Mutations in ALDH1A3 represent a frequent cause of microphthalmia/anophthalmia in consanguineous families.

Anophthalmia or microphthalmia (A/M), characterized by absent or small eye, can be unilateral or bilateral and represent developmental anomalies due to the mutations in several genes. Recently, mutations in aldehyde dehydrogenase family 1, member A3 (ALDH1A3) also known as retinaldehyde dehydrogenase 3, have been reported to cause A/M. Here, we screened a cohort of 75 patients with A/M and showed that mutations in ALDH1A3 occurred in six families. Based on this series, we estimate that mutations in ALDH1A3 represent a major cause of A/M in consanguineous families, and may be responsible for approximately 10% of the cases. Screening of this gene should be performed in a first line of investigation, together with SOX2.

A missense mutation in ALDH1A3 causes isolated microphthalmia/anophthalmia in nine individuals from an inbred Muslim kindred.

Nine affected individuals with isolated anophthalmia/microphthalmia from a large Muslim-inbred kindred were investigated. Assuming autosomal-recessive mode of inheritance, whole-genome linkage analysis, on DNA samples from four affected individuals, was undertaken. Homozygosity mapping techniques were employed and a 1.5-Mbp region, homozygous in all affected individuals, was delineated. The region contained nine genes, one of which, aldehyde dehydrogenase 1 (ALDH1A3), was a clear candidate. This gene seems to encode a key enzyme in the formation of a retinoic-acid gradient along the dorsoventral axis during an early eye development and the development of the olfactory system. Sanger sequence analysis revealed a missense mutation, causing a substitution of valine (Val) to methionine (Met) at position 71. Analyzing the p.Val71Met missense mutation using standard open access software (MutationTaster online, PolyPhen, SIFT/PROVEAN) predicts this variant to be damaging. Enzymatic activity, studied in vitro, showed no changes between the mutated and the wild-type ALDH1A3 protein.

Posterior microphthalmia and nanophthalmia in Tunisia caused by a founder c.1059_1066insC mutation of the PRSS56 gene.

Congenital microphthalmia (CMIC) is a common developmental ocular disorder characterized by a small, and sometimes malformed, eye. Posterior microphthalmia (PM) and nanophthalmia are two rare subtypes of isolated CMIC characterized by extreme hyperopia due to short axial length and elevated lens/eye volume ratio. While nanophthalmia is associated with a reduced size in both anterior and posterior segments, PM involves a normal-size anterior chamber but a small posterior segment. Several genes encoding transcription and non-transcription regulators have been identified in different forms of CMIC. MFRP gene mutations have, for instance, been associated with nanophthalmia, and mutations in the recently identified PRSS56 gene have been linked to PM. So far, these two forms of CMIC have been associated with 9 mutations in PRSS56. Of particular interest, a c.1059_1066insC mutation has recently been reported in four Tunisian families with isolated PM and one Tunisian family with nanophthalmia. Here, we performed a genome-wide scan using a high density single nucleotide polymorphism (SNP) array 50 K in a large consanguineous Tunisian family (PM7) affected with PM and identified the same causative disease mutation. A total of 24 polymorphic markers spanning the PRSS56 gene in 6 families originating from different regions of Tunisia were analyzed to investigate the origin of the c.1059_1066insC mutation and to determine whether it arose in a common ancestor. A highly significant disease-associated haplotype, spanning across the 146 kb of the 2q37.1 chromosome, was conserved in those families, suggesting that c.1059_1066insC arose from a common founder. The age of the mutation in this haplotype was estimated to be around 1,850 years. The identification of such 'founder effects' may greatly simplify diagnostic genetic screening and lead to better prognostic counseling.

ALDH1A3 mutations cause recessive anophthalmia and microphthalmia.

