Mutations in the LHX2 gene are not a frequent cause of micro/anophthalmia

Purpose: Microphthalmia and anophthalmia are at the severe end of the spectrum of abnormalities in ocular development. A few genes (orthodenticle homeobox 2 [OTX2], retina and anterior neural fold homeobox [RAX], SRY-box 2 [SOX2], CEH10 homeodomain-containing homolog [CHX10], and growth differentiation factor 6 [GDF6]) have been implicated mainly in isolated micro/anophthalmia but causative mutations of these genes explain less than a quarter of these developmental defects. The essential role of the LIM homeobox 2 (LHX2) transcription factor in early eye development has recently been documented. We postulated that mutations in this gene could lead to micro/anophthalmia, and thus performed molecular screening of its sequence in patients having micro/anophthalmia. Methods: Seventy patients having non-syndromic forms of colobomatous microphthalmia (n=25), isolated microphthalmia (n=18), or anophthalmia (n=17), and syndromic forms of micro/anophthalmia (n=10) were included in this study after negative molecular screening for OTX2, RAX, SOX2, and CHX10 mutations. Mutation screening of LHX2 was performed by direct sequencing of the coding sequences and intron/exon boundaries. Results: Two heterozygous variants of unknown significance (c.128C > G [p.Pro43Arg]; c.776C > A [p.Pro259Gln]) were identified in LHX2 among the 70 patients. These variations were not identified in a panel of 100 control patients of mixed origins. The variation c.776C > A (p.Pro259Gln) was considered as non pathogenic by in silico analysis, while the variation c.128C > G (p.Pro43Arg) considered as deleterious by in silico analysis and was inherited from the asymptomatic father. Conclusions: Mutations in LHX2 do not represent a frequent cause of micro/anophthalmia

Linkage analysis of high myopia susceptibility locus in 26 families

Purpose: We conducted a linkage analysis in high myopia families to replicate suggestive results from chromosome 7q36 using a model of autosomal dominant inheritance and genetic heterogeneity. We also performed a genome-wide scan to identify novel loci. Methods: Twenty-six families, with at least two high-myopic subjects (ie. refractive value in the less affected eye of -5 diopters) in each family, were included. Phenotypic examination included standard autorefractometry, ultrasonographic eye length measurement, and clinical confirmation of the non-syndromic character of the refractive disorder. Nine families were collected de novo including 136 available members of whom 34 were highly myopic subjects. Twenty new subjects were added in 5 of the 17 remaining families. A total of 233 subjects were submitted to a genome scan using ABI linkage mapping set LMSv2-MD-10, additional markers in all regions where preliminary LOD scores were greater than 1.5 were used. Multipoint parametric and non-parametric analyses were conducted with the software packages Genehunter 2.0 and Merlin 1.0.1. Two autosomal recessive, two autosomal dominant, and four autosomal additive models were used in the parametric linkage analyses. Results: No linkage was found using the subset of nine newly collected families. Study of the entire population of 26 families with a parametric model did not yield a significant LOD score (>3), even for the previously suggestive locus on 7q36. A non-parametric model demonstrated significant linkage to chromosome 7p15 in the entire population (Z-NPL=4.07, p=0.00002). The interval is 7.81 centiMorgans (cM) between markers D7S2458 and D7S2515. Conclusions: The significant interval reported here needs confirmation in other cohorts. Among possible susceptibility genes in the interval, certain candidates are likely to be involved in eye growth and development

Novel ABCC6 Mutations in Pseudoxanthoma Elasticum

Pseudoxanthoma elasticum (PXE) is a heritable connective tissue disorder caused by mutations in an ABC (ATP-Binding Cassette) transporter gene (ABCC6), which manifests with cutaneous, ophthalmologic, and cardiovascular findings. We studied a cohort of 19 families with PXE, and identified 16 different mutations, nine of which were novel variants. The mutation detection rate was about 77%. We found that arginine codon 518 was, with the previously described R1141X and EX23_29del, a recurrently mutated amino acid (11.5% of the mutations detected for each variant R518Q and R518X). No clear delineation of genotype/phenotype correlation was identified, and marked intra-familial variability of the disease was seen in one family. One family with pseudodominant inheritance displayed three distinct ABCC6 mutations, providing further evidence for the probable exclusive recessive transmission of PXE. These data contribute to the expanding database of ABCC6 mutations, to the description of phenotypic variability, and inheritance in PXE, and should be helpful for genetic counselling

