Marion A. Maw

The Deafness-Associated Mitochondrial DNA Mutation at Position 7445, Which Affects tRNASer(UCN) Precursor Processing, Has Long-Range Effects on NADH Dehydrogenase Subunit ND6 Gene Expression

ABSTRACT The pathogenetic mechanism of the deafness-associated mitochondrial DNA (mtDNA) T7445C mutation has been investigated in several lymphoblastoid cell lines from members of a New Zealand pedigree exhibiting the mutation in homoplasmic form and from control individuals. We show here that the mutation flanks the 3′ end of the tRNASer(UCN) gene sequence and affects the rate but not the sites of processing of the tRNA precursor. This causes an average reduction of ∼70% in the tRNASer(UCN) level and a decrease of ∼45% in protein synthesis rate in the cell lines analyzed. The data show a sharp threshold in the capacity of tRNASer(UCN) to support the wild-type protein synthesis rate, which corresponds to ∼40% of the control level of this tRNA. Strikingly, a 7445 mutation-associated marked reduction has been observed in the level of the mRNA for the NADH dehydrogenase (complex I) ND6 subunit gene, which is located ∼7 kbp upstream and is cotranscribed with the tRNASer(UCN) gene, with strong evidence pointing to a mechanistic link with the tRNA precursor processing defect. Such reduction significantly affects the rate of synthesis of the ND6 subunit and plays a determinant role in the deafness-associated respiratory phenotype of the mutant cell lines. In particular, it accounts for their specific, very significant decrease in glutamate- or malate-dependent O2 consumption. Furthermore, several homoplasmic mtDNA mutations affecting subunits of NADH dehydrogenase may play a synergistic role in the establishment of the respiratory phenotype of the mutant cells.

Mitochondrial A7445G mutation in two pedigrees with palmoplantar keratoderma and deafness

A New Zealand and a Scottish pedigree with maternally inherited sensorineural deafness were both previously shown to carry a heteroplasmic A7445G mutation in the mitochondrial genome. More detailed clinical examination of the New Zealand family showed that the hearing loss was progressive, with the severity of the overall loss and the frequencies most affected differing markedly between individuals of similar age, and showed that many relatives also had palmoplantar keratoderma. Review of the literature demonstrated three other large families with presumed autosomal dominant inheritance of palmoplantar keratoderma and hearing loss. In a United Kingdom pedigree the syndrome was transmitted by female and male parents, an inheritance pattern which made mitochondrial inheritance unlikely; however, in a Turkish and a Japanese pedigree the affected individuals were all maternally related. Subsequent analysis of the Japanese pedigree documented the same A7445G mitochondrial mutation as was previously found in the New Zealand and Scottish pedigrees. Other mitochondrial sequence variants previously reported in the New Zealand or Scottish pedigrees were absent from the Japanese pedigree which suggests that the A7445G mutation arose independently in all three pedigrees. To our knowledge palmoplantar keratoderma has not previously been associated with mitochondrial defects; however, the current findings suggest that the A7445G mutation is associated not only with progressive hearing loss but also with palmoplantar keratoderma. The penetrance and expressivity of both symptoms varied considerably between individuals in the Scottish and New Zealand Studies which suggests that additional environmental and/or genetic factors are involved.

Effects of presynaptic mutations on a postsynaptic Cacna1s calcium channel colocalized with mGluR6 at mouse photoreceptor ribbon synapses.

