User:Etchevers/Notebook/Conference notes/2008/11/18

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Notes for confocal training and ASHG 2008 interesting abstracts

Colocalization – if don’t see on screen, doesn’t mean there isn’t any.

Click on Profile button (or line scan) – click to draw a line vector. If colocalization, the two peak profiles ought to follow one another. If want to keep the information: this information can be published – but don’t just use “SAVE AS” but “File export”. (The free viewer does not have the ability to read the profiles like this.) Exports as a .tif. Choose image type not as raw data but “contents of image window – single plane”.

What if want to look beyond objects in line profile to the whole image? Create a histogram, then colocalization, to get quantitative information – my example is more that of exclusion – no superimposition. Check out anyhow. Have three quadrants like for a cell sort - place the quadrants to exclude the low signals in each channel. Better yet, use threshold method (with threshold button) and place all at maximum, choose one channel, then click on mask region 1 in white button. Cover up all the signal you consider to be a real signal, with white. Then do the same with other channel (region 2). Remember the intensity threshold from the first, then replace it, and remove masks.

Navigating and looking at feeble far red signal is not really very fun.

Module Z stack. Define 2 parameters – the interval and the upper and lower limits. Z slice – click optimal interval. If there is too much difference between the two colors, do optimal pinhole diameter to optimize – to the lower one. Could redo the palette then – best to get the two channels with the same pinhole optical slice size (1.0 um).

Next button – mark first/last. Go into fast x/y and with screw go up and down. GO up until don’t see anymore, mark first, go other way and mark last. Do a mean of 2 per image, then click on Gallery. If click the Data button, then get the deepness of the slice as well.

Then go to different buttons: Ortho to get x*z or y*z slices. Upper menu: 3D view has “Projection 3D” choose eg 64 projections (for full rotation) and then press the Slice button next to the projection in order to get different angles. Can save the gallery as well. Get the z button by pressing “slice”. If want the projection all smashed together, make projection maximum and 1 section. Can do File Export as .avi to keep the animation for Windows eg Powerpoint. Make sure source file for animation is in same folder as PP presentation.

Abstracts from ASHG I want to read closer

Identification of a novel mutation of OTX2 in a Danish patient with microphthalmia. K. Grønskov1, J. Ek1, A. Sand1, L. Lavard2, H. Jensen1, K. Brøndum-Nielsen1. 1) Kennedy Center, Glostrup, Denmark; 2) Glostrup Hospital, Glostrup, Denmark

The purpose of the study was to investigate OTX2 mutations as the genetic cause of microphthalmia/anophthalmia in a cohort of Danish patients. Microphthalmia is characterized by the presence of a small eye and anophthalmia of absence of an eye within the orbit. The prevalence is reported to be 3 to 30 in 100.000. Microphthalmia/ anophthalmia can occur isolated or as part of a syndrome. The cause can be chromosomal, monogenic or environmental. Of monogenic causes SOX2 is the major gene responsible for 10-15% of the cases. Other genes causing microphthalmia/anophthalmia are OTX2, PAX6, RAX, CHX10 and FOXE3, but only few mutations have been identified for each of these genes. OTX2 is a bicoid-type homeodomain containing transcription factor, expressed during development in the neural and sensory structures, in the eye, ear, nose and brain. The OTX2 gene consists of 3 exons that are alternatively spliced, resulting in at least two protein isoforms of 289 and 297 amino acids. We investigated 23 patients with microphthalmia/anophthalmia for mutations in OTX2. Mutation analysis of OTX2 was performed by direct sequencing of PCR amplified coding regions and MLPA analysis. We identified a two-basepair deletion (c.667_668delGG) in exon 3 of OTX2. This mutation is predicted to cause premature truncation of the protein (p.Gly223TYRfsX28). The patient presented with bilateral microphthalmia, agenesis of the corpus callosum, congenital hypothyroidism, ventricular septal defect, cryptorchidism and asymmetry of the lower extremities.

