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Microphthalmia, anophthalmia, and anterior segment dysgenesis are severe ocular developmental defects. There is a wide genetic heterogeneity leading to these ocular malformations. By using whole genome, exome and targeted sequencing in patients with ocular developmental anomalies, six biallelic pathogenic variants (including five novel variants) were identified in the PXDN gene in four families with microphthalmia and anterior segment dysgenesis. Only 11 different mutations (11 families) have been described in this gene to date. The phenotype of these patients is variable in severity, ranging from cataract and developmental glaucoma to complex microphthalmia. Interestingly, two unrelated patients of our series presented with an ocular phenotype including aniridia and microspherophakia. However, despite various phenotypic presentations and types of mutations, no genotype-phenotype correlation could be made. Thus, this work improves our knowledge of the recessive phenotype associated with biallelic variants in this gene and highlights the importance of screening PXDN in patients with anterior segment dysgenesis with or without microphthalmia.

The identification of genetic variants implicated in human developmental disorders has been revolutionized by second-generation sequencing combined with international pooling of cases. Here, we describe seven individuals who have diverse yet overlapping developmental anomalies, and who all have de novo missense FBXW11 variants identified by whole exome or whole genome sequencing and not reported in the gnomAD database. Their phenotypes include striking neurodevelopmental, digital, jaw, and eye anomalies, and in one individual, features resembling Noonan syndrome, a condition caused by dysregulated RAS signaling. FBXW11 encodes an F-box protein, part of the Skp1-cullin-F-box (SCF) ubiquitin ligase complex, involved in ubiquitination and proteasomal degradation and thus fundamental to many protein regulatory processes. FBXW11 targets include β-catenin and GLI transcription factors, key mediators of Wnt and Hh signaling, respectively, critical to digital, neurological, and eye development. Structural analyses indicate affected residues cluster at the surface of the loops of the substrate-binding domain of FBXW11, and the variants are predicted to destabilize the protein and/or its interactions. In situ hybridization studies on human and zebrafish embryonic tissues demonstrate FBXW11 is expressed in the developing eye, brain, mandibular processes, and limb buds or pectoral fins. Knockdown of the zebrafish FBXW11 orthologs fbxw11a and fbxw11b resulted in embryos with smaller, misshapen, and underdeveloped eyes and abnormal jaw and pectoral fin development. Our findings support the role of FBXW11 in multiple developmental processes, including those involving the brain, eye, digits, and jaw., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Two distinct syndromes arise from pathogenic variants in the X-linked gene BCOR (BCL-6 corepressor): oculofaciocardiodental (OFCD) syndrome, which affects females, and a severe microphthalmia ('Lenz'-type) syndrome affecting males. OFCD is an X-linked dominant syndrome caused by a variety of BCOR null mutations. As it manifests only in females, it is presumed to be lethal in males. The severe male X-linked recessive microphthalmia syndrome ('Lenz') usually includes developmental delay in addition to the eye findings and is caused by hypomorphic BCOR variants, mainly by a specific missense variant c.254C > T, p.(Pro85Leu). Here, we detail 16 new cases (11 females with 4 additional, genetically confirmed, affected female relatives; 5 male cases each with unaffected carrier mothers). We describe new variants and broaden the phenotypic description for OFCD to include neuropathy, muscle hypotonia, pituitary underdevelopment, brain atrophy, lipoma and the first description of childhood lymphoma in an OFCD case. Our male X-linked recessive cases show significant new phenotypes: developmental delay (without eye anomalies) in two affected half-brothers with a novel BCOR variant, and one male with high myopia, megalophthalmos, posterior embryotoxon, developmental delay, and heart and bony anomalies with a previously undescribed BCOR splice site variant. Our female OFCD cases and their affected female relatives showed variable features, but consistently had early onset cataracts. We show that a mosaic carrier mother manifested early cataract and dental anomalies. All female carriers of the male X-linked recessive cases for whom genetic confirmation was available showed skewed X-inactivation and were unaffected. In view of the extended phenotype, we suggest a new term of X-linked BCOR-related syndrome.