PURPOSE Photoreceptor ribbon synapses translate light-dependent changes of membrane potential into graded transmitter release via L-type voltage-dependent calcium channel (VDCC) activity. Functional abnormalities (e.g., a reduced electroretinogram b-wave), arising from mutations of presynaptic proteins, such as Bassoon and the VDCCalpha1 subunit Cacna1f, have been shown to altered transmitter release. L-type VDCCalpha1 subtype expression in wild-type and mutant mice was examined, to investigate the underlying pathologic mechanism. METHODS Two antisera against Cacna1f, and a Cacna1f mouse mutant (Cacna1fDeltaEx14-17) were generated. Immunocytochemistry for L-type VDCCalpha1 subunits and additional synaptic marker proteins was performed in wild-type, BassoonDeltaEx4-5 and Cacna1fDeltaEx14-17 mice. RESULTS Active zone staining at photoreceptor ribbon synapses with a panalpha1 antibody colocalized with staining for Cacna1f in wild-type mouse retina. Similarly, in the BassoonDeltaEx4-5 mouse, residual mislocalized staining for panalpha1 and Cacna1f showed colocalization. Unlike the presynaptic location of Cacna1f and panalpha1 antibody staining, the skeletal muscle VDCCalpha1 subunit Cacna1s was present postsynaptically at ON-bipolar cell dendrites, where it colocalized with metabotropic glutamate receptor 6 (mGluR6). Surprisingly, Cacna1s labeling was severely downregulated in the BassoonDeltaEx4-5 and Cacna1fDeltaEx14-17 mutants. Subsequent analyses revealed severely reduced ON-bipolar cell dendritic expression of the sarcoplasmic reticulum Ca(2+) ATPase Serca2 in both mouse mutants and of mGluR6 in the Cacna1fDeltaEx14-17 mutant. CONCLUSIONS Presynaptic mutations leading to reduced photoreceptor-to-bipolar cell signaling are associated with disturbances in protein expression within postsynaptic dendrites. Moreover, detection of Cacna1s and Serca2 in ON-bipolar cell dendrites in wild-type animals suggests a putative role in regulation of postsynaptic Ca(2+) flux.

Clinical manifestations of a unique X‐linked retinal disorder in a large New Zealand family with a novel mutation in CACNA1F, the gene responsible for CSNB2

Purpose: To describe the phenotype in a New Zealand family with an unusual severe X‐linked retinal disorder with a novel I745T mutation in CACNA1F, the gene responsible for incomplete congenital stationary night blindness (CSNB2).

Structural determinants of L-type channel activation in segment IIS6 revealed by a retinal disorder

The mechanism of channel opening for voltage-gated calcium channels is poorly understood. The importance of a conserved isoleucine residue in the pore-lining segment IIS6 has recently been highlighted by functional analyses of a mutation (I745T) in the CaV1.4 channel causing severe visual impairment (Hemara-Wahanui, A., Berjukow, S., Hope, C. I., Dearden, P. K., Wu, S. B., Wilson-Wheeler, J., Sharp, D. M., Lundon-Treweek, P., Clover, G. M., Hoda, J. C., Striessnig, J., Marksteiner, R., Hering, S., and Maw, M. A. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 7553–7558). In the present study we analyzed the influence of amino acids in segment IIS6 on gating of the CaV1.2 channel. Substitution of Ile-781, the CaV1.2 residue corresponding to Ile-745 in CaV1.4, by residues of different hydrophobicity, size and polarity shifted channel activation in the hyperpolarizing direction (I781P > I781T > I781N > I781A > I781L). As I781P caused the most dramatic shift (-37 mV), substitution with this amino acid was used to probe the role of other residues in IIS6 in the process of channel activation. Mutations revealed a high correlation between the midpoint voltages of activation and inactivation. A unique kinetic phenotype was observed for residues 779–782 (LAIA) located in the lower third of segment IIS6; a shift in the voltage dependence of activation was accompanied by a deceleration of activation at hyperpolarized potentials, a deceleration of deactivation at all potentials (I781P and I781T), and decreased inactivation. These findings indicate that Ile-781 substitutions both destabilize the closed conformation and stabilize the open conformation of CaV1.2. Moreover there may be a flexible center of helix bending at positions 779–782 of CaV1.2. These four residues are completely conserved in high voltage-activated calcium channels suggesting that these channels may share a common mechanism of gating.

Oguchi disease: suggestion of linkage to markers on chromosome 2q.

Oguchi disease is a rare autosomal recessive form of congenital stationary night blindness. The condition is associated with fundus discolouration and abnormally slow dark adaptation. Earlier studies suggested that the 48 kD protein S antigen may be involved in the recovery phase of light transduction. Previous cytogenetic and linkage studies have localised the S antigen gene (SAG) to chromosome 2q37.1. In the present study markers which map to distal chromosome 2q were typed in an inbred Oguchi pedigree. The segregation data obtained suggested that the affected subjects are homozygous by descent for a region between D2S172 and D2S345. An intragenic SAG polymorphism was homozygous in all affected people and a recombination event suggested that SAG maps proximal to D2S345. Collectively, these findings support the hypothesis that a defect in S antigen may be responsible for Oguchi disease.

