TRPV1 variants impair intracellular Ca2+ signaling and may confer susceptibility to malignant hyperthermia.

PURPOSE: Malignant hyperthermia (MH) is a pharmacogenetic disorder arising from uncontrolled muscle calcium release due to an abnormality in the sarcoplasmic reticulum (SR) calcium-release mechanism triggered by halogenated inhalational anesthetics. However, the molecular mechanisms involved are still incomplete. METHODS: We aimed to identify transient receptor potential vanilloid 1 (TRPV1) variants within the entire coding sequence in patients who developed sensitivity to MH of unknown etiology. In vitro and in vivo functional studies were performed in heterologous expression system, trpv1-/- mice, and a murine model of human MH. RESULTS: We identified TRPV1 variants in two patients and their heterologous expression in muscles of trpv1-/- mice strongly enhanced calcium release from SR upon halogenated anesthetic stimulation, suggesting they could be responsible for the MH phenotype. We confirmed the in vivo significance by using mice with a knock-in mutation (Y524S) in the type I ryanodine receptor (Ryr1), a mutation analogous to the Y522S mutation associated with MH in humans. We showed that the TRPV1 antagonist capsazepine slows the heat-induced hypermetabolic response in this model. CONCLUSION: We propose that TRPV1 contributes to MH and could represent an actionable therapeutic target for prevention of the pathology and also be responsible for MH sensitivity when mutated.

Functional Characterization and Rescue of a Deep Intronic Mutation in OCRL Gene Responsible for Lowe Syndrome.

Dent-2 disease and Lowe syndrome are two pathologies caused by mutations in inositol polyphosphate 5-phosphatase OCRL gene. Both conditions share proximal tubulopathy evolving to chronic kidney failure. Lowe syndrome is in addition defined by a bilateral congenital cataract, intellectual disability, and hypotonia. The pathology evolves in two decades to a severe condition with renal complications and a fatal issue. We describe here a proof of principle for a targeted gene therapy on a mutation of the OCRL gene that is associated with Lowe syndrome. The affected patient bears a deep intronic mutation inducing a pseudo-exon inclusion in the mRNA, leading to a OCRL-1 protein loss. An exon-skipping strategy was designed to correct the effect of the mutation in cultured cells. We show that a recombinant U7-modified small RNA efficiently triggered the restoration of normal OCRL expression at mRNA and protein levels in patient's fibroblasts. Moreover, the PI(4,5)P2 accumulation and cellular alterations that are hallmark of OCRL-1 dysfunction were also rescued. Altogether, we provide evidence that the restoration of OCRL-1 protein, even at a reduced level, through RNA-based therapy represents a potential therapeutic approach for patients with OCRL splice mutations.

OCRL-mutated fibroblasts from patients with Dent-2 disease exhibit INPP5B-independent phenotypic variability relatively to Lowe syndrome cells.

OCRL mutations are associated with both Lowe syndrome and Dent-2 disease, two rare X-linked conditions. Lowe syndrome is an oculo-cerebro-renal disorder, whereas Dent-2 patients mainly present renal proximal tubulopathy. Loss of OCRL-1, a phosphoinositide-5-phosphatase, leads in Lowe patients' fibroblasts to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) accumulation, with defects in F-actin network, α-actinin distribution and ciliogenesis, whereas fibroblasts of Dent-2 patients are still uncharacterized. To search for mechanisms linked to clinical variability observed between these two OCRL mutation-associated pathologies, we compared dermal fibroblasts from independent patients, four affected by Dent-2 disease and six with Lowe syndrome. For the first time, we describe that Dent-2 fibroblasts with OCRL loss-of-function (LOF) mutations exhibit decrease in actin stress fibers, appearance of punctate α-actinin signals and alteration in primary cilia formation. Interestingly, we quantified these phenotypes as clearly intermediate between Lowe and control fibroblasts, thus suggesting that levels of these defects correlate with clinical variations observed between patients with OCRL mutations. In addition, we show that Lowe and Dent-2 fibroblasts display similar PI(4,5)P2 accumulation levels. Finally, we analyzed INPP5B, a paralogous gene already reported to exhibit functional redundancy with OCRL, and report neither differences in its expression at RNA or protein levels, nor specific allelic variations between fibroblasts of patients. Altogether, we describe here differential phenotypes between fibroblasts from Lowe and Dent-2 patients, both associated with OCRL LOF mutations, we exclude direct roles of PI(4,5)P2 and INPP5B in this phenotypic variability and we underline potential key alterations leading to ocular and neurological clinical features in Lowe syndrome.

