[Show abstract][Hide abstract] ABSTRACT: Purpose:
To identify specific mutations causing North Carolina macular dystrophy (NCMD).
Whole-genome sequencing coupled with reverse transcription polymerase chain reaction (RT-PCR) analysis of gene expression in human retinal cells.
A total of 141 members of 12 families with NCMD and 261 unrelated control individuals.
Genome sequencing was performed on 8 affected individuals from 3 families affected with chromosome 6-linked NCMD (MCDR1) and 2 individuals affected with chromosome 5-linked NCMD (MCDR3). Variants observed in the MCDR1 locus with frequencies <1% in published databases were confirmed using Sanger sequencing. Confirmed variants absent from all published databases were sought in 8 additional MCDR1 families and 261 controls. The RT-PCR analysis of selected genes was performed in stem cell-derived human retinal cells.
Main outcome measures:
Co-segregation of rare genetic variants with disease phenotype.
Five sequenced individuals with MCDR1-linked NCMD shared a haplotype of 14 rare variants spanning 1 Mb of the disease-causing allele. One of these variants (V1) was absent from all published databases and all 261 controls, but was found in 5 additional NCMD kindreds. This variant lies in a DNase 1 hypersensitivity site (DHS) upstream of both the PRDM13 and CCNC genes. Sanger sequencing of 1 kb centered on V1 was performed in the remaining 4 NCMD probands, and 2 additional novel single nucleotide variants (V2 in 3 families and V3 in 1 family) were identified in the DHS within 134 bp of the location of V1. A complete duplication of the PRDM13 gene was also discovered in a single family (V4). The RT-PCR analysis of PRDM13 expression in developing retinal cells revealed marked developmental regulation. Next-generation sequencing of 2 individuals with MCDR3-linked NCMD revealed a 900-kb duplication that included the entire IRX1 gene (V5). The 5 mutations V1 to V5 segregated perfectly in the 102 affected and 39 unaffected members of the 12 NCMD families.
We identified 5 rare mutations, each capable of arresting human macular development. Four of these strongly implicate the involvement of PRDM13 in macular development, whereas the pathophysiologic mechanism of the fifth remains unknown but may involve the developmental dysregulation of IRX1.
[Show abstract][Hide abstract] ABSTRACT: Background
It has been recognized that cells do not respond equally to ultraviolet (UV) radiation but it is not clear whether this is due to genetic, biochemical or structural differences of the cells. We have a novel cybrid (cytoplasmic hybrids) model that allows us to analyze the contribution of mitochondrial DNA (mtDNA) to cellular response after exposure to sub-lethal dose of UV. mtDNA can be classified into haplogroups as defined by accumulations of specific single nucleotide polymorphisms (SNPs). Recent studies have shown that J haplogroup is high risk for age-related macular degeneration while the H haplogroup is protective. This study investigates gene expression responses in J cybrids versus H cybrids after exposure to sub-lethal doses of UV-radiation.
Cybrids were created by fusing platelets isolated from subjects with either H (n = 3) or J (n = 3) haplogroups with mitochondria-free (Rho0) ARPE-19 cells. The H and J cybrids were cultured for 24 hours, treated with 10 mJ of UV-radiation and cultured for an additional 120 hours. Untreated and treated cybrids were analyzed for growth rates and gene expression profiles. The UV-treated and untreated J cybrids had higher growth rates compared to H cybrids. Before treatment, J cybrids showed lower expression levels for CFH, CD55, IL-33, TGF-A, EFEMP-1, RARA, BCL2L13 and BBC3. At 120 hours after UV-treatment, the J cybrids had decreased CFH, RARA and BBC3 levels but increased CD55, IL-33 and EFEMP-1 compared to UV-treated H cybrids.
In cells with identical nuclei, the cellular response to sub-lethal UV-radiation is mediated in part by the mtDNA haplogroup. This supports the hypothesis that differences in growth rates and expression levels of complement, inflammation and apoptosis genes may result from population-specific, hereditary SNP variations in mtDNA. Therefore, when analyzing UV-induced damage in tissues, the mtDNA haplogroup background may be important to consider.
