Lessons learned from additional research analyses of unsolved clinical exome cases.

BACKGROUND: Given the rarity of most single-gene Mendelian disorders, concerted efforts of data exchange between clinical and scientific communities are critical to optimize molecular diagnosis and novel disease gene discovery. METHODS: We designed and implemented protocols for the study of cases for which a plausible molecular diagnosis was not achieved in a clinical genomics diagnostic laboratory (i.e. unsolved clinical exomes). Such cases were recruited to a research laboratory for further analyses, in order to potentially: (1) accelerate novel disease gene discovery; (2) increase the molecular diagnostic yield of whole exome sequencing (WES); and (3) gain insight into the genetic mechanisms of disease. Pilot project data included 74 families, consisting mostly of parent-offspring trios. Analyses performed on a research basis employed both WES from additional family members and complementary bioinformatics approaches and protocols. RESULTS: Analysis of all possible modes of Mendelian inheritance, focusing on both single nucleotide variants (SNV) and copy number variant (CNV) alleles, yielded a likely contributory variant in 36% (27/74) of cases. If one includes candidate genes with variants identified within a single family, a potential contributory variant was identified in a total of ~51% (38/74) of cases enrolled in this pilot study. The molecular diagnosis was achieved in 30/63 trios (47.6%). Besides this, the analysis workflow yielded evidence for pathogenic variants in disease-associated genes in 4/6 singleton cases (66.6%), 1/1 multiplex family involving three affected siblings, and 3/4 (75%) quartet families. Both the analytical pipeline and the collaborative efforts between the diagnostic and research laboratories provided insights that allowed recent disease gene discoveries (PURA, TANGO2, EMC1, GNB5, ATAD3A, and MIPEP) and increased the number of novel genes, defined in this study as genes identified in more than one family (DHX30 and EBF3). CONCLUSION: An efficient genomics pipeline in which clinical sequencing in a diagnostic laboratory is followed by the detailed reanalysis of unsolved cases in a research environment, supplemented with WES data from additional family members, and subject to adjuvant bioinformatics analyses including relaxed variant filtering parameters in informatics pipelines, can enhance the molecular diagnostic yield and provide mechanistic insights into Mendelian disorders. Implementing these approaches requires collaborative clinical molecular diagnostic and research efforts.

An exome sequencing study of Moebius syndrome including atypical cases reveals an individual with CFEOM3A and a TUBB3 mutation.

Moebius syndrome is characterized by congenital unilateral or bilateral facial and abducens nerve palsies (sixth and seventh cranial nerves) causing facial weakness, feeding difficulties, and restricted ocular movements. Abnormalities of the chest wall such as Poland anomaly and variable limb defects are frequently associated with this syndrome. Most cases are isolated; however, rare families with autosomal dominant transmission with incomplete penetrance and variable expressivity have been described. The genetic basis of this condition remains unknown. In a cohort study of nine individuals suspected to have Moebius syndrome (six typical, three atypical), we performed whole-exome sequencing to try to identify a commonly mutated gene. Although no such gene was identified and we did not find mutations in PLXND1 and REV3L, we found a de novo heterozygous mutation, p.E410K, in the gene encoding tubulin beta 3 class III (TUBB3), in an individual with atypical Moebius syndrome. This individual was diagnosed with near-complete ophthalmoplegia, agenesis of the corpus callosum, and absence of the septum pellucidum. No substantial limb abnormalities were noted. Mutations in TUBB3 have been associated with complex cortical dysplasia and other brain malformations and congenital fibrosis of extraocular muscles type 3A (CFEOM3A). Our report highlights the overlap of genetic etiology and clinical differences between CFEOM and Moebius syndrome and describes our approach to identifying candidate genes for typical and atypical Moebius syndrome.

Recurrent De Novo and Biallelic Variation of ATAD3A, Encoding a Mitochondrial Membrane Protein, Results in Distinct Neurological Syndromes.

