Utility of whole-exome sequencing for those near the end of the diagnostic odyssey: time to address gaps in care.

An accurate diagnosis is an integral component of patient care for children with rare genetic disease. Recent advances in sequencing, in particular whole-exome sequencing (WES), are identifying the genetic basis of disease for 25-40% of patients. The diagnostic rate is probably influenced by when in the diagnostic process WES is used. The Finding Of Rare Disease GEnes (FORGE) Canada project was a nation-wide effort to identify mutations for childhood-onset disorders using WES. Most children enrolled in the FORGE project were toward the end of the diagnostic odyssey. The two primary outcomes of FORGE were novel gene discovery and the identification of mutations in genes known to cause disease. In the latter instance, WES identified mutations in known disease genes for 105 of 362 families studied (29%), thereby informing the impact of WES in the setting of the diagnostic odyssey. Our analysis of this dataset showed that these known disease genes were not identified prior to WES enrollment for two key reasons: genetic heterogeneity associated with a clinical diagnosis and atypical presentation of known, clinically recognized diseases. What is becoming increasingly clear is that WES will be paradigm altering for patients and families with rare genetic diseases.

Identification of the gene for Nance-Horan syndrome (NHS).

BACKGROUND: The disease intervals for Nance-Horan syndrome (NHS [MIM 302350]) and X linked congenital cataract (CXN) overlap on Xp22. OBJECTIVE: To identify the gene or genes responsible for these diseases. METHODS: Families with NHS were ascertained. The refined locus for CXN was used to focus the search for candidate genes, which were screened by polymerase chain reaction and direct sequencing of potential exons and intron-exon splice sites. Genomic structures and homologies were determined using bioinformatics. Expression studies were undertaken using specific exonic primers to amplify human fetal cDNA and mouse RNA. RESULTS: A novel gene NHS, with no known function, was identified as causative for NHS. Protein truncating mutations were detected in all three NHS pedigrees, but no mutation was identified in a CXN family, raising the possibility that NHS and CXN may not be allelic. The NHS gene forms a new gene family with a closely related novel gene NHS-Like1 (NHSL1). NHS and NHSL1 lie in paralogous duplicated chromosomal intervals on Xp22 and 6q24, and NHSL1 is more broadly expressed than NHS in human fetal tissues. CONCLUSIONS: This study reports the independent identification of the gene causative for Nance-Horan syndrome and extends the number of mutations identified.

The phenotype of normal tension glaucoma patients with and without OPA1 polymorphisms.

AIM: Polymorphisms in OPA1, the gene responsible for autosomal dominant optic atrophy, were recently found to be strongly associated with normal tension glaucoma (NTG). The aim of this study was to determine whether OPA1 polymorphisms affect the phenotype of NTG patients. METHODS: A retrospective analysis was performed of 108 well characterised NTG patients who had been genotyped for OPA1 variations, and who had previously undergone automated perimetry and Heidelberg retina tomography (HRT). 25 NTG patients had the at-risk OPA1 genotype (IVS 8 +4 C/T; +32 T/C) and 83 NTG patients did not. Differences between groups were sought in a wide range of structural, psychophysical, and demographic factors. These included sex, age at diagnosis, family history of glaucoma, history of ischaemic risk factors and vasospasm, laterality of glaucoma, presenting and highest diurnal intraocular pressure (IOP), initial cup-disc (CD) ratio, baseline visual field global indices, and optic disc parameters as measured by HRT. For a subgroup of patients with at least 5 years of follow up and 10 visual field tests, pointwise linear regression analysis (PROGRESSOR for Windows software) was applied to the visual field series. RESULTS: There was no significant difference in the two groups with respect to sex, age at diagnosis, family history of glaucoma, history of ischaemic risk factors and vasospasm, or laterality of glaucoma. The comparison of IOP, CD ratio and visual field global indices, MD and CPSD in the two groups showed no significant difference. There were no differences in the mean values for any of the HRT parameters analysed. For the subgroup of patients with at least 5 years of follow up, there was also no significant difference in the number of patients with progressing locations, the mean number of progressing locations per subject, the mean slope of the progressing locations or the mean slope for whole visual field. CONCLUSIONS: The absence of phenotypic differences in normal tension glaucoma patients with and without the OPA1 polymorphisms IVS 8 +4 C/T; +32 T/C suggest that these OPA1 polymorphisms do not underlie any major phenotypic diversity in these patients.

A novel keratocan mutation causing autosomal recessive cornea plana.

