The dynamics of actin protrusions can be controlled by tip-localized myosin motors.

Class III myosins localize to inner ear hair cell stereocilia and are thought to be crucial for stereocilia length regulation. Mutations within the motor domain of MYO3A that disrupt its intrinsic motor properties have been associated with non-syndromic hearing loss, suggesting that the motor properties of MYO3A are critical for its function within stereocilia. In this study, we investigated the impact of a MYO3A hearing loss mutation, H442N, using both in vitro motor assays and cell biological studies. Our results demonstrate the mutation causes a dramatic increase in intrinsic motor properties, actin-activated ATPase and in vitro actin gliding velocity, as well as an increase in actin protrusion extension velocity. We propose that both "gain of function" and "loss of function" mutations in MYO3A can impair stereocilia length regulation, which is crucial for stereocilia formation during development and normal hearing. Furthermore, we generated chimeric MYO3A constructs that replace the MYO3A motor and neck domain with the motor and neck domain of other myosins. We found that duty ratio, fraction of ATPase cycle myosin is strongly bound to actin, is a critical motor property that dictates the ability to tip localize within filopodia. In addition, in vitro actin gliding velocities correlated extremely well with filopodial extension velocities over a wide range of gliding and extension velocities. Taken together, our data suggest a model in which tip-localized myosin motors exert force that slides the membrane tip-ward, which can combat membrane tension and enhance the actin polymerization rate that ultimately drives protrusion elongation.

Whole exome sequencing reveals pathogenic variants in MYO3A, MYO15A and COL9A3 and differential frequencies in ancestral alleles in hearing impairment genes among individuals from Cameroon.

There is scarcity of known gene variants of hearing impairment (HI) in African populations. This knowledge deficit is ultimately affecting the development of genetic diagnoses. We used whole exome sequencing to investigate gene variants, pathways of interactive genes and the fractions of ancestral overderived alleles for 159 HI genes among 18 Cameroonian patients with non-syndromic HI (NSHI) and 129 ethnically matched controls. Pathogenic and likely pathogenic (PLP) variants were found in MYO3A, MYO15A and COL9A3, with a resolution rate of 50% (9/18 patients). The study identified significant genetic differentiation in novel population-specific gene variants at FOXD4L2, DHRS2L6, RPL3L and VTN between HI patients and controls. These gene variants are found in functional/co-expressed interactive networks with other known HI-associated genes and in the same pathways with VTN being a hub protein, that is, focal adhesion pathway and regulation of the actin cytoskeleton (P-values <0.05). The results suggest that these novel population-specific gene variants are possible modifiers of the HI phenotypes. We found a high proportion of ancestral allele versus derived at low HI patients-specific minor allele frequency in the range of 0.0-0.1. The results showed a relatively low pickup rate of PLP variants in known genes in this group of Cameroonian patients with NSHI. In addition, findings may signal an evolutionary enrichment of some variants of HI genes in patients, as the result of polygenic adaptation, and suggest the possibility of multigenic influence on the phenotype of congenital HI, which deserves further investigations.

Knock-In Mice with Myo3a Y137C Mutation Displayed Progressive Hearing Loss and Hair Cell Degeneration in the Inner Ear.

Myo3a is expressed in cochlear hair cells and retinal cells and is responsible for human recessive hereditary nonsyndromic deafness (DFNB30). To investigate the mechanism of DFNB30-type deafness, we established a mouse model of Myo3a kinase domain Y137C mutation by using CRISPR/Cas9 system. No difference in hearing between 2-month-old Myo3a mutant mice and wild-type mice was observed. The hearing threshold of the ≥6-month-old mutant mice was significantly elevated compared with that of the wild-type mice. We observed degeneration in the inner ear hair cells of 6-month-old Myo3a mutant mice, and the degeneration became more severe at the age of 12 months. We also found structural abnormality in the cochlear hair cell stereocilia. Our results showed that Myo3a is essential for normal hearing by maintaining the intact structure of hair cell stereocilia, and the kinase domain plays a critical role in the normal functions of Myo3a. This mouse line is an excellent model for studying DFNB30-type deafness in humans.

