Khanh-Nhat Tran-Viet

Multiple rare SAPAP3 missense variants in trichotillomania and OCD

Obsessive–compulsive disorder (OCD) and the spectrum of associated conditions, such as trichotillomania (TTM), Tourette syndrome and body dysmorphic disorder, affect about 2–4% of the world population. 1,2 Clinically OCD spectrum disorders are characterized by persistent intrusive thoughts (obsessions), repetitive actions (compulsions) and excessive anxiety. Although heritability studies in OCD have shown a 3–12 times increased risk for first-degree relatives and twin studies revealed higher concordance amongst monozygotic twins (80–90%) compared to dizygotic twins (47–50%), the identification of the underlying risk-conferring genetic variation by means of classic genetic association studies has proven to be difficult.3 Recently, it has been shown that mice deficient of the postsynaptic synapse-associated protein 90 (SAP90)/postsynaptic density-95 (PSD95)-associated protein 3 (SAPAP3, also known as Dlgap3) develop an OCD-like phenotype, which includes compulsive grooming and increased anxiety. Interestingly, the phenotype of Sapap3 knock-out mice can be rescued by administering selective serotonin reuptake inhibitors.4 We hypothesized that rare variants in the human orthologue SAPAP3 could contribute to disorders in the OCD spectrum. To test this, we resequenced SAPAP3 in three case populations, including 77 unrelated TTM probands collected at Duke University, 44 OCD with TTM probands from National Institute of Mental Health (NIMH), and 44 OCD cases without TTM from NIMH.5,6 Controls were 48 OCD spectrum-negative subjects from NIMH6 and a psychiatric comparison sample of 138 subjects screened for depression but not specifically for OCD from Duke University.7 A board-certified psychiatrist saw all patients and controls and diagnoses met Diagnostic and Statistical Manual of Mental Disorders, 4th edn. criteria. Samples were collected under approved institutional review board protocols. In 165 cases and 178 controls the complete coding region and flanking intronic sequence of SAPAP3 was resequenced using standard capillary sequencing methods (Applied Biosystems, Foster City, CA, USA). We detected seven novel nonsynonymous heterozygous variants, with all but A189V occurring only once (Table 1; Figure 1). Thus, in total, heterozygous SAPAP3 variants were present in 4.2% of diagnosed TTM/OCD patients, but only in 1.1% of controls (two changes in Duke control samples, with one developing depression subsequent to entry into the study). The majority of changes presented missense mutations; one variant was an in-frame insertion of five amino acids, A148insGPAGA. In silico analysis of the missense variants applying PMut and PolyPhen predicted two, or three, respectively, variants as of functional relevance (Table 1). The remaining polymorphisms were considered benign, including the two changes detected in controls. Further, we genotyped 6 of the identified variants in the TTM/OCD subjects in an additional sample of 281 OCD cases and in 751 general population controls.6 R13C and P606T were found in one control each, whereas A189V was present in three controls. This suggests that these specific variants are not by themselves disease-causing abnormalities, but still leaves open the possibility that an aggregate of susceptibility variants may prove contributory to disease, as suggested for some other disorders including autism as well as OCD. The combined analyses of 2766 alleles showed that all changes are very rare, with minor allele frequencies between 0.00036 (T523K, K910R) and 0.002 (A189V). Figure 1 Identified rare nonsynonymous polymorphisms in synapse-associated protein 90/postsynaptic density-95-associated protein 3 (SAPAP3). (a) Schematic of SAPAP3, which consists of 10 coding exons (blue boxes). Seven rare changes were identified in trichotillomania ... Table 1 Identified rare variants in SAPAP3 and predicted functional relevance Available pedigrees from TTM/OCD mutation carriers were enriched for a diverse set of psychiatric conditions, including panic disorder, attention deficit hyperactivity disorder (ADHD), depression, bipolar disorder and substance abuse as well as OCD spectrum disorders (details are given in Supplementary Figure 1 and Supplementary Table 1). This situation is quite typical for psychiatric genetic studies and complicates allele segregation studies. Cosegregation of genotype and phenotype is also confounded by phenotypic penetrance rates, limited psychometric instruments and assortative mating. Thus, we consider it more significant to study the combined mutation load of SAPAP3 comparing cases to controls. Similar approaches were recently adopted by other studies.8 We observed a significant case–control association in our moderately sized sample (Fisher’s one-sided exact test P = 0.045). With generally still limited abilities to determine functional consequences of genetic variants, we speculate that the predicted moderate functional consequences (Table 1) are not detrimental for protein function but rather increase susceptibility for OCD spectrum behavior, possibly through permissive or epistatic interactions with additional genetic and environmental factors. A recent study estimated that up to 70% of low-frequency missense alleles in humans have mildly deleterious effects.9 The excess of rare mildly deleterious variants in any OCD risk gene could be promoted by an inefficient evolutionary selection against OCD risk alleles, which is supported by the high OCD spectrum frequency of 2–4% in the population, early disease onset and normal reproductive fitness. In summary, on the background of an intriguing Sapap3-OCD mouse model we suggest that the present data support a role for SAPAP3 in TTM and OCD. Expansion of our approach and modeling of rare genetic variants in SAPAP3 will be essential to further test this hypothesis.

