Serum N-glycomics of a novel CDG-IIb patient reveals aberrant IgG glycosylation.

Rare genetic mutations of the mannosyl-oligosaccharide glucosidase (MOGS) gene affecting the function of the mannosyl-oligosaccharide glucosidase (glucosidase I) are the cause of the congenital disorder of glycosylation IIb (CDG-IIb). Glucosidase I specifically removes the distal α1,2-linked glucose from the protein bound precursor N-glycan Glc3Man9GlcNAc2, which is the initial step of N-glycan maturation. Here, we comparatively analyzed N-glycosylation of the whole serum proteome, serum-derived immunoglobulin G (IgG), transferrin (TF), and α-1-antitrypsin (AAT) of a female patient who is compound heterozygous for 2 novel missense mutations in the MOGS gene, her heterozygous parents, and a sibling with wildtype genotype by multiplexed capillary gel electrophoresis coupled to laser induced fluorescence detection (xCGE-LIF) at unprecedented depth. Thereby, we detected the CDG-IIb-characteristic non-de-glucosylated N-glycans Glc3Man7-9GlcNAc2 as well as the free tetrasaccharide Glc3-Man in whole serum of the patient but not in the other family members. The N-glycan analysis of the serum proteome further revealed that relative intensities of IgG-specific complex type di-antennary N-glycans with core-fucosylation were considerably reduced in the patient's serum whereas TF- and AAT-characteristic sialylated di- and tri-antennary N-glycans were increased. This finding reflected the hypogammaglobulinemia diagnosed in the patient. We further detected aberrant oligo-mannose (Glc3Man7GlcNAc2) and hybrid type N-glycans on patient-derived IgGs and we attributed this defective glycosylation to be the reason for an increased IgG clearance. This mechanism can explain the hypogammaglobulinemia that is associated with CDG-IIb.

A novel likely pathogenic heterozygous HECW2 missense variant in a family with variable expressivity of neurodevelopmental delay, hypotonia, and epileptiform EEG patterns.

Pathogenic variants in HECW2 are extremely rare. So far, only 19 cases have been reported. They were associated with epilepsy, intellectual disability, absent language, hypotonia, and autism. As these cases were all de novo mutations, mostly presenting without identical variants, variable expressivity has never been investigated. Here, we describe the first family with the same novel variant in HECW2. A 19-year old female patient presented with bursts of generalized spike-wave discharges and intellectual disability. We performed next-generation-sequencing, to detect the genetic cause. Next-generation-sequencing revealed a novel likely pathogenic variant in HECW2 (c.3571C>T; p.Arg1191Trp) in the index patient, her mother and brother. They showed some similar phenotypic patterns with intellectual disability, hypotonia and generalized epileptiform patterns. However, the mother was less severely affected and epileptiform patterns were less frequent. The brother presented with additional autistic features. In contrast to previous cases, the speech of all individuals was only mildly impaired. This is the first case report of a family with the same novel likely pathogenic variant in HECW2 and as such provides insight into the phenotypic variability of this mutation. The expressivity of symptoms may be so mild that genetic and EEG analysis are needed to disclose the correct diagnosis.

Phenotypic expansion of the BPTF-related neurodevelopmental disorder with dysmorphic facies and distal limb anomalies.

Neurodevelopmental disorder with dysmorphic facies and distal limb anomalies (NEDDFL), defined primarily by developmental delay/intellectual disability, speech delay, postnatal microcephaly, and dysmorphic features, is a syndrome resulting from heterozygous variants in the dosage-sensitive bromodomain PHD finger chromatin remodeler transcription factor BPTF gene. To date, only 11 individuals with NEDDFL due to de novo BPTF variants have been described. To expand the NEDDFL phenotypic spectrum, we describe the clinical features in 25 novel individuals with 20 distinct, clinically relevant variants in BPTF, including four individuals with inherited changes in BPTF. In addition to the previously described features, individuals in this cohort exhibited mild brain abnormalities, seizures, scoliosis, and a variety of ophthalmologic complications. These results further support the broad and multi-faceted complications due to haploinsufficiency of BPTF.