PURPOSE: Mutations in keratocan (KERA), a small leucine-rich proteoglycan, have recently been shown to be responsible for cases of autosomal recessive cornea plana (CNA2). A consanguineous pedigree in which cornea plana cosegregated with microphthalmia was investigated by linkage analysis and direct sequencing. METHODS: Linkage was sought to polymorphic microsatellite markers distributed around the CNA2 and microphthalmia loci (arCMIC, adCMIC, NNO1, and CHX10) using PCR and nondenaturing polyacrylamide gel electrophoresis before KERA was directly sequenced for mutations. RESULTS: Positive lod scores were obtained with markers encompassing the CNA2 locus, the maximum two-point lod scores of 2.18 at recombination fraction theta = 0 was obtained with markers D12S95 and D12S327. Mutation screening of KERA revealed a novel single-nucleotide substitution at codon 215, which results in the substitution of lysine for threonine at the start of a highly conserved leucine-rich repeat motif. Structural modeling predicts that the motifs are stacked into an arched beta-sheet array and that the effect of the mutation is to alter the length and position of one of these motifs. CONCLUSIONS: This report describes a novel mutation in KERA that alters a highly conserved motif and is predicted to affect the tertiary structure of the molecule. Normal corneal function is dependent on the regular spacing of collagen fibrils, and the predicted alteration of the tertiary structure of KERA is the probable mechanism of the cornea plana phenotype.

Risk factors for development of post-trabeculectomy endophthalmitis.

BACKGROUND/AIMS: Although adjunctive use of antiproliferative agents improves the success rate of glaucoma filtration surgery it profoundly alters the morphology of the filtering bleb. In view of these structural changes, which have been suggested to predispose to bleb infection, the relative importance of potential risk factors in the development of post-trabeculectomy endophthalmitis was investigated. METHODS: A case-control study was performed on patients with post-trabeculectomy endophthalmitis presenting to a single academic centre over a 6 1/2 year period. Cases were diagnosed by the combination of vitreous and aqueous inflammation occurring 4 or more weeks postoperatively with control patients chosen by selecting the three patients undergoing trabeculectomy immediately following each index case. RESULTS: Analysis of these data, derived from 23 cases and 69 controls, demonstrated that an episode of blebitis and the presence of diabetes mellitus were statistically significantly associated with subsequent endophthalmitis (odds ratios (OR) 11.8, 95% CI: 2.21-88.31, p = 0.003 and OR 4.51, 95 % CI 1.02-20.29, p = 0.04 respectively). The data also suggest an association exists between antiproliferative use and endophthalmitis (OR 3.3, 95% CI 0.95-15.19, p = 0.07) as the time interval between filtration surgery and development of endophthalmitis was significantly shorter in patients treated with antiproliferative agents (p = 0.001). CONCLUSIONS: These results provide strong evidence of an increased risk of late endophthalmitis in patients who have diabetes mellitus or have had an episode of blebitis and suggest antiproliferative agents may also have an important role.

Chromosomal duplication involving the forkhead transcription factor gene FOXC1 causes iris hypoplasia and glaucoma.

The forkhead transcription factor gene FOXC1 (formerly FKHL7) is responsible for a number of glaucoma phenotypes in families in which the disease maps to 6p25, although mutations have not been found in all families in which the disease maps to this region. In a large pedigree with iris hypoplasia and glaucoma mapping to 6p25 (peak LOD score 6.20 [recombination fraction 0] at D6S967), no FOXC1 mutations were detected by direct sequencing. However, genotyping with microsatellite repeat markers suggested the presence of a chromosomal duplication that segregated with the disease phenotype. The duplication was confirmed in affected individuals by FISH with markers encompassing FOXC1. These results provide evidence of gene duplication causing developmental disease in humans, with increased gene dosage of either FOXC1 or other, as yet unknown genes within the duplicated segment being the probable mechanism responsible for the phenotype.

Investigation of beta defensin gene expression in the ocular anterior segment by semiquantitative RT-PCR.