Anophthalmia and microphthalmia (A/M) are early-eye-development anomalies resulting in absent or small ocular globes, respectively. A/M anomalies occur in syndromic or nonsyndromic forms. They are genetically heterogeneous, some mutations in some genes being responsible for both anophthalmia and microphthalmia. Using a combination of homozygosity mapping, exome sequencing, and Sanger sequencing, we identified homozygosity for one splice-site and two missense mutations in the gene encoding the A3 isoform of the aldehyde dehydrogenase 1 (ALDH1A3) in three consanguineous families segregating A/M with occasional orbital cystic, neurological, and cardiac anomalies. ALDH1A3 is a key enzyme in the formation of a retinoic acid gradient along the dorso-ventral axis during early eye development. Transitory expression of mutant ALDH1A3 open reading frames showed that both missense mutations reduce the accumulation of the enzyme, potentially leading to altered retinoic acid synthesis. Although the role of retinoic acid signaling in eye development is well established, our findings provide genetic evidence of a direct link between retinoic-acid-synthesis dysfunction and early-eye-development anomalies in humans.

The impairment of HCCS leads to MLS syndrome by activating a non-canonical cell death pathway in the brain and eyes.

Mitochondrial-dependent (intrinsic) programmed cell death (PCD) is an essential homoeostatic mechanism that selects bioenergetically proficient cells suitable for tissue/organ development. However, the link between mitochondrial dysfunction, intrinsic apoptosis and developmental anomalies has not been demonstrated to date. Now we provide the evidence that non-canonical mitochondrial dependent apoptosis explains the phenotype of microphthalmia with linear skin lesions (MLS), an X-linked developmental disorder caused by mutations in the holocytochrome c-type synthase (HCCS)gene [corrected]. By taking advantage of a medaka model that recapitulates the MLS phenotype we demonstrate that downregulation of hccs, an essential player of the mitochondrial respiratory chain (MRC), causes increased cell death via an apoptosome-independent caspase-9 activation in brain and eyes. We also show that the unconventional activation of caspase-9 occurs in the mitochondria and is triggered by MRC impairment and overproduction of reactive oxygen species (ROS). We thus propose that HCCS plays a key role in central nervous system (CNS) development by modulating a novel non-canonical start-up of cell death and provide the first experimental evidence for a mechanistic link between mitochondrial dysfunction, intrinsic apoptosis and developmental disorders.

Autosomal-recessive posterior microphthalmos is caused by mutations in PRSS56, a gene encoding a trypsin-like serine protease.

Posterior microphthalmos (MCOP) is a rare isolated developmental anomaly of the eye characterized by extreme hyperopia due to short axial length. The population of the Faroe Islands shows a high prevalence of an autosomal-recessive form (arMCOP) of the disease. Based on published linkage data, we refined the position of the disease locus (MCOP6) in an interval of 250 kb in chromosome 2q37.1 in two large Faroese families. We detected three different mutations in PRSS56. Patients of the Faroese families were either homozygous for c.926G>C (p.Trp309Ser) or compound heterozygous for c.926G>C and c.526C>G (p.Arg176Gly), whereas a homozygous 1 bp duplication (c.1066dupC) was identified in five patients with arMCOP from a consanguineous Tunisian family. In one patient with MCOP from the Faroe Islands and in another one from Turkey, no PRSS56 mutation was detected, suggesting nonallelic heterogeneity of the trait. Using RT-PCR, PRSS56 transcripts were detected in samples derived from the human adult retina, cornea, sclera, and optic nerve. The expression of the mouse ortholog could be first detected in the eye at E17 and was maintained into adulthood. The predicted PRSS56 protein is a 603 amino acid long secreted trypsin-like serine peptidase. The c.1066dupC is likely to result in a functional null allele, whereas the two point mutations predict the replacement of evolutionary conserved and functionally important residues. Molecular modeling of the p.Trp309Ser mutant suggests that both the affinity and reactivity of the enzyme toward in vivo protein substrates are likely to be substantially reduced.

Heparan sulfate deficiency in periocular mesenchyme causes microphthalmia and ciliary body dysgenesis.