ATPase family AAA-domain containing protein 3A (ATAD3A) is a nuclear-encoded mitochondrial membrane protein implicated in mitochondrial dynamics, nucleoid organization, protein translation, cell growth, and cholesterol metabolism. We identified a recurrent de novo ATAD3A c.1582C>T (p.Arg528Trp) variant by whole-exome sequencing (WES) in five unrelated individuals with a core phenotype of global developmental delay, hypotonia, optic atrophy, axonal neuropathy, and hypertrophic cardiomyopathy. We also describe two families with biallelic variants in ATAD3A, including a homozygous variant in two siblings, and biallelic ATAD3A deletions mediated by nonallelic homologous recombination (NAHR) between ATAD3A and gene family members ATAD3B and ATAD3C. Tissue-specific overexpression of borR534W, the Drosophila mutation homologous to the human c.1582C>T (p.Arg528Trp) variant, resulted in a dramatic decrease in mitochondrial content, aberrant mitochondrial morphology, and increased autophagy. Homozygous null bor larvae showed a significant decrease of mitochondria, while overexpression of borWT resulted in larger, elongated mitochondria. Finally, fibroblasts of an affected individual exhibited increased mitophagy. We conclude that the p.Arg528Trp variant functions through a dominant-negative mechanism that results in small mitochondria that trigger mitophagy, resulting in a reduction in mitochondrial content. ATAD3A variation represents an additional link between mitochondrial dynamics and recognizable neurological syndromes, as seen with MFN2, OPA1, DNM1L, and STAT2 mutations.

Novel Mutations in Two Saudi Patients with Congenital Retinal Dystrophy.

UNLABELLED: To report novel mutations in two Saudi children with clinical features of Leber congenital amaurosis (LCA) and Alström syndrome. CASE REPORTS: Case 1 was a child with phenotypic features of LCA including oculodigital sign, bilateral enophthalmos, nystagmus, pale disc, and retinal changes. Direct sequencing of the coding sequence of GUCY2D revealed a missense mutation affecting highly conserved position (c. 743C > T; p.S248 L). Case 2 describes a girl with marked nystagmus, photophobia, and retinal changes in both eyes with short and stubby fingers tapering at the distal phalanges. The electroretinograms were nonrecordable in each eye. She had a hearing aid in the left ear, mid-facial hypoplasia, bilateral enophthalmos, and insulin dependent diabetes. Mutation screening of candidates genes revealed a pathogenic mutation in ALMS1 gene (c. 8441C > A, p.S2814). Two novel mutations causing phenotypic LCA and Alström syndrome in Saudi patients from consanguineous families expand the genotypic spectrum of congenital retinal dystrophies.

Risk of cataract in persons with cytomegalovirus retinitis and the acquired immune deficiency syndrome.

OBJECTIVE: To evaluate cataract risk in eyes of patients with AIDS and cytomegalovirus (CMV) retinitis and to identify risk factors. DESIGN: Prospective cohort study. PARTICIPANTS: Patients with AIDS and CMV retinitis. METHODS: Patients 13 years of age and older were enrolled between 1998 and 2008. Demographic and clinical characteristics, slit-lamp biomicroscopy findings, and dilated ophthalmoscopy results were documented at quarterly visits. Cataract status was determined at the initial visit (prevalence) and at follow-up visits (incidence). MAIN OUTCOME MEASURES: For cataract, a high grade of lens opacity by biomicroscopy to which best-corrected visual acuity worse than 20/40 was attributed. Eyes that had undergone cataract surgery before enrollment or between visits also were counted as having cataract. RESULTS: Seven hundred twenty-nine eyes of 489 patients diagnosed with CMV retinitis were evaluated. Higher prevalence was observed for patients with bilateral versus unilateral CMV retinitis (adjusted odds ratio [aOR], 2.74; 95% confidence interval [CI], 1.76-4.26) and, among unilateral CMV retinitis cases, for eyes with retinitis versus without retinitis (15% vs. 1.4%; P<0.0001). The age-adjusted prevalence of cataract among CMV retinitis cases was higher than that in a population-based sample (P<0.0001). Cataract prevalence increased with age (aOR, 11.77; 95% CI, 2.28-60.65 for age ≥ 60 years vs. younger than 40 years) and longer duration of retinitis (aOR, 1.36; 95% CI, 1.20-1.54 per year). Among eyes with CMV retinitis initially free of cataract, the cataract incidence was 8.1%/eye-year (95% CI, 6.7%-10.0%). Prior retinal detachment was associated with higher cataract risk (if repaired with silicone oil: adjusted hazard ratio [aHR], 10.37; 95% CI, 6.51-16.52; otherwise: aHR, 2.90; 95% CI, 1.73-4.87). Large CMV retinitis lesions also were associated with higher risk of cataract (for involvement of 25-49% retinal area: aHR, 2.30; 95% CI, 1.51-3.50; for ≥ 50% involvement: aHR, 3.63; 95% CI, 2.18-6.04), each with respect to ≤ 24% involvement, as were anterior segment inflammation (aHR, 2.27; 95% CI, 1.59-3.25) and contralateral cataract (aHR, 2.52; 95% CI, 1.74-3.66). CONCLUSIONS: Cytomegalovirus retinitis is associated with a high absolute and relative risk of cataract. Among several risk factors, large retinal lesion size and use of silicone oil in retinal detachment repair are potentially modifiable, albeit not in all cases. Cataract is likely to be an increasingly important cause of visual morbidity in this population.