Functional evidence implicating a novel TOR1A mutation in idiopathic, late-onset focal dystonia

Background TOR1A encodes a chaperone-like AAA-ATPase whose ΔGAG (ΔE) mutation is responsible for an early onset, generalised dystonia syndrome. Because of the established role of the TOR1A gene in heritable generalised dystonia (DYT1), a potential genetic contribution of TOR1A to the more prevalent and diverse presentations of late onset, focal dystonia has been suggested. Results A novel TOR1A missense mutation (c.613T→A, p.F205I) in a patient with late onset, focal dystonia is reported. The mutation occurs in a highly evolutionarily conserved region encoding the AAA-ATPase domain. Expression assays revealed that expression of F205I or ΔE, but not wildtype TOR1A, produced frequent intracellular inclusions. Conclusions A novel, rare TOR1A variant has been identified in an individual with late onset, focal dystonia and evidence provided that the mutation impairs TOR1A function. Together these findings raise the possibility that this novel TOR1A variant may contribute to the expression of dystonia. In light of these findings, a more comprehensive genetic effort is warranted to identify the role of this and other rare TOR1A variants in the expression of late onset, focal dystonia.

Mutations in SCO2 Are Associated with Autosomal-Dominant High-Grade Myopia

Myopia, or near-sightedness, is an ocular refractive error of unfocused image quality in front of the retinal plane. Individuals with high-grade myopia (dioptric power greater than -6.00) are predisposed to ocular morbidities such as glaucoma, retinal detachment, and myopic maculopathy. Nonsyndromic, high-grade myopia is highly heritable, and to date multiple gene loci have been reported. We performed exome sequencing in 4 individuals from an 11-member family of European descent from the United States. Affected individuals had a mean dioptric spherical equivalent of -22.00 sphere. A premature stop codon mutation c.157C>T (p.Gln53*) cosegregating with disease was discovered within SCO2 that maps to chromosome 22q13.33. Subsequent analyses identified three additional mutations in three highly myopic unrelated individuals (c.341G>A, c.418G>A, and c.776C>T). To determine differential gene expression in a developmental mouse model, we induced myopia by applying a -15.00D lens over one eye. Messenger RNA levels of SCO2 were significantly downregulated in myopic mouse retinae. Immunohistochemistry in mouse eyes confirmed SCO2 protein localization in retina, retinal pigment epithelium, and sclera. SCO2 encodes for a copper homeostasis protein influential in mitochondrial cytochrome c oxidase activity. Copper deficiencies have been linked with photoreceptor loss and myopia with increased scleral wall elasticity. Retinal thinning has been reported with an SC02 variant. Human mutation identification with support from an induced myopic animal provides biological insights of myopic development.

