Dual AAV-based PCDH15 gene therapy achieves sustained rescue of visual function in a mouse model of Usher syndrome 1F.

Mutations in the PCDH15 gene, encoding protocadherin-15, are among the leading causes of Usher syndrome type 1 (USH1F), and account for up to 12% USH1 cases worldwide. A founder truncating variant of PCDH15 has a ∼2% carrier frequency in Ashkenazi Jews accounting for nearly 60% of their USH1 cases. Although cochlear implants can restore hearing perception in USH1 patients, presently there are no effective treatments for the vision loss due to retinitis pigmentosa. We established a founder allele-specific Pcdh15 knockin mouse model as a platform to ascertain therapeutic strategies. Using a dual-vector approach to circumvent the size limitation of adeno-associated virus, we observed robust expression of exogenous PCDH15 in the retinae of Pcdh15KI mice, sustained recovery of electroretinogram amplitudes and key retinoid oxime, substantially improved light-dependent translocation of phototransduction proteins, and enhanced levels of retinal pigment epithelium-derived enzymes. Thus, our data raise hope and pave the way for future gene therapy trials in USH1F subjects.

Syntaxin 4 is essential for hearing in human and zebrafish.

Congenital hearing impairment (HI) is a genetically highly heterogeneous disorder in which prompt recognition and intervention are crucial to optimize outcomes. In this study, we used exome sequencing to investigate a large consanguineous Pakistani family with eight affected individuals showing bilateral severe-to-profound HI. This identified a homozygous splice region variant in STX4 (c.232 + 6T>C), which causes exon skipping and a frameshift, that segregated with HI (two-point logarithm of odds (LOD) score = 5.9). STX4, a member of the syntaxin family, is a component of the SNARE machinery involved in several vesicle transport and recycling pathways. In silico analysis showed that murine orthologue Stx4a is highly and widespread expressed in the developing and adult inner ear. Immunofluorescent imaging revealed localization of STX4A in the cell body, cell membrane and stereocilia of inner and outer hair cells. Furthermore, a morpholino-based knockdown of stx4 in zebrafish showed an abnormal startle response, morphological and developmental defects, and a disrupted mechanotransduction function in neuromast hair cells measured via FM1-43 uptake. Our findings indicate that STX4 dysfunction leads to HI in humans and zebrafish and supports the evolutionary conserved role of STX4 in inner ear development and hair cell functioning.

Long-Term Anatomic and Visual Outcomes of Planned Preterm Delivery and Treatment of Norrie Disease.

We previously reported that planned preterm delivery at 34 weeks gestational age provided an opportunity to treat Norrie disease in the vasoproliferative phase, prevented infantile retinal detachment, and preserved functional vision without further treatment after infancy. Although retinal vascularization did not proceed postnatally, after 8 years of follow-up, the retinas remained attached, and rudimentary foveal development was observed by optical coherence tomography. Best corrected visual acuity gradually improved to 20/80 with both eyes, and visual fields and real-world visual performance were remarkably functional. Global development progressed appropriately, and no long-term sequelae of premature delivery were observed. [Ophthalmic Surg Lasers Imaging Retina 2022;53:464-467.].

Protocadherin 15 suppresses oligodendrocyte progenitor cell proliferation and promotes motility through distinct signalling pathways.

Oligodendrocyte progenitor cells (OPCs) express protocadherin 15 (Pcdh15), a member of the cadherin superfamily of transmembrane proteins. Little is known about the function of Pcdh15 in the central nervous system (CNS), however, Pcdh15 expression can predict glioma aggression and promote the separation of embryonic human OPCs immediately following a cell division. Herein, we show that Pcdh15 knockdown significantly increases extracellular signal-related kinase (ERK) phosphorylation and activation to enhance OPC proliferation in vitro. Furthermore, Pcdh15 knockdown elevates Cdc42-Arp2/3 signalling and impairs actin kinetics, reducing the frequency of lamellipodial extrusion and slowing filopodial withdrawal. Pcdh15 knockdown also reduces the number of processes supported by each OPC and new process generation. Our data indicate that Pcdh15 is a critical regulator of OPC proliferation and process motility, behaviours that characterise the function of these cells in the healthy CNS, and provide mechanistic insight into the role that Pcdh15 might play in glioma progression.