Mutations in CNTNAP1 and ADCY6 are responsible for severe arthrogryposis multiplex congenita with axoglial defects.

Non-syndromic arthrogryposis multiplex congenita (AMC) is characterized by multiple congenital contractures resulting from reduced fetal mobility. Genetic mapping and whole exome sequencing (WES) were performed in 31 multiplex and/or consanguineous undiagnosed AMC families. Although this approach identified known AMC genes, we here report pathogenic mutations in two new genes. Homozygous frameshift mutations in CNTNAP1 were found in four unrelated families. Patients showed a marked reduction in motor nerve conduction velocity (<10 m/s) and transmission electron microscopy (TEM) of sciatic nerve in the index cases revealed severe abnormalities of both nodes of Ranvier width and myelinated axons. CNTNAP1 encodes CASPR, an essential component of node of Ranvier domains which underlies saltatory conduction of action potentials along the myelinated axons, an important process for neuronal function. A homozygous missense mutation in adenylate cyclase 6 gene (ADCY6) was found in another family characterized by a lack of myelin in the peripheral nervous system (PNS) as determined by TEM. Morpholino knockdown of the zebrafish orthologs led to severe and specific defects in peripheral myelin in spite of the presence of Schwann cells. ADCY6 encodes a protein that belongs to the adenylate cyclase family responsible for the synthesis of cAMP. Elevation of cAMP can mimic axonal contact in vitro and upregulates myelinating signals. Our data indicate an essential and so far unknown role of ADCY6 in PNS myelination likely through the cAMP pathway. Mutations of genes encoding proteins of Ranvier domains or involved in myelination of Schwann cells are responsible for novel and severe human axoglial diseases.

Fine characterisation of a recombination hotspot at the DPY19L2 locus and resolution of the paradoxical excess of duplications over deletions in the general population.

We demonstrated previously that 75% of infertile men with round, acrosomeless spermatozoa (globozoospermia) had a homozygous 200-Kb deletion removing the totality of DPY19L2. We showed that this deletion occurred by Non-Allelic Homologous Recombination (NAHR) between two homologous 28-Kb Low Copy Repeats (LCRs) located on each side of the gene. The accepted NAHR model predicts that inter-chromatid and inter-chromosome NAHR create a deleted and a duplicated recombined allele, while intra-chromatid events only generate deletions. Therefore more deletions are expected to be produced de novo. Surprisingly, array CGH data show that, in the general population, DPY19L2 duplicated alleles are approximately three times as frequent as deleted alleles. In order to shed light on this paradox, we developed a sperm-based assay to measure the de novo rates of deletions and duplications at this locus. As predicted by the NAHR model, we identified an excess of de novo deletions over duplications. We calculated that the excess of de novo deletion was compensated by evolutionary loss, whereas duplications, not subjected to selection, increased gradually. Purifying selection against sterile, homozygous deleted men may be sufficient for this compensation, but heterozygously deleted men might also suffer a small fitness penalty. The recombined alleles were sequenced to pinpoint the localisation of the breakpoints. We analysed a total of 15 homozygous deleted patients and 17 heterozygous individuals carrying either a deletion (n = 4) or a duplication (n = 13). All but two alleles fell within a 1.2-Kb region central to the 28-Kb LCR, indicating that >90% of the NAHR took place in that region. We showed that a PRDM9 13-mer recognition sequence is located right in the centre of that region. Our results therefore strengthen the link between this consensus sequence and the occurrence of NAHR.

Homozygous mutation of AURKC yields large-headed polyploid spermatozoa and causes male infertility.