COL1A1 and COL2A1 Genes and Myopia Susceptibility: Evidence of Association and Suggestive Linkage to the COL2A1 Locus

PURPOSE Collagen involvement in myopia development via scleral remodeling is well-known. Recently, COL1A1 and COL2A1 gene polymorphisms were reported to be associated with high-grade and common myopia, respectively. This study was conducted to investigate whether these collagen genes are associated and/or genetically linked with myopia in large Caucasian family datasets. METHODS High-grade myopia was defined as <or=-5.00 D. Two independent datasets comprising 146 (Duke) and 130 (Cardiff) families with high-grade myopia participated in the association study. Allelic discrimination assays were performed on tagging SNPs for COL1A1 and COL2A1. The pedigree disequilibrium test (PDT) and the association test in the presence of linkage (APL) were used for association analyses. Linkage analyses for COL2A1 locus markers were performed with the Fastlink and Merlin programs in conjunction with data obtained from our collaborative whole-genome linkage study (254 families). RESULTS Significant association was identified between five SNPs (rs1034762, rs1635529, rs1793933, rs3803183, and rs17122571) of the COL2A1 locus and high-grade myopia (P < 0.045, minimum (min) P = 0.008) and with myopia status set at <or=-0.50 or -0.75 D (min P = 0.004) in the Duke dataset. The SNP rs1635529 also showed significant association in the Cardiff dataset (<or=-5.00 D, min P = 0.004; <or=-0.50 D, min P = 0.007). Linkage analyses showed suggestive linkage to the COL2A1 locus on 12q. No association was found between COL1A1 SNPs and any degree of myopia. CONCLUSIONS The COL2A1 gene was associated with high-grade myopia in two independent Caucasian family datasets. COL1A1 gene polymorphisms were not associated with myopia in our dataset, indicating possible heterogeneity across different ethnicities.

Genetic association of insulin-like growth factor-1 polymorphisms with high-grade myopia in an international family cohort.

PURPOSE Evidence from human myopia genetic mapping studies (MYP3 locus), modulated animal models, and observations of glycemic control in humans suggests that insulin-like growth factor (IGF)-1 plays a role in the control of eye growth. This study was conducted to determine whether IGF-1 polymorphisms are associated with myopia in a large, international dataset of Caucasian high-grade myopia pedigrees. METHODS Two hundred sixty-five multiplex families with 1391 subjects participated in the study. IGF-1 genotyping was performed with 13 selected tag single nucleotide polymorphisms (SNPs) using allelic discrimination assays. A family-based pedigree disequilibrium test (PDT) was performed to test for association. Myopia status was defined using sphere (SPH) or spherical equivalent (SE), and analyses assessed the association of (1) high-grade myopia (<or=-5.00 D), and (2) any myopia (<or=-0.50 D) with IGF-1 markers. Results were declared significant at P<or=0.0038 after Bonferroni correction. Q values that take into account multiple testing were also obtained. RESULTS In all, three SNPs-rs10860860, rs2946834, and rs6214-were present at P<0.05. SNP rs6214 showed positive association with both the high-grade- and any-myopia groups (P=2x10(-3) and P=2x10(-3), respectively) after correction for multiple testing. CONCLUSIONS The study supports a genetic association between IGF-1 and high-grade myopia. These findings are in line with recent evidence in an experimental myopia model showing that IGF-1 promotes ocular growth and axial myopia. IGF-1 may be a myopia candidate gene for further investigation.