Identification of Frameshift Variants in POLH Gene Causing Xeroderma Pigmentosum in Two Consanguineous Pakistani Families.

Xeroderma pigmentosum (XP) is a rare autosomal recessive genetic disorder characterized by severe sensitivity of skin to sunlight and an increased risk of skin cancer. XP variant (XPV), a milder subtype, is caused by variants in the POLH gene. POLH encodes an error-prone DNA-polymerase eta (pol eta) which performs translesion synthesis past ultraviolet photoproducts. The current study documents the clinical and genetic investigations of two large consanguineous Pakistani families affected with XPV. In family 1, whole exome sequencing (WES) revealed a novel frameshift variant, c.1723dupG (p.(Val575Glyfs*4)), of POLH, which is predicted to cause frameshift and premature truncation of the encoded enzyme. Indeed, our ex vivo studies in HEK293T cells confirmed the truncation of the encoded protein due to the c.1723dupG variant. In family 2, Sanger sequencing of POLH exons, revealed a recurrent nonsense variant, c.437dupA (p.Tyr146*). POLH forms a hetero-tetrameric POLZ complex with REV3L, REV7, POLD2 and POLD3. Next, we performed in silico analysis of POLH and other POLZ complex genes expression in publicly available single cell mRNAseq datasets from adult human healthy and aging skin. We found overlapping expression of POLH, REV3L and POLD2 in multiple cell types including differentiated and undifferentiated keratinocytes, pericytes and melanocytes in healthy skin. However, in aging human skin, POLH expression is reduced in compare to its POLZ complex partners. Insights from our study will facilitate counseling regarding the molecular and phenotypic landscape of POLH-related XPV.

Corneal Structural Changes in Congenital Glaucoma.

OBJECTIVE: To identify corneal structure differences on quantitative high-frequency ultrasound biomicroscopy (UBM) among subjects with congenital glaucoma compared with controls. METHODS: This prospective case-control study evaluated 180 UBM images from 44 eyes of 30 subjects (18 control and 12 glaucoma, mean age 5.2±8.0 years, range 0.2-25.8 years) enrolled in the Pediatric Anterior Segment Imaging and Innovation Study (PASIIS). ImageJ was used to quantify a comprehensive set of corneal structures according to 21 quantitative parameters. Statistical analysis compared corneal measurements in glaucoma subtypes and age-matched controls with significance testing and mixed effects models. RESULTS: Significant differences between congenital glaucoma cases and controls were identified in 16 of 21 measured parameters including angle-to-angle, central and peripheral corneal thicknesses, scleral integrated pixel density, anterior corneal radius of curvature, and posterior corneal radius of curvature. Eight parameters differed significantly between primary congenital glaucoma and glaucoma following congenital cataract surgery. CONCLUSION: Multiple measurable corneal structural differences exist between congenital glaucoma and control eyes, and between primary and secondary congenital glaucoma, including but not limited to corneal width and thickness. The structural differences can be quantified from UBM image analysis. Further studies are needed to determine whether corneal features associated with glaucoma can be used to diagnose or monitor progression of congenital glaucoma.

The role of CDHR3 in susceptibility to otitis media.