Quality of DNA Extracted from Mouthwashes

Background A cost effective, safe and efficient method of obtaining DNA samples is essential in large scale genetic analyses. Buccal cells are an attractive source of DNA, as their collection is non-invasive and can be carried out by mail. However, little attention has been given to the quality of DNA extracted from mouthwashes. Methodology Mouthwash-derived DNA was extracted from 500 subjects participating in a genetic study of high myopia. DNA quality was investigated using two standard techniques: agarose gel electrophoresis and quantitative polymerase chain reaction (qPCR). Principal Findings Whereas the majority of mouthwash-derived DNA samples showed a single band of high molecular weight DNA by gel electrophoresis, 8.9% (95% CI: 7.1–10.7%) of samples contained only a smear of low-to-medium molecular weight, degraded DNA. The odds of DNA degradation in a subject's second mouthwash sample, given degradation of the first, was significantly greater than one (OR = 3.13; 95% CI: 1.22–7.39; Fisher's test P = 0.009), suggesting that DNA degradation was at least partially a subject-specific phenomenon. Approximately 12.4% (95% CI: 10.4–14.4%) of mouthwash-derived DNA failed to PCR amplify efficiently (using an ~200 bp microsatellite marker). However, we found there was no significant difference in amplification success rate between DNA samples judged to be degraded or non-degraded by gel electrophoresis (Fisher's test P = 0.5). Conclusions This study demonstrated that DNA degradation affects a significant minority of saline mouthwashes, and that the phenomenon is partially subject-specific. Whilst the level of degradation did not significantly prevent successful amplification of short PCR fragments, previous studies suggest that such DNA degradation would compromise more demanding applications

Deletion upstream of MAB21L2 highlights the importance of evolutionarily conserved non-coding sequences for eye development

Anophthalmia, microphthalmia and coloboma (AMC) comprise a spectrum of developmental eye disorders, accounting for approximately 20% of childhood visual impairment. While non-coding regulatory sequences are increasingly recognised as contributing to disease burden, characterising their impact on gene function and phenotype remains challenging. Furthermore, little is known of the nature and extent of their contribution to AMC phenotypes. We report two families with variants in or near MAB21L2, a gene where genetic variants are known to cause AMC in humans and animal models. The first proband, presenting with microphthalmia and coloboma, has a likely pathogenic missense variant (c.338 G > C; p.[Trp113Ser]), segregating within the family. The second individual, presenting with microphthalmia, carries an ~ 113.5 kb homozygous deletion 19.38 kb upstream of MAB21L2. Modelling of the deletion results in transient small lens and coloboma as well as midbrain anomalies in zebrafish, and microphthalmia and coloboma in Xenopus tropicalis. Using conservation analysis, we identify 15 non-coding conserved elements (CEs) within the deleted region, while ChIP-seq data from mouse embryonic stem cells demonstrates that two of these (CE13 and 14) bind Otx2, a protein with an established role in eye development. Targeted disruption of CE14 in Xenopus tropicalis recapitulates an ocular coloboma phenotype, supporting its role in eye development. Together, our data provides insights into regulatory mechanisms underlying eye development and highlights the importance of non-coding sequences as a source of genetic diagnoses in AMC

Compositional diversity of rehabilitated tropical lands supports multiple ecosystem services and buffers uncertainties

High landscape diversity is assumed to increase the number and level of ecosystem services. However, the interactions between ecosystem service provision, disturbance and landscape composition are poorly understood. Here we present a novel approach to include uncertainty in the optimization of land allocation for improving the provision of multiple ecosystem services. We refer to the rehabilitation of abandoned agricultural lands in Ecuador including two types of both afforestation and pasture rehabilitation, together with a succession option. Our results show that high compositional landscape diversity supports multiple ecosystem services (multifunction effect). This implicitly provides a buffer against uncertainty. Our work shows that active integration of uncertainty is only important when optimizing single or highly correlated ecosystem services and that the multifunction effect on landscape diversity is stronger than the uncertainty effect. This is an important insight to support a land-use planning based on ecosystem services

Compositional diversity of rehabilitated tropical lands supports multiple ecosystem services and buffers uncertainties

High landscape diversity is assumed to increase the number and level of ecosystem services. However, the interactions between ecosystem service provision, disturbance and landscape composition are poorly understood. Here we present a novel approach to include uncertainty in the optimization of land allocation for improving the provision of multiple ecosystem services. We refer to the rehabilitation of abandoned agricultural lands in Ecuador including two types of both afforestation and pasture rehabilitation, together with a succession option. Our results show that high compositional landscape diversity supports multiple ecosystem services (multifunction effect). This implicitly provides a buffer against uncertainty. Our work shows that active integration of uncertainty is only important when optimizing single or highly correlated ecosystem services and that the multifunction effect on landscape diversity is stronger than the uncertainty effect. This is an important insight to support a land-use planning based on ecosystem services