Early-onset severe neuromuscular phenotype associated with compound heterozygosity for OPA1 mutations.

INTRODUCTION: Pathogenic mutations in the OPA1 gene are the most common identifiable cause of autosomal dominant optic atrophy (DOA), which is characterized by selective retinal ganglion cell loss, a distinctive pattern of temporal pallor of the optic nerve and a typical color vision deficit, with variable effects on visual acuity. Haploinsufficiency has been suggested as the major pathogenic mechanism for DOA. Here we present two siblings with severe ataxia, hypotonia, gastrointestinal dysmotility, dysphagia, and severe, early-onset optic atrophy who were found to be compound heterozygotes for two pathogenic OPA1 mutations. This example expands the clinical phenotype of OPA1-associated disorders and provides additional evidence for semi-dominant inheritance. METHODS AND RESULTS: Molecular analysis of the OPA1 gene in this family by Sanger sequencing revealed compound heterozygosity for two mutations in trans configuration, a p.I382 M missense mutation and a p.V903GfsX3 frameshift deletion in both affected siblings. Electron microscopy of a skeletal muscle biopsy of the older sibling revealed dense osmiophilic bodies within the mitochondria. Mitochondrial DNA (mtDNA) content was within normal limits, and electron transport chain analysis showed no deficiencies of the mitochondrial respiratory chain enzymes. Multiple mtDNA deletions were not found. CONCLUSION: Compound heterozygosity of pathogenic OPA1 mutations may cause severe neuromuscular phenotypes in addition to early-onset optic atrophy. While a role for OPA1 in mtDNA maintenance has been discussed, compound biallelic pathogenic OPA1 mutations in our patients did not result in altered mtDNA copy number, mtDNA deletions, or deficiencies of the electron transport chain, despite the severe clinical phenotype.

SMAD4 mutation segregating in a family with juvenile polyposis, aortopathy, and mitral valve dysfunction.

Juvenile polyposis syndrome (JPS) is caused by heterozygous mutations in either SMAD4 or BMPR1A. Individuals with JPS due to mutations in SMAD4 are at greater risk to manifest signs of hereditary hemorrhagic telangiectasia (HHT). HHT is caused by either mutations in SMAD4 or other genes that modulate transforming growth factor-beta (TGFß) signaling. Additional genes in the TGFß network include FBN1, TGFBR1, and TGFBR2, mutations of which cause either Marfan syndrome (MFS) or Loeys-Dietz syndrome (LDS), respectively. As SMAD4, FBN1, and TGFBR1/2 map to different regions of the genome, disorders associated with mutations in these genes are not expected to co-segregate in a family. We report an individual whose family history was positive for aortopathy, mitral valve dysfunction, and JPS. Mutation analysis of SMAD4 implicates this gene for these phenotypes in this family. Although SMAD4 is among several genes in the TGFß network, and although prior single case reports have described large vessel aneurysms in HHT, this is the first description of aortic and mitral disease presenting with JPS. This observation suggests that, in addition to HHT, individuals with SMAD4 mutations may be at risk for aortic dilation and mitral valve dysfunction. We emphasize the importance of comprehensive review of the medical history prior to molecular testing, especially in an asymptomatic patient.