Otitis media (OM) is common in young children and can cause hearing loss and speech, language, and developmental delays. OM has high heritability; however, little is known about OM-related molecular and genetic processes. CDHR3 was previously identified as a locus for OM susceptibility, but to date, studies have focused on how the CDHR3 p.Cys529Tyr variant increases epithelial binding of rhinovirus-C and risk for lung or sinus pathology. In order to further delineate a role for CDHR3 in OM, we performed the following: exome sequencing using DNA samples from OM-affected individuals from 257 multi-ethnic families; Sanger sequencing, logistic regression and transmission disequilibrium tests for 407 US trios or probands with OM; 16S rRNA sequencing and analysis for middle ear and nasopharyngeal samples; and single-cell RNA sequencing and differential expression analyses for mouse middle ear. From exome sequence data, we identified a novel pathogenic CDHR3 splice variant that co-segregates with OM in US and Finnish families. Additionally, a frameshift and six missense rare or low-frequency variants were identified in Finnish probands. In US probands, the CDHR3 p.Cys529Tyr variant was associated with the absence of middle ear fluid at surgery and also with increased relative abundance of Lysobacter in the nasopharynx and Streptomyces in the middle ear. Consistent with published data on airway epithelial cells and our RNA-sequence data from human middle ear tissues, Cdhr3 expression is restricted to ciliated epithelial cells of the middle ear and is downregulated after acute OM. Overall, these findings suggest a critical role for CDHR3 in OM susceptibility. KEY MESSAGES: • Novel rare or low-frequency CDHR3 variants putatively confer risk for otitis media. • Pathogenic variant CDHR3 c.1653 + 3G > A was found in nine families with otitis media. • CDHR3 p.Cys529Tyr was associated with lack of effusion and bacterial otopathogens. • Cdhr3 expression was limited to ciliated epithelial cells in mouse middle ear. • Cdhr3 was downregulated 3 h after infection of mouse middle ear.

CIB2 regulates mTORC1 signaling and is essential for autophagy and visual function.

Age-related macular degeneration (AMD) is a multifactorial neurodegenerative disorder. Although molecular mechanisms remain elusive, deficits in autophagy have been associated with AMD. Here we show that deficiency of calcium and integrin binding protein 2 (CIB2) in mice, leads to age-related pathologies, including sub-retinal pigment epithelium (RPE) deposits, marked accumulation of drusen markers APOE, C3, Aß, and esterified cholesterol, and impaired visual function, which can be rescued using exogenous retinoids. Cib2 mutant mice exhibit reduced lysosomal capacity and autophagic clearance, and increased mTORC1 signaling-a negative regulator of autophagy. We observe concordant molecular deficits in dry-AMD RPE/choroid post-mortem human tissues. Mechanistically, CIB2 negatively regulates mTORC1 by preferentially binding to 'nucleotide empty' or inactive GDP-loaded Rheb. Upregulated mTORC1 signaling has been implicated in lymphangioleiomyomatosis (LAM) cancer. Over-expressing CIB2 in LAM patient-derived fibroblasts downregulates hyperactive mTORC1 signaling. Thus, our findings have significant implications for treatment of AMD and other mTORC1 hyperactivity-associated disorders.

Identification of Hearing Loss-Associated Variants of PTPRQ, MYO15A, and SERPINB6 in Pakistani Families.

The inner ear is an essential part of a well-developed and well-coordinated hearing system. However, hearing loss can make communication and interaction more difficult. Inherited hearing loss (HL) can occur from pathogenic genetic variants that negatively alter the intricate inner ear sensory mechanism. Recessively inherited forms of HL are highly heterogeneous and account for a majority of prelingual deafness. The current study is designed to investigate genetic causes of HL in three consanguineous Pakistani families. After IRB approval, the clinical history and pure tone audiometric data was obtained for the clinical diagnosis of HL segregating in these three Pakistani families. We performed whole exome sequencing (WES) followed by Sanger sequencing in order to identify and validate the HL-associated pathogenic variants, respectively. The 3-D molecular modeling and the Ramachandran analysis of the identified missense variants were compiled to evaluate the impact of the variants on the encoded proteins. Clinical evaluation revealed prelingual severe to profound sensorineural HL segregating among the affected individuals in all three families. Genetic analysis revealed segregation of several novel variants associated with HL, including a canonical splice-site variant (c.55-2A>G) of PTPRQ in family GCFHL-01, a missense variant [c.1079G>A; p.(Arg360Gln)] of SERPINB6 in family LUHL-01, and an insertion variant (c.10208-10211insCCACCAGGCCCGTGCCTC) within MYO15A in family LUHL-011. All the identified variants had very low frequencies in the control databases. The molecular modeling of p.Arg360Gln missense variant also predicted impaired folding of SERPINB6 protein. This study reports the identification of novel disease-causing variants in three known deafness genes and further highlights the genetic heterogeneity of HL in Pakistani population.