PLoS ONE 06/2014; 9(6):e99003. DOI:10.1371/journal.pone.0099003 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: It has been recognized that cells do not respond equally to ultraviolet (UV) radiation but it is not clear whether this is due to genetic, biochemical or structural differences of the cells. We have a novel cybrid (cytoplasmic hybrids) model that allows us to analyze the contribution of mitochondrial DNA (mtDNA) to cellular response after exposure to sub-lethal dose of UV. mtDNA can be classified into haplogroups as defined by accumulations of specific single nucleotide polymorphisms (SNPs). Recent studies have shown that J haplogroup is high risk for age-related macular degeneration while the H haplogroup is protective. This study investigates gene expression responses in J cybrids versus H cybrids after exposure to sub-lethal doses of UV-radiation. METHODOLOGY/PRINCIPAL FINDINGS: Cybrids were created by fusing platelets isolated from subjects with either H (n = 3) or J (n = 3) haplogroups with mitochondria-free (Rho0) ARPE-19 cells. The H and J cybrids were cultured for 24 hours, treated with 10 mJ of UV-radiation and cultured for an additional 120 hours. Untreated and treated cybrids were analyzed for growth rates and gene expression profiles. The UV-treated and untreated J cybrids had higher growth rates compared to H cybrids. Before treatment, J cybrids showed lower expression levels for CFH, CD55, IL-33, TGF-A, EFEMP-1, RARA, BCL2L13 and BBC3. At 120 hours after UV-treatment, the J cybrids had decreased CFH, RARA and BBC3 levels but increased CD55, IL-33 and EFEMP-1 compared to UV-treated H cybrids. CONCLUSION/SIGNIFICANCE: In cells with identical nuclei, the cellular response to sub-lethal UV-radiation is mediated in part by the mtDNA haplogroup. This supports the hypothesis that differences in growth rates and expression levels of complement, inflammation and apoptosis genes may result from population-specific, hereditary SNP variations in mtDNA. Therefore, when analyzing UV-induced damage in tissues, the mtDNA haplogroup background may be important to consider. FAU - Malik, Deepika
[Show abstract][Hide abstract] ABSTRACT: Background
Age-related macular degeneration (AMD) is the leading cause of vision loss in developed countries. While linked to genetic polymorphisms in the complement pathway, there are many individuals with high risk alleles that do not develop AMD, suggesting that other "modifiers" may be involved. Mitochondrial (mt) haplogroups, defined by accumulations of specific mtDNA single nucleotide polymorphisms (SNPs) which represent population origins, may be one such modifier. J haplogroup has been associated with high risk for AMD while the H haplogroup is protective. It has been difficult to assign biological consequences for haplogroups so we created human ARPE-19 cybrids (cytoplasmic hybrids), which have identical nuclei but mitochondria of either J or H haplogroups, to investigate their effects upon bioenergetics and molecular pathways.Methodology/Principal FindingsJ cybrids have altered bioenergetic profiles compared to H cybrids. Q-PCR analyses, show significantly lower expression levels for seven respiratory complex genes encoded by mtDNA. J and H cybrids have significantly altered expression of eight nuclear genes of the alternative complement, inflammation and apoptosis pathways. Sequencing of the entire mtDNA DNA was carried out for all the cybrids to identify haplogroup and non haplogroup defining SNPs.Conclusion/SignificancemtDNA can mediate cellular bioenergetics and expression levels of nuclear genes related to complement, inflammation and apoptosis. Sequencing data suggest that observed effects are not due to rare mtDNA variants but rather the combination of SNPs representing the J versus H haplogroups. These findings represent a paradigm shift in our concepts of mitochondrial-nuclear interactions.
Human Molecular Genetics 02/2014; 23(13). DOI:10.1093/hmg/ddu065 · 6.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The geographic origins of populations can be identified by their maternally inherited mitochondrial DNA (mtDNA) haplogroups. This study compared human cybrids (cytoplasmic hybrids), which are cell lines with identical nuclei but mitochondria from different individuals with mtDNA from either the H haplogroup or L haplogroup backgrounds. The most common European haplogroup is H while individuals of maternal African origin are of the L haplogroup. Despite lower mtDNA copy numbers, L cybrids had higher expression levels for nine mtDNA-encoded respiratory complex genes, decreased ATP turnover rates and lower levels of ROS production, parameters which are consistent with more efficient oxidative phosphorylation. Surprisingly, GeneChip arrays showed that the L and H cybrids had major differences in expression of genes of the canonical complement system (5 genes), dermatan/chondroitin sulfate biosynthesis (5 genes) and CCR3 signaling (9 genes). Quantitative nuclear gene expression studies confirmed that L cybrids had (a) lower expression levels of complement pathway and innate immunity genes and (b) increased levels of inflammation-related signaling genes, which are critical in human diseases. Our data support the hypothesis that mtDNA haplogroups representing populations from different geographic origins may play a role in differential susceptibilities to diseases.
