Bi-allelic variants in WNT7B disrupt the development of multiple organs in humans.

BACKGROUND: Pulmonary hypoplasia, Diaphragmatic anomalies, Anophthalmia/microphthalmia and Cardiac defects delineate the PDAC syndrome. We aim to identify the cause of PDAC syndrome in patients who do not carry pathogenic variants in RARB and STRA6, which have been previously associated with this disorder. METHODS: We sequenced the exome of patients with unexplained PDAC syndrome and performed functional validation of candidate variants. RESULTS: We identified bi-allelic variants in WNT7B in fetuses with PDAC syndrome from two unrelated families. In one family, the fetus was homozygous for the c.292C>T (p.(Arg98*)) variant whereas the fetuses from the other family were compound heterozygous for the variants c.225C>G (p.(Tyr75*)) and c.562G>A (p.(Gly188Ser)). Finally, a molecular autopsy by proxy in a consanguineous couple that lost two babies due to lung hypoplasia revealed that both parents carry the p.(Arg98*) variant. Using a WNT signalling canonical luciferase assay, we demonstrated that the identified variants are deleterious. In addition, we found that wnt7bb mutant zebrafish display a defect of the swimbladder, an air-filled organ that is a structural homolog of the mammalian lung, suggesting that the function of WNT7B has been conserved during evolution for the development of these structures. CONCLUSION: Our findings indicate that defective WNT7B function underlies a form of lung hypoplasia that is associated with the PDAC syndrome, and provide evidence for involvement of the WNT-ß-catenin pathway in human lung, tracheal, ocular, cardiac, and renal development.

A Central Role of Telomere Dysfunction in the Formation of a Unique Translocation within the Sub-Telomere Region Resulting in Duplication and Partial Trisomy.

Telomeres play a major role in maintaining genome stability and integrity. Putative involvement of telomere dysfunction in the formation of various types of chromosomal aberrations is an area of active research. Here, we report a case of a six-month-old boy with a chromosomal gain encompassing the 11q22.3q25 region identified by SNP array analysis. The size of the duplication is 26.7 Mb and contains 170 genes (OMIM). The duplication results in partial trisomy of the region in question with clinical consequences, including bilateral renal dysplasia, delayed development, and a heart defect. Moreover, the karyotype determined by R-banding and chromosome painting as well as by hybridization with specific sub-telomere probes revealed the presence of an unbalanced t(9;11)(p24;q22.3) translocation with a unique breakpoint involving the sub-telomere region of the short arm of chromosome 9. The karyotypes of the parents were normal. Telomere integrity in circulating lymphocytes from the child and from his parents was assessed using an automated high-throughput method based on fluorescence in situ hybridization (FISH) with telomere- and centromere-specific PNA probes followed by M-FISH multicolor karyotyping. Very short telomeres, as well as an increased frequency of telomere loss and formation of telomere doublets, were detected in the child's cells. Interestingly, similar telomere profiles were found in the circulating lymphocytes of the father. Moreover, an assessment of clonal telomere aberrations identified chromosomes 9 and 11 with particularly high frequencies of such aberrations. These findings strongly suggest that telomere dysfunction plays a central role in the formation of this specific unbalanced chromosome rearrangement via chromosome end-to-end fusion and breakage-fusion-bridge cycles.

Telomere aberrations, including telomere loss, doublets, and extreme shortening, are increased in patients with infertility.

OBJECTIVE: To test the hypothesis that telomere shortening and/or loss are risk factors for infertility. DESIGN: Retrospective analysis of the telomere status in patients with infertility using conventional cytogenetic data collected prospectively. SETTING: Academic centers. PATIENT(S): Cytogenetic slides with cultured peripheral lymphocytes from 50 patients undergoing fertility treatment and 150 healthy donors, including 100 donors matched for age. INTERVENTION(S): Cytogenetic slides were used to detect chromosomal and telomere aberrations. MAIN OUTCOME MEASURE(S): Telomere length and telomere aberrations were analyzed after telomere and centromere staining. RESULT(S): The mean telomere length of patients consulting for infertility was significantly less than that of healthy donors of similar age. Moreover, patients with infertility showed significantly more extreme telomere loss and telomere doublet formation than healthy controls. Telomere shortening and/or telomere aberrations were more pronounced in patients with structural chromosomal aberrations. Dicentric chromosomes were identified in 6/13 patients, with constitutional chromosomal aberrations leading to chromosomal instability that correlated with chromosomal end-to-end fusions. CONCLUSION(S): Our findings demonstrate the feasibility of analyzing telomere aberrations in addition to chromosomal aberrations, using cytogenetic slides. Telomere attrition and/or dysfunction represent the main common cytogenetic characteristic of patients with infertility, leading to potential implications for fertility assessment. Pending further studies, these techniques that correlate the outcome of assisted reproduction and telomere integrity status may represent a novel and useful diagnostic and/or prognostic tool for medical care in this field.

