A genomic rearrangement resulting in a tandem duplication is associated with split hand-split foot malformation 3 (SHFM3) at 10q24

Split hand-split foot malformation (SHFM) is characterized by hypoplasia/aplasia of the central digits with fusion of the remaining digits. SHFM is usually an autosomal dominant condition and at least five loci have been identified in humans. Mutation analysis of the DACTYLIN gene, suspected to be responsible for SHFM3 in chromosome 10q24, was conducted in seven SHFM patients. We screened the coding region of DACTYLIN by single-strand conformation polymorphism and sequencing, and found no point mutations. However, Southern, pulsed field gel electrophoresis and dosage analyses demonstrated a complex rearrangement associated with a ∼0.5 Mb tandem duplication in all the patients. The distal and proximal breakpoints were within an 80 and 130 kb region, respectively. This duplicated region contained a disrupted extra copy of the DACTYLIN gene and the entire LBX1 and β-TRCP genes, known to be involved in limb development. The possible role of these genes in the SHFM3 phenotype is discusse

MINPP1 prevents intracellular accumulation of the chelator inositol hexakisphosphate and is mutated in Pontocerebellar Hypoplasia

Inositol polyphosphates are vital metabolic and secondary messengers, involved in diverse cellular functions. Therefore, tight regulation of inositol polyphosphate metabolism is essential for proper cell physiology. Here, we describe an early-onset neurodegenerative syndrome caused by loss-of-function mutations in the multiple inositol-polyphosphate phosphatase 1 gene (MINPP1). Patients are found to have a distinct type of Pontocerebellar Hypoplasia with typical basal ganglia involvement on neuroimaging. We find that patient-derived and genome edited MINPP1−/− induced stem cells exhibit an inefficient neuronal differentiation combined with an increased cell death. MINPP1 deficiency results in an intracellular imbalance of the inositol polyphosphate metabolism. This metabolic defect is characterized by an accumulation of highly phosphorylated inositols, mostly inositol hexakisphosphate (IP6), detected in HEK293 cells, fibroblasts, iPSCs and differentiating neurons lacking MINPP1. In mutant cells, higher IP6 level is expected to be associated with an increased chelation of intracellular cations, such as iron or calcium, resulting in decreased levels of available ions. These data suggest the involvement of IP6-mediated chelation on Pontocerebellar Hypoplasia disease pathology and thereby highlight the critical role of MINPP1 in the regulation of human brain development and homeostasis

Clinical evidence for a mandibular to maxillary transformation in auriculocondylar syndrome

Université Sorbonne Paris-Cité Pôle - project number SPC/JFG/2013-031ANR - project EvoDevoMut 2010E-Rare CRANIRARE gran

Clinical, biochemical and genetic spectrum of 70 patients with ACAD9 deficiency: Is riboflavin supplementation effective?

Background: Mitochondrial acyl-CoA dehydrogenase family member 9 (ACAD9) is essential for the assembly of mitochondrial respiratory chain complex I. Disease causing biallelic variants in ACAD9 have been reported in individuals presenting with lactic acidosis and cardiomyopathy. Results: We describe the genetic, clinical and biochemical findings in a cohort of 70 patients, of whom 29 previously unpublished. We found 34 known and 18 previously unreported variants in ACAD9. No patients harbored biallelic loss of function mutations, indicating that this combination is unlikely to be compatible with life. Causal pathogenic variants were distributed throughout the entire gene, and there was no obvious genotype-phenotype correlation. Most of the patients presented in the first year of life. For this subgroup the survival was poor (50% not surviving the first 2 years) comparing to patients with a later presentation (more than 90% surviving 10 years). The most common clinical findings were cardiomyopathy (85%), muscular weakness (75%) and exercise intolerance (72%). Interestingly, severe intellectual deficits were only reported in one patient and

Mouse Studies to Shape Clinical Trials for Mitochondrial Diseases: High Fat Diet in Harlequin Mice

BACKGROUND: Therapeutic options in human mitochondrial oxidative phosphorylation (OXPHOS) diseases have been poorly evaluated mostly because of the scarcity of cohorts and the inter-individual variability of disease progression. Thus, while a high fat diet (HFD) is often recommended, data regarding efficacy are limited. Our objectives were 1) to determine our ability to evaluate therapeutic options in the Harlequin OXPHOS complex I (CI)-deficient mice, in the context of a mitochondrial disease with human hallmarks and 2) to assess the effects of a HFD. METHODS AND FINDINGS: Before launching long and expensive animal studies, we showed that palmitate afforded long-term death-protection in 3 CI-mutant human fibroblasts cell lines. We next demonstrated that using the Harlequin mouse, it was possible to draw solid conclusions on the efficacy of a 5-month-HFD on neurodegenerative symptoms. Moreover, we could identify a group of highly responsive animals, echoing the high variability of the disease progression in Harlequin mice. CONCLUSIONS: These results suggest that a reduced number of patients with identical genetic disease should be sufficient to reach firm conclusions as far as the potential existence of responders and non responders is recognized. They also positively prefigure HFD-trials in OXPHOS-deficient patients