Potential involvement of more than one locus in trait manifestation for individuals with Leber congenital amaurosis.

Leber congenital amaurosis (LCA) is a clinically and genetically heterogeneous retinal dystrophy. The causes of LCA have been unraveled partially at the molecular level. At least 14 genes have been reported that, when mutated, result in LCA. To understand the roles of the known genes in LCA, a group of outbred subjects from 60 apparently either recessive families, with one or more affected individuals, or isolated patients were evaluated. One affected individual from each family underwent comprehensive mutational analysis by direct DNA sequencing of all coding regions and splice junctions of 13 LCA genes. Mutations were identified in 70% of individuals. CEP290 made the largest contribution to the identified mutations, providing 43% of those mutant alleles. We identified seven families in which affected individuals with two mutant alleles, sufficient to cause disease, had an additional mutation at a second LCA locus. Our findings suggest that mutational load can be important to penetrance of the LCA phenotype.

Exome capture sequencing identifies a novel mutation in BBS4.

PURPOSE: Leber congenital amaurosis (LCA) is one of the most severe eye dystrophies characterized by severe vision loss at an early stage and accounts for approximately 5% of all retinal dystrophies. The purpose of this study was to identify a novel LCA disease allele or gene and to develop an approach combining genetic mapping with whole exome sequencing. METHODS: Three patients from King Khaled Eye Specialist Hospital (KKESH205) underwent whole genome single nucleotide polymorphism genotyping, and a single candidate region was identified. Taking advantage of next-generation high-throughput DNA sequencing technologies, whole exome capture sequencing was performed on patient KKESH205#7. Sanger direct sequencing was used during the validation step. The zebrafish model was used to examine the function of the mutant allele. RESULTS: A novel missense mutation in Bardet-Biedl syndrome 4 protein (BBS4) was identified in a consanguineous family from Saudi Arabia. This missense mutation in the fifth exon (c.253G>C;p.E85Q) of BBS4 is likely a disease-causing mutation as it segregates with the disease. The mutation is not found in the single nucleotide polymorphism (SNP) database, the 1000 Genomes Project, or matching normal controls. Functional analysis of this mutation in zebrafish indicates that the G253C allele is pathogenic. Coinjection of the G253C allele cannot rescue the mislocalization of rhodopsin in the retina when BBS4 is knocked down by morpholino injection. Immunofluorescence analysis in cell culture shows that this missense mutation in BBS4 does not cause obvious defects in protein expression or pericentriolar localization. CONCLUSIONS: This mutation likely mainly reduces or abolishes BBS4 function in the retina. Further studies of this allele will provide important insights concerning the pleiotropic nature of BBS4 function.

Potocki-Shaffer syndrome: comprehensive clinical assessment, review of the literature, and proposals for medical management.

Potocki-Shaffer syndrome is a rare contiguous gene deletion syndrome due to haploinsufficiency of the 11p11.2p12 region and is characterized by craniofacial abnormalities, developmental delay, intellectual disability, multiple exostoses, and biparietal foramina. In this study, six patients with the Potocki-Shaffer syndrome were identified and evaluated using a multidisciplinary protocol that included assessments by a geneticist, ophthalmologist, otolaryngologist, orthopedist, nephrologist, audiologist, and neuropsychologist. Diagnostic studies included skeletal survey, magnetic resonance imaging of the brain, renal ultrasound, complete blood count, comprehensive metabolic panel, thyroid studies, and urinalysis. Using array comparative genomic hybridization, we further characterized the deletion in five of these patients. The results of these evaluations were combined with a comprehensive review of reported cases. Our data highlight the characteristic facial features, biparietal foramina, moderate-to-severe developmental delay and intellectual disability, myopia and strabismus, and multiple exostoses seen with this disorder. We also identify for the first time an association of Potocki-Shaffer syndrome with sensorineural hearing loss and autistic behaviors. Finally, we provide recommendations for the health maintenance of patients with Potocki-Shaffer syndrome.

