Identification of a novel splice-site mutation in the Lebercilin (LCA5) gene causing Leber congenital amaurosis

Purpose: Leber congenital amaurosis (LCA) is one of the most common causes of hereditary blindness in infants. To date, mutations in 13 known genes and at two other loci have been implicated in LCA causation. An examination of the known genes highlights several processes which, when defective, cause LCA, including photoreceptor development and maintenance, phototransduction, vitamin A metabolism, and protein trafficking. In addition, it has been known for some time that defects in sensory cilia can cause syndromes involving hereditary blindness. More recently evidence has come to light that non-syndromic LCA can also be a “ciliopathy.” Methods: Here we present a homozygosity mapping analysis in a consanguineous sibship that led to the identification of a mutation in the recently discovered LCA5 gene. Homozygosity mapping was done using Affymetrix 10K Xba I Gene Chip and a 24.5cM region on chromosome 6 (6q12- q16.3) was identified to be significantly homozygous. The LCA5 gene on this region was sequenced and cDNA sequencing also done to characterize the mutation. Results: A c.955G>A missense mutation in the last base of exon 6 causing disruption of the splice donor site was identified in both the affected sibs. Since there is a second consensus splice donor sequence 5 bp into the adjacent intron, this mutation results in a transcript with a 5 bp insertion of intronic sequence, leading to a frameshift and premature truncation. Conclusions: We report a missense mutation functionally altering the splice donor site and leading to a truncated protein. This is the second report of LCA5 mutations causing LCA. It may also be significant that one affected child died at eleven months of age due to asphyxia during sleep. To date the only phenotype unambiguously associated with mutations in this gene is LCA. However the LCA5 gene is known to be expressed in nasopharynx, trachea and lungs and was originally identified in the proteome of bronchial epithelium ciliary axonemes. The cause of death in this child may therefore imply that LCA5 mutations can in fact cause a wider spectrum of phenotypes including respiratory disease

Association of polymorphisms in the intron of TCF4 gene to late-onset Fuchs endothelial corneal dystrophy: An Indian cohort study

Purpose: Fuchs endothelial corneal dystrophy (FECD) is a progressive degenerative disease of the corneal endothelium. It is genetically heterogeneous and follows either an autosomal dominant or sporadic pattern of inheritance. Here, we have explored the association of four previously reported intronic single nucleotide polymorphisms and intronic CTG repeat expansions in TCF4 gene to FECD in an Indian cohort. Methods: The cohort consisting of 52 sporadic late-onset cases, 5 early-onset cases, and 148 controls was taken for the study. rs2286812 and rs613872 were genotyped by allele specific polymerase chain reaction (ASPCR) and PCR-based restriction digestion, respectively; rs17595731 and rs9954153 were genotyped by Taqman assay using real-time PCR. The quantitative assessment of the CTG repeat region was performed by PCR/Sanger DNA sequencing. The repeats were assessed qualitatively by short tandem repeat and triplet repeat primed PCR assays. The statistical analysis was performed using two-tailed Fisher's exact probability test. Results: SNPsrs613872 (G/T) for the 'G' allele (P value: 4.57 × 10−5) and rs17595731 (C/T) for the 'C' allele (P value: 1.87 × 10−5), respectively, showed a significant association to sporadic late-onset FECD. CTG repeat expansions were found to be associated with FECD with a P value = 2.4 × 10−3. Conclusion: rs613872, rs17595731, and CTG repeat expansions in intronic region of TCF4 are associated with increased risk of sporadic late-onset FECD in the Indian cohort studied

Molecular profiling of complete congenital stationary night blindness: a pilot study on an Indian cohort

International audiencePURPOSE:Congenital stationary night blindness (CSNB) is a non-progressive retinal disorder that shows genetic and clinical heterogeneity. CSNB is inherited as an autosomal recessive, autosomal dominant, or X-linked recessive trait and shows a good genotype-phenotype correlation. Clinically, CSNB is classified as the Riggs type and the Schubert-Bornschein type. The latter form is further sub-classified into complete and incomplete forms based on specific waveforms on the electroretinogram (ERG). There are no molecular genetic data for CSNB in the Indian population. Therefore, we present for the first time molecular profiling of eight families with complete CSNB (cCSNB).METHODS:The index patients and their other affected family members were comprehensively evaluated for the phenotype, including complete ophthalmic evaluation, ERG, fundus autofluorescence, optical coherence tomography, and color vision test. The known gene defects for cCSNB, LRIT3, TRPM1, GRM6, GPR179, and NYX, were screened by PCR direct sequencing. Bioinformatic analyses were performed using SIFT and PolyPhen for the identified missense mutations.RESULTS:All eight affected index patients and affected family members were identified as having cCSNB based on their ERG waveforms. Mutations in the TRPM1 gene were identified in six index patients. The two remaining index patients each carried a GPR179 and GRM6 mutation. Seven of the patients revealed homozygous mutations, while one patient showed a compound heterozygous mutation. Six of the eight mutations identified are novel.CONCLUSIONS:This is the first report on molecular profiling of candidate genes in CSNB in an Indian cohort. As shown for other cohorts, TRPM1 seems to be a major gene defect in patients with cCSNB in India

Homozygosity Mapping in Leber Congenital Amaurosis and Autosomal Recessive Retinitis Pigmentosa in South Indian Families