Impact of the Motor and Tail Domains of Class III Myosins on Regulating the Formation and Elongation of Actin Protrusions.

Class III myosins (MYO3A and MYO3B) are proposed to function as transporters as well as length and ultrastructure regulators within stable actin-based protrusions such as stereocilia and calycal processes. MYO3A differs from MYO3B in that it contains an extended tail domain with an additional actin-binding motif. We examined how the properties of the motor and tail domains of human class III myosins impact their ability to enhance the formation and elongation of actin protrusions. Direct examination of the motor and enzymatic properties of human MYO3A and MYO3B revealed that MYO3A is a 2-fold faster motor with enhanced ATPase activity and actin affinity. A chimera in which the MYO3A tail was fused to the MYO3B motor demonstrated that motor activity correlates with formation and elongation of actin protrusions. We demonstrate that removal of individual exons (30-34) in the MYO3A tail does not prevent filopodia tip localization but abolishes the ability to enhance actin protrusion formation and elongation in COS7 cells. Interestingly, our results demonstrate that MYO3A slows filopodia dynamics and enhances filopodia lifetime in COS7 cells. We also demonstrate that MYO3A is more efficient than MYO3B at increasing formation and elongation of stable microvilli on the surface of cultured epithelial cells. We propose that the unique features of MYO3A, enhanced motor activity, and an extended tail with tail actin-binding motif, allow it to play an important role in stable actin protrusion length and ultrastructure maintenance.

MYO3A Causes Human Dominant Deafness and Interacts with Protocadherin 15-CD2 Isoform.

Hereditary hearing loss (HL) is characterized by both allelic and locus genetic heterogeneity. Both recessive and dominant forms of HL may be caused by different mutations in the same deafness gene. In a family with post-lingual progressive non-syndromic deafness, whole-exome sequencing of genomic DNA from five hearing-impaired relatives revealed a single variant, p.Gly488Glu (rs145970949:G>A) in MYO3A, co-segregating with HL as an autosomal dominant trait. This amino acid change, predicted to be pathogenic, alters a highly conserved residue in the motor domain of MYO3A. The mutation severely alters the ATPase activity and motility of the protein in vitro, and the mutant protein fails to accumulate in the filopodia tips in COS7 cells. However, the mutant MYO3A was able to reach the tips of organotypic inner ear culture hair cell stereocilia, raising the possibility of a local effect on positioning of the mechanoelectrical transduction (MET) complex at the stereocilia tips. To address this hypothesis, we investigated the interaction of MYO3A with the cytosolic tail of the integral tip-link protein protocadherin 15 (PCDH15), a core component of MET complex. Interestingly, we uncovered a novel interaction between MYO3A and PCDH15 shedding new light on the function of myosin IIIA at stereocilia tips.

Phosphorylation of the kinase domain regulates autophosphorylation of myosin IIIA and its translocation in microvilli.

Motor activity of myosin III is regulated by autophosphorylation. To investigate the role of the kinase activity on the transporter function of myosin IIIA (Myo3A), we identified the phosphorylation sites of kinase domain (KD), which is responsible for the regulation of kinase activity and thus motor function. Using mass spectrometry, we identified six phosphorylation sites in the KD, which are highly conserved among class III myosins and Ste20-related misshapen (Msn) kinases. Two predominant sites, Thr¹84 and Thr¹88, in KD are important for phosphorylation of the KD as well as the motor domain, which regulates the affinity for actin. In the Caco2 cells, the full-length human Myo3A (hMyo3AFull) markedly enlarged the microvilli, although it did not show discrete localization within the microvilli. On the other hand, hMyo3AFull(T184A) and hMyo3AFull(T188A) both showed clear localization at the microvilli tips. Our results suggest that Myo3A induces large actin bundle formation to form microvilli, and phosphorylation of KD at Thr¹84 and Thr¹88 is critical for the kinase activity of Myo3A, and regulation of Myo3A translocation to the tip of microvilli. Retinal extracts potently dephosphorylate both KD and motor domain without IQ motifs (MDIQo), which was inhibited by okadaic acid (OA) with nanomolar range and by tautomycetin (TMC) with micromolar range. The results suggest that Myo3A phosphatase is protein phosphatase type 2A (PP2A). Supporting this result, recombinant PP2Ac potently dephosphorylates both KD and MDIQo. We propose that the phosphorylation-dephosphorylation mechanism plays an essential role in mediating the transport and actin bundle formation and stability functions of hMyo3A.

