Stéphane Blanchard

A defect in harmonin, a PDZ domain-containing protein expressed in the inner ear sensory hair cells, underlies Usher syndrome type 1C

Usher syndrome type 1 (USH1) is an autosomal recessive sensory defect involving congenital profound sensorineural deafness, vestibular dysfunction and blindness (due to progressive retinitis pigmentosa). Six different USH1 loci have been reported. So far, only MYO7A (USH1B), encoding myosin VIIA (ref. 2), has been identified as a gene whose mutation causes the disease. Here, we report a gene underlying USH1C (MIM 276904), a USH1 subtype described in a population of Acadian descendants from Louisiana and in a Lebanese family. We identified this gene (USH1C), encoding a PDZ-domain–containing protein, harmonin, in a subtracted mouse cDNA library derived from inner ear sensory areas. In patients we found a splice-site mutation, a frameshift mutation and the expansion of an intronic variable number of tandem repeat (VNTR). We showed that, in the mouse inner ear, only the sensory hair cells express harmonin. The inner ear Ush1c transcripts predicted several harmonin isoforms, some containing an additional coiled-coil domain and a proline- and serine-rich region. As several of these transcripts were absent from the eye, we propose that USH1C also underlies the DFNB18 form of isolated deafness.

MyRIP, a novel Rab effector, enables myosin VIIa recruitment to retinal melanosomes

Defects of the myosin VIIa motor protein cause deafness and retinal anomalies in humans and mice. We report on the identification of a novel myosin‐VIIa‐interacting protein that we have named MyRIP (myosin‐VIIa‐ and Rab‐interacting protein), since it also binds to Rab27A in a GTP‐dependent manner. In the retinal pigment epithelium cells, MyRIP, myosin VIIa and Rab27A are associated with melanosomes. In transfected PC12 cells, overexpression of MyRIP was shown to interfere with the myosin VIIa tail localization. We propose that a molecular complex composed of Rab27A, MyRIP and myosin VIIa bridges retinal melanosomes to the actin cytoskeleton and thereby mediates the local trafficking of these organelles. The defect of this molecular complex is likely to account for the perinuclear mislocalization of the melanosomes observed in the retinal pigment epithelium cells of myosinVIIa‐defective mice.

Mutations in a new gene encoding a protein of the hair bundle cause non-syndromic deafness at the DFNB16 locus.

Hearing impairment affects about 1 in 1,000 children at birth. Approximately 70 loci implicated in non-syndromic forms of deafness have been reported in humans and 24 causative genes have been identified (see also http://www.uia.ac.be/dnalab/hhh). We report a mouse transcript, isolated by a candidate deafness gene approach, that is expressed almost exclusively in the inner ear. Genomic analysis shows that the human ortholog STRC (so called owing to the name we have given its protein—stereocilin), which is located on chromosome 15q15, contains 29 exons encompassing approximately 19 kb. STRC is tandemly duplicated, with the coding sequence of the second copy interrupted by a stop codon in exon 20. We have identified two frameshift mutations and a large deletion in the copy containing 29 coding exons in two families affected by autosomal recessive non-syndromal sensorineural deafness linked to the DFNB16 locus. Stereocilin is made up of 1,809 amino acids, and contains a putative signal petide and several hydrophobic segments. Using immunohistolabeling, we demonstrate that, in the mouse inner ear, stereocilin is expressed only in the sensory hair cells and is associated with the stereocilia, the stiff microvilli forming the structure for mechanoreception of sound stimulation.

Alsin/Rac1 signaling controls survival and growth of spinal motoneurons.

Recessive mutations in alsin, a guanine‐nucleotide exchange factor for the GTPases Rab5 and Rac1, cause juvenile amyotrophic lateral sclerosis (ALS2) and related motoneuron disorders. Alsin function in motoneurons remained unclear because alsin knock‐out mice do not develop overt signs of motoneuron degeneration.