The heparan sulfate (HS) is a component of proteoglycans in the extracellular matrix and on cell surfaces, modulating developmental processes. The aim of this study is to investigate whether the defect of HS in the periocular mesenchyme impairs ocular morphogenesis. First, using Protein 0-Cre transgenic mice, we ablated Ext1, which encodes an indispensable enzyme for HS synthesis, in the developing periocular mesenchyme. The expression of Ext1 messenger RNA (mRNA) and HS were observed by RT-PCR and immunohistochemistry, respectively. The phenotypes in the mutant were evaluated by light microscopy and immunohistochemistry for cellular makers. Second, the distribution of the mutant periocular mesenchymal cells was tracked using a Rosa26 Cre-reporter gene. No mutant embryos (Protein 0-Cre;Ext1(flox/flox)) were identified after embryonic day 14.5 (E14.5). RT-PCR showed that an intense band amplified from Ext1 was observed in cDNAs from the control periocular mesenchymal cells at E13.5; however, the band for Ext1 was hardly detectable in cDNA from the mutant embryo, indicating that the mRNA was missing in the mutant periocular mesenchyme at E13.5. The HS expression was disrupted in the periocular mesenchyme of the mutant ocular tissues. The HS deficiency resulted in microphthalmia with reduced axial lengths, lens diameters, and vitreous sizes compared with the littermate eyes. The mutant embryos showed agenesis of the anterior chamber, where cells expressing Cre recombinase were distributed. Moreover, the mutants showed phenotypic alterations in the neural ectoderm including dysgenesis of the presumptive ciliary body and agenesis of the optic nerve head. These findings demonstrate that HS in the periocular mesenchyme plays a critical role in normal ocular morphogenesis, indicating reciprocal interactions between the periocular mesenchyme and the neural ectoderm.

Mutations of the mitochondrial holocytochrome c-type synthase in X-linked dominant microphthalmia with linear skin defects syndrome.

The microphthalmia with linear skin defects syndrome (MLS, or MIDAS) is an X-linked dominant male-lethal disorder almost invariably associated with segmental monosomy of the Xp22 region. In two female patients, from two families, with MLS and a normal karyotype, we identified heterozygous de novo point mutations--a missense mutation (p.R217C) and a nonsense mutation (p.R197X)--in the HCCS gene. HCCS encodes the mitochondrial holocytochrome c-type synthase that functions as heme lyase by covalently adding the prosthetic heme group to both apocytochrome c and c(1). We investigated a third family, displaying phenotypic variability, in which the mother and two of her daughters carry an 8.6-kb submicroscopic deletion encompassing part of the HCCS gene. Functional analysis demonstrates that both mutant proteins (R217C and Delta 197-268) were unable to complement a Saccharomyces cerevisiae mutant deficient for the HCCS orthologue Cyc3p, in contrast to wild-type HCCS. Moreover, ectopically expressed HCCS wild-type and the R217C mutant protein are targeted to mitochondria in CHO-K1 cells, whereas the C-terminal-truncated Delta 197-268 mutant failed to be sorted to mitochondria. Cytochrome c, the final product of holocytochrome c-type synthase activity, is implicated in both oxidative phosphorylation (OXPHOS) and apoptosis. We hypothesize that the inability of HCCS-deficient cells to undergo cytochrome c-mediated apoptosis may push cell death toward necrosis that gives rise to severe deterioration of the affected tissues. In summary, we suggest that disturbance of both OXPHOS and the balance between apoptosis and necrosis, as well as the X-inactivation pattern, may contribute to the variable phenotype observed in patients with MLS.

Development of cataractous macrophthalmia in mice expressing an active MEK1 in the lens.

PURPOSE: To characterize the extracellular signal-regulated kinase (ERK) pathway in the lens and to try to understand how this pathway contributes to lens function and cataractogenesis. METHODS: The members of the ERK pathway in the lens were examined by Western blotting, immunohistochemical staining, and kinase assay. A gain-of-function approach was used to perturb the ERK pathway in the lenses of transgenic mice via expression of a constitutively active mutant of the mitogen-activated protein kinase kinase 1 (MEK1(E)), the direct upstream kinase of the ERK1 and ERK2 kinases, under the alphaA-crystallin promoter. RESULTS: The presence of an active ERK pathway was found in lens epithelial cells and in differentiating fibers. Transgenic mice that expressed MEK1(E) developed postnatal cataracts as well as macrophthalmia. Distinct morphologic alterations, such as lens enlargement, swelling fiber cells, enlarged extracellular space, and vacuole formation, were observed in the lenses of these transgenic mice. A significant increase in the glucose transporter 1 (GLUT1) level, as well as in the glucose level, was detected in the lens. CONCLUSIONS: The MAP kinase pathway is involved in the regulation of glucose metabolism and balance in the mouse lens. Moreover, the alteration of MAP kinase activity in the lens is sufficient to cause cataract formation with enlarged extracellular space and vacuoles in the differentiating fibers. This transgenic mouse may provide a useful model for understanding the mechanism(s) for some aspects of human cataracts.