MN1 C-terminal truncation syndrome is a novel neurodevelopmental and craniofacial disorder with partial rhombencephalosynapsis.

MN1 encodes a transcriptional co-regulator without homology to other proteins, previously implicated in acute myeloid leukaemia and development of the palate. Large deletions encompassing MN1 have been reported in individuals with variable neurodevelopmental anomalies and non-specific facial features. We identified a cluster of de novo truncating mutations in MN1 in a cohort of 23 individuals with strikingly similar dysmorphic facial features, especially midface hypoplasia, and intellectual disability with severe expressive language delay. Imaging revealed an atypical form of rhombencephalosynapsis, a distinctive brain malformation characterized by partial or complete loss of the cerebellar vermis with fusion of the cerebellar hemispheres, in 8/10 individuals. Rhombencephalosynapsis has no previously known definitive genetic or environmental causes. Other frequent features included perisylvian polymicrogyria, abnormal posterior clinoid processes and persistent trigeminal artery. MN1 is encoded by only two exons. All mutations, including the recurrent variant p.Arg1295* observed in 8/21 probands, fall in the terminal exon or the extreme 3' region of exon 1, and are therefore predicted to result in escape from nonsense-mediated mRNA decay. This was confirmed in fibroblasts from three individuals. We propose that the condition described here, MN1 C-terminal truncation (MCTT) syndrome, is not due to MN1 haploinsufficiency but rather is the result of dominantly acting C-terminally truncated MN1 protein. Our data show that MN1 plays a critical role in human craniofacial and brain development, and opens the door to understanding the biological mechanisms underlying rhombencephalosynapsis.

Meis1 coordinates a network of genes implicated in eye development and microphthalmia.

Microphthalmos is a rare congenital anomaly characterized by reduced eye size and visual deficits of variable degree. Sporadic and hereditary microphthalmos have been associated with heterozygous mutations in genes fundamental for eye development. Yet, many cases are idiopathic or await the identification of molecular causes. Here we show that haploinsufficiency of Meis1, which encodes a transcription factor with evolutionarily conserved expression in the embryonic trunk, brain and sensory organs, including the eye, causes microphthalmic traits and visual impairment in adult mice. By combining analysis of Meis1 loss-of-function and conditional Meis1 functional rescue with ChIP-seq and RNA-seq approaches we show that, in contrast to its preferential association with Hox-Pbx BSs in the trunk, Meis1 binds to Hox/Pbx-independent sites during optic cup development. In the eye primordium, Meis1 coordinates, in a dose-dependent manner, retinal proliferation and differentiation by regulating genes responsible for human microphthalmia and components of the Notch signaling pathway. In addition, Meis1 is required for eye patterning by controlling a set of eye territory-specific transcription factors, so that in Meis1(-/-) embryos boundaries among the different eye territories are shifted or blurred. We propose that Meis1 is at the core of a genetic network implicated in eye patterning/microphthalmia, and represents an additional candidate for syndromic cases of these ocular malformations.

Neutralizing IL-17 protects the optic nerve from autoimmune pathology and prevents retinal nerve fiber layer atrophy during experimental autoimmune encephalomyelitis.