Townes‐Brocks syndrome: Detection of a SALL1 mutation hot spot and evidence for a position effect in one patient

Townes‐Brocks syndrome (TBS) is an autosomal dominant developmental disorder characterized by anal and thumb malformations and by ear anomalies that can affect the three compartments and usually lead to hearing loss. The gene underlying TBS, SALL1, is a human homolog of the Drosophila spalt gene which encodes a transcription factor. A search for SALL1 mutations undertaken in 11 unrelated affected individuals (five familial and six sporadic cases) led to the detection of mutations in nine of them. One nonsense and six different novel frameshift mutations, all located in the second exon, were identified. Together with the previously reported mutations [Kohlhase et al., 1999], they establish that TBS results from haploinsufficiency. The finding of de novo mutations in the sporadic cases is consistent with the proposed complete penetrance of the disease. Moreover, the occurrence of the same 826C>T transition in a CG dimer, in three sporadic cases from the present series and three sporadic cases from the other series [Kohlhase et al., 1999] (i.e., six of the eight mutations identified in sporadic cases), reveals the existence of a mutation hotspot. Six different SALL1 polymorphisms were identified in the course of the present study, three of which are clustered in a particular region of the gene that encodes a stretch of serine residues. Finally, the chromosome 16 breakpoint of a t(5;16)(p15.3;q12.1) translocation carried by a TBS‐affected individual was mapped at least 180 kb telomeric to SALL1, thus indicating that a position effect underlies the disease in this individual. Hum Mutat 14:377–386, 1999.

A Bragg glass phase in the vortex lattice of a type II superconductor.

Although crystals are usually quite stable, they are sensitive to a disordered environment: even an infinitesimal amount of impurities can lead to the destruction of crystalline order. The resulting state of matter has been a long-standing puzzle. Until recently it was believed to be an amorphous state in which the crystal would break into ‘crystallites’. But a different theory predicts the existence of a novel phase of matter: the so-called Bragg glass, which is a glass and yet nearly as ordered as a perfect crystal. The ‘lattice’ of vortices that contain magnetic flux in type II superconductors provide a good system to investigate these ideas. Here we show that neutron-diffraction data of the vortex lattice provides unambiguous evidence for a weak, power-law decay of the crystalline order characteristic of a Bragg glass. The theory also predicts accurately the electrical transport properties of superconductors; it naturally explains the observed phase transitions and the dramatic jumps in the critical current associated with the melting of the Bragg glass. Moreover, the model explains experiments as diverse as X-ray scattering in disordered liquid crystals and the conductivity of electronic crystals.

Modeling neuronal defects associated with a lysosomal disorder using patient-derived induced pluripotent stem cells

By providing access to affected neurons, human induced pluripotent stem cells (iPSc) offer a unique opportunity to model human neurodegenerative diseases. We generated human iPSc from the skin fibroblasts of children with mucopolysaccharidosis type IIIB. In this fatal lysosomal storage disease, defective α-N-acetylglucosaminidase interrupts the degradation of heparan sulfate (HS) proteoglycans and induces cell disorders predominating in the central nervous system, causing relentless progression toward severe mental retardation. Partially digested proteoglycans, which affect fibroblast growth factor signaling, accumulated in patient cells. They impaired isolation of emerging iPSc unless exogenous supply of the missing enzyme cleared storage and restored cell proliferation. After several passages, patient iPSc starved of an exogenous enzyme continued to proliferate in the presence of fibroblast growth factor despite HS accumulation. Survival and neural differentiation of patient iPSc were comparable with unaffected controls. Whereas cell pathology was modest in floating neurosphere cultures, undifferentiated patient iPSc and their neuronal progeny expressed cell disorders consisting of storage vesicles and severe disorganization of Golgi ribbons associated with modified expression of the Golgi matrix protein GM130. Gene expression profiling in neural stem cells pointed to alterations of extracellular matrix constituents and cell-matrix interactions, whereas genes associated with lysosome or Golgi apparatus functions were downregulated. Taken together, these results suggest defective responses of patient undifferentiated stem cells and neurons to environmental cues, which possibly affect Golgi organization, cell migration and neuritogenesis. This could have potential consequences on post-natal neurological development, once HS proteoglycan accumulation becomes prominent in the affected child brain.

Identification of three novel mutations in the USH1C gene and detection of thirty-one polymorphisms used for haplotype analysis.