The World Health Organization conservatively estimates that 80 million people suffer from infertility worldwide. Male factors are believed to be responsible for 20-50% of all infertility cases, but microdeletions of the Y chromosome are the only genetic defects altering human spermatogenesis that have been reported repeatedly. We focused our work on infertile men with a normal somatic karyotype but typical spermatozoa mainly characterized by large heads, a variable number of tails and an increased chromosomal content (OMIM 243060). We performed a genome-wide microsatellite scan on ten infertile men presenting this characteristic phenotype. In all of these men, we identified a common region of homozygosity harboring the aurora kinase C gene (AURKC) with a single nucleotide deletion in the AURKC coding sequence. In addition, we show that this founder mutation results in premature termination of translation, yielding a truncated protein that lacks the kinase domain. We conclude that the absence of AURKC causes male infertility owing to the production of large-headed multiflagellar polyploid spermatozoa.

The neuronal endopeptidase ECEL1 is associated with a distinct form of recessive distal arthrogryposis.

Distal arthrogryposis (DA) is a heterogeneous subgroup of arthrogryposis multiplex congenita (AMC), a large family of disorders characterized by multiple congenital joint limitations due to reduced fetal movements. DA is mainly characterized by contractures afflicting especially the distal extremities without overt muscular or neurological signs. Although a limited number of genes mostly implicated in the contractile apparatus have been identified in DA, most patients failed to show mutations in currently known genes. Using a pangenomic approach, we demonstrated linkage of DA to chromosome 2q37 in two consanguineous families and the endothelin-converting enzyme like 1 (ECEL1) gene present in this region was associated with DA. Screening of a panel of 20 families with non-specific DA identified seven homozygous or compound heterozygous mutations of ECEL1 in a total of six families. Mutations resulted mostly in the absence of protein. ECEL1 is a neuronal endopeptidase predominantly expressed in the central nervous system and brain structures during fetal life in mice and human. ECEL1 plays a major role in intramuscular axonal branching of motor neurons in skeletal muscle during embryogenesis. A detailed review of clinical findings of DA patients with ECEL1 mutations revealed a homogeneous and recognizable phenotype characterized by limited knee flexion, flexed third to fifth fingers and severe muscle atrophy predominant on lower limbs and tongue that suggested a common pathogenic mechanism. We described a new and homogenous phenotype of DA associated with ECEL1 that resulted in symptoms involving rather the peripheral than the central nervous system and suggesting a developmental dysfunction.

Coagulation Factor XII Gene Mutation in Brazilian Families with Hereditary Angioedema with Normal C1 Inhibitor.

BACKGROUND: Hereditary angioedema (HAE) with normal C1 inhibitor (C1-INH) is a rare disorder. Mutations of the gene encoding coagulation factor XII have been identified in a subset of patients with this condition. Our aim was to investigate mutations in the F12 gene in patients with HAE with normal C1-INH from Brazil. METHODS: We studied 5 Brazilian families with index female patients who presented with recurrent angioedema with normal C1-INH and C4 levels. Genomic DNA was isolated from whole blood and PCR was performed. Mutations were detected by the sequencing of exon 9 of the F12 gene and allelic discrimination. RESULTS: The c.983C>A (p.Thr328Lys) mutation was identified in 16 subjects, from 4 of the 5 families studied, including 8 patients with symptoms of HAE with normal C1-INH (87.5% women) and 8 subjects asymptomatic for HAE (25% women). Mean age at onset of symptoms among the FXII-HAE patients was 13.8 years (range 6-25 years). Recurrent abdominal pain (100%) and subcutaneous angioedema (87.5%) were the most frequent clinical presentations. Two patients presented with associated laryngeal edema. In keeping with previous observations in patients with both C1-INH-HAE and HAE with normal C1-INH, all 7 women with FXII-HAE reported triggering or worsening of symptoms upon intake of estrogen-containing oral contraceptives and/or pregnancy. CONCLUSIONS: We report for the first time in Brazil a mutation in the F12 gene as a likely cause of HAE with normal C1-INH in patients with recurrent attacks of angioedema and/or abdominal pain. A higher frequency of abdominal pain attacks and onset of symptoms at a younger age were observed among Brazilian patients when compared to those from other parts of the world.

Congenital myopathy-causing tropomyosin mutations induce thin filament dysfunction via distinct physiological mechanisms.