Telomere and Centromere Staining Followed by M-FISH Improves Diagnosis of Chromosomal Instability and Its Clinical Utility.

Dicentric chromosomes are a relevant marker of chromosomal instability. Their appearance is associated with telomere dysfunction, leading to cancer progression and a poor clinical outcome. Here, we present Telomere and Centromere staining followed by M-FISH (TC+M-FISH) for improved detection of telomere dysfunction and the identification of dicentric chromosomes in cancer patients and various genetic syndromes. Significant telomere length shortening and significantly higher frequencies of telomere loss and deletion were found in the peripheral lymphocytes of patients with cancer and genetic syndromes relative to similar age-matched healthy donors. We assessed our technique against conventional cytogenetics for the detection of dicentric chromosomes by subjecting metaphase preparations to both approaches. We identified dicentric chromosomes in 28/50 cancer patients and 21/44 genetic syndrome patients using our approach, but only 7/50 and 12/44, respectively, using standard cytogenetics. We ascribe this discrepancy to the identification of the unique configuration of dicentric chromosomes. We observed significantly higher frequencies of telomere loss and deletion in patients with dicentric chromosomes (p < 10-4). TC+M-FISH analysis is superior to classical cytogenetics for the detection of chromosomal instability. Our approach is a relatively simple but useful tool for documenting telomere dysfunction and chromosomal instability with the potential to become a standard additional diagnostic tool in medical genetics and the clinic.

Biallelic variants in the transcription factor PAX7 are a new genetic cause of myopathy.

PURPOSE: Skeletal muscle growth and regeneration rely on muscle stem cells, called satellite cells. Specific transcription factors, particularly PAX7, are key regulators of the function of these cells. Knockout of this factor in mice leads to poor postnatal survival; however, the consequences of a lack of PAX7 in humans have not been established. METHODS: Here, we study five individuals with myopathy of variable severity from four unrelated consanguineous couples. Exome sequencing identified pathogenic variants in the PAX7 gene. Clinical examination, laboratory tests, and muscle biopsies were performed to characterize the disease. RESULTS: The disease was characterized by hypotonia, ptosis, muscular atrophy, scoliosis, and mildly dysmorphic facial features. The disease spectrum ranged from mild to severe and appears to be progressive. Muscle biopsies showed the presence of atrophic fibers and fibroadipose tissue replacement, with the absence of myofiber necrosis. A lack of PAX7 expression was associated with satellite cell pool exhaustion; however, the presence of residual myoblasts together with regenerating myofibers suggest that a population of PAX7-independent myogenic cells partially contributes to muscle regeneration. CONCLUSION: These findings show that biallelic variants in the master transcription factor PAX7 cause a new type of myopathy that specifically affects satellite cell survival.

Further delineation of the MECP2 duplication syndrome phenotype in 59 French male patients, with a particular focus on morphological and neurological features.

The Xq28 duplication involving the MECP2 gene (MECP2 duplication) has been mainly described in male patients with severe developmental delay (DD) associated with spasticity, stereotypic movements and recurrent infections. Nevertheless, only a few series have been published. We aimed to better describe the phenotype of this condition, with a focus on morphological and neurological features. Through a national collaborative study, we report a large French series of 59 affected males with interstitial MECP2 duplication. Most of the patients (93%) shared similar facial features, which evolved with age (midface hypoplasia, narrow and prominent nasal bridge, thick lower lip, large prominent ears), thick hair, livedo of the limbs, tapered fingers, small feet and vasomotor troubles. Early hypotonia and global DD were constant, with 21% of patients unable to walk. In patients able to stand, lower limbs weakness and spasticity led to a singular standing habitus: flexion of the knees, broad-based stance with pseudo-ataxic gait. Scoliosis was frequent (53%), such as divergent strabismus (76%) and hypermetropia (54%), stereotypic movements (89%), without obvious social withdrawal and decreased pain sensitivity (78%). Most of the patients did not develop expressive language, 35% saying few words. Epilepsy was frequent (59%), with a mean onset around 7.4 years of age, and often (62%) drug-resistant. Other medical issues were frequent: constipation (78%), and recurrent infections (89%), mainly lung. We delineate the clinical phenotype of MECP2 duplication syndrome in a large series of 59 males. Pulmonary hypertension appeared as a cause of early death in these patients, advocating its screening early in life.

High Rate of Recurrent De Novo Mutations in Developmental and Epileptic Encephalopathies.

