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Herein, we report the screening of a large panel of genes in a series of 80 fetuses with congenital heart defects (CHDs) and/or heterotaxy and no cytogenetic anomalies. There were 49 males (61%/39%), with a family history in 28 cases (35%) and no parental consanguinity in 77 cases (96%). All fetuses had complex CHD except one who had heterotaxy and midline anomalies while 52 cases (65%) had heterotaxy in addition to CHD. Altogether, 29 cases (36%) had extracardiac and extra-heterotaxy anomalies. A pathogenic variant was found in 10/80 (12.5%) cases with a higher percentage in the heterotaxy group (8/52 cases, 15%) compared with the non-heterotaxy group (2/28 cases, 7%), and in 3 cases with extracardiac and extra-heterotaxy anomalies (3/29, 10%). The inheritance was recessive in six genes (DNAI1, GDF1, MMP21, MYH6, NEK8, and ZIC3) and dominant in two genes (SHH and TAB2). A homozygous pathogenic variant was found in three cases including only one case with known consanguinity. In conclusion, after removing fetuses with cytogenetic anomalies, next-generation sequencing discovered a causal variant in 12.5% of fetal cases with CHD and/or heterotaxy. Genetic counseling for future pregnancies was greatly improved. Surprisingly, unexpected consanguinity accounts for 20% of cases with identified pathogenic variants., (© 2020 Wiley Periodicals LLC.)

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., (© 2019 Wiley Periodicals, Inc.)

Pathogenic complex genomic rearrangements are being increasingly characterized at the nucleotide level, providing unprecedented opportunities to evaluate the complexities of mutational mechanisms. Here, we report the molecular characterization of a complex duplication-triplication rearrangement involving exons 45-60 of the DMD gene. Inverted repeats facilitated this complex rearrangement, which shares common genomic organization with the recently described duplication-inverted triplication-duplication (DUP-TRP/INV-DUP) events; specifically, a 690-kb region comprising DMD exons from 45 to 60 was duplicated in tandem, and another 46-kb segment containing exon 51 was inserted inversely in between them. Taking into consideration (1) the presence of a predicted PRDM9 binding site in the near vicinity of the junction involving two inverted L1 elements and (2) the inherent properties of X-Y chromosome recombination during male meiosis, we proposed an alternative two-step model for the generation of this X-linked DMD DUP-TRP/INV-DUP event., (© 2013 WILEY PERIODICALS, INC.)

We report on two unprecedented cases of pseudoexon (PE) activation in the DMD gene resulting from pure intronic double-deletion events that possibly involve microhomology-mediated mechanisms. Array comparative genomic hybridization analysis and direct genomic sequencing allowed us to elucidate the causes of the pathological PE inclusion detected in the RNA of the patients. In the first case (Duchenne phenotype), we showed that the inserted 387-bp PE was originated from an inverted ∼57 kb genomic region of intron 44 flanked by two deleted ∼52 kb and ∼1 kb segments. In the second case (Becker phenotype), we identified in intron 56 two small deletions of 592 bp (del 1) and 29 bp (del 2) directly flanking a 166-bp PE located in very close proximity (134 bp) to exon 57. The key role of del 1 in PE activation was established by using splicing reporter minigenes. However, the analysis of mutant constructs failed to identify cis elements that regulate the inclusion of the PE and suggested that other splicing regulatory factors may be involved such as RNA structure. Our study introduces a new class of mutations in the DMD gene and emphasizes the potential role of underdetected intronic rearrangements in human diseases., (© 2011 Wiley-Liss, Inc.)

In six index cases/families referred for Marfan syndrome (MFS) molecular diagnosis, we identified six novel mutations in the FBN1 gene: c.1753G>C (p.Gly585Arg), c.2456G>A (p.Gly819Glu), c.4981G>A (p.Gly1661Arg), c.5339G>A (p.Gly1780Glu), c.6418G>A (p.Gly2140Arg) and c.6419G>A (p.Gly2140Glu). These variants, predicted to result in Glycine substitutions are located at the third position of a 4 amino acids loop-region of calcium-binding Epidermal Growth Factor-like (cb-EGF) fibrillin-1 domains 5, 9, 24, 25 and 32. Familial segregation studies showing cosegregation with MFS manifestations or de novo inheritance in addition to in silico analyses (conservation, 3D modeling) suggest evidence for a crucial role of the respective Glycine positions. Extending these analyses to all Glycine residue at position 3 of this 4 residues loop in fibrillin-1 cb-EGF with the UMD predictor tool and alignment of 2038 available related sequences strongly support a steric strain that only allows Glycine or even Alanine residues for domain structure maintenance and for the fibrillin functions. Our data compared with those of the literature strongly suggest the existence of a cb-EGF domain subtype with implications for related diseases.