Genetic Causes of Oculocutaneous Albinism in Pakistani Population.

Melanin pigment helps protect our body from broad wavelength solar radiation and skin cancer. Among other pigmentation disorders in humans, albinism is reported to manifest in both syndromic and nonsyndromic forms as well as with varying inheritance patterns. Oculocutaneous albinism (OCA), an autosomal recessive nonsyndromic form of albinism, presents as partial to complete loss of melanin in the skin, hair, and iris. OCA has been known to be caused by pathogenic variants in seven different genes, so far, according to all the currently published population studies. However, the detection rate of alleles causing OCA varies from 50% to 90%. One of the significant challenges of uncovering the pathological variant underlying disease etiology is inter- and intra-familial locus heterogeneity. This problem is especially pertinent in highly inbred populations. As examples of such familial locus heterogeneity, we present nine consanguineous Pakistani families with segregating OCA due to variants in one or two different known albinism-associated genes. All of the identified variants are predicted to be pathogenic, which was corroborated by several in silico algorithms and association with diverse clinical phenotypes. We report an individual affected with OCA carries heterozygous, likely pathogenic variants in TYR and OCA2, raising the question of a possible digenic inheritance. Altogether, our study highlights the significance of exome sequencing for the complete genetic diagnosis of inbred families and provides the ramifications of potential genetic interaction and digenic inheritance of variants in the TYR and OCA2 genes.

Identification and Computational Analysis of Novel Pathogenic Variants in Pakistani Families with Diverse Epidermolysis Bullosa Phenotypes.

Epidermolysis bullosa (EB) includes a group of rare gesnodermatoses that result in blistering and erosions of the skin and mucous membranes. Genetically, pathogenic variants in around 20 genes are known to alter the structural and functional integrity of intraepidermal adhesion and dermo-epidermal anchorage, leading to four different types of EB. Here we report the underlying genetic causes of EB phenotypes segregating in seven large consanguineous families, recruited from different regions of Pakistan. Whole exome sequencing, followed by segregation analysis of candidate variants through Sanger sequencing, identified eight pathogenic variants, including three novel (ITGB4: c.1285G>T, and c.3373G>A; PLEC: c.1828A>G) and five previously reported variants (COL7A1: c.6209G>A, and c.1573C>T; FERMT1: c.676insC; LAMA3: c.151insG; LAMB3: c.1705C>T). All identified variants were either absent or had very low frequencies in the control databases. Our in-silico analyses and 3-dimensional (3D) molecular modeling support the deleterious impact of these variants on the encoded proteins. Intriguingly, we report the first case of a recessively inherited form of rare EBS-Ogna associated with a homozygous variant in the PLEC gene. Our study highlights the clinical and genetic diversity of EB in the Pakistani population and expands the mutation spectrum of EB; it could also be useful for prenatal diagnosis and genetic counseling of the affected families.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Otitis media susceptibility and shifts in the head and neck microbiome due to SPINK5 variants.