In humans, congenital myopathy-linked tropomyosin mutations lead to skeletal muscle dysfunction, but the cellular and molecular mechanisms underlying such dysfunction remain obscure. Recent studies have suggested a unifying mechanism by which tropomyosin mutations partially inhibit thin filament activation and prevent proper formation and cycling of myosin cross-bridges, inducing force deficits at the fiber and whole-muscle levels. Here, we aimed to verify this mechanism using single membrane-permeabilized fibers from patients with three tropomyosin mutations (TPM2-null, TPM3-R167H and TPM2-E181K) and measuring a broad range of parameters. Interestingly, we identified two divergent, mutation-specific pathophysiological mechanisms. (i) The TPM2-null and TPM3-R167H mutations both decreased cooperative thin filament activation in combination with reductions in the myosin cross-bridge number and force production. The TPM3-R167H mutation also induced a concomitant reduction in thin filament length. (ii) In contrast, the TPM2-E181K mutation increased thin filament activation, cross-bridge binding and force generation. In the former mechanism, modulating thin filament activation by administering troponin activators (CK-1909178 and EMD 57033) to single membrane-permeabilized fibers carrying tropomyosin mutations rescued the thin filament activation defect associated with the pathophysiology. Therefore, administration of troponin activators may constitute a promising therapeutic approach in the future.

Absence of triadin, a protein of the calcium release complex, is responsible for cardiac arrhythmia with sudden death in human.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease so far related to mutations in the cardiac ryanodine receptor (RYR2) or the cardiac calsequestrin (CASQ2) genes. Because mutations in RYR2 or in CASQ2 are not retrieved in all CPVT cases, we searched for mutations in the physiological protein partners of RyR2 and CSQ2 in a large cohort of CPVT patients with no detected mutation in these two genes. Based on a candidate gene approach, we focused our investigations on triadin and junctin, two proteins that link RyR2 and CSQ2. Mutations in the triadin (TRDN) and in the junctin (ASPH) genes were searched in a cohort of 97 CPVT patients. We identified three mutations in triadin which cosegregated with the disease on a recessive mode of transmission in two families, but no mutation was found in junctin. Two TRDN mutations, a 4 bp deletion and a nonsense mutation, resulted in premature stop codons; the third mutation, a p.T59R missense mutation, was further studied. Expression of the p.T59R mutant in COS-7 cells resulted in intracellular retention and degradation of the mutant protein. This was confirmed after in vivo expression of the mutant triadin in triadin knock-out mice by viral transduction. In this work, we identified TRDN as a new gene responsible for an autosomal recessive form of CPVT. The mutations identified in the two families lead to the absence of the protein, thereby demonstrating the importance of triadin for the normal function of the cardiac calcium release complex in humans.

A recurrent deletion of DPY19L2 causes infertility in man by blocking sperm head elongation and acrosome formation.

An increasing number of couples require medical assistance to achieve a pregnancy, and more than 2% of the births in Western countries now result from assisted reproductive technologies. To identify genetic variants responsible for male infertility, we performed a whole-genome SNP scan on patients presenting with total globozoospermia, a primary infertility phenotype characterized by the presence of 100% round acrosomeless spermatozoa in the ejaculate. This strategy allowed us to identify in most patients (15/20) a 200 kb homozygous deletion encompassing only DPY19L2, which is highly expressed in the testis. Although there was no known function for DPY19L2 in humans, previous work indicated that its ortholog in C. elegans is involved in cell polarity. In man, the DPY19L2 region has been described as a copy-number variant (CNV) found to be duplicated and heterozygously deleted in healthy individuals. We show here that the breakpoints of the deletions are located on a highly homologous 28 kb low copy repeat (LCR) sequence present on each side of DPY19L2, indicating that the identified deletions were probably produced by nonallelic homologous recombination (NAHR) between these two regions. We demonstrate that patients with globozoospermia have a homozygous deletion of DPY19L2, thus indicating that DPY19L2 is necessary in men for sperm head elongation and acrosome formation. A molecular diagnosis can now be proposed to affected men; the presence of the deletion confirms the diagnosis of globozoospermia and assigns a poor prognosis for the success of in vitro fertilization.

