Andrea Molinari

Localization of a Gene for Keratoconus to a 5.6-Mb Interval on 13q32

PURPOSE Keratoconus (KTCN) is a noninflammatory thinning and anterior protrusion of the cornea that results in steepening and distortion of the cornea, altered refractive powers, and reduced visual acuity. Several loci responsible for a familial form of KTCN have been mapped, however; no mutations in any genes have been identified for any of these loci. There is also evidence that VSX1 and SOD1 may be involved in the etiology of KTCN. The purpose of this study was to verify the available data and to identify a new keratoconus susceptibility locus. METHODS KTCN without other ocular or systemic features was diagnosed in 18 families. VSX1 and SOD1 sequencing was performed on affected individuals and control subjects. Genomewide linkage analysis was then performed in all families using polymorphic microsatellite markers with an average spacing of 5 cM. Next, single-nucleotide polymorphism (SNP) arrays, fluorescence in situ hybridization (FISH) analysis, and a comparative genomic hybridization array were used in one family to assess a candidate region on 13q32. RESULTS All previously reported KTCN loci were excluded. VSX1 and SOD1 were sequenced, and no potentially functional variants were found. One KTCN family yielded a maximum multipoint parametric LOD score of 4.1 and multipoint nonparametric linkage (NPL) LOD score of 3.2. Multipoint linkage and haplotype analysis narrowed the locus to a 5.6-Mb region between the SNPs rs9516572 and rs3825523 on 13q32. CONCLUSIONS The results exclude VSX1 and SOD1 as potential disease-causing genes in these families and localize a novel gene for keratoconus to a 5.6-Mb interval on 13q32.

Novel mutation and three other sequence variants segregating with phenotype at keratoconus 13q32 susceptibility locus.

Keratoconus (KTCN), a non-inflammatory corneal disorder characterized by stromal thinning, represents a major cause of corneal transplantations. Genetic and environmental factors have a role in the etiology of this complex disease. Previously reported linkage analysis revealed that chromosomal region 13q32 is likely to contain causative gene(s) for familial KTCN. Consequently, we have chosen eight positional candidate genes in this region: MBNL1, IPO5, FARP1, RNF113B, STK24, DOCK9, ZIC5 and ZIC2, and sequenced all of them in 51 individuals from Ecuadorian KTCN families and 105 matching controls. The mutation screening identified one mutation and three sequence variants showing 100% segregation under a dominant model with KTCN phenotype in one large Ecuadorian family. These substitutions were found in three different genes: c.2262A>C (p.Gln754His) and c.720+43A>G in DOCK9; c.2377-132A>C in IPO5 and c.1053+29G>C in STK24. PolyPhen analyses predicted that c.2262A>C (Gln754His) is possibly damaging for the protein function and structure. Our results suggest that c.2262A>C (p.Gln754His) mutation in DOCK9 may contribute to the KTCN phenotype in the large KTCN-014 family.

Cytochrome P4501B1 mutations cause only part of primary congenital glaucoma in Ecuador

Purpose: To determine the role of cytochrome P4501B1 ( CYP1B1 ) mutations in causing primary congenital glaucoma (PCG) in a cohort of Native Americans from Quito, Ecuador. Materials and methods: Seventeen patients with PCG from 15 Native American families were recruited from the Ophthalmology Clinic at Hospital Metropolitano, Quito, Ecuador. Experienced ophthalmologists examined all affected study subjects. Purified DNA was prepared from peripheral blood samples and CYP1B1 coding exons (exons 2 and 3) were amplified and sequenced. Southern blot was performed only on those affected patients who showed no mutations in the CYP1B1 coding exons. Results: The molecular basis of PCG in two families was determined: two novel mutations (a deletion and a point mutation) and one novel polymorphism in CYP1B1 were identified in addition to a previously described single amino acid substitution. Southern blot analyses on whole genomic DNA from affected individuals in whom no mutations were identified by the direct PCR/sequencing approach did not detect any large rearrangements or mutations outside the coding region. Conclusion: These findings suggest that mutations in CYP1B1 are not a major cause of PCG in this population and that at least one additional locus for this condition is responsible for most cases. Further, the PCG phenotype did not correlate readily with the molecular basis of the disorder, suggesting that careful clinical analysis of the phenotype cannot predict the molecular basis of the disease with accuracy.

Substitution at IL1RN and deletion at SLC4A11 segregating with phenotype in familial keratoconus.