Neuroimaging aspects of Aicardi syndrome.

Aicardi syndrome is a rare neurodevelopmental disorder characterized by congenital chorioretinal lacunae, corpus callosum dysgenesis, seizures, polymicrogyria, cerebral heterotopias, intracranial cysts, and costovertebral defects. Cerebellar abnormalities have been described occasionally. Aicardi syndrome is sporadic and has been observed only in females and 47,XXY males. Therefore, it is thought to result from a mutation in an X-linked gene. Improved definition of the clinical phenotype should focus the selection of functional candidate genes for mutation analysis. Because central nervous system abnormalities are the most prominent component of the phenotype, we performed a detailed characterization of abnormalities identified on magnetic resonance neuroimaging studies from 23 girls with Aicardi syndrome, the largest cohort to undergo such review by a single group of investigators. All patients had polymicrogyria that was predominantly frontal and perisylvian and often associated with underopercularization. Periventricular nodular heterotopias, present in all patients, were more frequent than previously reported; 10 had single and 11 had multiple intracranial cysts. Posterior fossa abnormalities were also more frequent than previously described. Cerebellar abnormalities were noted in 95% of studies where they could be evaluated. As a novel finding, we noted tectal enlargement in 10 patients. Since mildly affected girls with variable callosal dysgenesis have now been reported, the constellation of frontal-dominant and perisylvian polymicrogyria, periventricular nodular heterotopias, intracranial cysts, and posterior fossa abnormalities, including tectal enlargement, should prompt consideration of the diagnosis of Aicardi syndrome. We further propose that improved characterization of the neurological phenotype will benefit the selection of candidate genes for mutation analysis.

Hypomorphic mutations in syndromic encephalocele genes are associated with Bardet-Biedl syndrome.

Meckel-Gruber syndrome (MKS) is a genetically heterogeneous, neonatally lethal malformation and the most common form of syndromic neural tube defect (NTD). To date, several MKS-associated genes have been identified whose protein products affect ciliary function. Here we show that mutations in MKS1, MKS3 and CEP290 (also known as NPHP6) either can cause Bardet-Biedl syndrome (BBS) or may have a potential epistatic effect on mutations in known BBS-associated loci. Five of six families with both MKS1 and BBS mutations manifested seizures, a feature that is not a typical component of either syndrome. Functional studies in zebrafish showed that mks1 is necessary for gastrulation movements and that it interacts genetically with known bbs genes. Similarly, we found two families with missense or splice mutations in MKS3, in one of which the affected individual also bears a homozygous nonsense mutation in CEP290 that is likely to truncate the C terminus of the protein. These data extend the genetic stratification of ciliopathies and suggest that BBS and MKS, although distinct clinically, are allelic forms of the same molecular spectrum.

Generalized metabolic bone disease in Neurofibromatosis type I.

Skeletal abnormalities are a recognized component of Neurofibromatosis type I (NF1) but a generalized metabolic bone defect in NF1 has not been fully characterized thus far. The purpose of this study was to characterize at the densitometric, biochemical and pathological level the bone involvement in NF1 patients. Using dual energy X-ray absorptiometry (DXA) we analyzed bone status in 73 unselected NF1 subjects, 26 males and 47 females, mainly children and adolescents (mean age: 16.6 years). In a subgroup of subjects with low bone mass, we measured indices of calcium-phosphate metabolism, bone turnover, and bone density before and after vitamin D and calcium treatment. We found statistically significant and generalized reduction in bone mass with the mean lumbar bone mineral density (BMD) z-score being -1.38+/-1.05 (CI 95% -1.62 to -1.13), and whole body bone mineral content (BMC) z-score -0.61+/-1.19 (CI 95% -0.94 to -0.29), both significantly reduced compared to normal controls (p<.001). PTH was moderately elevated and after 4 months of supplemental therapy with calcium and vitamin D, it decreased to the normal range. However, BMD z-scores did not significantly improve after 2 years of follow-up. Histological analysis of bone samples from NF1 patients revealed substantial alteration of bone microarchitecture due mainly to reduced trabecular bone. Our observations are consistent with a generalized bone metabolic defect due to loss of the function of neurofibromin. Early identification of patients with osteoporosis may permit more timely and aggressive treatments to prevent the likely substantial morbidity associated with increased fracture risk later in life.