Delineation of the 3p14.1p13 microdeletion associated with syndromic distal limb contractures.

Distal limb contractures (DLC) represent a heterogeneous clinical and genetic condition. Overall, 20-25% of the DLC are caused by mutations in genes encoding the muscle contractile apparatus. Large interstitial deletions of the 3p have already been diagnosed by standard chromosomal analysis, but not associated with a specific phenotype. We report on four patients with syndromic DLC presenting with a de novo 3p14.1p13 microdeletion. The clinical features associated multiple contractures, feeding problems, developmental delay, and intellectual disability. Facial dysmorphism was constant with low-set posteriorly rotated ears and blepharophimosis. Review of previously reported cases with a precise mapping of the deletions, documented a 250 kb smallest region of overlap (SRO) necessary for DLC. This region contained one gene, EIF4E3, the first three exons of the FOXP1 gene, and an intronic enhancer of FOXP1 named hs1149. Sanger sequencing and locus quantification of hs1149, EIF4E3, and FOXP1 in a cohort of 11 French patients affected by DLC appeared normal. In conclusion, we delineate a new microdeletion syndrome involving the 3p14.1p13 locus and associated with DLC and severe developmental delay.

Oxidative stress and successful antioxidant treatment in models of RYR1-related myopathy.

The skeletal muscle ryanodine receptor is an essential component of the excitation-contraction coupling apparatus. Mutations in RYR1 are associated with several congenital myopathies (termed RYR1-related myopathies) that are the most common non-dystrophic muscle diseases of childhood. Currently, no treatments exist for these disorders. Although the primary pathogenic abnormality involves defective excitation-contraction coupling, other abnormalities likely play a role in disease pathogenesis. In an effort to discover novel pathogenic mechanisms, we analysed two complementary models of RYR1-related myopathies, the relatively relaxed zebrafish and cultured myotubes from patients with RYR1-related myopathies. Expression array analysis in the zebrafish disclosed significant abnormalities in pathways associated with cellular stress. Subsequent studies focused on oxidative stress in relatively relaxed zebrafish and RYR1-related myopathy myotubes and demonstrated increased oxidant activity, the presence of oxidative stress markers, excessive production of oxidants by mitochondria and diminished survival under oxidant conditions. Exposure to the antioxidant N-acetylcysteine reduced oxidative stress and improved survival in the RYR1-related myopathies human myotubes ex vivo and led to significant restoration of aspects of muscle function in the relatively relaxed zebrafish, thereby confirming its efficacy in vivo. We conclude that oxidative stress is an important pathophysiological mechanism in RYR1-related myopathies and that N-acetylcysteine is a successful treatment modality ex vivo and in a vertebrate disease model. We propose that N-acetylcysteine represents the first potential therapeutic strategy for these debilitating muscle diseases.

Two closely related endocytic proteins that share a common OCRL-binding motif with APPL1.

Mutations of the inositol 5' phosphatase oculocerebrorenal syndrome of Lowe (OCRL) give rise to the congenital X-linked disorders oculocerebrorenal syndrome of Lowe and Dent disease, two conditions giving rise to abnormal kidney proximal tubule reabsorption, and additional nervous system and ocular defects in the case of Lowe syndrome. Here, we identify two closely related endocytic proteins, Ses1 and Ses2, which interact with the ASH-RhoGAP-like (ASPM-SPD-2-Hydin homology and Rho-GTPase Activating Domain-like) domain of OCRL. The interaction is mediated by a short amino acid motif similar to that used by the rab-5 effector APPL1 (Adaptor Protein containing pleckstrin homology [PH] domain, PTB domain and Leucine zipper motif 1) APPL1 for OCRL binding. Ses binding is mutually exclusive with APPL1 binding, and is disrupted by the same missense mutations in the ASH-RhoGAP-like domain that also disrupt APPL1 binding. Like APPL1, Ses1 and -2 are localized on endosomes but reside on different endosomal subpopulations. These findings define a consensus motif (which we have called a phenylalanine and histidine [F&H] motif) for OCRL binding and are consistent with a scenario in which Lowe syndrome and Dent disease result from perturbations at multiple sites within the endocytic pathway.

