrenal glands, and due to germline mutations in the VHL tumor suppressor gene. About 5% of patients with a typical VHL phenotype have no mutation detected by conventional techniques, so a postzygotic VHL mosaicism can be suspected. The aim of this study was therefore to implement a next-generation sequencing (NGS) strategy for VHL mosaic mutation detection, including an optimization of the original Personal Genome Machine design by enrichment with oligonucleotides corresponding to amplicons with insufficient depth of coverage. Two complementary strategies were developed for the confirmation of mosaic mutations identified by NGS, SNaPshot for variants present at an allelic ratio greater than 5%, and droplet digital PCR for allelic ratio above 1%. VHL mutant plasmids were generated to assess VHL mosaic mutation detection in different exons and to set up an internal quality control that could be included in each run or regularly to validate the assay. This strategy was applied to 47 patients with a suggestive or clinical VHL disease, and mosaic mutations were identified in 8.5% of patients. In conclusion, NGS technologies combined with SNaPshot or droplet digital PCR allow the detection and confirmation of mosaic mutations in a clinical laboratory setting.

[Use of blinatumomab in children acute lymphoblastic leukemia in the Grand Ouest interregion: A chance for all]. / Expérience du blinatumomab dans les leucémies aiguës lymphoblastiques de l'enfant et de l'adolescent dans l'interrégion Grand Ouest : une chance pour tous.

INTRODUCTION: Relapsed/refractory acute lymphoblastic leukemia (ALL) in children has a pejorative prognosis and justifies to be treated by hematopoietic stem cell transplantation (HSCT). A minimal residual disease (MRD) before transplantation is a major part of prognosis. Blinatumomab, a bispecific antibody CD19+/CD3+, allowed to achieve a cytologic and molecular complete remission in adults with refractory B-precursor ALL. This retrospective study analyses results from a pediatric cohort treated by blinatumomab thanks to an interregional structuring consortium. PATIENTS AND METHODS: Patients between 0 and 23 years old, from the 7 centers of the french "Grand Ouest" interregional network, treated by blinatumomab for a relapsed or refractory ALL, from January 2015 to January 2018, were included. The efficiency of blinatumomab was assessed in terms of complete remission, minimal residual disease, overall survival, and tolerability of treatment. RESULTS: Thirteen of 18 patients achieved a complete remission, with negative minimal residual disease for ten of them. Fourteen patients proceeded to stem cell transplantation,. Eight out of 14 patients obtained long term remission after HSCT. As far as tolerance is concerned, no serious adverse event, neurological or psychiatric disorder, was observed. CONCLUSION: Thanks to an interregional network collaboration, all children with high risk ALL coming from the western french interregion could be treated by blinatumomab. Blinatumomab offered good hematological conditions to undergo HSCT with a good tolerability.

Novel KHDRBS1-NTRK3 rearrangement in a congenital pediatric CD34-positive skin tumor: a case report.

Cutaneous spindle-cell neoplasms in adults as well as children represent a frequent dilemma for pathologists. Along this neoplasm spectrum, the differential diagnosis with CD34-positive proliferations can be challenging, particularly concerning neoplasms of fibrohistiocytic and fibroblastic lineages. In children, cutaneous and superficial soft-tissue neoplasms with CD34-positive spindle cells are associated with benign to intermediate malignancy potential and include lipofibromatosis, plaque-like CD34-positive dermal fibroma, fibroblastic connective tissue nevus, and congenital dermatofibrosarcoma protuberans. Molecular biology has been valuable in showing dermatofibrosarcoma protuberans and infantile fibrosarcoma that are characterized by COL1A1-PDGFB and ETV6-NTRK3 rearrangements respectively. We report a case of congenital CD34-positive dermohypodermal spindle-cell neoplasm occurring in a female infant and harboring a novel KHDRBS1-NTRK3 fusion. This tumor could belong to a new subgroup of pediatric cutaneous spindle-cell neoplasms, be an atypical presentation of a plaque-like CD34-positive dermal fibroma, of a fibroblastic connective tissue nevus, or represent a dermatofibrosarcoma protuberans with an alternative gene rearrangement.

Holt-Oram syndrome: clinical and molecular description of 78 patients with TBX5 variants.