Scan of 640 SNPs of 43 candidate cleft lip or palate genes in the nonsyndromic cleft lip or palate patients of Lithuania. V. Kucinskas, L. Ambrozaityte, A. Matuleviciene, E. Preiksaitiene. Human & Medical Gen, Vilnius Univ, Vilnius, Lithuania

Orofacial clefts represent complex phenotype and reflect a breakage in the normal mechanisms during embryological development of the face. The incidence of cleft lip and/or cleft palate (CL/P) in the population of Lithuania is 1 in 544 newborns. As complex diseases may be caused by different causal mechanisms, many genes are considered as candidate loci for nonsyndromic CL/P responsible for this malformation. 104 triads of Lithuania of a child affected with nonsyndromic cleft lip and/or palate or cleft palate only and both his/her parents were included in this study. DNA microarray of 640 SNPs of 43 candidate cleft lip or palate genes was designed and produced by AsperBiotech, Estonia. The SNPs in the DNA microarray are distributed within and outside the genes. This experiment was carried out using arrayed primer extension - based genotyping technology (APEX-2). 20 SNPs were excluded from further analysis in the population of Lithuania having low call rate. Association statistics was performed by TDT/S-TDT, introduced by Spielman et al. 1993. This study showed statistically significant association of 18 SNPs (i.e. 2.9% of the investigated SNPs) to nonsyndromic cleft lip or palate. Six SNPs are at oddment distribution of the genes on the microarray - CDH1 gene (rs7188750 p=0.033), FOXE1 gene (rs973473 p=0.0338), LHX8 gene (rs17096272 p=0.003), MMP3 gene (rs629946 p=0.019), MMP9 gene (rs6032619 p=0.035), MSX2 gene (rs17063892 p=0.014). Three associated SNPs are of BMP2 gene - (rs17731603 p=0.014; rs6077060 p=0.039; rs235730 p=0.034). Most significant results involve/include FGF1, FGF2 and FGFR1 genes where nine SNPs in total show statistically significant results - (rs10064637 p=0.041; rs33995 p=0.004; rs10070885 p=0.004; rs249923 p=0.028; rs2034461 p=0.012; rs308434 p=0.012; rs308395 p=0.041; rs3804158 p=0.045; rs6987534 p=0.048). First results of our study suggest significant association of 18 allelic variants in ten out of 43 investigated genes that are possibly contributing to the aetiology and pathogenesis of nonsyndromic cleft lip or palate.

Does dysregulation of the PDGFRA gene cause anomalies of the human pulmonary veins? Combining evidence from TAPVR genetics and model organisms. S.B. Bleyl1, Y. Saijoh2, K. Shiota3, S. Klewer4, G.C. Schoenwolf2. 1) Dept Pediatrics, Univ Utah, Salt Lake City, UT; 2) Dept Neurobiology Anatomy,Univ Utah, Salt Lake City, UT; 3) Dept Anatomy Dev Biol, Kyoto Univ, Kyoto, Japan; 4) Dept Pediatric Cardiology, Univ Arizona, Tucson, AZ

Total anomalous pulmonary venous return (TAPVR), is a life-threatening congenital heart defect inherited as a multifactorial trait via complex genetic and/or environmental factors. We mapped the first locus for isolated TAPVR to a 2.4 Mb interval of chromosome 4q12 by linkage and founder effect mapping, but a causative gene could not be identified by mutation analysis. While the embryology of normal "heart stalk" and pulmonary vein is well described, little is known about the embryogenesis or molecular pathogenesis of TAPVR. Indeed, no animal models for TAPVR have previously been reported. Here we report further mapping in the original TAPVR founder kindreds and several new extended TAPVR kindreds using 40 tag SNPs spanning a region of shared STR haplotypes. Alignment of phased haplotypes between kindreds implicates a narrow interval within the PDGFRA-KIT intragenic region, suggesting defective regulation of a neighboring gene(s) in the development of TAPVR. Using in situ hybridization in mouse and chick embryos, we found that PDGFRA and its ligand PDGFA are expressed in a temporal and spatial pattern consistent with a role heart stalk remodeling. We then used an in ovo function blocking assay in chick and a conditional knockout approach in mouse to knock down PDGFRA expression in the developing heart stalk and compared the morphology of the heart stalk and pulmonary veins using histological sections and 3D reconstruction. We observed that loss of PDGFRA function in both organisms can cause TAPVR, but with low penetrance (~10%) reminiscent of that observed in our human TAPVR kindreds. These animal models of TAPVR, the first reported, provide important insight into the pathogenesis of anomalous pulmonary vein development. Taken together, these data from human mapping and animal models support a role for PDGF-signaling in the normal development of the pulmonary veins, and in the pathogenesis of TAPVR.