Myosin 3A kinase activity is regulated by phosphorylation of the kinase domain activation loop.

Class III myosins are unique members of the myosin superfamily in that they contain both a motor and kinase domain. We have found that motor activity is decreased by autophosphorylation, although little is known about the regulation of the kinase domain. We demonstrate by mass spectrometry that Thr-178 and Thr-184 in the kinase domain activation loop and two threonines in the loop 2 region of the motor domain are autophosphorylated (Thr-908 and Thr-919). The kinase activity of MYO3A 2IQ with the phosphomimic (T184E) or phosphoblock (T184A) mutations demonstrates that kinase activity is reduced 30-fold as a result of the T184A mutation, although the Thr-178 site only had a minor impact on kinase activity. Interestingly, the actin-activated ATPase activity of MYO3A 2IQ is slightly reduced as a result of the T178A and T184A mutations suggesting coupling between motor and kinase domains. Full-length GFP-tagged T184A and T184E MYO3A constructs transfected into COS7 cells do not disrupt the ability of MYO3A to localize to filopodia structures. In addition, we demonstrate that T184E MYO3A reduces filopodia elongation in the presence of espin-1, whereas T184A enhances filopodia elongation in a similar fashion to kinase-dead MYO3A. Our results suggest that as MYO3A accumulates at the tips of actin protrusions, autophosphorylation of Thr-184 enhances kinase activity resulting in phosphorylation of the MYO3A motor and reducing motor activity. The differential regulation of the kinase and motor activities allows for MYO3A to precisely self-regulate its concentration in the actin bundle-based structures of cells.

Regulation of arrestin translocation by Ca2+ and myosin III in Drosophila photoreceptors.

Upon illumination several phototransduction proteins translocate between cell body and photosensory compartments. In Drosophila photoreceptors arrestin (Arr2) translocates from cell body to the microvillar rhabdomere down a diffusion gradient created by binding of Arr2 to photo-isomerized metarhodopsin. Translocation is profoundly slowed in mutants of key phototransduction proteins including phospholipase C (PLC) and the Ca(2+)-permeable transient receptor potential channel (TRP), but how the phototransduction cascade accelerates Arr2 translocation is unknown. Using real-time fluorescent imaging of Arr2-green fluorescent protein translocation in dissociated ommatidia, we show that translocation is profoundly slowed in Ca(2+)-free solutions. Conversely, in a blind PLC mutant with ∼100-fold slower translocation, rapid translocation was rescued by the Ca(2+) ionophore, ionomycin. In mutants lacking NINAC (calmodulin [CaM] binding myosin III) in the cell body, translocation remained rapid even in Ca(2+)-free solutions. Immunolabeling revealed that Arr2 in the cell body colocalized with NINAC in the dark. In intact eyes, the impaired translocation found in trp mutants was rescued in ninaC;trp double mutants. Nevertheless, translocation following prolonged dark adaptation was significantly slower in ninaC mutants, than in wild type: a difference that was reflected in the slow decay of the electroretinogram. The results suggest that cytosolic NINAC is a Ca(2+)-dependent binding target for Arr2, which protects Arr2 from immobilization by a second potential sink that sequesters and releases arrestin on a much slower timescale. We propose that rapid Ca(2+)/CaM-dependent release of Arr2 from NINAC upon Ca(2+) influx accounts for the acceleration of translocation by phototransduction.

Myopodin isoforms alter the chemokinetic response of PC3 cells in response to different migration stimuli via differential effects on Rho-ROCK signaling pathways.