Nuclear Outsourcing of RNA Interference Components to Human Mitochondria

MicroRNAs (miRNAs) are small non-coding RNAs that associate with Argonaute proteins to regulate gene expression at the post-transcriptional level in the cytoplasm. However, recent studies have reported that some miRNAs localize to and function in other cellular compartments. Mitochondria harbour their own genetic system that may be a potential site for miRNA mediated post-transcriptional regulation. We aimed at investigating whether nuclear-encoded miRNAs can localize to and function in human mitochondria. To enable identification of mitochondrial-enriched miRNAs, we profiled the mitochondrial and cytosolic RNA fractions from the same HeLa cells by miRNA microarray analysis. Mitochondria were purified using a combination of cell fractionation and immunoisolation, and assessed for the lack of protein and RNA contaminants. We found 57 miRNAs differentially expressed in HeLa mitochondria and cytosol. Of these 57, a signature of 13 nuclear-encoded miRNAs was reproducibly enriched in mitochondrial RNA and validated by RT-PCR for hsa-miR-494, hsa-miR-1275 and hsa-miR-1974. The significance of their mitochondrial localization was investigated by characterizing their genomic context, cross-species conservation and instrinsic features such as their size and thermodynamic parameters. Interestingly, the specificities of mitochondrial versus cytosolic miRNAs were underlined by significantly different structural and thermodynamic parameters. Computational targeting analysis of most mitochondrial miRNAs revealed not only nuclear but also mitochondrial-encoded targets. The functional relevance of miRNAs in mitochondria was supported by the finding of Argonaute 2 localization to mitochondria revealed by immunoblotting and confocal microscopy, and further validated by the co-immunoprecipitation of the mitochondrial transcript COX3. This study provides the first comprehensive view of the localization of RNA interference components to the mitochondria. Our data outline the molecular bases for a novel layer of crosstalk between nucleus and mitochondria through a specific subset of human miRNAs that we termed ‘mitomiRs’

Expanding the clinical spectrum of hereditary fibrosing poikiloderma with tendon contractures, myopathy and pulmonary fibrosis due to <i>FAM111B </i>mutations

BACKGROUND: Hereditary Fibrosing Poikiloderma (HFP) with tendon contractures, myopathy and pulmonary fibrosis (POIKTMP [MIM 615704]) is a very recently described entity of syndromic inherited poikiloderma. Previously by using whole exome sequencing in five families, we identified the causative gene, FAM111B (NM_198947.3), the function of which is still unknown. Our objective in this study was to better define the specific features of POIKTMP through a larger series of patients. METHODS: Clinical and molecular data of two families and eight independent sporadic cases, including six new cases, were collected. RESULTS: Key features consist of: (i) early-onset poikiloderma, hypotrichosis and hypohidrosis; (ii) multiple contractures, in particular triceps surae muscle contractures; (iii) diffuse progressive muscular weakness; (iv) pulmonary fibrosis in adulthood and (v) other features including exocrine pancreatic insufficiency, liver impairment and growth retardation. Muscle magnetic resonance imaging was informative and showed muscle atrophy and fatty infiltration. Histological examination of skeletal muscle revealed extensive fibroadipose tissue infiltration. Microscopy of the skin showed a scleroderma-like aspect with fibrosis and alterations of the elastic network. FAM111B gene analysis identified five different missense variants (two recurrent mutations were found respectively in three and four independent families). All the mutations were predicted to localize in the trypsin-like cysteine/serine peptidase domain of the protein. We suggest gain-of-function or dominant-negative mutations resulting in FAM111B enzymatic activity changes. CONCLUSIONS: HFP with tendon contractures, myopathy and pulmonary fibrosis, is a multisystemic disorder due to autosomal dominant FAM111B mutations. Future functional studies will help in understanding the specific pathological process of this fibrosing disorder

rs5888 Variant of SCARB1 Gene Is a Possible Susceptibility Factor for Age-Related Macular Degeneration