The Integration Of Transcriptional Profiling And Functional Genetics Reveals RBM24 To Be A Novel Candidate For Cardiac Defects. R. Miller1, A. Bertoli-Avella3, B. de Graaf3, M. Wessels3, J. Gearhart4, D. McGaughey1,4, A. McCallion1,2. 1) McKusick-Nathans Inst Gen Med, Johns Hopkins Univ, Baltimore, MD; 2) Department of Molecular and Comparative Pathobiology, Johns Hopkins Univ, Baltimore, MD; 3) Departrments of Clinical Genetics, Erasmus Medical Centre, 3016 AH Rotterdam, The Netherlands; 4) Institute for Cell Engineering, Johns Hopkins Univ, Baltimore, MD

Cardiogenesis is the result of a complex and coordinated series of events. Perturbations of these processes can lead to congenital heart defects, the most prevalent of all birth defects (7/1000 live births). To better understand cardiac development, we determined the transcriptional profile of mouse embryonic stem cells (mESCs) as they differentiate along a cardiac lineage. By comparing the profile of differentiating cardiomyocytes (DCMs) with time-matched non-DCMs and undifferentiated mESCs we have identified genes whose expression is enriched in DCMs. We have determined the temporal/spatial expression of 31/133 (23%) novel candidates identified in this screen by RNA in situ hybridization at key points during cardiogenesis (E7.5, E8.5, E9.5). All candidates evaluated were expressed in key cardiac structures, with 9/31 detected as early as the formation of the cardiac crescent. One gene identified in this study, Rbm24, was selected for functional evaluation in zebrafish. The zebrafish homolog, zgc:136803 (rbm24, 92.6% identity by AA comparison) recapitulates the cardiac and somitic expression observed in mouse. Injection with translation blocking morpholinos against rbm24 resulted in cardiac looping defects and cardiac edema as well as defects in somite formation. Co-injection of the full-length transcript with the morpholino resulted in phenotype rescue of ≥62%of injected embryos. Importantly, the human ortholog (RBM24) lies on chromosome 6p22.3, within an interval linked to the segregation of congenital cardiac abnormalities (6p24.3-21.2) in a three-generation European family (nine affecteds). We will discuss this work, our ongoing mutation detection efforts at this locus, and associated functional evaluation of RBM24 coding and non-coding sequences.

Hypothalamic-pituitary defects in Chd7 deficient mice suggest critical roles for CHD7 in endocrine tissues in human CHARGE syndrome. D.M. Martin, E.A. Hurd, W.S. Layman. Pediatrics & Human Genetics, University of Michigan Medical Center, Ann Arbor, MI.

CHARGE syndrome is a multiple congenital anomaly condition characterized by ocular coloboma, heart defects, atresia of the choanae, retarded growth and development, genital hypoplasia, and characteristic ear abnormalities. CHARGE has variable phenotypic features which are incompletely penetrant. Many patients with CHARGE display delayed growth, which may be related to early feeding issues, cardiac disease, and/or endocrine dysfunction. Growth hormone (GH) deficiency and other endocrine defects including hypogonadotropic hypogonadism and hypothyroidism have been reported in children with CHARGE. Reduced testosterone levels and pubertal delay are common in boys with CHARGE, and girls display no luteinizing hormone (LH) or follicle-stimulating hormone (FSH) response to GnRH stimulation. CHD7, a member of the CHD family of chromatin remodeling proteins, is mutated in 60-80% of individuals with CHARGE. Members of the CHD family have pivotal roles in chromatin assembly and regulation of gene expression. Based on these observations, we hypothesized that loss of CHD7 disrupts hypothalamic-pituitary signaling during development. To analyze the roles of CHD7 in endocrine function, we generated mice carrying a Chd7Gt allele derived from Chd7 deficient gene trapped lacZ reporter embryonic stem cells. Chd7Gt/+ mice exhibit growth delays with onset around postnatal day 7. β-galactosidase expression and immunohistochemistry in Chd7Gt/+ mice showed Chd7 expression in the embryonic pituitary and hypothalamus. We found no apparent defects in embryonic pituitary morphology of Chd7Gt/+ mice. However, immunofluorescence showed increased GH-positive cells, decreased LH-positive cells, and variable expression of adrenocorticotropin (ACTH) and thyroid-stimulating hormone (TSH) in the late gestation (e18.5) Chd7Gt/+ pituitary. Serum IGF-1 was significantly reduced in six week old male Chd7Gt/+ mice. These studies suggest that primary defects in hypothalamic-pituitary signaling may underlie the endocrine dysfunction in human CHARGE syndrome.