[Show abstract][Hide abstract] ABSTRACT: Mitochondrial dysfunction is associated with the development and progression of age-related macular degeneration (AMD). Recent studies using populations from the United States and Australia have demonstrated that AMD is associated with mitochondrial (mt) DNA haplogroups (as defined by combinations of mtDNA polymorphisms) that represent Northern European Caucasians. The aim of this study was to use the cytoplasmic hybrid (cybrid) model to investigate the molecular and biological functional consequences that occur when comparing the mtDNA H haplogroup (protective for AMD) versus J haplogroup (high risk for AMD).
Cybrids were created by introducing mitochondria from individuals with either H or J haplogroups into a human retinal epithelial cell line (ARPE-19) that was devoid of mitochondrial DNA (Rho0). In cybrid lines, all of the cells carry the same nuclear genes but vary in mtDNA content. The J cybrids had significantly lower levels of ATP and reactive oxygen/nitrogen species production, but increased lactate levels and rates of growth. Q-PCR analyses showed J cybrids had decreased expressions for CFH, C3, and EFEMP1 genes, high risk genes for AMD, and higher expression for MYO7A, a gene associated with retinal degeneration in Usher type IB syndrome. The H and J cybrids also have comparatively altered expression of nuclear genes involved in pathways for cell signaling, inflammation, and metabolism.
Our findings demonstrate that mtDNA haplogroup variants mediate not only energy production and cell growth, but also cell signaling for major molecular pathways. These data support the hypothesis that mtDNA variants play important roles in numerous cellular functions and disease processes, including AMD.
PLoS ONE 01/2013; 8(1):e54339. DOI:10.1371/journal.pone.0054339 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
Age-related macular degeneration (AMD) is the leading cause of vision loss in elderly, Caucasian populations. There is strong evidence that mitochondrial dysfunction and oxidative stress play a role in the cell death found in AMD retinas. The purpose of this study was to examine the association of the Caucasian mitochondrial JTU haplogroup cluster with AMD. We also assessed for gender bias and additive risk with known high risk nuclear gene SNPs, ARMS2/LOC387715 (G > T; Ala69Ser, rs10490924) and CFH (T > C; Try402His, rs1061170).
Total DNA was isolated from 162 AMD subjects and 164 age-matched control subjects located in Los Angeles, California, USA. Polymerase chain reaction (PCR) and restriction enzyme digestion were used to identify the J, U, T, and H mitochondrial haplogroups and the ARMS2-rs10490924 and CFH-rs1061170 SNPs. PCR amplified products were sequenced to verify the nucleotide substitutions for the haplogroups and ARMS2 gene.
The JTU haplogroup cluster occurred in 34% (55/162) of AMD subjects versus 15% (24/164) of normal (OR = 2.99; p = 0.0001). This association was slightly greater in males (OR = 3.98, p = 0.005) than the female population (OR = 3.02, p = 0.001). Assuming a dominant effect, the risk alleles for the ARMS2 (rs10490924; p = 0.00001) and CFH (rs1061170; p = 0.027) SNPs were significantly associated with total AMD populations. We found there was no additive risk for the ARMS2 (rs10490924) or CFH (rs1061170) SNPs on the JTU haplogroup background.
There is a strong association of the JTU haplogroup cluster with AMD. In our Southern California population, the ARMS2 (rs10490924) and CFH (rs1061170) genes were significantly but independently associated with AMD. SNPs defining the JTU mitochondrial haplogroup cluster may change the retinal bioenergetics and play a significant role in the pathogenesis of AMD.