AIM: To determine if beta defensins are expressed in the anterior segment of the eye and to determine the temporal pattern of expression using a real time semiquantitative reverse transcription polymerase chain reaction (RT-PCR). METHODS: Ocular tissue (corneal epithelium, conjunctiva, iris, and lens capsule) was collected from 23 patients undergoing surgery. Serial corneal or conjunctival impression cytology was performed on a separate group of 10 patients undergoing corneal tunnel phacoemulsification or trabeculectomy. The samples were analysed for beta defensin mRNA by semiquantitative RT-PCR and the mRNA standardised for cell numbers. RESULTS: RT-PCR amplified beta defensin 1 mRNA from all lens capsule (six) and corneal (five) samples and all but one of the conjunctival (six) and iris samples (six). beta Defensin 2 mRNA was amplified from three of five corneal, two of six conjunctival, and none of the iris or capsule samples. The impression cytology samples demonstrated a decline in defensin expression over the three time points studied. There were no false positive results from either the no-RT or negative control samples. CONCLUSIONS: This preliminary study confirms that natural antibacterial peptides are expressed in the anterior segment of the eye. There appears to be a pattern to the expression with inducible beta defensin 2 not expressed intraocularly and higher levels of beta defensin 1 than beta defensin 2 expressed in extraocular tissue. The implication is that beta defensin 1 is constitutively produced in ocular tissues and represents a key component of the innate immune system.

Polymerase chain reaction analysis of corneal epithelial and tear samples in the diagnosis of Acanthamoeba keratitis.

PURPOSE: Acanthamoeba is an uncommon cause of corneal infection in which the best visual outcome follows prompt diagnosis and a long course of appropriate antimicrobial therapy. Because conventional detection techniques for Acanthamoeba have certain limitations, we investigated the ability of the polymerase chain reaction (PCR) to confirm the clinical diagnosis of Acanthamoeba keratitis, with the ultimate aim of achieving early diagnosis. METHODS: Using two different pairs of primers, PCR was performed on representative cultured Acanthamoeba isolates to confirm the assay's ability to amplify Acanthamoeba DNA from a wide range of acanthamoebae. Subsequently, corneal epithelial samples from 19 patients and tear samples from 12 patients with Acanthamoeba keratitis were analyzed by PCR for the presence of Acanthamoeba DNA. RESULTS: Acanthamoeba DNA was amplified by PCR from 16 (84%) of 19 corneal epithelial samples, whereas Acanthamoeba was cultured from 10 samples (53%), all of which were PCR positive. Tear samples from 8 (66%) of 12 patients were positive on PCR testing, and one tear sample was PCR positive, whereas the corresponding epithelial biopsy had yielded a negative PCR result. Samples from culture-positive patients were positive on PCR testing more frequently than those from culture-negative patients (10/10 culture-positive corneal epithelial and 5/7 [71%] culture-positive initial tear samples versus 6/9 [66%] culture-negative corneal epithelial and 2/5 [40%] culture-negative tear samples). All control epithelial (n = 15) and tear (n = 15) samples yielded negative results. CONCLUSIONS: PCR was a more sensitive diagnostic test than a culture for Acanthamoeba keratitis, and the use of two different primers achieved better sensitivity than a single set. A PCR of a tear sample also may be a useful complementary test and, in combination with PCR of epithelial samples, would prove particularly helpful in confirming the clinical diagnosis in culture-negative cases.

Acanthamoeba keratitis: multicentre survey in England 1992-6. National Acanthamoeba Keratitis Study Group.

AIM: To investigate the frequency, outcomes, and risk factors for acanthamoeba keratitis (AK) in England during the past 4 years. METHODS: An ophthalmologist in 12 of the 14 regional health authorities (RHAs) coordinated identification of patients in their region presenting with AK between 1 October 1992 and 30 September 1996. Clinical and postal patient questionnaire data were analysed. RESULTS: 243 patients (259 eyes) with an AK diagnosis were identified, equating to an annualised incidence of 0.14 per 100,000 individuals. UK resident patients for each year numbered 50, 71, 73, and 32 respectively. Among patients with sufficient data 170/237 (72%) were diagnosed early (within 30 days of presentation), 197/218 (90%) were treated with polyhexamethyl biguanide and/or chlorhexidine, and 40/243 (16%) underwent surgery. Visual acuities of 6/12 or better were achieved by 222/259 (86%) eyes, including 84 eyes of patients under review or lost to follow up. Non-contact lens (CL) wearers were associated with delayed diagnosis, increased need for surgery and a poorer visual outcome (only 10/18 eyes, 56%, achieved 6/12 acuity). 225/243 (93%) patients were CL wearers, and 205/243 (84%) were soft CL (SCL) users. Among SCL user respondents, previously identified risk factors--swimming with CL (47/138, 34%), non-sterile CL rinsing (11/138, 8%), omitted disinfection (85/138, 62%), and chlorine release disinfection (65/138, 47%)--were identified for 125/138 (91%) patients. CONCLUSIONS: Earlier diagnosis and more effective medical therapy have improved the prognosis for most AK patients. The study demonstrates the highly preventable nature of the disease: 91% of the SCL wearers could have avoided the disease by refraining from inadvisable practices, and a marked fall in frequency was seen after intensive media attention to AK, possibly in conjunction with increasing penetrance of new CL products. Since the frequency of AK appears to be largely determined by the ever changing trends in CL use, continued monitoring is indicated.