Waved with open eyes (woe) phenotype caused by a mutation in Adam17. E.L. Hassemer1, R.R. Dubielzig2, C. Zeiss3, B. Chang4, D.J. Sidjanin1. 1) The Department of Cell Biology, Neurobiology and Antomy, Medical College of Wisconsin, Milwaukee, WI 53226; 2) School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI 53706; 3) School of Medicine, Yale University, New Haven, CT 06520; 4) The Jackson Laboratories, Bar Harbor, ME 04609

Waved with open eyes (woe) is an autosomal recessive mouse mutant that arose spontaneously on the C57BL/6J background. Phenotypically, the woe mice have a wavy coat, microphthalmia/anophthalmia and corneal opacities. In order to map the woe locus, homozygote woe mice were backcrossed to C3H mice to generate 138 F2 progeny. A genome wide scan mapped the woe locus to the proximal arm of mouse chromosome 12. Evaluation of the woe critical region identified Adam17 as a candidate gene. The role of Adam17 is in shedding the soluble forms of TNF-α, TGF-α and other proteins from their membrane-bound precursors. The Adam17-/- mice are phenotypically similar to the woe mice; however, Adam17-/- mice die shortly after birth. The Adam17 sequence analysis in woe mice revealed a C794T substitution that leads to a Thr265Met change in an evolutionary conserved residue. To prove that the Adam17Thr265Met substitution is responsible for the woe phenotype, a complementation breeding was set up with an Adam17+/- mouse. The Adam17 null allele rescued the lethality seen in the Adam17-/- mouse, but did not rescue the eye and fur phenotype proving the mutation. Western blot analysis did not detect different levels of Adam17 in wild-type and woe tissues. In-vitro expression and subcellular localization of Adam17Thr265Met in COS-7 cells showed no difference in the subcellular localization between Adam17Thr265 and Adam17Thr265Met; however, evaluation of the free TNF-α in sera of woe and wild-type mice via ELISA showed a slight decrease in Adam17Thr265Met shedding activity indicating that the mutation might be affecting other Adam17 specific substrates (e.g. TGF-α). Our data suggest that woe is a hypomorphic mutation in Adam17 where the Thr265Met substitution results in lowering the metalloprotease enzymatic function. Evaluation of the role of Adam17 in ocular development is currently in progress.

Identification of a new gene involved in cleft lip using the molecular analysis of an apparently balanced chromosome translocation. HG. Kim1, K. Norris2, AS. Kulharya2, LC. Layman1. 1) Section of Reproductive Endocrinology, Infertility, & Genetics, Dept Ob/Gyn, Institute of Molecular Medicine and Genetics, Medical College of Georgia; 2) Depts. Pediatrics & Pathology, Medical College of Georgia, Augusta, GA

Cleft lip and/or palate (CL/P) is a congenital developmental anomaly known to have a strong genetic component. CL/P occurs either as an isolated malformation (nonsyndromic) or in association with other developmental anomalies (syndromic). It affects approximately one in every 500 births worldwide, making it the one of the most common major birth defects. Isolated CL/P is genetically heterogeneous and its genetic ethiology has been investigated extensively for many years. Although mutations in genes such as MSX1, TBX10, PVRL1, IRF6, FGFR1, and SUMO1 explain the most commonly encountered etiologies in CL/P, they only constitute about 20% of the molecular basis in these patients, suggesting that other genes have to be involved in the pathogenesis of CL/P. In a number of genetic diseases, structural chromosomal changes that segregate with the disease phenotype have served to map causative genes to specific chromosome regions. In this regard, Mendelian cytogenetics refers to the association between chromosomal rearrangements and single gene disorders. De novo chromosome translocations have been most widely used for the mapping and cloning of disease genes, in which translocation breakpoints provided important information about the gene location. To identify a new CL/P gene by positional cloning, we investigated a male cleft lip patient with an apparently balanced de novo translocation t(10;14)(p14;q31). CGH arrays did not detect additional chromosomal rearrangements, and by performing FISH we narrowed the 10p14 translocation breakpoint to 11.1 Mb containing 39 known genes between RP11-164N21 and CTD-2382H16. The breakpoint of 14q31 was refined to 14.7 Mb , which harbors 66 genes between RP11- 929J14 and RP11-353F5. Thus further refining of both breakpoint regions in our balanced translocation patient is warranted to identify a new causative gene for CL/P.