Optic neuritis is a common inflammatory manifestation of multiple sclerosis (MS). In experimental autoimmune encephalomyelitis (EAE), the optic nerve is affected as well. Here, we investigated whether autoimmune inflammation in the optic nerve is distinct from inflammation in other parts of the central nervous system (CNS). In our study, inflammatory infiltrates in the optic nerve and the brain were characterized by a high fraction of Ly6G(+) granulocytes whereas in the spinal cord, macrophage infiltrates were predominant. At the peak of disease, IL-17 mRNA abundance was highest in the optic nerve as compared with other parts of the CNS. The ratio of IL-17 vs IFN-γ producing CD4(+) T cells was higher in the optic nerve and brain than in the spinal cord and more effector CD4(+) T cells were committed to the Th17 transcriptional program in the optic nerve than in the spinal cord. IL-17 producing γδ T cells but rather not Ly6G(+) granulocytes themselves contributed to IL-17 production. Optical coherence tomography (OCT) studies on murine eyes revealed a decline in thickness of the retinal nerve fiber layer (RNFL) and the common layer of ganglion cells and inner plexiform layer (GCL+) after the recovery from motor symptoms indicating that autoimmune inflammation induced a significant atrophy of optic nerve fibers during EAE. Neutralization of IL-17 by treatment with anti-IL-17 antibodies reduced but did not abrogate motor symptoms of EAE. However, RNFL and GCL+ atrophy were completely prevented by blocking IL-17. Thus, the optic nerve compartment is particularly prone to support IL-17 mediated inflammatory responses during CNS autoimmunity and structural integrity of the retina can be preserved by neutralizing IL-17.

Longitudinal fundus and retinal studies with SD-OCT: a comparison of five mouse inbred strains.

Spectral domain optical coherence tomography (SD-OCT) has recently been established as a method for in vivo imaging of fundus and retina in the mouse. It enables more effective studies of retinal diseases including investigations of etiopathologic mechanisms. In order to learn more about longitudinal fundus development and to enable recognition of disease-associated irregularities, we performed confocal scanning laser ophthalmoscopy (cSLO) and SD-OCT measurements in the inbred strains C57BL/6J, C3HeB/FeJ, FVB/NCrl, BALB/cByJ, and 129S2/SvJ when they were between 2 and 6 months of age. In general, cSLO and SD-OCT data did not reveal sex-specific or unilateral differences. C3HeB/FeJ and FVB/NCrl mice showed diffuse choroidal dysplasia. Choroidal vein-like structures appeared as dark fundus stripes in C3HeB/FeJ. In FVB/NCrl, fundus fleck accumulation was found. In contrast, only minor time-dependent changes of fundus appearance were observed in C57BL/6J, BALB/cByJ, and 129S2/SvJ. This was also found for individual fundic main blood vessel patterns in all inbred strains. Vessel numbers varied between 6 and 13 in C57BL/6J. This was comparable in most cases. We further found that retinae were significantly thicker in C57BL/6J compared to the other strains. Total retinal thickness generally did not change between 2 and 6 months of age. As a conclusion, our results indicate lifelong pathologic processes in C3HeB/FeJ and FVB/NCrl that affect choroid and orbital tissues. Inbred strains with regular retinal development did not reveal major time-dependent variations of fundus appearance, blood vessel pattern, or retinal thickness. Consequently, progressive changes of these parameters are suitable indicators for pathologic outliers.

Lens density tracking in mice by Scheimpflug imaging.

Scheimpflug imaging has recently been established for in vivo imaging of the anterior eye segment and quantitative determination of lens transparency in the mouse. This enables more effective investigations of cataract formation with the mouse model, including longitudinal studies. In order to enable recognition of disease-associated irregularities, we performed Scheimpflug measurements with the common laboratory inbred lines C57BL/6J, C3HeB/FeJ, FVB/NCrl, BALB/cByJ, and 129/SvJ in a period between 2 and 12 months of age. C57BL/6J mice showed lowest mean lens densities during the test period. Progressive cortical lens opacification was generally observed, with the earliest onset in C57BBL/6J, C3HeB/FeJ, and 129/SvJ, between 2 and 6 months after birth. Moreover, lenses of these inbred lines developed nuclear opacities. Calculated mean lens density significantly increased between 6 and 12 months of age in all inbred strains except 129/SvJ. Lens densities (and the corresponding standard deviations) of FVB/NCrl and 129/SvJ increased most likely because of differences in the genetic background. Albinism as confounder might be excluded since the albino Balb/cByJ mice are more similar to the C57BL/6J or C3Heb/FeJ mice. We further identified strain-specific anterior lens opacities (C57BL/6J) and cloudy corneal lesions (C57BL/6J, FVB/NCrl, and BALB/cByJ) at later stages. In conclusion, our results indicate that there are lifelong opacification processes in the mouse lens. The highest lens transparency and a dark coat color, which prevents interference from light reflections, make mice with the C57BL/6J background most suitable for cataract research by Scheimpflug imaging. We show that lens densitometry by Scheimpflug imaging in mouse eyes can resolve differences of less than 1 %, making it possible to detect differences in cataract development in different mouse strains, even if they are small.