Identification of a new TWIST mutation (7p21) with variable eyelid manifestations supports locus homogeneity of BPES at 3q22

Editor—Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) is an autosomal dominant disorder of eyelid development defined by small palpebral fissures, epicanthus inversus, and ptosis.1 2 BPES type I (OMIM 110100) is characterised by female infertility, whereas BPES type II (OMIM 601649) is transmitted by both females and males. Most cases of BPES types I and II map to chromosome 3q22-q23 ( BPES1 ).3-7However, a second locus ( BPES2 ) was reported in the chromosome 7p13-p21 region on the basis of patients presenting with eyelid anomalies carrying chromosomal abnormalities in the 7p21 region8-11 and the further linkage data of a large Indian family diagnosed initially with BPES type II.7 The TWIST gene, mapped on chromosome 7p21, codes for a transcription factor with a bHLH domain.12 TWIST mutations13-18 have been reported in the heterozygous state in patients presenting with the Saethre-Chotzen syndrome (SCS, OMIM 101400). This disorder is a common autosomal dominant form of syndromic craniosynostosis defined by craniostenosis, minor limb and ear abnormalities, and frequent ptosis of the eyelids.19 In the present study, molecular genetic analysis at TWIST and subsequent clinical re-evaluation of the Indian family …

Photoreceptor Degeneration in Two Mouse Models for Congenital Stationary Night Blindness Type 2

Light-dependent conductance changes of voltage-gated Cav1.4 channels regulate neurotransmitter release at photoreceptor ribbon synapses. Mutations in the human CACNA1F gene encoding the α1F subunit of Cav1.4 channels cause an incomplete form of X-linked congenital stationary night blindness (CSNB2). Many CACNA1F mutations are loss-of-function mutations resulting in non-functional Cav1.4 channels, but some mutations alter the channels’ gating properties and, presumably, disturb Ca2+ influx at photoreceptor ribbon synapses. Notably, a CACNA1F mutation (I745T) was identified in a family with an uncommonly severe CSNB2-like phenotype, and, when expressed in a heterologous system, the mutation was shown to shift the voltage-dependence of channel activation, representing a gain-of-function. To gain insight into the pathomechanism that could explain the severity of this disorder, we generated a mouse model with the corresponding mutation in the murine Cacna1f gene (I756T) and compared it with a mouse model carrying a loss-of-function mutation (ΔEx14–17) in a longitudinal study up to eight months of age. In ΔEx14–17 mutants, the b-wave in the electroretinogram was absent, photoreceptor ribbon synapses were abnormal, and Ca2+ responses to depolarization of photoreceptor terminals were undetectable. In contrast, I756T mutants had a reduced scotopic b-wave, some intact rod ribbon synapses, and a strong, though abnormal, Ca2+ response to depolarization. Both mutants showed a progressive photoreceptor loss, but degeneration was more severe and significantly enhanced in the I756T mutants compared to the ΔEx14–17 mutants.

Connexin26 deafness in several interconnected families

Mutations in the connexin26 gene are the basis of much autosomal recessive sensorineural deafness. There is a high frequency of mutant alleles, largely accounted for by one common mutation, 35delG. We have studied a group of families, who had been brought together through marriages between Deaf persons, in which there are more than 30 Deaf people in four generations. We show that many of the several cases of deafness are the result of 35delG homozygosity or 35delG/Q57X compound heterozygosity at the connexin26 locus. A considerable range of audiographic phenotypes was observed. The combined effects of a high population frequency of mutant alleles, and of positive assortative marriage among the Deaf, led to an infrequently observed recessive pedigree pattern.

The effect on mitochondrial function of the tRNA Ser(UCN)/COI A7445G mtDNA point mutation associated with maternally-inherited sensorineural deafness

An A to G point mutation at nucleotide position 7445 in the mitochondrial DNA genome is associated with maternally‐inherited sensorineural deafness in two separate human pedigrees. To determine whether this point mutation [tRNASer(UCN)/COI A7445G] led to sensorineural deafness by affecting cellular energy metabolism we investigated the bioenergetic function of mitochondria in lymphoblastoid cultures established from these patients. Even though essentially all of the mitochondrial DNA in these cells contained the mutation at nucleotide position 7445, there was no effect on a number of mitochondrial bioenergetic functions (mitochondrial content, membrane potential in both intact and digitonin‐permeabilised cells, cellular ATP/ADP ratios and respiratory enzyme activity) when compared with control cells. The implications of these findings for both the aetiology of the sensorineural deafness associated with the A7445G mtDNA mutation, and the role of bioenergetic defects in mitochondrial DNA diseases in general, are discussed.