Association of Cleft Lip/Palate with Mutations in FGFR2 Found in Breast Cancer Patients. B.N. Erickson1, K. Christensen3, M.L. Marazita4, J.C. Murray1,2. 1) Molecular and Cellular Biology, University of Iowa, Iowa City, IA; 2) Pediatrics, University of Iowa, Iowa City, IA; 3) Epidemiology, University of Southern Denmark, Odense, Denmark; 4) Oral Biology, University of Pittsburgh, Pittsburgh, PA

Background: Understanding the lifelong clinical course of children born with congenital malformations is important to anticipate and prevent treatable causes of morbidity and mortality. Epidemiologic data has shown an increased incidence of breast cancer in women born with non-syndromic cleft with or without cleft palate (CL/P). Population studies have linked breast cancer risk to a CL/P candidate gene, FGFR2. Objective: Investigate single nucleotide polymorphisms (SNPs) in FGFR2 for evidence of association in CL/P families. Methods: Seven SNPs in a highly conserved region of intron 2 in FGFR2 were genotyped using TaqMan assays. We genotyped 221 unrelated, multiplex Filipino families (5903 individuals), 516 unrelated Danish families (2293 individuals), and 359 unrelated Iowa families (1259 individuals). Family based association testing for individual SNPs (FBAT) and haplotypes (HBAT) were used to identify association between CL/P and FGFR2 SNPs. Results: FBAT analysis found no individual SNP in association with CL/P. HBAT found the CC haplotype at rs3750817 and rs2981582 is significantly associated with CL/P (p=0.0075) When a third SNP is added to the HBAT analysis (CCT haplotype at rs3750817, rs2981582 and rs7896565), significant association with CL/P was observed (p=0.0067). Conclusions: This study provides evidence that SNPs in FGFR2 are associated with CL/P. Haplotypes of SNPs previously associated with breast cancer (rs3750817 and rs2981582) along with rs7896565 were significantly associated with CL/P. This work provides some evidence that SNPs in intron 2 of FGFR2 are associated in both CL/P and breast cancer. Future work will continue to narrow in on the region of FGFR2 association with CL/P and cellular studies may provide clues as to how CL/P relates to breast cancer risk later in life.

AXIN2, Orofacial clefts and positive family history for cancer. R. Menezes1, M.L. Marazita1, T.G. McHenry1, M.E. Cooper1, K. Bardi1, C. Brandon1, A. Letra1, R.A. Martin2, A.R Vieira1. 1) Dept of Oral Biology, Center for Craniofacial and Dental Genetics, University of Pittsburgh, Pittsburgh, PA; 2) Dept of Pediatrics, Division of Medical Genetics, St. Louis University School of Medicine, St. Louis, Missouri

Background: Cancer and congenital malformations may occasionally have a common etiology. We investigated if families segregating orofacial clefts (CL/P) presented increased cancer incidence when compared to control families. Methods: We assessed 75 CL/P families and 93 control families of Caucasian ethnicity from Pittsburgh with regards to positive history of cancer. Chi-square and Fisher exact tests were used to determine statistically significant differences between both groups with alpha of 0.05. Then, we performed molecular studies with genes in which mutations have been independently associated with both cancer and craniofacial anomalies in a total of 111 families presenting isolated CL/P from Pittsburgh and St. Louis. Thirteen SNPs in eight candidate genes (AXIN2, CDH1, FGF3, FGF7, FGF10, FGF18, FGFR1 and FGFR2) were genotyped using Taqman chemistry and transmission distortion was analyzed using the Family Based Association Test (FBAT). Results: CL/P families reported positive family history of cancer more often than control families (p=0.0002), and had higher rates of specific cancer types, including colon (p=0.0009), brain (p=0.003), leukemia (p=0.005), breast (p=0.009), prostate (p=0.01), skin (p=0.01), lung (p=0.02), and liver (0.02). Over transmission of a marker allele in AXIN2 was detected in CL/P probands (p=0.003). Conclusion: Families segregating CL/P may have an increased susceptibility for cancer, notably colon cancer. Further, AXIN2, a gene that when mutated increases susceptibility to colon cancer, is also associated with CL/P. Individuals detected at a higher risk for disease predisposition could be able to adopt a better lifestyle avoiding exposure to other risk factors that may interact with the individual's genotype. NIH grants: R21-DE016718, R01-DE016148 and P50-DE016215.

