Christel Thauvin

KLHL3 mutations cause familial hyperkalemic hypertension by impairing ion transport in the distal nephron

Familial hyperkalemic hypertension (FHHt) is a Mendelian form of arterial hypertension that is partially explained by mutations in WNK1 and WNK4 that lead to increased activity of the Na+-Cl− cotransporter (NCC) in the distal nephron. Using combined linkage analysis and whole-exome sequencing in two families, we identified KLHL3 as a third gene responsible for FHHt. Direct sequencing of 43 other affected individuals revealed 11 additional missense mutations that were associated with heterogeneous phenotypes and diverse modes of inheritance. Polymorphisms at KLHL3 were not associated with blood pressure. The KLHL3 protein belongs to the BTB-BACK-kelch family of actin-binding proteins that recruit substrates for Cullin3-based ubiquitin ligase complexes. KLHL3 is coexpressed with NCC and downregulates NCC expression at the cell surface. Our study establishes a role for KLHL3 as a new member of the complex signaling pathway regulating ion homeostasis in the distal nephron and indirectly blood pressure.

Complete exon sequencing of all known Usher syndrome genes greatly improves molecular diagnosis

BackgroundUsher syndrome (USH) combines sensorineural deafness with blindness. It is inherited in an autosomal recessive mode. Early diagnosis is critical for adapted educational and patient management choices, and for genetic counseling. To date, nine causative genes have been identified for the three clinical subtypes (USH1, USH2 and USH3). Current diagnostic strategies make use of a genotyping microarray that is based on the previously reported mutations. The purpose of this study was to design a more accurate molecular diagnosis tool.MethodsWe sequenced the 366 coding exons and flanking regions of the nine known USH genes, in 54 USH patients (27 USH1, 21 USH2 and 6 USH3).ResultsBiallelic mutations were detected in 39 patients (72%) and monoallelic mutations in an additional 10 patients (18.5%). In addition to biallelic mutations in one of the USH genes, presumably pathogenic mutations in another USH gene were detected in seven patients (13%), and another patient carried monoallelic mutations in three different USH genes. Notably, none of the USH3 patients carried detectable mutations in the only known USH3 gene, whereas they all carried mutations in USH2 genes. Most importantly, the currently used microarray would have detected only 30 of the 81 different mutations that we found, of which 39 (48%) were novel.ConclusionsBased on these results, complete exon sequencing of the currently known USH genes stands as a definite improvement for molecular diagnosis of this disease, which is of utmost importance in the perspective of gene therapy.

Mutations in FAM111B Cause Hereditary Fibrosing Poikiloderma with Tendon Contracture, Myopathy, and Pulmonary Fibrosis

Congenital poikiloderma is characterized by a combination of mottled pigmentation, telangiectasia, and epidermal atrophy in the first few months of life. We have previously described a South African European-descent family affected by a rare autosomal-dominant form of hereditary fibrosing poikiloderma accompanied by tendon contracture, myopathy, and pulmonary fibrosis. Here, we report the identification of causative mutations in FAM111B by whole-exome sequencing. In total, three FAM111B missense mutations were identified in five kindreds of different ethnic backgrounds. The mutation segregated with the disease in one large pedigree, and mutations were de novo in two other pedigrees. All three mutations were absent from public databases and were not observed on Sanger sequencing of 388 ethnically matched control subjects. The three single-nucleotide mutations code for amino acid changes that are clustered within a putative trypsin-like cysteine/serine peptidase domain of FAM111B. These findings provide evidence of the involvement of FAM111B in congenital poikiloderma and multisystem fibrosis.