Identification of three novel NHS mutations in families with Nance-Horan syndrome.

PURPOSE: Nance-Horan Syndrome (NHS) is an infrequent and often overlooked X-linked disorder characterized by dense congenital cataracts, microphthalmia, and dental abnormalities. The syndrome is caused by mutations in the NHS gene, whose function is not known. The purpose of this study was to identify the frequency and distribution of NHS gene mutations and compare genotype with Nance-Horan phenotype in five North American NHS families. METHODS: Genomic DNA was isolated from white blood cells from NHS patients and family members. The NHS gene coding region and its splice site donor and acceptor regions were amplified from genomic DNA by PCR, and the amplicons were sequenced directly. RESULTS: We identified three unique NHS coding region mutations in these NHS families. CONCLUSIONS: This report extends the number of unique identified NHS mutations to 14.

Rhinologic manifestations of ectodermal dysplasia.

BACKGROUND: The Ectodermal Dysplasias (ED) are a group of uncommon genetic disorders characterized by deficient development of tissues derived from ectoderm and mesoderm. Abnormalities of the respiratory epithelium prevent normal mucociliary function. We explored the prevalence and severity of sinonasal features in affected individuals. METHODS: Thirty-four subjects with ED were evaluated. A focused history and thorough nasal examination including flexible nasopharyngoscopy were performed. Patients and caregivers rated the severity of rhinologic symptoms with a standard scale. Standardized smell tests were also performed. RESULTS: The study included 21 males and 13 females whose mean age was 18.8 years (range, 4 months to 85 years). The majority (79%) had Hypohydrotic ED. Nearly half of the individuals evaluated reported moderate to severe nasal obstruction. Allergic symptoms were present in 44% of individuals whereas 41% had a history of sinusitis. Nasal concretions were significantly more troublesome in those aged 15 and under (p = 0.028). Eighteen percent had undergone sinonasal surgery. On examination, normal intranasal structures were identifiable in all patients. However, dry mucosae, prominent crusts, and concretions were found in most (79%). A deviated nasal septum was present in 23% of individuals and a saddle nose deformity in 18. Smell tests were normal for age in all subjects. CONCLUSION: Rhinologic issues are prevalent and problematic in individuals with ED. Management should focus on humidification, nasal debridement, and tailored therapy for allergic rhinitis and sinusitis. Increased awareness of the sinonasal concerns in ED will facilitate diagnosis and improve the care of these patients.

Of mice and men: tyrosinase modification of congenital glaucoma in mice but not in humans.

PURPOSE: Primary congenital glaucoma (PCG) is an autosomal recessive ocular trait caused by mutations in the gene for cytochrome P4501B1 (CYP1B1). Although PCG is often considered to be fully penetrant, the disease shows 50% penetrance in some Saudi Arabian families. The familial segregation of the nonpenetrance suggests a genetic modifier. Recently, tyrosinase (Tyr) deficiency was found to worsen the drainage structure/ocular dysgenesis phenotype of Cyp1b1-/- mice, suggesting that Tyr is a modifier of the phenotype. In the current study, tyrosinase (TYR) was investigated in human PCG. METHODS: A genome-wide screen, a single nucleotide polymorphism (SNP) analysis in the TYR chromosomal region 11q13-q21, and sequencing of the TYR gene was performed with individuals from Saudi Arabian families with multiple, clinically confirmed, molecularly proven, nonpenetrant members. RESULTS: The study outcome did not support TYR as a modifier of the PCG phenotype in this population. The sequencing data showed no TYR mutations in the nonpenetrant family members and no difference in polymorphism frequencies between nonpenetrant or fully penetrant families. CONCLUSIONS: TYR is not a modifier of the CYP1B1-associated PCG phenotype in the Saudi Arabian population.

ABCA4 mutations causing mislocalization are found frequently in patients with severe retinal dystrophies.