Associated malformations in cases with congenital diaphragmatic hernia. C. Stoll, Y. Alembik, B. Dott, M.P. Roth. Genetique Medicale,Faculte de Medecine,Strasbourg, France

The etiology of congenital diaphragmatic hernia (CDH) is unclear and its pathogenesis is controversial. Because previous reports have inconsistently noted the type and frequency of malformations associated with CDH, we assessed these associated malformations ascertained between 1979 and 2003 in 334,262 consecutive births. Of the 115 patients with the most common type of CDH , the posterolateral, or Bochdalek-type, hernia , 70 (60.8%) had associated malformations. These included patients with chromosomal abnormalities (21, 30.0%); non-chromosomal syndromes including Fryns syndrome, fetal alcoholism syndrome, De Lange syndrome, CHARGE syndrome, Fraser syndrome, Goldenhar syndrome, Smith-Lemli-Opitz syndrome, multiple pterygium syndrome, Noonan syndrome, and spondylocostal dysostosis; malformation sequences including laterality sequence, and ectopia cordis; malformation complexes including limb body wall complex and non syndromic multiple congenital anomalies (MCA) (30, 42.9%). Malformations of the cardiovascular system (42, 27.5%), urogenital system (27, 17.7%), musculoskeletal system (24, 15.7%), and central nervous system (15, 9.8%) were the most common other congenital malformations. We observed specific patterns of malformations associated with CDH which emphasizes the need to evaluate all patients with CDH for possible associated malformations. Geneticists and pediatricians should be aware that the malformations associated with CDH can be often classified into a recognizable malformation syndrome or pattern (57.1%).

Significant Results of CNV Analysis of Myopia in Schoolchildren. A. Dellinger1, T.L. Young1,3, M. Seielstad2, L.K. Goh3, S.M. Saw4,5, Y.J. Li1. 1) Ctr Human Genetics, Duke Univ Medical Ctr, Durham, NC; 2) Genome Inst. of Singapore; 3) Duke Singapore Graduate Med School; 4) Dept of Community, Occupational, and Family Med, Nat. Univ. of Singapore; 5) Singapore Eye Research Institute

The Singapore Cohort study Of the Risk factors for Myopia (SCORM) followed the ocular development of over 1000 Singapore schoolchildren over several years. Illumina 550K SNP arrays were used to analyze the copy number variation (CNV) content of 1027 SCORM samples, of which 123 were normal, 109 were hyperopic, 730 were myopic, and 65 had unknown phenotype. We classified phenotype as: hyperopia (>0.50 diopters (D)), normal (-0.5 < D <= 0.5), low myopia (-3.00 < D <= -0.5), medium myopia (-6.0 < D <= -3.0), and high myopia (D <= -6.0) at the most recent visit. Nexus CGHTM was used to detect CNVs in the samples. Plink was used to perform tests of association. There is a trend of increasing numbers of CNVs per individual with increase in myopia severity- 33.9, 36.7, 37.8 for low, medium and high myopia cases, respectively, compared with 28.3 for controls (normal vision). Searching for CNVs of greater frequency in high myopia cases (n=93) than in controls (n=123) at uncorrected p < 0.05 showed: CNVs, including a gene, in MYP4 and MYP10 myopia loci; a CNV in a region implicated in glaucoma; and four genes involved in neurological diseases. Comparing all myopia cases to controls, CNVs were overrepresented (p < 0.05) in controls in myopia loci MYP3, MYP5, MYP6, MYP10, and MYP14. Genes in these CNVs primarily have neurological and developmental roles. Another gene involved in retinitis pigmentosa and retinal degeneration is significant in myopia when comparing high myopia vs. controls and medium+high myopia vs. controls. Deletion CNVs cover 156Kb of this gene. The region of the CNV with the most significant p-value (p < 0.004) is present in 7.5% of high myopia cases, 3% of both medium and low myopia cases, and 0% in controls. Use of additional CNV detection methods and qPCR to confirm these CNVs are warranted.