BMC Medical Genetics 01/2013; 14(1):4. DOI:10.1186/1471-2350-14-4 · 2.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Enhanced S Cone Syndrome is a rare autosomal recessive disorder characterized clinically by an absence of rod function, a replacement of most L and M cone function by S cone activity (Goldmann-Favre Syndrome) and by variable degrees of retinal degeneration in different families. The causative gene, nuclear receptor subfamily 2, group E, member 3 (NR2E3), controls the developmental sequence for rods and cones. The purpose of this study was to compare the nature and implications of mutations in two subjects with Enhanced S Cone Syndrome who have significantly different degrees of degenerative damage.
A direct sequencing approach was used to identify the mutations. Genomic DNA was amplified from all the exons of NR2E3 and used as a template for sequencing. Of the two families studied, Case 1 is of Persian ethnicity while Case 2 is Brazilian. A total of six individuals within the two families were studied.
Case 1 (original propositus of the syndrome) has the characteristic developmental rod/cone abnormality with large amplitude electroretinogram responses and no retinal degeneration. She was homozygous for a novel mutation, c.[del196-201del6] (p.G66-C67del), which lies entirely within the P-box for this gene. By comparison, Case 2 had Goldmann-Favre Syndrome with retinal degeneration and low electroretinogram signals. She was a compound heterozygote for c.[119-2A>C]+[del194-202del9] (p.N65-C67del), mutations that have been reported previously. Her second mutation overlaps that of Case 1 within the P-box.
The novel in-frame homozygous deletion of Case 1, within the P-box motif of the DNA binding domain, caused a developmental abnormality without retinal degeneration. Case 2, with more traditional Goldmann-Favre Syndrome with retinal degeneration, was a compound heterozygote where one allele had a similar P-box deletion but the other was a splicing defect. Case 1 is the first reported homozygous deletion within the P-box. This is the first report of NR2E3 mutations in a Persian and a Brazilian family.
[Show abstract][Hide abstract] ABSTRACT: To determine mitochondrial (mt)DNA variants in AMD and age-matched normal retinas.
Total DNA was isolated from retinas (AMD, n = 13; age-matched normal, n = 13), choroid (AMD, n = 3), and blood (AMD, n = 138; normal, n = 133). Long-extension-polymerase chain reaction amplified the full-length ( approximately 16.2 kb) mtDNA genome. Retinal mtDNA was sequenced for nucleotide variants and length heteroplasmy. Pyrosequencing was performed on heteroplasmic mtDNA. PCR amplification and enzyme digestions were used to analyze for nucleotide changes.
Retinal mtDNA had a greater number of rearrangements and deletions than did blood mtDNA in normal samples (9.3 +/- 1.78 vs. 3 +/- 1.18, P = 0.019), and AMD samples (14.33 +/- 1.96 vs. 5.2 +/- 0.80, P = 0.0031. Five (55%) of 9 AMD patients had unreported SNPs, and 2 (16.6%) of 12 of the normal group did. The mtDNA coding region had 20 SNPs that produced amino acid changes. The noncoding MT-Dloop region had nucleotide heteroplasmy and length heteroplasmy. There were more SNPs per person in the AMD population than in the older (P = 0.003) and younger (P = 0.05) normal subjects. The C12557T (T-I) in the MT-ND5 gene was present in two AMD subjects (2/138) but was absent in the normal (0/133). Common mutations for Leber's hereditary optic neuropathy (LHON: G11778A; T14484C; and G3460A) were not present in AMD samples.
AMD subjects have high levels of large mtDNA deletions/rearrangements in the retinas, unreported and amino acid-changing SNPs in the coding genome, and a greater number of SNPs per person in the noncoding MT-Dloop region. These mtDNA variants could diminish energy production efficiency, alter the mtDNA copy numbers and/or impact transcription in AMD retinas.
[Show abstract][Hide abstract] ABSTRACT: We reported previously a 7-base intronic deletion close to the intron/exon junction of the SOD1 gene in 2 separate families with an autosomal-dominant form of keratoconus. The goal of this study was to determine if the 2 families (families A and H) shared a common haplotype by identifying closely linked new microsatellite markers flanking the SOD1 gene.