CYP1B1, MYOC, and LTBP2 Mutations in Primary Congenital Glaucoma Patients in the United States

PURPOSE To screen primary congenital glaucoma patients in the United States for sequence variants within the CYP1B1, LTBP2, and MYOC genes using Sanger and whole exome sequencing. DESIGN Retrospective case-control study. METHODS Fifty-seven primary congenital glaucoma patients (47 families), 71 unaffected family members of the primary congenital glaucoma probands, and 101 healthy unrelated individuals were recruited from a single institution. Sanger sequencing of the primary congenital glaucoma gene, CYP1B1, was performed on 47 proband deoxyribonucleic acid samples. Simultaneously, whole exome sequencing was conducted on 3 families, each including more than 1 affected individual. Concurrently, 33 of 47 primary congenital glaucoma probands with extended family deoxyribonucleic acid samples were screened for LTBP2 and MYOC gene mutations. Exome-sequenced variations were validated by additional Sanger sequencing to confirm segregation of filtered disease-causing single nucleotide variations. RESULTS Seven primary congenital glaucoma families (14.9%) manifested disease phenotypes attributable to CYP1B1 mutations. One primary congenital glaucoma family possessed homozygous mutant alleles, whereas 6 families carried compound heterozygous mutations. Five novel combinations of compound heterozygous mutations were identified, of which 2 combinations were found with whole exome sequencing. No disease-causing mutations within the LTBP2 and MYOC genes were discovered. CONCLUSIONS This study analyzed CYP1B1, LTBP2, and MYOC mutations in a cohort of primary congenital glaucoma patients from the United States, applying whole exome sequencing as a complementary tool to Sanger sequencing. Whole exome sequencing, coupled with Sanger sequencing, may identify novel genes in primary congenital glaucoma patients who have no mutations in known primary congenital glaucoma genes.

Mutational hot spot potential of a novel base pair mutation of the CSPG2 gene in a family with Wagner syndrome.

OBJECTIVE To report a 3-generation white family clinically diagnosed variably with Wagner, Stickler, and Jansen syndromes and screened for sequence variants in the COL2A1 and CSPG2 genes. Wagner syndrome is an autosomal dominant vitreoretinopathy with a predisposition to retinal detachment and cataracts. It has significant phenotypic overlap with allelic Jansen syndrome and ocular Stickler syndrome type 1. Sticker syndrome type 1 maps to chromosome 12q13.11-q13.2, with associated COL2A1 gene mutations. Wagner syndrome maps to chromosome 5q13-q14 and is associated with mutations in CSPG2 encoding versican, a proteoglycan present in human vitreous. METHODS Genomic DNA samples derived from venous blood were collected from all family members. Complete sequencing of COL2A1 was performed on a proband. Primers for polymerase chain reaction and sequencing were designed to cover all exon and intron-exon boundaries. Direct sequencing of CSPG2 was performed on all family member samples. RESULTS No detectable COL2A1 mutations were noted, making the diagnosis of ocular Stickler syndrome highly unlikely for this family. A unique base pair substitution (c.9265 + 1G>T) in intron 8 of the CSPG2 gene cosegregating with disease status was identified. This mutation occurred in a highly conserved previously reported splice site with a similar base pair substitution (G>A). Direct sequencing of this splice site mutation in 107 unrelated external controls revealed no variants, supporting the rarity of this base pair change and its causation in Wagner syndrome. This novel base pair substitution is thought to cause the deletion of exon 8 and formation of a truncated protein product. CONCLUSION Mutation screening of CSPG2 in autosomal dominant vitreoretinopathy families is important for accurate diagnosis. CLINICAL RELEVANCE This study underscores the importance of obtaining extensive pedigree information and comparative ophthalmologic clinical information, as the phenotypic findings may vary greatly among independent family members. The study also affirms the paradigm shift from diagnosis assignment based on eponyms to that based on gene mutation type.

Whole exome sequencing identifies a mutation for a novel form of corneal intraepithelial dyskeratosis