Identification of copy number variants implicated in the development of neural tube defects. K. Soldano1, A. Dellinger1, D.S. Stamm2, A. Trott1, N. Ellis1, D.G. Siegel1, H. Cope1, P. Xu1, C.F. Potocky1, C.S. Haynes1, T.M. George3, A.E. Ashley-Koch1, S.G. Gregory1. 1) Center for Human Genetics, Duke University Medical Center, Durham, NC; 2) University of California, Davis School of Medicine, Sacramento, CA; 3) Dell Children's Medical Center of Central Texas, Austin, TX

Neural tube defects (NTDs) are among the most serious congenital birth defects and result from the neural tube failing to close during the first three to four weeks of fetal development. NTDs are a phenotypically heterogeneous group of disorders with a large spectrum of clinical presentation and degree of impairment. The most common NTD presentations are lumbosacral myelomenginocele (also known as spina bifida) and anencephaly. In the US, spina bifida and anencephaly occurs in 19.6 per 100,000 live births and 10.4 per 100,000 live births, respectively. Non-syndromic NTDs comprise the majority of NTDs, and are thought to have a multifactorial etiology with a complex interplay of genetic and environmental factors. We recently completed a genome wide association screen (GWAS) of 50 families affected with cranial NTDs using the Illumina Infinium HumanHap300 genotyping microarray. As part of this analysis we used the GWAS data to identify copy number variants (CNVs), genomic deletions or duplications, which may harbor genes that contribute to NTDs. This was achieved using Nexus Copy Number software (BioDiscovery, Inc.). The most interesting findings amongst the detailed analysis, consisting of 49 anencephaly affected individuals, 2 affected siblings, 36 unaffected siblings and 50 parents, were de novo rearrangements containing ERBB4 and CNTNAP2, and familial CNVs that were independent of known CNVs in the AKT3 gene. Genomic rearrangements were also detected in HDAC9, FRG2C, and the KIR gene cluster although those genomic regions contain known CNVs. AKT3, CNTNAP2, HDAC9 and ERBB4 could be relevant to the etiology of NTDs because they are expressed in the fetal brain and may play a role in the developing neural tube.

INADL and MYT1L are associated with risk for anencephaly. C.F. Potocky1, N. Ellis1, A. Trott1, C.S. Haynes1, D.G. Siegel1, H. Cope1, K. Soldano1, D.S. Stamm2, A. Dellinger1, P. Xu1, T.M. George3, S.G. Gregory1, A.E. Ashley-Koch1. 1) Center for Human Genetics, Duke Medical Center, Durham, NC; 2) University of California, Davis School of Medicine, Sacramento, CA; 3) Dell Children's Medical Center of Central Texas, Austin, TX

We previously identified INADL and MYT1L as novel candidate genes for anencephaly by a genome wide association study (GWAS) (Stamm et al., 2007) and differential expression in fetal neuronal longSAGE libraries (Xu et al., 2007). InaDL (inactivation no afterpotential D-like) helps control epithelial migration (Shin et al. 2007) and is critical in establishing cell polarity (Michel et al. 2005; Li et al. 2004). Myt1L (myelin transcription factor 1-like) is a zinc finger DNA binding protein. Both Myt1 and Myt1L are found in neurons at early stages of differentiation (Bellefroid et al. 1996; Jiang et al 1996; Kim et al. 1997; Weiner and Chun 1997; Yee and Yu 1998). Here we report fine mapping of these genes in an expanded dataset. The initial GWAS dataset contained 45 anencephaly families and 5 families with other cranial neural tube defects (NTDs). The follow-up data set was comprised of 86 anencephalic families and 22 families with other cranial NTDs, inclusive of the GWAS families. Three affection models were considered for analysis: anencephaly only, all cranial NTDs, and any NTD regardless of lesion location. These phenotypes were analyzed for association with 26 SNPs over a 400kb region including INADL and 26 SNPs over a 550kb region including MYT1L using APL and hAPL. Two nonsynonymous coding SNPs within INADL were significantly associated (rs1056513 and rs1134767, APL p<0.01 for both SNPs in all 3 models). One of these coding SNPs, rs1134767, creates a highly significant haplotype with rs6697273, the INADL SNP originally identified in the GWAS (hAPL p<0.001 for all 3 models). Association was also confirmed in MYT1L with rs12470297 showing significant association, particularly in the anencephaly subset (APL p=0.0007). In conclusion, fine mapping confirmed INADL and MYT1L are associated with risk for NTDs and identified two nonsynonymous coding SNPs with significant association to cranial NTDs.

  • Heather 12:31, 18 November 2008 (EST):