Background: Congenital cataract displays large phenotypic (syndromic and isolated cataracts) and genetic heterogeneity. Mutations in several transcription factors involved in eye development, like PITX3, have been associated with congenital cataracts and anterior segment mesenchymal disorders., Materials and Methods: Targeted sequencing of 187 genes involved in ocular development was performed in 96 patients with mainly anophthalmia and microphthalmia. Additionally, Sanger sequencing analysis of PITX3 was performed on a second cohort of 32 index cases with congenital cataract and Peters anomaly and/or sclereocornea., Results: We described five families with four different PITX3 mutations, two of which were novel. In Family 1, the heterozygous recurrent c.640_656dup (p.Gly220Profs*95) mutation cosegregated with eye anomalies ranging from congenital cataract to Peters anomaly. In Family 2, the novel c.669del [p.(Leu225Trpfs*84)] mutation cosegregated with dominantly inherited eye anomalies ranging from posterior embryotoxon to congenital cataract in heterozygous carriers and congenital sclereocornea and cataract in a patient homozygous for this mutation. In Family 3, we identified the recurrent heterozygous c.640_656dup (p.Gly220Profs*95) mutation segregating with congenital cataract. In Family 4, the de novo c.582del [p.(Ile194Metfs*115)] mutation was identified in a patient with congenital cataract, microphthalmia, developmental delay and autism. In Family 5, the c.38G>A (p.Ser13Asn) mutation segregated dominantly in a family with Peters anomaly, which is a novel phenotype associated with the c.38G>A variant compared with the previously reported isolated congenital cataract., Conclusions: Our study unveils different phenotypes associated with known and novel mutations in PITX3, which will improve the genetic counselling of patients and their families.

A consanguineous family from Pakistan was ascertained to have a novel deafness-dystonia syndrome with motor regression, ichthyosis-like features and signs of sensory neuropathy. By applying a combined strategy of linkage analysis and whole-exome sequencing in the presented family, a homozygous nonsense mutation, c.4G>T (p.Glu2*), in FITM2 was identified. FITM2 and its paralog FITM1 constitute an evolutionary conserved protein family involved in partitioning of triglycerides into cellular lipid droplets. Despite the role of FITM2 in neutral lipid storage and metabolism, no indications for lipodystrophy were observed in the affected individuals. In order to obtain independent evidence for the involvement of FITM2 in the human pathology, downregulation of the single Fitm ortholog, CG10671, in Drosophila melanogaster was pursued using RNA interference. Characteristics of the syndrome, including progressive locomotor impairment, hearing loss and disturbed sensory functions, were recapitulated in Drosophila, which supports the causative nature of the FITM2 mutation. Mutation-based genetic counseling can now be provided to the family and insight is obtained into the potential impact of genetic variation in FITM2., (© 2017. Published by The Company of Biologists Ltd.)

Hearing impairment (HI) is genetically heterogeneous which hampers genetic counseling and molecular diagnosis. Testing of several single HI-related genes is laborious and expensive. In this study, we evaluate the diagnostic utility of whole-exome sequencing (WES) targeting a panel of HI-related genes. Two hundred index patients, mostly of Dutch origin, with presumed hereditary HI underwent WES followed by targeted analysis of an HI gene panel of 120 genes. We found causative variants underlying the HI in 67 of 200 patients (33.5%). Eight of these patients have a large homozygous deletion involving STRC, OTOA or USH2A, which could only be identified by copy number variation detection. Variants of uncertain significance were found in 10 patients (5.0%). In the remaining 123 cases, no potentially causative variants were detected (61.5%). In our patient cohort, causative variants in GJB2, USH2A, MYO15A and STRC, and in MYO6 were the leading causes for autosomal recessive and dominant HI, respectively. Segregation analysis and functional analyses of variants of uncertain significance will probably further increase the diagnostic yield of WES.