MLPA and sequence analysis of DPY19L2 reveals point mutations causing globozoospermia.

STUDY QUESTION: Do DPY19L2 heterozygous deletions and point mutations account for some cases of globozoospermia? SUMMARY ANSWER: Two DPY19L2 heterozygous deletions and three point mutations were identified, thus further confirming that genetic alterations of the DPY19L2 gene are the main cause of globozoospermia and indicating that DPY19L2 molecular diagnostics should not be stopped in the absence of a homozygous gene deletion. WHAT IS KNOWN ALREADY: Globozoospermia is a rare phenotype of primary male infertility characterized by the production of a majority of round-headed spermatozoa without acrosome. We demonstrated previously that most cases in man were caused by a recurrent homozygous deletion of the totality of the DPY19L2 gene, preventing sperm head elongation and acrosome formation. In mammals, DPY19L2 has three paralogs of yet unknown function and one highly homologous pseudogene showing >95% sequence identity with DPY19L2. Specific amplification and sequencing of DPY19L2 have so far been hampered by the presence of this pseudogene which has greatly complicated specific amplification and sequencing. STUDY DESIGN, SIZE, DURATION: In this cohort study, 34 patients presenting with globozoospermia were recruited during routine infertility treatment in infertility centers in France and Tunisia between January 2008 and December 2011. The molecular variants identified in patients were screened in 200 individuals from the general population to exclude frequent non-pathological polymorphisms. PARTICIPANTS/MATERIALS, SETTING, METHODS: We developed a Multiplex Ligation-dependent Probe Amplification test to detect the presence of heterozygous deletions and identified the conditions to specifically amplify and sequence the 22 exons and intronic boundaries of the DPY19L2 gene. The pathogenicity of the identified mutations and their action on the protein were evaluated in silico. MAIN RESULTS AND THE ROLE OF CHANCE: There were 23 patients who were homozygous for the DPY19L2 deletion (67.6%). Only eight of the eleven non-homozygously deleted patients could be sequenced due to poor DNA quality of three patients. Two patients were compound heterozygous carrying one DPY19L2 deleted allele associated respectively with a nonsense (p.Q342*) and a missense mutation (p.R290H). One patient was homozygous for p.M358K, another missense mutation affecting a highly conserved amino acid. Due to the localization of this mutation and the physicochemical properties of the substituted amino acids, we believe that this variant is likely to disrupt one of the protein transmembrane domains and destabilize the protein. Overall, 84% of the fully analysed patients (n = 31) had a molecular alteration of DPY19L2. There was no clear phenotypic difference between the homozygous deleted individual, patients carrying a point mutation and undiagnosed patients. LIMITATIONS, REASONS FOR CAUTION: Globally poor fertilization rates are observed after intracytoplasmic sperm injection of round spermatozoa. Further work is needed to assess whether DPY19L2 mutated patients present a better or worse prognostic than the non-diagnosed patients. Evaluation of the potential benefit of treatment with a calcium ionophore, described to improve fertilization, should be evaluated in these two groups. WIDER IMPLICATIONS OF THE FINDINGS: In previous work, deletions of DPY19L2 had only been identified in North African patients. Here we have identified DPY19L2 deletions and point mutations in European patients, indicating that globozoospemia caused by a molecular defect of DPY19L2 can be expected in individuals from any ethnic background. STUDY FUNDING/COMPETING INTEREST(S): None of the authors have any competing interest. This work is part of the project 'Identification and Characterization of Genes Involved in Infertility (ICG2I)' funded by the program GENOPAT 2009 from the French Research Agency (ANR).

Identification of a new recurrent aurora kinase C mutation in both European and African men with macrozoospermia.