The gene encoding myopodin, an actin binding protein, is commonly deleted in invasive, but not in indolent, prostate cancers. There are conflicting reports on the effects of myopodin expression on prostate cancer cell migration and invasion. The recent recognition that myopodin is expressed as four different isoforms further complicates our understanding of how this potentially important invasive prostate cancer biomarker affects tumor cell migration and invasion. We now show that myopodin affects the chemokinetic, rather than the chemotactic, properties of PC3 prostate cancer cells. Furthermore, all myopodin isoforms can either increase or decrease PC3 cell migration in response to different chemokinetic stimuli. These migration properties were reflected by differences in cell morphology and the relative dependence on Rho-ROCK signaling pathways induced by the environmental stimuli. Truncation analysis determined that a unique 9-residue C-terminal sequence in the shortest isoform and the conserved, PDZ domain-containing N-terminal region of the long isoforms both contribute to the ability of myopodin to alter the response of PC3 cells to chemokinetic stimuli. Matrigel invasion assays also indicated that myopodin primarily affects the migration, rather than the invasion, properties of PC3 cells. The correlation between loss of myopodin expression and invasive prostate cancer therefore reflects complex myopodin interactions with pathways that regulate the cellular migration response to diverse signals that may be present in a tumor microenvironment.

Molecular insights into MYO3A kinase domain variants explain variability in both severity and progression of DFNB30 hearing impairment.

Hereditary hearing impairment (HI) is a common disease with the highest incidence among sensory defects. Several genes have been identified to affect stereocilia structure causing HI, including the unconventional myosin3A. Interestingly, we noticed that variants in MYO3A gene have been previously found to cause variable HI onset and severity. Using clinical exome sequencing, we identified a novel pathogenic variant p.(Lys50Arg) in the MYO3A kinase domain (MYO3A-KD). Previous in vitro studies supported its damaging effect as a 'kinase-dead' mutant. We further analyzed this variation through molecular dynamics which predicts that changes in flexibility of MYO3A structure would influence the protein-ATP binding properties. This Lys50Arg mutation segregated with congenital profound non-syndromic HI. To better investigate this variability, we collected previously identified MYO3A-KDs variants, p.(Tyr129Cys), p.(His142Gln) and p.(Pro189Thr), and built both wild type and mutant 3 D MYO3A-KD models to assess their impact on the protein structure and function. Our results suggest that KD mutations could either cause a congenital profound form of HI, when particularly affecting the kinase activity and preventing the auto-phosphorylation of the motor, or a late onset and progressive form, when partially or completely inactivating the MYO3A protein. In conclusion, we report a novel pathogenic variant affecting the ATP-binding site within the MYO3A-KD causing congenital profound HI. Through computational approaches we provide a deeper understanding on the correlation between the effects of MYO3A-KD mutations and the variable hearing phenotypes. To the best of our knowledge this is the first study to correlate mutations' genotypes with the variable phenotypes of DFNB30.Communicated by Ramaswamy H. Sarma.

Frequency and origin of the c.2090T>G p.(Leu697Trp) MYO3A variant associated with autosomal dominant hearing loss.

We recently described a novel missense variant [c.2090T>G:p.(Leu697Trp)] in the MYO3A gene, found in two Brazilian families with late-onset autosomal dominant nonsyndromic hearing loss (ADNSHL). Since then, with the objective of evaluating its contribution to ADNSHL in Brazil, the variant was screened in additional 101 pedigrees with probable ADNSHL without conclusive molecular diagnosis. The variant was found in three additional families, explaining 3/101 (~3%) of cases with ADNSHL in our Brazilian pedigree collection. In order to identify the origin of the variant, 21 individuals from the five families were genotyped with a high-density SNP array (~600 K SNPs- Axiom Human Origins; ThermoFisher). The identity by descent (IBD) approach revealed that many pairs of individuals from the different families have a kinship coefficient equivalent to that of second cousins, and all share a minimum haplotype of ~607 kb which includes the c.2090T>G variant suggesting it probably arose in a common ancestor. We inferred that the mutation occurred in a chromosomal segment of European ancestry and the time since the most common ancestor was estimated in 1100 years (CI = 775-1425). This variant was also reported in a Dutch family, which shares a 87,121 bp haplotype with the Brazilian samples, suggesting that Dutch colonists may have brought it to Northeastern Brazil in the 17th century. Therefore, the present study opens new avenues to investigate this variant not only in Brazilians but also in European families with ADNSHL.