UMD-DMD France is a knowledgebase developed through a multicenter academic effort to provide an up-to-date resource of curated information covering all identified mutations in patients with a dystrophinopathy. The current release includes 2,411 entries consisting in 2,084 independent mutational events identified in 2,046 male patients and 38 expressing females, which corresponds to an estimated number of 39 people per million with a genetic diagnosis of dystrophinopathy in France. Mutations consist in 1,404 large deletions, 215 large duplications, and 465 small rearrangements, of which 39.8% are nonsense mutations. The reading frame rule holds true for 96% of the DMD patients and 93% of the BMD patients. Quality control relies on the curation by four experts for the DMD gene and related diseases. Data on dystrophin and RNA analysis, phenotypic groups, and transmission are also available. About 24% of the mutations are de novo events. This national centralized resource will contribute to a greater understanding of prevalence of dystrophinopathies in France, and in particular, of the true frequency of BMD, which was found to be almost half (43%) that of DMD. UMD-DMD is a searchable anonymous database that includes numerous newly developed tools, which can benefit to all the scientific community interested in dystrophinopathies. Dedicated functions for genotype-based therapies allowed the prediction of a new multiexon skipping (del 45-53) potentially applicable to 53% of the deleted DMD patients. Finally, such a national database will prove to be useful to implement the international global DMD patients' registries under development.

Purpose: To evaluate aortic elasticity with MRI on young asymptomatic individuals with mutation of the smooth muscle myosin heavy chain in whom aortic enlargement is not present., Materials and Methods: Aortic compliance, aortic distensibility, and pulse wave velocity (PWV) were semiautomatically measured from MRI in 8 asymptomatic subjects having a mutation of the MYH11 gene (M+) and 21 nonmutated relatives (M-) of similar age, sex, and blood pressure characteristics., Results: Despite a similar aortic diameter in both groups, the aortic compliance and distensibility were significantly lower in M+ subjects compared with M- (0.84+/-0.33 versus 2.03+/-0.54 mm2/mmHg, 1.18+/-0.62 10(-3) versus 5.11+/-1.58 10(-3) mmHg(-1), respectively), and PWV was significantly higher (5.35+/-1.53 versus 3.60+/-0.64 m.s(-1)). A threshold aortic compliance value of 1.3 mm2/mmHg separated the two groups. The receiver operating characteristics curve analysis indicated an optimal threshold of 2.9 10(-3) mmHg(-1) for aortic distensibility (sensitivity: 87.5%, specificity: 90%), and of 4.4 m.s(-1) for PWV (sensitivity: 75%, specificity: 100%)., Conclusion: Young asymptomatic adults with MYH11 mutation have an aortic compliance impairment which is not detectable by the sole measurement of the aortic size. Aortic compliance measurement might be part of routine examination in patients suspected of inherited aortic disease even with a normal aortic diameter., (Copyright (c) 2008 Wiley-Liss, Inc.)

Mazabraud syndrome is a rare sporadic disorder, mainly characterized by bone fibrous dysplasia and intramuscular myxomas. We report here two new cases of Mazabraud syndrome. One of our patients (Patient 1) also had café-au-lait spots and multinodular goiter suggestive of McCune-Albright syndrome. We review the 37 previously reported cases with Mazabraud syndrome and discuss the 6/37 patients with criteria of Mazabraud and McCune-Albright syndromes. Based on the clinical overlap between the two syndromes, we tested the GNAS1 gene in blood leukocytes and skin fibroblasts of Patient 1, but found no evidence of an activating mutation in the GNAS1 gene., (Copyright 2001 Wiley-Liss, Inc.)