STUDY QUESTION: Can we identify new sequence variants in the aurora kinase C gene (AURKC) of patients with macrozoospermia and establish a genotype-phenotype correlation? SUMMARY ANSWER: We identified a new non-sense mutation, p.Y248*, that represents 13% of all mutant alleles. There was no difference in the phenotype of individuals carrying this new mutation versus the initially described and main mutation c.144delC. WHAT IS KNOWN ALREADY: The absence of a functional AURKC gene causes primary infertility in men by blocking the first meiotic division and leading to the production of tetraploid large-headed spermatozoa. We previously demonstrated that most affected men were of North African origin and carried a homozygous truncating mutation (c.144delC). STUDY DESIGN, SIZE, DURATION: This is a retrospective study carried out on patients consulting for infertility and described as having >5% large-headed spermatozoa. A total of 87 patients are presented here, 43 patients were published previously and 44 are new patients recruited between January 2008 and December 2011. PARTICIPANTS/MATERIALS, SETTING, METHODS: All patients consulted for primary infertility in fertility clinics in France (n = 44), Tunisia (n = 30), Morocco (n = 9) or Algeria (n = 4). Sperm analysis was carried out in the recruiting fertility clinics and all molecular analyses were performed at Grenoble teaching hospital. DNA was extracted from blood or saliva and the seven AURKC exons were sequenced. RT-PCR was carried out on transcripts extracted from leukocytes from one patient homozygous for p.Y248*. Microsatellite analysis was performed on all p.Y248* patients to evaluate the age of this new mutation. MAIN RESULTS AND THE ROLE OF CHANCE: We identified a new non-sense mutation, p.Y248*, in 10 unrelated individuals of European (n = 4) and North African origin (n = 6). We show that this new variant represents 13% of all mutant alleles and that the initially described c.144delC variant accounts for almost all of the remaining mutated alleles (85.5%). No mutated transcripts could be detected by RT-PCR suggesting a specific degradation of the mutant transcripts by non-sense mediated mRNA decay. A rare variant located in the 3' untranslated region was found to strictly co-segregate with p.Y248*, demonstrating a founding effect. Microsatellite analysis confirmed this linkage and allowed us to estimate a mutational age of between 925 and 1325 years, predating the c.144delC variant predicted by the same method to have arisen 250-650 years ago. Patients with no identified AURKC mutation (n = 15) have significantly improved parameters in terms of vitality and concentration of normal spermatozoa, and a decreased rate of spermatozoa with a large head and multiple flagella (P < 0.001). LIMITATIONS, REASONS FOR CAUTION: Despite adherence to the World Health Organization guidelines, large variations in most characteristic sperm parameters were observed, even for patients with the same homozygous mutation. We believe that is mainly related to inter-laboratory variability in sperm parameter scoring. This prevented us from establishing clear-cut values to indicate a need for molecular analysis of patients with macrozoospermia. WIDER IMPLICATIONS OF THE FINDINGS: This study confirms yet again the importance of AURKC mutations in the aetiology of macrozoospermia. Although a large majority of patients are of North African origin, we have now identified European patients carrying a new non-sense mutation indicating that a diagnosis of absence of a functional AURKC gene should not be ruled out for non-Magrebian individuals. Indirect evidence indicates that AURKC might be playing a role in the meiotic spindle assembly checkpoint (SAC) during meiosis. We postulate that heterozygous men might have a more relaxed SAC leading to a more abundant sperm production and a reproductive advantage. This could be the reason for the rapid accumulation of the two AURKC mutations we observe in North African individuals. STUDY FUNDING/COMPETING INTEREST(S): None of the authors have any competing interest. This work is part of the project 'Identification and Characterization of Genes Involved in Infertility (ICG2I)' funded by the programme GENOPAT 2009 from the French Research Agency (ANR).

[The high phenotypic variability of RYR1 gene mutations]. / La grande variabilité phénotypique des mutations du gène RYR1.

The RYR1 gene encodes the ryanodine-receptor 1, a key protein in the excitation-contraction coupling that takes place in muscle fibers. This receptor is the main channel responsible for calcium release from the endoplasmic reticulum [1]. A number of clinical phenotypes are linked to various mutations in this large gene as shown in a compilation established by ORPHANET (see table). In this work we describe two distinct, somewhat misleading, phenotypes in relation to pathogenic variants in this gene.

Title: La grande variabilité phénotypique des mutations du gène RYR1. Abstract: Le gène RYR1 (Ryanodine-Receptor-1) code pour une protéine-clé dans le processus de couplage excitation-contraction de la fibre musculaire. Ce récepteur est le principal canal de libération du calcium à partir du réticulum endoplasmique [1]. Un certain nombre de phénotypes cliniques sont imputables aux mutations de ce gène de grande taille comme rappelé dans la liste établie par ORPHANET (voir Encadré). Nous décrivons, dans ce travail, deux phénotypes distincts, et trompeurs à certains égards, en rapport avec des mutations de ce gène.