Deafness mutation in the MYO3A motor domain impairs actin protrusion elongation mechanism.

Class III myosins are actin-based motors proposed to transport cargo to the distal tips of stereocilia in the inner ear hair cells and/or to participate in stereocilia length regulation, which is especially important during development. Mutations in the MYO3A gene are associated with delayed onset deafness. A previous study demonstrated that L697W, a dominant deafness mutation, disrupts MYO3A ATPase and motor properties but does not impair its ability to localize to the tips of actin protrusions. In the current study, we characterized the transient kinetic mechanism of the L697W motor ATPase cycle. Our kinetic analysis demonstrates that the mutation slows the ADP release and ATP hydrolysis steps, which results in a slight reduction in the duty ratio and slows detachment kinetics. Fluorescence recovery after photobleaching (FRAP) of filopodia tip localized L697W and WT MYO3A in COS-7 cells revealed that the mutant does not alter turnover or average intensity at the actin protrusion tips. We demonstrate that the mutation slows filopodia extension velocity in COS-7 cells which correlates with its twofold slower in vitro actin gliding velocity. Overall, this work allowed us to propose a model for how the motor properties of MYO3A are crucial for facilitating actin protrusion length regulation.

Intermolecular autophosphorylation regulates myosin IIIa activity and localization in parallel actin bundles.

Myosin IIIa (Myo3A) transports cargo to the distal end of actin protrusions and contains a kinase domain that is thought to autoregulate its activity. Because Myo3A tends to cluster at the tips of actin protrusions, we investigated whether intermolecular phosphorylation could regulate Myo3A biochemical activity, cellular localization, and cellular function. Inactivation of Myo3A 2IQ kinase domain with the point mutation K50R did not alter maximal ATPase activity, whereas phosphorylation of Myo3A 2IQ resulted in reduced maximal ATPase activity and actin affinity. The rate and degree of Myo3A 2IQ autophosphorylation was unchanged by the presence of actin but was found to be dependent upon Myo3A 2IQ concentration within the range of 0.1 to 1.2 µm, indicating intermolecular autophosphorylation. In cultured cells, we observed that the filopodial tip localization of Myo3A lacking the kinase domain decreased when co-expressed with kinase-active, full-length Myo3A. The cellular consequence of reduced Myo3A tip localization was decreased filopodial density along the cell periphery, identifying a novel cellular function for Myo3A in mediating the formation and stability of actin-based protrusions. Our results suggest that Myo3A motor activity is regulated through a mechanism involving concentration-dependent autophosphorylation. We suggest that this regulatory mechanism plays an essential role in mediating the transport and actin bundle formation/stability functions of Myo3A.

Mouse class III myosins: kinase activity and phosphorylation sites.

As class III unconventional myosins are motor proteins with an N-terminal kinase domain, it seems likely they play a role in both signaling and actin based transport. A growing body of evidence indicates that the motor functions of human class IIIA myosin, which has been implicated in progressive hearing loss, are modulated by intermolecular autophosphorylation. However, the phosphorylation sites have not been identified. We studied the kinase activity and phosphorylation sites of mouse class III myosins, mMyo3A and 3B, which are highly similar to their human orthologs. We demonstrate that the kinase domains of mMyo3A and 3B are active kinases, and that they have similar, if not identical, substrate specificities. We show that the kinase domains of these proteins autophosphorylate, and that they can phosphorylate sites within their myosin and tail domains. Using liquid chromatography-mass spectrometry, we identified phosphorylated sites in the kinase, myosin motor and tail domains of both mMyo3A and 3B. Most of the phosphorylated sites we identified and their consensus phosphorylation motifs are highly conserved among vertebrate class III myosins, including human class III myosins. Our findings are a major step toward understanding how the functions of class III myosins are regulated by phosphorylation.