BACKGROUND: Otitis media (OM) susceptibility has significant heritability; however, the role of rare variants in OM is mostly unknown. Our goal is to identify novel rare variants that confer OM susceptibility. METHODS: We performed exome and Sanger sequencing of >1000 DNA samples from 551 multiethnic families with OM and unrelated individuals, RNA-sequencing and microbiome sequencing and analyses of swabs from the outer ear, middle ear, nasopharynx and oral cavity. We also examined protein localisation and gene expression in infected and healthy middle ear tissues. RESULTS: A large, intermarried pedigree that includes 81 OM-affected and 53 unaffected individuals cosegregates two known rare A2ML1 variants, a common FUT2 variant and a rare, novel pathogenic variant c.1682A>G (p.Glu561Gly) within SPINK5 (LOD=4.09). Carriage of the SPINK5 missense variant resulted in increased relative abundance of Microbacteriaceae in the middle ear, along with occurrence of Microbacteriaceae in the outer ear and oral cavity but not the nasopharynx. Eight additional novel SPINK5 variants were identified in 12 families and individuals with OM. A role for SPINK5 in OM susceptibility is further supported by lower RNA counts in variant carriers, strong SPINK5 localisation in outer ear skin, faint localisation to middle ear mucosa and eardrum and increased SPINK5 expression in human cholesteatoma. CONCLUSION: SPINK5 variants confer susceptibility to non-syndromic OM. These variants potentially contribute to middle ear pathology through breakdown of mucosal and epithelial barriers, immunodeficiency such as poor vaccination response, alteration of head and neck microbiota and facilitation of entry of opportunistic pathogens into the middle ear.

Pathogenic variants of AIPL1, MERTK, GUCY2D, and FOXE3 in Pakistani families with clinically heterogeneous eye diseases.

Significant number out of 2.2 billion vision impairments in the world can be attributed to genetics. The current study is aimed to decipher the genetic basis of Leber congenital Amaurosis (LCA), Anterior Segment dysgenesis (ASD), and Retinitis Pigmentosa (RP), segregating in four large consanguineous Pakistani families. The exome sequencing followed by segregation analysis via Sanger sequencing revealed the LCA phenotypes segregating in families GCUF01 and GCUF04 can be attributed to c.465G>T (p.(Gln155His)) missense and novel c.139_140delinsA p.(Pro47Trhfster38) frameshift variant of AIPL1 and GUCY2D, respectively. The c.1843A>T (p.(Lys615*) truncating allele of MERTK is homozygous in all the affected individuals, presumably suffering with RP, of the GCUF02 family. Meanwhile, co-segregation of the ASD phenotype and the c.289A>G (p.(Ile97Val)) variant of FOXE3 was found in the GCUF06 family. All the identified variants were either absent or present in very low frequencies in the control databases. Our in-silico analyses and 3D molecular modeling support the deleterious impact of these variants on the encoded proteins. Variants identified in MERTK, GUCY2D, and FOXE3 were categorized as "pathogenic" or "likely pathogenic", while the missense variant found in AIPL1 was deemed to have "uncertain significance" based upon the variant pathogenicity guidelines from the American College of Medical Genetics and Genomics (ACMG). This paper highlights the genetic diversity of vision disorders in the Pakistani population and reports the identification of four novel mutations in families who segregate clinically heterogeneous eye diseases. Our results give insight into the genotype-phenotype correlations of AIPL1, FOXE3, MERTK, and GUCY2D variants.

Molecular characterization of SLC24A5 variants and evaluation of Nitisinone treatment efficacy in a zebrafish model of OCA6.

Skin pigmentation is a highly heterogeneous trait with diverse consequences worldwide. SLC24A5, encoding a potent K+ -dependent Na+ /Ca2+ exchanger, is among the known color-coding genes that participate in melanogenesis by maintaining pH in melanosomes. Deficient SLC24A5 activity results in oculocutaneous albinism (OCA) type 6 in humans. In this study, by utilizing a exome sequencing (ES) approach, we identified two new variants [p. (Gly110Arg) and p. (IIe189Ilefs*1)] of SLC24A5 cosegregating with the OCA phenotype, including nystagmus, strabismus, foveal hypoplasia, albinotic fundus, and vision impairment, in three large consanguineous Pakistani families. Both of these variants failed to rescue the pigmentation in zebrafish slc24a5 morphants, confirming the pathogenic effects of the variants. We also phenotypically characterized a commercially available zebrafish mutant line (slc24a5ko ) that harbors a nonsense (p.Tyr208*) allele of slc24a5. Similar to morphants, homozygous slc24a5ko mutants had significantly reduced melanin content and pigmentation. Next, we used these slc24a5ko zebrafish mutants to test the efficacy of nitisinone, a compound known to increase ocular and fur pigmentation in OCA1 (TYR) mutant mice. Treatment of slc24a5ko mutant zebrafish embryos with varying doses of nitisinone did not improve melanin production and pigmentation, suggesting that treatment with nitisinone is unlikely to be therapeutic in OCA6 patients.