Total genomic DNA was extracted from the blood of available members of families A and H. The DNA was amplified by polymerase chain reaction and digested with HpyCH4 III. A genomic contig was first constructed flanking the human SOD1 gene on chromosome 21q22.1-21q22.11. New polymorphic microsatellite markers were identified. All available individuals from the 2 families were genotyped using a set of 7 different markers (SOD1NU10, SOD1NU1, SOD1NU2, SOD1NU3, SOD1NU13, SOD1NU8, and SOD1NU9) to identify phase and the disease haplotype was constructed.
Five of the 7 markers are novel (SOD1NU1, SOD1NU2, SOD1NU3, SOD1NU8, and SOD1NU9). Family A is a 3-generation family and the disease haplotype was inferred based on segregation data for 7 different markers. Family H shared only 3 of the disease-associated alleles (SOD1NU1, SOD1NU2, and SOD1NU13) compared with family A.
Based on the dissimilarity of disease-associated alleles, the 2 families do not appear to share the same haplotype and therefore are not closely related. This strongly supports the uniqueness of the 7-base deletion in intron 2 of the SOD1 gene to the keratoconus phenotype.
[Show abstract][Hide abstract] ABSTRACT: To study the effects of hydrogen peroxide exposure on mitochondrial DNA (mtDNA) in cultured human corneal epithelial cells. In addition, we compared the integrity of mtDNA found in epithelial cells isolated from keratoconus (KC) and normal (NL) corneas.
Telomerase immortalized human corneal epithelial cell line (hTCEpi) were cultured at pH 7.0 or pH 5.0 with or without 200 microM hydrogen peroxide (H2O2). Immunohistochemistry with a marker for oxidative damage, 8-hydroxy-2'-deoxyguanosine (8-OH-dG), was performed on KC and NL corneas (n = 10). Epithelial cells were isolated from KC corneas (n = 5) and NL corneas (n = 7). Total DNA was extracted, and the mtDNA was analyzed by long extension polymerase chain reaction (LX-PCR). The ratios of mtDNA to nuclear DNA were measured by PCR. The mtDNA control regions were PCR amplified and sequenced.
In the epithelial cell cultures, the full-length LX-PCR mtDNA decreased 54% and 44% in the H2O2 + pH7 cultures and H2O2 + pH5 cultures, respectively. 8-OH-dG was present in all layers of KC epithelial cells but only in superficial layers of NL epithelial cells. The isolated KC and NL epithelial cells had comparable levels of full-length LX-PCR mtDNA (16.2 kb) and smaller sized mtDNA bands (4.3 +/- 0.99 vs 4.0 +/- 0.83 bands per individual, respectively). There were no significant differences in the control region nucleotide sequences in KC and NL epithelia.
Hydrogen peroxide can significantly degrade LX-PCR mtDNA in vitro. Although the KC epithelium showed a higher degree of oxidative damage, the levels of mtDNA damage in NL and KC epithelial cells were similar to each other.
[Show abstract][Hide abstract] ABSTRACT: To examine the mtDNA control regions in normal and age-related macular degeneration (AMD) retinas. To identify the mtDNA variations associated with AMD.
Retinas from 10 normal and 11 AMD globes were isolated and analyzed for mtDNA rearrangements by long extension-polymerase chain reaction (LX-PCR) and for the nature and frequency of single-nucleotide polymorphisms (SNPs) in the mtDNA control region by direct sequencing. Blood DNA was extracted from 99 AMD and 92 age-matched control subjects. The sequence variations that define haplogroups H, I, J, K, T, V, X, and U were characterized by PCR, restriction enzyme digestion, and/or sequencing.
LX-PCR of retinal mtDNAs revealed high levels of rearrangements in the patients with AMD and the control subjects, consistent with the decline in mitochondrial function with age. However, the AMD retinas had higher oxidized DNA levels and a higher number of SNPs than controls (P = 0.02). The control region SNPs T16126C and A73G, commonly found in haplogroups J and T, were more frequent in the AMD retinas than in normal retinas. The associations between AMD and haplogroups J and T were confirmed and extended by analysis of blood DNA. SNPs at position a T16126C (J; odds ratio [OR] = 3.66), T16126C+G13368A (JT; OR = 10.27), A4917G+A73G (T4; OR = 5), and T3197C+A12308G (U5; OR = infinity), were all strongly associated with AMD.