Holt-Oram syndrome (HOS) is an autosomal dominant condition characterised by the association of congenital heart defect (CHD), with or without rhythm disturbances and radial defects, due to TBX5 variants. The diagnosis is challenged by the variability of expression and the large phenotypic overlap with other conditions, like Okihiro syndrome, TAR syndrome or Fanconi disease. We retrospectively reviewed 212 patients referred for suspicion of HOS between 2002 and 2014, who underwent TBX5 screening. A TBX5 variant has been identified in 78 patients, representing the largest molecular series ever described. In the cohort, 61 met the previously described diagnostic criteria and 17 have been considered with an uncertain HOS diagnosis. A CHD was present in 91% of the patients with a TBX5 variant, atrial septal defects being the most common (61.5%). The genotype-phenotype study highlights the importance of some critical features in HOS: the septal characteristic of the CHD, the bilateral and asymmetric characteristics of the radial defect and the presence of shoulder or elbow mobility defect. Besides, 21 patients presented with an overlapping condition. Among them, 13 had a typical HOS presentation. We discuss the strategies that could be adopted to improve the molecular delineation of the remaining typical patients.

Should We Pay Attention to the Delay Before Admission to a Pediatric Intensive Care Unit for Children With Cancer? Impact on 1-Month Mortality. A Report From the French Children's Oncology Study Group, GOCE.

Acute complications requiring admission to pediatric intensive care unit (PICU) are frequent for children with cancer. Our objective was to determine early prognostic factors of mortality in a cohort of children with cancer hospitalized in PICU for acute complications and particularly to assess whether the delay before admission to a PICU is an early predictor of mortality. We conduct a retrospective multicenter analysis. All patients transferred in PICU for acute complications between January 2002 and December 2012 were included. One-month mortality of the 224 patients analyzed was 24.5%. Delay before PICU admission was a significant prognostic factor of 1-month mortality with nonsurvivors experiencing a longer median delay than survivors (24 vs. 12 h, respectively, P<0.05). Time from diagnosis to PICU admission (P<0.001), hematopoietic stem cell transplant (P<0.05), the duration of neutropenia (P<0.01), infection type (P<0.001), number of organ dysfunctions (P<0.001), and reaching any grade 4 toxicity before PICU admission (P<0.001) also affected mortality rate at 1-month post-PICU discharge. In the multivariate analysis, only reaching any grade 4 toxicity before PICU admission influenced 1-month mortality (odds ratio, 2.30; 95% confidence interval, 1.07-4.96; P<0.05). These results suggest that PICU admission before severe impairment leads to a better outcome for children with cancer.

Blepharocheilodontic syndrome is a CDH1 pathway-related disorder due to mutations in CDH1 and CTNND1.

PURPOSE: Blepharocheilodontic (BCD) syndrome is a rare autosomal dominant condition characterized by eyelid malformations, cleft lip/palate, and ectodermal dysplasia. The molecular basis of BCD syndrome remains unknown. METHODS: We recruited 11 patients from 8 families and performed exome sequencing for 5 families with de novo BCD syndrome cases and targeted Sanger sequencing in the 3 remaining families. RESULTS: We identified five CDH1 heterozygous missense mutations and three CTNND1 heterozygous truncating mutations leading to loss-of-function or haploinsufficiency. Establishment of detailed genotype-phenotype correlations was not possible because of the size of the cohort; however, the phenotype seems to appear more severe in case of CDH1 mutations. Functional analysis of CDH1 mutations confirmed their deleterious impact and suggested accelerated E-cadherin degradation. CONCLUSION: Mutations in CDH1 encoding the E-cadherin were previously reported in hereditary diffuse gastric cancer as well as in nonsyndromic cleft lip/palate. Mutations in CTNND1 have never been reported before. The encoded protein, p120ctn, prevents E-cadherin endocytosis and stabilizes its localization at the cell surface. Conditional deletion of Cdh1 and Ctnnd1 in various animal models induces features reminiscent of BCD syndrome and underlines critical role of the E-cadherin-p120ctn interaction in eyelid, craniofacial, and tooth development. Our data assert BCD syndrome as a CDH1 pathway-related disorder due to mutations in CDH1 and CTNND1 and widen the phenotypic spectrum of E-cadherin anomalies.Genet Med advance online publication 09 March 2017.

Nail-Patella Syndrome: clinical and molecular data in 55 families raising the hypothesis of a genetic heterogeneity.

Nail-Patella Syndrome (NPS) is a rare autosomal dominant condition comprising nail and skeletal anomalies. Skeletal features include dysplastic patellae and iliac horns, as well as scapula and elbow dysplasia. Nephropathy and glaucoma or intra-ocular hypertension can sometimes be present. NPS is due to variants affecting function in LMX1B, which encodes a LIM-homeodomain protein critical for limb, kidney and eye development. We describe the phenotype and the molecular data of 55 index patients and their 39 relatives presenting with typical NPS. We identified 38 different LMX1B anomalies, 19 of which were not reported before. In our series, 9% of families are not carriers of a LMX1B genomic alteration after extensive study of the coding and non-coding regions of the gene. One of the families showed no linkage to the LMX1B locus, raising the hypothesis of a genetic heterogeneity.