Delineation of Homozygous Variants Associated with Prelingual Sensorineural Hearing Loss in Pakistani Families.

Hearing loss is a genetically heterogeneous disorder affecting approximately 360 million people worldwide and is among the most common sensorineural disorders. Here, we report a genetic analysis of seven large consanguineous families segregating prelingual sensorineural hearing loss. Whole-exome sequencing (WES) revealed seven different pathogenic variants segregating with hearing loss in these families, three novel variants (c.1204G>A, c.322G>T, and c.5587C>T) in TMPRSS3, ESRRB, and OTOF, and four previously reported variants (c.208C>T, c.6371G>A, c.226G>A, and c.494C>T) in LRTOMT, MYO15A, KCNE1, and LHFPL5, respectively. All identified variants had very low frequencies in the control databases and were predicted to have pathogenic effects on the encoded proteins. In addition to being familial, we also found intersibship locus heterogeneity in the evaluated families. The known pathogenic c.226C>T variant identified in KCNE1 only segregates with the hearing loss phenotype in a subset of affected members of the family GCNF21. This study further highlights the challenges of identifying disease-causing variants for highly heterogeneous disorders and reports the identification of three novel and four previously reported variants in seven known deafness genes.

Identities and frequencies of variants in CYP1B1 causing primary congenital glaucoma in Pakistan.

Purpose: Primary congenital glaucoma (PCG) is a clinically and genetically heterogeneous disease. The present study was undertaken to find the genetic causes of PCG segregating in 36 large consanguineous Pakistani families. Methods: Ophthalmic examination including fundoscopy, or slit-lamp microscopy was performed to clinically characterize the PCG phenotype. Genomic nucleotide sequences of the CYP1B1 and LTBP2 genes were analyzed with either Sanger or whole exome sequencing. In silico prediction programs were used to assess the pathogenicity of identified alleles. ClustalW alignments were performed to determine evolutionary conservation, and three-dimensional (3D) modeling was performed using HOPE and Phyre2 software. Results: Among the known loci, mutations in CYP1B1 and LTBP2 are the common causes of PCG. Therefore, we analyzed the genomic nucleotide sequences of CYP1B1 and LTBP2, and detected probable pathogenic variants cosegregating with PCG in 14 families. These included the three novel (c.542T>A, c.1436A>G, and c.1325delC) and five known (c.868dupC, c.1168C>T, c.1169G>A, c.1209InsTCATGCCACC, and c.1310C>T) variants in CYP1B1. Two of the novel variants are missense substitutions [p.(Leu181Gln), p.(Gln479Arg)], which replaced evolutionary conserved amino acids, and are predicted to be pathogenic by various in silico programs, while the third variant (c.1325delC) is predicted to cause reading frameshift and premature truncation of the protein. A single mutation, p.(Arg390His), causes PCG in six (~43%) of the 14 CYP1B1 mutations harboring families, and thus, is the most common variant in this cohort. Surprisingly, we did not find any LTBP2 pathogenic variants in the families, which further supports the genetic heterogeneity of PCG in the Pakistani population. Conclusions: In conclusion, results of the present study enhance our understanding of the genetic basis of PCG, support the notion of a genetic modifier of CYP1B1, and contribute to the development of genetic testing protocols and genetic counseling for PCG in Pakistani families.

FAM92A Underlies Nonsyndromic Postaxial Polydactyly in Humans and an Abnormal Limb and Digit Skeletal Phenotype in Mice.