Novel small-eye allele in paired box gene 6 (Pax6) is caused by a point mutation in intron 7 and creates a new exon.

PURPOSE: Within a mutagenesis screen, we identified the new mouse mutant Aey80 with small eyes; homozygous mutants were not obtained. The aim of the study was its molecular characterization. METHODS: We analyzed the offspring of paternally N-ethyl-N-nitrosourea (ENU)-treated C3HeB/FeJ mice for dysmorphology parameters, which can be observed with the naked eye. The Aey80 mutant (abnormality of the eye) was further characterized with laser interference biometry, Scheimpflug imaging, and optical coherence tomography. Linkage analysis of the Aey80 mutant was performed using a panel of single nucleotide polymorphisms different among C3HeB/FeJ and C57BL/6J mice. The Aey80 mutation was identified with sequence analysis of the positional candidate gene. RESULTS: We identified a new mutant characterized by an obvious small-eye phenotype; homozygotes are not viable after birth. Embryos at embryonic day 15.5 demonstrate a clear gene-dosage effect: Heterozygotes have small eyes, whereas homozygous mutants do not have eyes. In adult mice, the lenses and the entire eyes of the heterozygous mutants were significantly smaller than those of the wild-types (p<0.01). No other ocular phenotypes were observed; the lenses were fully transparent, and no adhesion to the cornea was observed. The mutation was mapped to chromosome 2; markers between 70.8 MB and 129.5 MB showed significant linkage to the mutation resulting in paired box gene 6 (Pax6) as an excellent candidate gene. We amplified cDNAs from the embryonic eyes and observed an additional band while amplifying the region corresponding to exons 7 and 8. The additional band included an alternative exon of 141 bp, which was associated with a G->A exchange four bases downstream of the end of the alternative exon. The alternative exon in the mutants is predicted to encode 30 novel amino acids and three stop codons. This alternative exon kept the paired domain intact but led to a loss of the homeodomain and the C-terminal proline-serine-threonine (PST) domain. The mutation cosegregated in the mutant line, since all five additional small-eyed mice from this line showed the same mutation. A general polymorphism at the mutated site was excluded with sequence analysis of seven other wild-type mouse strains different from C3HeB/FeJ. CONCLUSIONS: These findings demonstrate a novel allele of the paired box gene 6 (Pax6) that affects lens development in a semidominant manner leading to a classical small-eye phenotype. However, the site of the mutation more than 1 kb downstream of exon 7 and resulting in an alternative exon is quite unusual. It indicates the importance of sequence analysis of cDNA for mutation detection; mutations like this are unlikely to be identified by analyzing genomic sequences only. Moreover, this particular mutation demonstrates how a novel exon can be created by only a single base-pair exchange.

Cytochrome c oxidase subunit 4 isoform 2-knockout mice show reduced enzyme activity, airway hyporeactivity, and lung pathology.

Cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial electron transport chain. The purpose of this study was to analyze the function of lung-specific cytochrome c oxidase subunit 4 isoform 2 (COX4i2) in vitro and in COX4i2-knockout mice in vivo. COX was isolated from cow lung and liver as control and functionally analyzed. COX4i2-knockout mice were generated and the effect of the gene knockout was determined, including COX activity, tissue energy levels, noninvasive and invasive lung function, and lung pathology. These studies were complemented by a comprehensive functional screen performed at the German Mouse Clinic (Neuherberg, Germany). We show that isolated cow lung COX containing COX4i2 is about twice as active (88 and 102% increased activity in the presence of allosteric activator ADP and inhibitor ATP, respectively) as liver COX, which lacks COX4i2. In COX4i2-knockout mice, lung COX activity and cellular ATP levels were significantly reduced (-50 and -29%, respectively). Knockout mice showed decreased airway responsiveness (60% reduced P(enh) and 58% reduced airway resistance upon challenge with 25 and 100 mg methacholine, respectively), and they developed a lung pathology deteriorating with age that included the appearance of Charcot-Leyden crystals. In addition, there was an interesting sex-specific phenotype, in which the knockout females showed reduced lean mass (-12%), reduced total oxygen consumption rate (-8%), improved glucose tolerance, and reduced grip force (-14%) compared to wild-type females. Our data suggest that high activity lung COX is a central determinant of airway function and is required for maximal airway responsiveness and healthy lung function. Since airway constriction requires energy, we propose a model in which reduced tissue ATP levels explain protection from airway hyperresponsiveness, i.e., absence of COX4i2 leads to reduced lung COX activity and ATP levels, which results in impaired airway constriction and thus reduced airway responsiveness; long-term lung pathology develops in the knockout mice due to impairment of energy-costly lung maintenance processes; and therefore, we propose mitochondrial oxidative phosphorylation as a novel target for the treatment of respiratory diseases, such as asthma.

Requirement of the RNA-editing enzyme ADAR2 for normal physiology in mice.

ADAR2, an RNA editing enzyme that converts specific adenosines to inosines in certain pre-mRNAs, often leading to amino acid substitutions in the encoded proteins, is mainly expressed in brain. Of all ADAR2-mediated edits, a single one in the pre-mRNA of the AMPA receptor subunit GluA2 is essential for survival. Hence, early postnatal death of mice lacking ADAR2 is averted when the critical edit is engineered into both GluA2 encoding Gria2 alleles. Adar2(-/-)/Gria2(R/R) mice display normal appearance and life span, but the general phenotypic effects of global lack of ADAR2 have remained unexplored. Here we have employed the Adar2(-/-)/Gria2(R/R) mouse line, and Gria2(R/R) mice as controls, to study the phenotypic consequences of loss of all ADAR2-mediated edits except the critical one in GluA2. Our extended phenotypic analysis covering ∼320 parameters identified significant changes related to absence of ADAR2 in behavior, hearing ability, allergy parameters and transcript profiles of brain.

Visual capabilities and cortical maps in BALB/c mice.

By combining behavioural analyses with intrinsic signal optical imaging, we analysed visual performance and visual cortical activity in the albino mouse strain BALB/c, which is increasingly being used as an animal model of neuropsychological disorders. Visual acuity, as measured by a virtual-reality optomotor system, was 0.12 cycles per degree (cyc/deg) in BALB/c mice and 0.39 cyc/deg in pigmented C57BL/6 mice. Surprisingly, BALB/c mice showed reflexive head movements against the direction of the rotating stimulus. Contrast sensitivity was significantly lower in BALB/c mice (45% contrast at 0.064 cyc/deg) than in C57BL/6 mice (6% contrast). In the visual water task, visual acuity was 0.3 cyc/deg in BALB/c mice and 0.59 cyc/deg in C57BL/6 mice. Thus, the visual performance of BALB/c mice was significantly impaired in both behavioural tests - visual acuity was ∼ 0.3 cyc/deg lower than in C57BL/6 mice, and contrast sensitivity was reduced by a factor of ∼ 8. In BALB/c mice, visual cortical maps induced by stimulation of the contralateral eye were normal in both activation strength and retinotopic map quality. In contrast, maps induced by ipsilateral eye stimulation differed significantly between the strains - activity in a region representing 15° to 19° elevation in the visual field was significantly weaker in BALB/c mice than in C57BL/6 mice. Taken together, our observations show that BALB/c mice, like the albino animals of other species, have a significantly lower visual performance than C57BL/6 mice and a modified cortical representation of the ipsilateral eye that may impair stereopsis. Thus, our results caution against disregarding vision as a confounding factor in behavioural tests of neuropsychological disorders.