Major genetic factors for age-related macular degeneration (AMD) have recently been identified as susceptibility risk factors, including variants in the CFH gene and the ARMS2 LOC387715/HTRA1locus. Our purpose was to perform a case-control study in two populations among individuals who did not carry risk variants for CFHY402H and LOC387715 A69S (ARMS2), called “study” individuals, in order to identify new genetic risk factors. Based on a candidate gene approach, we analyzed SNP rs5888 of the SCARB1 gene, coding for SRBI, which is involved in the lipid and lutein pathways. This study was conducted in a French series of 1241 AMD patients and 297 controls, and in a North American series of 1257 patients with advanced AMD and 1732 controls. Among these individuals, we identified 61 French patients, 77 French controls, 85 North American patients and 338 North American controls who did not carry the CFH nor ARMS2 polymorphisms. An association between AMD and the SCARB1 gene was seen among the study subjects. The genotypic distribution of the rs5888 polymorphism was significantly different between cases and controls in the French population (p<0.006). Heterozygosity at the rs5888 SNP increased risk of AMD compared to the CC genotypes in the French study population (odds ratio (OR) = 3.5, CI95%: 1.4–8.9, p<0.01) and after pooling the 2 populations (OR = 2.9, 95% CI: 1.6–5.3, p<0.002). Subgroup analysis in exudative forms of AMD revealed a pooled OR of 3.6 for individuals heterozygous for rs5888 (95% CI: 1.7–7.6, p<0.0015). These results suggest the possible contribution of SCARB1, a new genetic factor in AMD, and implicate a role for cholesterol and antioxidant micronutrient (lutein and vitamin E) metabolism in AMD

ISL1 Directly Regulates FGF10 Transcription during Human Cardiac Outflow Formation

The LIM homeodomain gene Islet-1 (ISL1) encodes a transcription factor that has been associated with the multipotency of human cardiac progenitors, and in mice enables the correct deployment of second heart field (SHF) cells to become the myocardium of atria, right ventricle and outflow tract. Other markers have been identified that characterize subdomains of the SHF, such as the fibroblast growth factor Fgf10 in its anterior region. While functional evidence of its essential contribution has been demonstrated in many vertebrate species, SHF expression of Isl1 has been shown in only some models. We examined the relationship between human ISL1 and FGF10 within the embryonic time window during which the linear heart tube remodels into four chambers. ISL1 transcription demarcated an anatomical region supporting the conserved existence of a SHF in humans, and transcription factors of the GATA family were co-expressed therein. In conjunction, we identified a novel enhancer containing a highly conserved ISL1 consensus binding site within the FGF10 first intron. ChIP and EMSA demonstrated its direct occupation by ISL1. Transcription mediated by ISL1 from this FGF10 intronic element was enhanced by the presence of GATA4 and TBX20 cardiac transcription factors. Finally, transgenic mice confirmed that endogenous factors bound the human FGF10 intronic enhancer to drive reporter expression in the developing cardiac outflow tract. These findings highlight the interest of examining developmental regulatory networks directly in human tissues, when possible, to assess candidate non-coding regions that may be responsible for congenital malformations

Phenotypic spectrum and transcriptomic profile associated with germline variants in TRAF7

PURPOSE: Somatic variants in tumor necrosis factor receptor-associated factor 7 (TRAF7) cause meningioma, while germline variants have recently been identified in seven patients with developmental delay and cardiac, facial, and digital anomalies. We aimed to define the clinical and mutational spectrum associated with TRAF7 germline variants in a large series of patients, and to determine the molecular effects of the variants through transcriptomic analysis of patient fibroblasts. METHODS: We performed exome, targeted capture, and Sanger sequencing of patients with undiagnosed developmental disorders, in multiple independent diagnostic or research centers. Phenotypic and mutational comparisons were facilitated through data exchange platforms. Whole-transcriptome sequencing was performed on RNA from patient- and control-derived fibroblasts. RESULTS: We identified heterozygous missense variants in TRAF7 as the cause of a developmental delay-malformation syndrome in 45 patients. Major features include a recognizable facial gestalt (characterized in particular by blepharophimosis), short neck, pectus carinatum, digital deviations, and patent ductus arteriosus. Almost all variants occur in the WD40 repeats and most are recurrent. Several differentially expressed genes were identified in patient fibroblasts. CONCLUSION: We provide the first large-scale analysis of the clinical and mutational spectrum associated with the TRAF7 developmental syndrome, and we shed light on its molecular etiology through transcriptome studies