Usher syndrome (USH) is a clinically and genetically heterogeneous autosomal recessive disorder in which sensorineural hearing loss is associated with retinitis pigmentosa. Usher syndrome type 1, the most severe form, is characterized by profound congenital deafness, vestibular dysfunction, and prepubertal onset of retinitis pigmentosa. Six different USH1 genes have so far been mapped, of which two have already been identified. MYO7A, encoding the unconventional myosin VIIA, underlies USH1B. Recently, the USH1C gene was shown to encode harmonin, a PDZ domain‐containing protein. A previous screening of 18 unrelated USH1 patients, without a detected MYO7A mutation, for the three USH1C mutations described to date had demonstrated the presence of the 238–239insC mutation in the heterozygous state in four of them. A complete USH1C mutation screening in these four carriers of the 238–239insC mutation resulted in the detection of the second mutation in all the individuals, and the identification of three novel mutations, namely two splice site mutations (IVS1+1G>T and IVS5+1G>A) and a nonsense mutation (R31X). Thirty‐one polymorphisms were detected in the USH1C gene. We observed that the E519D substitution is non‐pathogenic, which is of particular interest for molecular diagnosis. Our analysis indicated that all the carriers of the 238–239insC mutation share a common haplotype. A different common haplotype was found in the two IVS1+1G>T carriers. Future studies of additional carriers and non‐carriers should document the here proposed founder effect of these two mutations. Hum Mutat 17:34–41, 2001.

PHR1, an integral membrane protein of the inner ear sensory cells, directly interacts with myosin 1c and myosin VIIa

By using the yeast two-hybrid technique, we identified a candidate protein ligand of the myosin 1c tail, PHR1, and found that this protein can also bind to the myosin VIIa tail. PHR1 is an integral membrane protein that contains a pleckstrin homology (PH) domain. Myosin 1c and myosin VIIa are two unconventional myosins present in the inner ear sensory cells. We showed that PHR1 immunoprecipitates with either myosin tail by using protein extracts from cotransfected HEK293 cells. In vitro binding assays confirmed that PHR1 directly interacts with these two myosins. In both cases the binding involves the PH domain. In vitro interactions between PHR1 and the myosin tails were not affected by the presence or absence of Ca2+ and calmodulin. Finally, we found that PHR1 is able to dimerise. As PHR1 is expressed in the vestibular and cochlear sensory cells, its direct interactions with the myosin 1c and VIIa tails are likely to play a role in anchoring the actin cytoskeleton to the plasma membrane of these cells. Moreover, as both myosins have been implicated in the mechanotransduction slow adaptation process that takes place in the hair bundles, we propose that PHR1 is also involved in this process.

Forced expression of the motor neuron determinant HB9 in neural stem cells affects neurogenesis.

In contrast to mouse embryonic stem cells and in spite of overlapping gene expression profiles, neural stem cells (NSCs) isolated from the embryonic spinal cord do not respond to physiological morphogenetic stimuli provided by Sonic hedgehog and retinoic acid and do not generate motor neurons upon differentiation. Transcription factors expressed in motor neuron progenitors during embryogenesis include Pax6, Ngn2, Nkx6.1 and Olig2, whose expression precedes that of factors specifying motor neuron fate, including HB9, Islet1 and LIM3. We showed that all these factors were present in neural progenitors derived from mouse ES cells, whereas NSCs derived from the rat embryonic spinal cord expressed neither HB9 nor Islet1 and contained low levels of Nkx6.1 and LIM3. We constructed a lentivirus vector to express HB9 and GFP in NSCs and examined the consequences of HB9 expression on other transcription factors and cell differentiation. Compared to cell expressing GFP alone, NSCs expressing GFP and HB9 cycled less rapidly, downregulated Pax6 and Ngn2 mRNA levels, produced higher proportions of neurons in vitro and lower numbers of neurons after transplantation in the spinal cord of recipient rats. Oligodendrocytic and astrocytic differentiations were not affected. HB9 expressing NSCs did not express Islet1 or upregulate LIM3. They neither responded to Sonic hedgehog and retinoic acid nor produced cholinergic neurons. We concluded that forced HB9 expression affected neurogenesis but was not sufficient to confer motor neuron fate to NSCs.