PURPOSE Keratoconus (KTCN) is a thinning and anterior protrusion of the cornea that results in altered refractive powers and loss of visual acuity. Despite numerous studies, the reasons for development and progression of KTCN remain unknown. Genetic studies have led to identification of several loci linked with KTCN, including a locus in one multigenerational Ecuadorian family. The purpose of this study was to identify sequence variants in candidate genes segregating with the KTCN phenotype in another Ecuadorian family. METHODS Nonparametric linkage analysis was performed in Ecuadorian family KTCN-019. Candidate genes IL1A, IL1B, IL1RN, and SLC4A11 were selected and examined in this family by direct sequencing of all exons, promoters, and intron-exon junctions. RESULTS Two novel suggestive loci were identified in 2q13-q14.3 and 20p13-p12.2. Screening of the candidate genes revealed 66 sequence variants, including five novel variants, in both coding and noncoding regions. The substitution c.214+242C > T in the IL1RN gene was observed in all affected individuals and three apparently unaffected family members. The novel deletion of 54 nucleotides in position c.2558+149_2558+203 in SLC4A11 was observed in all patients but one, as well as two healthy individuals and one person with an unknown phenotype. CONCLUSIONS The analyses of selected genes have led to identification of numerous sequence variants in the examined Ecuadorian family. Both substitution c.214+242C > T in IL1RN and novel deletion c.2558+149_2558+203del54 in SLC4A11 were observed significantly more frequently in family members with KTCN (P = 0.004525 and P = 0.00761, respectively), suggesting involvement of these two genes in KTCN etiology in the studied family.

Variant c.2262A>C in DOCK9 leads to exon skipping in keratoconus family

PURPOSE Keratoconus (KTCN) is a degenerative disorder of the eye that is characterized by a conical shape and thinning of the cornea, resulting in impaired visual function. Previously, we identified heterozygous single base-pair substitutions in DOCK9, IPO5, and STK24, showing concurrent 100% segregation with the affected phenotype in an Ecuadorian family. As the pathogenic consequences of these variants were not obvious, we performed in vitro splicing analyses to determine their functional significance. METHODS We generated expression constructs using patient DNA as a template corresponding to the wild-type and mutant alleles of DOCK9, IPO5, and STK24. After transfecting HeLa cells with each construct, total RNA samples were extracted, reverse transcribed, and amplified using specific primers. RESULTS In vitro splicing analysis revealed that only c.2262A>C in exon 20 of DOCK9 led to aberrant splicing, resulting in the changed ratio between two protein isoforms: a normal transcript and a transcript with exon skipping. The exon skipping causes a premature stop codon, disrupting the functional domains of DOCK9 protein, which may alter the biological role of DOCK9 as a Cdc42 activator. CONCLUSIONS Based on in vitro results, we demonstrated that c.2262A>C substitution in DOCK9, previously identified in KTCN-affected members of an Ecuadorian family, leads to a splicing aberration. However, because the mutation effect was observed in vitro, a definitive relationship between DOCK9 and KTCN phenotype could not be established. Our results indicate that further elucidation of the causes of KTCN is needed.

Variants in SKP1 , PROB1 , and IL17B genes at keratoconus 5q31.1–q35.3 susceptibility locus identified by whole-exome sequencing

Keratoconus (KTCN) is a protrusion and thinning of the cornea, resulting in impairment of visual function. The extreme genetic heterogeneity makes it difficult to discover factors unambiguously influencing the KTCN phenotype. In this study, we used whole-exome sequencing (WES) and Sanger sequencing to reduce the number of candidate genes at the 5q31.1–q35.3 locus and to prioritize other potentially relevant variants in an Ecuadorian family with KTCN. We applied WES in two affected KTCN individuals from the Ecuadorian family that showed a suggestive linkage between the KTCN phenotype and the 5q31.1–q35.3 locus. Putative variants identified by WES were further evaluated in this family using Sanger sequencing. Exome capture discovered a total of 173 rare (minor allele frequency <0.001 in control population) nonsynonymous variants in both affected individuals. Among them, 16 SNVs were selected for further evaluation. Segregation analysis revealed that variants c.475T>G in SKP1, c.671G>A in PROB1, and c.527G>A in IL17B in the 5q31.1–q35.3 linkage region, and c.850G>A in HKDC1 in the 10q22 locus completely segregated with the phenotype in the studied KTCN family. We demonstrate that a combination of various techniques significantly narrowed the studied genomic region and reduced the list of the putative exonic variants. Moreover, since this locus overlapped two other chromosomal regions previously recognized in distinct KTCN studies, our findings suggest that this 5q31.1–q35.3 locus might be linked with KTCN.