Linkage analysis combined with whole-exome sequencing in a large family with congenital and stable non-syndromic unilateral and asymmetric hearing loss (NS-UHL/AHL) revealed a heterozygous truncating mutation, c.286_303delinsT (p.Ser96Ter), in KITLG. This mutation co-segregated with NS-UHL/AHL as a dominant trait with reduced penetrance. By screening a panel of probands with NS-UHL/AHL, we found an additional mutation, c.200_202del (p.His67_Cys68delinsArg). In vitro studies revealed that the p.His67_Cys68delinsArg transmembrane isoform of KITLG is not detectable at the cell membrane, supporting pathogenicity. KITLG encodes a ligand for the KIT receptor. Also, KITLG-KIT signaling and MITF are suggested to mutually interact in melanocyte development. Because mutations in MITF are causative of Waardenburg syndrome type 2 (WS2), we screened KITLG in suspected WS2-affected probands. A heterozygous missense mutation, c.310C>G (p.Leu104Val), that segregated with WS2 was identified in a small family. In vitro studies revealed that the p.Leu104Val transmembrane isoform of KITLG is located at the cell membrane, as is wild-type KITLG. However, in culture media of transfected cells, the p.Leu104Val soluble isoform of KITLG was reduced, and no soluble p.His67_Cys68delinsArg and p.Ser96Ter KITLG could be detected. These data suggest that mutations in KITLG associated with NS-UHL/AHL have a loss-of-function effect. We speculate that the mechanism of the mutation underlying WS2 and leading to membrane incorporation and reduced secretion of KITLG occurs via a dominant-negative or gain-of-function effect. Our study unveils different phenotypes associated with KITLG, previously associated with pigmentation abnormalities, and will thereby improve the genetic counseling given to individuals with KITLG variants., (Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Already 40 genes have been identified for autosomal-recessive nonsyndromic hearing impairment (arNSHI); however, many more genes are still to be identified. In a Dutch family segregating arNSHI, homozygosity mapping revealed a 2.4 Mb homozygous region on chromosome 11 in p15.1-15.2, which partially overlapped with the previously described DFNB18 locus. However, no putative pathogenic variants were found in USH1C, the gene mutated in DFNB18 hearing impairment. The homozygous region contained 12 additional annotated genes including OTOG, the gene encoding otogelin, a component of the tectorial membrane. It is thought that otogelin contributes to the stability and strength of this membrane through interaction or stabilization of its constituent fibers. The murine orthologous gene was already known to cause hearing loss when defective. Analysis of OTOG in the Dutch family revealed a homozygous 1 bp deletion, c.5508delC, which leads to a shift in the reading frame and a premature stop codon, p.Ala1838ProfsX31. Further screening of 60 unrelated probands from Spanish arNSHI families detected compound heterozygous OTOG mutations in one family, c.6347C>T (p.Pro2116Leu) and c. 6559C>T (p.Arg2187X). The missense mutation p.Pro2116Leu affects a highly conserved residue in the fourth von Willebrand factor type D domain of otogelin. The subjects with OTOG mutations have a moderate hearing impairment, which can be associated with vestibular dysfunction. The flat to shallow "U" or slightly downsloping shaped audiograms closely resembled audiograms of individuals with recessive mutations in the gene encoding α-tectorin, another component of the tectorial membrane. This distinctive phenotype may represent a clue to orientate the molecular diagnosis., (Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Hereditary hearing loss is characterized by a high degree of genetic heterogeneity. Here we present OTOGL mutations, a homozygous one base pair deletion (c.1430 delT) causing a frameshift (p.Val477Glufs(∗)25) in a large consanguineous family and two compound heterozygous mutations, c.547C>T (p.Arg183(∗)) and c.5238+5G>A, in a nonconsanguineous family with moderate nonsyndromic sensorineural hearing loss. OTOGL maps to the DFNB84 locus at 12q21.31 and encodes otogelin-like, which has structural similarities to the epithelial-secreted mucin protein family. We demonstrate that Otogl is expressed in the inner ear of vertebrates with a transcription level that is high in embryonic, lower in neonatal, and much lower in adult stages. Otogelin-like is localized to the acellular membranes of the cochlea and the vestibular system and to a variety of inner ear cells located underneath these membranes. Knocking down of otogl with morpholinos in zebrafish leads to sensorineural hearing loss and anatomical changes in the inner ear, supporting that otogelin-like is essential for normal inner ear function. We propose that OTOGL mutations affect the production and/or function of acellular structures of the inner ear, which ultimately leads to sensorineural hearing loss., (Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

CaBPs are a family of Ca(2+)-binding proteins related to calmodulin and are localized in the brain and sensory organs, including the retina and cochlea. Although their physiological roles are not yet fully elucidated, CaBPs modulate Ca(2+) signaling through effectors such as voltage-gated Ca(v) Ca(2+) channels. In this study, we identified a splice-site mutation (c.637+1G>T) in Ca(2+)-binding protein 2 (CABP2) in three consanguineous Iranian families affected by moderate-to-severe hearing loss. This mutation, most likely a founder mutation, probably leads to skipping of exon 6 and premature truncation of the protein (p.Phe164Serfs(∗)4). Compared with wild-type CaBP2, the truncated CaBP2 showed altered Ca(2+) binding in isothermal titration calorimetry and less potent regulation of Ca(v)1.3 Ca(2+) channels. We show that genetic defects in CABP2 cause moderate-to-severe sensorineural hearing impairment. The mutation might cause a hypofunctional CaBP2 defective in Ca(2+) sensing and effector regulation in the inner ear., (Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Context: Genomic imprinting is the modification of the genome so that genes from only one (rather than two) of the parental alleles are expressed. The mechanism underlying imprinting is epigenetic, occurring via changes in DNA methylation and histone modifications rather than through alterations in the DNA sequence. To date, nine different imprinting disorders have been clinically and genetically identified and a considerable research effort has been focused on determining the cause of the corresponding methylation defects., Objective: Our objective was to identify multilocus imprinting defects and characterize any mutations in trans-acting genes in patients with pseudohypoparathyroidism (PHP) caused by epigenetic alterations at GNAS locus., Design: We have investigated multilocus imprinting defects in 22 PHP patients with aberrant methylation at the GNAS locus not due to previously described deletions or to paternal uniparental disomy (UPD) of chromosome 20., Results: We found that, in contrast to what has been described in growth disorders, multilocus hypomethylation is an uncommon event in PHP patients. We were also unable to identify any genetic alteration causative of the epigenetic defects in the currently known methylation regulatory genes., Conclusion: Our work suggests that a trans-acting gene regulating the establishment or maintenance of imprinting at GNAS locus, if it exists, should be specific to PHP cases caused by epigenetic defects at GNAS.