Innovations in phenotyping of mouse models in the German Mouse Clinic.

Under the label of the German Mouse Clinic (GMC), a concept has been developed and implemented that allows the better understanding of human diseases on the pathophysiological and molecular level. This includes better understanding of the crosstalk between different organs, pleiotropy of genes, and the systemic impact of envirotypes and drugs. In the GMC, experts from various fields of mouse genetics and physiology, in close collaboration with clinicians, work side by side under one roof. The GMC is an open-access platform for the scientific community by providing phenotypic analysis in bilateral collaborations ("bottom-up projects") and as a partner and driver in international large-scale biology projects ("top-down projects"). Furthermore, technology development is a major topic in the GMC. Innovative techniques for primary and secondary screens are developed and implemented into the phenotyping pipelines (e.g., detection of volatile organic compounds, VOCs).

Mice with an induced mutation in collagen 8A2 develop larger eyes and are resistant to retinal ganglion cell damage in an experimental glaucoma model.

PURPOSE: To study susceptibility to glaucoma injury as it may be affected by mutations in ocular connective tissue components. METHODS: Mice homozygous for an N-ethyl-N-nitrosourea induced G257D exchange (Gly to Asp) missense mutation (Aca23) in their collagen 8A2 gene were studied to measure intraocular pressure (IOP), axial length and width, number of retinal ganglion cells (RGC), and inflation responses. Three month old homozygous Aca23 mutant and wild type (WT) mice had 6 weeks exposure to elevated IOP induced by polystyrene microbead injection. Additional Aca23 and matched controls were studied at ages of 10 and 18 months. RESULTS: Aca23 mice had no significant difference from WT in IOP level, and in both strains IOP rose with age. In multivariable models, axial length and width were significantly larger in Aca23 than WT, became larger with age, and were larger after exposure to glaucoma (n=227 mice). From inflation test data, the estimates of scleral stress resultants in Aca23 mice were similar to age-matched and younger WT C57BL/6 (B6) mice, while the strain estimates for Aca23 were significantly less than those for either WT group in the mid-sclera and in some of the more anterior scleral measures (p<0.001; n=29, 22, 20 eyes in Aca23, older WT, younger WT, respectively). With chronic IOP elevation, Aca23 eyes increased 9% in length and 7% in width, compared to untreated fellow eyes (p<0.05, <0.01). With similar elevated IOP exposure, WT eyes enlarged proportionately twice as much as Aca23, increasing in length by 18% and in nasal-temporal width by 13% (both p<0.001, Mann-Whitney test). In 4 month old control optic nerves, mean RGC axon number was not different in Aca23 and WT (46,905±7,592, 43,628±11,162, respectively; p=0.43, Mann-Whitney test, n=37 and 29). With chronic glaucoma, Aca23 mice had a mean axon loss of only 0.57±17%, while WT mice lost 21±31% (median loss: 1% versus 10%, n=37, 29, respectively; p=0.001; multivariable model adjusting for positive integral IOP exposure). CONCLUSIONS: The Aca23 mutation in collagen 8α2 is the first gene defect found to alter susceptibility to experimental glaucoma, reducing RGC loss possibly due to differences in mechanical behavior of the sclera. Detailed study of the specific changes in scleral connective tissue composition and responses to chronic IOP elevation in this strain could produce new therapeutic targets for RGC neuroprotection.

Mouse phenotyping.