Developmental and epileptic encephalopathy (DEE) is a group of conditions characterized by the co-occurrence of epilepsy and intellectual disability (ID), typically with developmental plateauing or regression associated with frequent epileptiform activity. The cause of DEE remains unknown in the majority of cases. We performed whole-genome sequencing (WGS) in 197 individuals with unexplained DEE and pharmaco-resistant seizures and in their unaffected parents. We focused our attention on de novo mutations (DNMs) and identified candidate genes containing such variants. We sought to identify additional subjects with DNMs in these genes by performing targeted sequencing in another series of individuals with DEE and by mining various sequencing datasets. We also performed meta-analyses to document enrichment of DNMs in candidate genes by leveraging our WGS dataset with those of several DEE and ID series. By combining these strategies, we were able to provide a causal link between DEE and the following genes: NTRK2, GABRB2, CLTC, DHDDS, NUS1, RAB11A, GABBR2, and SNAP25. Overall, we established a molecular diagnosis in 63/197 (32%) individuals in our WGS series. The main cause of DEE in these individuals was de novo point mutations (53/63 solved cases), followed by inherited mutations (6/63 solved cases) and de novo CNVs (4/63 solved cases). De novo missense variants explained a larger proportion of individuals in our series than in other series that were primarily ascertained because of ID. Moreover, these DNMs were more frequently recurrent than those identified in ID series. These observations indicate that the genetic landscape of DEE might be different from that of ID without epilepsy.

Affected female carriers of MTM1 mutations display a wide spectrum of clinical and pathological involvement: delineating diagnostic clues.

X-linked myotubular myopathy (XLMTM), a severe congenital myopathy, is caused by mutations in the MTM1 gene located on the X chromosome. A majority of affected males die in the early postnatal period, whereas female carriers are believed to be usually asymptomatic. Nevertheless, several affected females have been reported. To assess the phenotypic and pathological spectra of carrier females and to delineate diagnostic clues, we characterized 17 new unrelated affected females and performed a detailed comparison with previously reported cases at the clinical, muscle imaging, histological, ultrastructural and molecular levels. Taken together, the analysis of this large cohort of 43 cases highlights a wide spectrum of clinical severity ranging from severe neonatal and generalized weakness, similar to XLMTM male, to milder adult forms. Several females show a decline in respiratory function. Asymmetric weakness is a noteworthy frequent specific feature potentially correlated to an increased prevalence of highly skewed X inactivation. Asymmetry of growth was also noted. Other diagnostic clues include facial weakness, ptosis and ophthalmoplegia, skeletal and joint abnormalities, and histopathological signs that are hallmarks of centronuclear myopathy such as centralized nuclei and necklace fibers. The histopathological findings also demonstrate a general disorganization of muscle structure in addition to these specific hallmarks. Thus, MTM1 mutations in carrier females define a specific myopathy, which may be independent of the presence of an XLMTM male in the family. As several of the reported affected females carry large heterozygous MTM1 deletions not detectable by Sanger sequencing, and as milder phenotypes present as adult-onset limb-girdle myopathy, the prevalence of this myopathy is likely to be greatly underestimated. This report should aid diagnosis and thus the clinical management and genetic counseling of MTM1 carrier females. Furthermore, the clinical and pathological history of this cohort may be useful for therapeutic projects in males with XLMTM, as it illustrates the spectrum of possible evolution of the disease in patients surviving long term.

Uncommon nucleotide excision repair phenotypes revealed by targeted high-throughput sequencing.

BACKGROUND: Deficient nucleotide excision repair (NER) activity causes a variety of autosomal recessive diseases including xeroderma pigmentosum (XP) a disorder which pre-disposes to skin cancer, and the severe multisystem condition known as Cockayne syndrome (CS). In view of the clinical overlap between NER-related disorders, as well as the existence of multiple phenotypes and the numerous genes involved, we developed a new diagnostic approach based on the enrichment of 16 NER-related genes by multiplex amplification coupled with next-generation sequencing (NGS). METHODS: Our test cohort consisted of 11 DNA samples, all with known mutations and/or non pathogenic SNPs in two of the tested genes. We then used the same technique to analyse samples from a prospective cohort of 40 patients. Multiplex amplification and sequencing were performed using AmpliSeq protocol on the Ion Torrent PGM (Life Technologies). RESULTS: We identified causative mutations in 17 out of the 40 patients (43%). Four patients showed biallelic mutations in the ERCC6(CSB) gene, five in the ERCC8(CSA) gene: most of them had classical CS features but some had very mild and incomplete phenotypes. A small cohort of 4 unrelated classic XP patients from the Basque country (Northern Spain) revealed a common splicing mutation in POLH (XP-variant), demonstrating a new founder effect in this population. Interestingly, our results also found ERCC2(XPD), ERCC3(XPB) or ERCC5(XPG) mutations in two cases of UV-sensitive syndrome and in two cases with mixed XP/CS phenotypes. CONCLUSIONS: Our study confirms that NGS is an efficient technique for the analysis of NER-related disorders on a molecular level. It is particularly useful for phenotypes with combined features or unusually mild symptoms. Targeted NGS used in conjunction with DNA repair functional tests and precise clinical evaluation permits rapid and cost-effective diagnosis in patients with NER-defects.