ABCA4, also called ABCR, is a retinal-specific member of the ATP-binding cassette (ABC) family that functions in photoreceptor outer segments as a flipase of all-trans retinal. Homozygous and compound heterozygous ABCA4 mutations are associated with various autosomal recessive retinal dystrophies, whereas heterozygous ABCA4 mutations have been associated with dominant susceptibility to age-related macular degeneration in both humans and mice. We analyzed a cohort of 29 arRP families for the mutations in ABCA4 with a commercial microarray, ABCR-400 in addition to direct sequencing and segregation analysis, and identified both mutant alleles in two families (7%): compound heterozygosity for missense (R602W) and nonsense (R408X) alleles and homozygosity for a complex [L541P; A1038V] allele. The missense mutations were analyzed functionally in the photoreceptors of Xenopus laevis tadpoles, which revealed mislocalization of ABCA4 protein. These mutations cause retention of ABCA4 in the photoreceptor inner segment, likely by impairing correct folding, resulting in the total absence of physiologic protein function. Patients with different retinal dystrophies harboring two misfolding alleles exhibit early age-of-onset (AO) (5-12 years) of retinal disease. Our data suggest that a class of ABCA4 mutants may be an important determinant of the AO of disease.

Molecular basis of Peters anomaly in Saudi Arabia.

Peters anomaly (PA) and primary congenital glaucoma (PCG) are genetically and phenotypically distinct conditions. Mutations in cytochrome P4501B1 (CYP1B1) are the most common cause of PCG in Saudi Arabia. Recent evidence suggests that there may be common genetic factors to these conditions. To determine the molecular basis of PA, 11 study subjects with PA from 10 Saudi Arabian families were recruited. Experienced ophthalmologists examined all affected subjects and most of their available unaffected relatives. The diagnosis of PA was confirmed by pathological examination of excised corneal buttons in seven subjects. The coding exons of FOXC1, PITX2, and PAX6 were screened and those of CYP1B1 and FOXE3 sequenced. Homozygous CYP1B1 mutations were identified in six individuals in five families. Five individuals were homozygous for G61E and one was homozygous for 143del10. No mutations were identified in FOXC1, PITX2, PAX6, or FOXE3. The clinical or pathologic phenotype of the subjects with CYP1B1 mutations was not different from that of the other PA patients in this study. Two families included at least one individual with homozygous CYP1B1 mutations and no ocular anomalies (nonpenetrant). Mutations in CYP1B1 may be a substantive cause for PA in this population. Thus, PA and PCG may share a common molecular pathophysiology. Indeed, PA and PCG may share the same spectrum of anterior segment dysgenesis. Finally, the occurrence of PA, PCG, and unaffected individuals with identical homozygous CYP1B1 mutations in the same sibship suggests the presence of modifiers that modulate the clinical severity of the phenotypic expression of the same CYP1B1 mutation(s).

Mutations in a member of the Ras superfamily of small GTP-binding proteins causes Bardet-Biedl syndrome.

RAB, ADP-ribosylation factors (ARFs) and ARF-like (ARL) proteins belong to the Ras superfamily of small GTP-binding proteins and are essential for various membrane-associated intracellular trafficking processes. None of the approximately 50 known members of this family are linked to human disease. Using a bioinformatic screen for ciliary genes in combination with mutational analyses, we identified ARL6 as the gene underlying Bardet-Biedl syndrome type 3, a multisystemic disorder characterized by obesity, blindness, polydactyly, renal abnormalities and cognitive impairment. We uncovered four different homozygous substitutions in ARL6 in four unrelated families affected with Bardet-Biedl syndrome, two of which disrupt a threonine residue important for GTP binding and function of several related small GTP-binding proteins. Analysis of the Caenorhabditis elegans ARL6 homolog indicates that it is specifically expressed in ciliated cells, and that, in addition to the postulated cytoplasmic functions of ARL proteins, it undergoes intraflagellar transport. These findings implicate a small GTP-binding protein in ciliary transport and the pathogenesis of a pleiotropic disorder.

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

PURPOSE: To determine the role of cytochrome P450IBI (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 CYPIBI 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.