Background Corneal intraepithelial dyskeratosis is an extremely rare condition. The classical form, affecting Native American Haliwa-Saponi tribe members, is called hereditary benign intraepithelial dyskeratosis (HBID). Herein, we present a new form of corneal intraepithelial dyskeratosis for which we identified the causative gene by using deep sequencing technology. Methods and results A seven member Caucasian French family with two corneal intraepithelial dyskeratosis affected individuals (6-year-old proband and his mother) was ascertained. The proband presented with bilateral complete corneal opacification and dyskeratosis. Palmoplantar hyperkeratosis and laryngeal dyskeratosis were associated with the phenotype. Histopathology studies of cornea and vocal cord biopsies showed dyskeratotic keratinisation. Quantitative PCR ruled out 4q35 duplication, classically described in HBID cases. Next generation sequencing with mean coverage of 50× using the Illumina Hi Seq and whole exome capture processing was performed. Sequence reads were aligned, and screened for single nucleotide variants and insertion/deletion calls. In-house pipeline filtering analyses and comparisons with available databases were performed. A novel missense mutation M77T was discovered for the gene NLRP1 which maps to chromosome 17p13.2. This was a de novo mutation in the probands mother, following segregation in the family, and not found in 738 control DNA samples. NLRP1 expression was determined in adult corneal epithelium. The amino acid change was found to destabilise significantly the protein structure. Conclusions We describe a new corneal intraepithelial dyskeratosis and how we identified its causative gene. The NLRP1 gene product is implicated in inflammation, autoimmune disorders, and caspase mediated apoptosis. NLRP1 polymorphisms are associated with various diseases.

Scleral micro-RNA signatures in adult and fetal eyes.

Introduction In human eyes, ocular enlargement/growth reflects active extracellular matrix remodeling of the outer scleral shell. Micro-RNAs are small non-coding RNAs that regulate gene expression by base pairing with target sequences. They serve as nodes of signaling networks. We hypothesized that the sclera, like most tissues, expresses micro-RNAs, some of which modulate genes regulating ocular growth. In this study, the scleral micro-RNA expression profile of rapidly growing human fetal eyes was compared with that of stable adult donor eyes using high-throughput microarray and quantitative PCR analyses. Methods Scleral samples from normal human fetal (24 wk) and normal adult donor eyes were obtained (n=4 to 6, each group), and RNA extracted. Genome-wide micro-RNA profiling was performed using the Agilent micro-RNA microarray platform. Micro-RNA target predictions were obtained using Microcosm, TargetScan and PicTar algorithms. TaqMan® micro-RNA assays targeting micro-RNAs showing either highest significance, detection, or fold differences, and collagen specificity, were applied to scleral samples from posterior and peripheral ocular regions (n=7, each group). Microarray data were analyzed using R, and quantitative PCR data with 2^-deltaCt methods. Results Human sclera was found to express micro-RNAs, and comparison of microarray results for adult and fetal samples revealed many to be differentially expressed (p<0.01, min p= 6.5x1011). Specifically, fetal sclera showed increased expression of mir-214, let-7c, let-7e, mir-103, mir-107, and mir-98 (1.5 to 4 fold changes, p<0.01). However, no significant regionally specific differences .i.e., posterior vs. peripheral sclera, were observed for either adult or fetal samples. Conclusion For the first time, micro-RNA expression has been catalogued in human sclera. Some micro-RNAs show age-related differential regulation, higher in the sclera of rapidly growing fetal eyes, consistent with a role in ocular growth regulation. Thus micro-RNAs represent potential targets for ocular growth manipulation, related to myopia and/or other disorders such as scleral ectasia.

Sequencing Analysis of the ATOH7 Gene in Individuals with Optic Nerve Hypoplasia

Abstract Background: The Atonal Homolog 7 (ATOH7) gene has been implicated in association studies with optic nerve head diameter size. Hence, we screened optic nerve hypoplasia (ONH) patient DNA samples from Australia, France, and the United States for sequence variants in theATOH7 gene using Sanger sequencing. Methods: Sanger sequencing of theATOH7 gene was performed on 34 affected individual DNA samples. Sequencing was also carried out in three unaffected family members to confirm segregation of identified single nucleotide variations. Results: Seven sequence variations were identified in ATOH7. No disease-causing sequence changes in the ATOH7 gene was discovered in the ONH patient samples. Conclusions: Mutations within the ATOH7 gene are not implicated in the pathogenesis of optic nerve hypoplasia in our patient cohort.