Variable expressivity of the clinical and biochemical phenotype associated with the apolipoprotein E p.Leu149del mutation

Splenomegaly with sea-blue histiocytes, thrombocytopenia and hypertriglyceridemia is a very rare association that has been described in only one report to date. The molecular defect in the two reported patients consists in a deletion of a leucine at position 149 in the receptor-binding region of the apoE molecule. Here, we report on another family in whom the proband and his brother were diagnosed with splenomegaly, thrombocytopenia and hypertriglyceridemia. An apoE p.Leu149del mutation was found in both subjects. A large β band in the VLDL fraction and elevated VLDL cholesterol-to-plasma triglyceride ratio was observed in the proband only. Their mother, presenting with isolated hypertriglyceridemia, also carried the same p.Leu149del mutation. The coexistence of factors facilitating the development of hypertriglyceridemia and/or low HDL-cholesterol level could explain why the proband and his brother developed a splenomegaly with thrombocytopenia, whereas the mother did not. Moreover, the presence of an apoE2 allele in the proband likely explains the more severe phenotype we observed in this subject. In conclusion, the apoE p.Leu149del mutation results in a very striking phenotype including one or all symptoms among splenomegaly, thrombocytopenia and hypertriglyceridemia, and should be considered as a differential diagnosis of storage disorders in the causes of splenomegaly with sea-blue histiocytes.

Autosomal-recessive SASH1 variants associated with a new genodermatosis with pigmentation defects, palmoplantar keratoderma and skin carcinoma.

SASH1 (SAM and SH3 domain-containing protein 1) is a tumor suppressor gene involved in the tumorigenesis of a spectrum of solid cancers. Heterozygous SASH1 variants are known to cause autosomal-dominant dyschromatosis. Homozygosity mapping and whole-exome sequencing were performed in a consanguineous Moroccan family with two affected siblings presenting an unclassified phenotype associating an abnormal pigmentation pattern (hypo- and hyperpigmented macules of the trunk and face and areas of reticular hypo- and hyperpigmentation of the extremities), alopecia, palmoplantar keratoderma, ungueal dystrophy and recurrent spinocellular carcinoma. We identified a homozygous variant in SASH1 (c.1849G>A; p.Glu617Lys) in both affected individuals. Wound-healing assay showed that the patient’s fibroblasts were better able than control fibroblasts to migrate. Following the identification of SASH1 heterozygous variants in dyschromatosis, we used reverse phenotyping to show that autosomal-recessive variants of this gene could be responsible for an overlapping but more complex phenotype that affected skin appendages. SASH1 should be added to the list of genes responsible for autosomal-dominant and -recessive genodermatosis, with no phenotype in heterozygous patients in the recessive form, and to the list of genes responsible for a predisposition to skin cancer.

A systematic variant screening in familial cases of congenital heart defects demonstrates the usefulness of molecular genetics in this field

The etiology of congenital heart defect (CHD) combines environmental and genetic factors. So far, there were studies reporting on the screening of a single gene on unselected CHD or on familial cases selected for specific CHD types. Our goal was to systematically screen a proband of familial cases of CHD on a set of genetic tests to evaluate the prevalence of disease-causing variant identification. A systematic screening of GATA4, NKX2-5, ZIC3 and Multiplex ligation-dependent probe amplification (MLPA) P311 Kit was setup on the proband of 154 families with at least two cases of non-syndromic CHD. Additionally, ELN screening was performed on families with supravalvular arterial stenosis. Twenty-two variants were found, but segregation analysis confirmed unambiguously the causality of 16 variants: GATA4 (1 ×), NKX2-5 (6 ×), ZIC3 (3 ×), MLPA (2 ×) and ELN (4 ×). Therefore, this approach was able to identify the causal variant in 10.4% of familial CHD cases. This study demonstrated the existence of a de novo variant even in familial CHD cases and the impact of CHD variants on adult cardiac condition even in the absence of CHD. This study showed that the systematic screening of genetic factors is useful in familial CHD cases with up to 10.4% elucidated cases. When successful, it drastically improved genetic counseling by discovering unaffected variant carriers who are at risk of transmitting their variant and are also exposed to develop cardiac complications during adulthood thus prompting long-term cardiac follow-up. This study provides an important baseline at dawning of the next-generation sequencing era.