<div><p>Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP) are retinal degenerative diseases which cause severe retinal dystrophy affecting the photoreceptors. LCA is predominantly inherited as an autosomal recessive trait and contributes to 5% of all retinal dystrophies; whereas RP is inherited by all the Mendelian pattern of inheritance and both are leading causes of visual impairment in children and young adults. Homozygosity mapping is an efficient strategy for mapping both known and novel disease loci in recessive conditions, especially in a consanguineous mating, exploiting the fact that the regions adjacent to the disease locus will also be homozygous by descent in such inbred children. Here we have studied eleven consanguineous LCA and one autosomal recessive RP (arRP) south Indian families to know the prevalence of mutations in known genes and also to know the involvement of novel loci, if any. Complete ophthalmic examination was done for all the affected individuals including electroretinogram, fundus photograph, fundus autofluorescence, and optical coherence tomography. Homozygosity mapping using Affymetrix 250K HMA GeneChip on eleven LCA families followed by screening of candidate gene(s) in the homozygous block identified mutations in ten families; <i>AIPL1</i> – 3 families, <i>RPE65</i>- 2 families, <i>GUCY2D</i>, <i>CRB1</i>, <i>RDH12</i>, <i>IQCB1</i> and <i>SPATA7</i> in one family each, respectively. Six of the ten (60%) mutations identified are novel. Homozygosity mapping using Affymetrix 10K HMA GeneChip on the arRP family identified a novel nonsense mutation in <i>MERTK</i>. The mutations segregated within the family and was absent in 200 control chromosomes screened. In one of the eleven LCA families, the causative gene/mutation was not identified but many homozygous blocks were noted indicating that a possible novel locus/gene might be involved. The genotype and phenotype features, especially the fundus changes for <i>AIPL1</i>, <i>RPE65</i>, <i>CRB1</i>, <i>RDH12</i> genes were as reported earlier.</p></div

Fundus photographs.

<p>Fig 3a A 10yrs old female with c.824G>A p.(Trp278*) mutation in <i>AIPL1</i> (LCA-5 family) showed normal disc, attenuated vessels, (arrow mark indicates) yellow patches in macula. Fig 3b A 14yrs old male with c.824G>A p.(Trp278*) mutation in <i>AIPL1</i> (LCA-5 family, elder sibling) showed normal disc, attenuated vessels, (arrow mark indicates) black pigments in macula. Fig 3c A 18 yrs old female with c.850+1G>T (r.spl?) mutation in <i>RPE65</i> (LCA-1 family) showed pallor disc, attenuated vessels with scar in the macula, peripheral RPE mottling (marked with arrow) Fig 3d A 28yrs old male with c.1409C>T p.(Pro470Leu) mutation in <i>RPE65</i> (LCA-9 family) showed pallor disc, attenuated vessels, normal macula, with salt and pepper fundus. Arrow mark shows distinct pin head size yellow white dot like spots at the posterior pole. Fig 3e A 14 yrs old female with c.2971G>A p.(Gly991Arg) mutation in <i>CRB1</i> (LCA-2 family) showed coin shaped pigment clumps and greyish atrophic changes seen in the macula, (arrow mark indicates the macula) Fig 3f A 18 yrs old female with c.2971G>A p.(Gly991Arg) mutation in <i>CRB1</i> (LCA-2 family, elder sibling) showed pale disc, attenuated vessels, atrophic macula with nummular pigment clumps and greyish atrophic reflex (arrow mark indicates the macula) Fig 3g A 19yrs old male female with c.2971G>A p.(Gly991Arg) mutation in <i>CRB1</i> (LCA-2 family, eldest sibling) showed coin shaped pigment clumps seen in the background (arrow mark indicates the coin shaped clumps) All the three affected siblings show progressive changes in macula with age for <i>CRB1</i> mutation positive family. Fig 3h, 3i, 3j A 24 yrs old female, a 25 yrs old female and a 32 yrs old female with c.721C>T p.(Gln 241*) mutation in <i>MERTK</i> (arRP1 family) showing mild, milder and marked features of RP, respectively. Progressive changes with age in the macula are observed.</p

Total number of homozygous blocks ≥ 1Mb in the LCA and arRP families.

<p>Total number of homozygous blocks ≥ 1Mb in the LCA and arRP families.</p

Segregation analysis.

<p>1a:arRP1 <i>MERTK</i> c.721C>T, 1b:LCA-1 <i>RPE65</i> c.850+1G>T, 1c: LCA-2 <i>CRB1</i> c.3307G>A, 1d:LCA-3 <i>GUCY2D</i> c.994delC, 1e:LCA-4 <i>IQCB1</i> c.1278+6T>A, 1f:LCA-5 <i>AIPL1</i> c.824G>A, 1g: LCA-7 <i>RDH12</i> c.344-8C>T, 1h:LCA-8 <i>AIPL1</i> c.247G>A, 1i:LCA-9 <i>RPE65</i> c.1409C>T, 1j:LCA-10 <i>AIPL1</i> c.613_622 delATCATCTGCC, 1k:LCA-11 <i>SPATA7</i> c.913-2A>G. The arrow indicates the index case. The filled in circles and squares are affected females and males respectively. [M];[M]–affected with homozygous mutation, [M]; [=] –carries for any given mutation and [=]; [=] –wild type. Lines above the individual indicate availability of genotype.</p

2% Agarose gel electrophoresis showing cDNA amplification of exon 11–13 of <i>IQCB1</i>.

<p>Lane 1-100bp ladder, Lane 3- Affected index case, Lane 5 & 7—Carrier parents, Lane 9—Control, Lane 2, 4, 6, 8—empty wells Fig 2b Eletrophoretogram trace showing the amplified cDNA of control and proband. In proband exon 11 is followed by exon 13 and exon 12 is completely deleted, whereas in control, exon 11, 12 and 13 is continuous. The end of exon 11 is marked in both the phoretograms.</p

Mutations identified in LCA families and the arRP family.

<p>Mutations identified in LCA families and the arRP family.</p