Association between nonadministration of subconjunctival cefuroxime and postoperative endophthalmitis.

PURPOSE: To determine whether not administering subconjunctival cefuroxime during cataract surgery is associated with postoperative endophthalmitis. SETTING: Southampton Eye Unit, Southampton General Hospital, England. METHODS: This retrospective, case-control study comprised nine patients who developed endophthalmitis after cataract surgery in a single ophthalmic unit over a 21 month period. Ten control patients for each case were randomly chosen from patients having cataract surgery within 1 week of the endophthalmitis case. RESULTS: None of the nine endophthalmitis patients received peroperative subconjunctival cefuroxime compared with 43 of 90 control patients (47.8%) (P = 0.008). No other variables were found to be associated with development of endophthalmitis in this study. CONCLUSION: Nonadministration of subconjunctival cefuroxime was associated with subsequent endophthalmitis. A further study to determine whether the observed association is causal is therefore warranted.

Half-life of intracameral gentamicin after phacoemulsification.

PURPOSE: To determine the half-life of intracameral gentamicin administered during phacoemulsification. SETTING: Southampton Eye Unit, Southampton General Hospital, England. METHODS: Thirty-one patients having scleral tunnel phacoemulsification were given intracameral gentamicin in the irrigation fluid. Samples of fluid were taken from the anterior chamber at the end of the operation and at various times postoperatively. The concentration of gentamicin in the samples was determined by fluorescence polarization immunoassay and the half-life calculated for a single compartment model using a peeling algorithm. RESULTS: The concentration of gentamicin in the anterior chamber after phacoemulsification decreased by half every 51 minutes (95% confidence interval, 42 to 66 minutes). CONCLUSION: Intracameral gentamicin was cleared from the anterior chamber after phacoemulsification at a rate that prevents the maintainance of the bactericidal levels required for reliable antibiotic prophylaxis.

Multipoint linkage analysis of the short arm of chromosome 11 in non-insulin dependent diabetes including maturity onset diabetes of youth.

Members of three families with maturity onset diabetes of youth (MODY) and seven with "common" type 2 diabetes were typed for six DNA markers (H-RAS, INS, HBBC, PTH, CALC1, CAT) on the short arm of chromosome 11. Using conventional pairwise linkage analysis, close linkage in the MODY families was excluded for all six markers. By multipoint analysis and a genetic map of the short arm of chromosome 11, MODY was excluded from a region of at least 35 and up to 60 centiMorgans (cM) on the short arm of chromosome 11. Multipoint analysis in the type 2 families also excludes linkage to the INS, H-RAS region of at least 3 and up to 30 cM. This study using multipoint linkage analysis in non-insulin dependent diabetes provides strong evidence against a role for mutations in or around the insulin gene in the causation of MODY or type 2 diabetes in the families studied.

Regional localization of the autosomal dominant polycystic kidney disease locus.

The localization of the autosomal dominant polycystic kidney disease locus (PKD1) within an array of anonymous polymorphic DNA sequences on chromosome 16 band p13 was determined by multipoint mapping. Nine polymorphic DNA markers, including two hypervariable sequences, were used to study 19 PKD1 and 21 reference families. PKD1 was found to lie proximal to the 3' and 5' hypervariable regions of alpha-globin and distal to the anonymous sequence CRI-0327. Somatic cell hybrid mapping places PKD1 within the region 16p13.11-16pter. The availability of an array of linked markers which bracket the PKD1 locus provides a framework for further attempts to identify the PKD1 gene and offers an improved method of presymptomatic diagnosis of the disease.

Mutations for the autosomal recessive and autosomal dominant forms of polycystic kidney disease are not allelic.

The autosomal dominant form of polycystic kidney disease (ADPKD) has been linked to the alpha-globin gene locus on 16p. Linkage studies between the autosomal recessive type (ARPKD) and the 3' HVR of the alpha-globin gene cluster showed that the ARPKD and ADPKD are not allelic.