Neuronal and inducible nitric oxide synthase distribution in retinas of microphthalmic mice.

A study about the distribution of neuronal nitric oxide synthase (n-NOS) and inducible NOS (i-NOS) was performed in microphthalmic retinas at postnatal day (pd) 2 and pd 7. Neither n-NOS nor i-NOS immunoresponse could be detected at pd 2. At pd 7, i-NOS activity was revealed in the retinas of mi/mi littermates but not in those of heterozygotes or wild-type littermates. The expression of n-NOS corresponded to the postnatal retinal development. Retinal rosettes were strongly labelled.

Expression of tyrosinase and the tyrosinase related proteins in the Mitfvit (vitiligo) mouse eye: implications for the function of the microphthalmia transcription factor.

Mitf (Microphthalmia transcription factor), a basic-helix-loop-helix zipper protein, encoded at the microphthalmia (Mitf) locus, regulates the transcription of the gene encoding tyrosinase, the rate-limiting enzyme in melanin biosynthesis, by binding the DNA sequence CATGTG. This binding site is present also in the genes encoding two tyrosinase related proteins, TRP-1 and TRP-2. To gain insight into the function of Mitf in vivo, we determined whether there was a difference in the levels of these proteins in the RPE/choroid of the vitiligo (Mitfvit) mouse, in which there is a mutation of the Mitf gene. This mouse has alteration of RPE pigmentation and function that presumably leads to slow progressive loss of photoreceptor cells. The RPE/choroid was dissected from eyes of vitiligo and C57BL/6 wild-type mice at postnatal ages 2, 4, 7, 10, 14, 21 and 42 days. Extracts of pooled tissues were subjected to electrophoresis and immunoblotting. The levels of tyrosinase, TRP-1 and TRP-2 were determined densitometrically following immunodetection with rabbit antipeptide antisera. In addition, the tyrosine hydroxylase activity of tyrosinase as assayed radiometrically. Levels of TRP-1 were 3-7 fold greater in control RPE/choroid compared with mutants. This marked difference in protein level was observed at the earliest age examined (P2) and persisted throughout the first two weeks. Tyrosinase levels in mutants were similar to controls at P2 and P4, but were reduced at P10 and beyond. Tyrosinase activity was diminished also in mutants by P10. Levels of TRP-2 were similar between mutants and controls, although the typical decrease seen in controls after P14 was attenuated in the mutant mice. There is a significant reduction in the level of TRP-1 in the RPE/choroid of the Mitfvit mouse. The data suggests that transcription of the gene encoding TRP-1 is extremely dependent upon functional Mitf. It provides in vivo evidence that Mitf regulates the transcription of the gene encoding TRP-1 as well as tyrosinase.

The regulation of tyrosinase gene transcription.

Tyrosinase is one of the key enzymes essential for melanogenesis. The control of its activity rests in part at the level of transcriptional regulation. The 5' promoter regions of the human, mouse, chicken, quail, snapping turtle, and frog tyrosinase sequences have been isolated and the mechanisms regulating the activity of these sequences are beginning to be elucidated. This review provides an update on the following aspects of tyrosinase gene regulation: basal promoter elements that determine the site of transcription initiation for RNA polymerase II; the cis-acting elements and DNA-binding factors that mediate melanocyte-specific expression of the tyrosinase gene; promoter elements involved in the temporal control of tyrosinase gene expression; additional elements that may be required to achieve wild-type levels of gene expression; and specific elements that may be required for modulation of tyrosinase gene expression in response to humoral factors or external stimuli that are known to influence the amounts of melanin synthesized by fully differentiated melanocytes. The wild type expression of tyrosinase is the result of the interaction of many different factors and it is becoming evident that certain elements and factors play more than one role in this process.

Diurnal fluctuation in the enzyme activity and the messenger RNA level of pineal serotonin N-acetyltransferase in normal and hereditary microphthalmic rats.