Model organisms like the mouse are important tools to learn more about gene function in man. Within the last 20 years many mutant mouse lines have been generated by different methods such as ENU mutagenesis, constitutive and conditional knock-out approaches, knock-down, introduction of human genes, and knock-in techniques, thus creating models which mimic human conditions. Due to pleiotropic effects, one gene may have different functions in different organ systems or time points during development. Therefore mutant mouse lines have to be phenotyped comprehensively in a highly standardized manner to enable the detection of phenotypes which might otherwise remain hidden. The German Mouse Clinic (GMC) has been established at the Helmholtz Zentrum München as a phenotyping platform with open access to the scientific community (www.mousclinic.de; [1]). The GMC is a member of the EUMODIC consortium which created the European standard workflow EMPReSSslim for the systemic phenotyping of mouse models (http://www.eumodic.org/[2]).

KBG syndrome: videoconferencing and use of artificial intelligence driven facial phenotyping in 25 new patients.

Genetic variants in Ankyrin Repeat Domain 11 (ANKRD11) and deletions in 16q24.3 are known to cause KBG syndrome, a rare syndrome associated with craniofacial, intellectual, and neurobehavioral anomalies. We report 25 unpublished individuals from 22 families with molecularly confirmed diagnoses. Twelve individuals have de novo variants, three have inherited variants, and one is inherited from a parent with low-level mosaicism. The mode of inheritance was unknown for nine individuals. Twenty are truncating variants, and the remaining five are missense (three of which are found in one family). We present a protocol emphasizing the use of videoconference and artificial intelligence (AI) in collecting and analyzing data for this rare syndrome. A single clinician interviewed 25 individuals throughout eight countries. Participants' medical records were reviewed, and data was uploaded to the Human Disease Gene website using Human Phenotype Ontology (HPO) terms. Photos of the participants were analyzed by the GestaltMatcher and DeepGestalt, Face2Gene platform (FDNA Inc, USA) algorithms. Within our cohort, common traits included short stature, macrodontia, anteverted nares, wide nasal bridge, wide nasal base, thick eyebrows, synophrys and hypertelorism. Behavioral issues and global developmental delays were widely present. Neurologic abnormalities including seizures and/or EEG abnormalities were common (44%), suggesting that early detection and seizure prophylaxis could be an important point of intervention. Almost a quarter (24%) were diagnosed with attention deficit hyperactivity disorder and 28% were diagnosed with autism spectrum disorder. Based on the data, we provide a set of recommendations regarding diagnostic and treatment approaches for KBG syndrome.

Microphakia and congenital cataract formation in a novel Lim2(C51R) mutant mouse.

PURPOSE: Within a mutagenesis screen, we identified the new mouse mutant Aca47 with small lenses and reduced axial eye lengths. The aim of the actual study was the molecular and morphological characterization of the mouse mutant Aca47. METHODS: We analyzed the offspring of paternally N-ethyl-N-nitrosourea (ENU) treated C57BL/6J mice for eye-size parameters by non-invasive in vivo laser interference biometry. Linkage analysis of the eye size mutant Aca47 was performed using single nucleotide polymorphisms and microsatellite markers. The Aca47 mutation was identified by sequence analysis of positional candidate genes. A general polymorphism at the mutated site was excluded by restriction analysis. Eyes of the Aca47 mouse mutant were characterized by histology. Visual properties were examined in the virtual drum. RESULTS: We identified a new mutant characterized by a significantly smaller lens and reduced axial eye length without any changes for cornea thickness, anterior chamber depth or aqueous humor size. The smaller size of lens was more pronounced in the homozygous mutants, which further developed congenital cataracts in the lens nucleus. The mutation was mapped to chromosome 7 between the markers D7Mit247 and D7Mit81. Using a positional candidate approach, the lens intrinsic integral membrane protein MP19 encoding gene Lim2 was sequenced; a T → C exchange at cDNA position 151 leads to a cysteine-to-arginine substitution at position 51 of the Lim2 protein. Eye histology of adult heterozygous mutants did not show alterations on the cellular level. However, homozygous lenses revealed irregularly arranged lens fiber layers in the cortex. Virtual vision tests indicated that visual properties are not affected by reduced eye size of heterozygous individuals. CONCLUSIONS: These findings demonstrate a novel missense mutation in the Lim2 gene that affects lens development in a semidominant manner. Since homozygous mutants develop congenital lens opacities, this line can be used as a model for inherited cataract formation in humans.