AMD retinas exhibited increased mtDNA control region SNPs compared to normal retinas. This correlated with an increased frequency of mtDNA SNPs associated with haplogroups J, T and U in patients with AMD. These results implicate mitochondrial alterations in the etiology of AMD.
[Show abstract][Hide abstract] ABSTRACT: To clinically characterize the retinal abnormalities and identify the mutation causing an autosomal dominant cone degeneration in an African American family.
Clinical characterization of family members using fundus photography, fluorescein angiography, and electrophysiological testing. Standard molecular genetic methods were used, including segregation analysis and DNA sequencing of candidate genes. Genetic mutation screening was performed in 20 individuals: 10 clinically unaffected and 10 affected.
The affected family members had findings consistent with a primary cone degeneration. A novel mutation, P575L, was found in exon 8 of the GUCY2D gene in 12 members of this family.
In addition to finding a previously undescribed mutation in GUCY2D, 2 of the family members who were thought to be unaffected through routine clinical examinations also had this mutation. These findings suggest that autosomal dominant cone degeneration in this family demonstrated age-dependent penetrance, which appears incomplete. This is the first African American family reported with a mutation in GUCY2D. Because the disease in this family and the one we previously described is primarily a cone degeneration, this disease should be more properly classified as cone degeneration and be called cone degeneration 2.
This study helps to expand the phenotype of the disease and help clinicians identify patients with cone degenerations.
Archives of Ophthalmology 04/2008; 126(3):397-403. DOI:10.1001/archopht.126.3.397 · 4.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To screen superoxide dismutase 1 (SOD1) on chromosome 21 as a possible candidate gene for familial keratoconus (KC).
Total genomic DNA was extracted from the blood of 15 different KC families and 156 unaffected subjects. All five exons of the SOD1 gene were sequenced. For a rapid screening test, DNA was amplified by polymerase chain reaction (PCR), digested with HpyCH4 III or analyzed by radioactively end-labeled exon PCR. RNA was extracted from leukocytes and reverse transcribed to cDNA, and the PCR was amplified for splice variants. Some samples were cloned and sequenced.
A heterozygous genomic 7-base deletion in intron 2 of the SOD1 gene was identified in two KC families (pedigrees 1 and 6). The deletion segregated within pedigree 1 and was absent in 312 chromosomes from normal individuals. RNA from the proband of pedigree 1 showed that in addition to the wild-type transcript, two other transcripts were expressed for the CuZn SOD (SOD1) gene: lacking entire exon 2 (LE2) and lacking entire exon 2 and entire exon 3 (LE2E3).
A unique genomic deletion within intron 2 close to the 5' splice junction of the SOD1 gene was identified in three patients with KC. Moreover, mRNA from one affected individual also had two transcript splice variants (LE2 and LE2E3) that others have shown to code for proteins lacking the active site of the SOD1 enzyme. Further studies should be conducted to determine whether a causal relationship exists between these two events that may increase oxidative stress and be associated with KC.
[Show abstract][Hide abstract] ABSTRACT: To perform candidate gene screening for posterior polymorphous corneal dystrophy (PPCD). The initial 3 genes chosen, ID1, BCL2L1, and VSX1, lie within the region on chromosome 20 to which the PPCD gene has been linked, and mutations in VSX1 have previously been identified in patients with PPCD.
DNA extraction, PCR amplification, and direct sequencing of the VSX1, BCL2L1, and ID1 genes were performed in 14 affected patients (12 families) as well as in unaffected family members and healthy control subjects.
No coding region mutations in the BCL2L1 or ID1 genes were identified in affected patients. In the VSX1 gene, the previously identified Gly160Asp missense change was not present in any of our 12 probands, and the Asp144Glu mutation was identified in 1 affected patient as well as 1 unaffected control individual. Additionally, 2 synonymous substitutions were identified, Ala182Ala (8 affected patients from 8 families) and Gly239Gly (1 affected patient and 1 unaffected patient from the same family). In the ID1 gene, the synonymous substitution Gly216Gly was observed in 2 affected patients (2 families) who also demonstrated a single nucleotide change in both the 5'UTR (2129T>C) and 3'UTR (3267A>G). Another 5'UTR change, 2177T>C, was identified in 1 affected patient and his unaffected parent, both of whom also demonstrated the 2129T>C and 3267A>G changes.