The enzyme activity and the messenger RNA level of pineal serotonin N-acetyltransferase were more than 20- and 50-fold higher, respectively, in the dark period than in the light period in normal rats. In hereditary microphthalmic rats, however, the serotonin N-acetyltransferase activity and its mRNA level did not undergo a great diurnal change through the light and dark periods. These results indicate that the diurnal rhythms of the activity and the mRNA level of serotonin N-acetyltransferase are not detected in the pineal gland of hereditary blind rats, suggesting free-running rhythms in individual animals due to desynchronization of their circadian rhythms by a lack of their optic nerve.

Cloning and characterization of a putative human holocytochrome c-type synthetase gene (HCCS) isolated from the critical region for microphthalmia with linear skin defects (MLS).

Microphthalmia with linear skin defects syndrome (MLS) is an X-linked male-lethal disorder associated with X chromosomal rearrangements resulting in monosomy from Xpter to Xp22. Features include micro- phthalmia, sclerocornea, linear skin defects, and agenesis of the corpus callosum. Using a cross-species conservation strategy, an expressed sequence from the 450- to the 550-kb MLS critical region on Xp22 was identified by screening a human embryo cDNA library. Northern analysis revealed a transcript of approximately 2.6 kb in all tissues examined, with weaker expression of approximately 1.2- and approximately 5.2-kb transcripts. The strongest expression was observed in heart and skeletal muscle. Sequence analysis of a 3-kb cDNA contig revealed an 807-bp open reading frame encoding a putative 268-amino-acid protein. Comparison of the sequence with sequences in the databases revealed homology with holocytochrome c-type synthetases, which catalyze the covalent addition of a heme group onto c-type cytochromes in the mitochondria. The c-type cytochromes are required for proper functioning of the electron transport pathway. The human gene (HGMW-approved symbol HCCS) and the corresponding murine gene characterized in this paper are the first mammalian holocytochrome c-type synthetases to be described in the literature. Because of the lack of a neuromuscular phenotype in MLS, it is uncertain whether the deletion of a mitochondrial holocytochrome synthetase would contribute to the phenotype seen in MLS. The expression pattern of this gene and knowledge about the function of holocytochrome synthetases, however, suggest that it is a good candidate for X-linked encephalomyopathies typically associated with mitochondrial dysfunction.

Elevated lactic acid dehydrogenase in aqueous humor in an eye without retinoblastoma.

A 14-month-old boy had a microphthalmic left eye. An anterior chamber paracentesis was performed six days after examination under anesthesia. Lactic acid dehydrogenase (LDH) level in the aqueous humor was 1,158 mu and was 202 mu in the serum. The aqueous humor/serum LDH ratio was 5.73. This eye was eventually enucleated and found to have retinal dysplasia and diffuse hemorrhagic necrosis. There was no eividence of retinoblastoma. The elevated LDH level in aqueous humor was probably secondary to the ocular tissue necrosis and hemorrhage. This false-positive result emphasizes the limitation of using the aqueous humor/serum LDH ratio to distinguish eyes with retinoblastoma from those without retinoblastoma.

[Enzymatic studies in lenses of different breeds of mice (author's transl)]. / Fermentchemische Untersuchungen and Linsen verschiedener Mäusestämme

Leucinaminopeptidase (LAP), lactatedehydrogenase (LDH) and glucose-6-phosphatdehydrogenase (GPDH) were analysed in the lenses of two different breeds of mice. Compared to the lenses of a control stock (Agnes Blum, Jena), in the healthy seeming, heterozygous mice the same activities of LAP, LDH, and GPDH were found. But in the breed with evident microphthalmos and cataract LDH was increased. Possible connections are discused.

Linkage studies in Lenz microphthalmia.

Glucose-6-phosphate dehydrogenase (G6PD) phenotype studies were done on a black family with X-linked heredofamilial bilateral microphthalmia (HBM). Three crossovers and three non-crossovers were detected in three informative matings of four generations yielding a recombination value of 0.5. These findings do not provide evidence for linkage between the G6PD and HBM loci, suggesting either that the G6PD and HBM loci are far apart on the X chromosome or that HBM in this family is inherited as an autosomal dominant male sex-limited trait.