Polydactyly is a common congenital anomaly of the hand and foot. Postaxial polydactyly (PAP) is characterized by one or more posterior or postaxial digits. In a Pakistani family with autosomal recessive nonsyndromic postaxial polydactyly type A (PAPA), we performed genomewide genotyping, linkage analysis, and exome and Sanger sequencing. Exome sequencing revealed a homozygous nonsense variant (c.478C>T, p.[Arg160*]) in the FAM92A gene within the mapped region on 8q21.13-q24.12 that segregated with the PAPA phenotype. We found that FAM92A is expressed in the developing mouse limb and E11.5 limb bud including the progress zone and the apical ectodermal ridge, where it strongly localizes at the cilia level, suggesting an important role in limb patterning. The identified variant leads to a loss of the FAM92A/Chibby1 complex that is crucial for ciliogenesis and impairs the recruitment and the colocalization of FAM92A with Chibby1 at the base of the cilia. In addition, we show that Fam92a-/- homozygous mice also exhibit an abnormal digit morphology, including metatarsal osteomas and polysyndactyly, in addition to distinct abnormalities on the deltoid tuberosity of their humeri. In conclusion, we present a new nonsyndromic PAPA ciliopathy due to a loss-of-function variant in FAM92A. © 2018 American Society for Bone and Mineral Research.

Modifier variant of METTL13 suppresses human GAB1-associated profound deafness.

A modifier variant can abrogate the risk of a monogenic disorder. DFNM1 is a locus on chromosome 1 encoding a dominant suppressor of human DFNB26 recessive, profound deafness. Here, we report that DFNB26 is associated with a substitution (p.Gly116Glu) in the pleckstrin homology domain of GRB2-associated binding protein 1 (GAB1), an essential scaffold in the MET proto-oncogene, receptor tyrosine kinase/HGF (MET/HGF) pathway. A dominant substitution (p.Arg544Gln) of METTL13, encoding a predicted methyltransferase, is the DFNM1 suppressor of GAB1-associated deafness. In zebrafish, human METTL13 mRNA harboring the modifier allele rescued the GAB1-associated morphant phenotype. In mice, GAB1 and METTL13 colocalized in auditory sensory neurons, and METTL13 coimmunoprecipitated with GAB1 and SPRY2, indicating at least a tripartite complex. Expression of MET-signaling genes in human lymphoblastoid cells of individuals homozygous for p.Gly116Glu GAB1 revealed dysregulation of HGF, MET, SHP2, and SPRY2, all of which have reported variants associated with deafness. However, SPRY2 was not dysregulated in normal-hearing humans homozygous for both the GAB1 DFNB26 deafness variant and the dominant METTL13 deafness suppressor, indicating a plausible mechanism of suppression. Identification of METTL13-based modification of MET signaling offers a potential therapeutic strategy for a wide range of associated hearing disorders. Furthermore, MET signaling is essential for diverse functions in many tissues including the inner ear. Therefore, identification of the modifier of MET signaling is likely to have broad clinical implications.

Mutational Spectrum of MYO15A and the Molecular Mechanisms of DFNB3 Human Deafness.

Deafness in humans is a common neurosensory disorder and is genetically heterogeneous. Across diverse ethnic groups, mutations of MYO15A at the DFNB3 locus appear to be the third or fourth most common cause of autosomal-recessive, nonsyndromic deafness. In 49 of the 67 exons of MYO15A, there are currently 192 recessive mutations identified, including 14 novel mutations reported here. These mutations are distributed uniformly across MYO15A with one enigmatic exception; the alternatively spliced giant exon 2, encoding 1,233 residues, has 17 truncating mutations but no convincing deafness-causing missense mutations. MYO15A encodes three distinct isoform classes, one of which is 395 kDa (3,530 residues), the largest member of the myosin superfamily of molecular motors. Studies of Myo15 mouse models that recapitulate DFNB3 revealed two different pathogenic mechanisms of hearing loss. In the inner ear, myosin 15 is necessary both for the development and the long-term maintenance of stereocilia, mechanosensory sound-transducing organelles that extend from the apical surface of hair cells. The goal of this Mutation Update is to provide a comprehensive review of mutations and functions of MYO15A.