None of the 12 probands with PPCD demonstrated the previously described Gly160Asp mutation within the VSX1 gene. The Asp144Glu missense change, present in an affected patient as well as an unaffected control individual, appears to be a rare polymorphism, not a disease-causing mutation. No coding region changes were identified in the ID1 or BCL2L1 genes. Therefore, although we report a number of novel polymorphisms in the VSX1 and ID1 genes, the failure to identify any sequence variants that sort with the disease phenotype suggests that other genetic factors are involved in PPCD.
[Show abstract][Hide abstract] ABSTRACT: To report a phenotypic variant of lattice corneal dystrophy associated with two missense changes, Ala546Asp and Pro551Gln, in the transforming growth factor-beta-induced gene (TGFBI).
Genomic DNA was obtained from the proband as well as affected and unaffected family members. Exons 4, 11, 12, and 14 of the TGFBI gene were amplified and sequenced. Additionally, a corneal button excised from the proband was examined by light and transmission electron microscopy. Haplotype analysis was performed on the proband's family and members of a previously identified pedigree with the same TGFBI gene missense changes.
Bilateral, symmetric, radially arranged, branching refractile lines within and surrounding an area of central anterior stromal haze were noted in the proband. Multiple polymorphic, refractile deposits were noted in the mid and posterior stroma in both the proband and her daughter. Light and electron microscopic analyses demonstrated amyloid and excluded the presence of deposits characteristic of granular corneal dystrophy. Screening of TGFBI exon 12 in the proband and her affected daughter revealed two missense changes, Ala546Asp and Pro551Gln (both absent in 250 control chromosomes). Haplotype analysis suggested that the mutations in this family and in a previously identified pedigree reflect a founder effect, rather than an independent occurrence.
We present a phenotypic variant of lattice corneal dystrophy associated with the Ala546Asp and Pro551Gln missense changes in exon 12 of the TGFBI gene. A common ancestor appears to account for the missense mutations observed in this pedigree and in a previously reported family.
American Journal of Ophthalmology 12/2004; 138(5):772-81. DOI:10.1016/j.ajo.2004.06.021 · 3.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To further characterize the mutations within the CHST6 gene responsible for causing macular corneal dystrophy in a cohort of affected patients from the United States.
Genomic DNA was extracted from buccal epithelium of 16 affected patients (14 families), 17 unaffected relatives, and 127 controls, followed by polymerase chain reaction amplification and direct sequencing of the CHST6 coding region. Subtyping of affected patients into type I and II macular corneal dystrophy was performed by measuring antigenic keratan sulfate (AgKS) serum levels. Haplotype analysis was performed in families that demonstrated common mutations.
CHST6 coding region analysis in 10 patients identified as having type I macular corneal dystrophy revealed 10 sequence changes: eight missense mutations, four of which are novel (Met104Val, Tyr110Cys, Gln122Pro, and Leu276Pro) and four of which have been reported previously (Ser51Leu, Pro72Ser, Cys102Gly, and Leu200Arg); one novel homozygous nonsense mutation in two patients from a single family (c. 1683C>T, Gln331X); and one frameshift mutation in a heterozygous state in a single patient (c.1744_1751dupGTGCGCTG). Mutation analysis in the four patients identified as having type II macular corneal dystrophy (serum samples were not obtained from two affected patients) revealed three patients heterozygous for either the c.923G>C, c.969C>A, or c.1519T>C sequence changes. The fourth patient was compound heterozygous for c.969C>A and c.1291T>G. None of these changes was observed in 127 control individuals. Haplotype analysis using microsatellite markers flanking the CHST6 gene did not reveal a common founder for the Leu200Arg (1291T>G) missense mutation, present in five families, identifying this position as a mutation hot-spot.
A variety of previously unreported mutations in the coding region of the CHST6 gene are associated with type I macular corneal dystrophy in a cohort of patients from the United States.
American Journal of Ophthalmology 03/2004; 137(3):465-73. DOI:10.1016/j.ajo.2003.09.036 · 3.87 Impact Factor