From Lowe syndrome to Dent disease: correlations between mutations of the OCRL1 gene and clinical and biochemical phenotypes.

Mutations of OCRL1 are associated with both the Lowe oculocerebrorenal syndrome, a multisystemic and Dent-2 disease, a renal tubulopathy. We have identified a mutation in 130 Lowe syndrome families and 6 affected by Dent-2 disease with 51 of these mutations being novel. No founding effect was evidenced for recurrent mutations. Two mutations initially reported as causing Dent-2 disease were identified in patients, including two brothers, presenting with Lowe syndrome thus extending the clinical variability of OCRL1 mutations. mRNA levels, protein content, and PiP(2) -ase activities were analyzed in patient's fibroblasts. Although mRNA levels were normal in cells harboring a missense mutation, the OCRL1 content was markedly lowered, suggesting that enzymatic deficiency resulted mainly from protein degradation rather than from a catalytic inactivation. Analysis of a splicing mutation that led to the elimination of the initiation codon evidenced the presence of shortened forms of OCRL1 that might result from the use of alternative initiation codons. The specific mapping of the frameshift and nonsense mutations, exclusively identified in exons 1-7 and exons 8-23, respectively, for Dent disease and Lowe syndrome together with the possible use of alternative initiation codons might be related to their clinical expression, that is, Lowe syndrome or Dent-2 disease.

Functional analysis reveals splicing mutations of the CASQ2 gene in patients with CPVT: implication for genetic counselling and clinical management.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare and severe arrhythmogenic disorder. Although usually transmitted in a recessive form, few cases of dominant mutations have been reported. Thirteen mutations in the CASQ2 gene have been reported so far in association with CPVT. We performed molecular analysis of the CASQ2 gene in 43 probands with CPVT and identified eight mutations in five patients. Six mutations were novel: one was a single nucleotide deletion, three affected consensus splice sites, and two had unknown consequences: the c.939 + 5G>C and the synonymous c.381C>T variations. We demonstrated that these two variations affected CASQ2 splicing using a splicing minigene assay. These data increased significantly the number of CASQ2 mutations described in association with CPVT, revealed the high prevalence of splicing and truncating mutations in this gene and brought new insight regarding the dominant inheritance of the disease. Moreover, our report of the first splicing abnormalities in CASQ2 caused by intronic mutation or synonymous change underlines the absolute necessity to perform extensive molecular analysis for genetic diagnosis and counseling of CPVT.

The Aurora Kinase C c.144delC mutation causes meiosis I arrest in men and is frequent in the North African population.

Infertility concerns a minimum of 70 million couples worldwide. An important proportion of cases is believed to have a genetic component, yet few causal genes have been identified so far. In a previous study, we demonstrated that a homozygous mutation (c.144delC) in the Aurora Kinase C (AURKC) gene led to the production of large-headed polyploid multi-flagellar spermatozoa, a primary infertility phenotype mainly observed in North Africans. We now want to estimate the prevalence of the defect, to improve our understanding of AURKC physiopathology in spermatogenesis and assess its implication in oogenesis. A carrier frequency of 1/50 was established from individuals from the Maghrebian general population, comparable to that of Y-microdeletions, thus far the only known recurrent genetic event altering spermatogenesis. A total of 62 patients were genotyped, all who had a typical phenotype with close to 100% large-headed spermatozoa were homozygously mutated (n = 32), whereas no AURKC mutations were detected in the others. Two homozygous females were identified; both were fertile indicating that AURKC is not indispensible in oogenesis. Previous FISH results had showed a great chromosomal heterogeneity in these patient's spermatozoa. We demonstrate here by flow cytometry that all spermatozoa have in fact a homogeneous 4C DNA content and are thus all blocked before the first meiotic division. Our data thus indicate that a functional AURKC protein is necessary for male meiotic cytokinesis while its absence does not impair oogenesis.