A mouse model for human hearing loss DFNB30 due to loss of function of myosin IIIA.

The motor protein myosin IIIA is critical for maintenance of normal hearing. Homozygosity and compound heterozygosity for loss-of-function mutations in MYO3A, which encodes myosin IIIA, are responsible for inherited human progressive hearing loss DFNB30. To further evaluate this hearing loss, we constructed a mouse model, Myo3a(KI/KI), that harbors the mutation equivalent to the nonsense allele responsible for the most severe human phenotype. Myo3a(KI/KI) mice were compared to their wild-type littermates. Myosin IIIA, with a unique N-terminal kinase domain and a C-terminal actin-binding domain, localizes to the tips of stereocilia in wild-type mice but is absent in the mutant. The phenotype of the Myo3a(KI/KI) mouse parallels the phenotype of human DFNB30. Hearing loss, as measured by auditory brainstem response, is reduced and progresses significantly with age. Vestibular function is normal. Outer hair cells of Myo3a(KI/KI) mice degenerate with age in a pattern consistent with their progressive hearing loss.

Effect of phosphorylation in the motor domain of human myosin IIIA on its ATP hydrolysis cycle.

Previous findings suggested that the motor activity of human myosin IIIA (HM3A) is influenced by phosphorylation [Kambara, T., et al. (2006) J. Biol. Chem. 281, 37291-37301]; however, how phosphorylation controls the motor activity of HM3A is obscure. In this study, we clarify the kinetic basis of the effect of phosphorylation on the ATP hydrolysis cycle of the motor domain of HM3A (huM3AMD). The affinity of human myosin IIIA for filamentous actin in the presence of ATP is more than 100-fold decreased by phosphorylation, while the maximum rate of ATP turnover is virtually unchanged. The rate of release of ADP from acto-phosphorylated huM3AMD is 6-fold greater than the overall cycle rate, and thus not a rate-determining step. The rate constant of the ATP hydrolysis step of the actin-dissociated form is markedly increased by phosphorylation by 30-fold. The dissociation constant for dissociation of the ATP-bound form of huM3AMD from actin is greatly increased by phosphorylation, and this result agrees well with the significant increase in the K(actin) value of the steady-state ATPase reaction. The rate constant of the P(i) off step is greater than 60 s(-1), suggesting that this step does not limit the overall ATP hydrolysis cycle rate. Our kinetic model indicates that phosphorylation induces the dissociation of huM3AMD from actin during the ATP hydrolysis cycle, and this is due to the phosphorylation-dependent marked decrease in the affinity of huM3AMD.ATP for actin and the increase in the ATP hydrolysis rate of huM3AMD in the actin-dissociated state. These results suggest that the phosphorylation of myosin IIIA significantly lowers the duty ratio, which may influence the cargo transporting ability of the native form of myosin IIIA that contains the ATP-independent actin binding site in the tail.

A novel missense variant in MYO3A is associated with autosomal dominant high-frequency hearing loss in a German family.

BACKGROUND: MYO3A, encoding the myosin IIIA protein, is associated with autosomal recessive and autosomal dominant nonsyndromic hearing loss. To date, only two missense variants located in the motor-head domain of MYO3A have been described in autosomal dominant families with progressive, mild-to-profound sensorineural hearing loss. These variants alter the ATPase activity of myosin IIIA. METHODS: Exome sequencing of a proband from a three-generation German family with prelingual, moderate-to-profound, high-frequency hearing loss was performed. Segregation analysis confirmed a dominant inheritance pattern. Regression analysis of mean hearing level thresholds per individual and ear was performed at high-, mid-, and low-frequencies. RESULTS: A novel heterozygous missense variant c.716T>C, p.(Leu239Pro) in the kinase domain of MYO3A was identified that is predicted in silico as disease causing. High-frequency, progressive hearing loss was identified. CONCLUSION: Correlation analysis of pure-tone hearing thresholds revealed progressive hearing loss, especially in the high-frequencies. In the present study, we report the first dominant likely pathogenic variant in MYO3A in a European family and further support MYO3A as an autosomal dominant hearing loss gene.