The expanding spectrum of COL2A1 gene variants IN 136 patients with a skeletal dysplasia phenotype.

Heterozygous COL2A1 variants cause a wide spectrum of skeletal dysplasia termed type II collagenopathies. We assessed the impact of this gene in our French series. A decision tree was applied to select 136 probands (71 Stickler cases, 21 Spondyloepiphyseal dysplasia congenita cases, 11 Kniest dysplasia cases, and 34 other dysplasia cases) before molecular diagnosis by Sanger sequencing. We identified 66 different variants among the 71 positive patients. Among those patients, 18 belonged to multiplex families and 53 were sporadic. Most variants (38/44, 86%) were located in the triple helical domain of the collagen chain and glycine substitutions were mainly observed in severe phenotypes, whereas arginine to cysteine changes were more often encountered in moderate phenotypes. This series of skeletal dysplasia is one of the largest reported so far, adding 44 novel variants (15%) to published data. We have confirmed that about half of our Stickler patients (46%) carried a COL2A1 variant, and that the molecular spectrum was different across the phenotypes. To further address the question of genotype–phenotype correlation, we plan to screen our patients for other candidate genes using a targeted next-generation sequencing approach.

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

BackgroundHereditary 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.MethodsClinical and molecular data of two families and eight independent sporadic cases, including six new cases, were collected.ResultsKey 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.ConclusionsHFP 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.

Pathogenic DDX3X mutations impair RNA metabolism and neurogenesis during fetal cortical development

De novo germline mutations in the RNA helicase DDX3X account for 1-3% of unexplained intellectual disability (ID) cases in females, and are associated with autism, brain malformations, and epilepsy. Yet, the developmental and molecular mechanisms by which DDX3X mutations impair brain function are unknown. Here we use human and mouse genetics, and cell biological and biochemical approaches to elucidate mechanisms by which pathogenic DDX3X variants disrupt brain development. We report the largest clinical cohort to date with DDX3X mutations (n=78), demonstrating a striking correlation between recurrent dominant missense mutations, polymicrogyria, and the most severe clinical outcomes. We show that Ddx3x controls cortical development by regulating neuronal generation and migration. Severe DDX3X missense mutations profoundly disrupt RNA helicase activity and induce ectopic RNA-protein granules and aberrant translation in neural progenitors and neurons. Together, our study demonstrates novel mechanisms underlying DDX3X syndrome, and highlights roles for RNA-protein aggregates in the pathogenesis of neurodevelopmental disease.

G.P.156

Hereditary Fibrosing Poikiloderma (HFP) with tendon contracture and pulmonary fibrosis has been described in a South-African family with autosomal dominant inheritance by Khumalo et al. in 2006. Recently, we confirmed the existence of this new syndrome by reporting four additional cases and identified the causative gene called FAM111B (NM_198947.3) by whole exome sequencing. Here we report the clinical and molecular data of seven independent patients: four males and three females (ages 3–38). Key features consist of: (i) congenital poikiloderma, hypotrichosis, hypohidrosis; (ii) muscle contractures in particular triceps surae and/or biceps brachii muscle contractures; (iii) progressive muscular weakness with a proximal and distal pattern and (iv) progressive pulmonary fibrosis. Muscle MRI showed extensive fatty infiltration confirmed by muscle biopsy. Histological examination of skeletal muscle revealed a partial loss of muscle tissue associated with extensive fibrofatty tissue infiltration regardless of age. There was no indication of denervation, necrosis, or inflammation. Microscopy of the skin showed a sclerodermiform aspect with fibrosis and alterations of the elastic network. FAM111B gene analysis identified five different missense variants (two mutations were shared respectively by three and two patients). 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. Functional studies are ongoing to better understand the pathophysiology of this entity. In conclusion, HFP with tendon contracture, myopathy and pulmonary fibrosis, a phenotypically recognisable syndrome, is caused by autosomal dominant mutations in FAM111B gene. These findings provide genetic evidence for a new pathogenetic pathway for muscle impairment.