Identification of a New Genetic Mutation Associated With Peters Anomaly.

PURPOSE: To report a new genetic mutation in the COL4A1 gene, which was identified in a baby girl with Peters anomaly (PA), a rare anterior segment mesenchymal dysgenesis, which is characterized by unilateral or bilateral corneal opacities often accompanied by glaucoma, cataract, and systemic malformations and associated with various genetic mutations. METHODS: Ophthalmologic examination of one baby girl and whole exome sequencing and Sanger sequencing of blood samples of the child and her biological parents were performed. RESULTS: Ophthalmologic examination led to the diagnosis of PA type I in the baby girl. Whole exome sequencing and Sanger sequencing identified the de novo mutation c.181_189delinsAGGTTTCCG; p.Gly61Arg in the COL4A1 gene in the child, whereas no other putatively causative variants in established genes associated with anterior segment dysgenesis were present. CONCLUSIONS: PA might be associated with the mutation c.181_189delinsAGGTTTCCG; p.Gly61Arg in the COL4A1 gene. The COL4A1 gene encodes for collagen IVα1, an essential component of basal membranes, and mutations are associated with an increased risk for renal and cerebrovascular disorders and stroke. This should be considered when advising and monitoring patients.

Imbalances in the eye lens proteome are linked to cataract formation.

The prevalent model for cataract formation in the eye lens posits that damaged crystallin proteins form light-scattering aggregates. The α-crystallins are thought to counteract this process as chaperones by sequestering misfolded crystallin proteins. In this scenario, chaperone pool depletion would result in lens opacification. Here we analyze lenses from different mouse strains that develop early-onset cataract due to point mutations in α-, ß-, or γ-crystallin proteins. We find that these mutant crystallins are unstable in vitro; in the lens, their levels are substantially reduced, and they do not accumulate in the water-insoluble fraction. Instead, all the other crystallin proteins, including the α-crystallins, are found to precipitate. The changes in protein composition and spatial organization of the crystallins observed in the mutant lenses suggest that the imbalance in the lenticular proteome and altered crystallin interactions are the bases for cataract formation, rather than the aggregation propensity of the mutant crystallins.

Mutation in Bmpr1b Leads to Optic Disc Coloboma and Ventral Retinal Gliosis in Mice.

Purpose: The clinical phenotype of retinal gliosis occurs in different forms; here, we characterize one novel genetic feature, (i.e., signaling via BMP-receptor 1b). Methods: Mouse mutants were generated within a recessive ENU mutagenesis screen; the underlying mutation was identified by linkage analysis and Sanger sequencing. The eye phenotype was characterized by fundoscopy, optical coherence tomography, optokinetic drum, electroretinography, and visual evoked potentials, by histology, immunohistology, and electron-microscopy. Results: The mutation affects intron 10 of the Bmpr1b gene, which is causative for skipping of exon 10. The expression levels of pSMAD1/5/8 were reduced in the mutant retina. The loss of BMPR1B-mediated signaling leads to optic nerve coloboma, gliosis in the optic nerve head and ventral retina, defective optic nerve axons, and irregular retinal vessels. The ventral retinal gliosis is proliferative and hypertrophic, which is concomitant with neuronal delamination and the reduction of retinal ganglion cells (RGCs); it is dominated by activated astrocytes overexpressing PAX2 and SOX2 but not PAX6, indicating that they may retain properties of gliogenic precursor cells. The expression pattern of PAX2 in the optic nerve head and ventral retina is altered during embryonic development. These events finally result in reduced electrical transmission of the retina and optic nerve and significantly reduced visual acuity. Conclusions: Our study demonstrates that BMPR1B is necessary for the development of the optic nerve and ventral retina. This study could also indicate a new mechanism in the formation of retinal gliosis; it opens new routes for its treatment eventually preventing scar formation in the retina.