Mutations in the tail domain of MYH3 contributes to atrial septal defect.

Atrial septal defect (ASD) is one of the most common congenital heart defects diagnosed in children. Sarcomeric genes has been attributed to ASD and knockdown of MYH3 functionally homologues gene in chick models indicated abnormal atrial septal development. Here, we report for the first time, a case-control study investigating the role of MYH3 among non-syndromic ASD patients in contributing to septal development. Four amplicons which will amplifies the 40 kb MYH3 were designed and amplified using long range-PCR. The amplicons were then sequenced using indexed paired-end libraries on the MiSeq platform. The STREGA guidelines were applied for planning and reporting. The non-synonymous c. 3574G>A (p.Ala1192Thr) [p = 0.001, OR = 2.30 (1.36-3.87)] located within the tail domain indicated a highly conserved protein region. The mutant model of c. 3574G>A (p.Ala1192Thr) showed high root mean square deviation (RMSD) values compared to the wild model. To our knowledge, this is the first study to provide compelling evidence on the pathogenesis of MYH3 variants towards ASD hence, suggesting the crucial role of non-synonymous variants in the tail domain of MYH3 towards atrial septal development. It is hoped that this gene can be used as panel for diagnosis of ASD in future.

Recessive marfanoid syndrome with herniation associated with a homozygous mutation in Fibulin-3.

We have previously reported on a consanguineous family where 2 siblings, a girl and a boy, presented with tall stature, long and triangular faces, prominent forehead, telecanthus, ptosis, everted lower eyelids, downslanting palpebral fissures, large ears, high arched palate, long arm span, arachnodactyly, advanced bone age, joint laxity, pectus excavatum, inguinal hernia, and myopia, suggestive of a new subtype of connective tissue disorder (Megarbane et al. AJMG, 2012; 158(A)5: 1185-1189). On clinical follow-up, both patients had multiple inguinal, crural, and abdominal herniae, intestinal occlusions, several huge diverticula throughout the gut and the bladder, and rectal prolapse. In addition, the girl had a mild hearing impairment, and the boy a left diaphragmatic hernia. Here we describe the molecular characterization of this disorder using Whole Exome Sequencing, revealing, in both siblings, a novel homozygous missense variant in the EFEMP1 gene, c.163T > C; p.(Cys55Arg) whose homozygous by descent, autosomal recessive transmission was confirmed through segregation analysis by Sanger sequencing. In addition, the girl exhibited a homozygous mutation in the MYO3A gene, c.1370_1371delGA; p.(Arg457Asnfs*25), associated with non-syndromic deafness. The siblings were also found to harbor a homozygous nonsense variant in the VCPKMT gene. We review the literature and discuss our updated clinical and molecular findings that suggest EFEMP1 to be the probable candidate gene implicated in this novel connective tissue disease.

Cloning and distribution of myosin 3B in the mouse retina: differential distribution in cone outer segments.

Class III myosins are important for the function and survival of photoreceptors and ciliary hair cells. Although vertebrates possess two class III myosin genes, myo3A and myo3B, recent studies have focused on Myo3A because mutations in the human gene are implicated in progressive hearing loss. Myo3B may compensate for defects in Myo3A, yet little is known about its distribution and function. This study focuses on Myo3B expression in the mouse retina. We cloned two variants of myo3B from mouse retina and determined that they are expressed early in retinal development. In this study we show for the first time in a mammal that both Myo3B and Myo3A proteins are present in inner segments of all photoreceptors. Myo3B is also present in outer segments of S opsin-immunoreactive cones but not M opsin dominant cones. Myo3B is also detected in rare cells of the inner nuclear layer and some ganglion cells. Myo3B may have diverse roles in retinal neurons. In photoreceptor inner segments Myo3B is positioned appropriately to prevent photoreceptor loss of function caused by Myo3A defects.