HIGH ABUNDANCE mtDNA RECOMBINANTS IN HETEROPLASMIC … · 2019-10-22 · of HOXA1 function. Our results demonstrate a new function for HOXA1 in vascular patterning that had not been

HIGH ABUNDANCE mtDNA RECOMBINANTS IN HETEROPLASMIC MICE: Implications for AssistedReproduction and Therapeutic Cloning. D.C. Wallace, M. Crimi. Center for Molecular and Mitochondrial Medicineand Genetics (MAMMAG), University of California, Irvine, Irvine, CA 92697-3940.

To determine if mammalian mtDNAs can recombine when mixed in the same cytoplasm, we prepared mice usingfemale ES cell cybrids that were heteroplasmic for the NZB and the Common inbred mtDNAs. These mtDNAs differ in109 nucleotides (nt), including a BamHI polymorphism at nt 4277. The two mtDNAs were maintained in theheteroplasmic state through back crosses to C57BL/6 males. At generations 12 through 15, the proportion of the twomtDNAs was determined using the BamHI polymorphism, revealing three classes of mice: those which maintainedmore than 30%, less than 30%, and no detectable NZB mtDNAs. Heteroplasmy was confirmed in heart, brain, liver,testis, muscle, ovarian, kidney, though it was highest in liver. PCR amplification, cloning and sequencing of nt 3422 to5552 from liver and ovary mtDNAs revealed that 4/9 liver and 1/7 ovary mtDNAs contained one of more crossoversbetween the NZB and Common mtDNAs. This extraordinary finding was confirmed by purifying the liver mtDNA froma heteroplasmic animal using two sequential CsCl-ethidium bromide gradients, amplification, cloning and sequencingand finding that 4 of 14 clones were recombinants, with the sequence shifting from the Common mtDNA to the NZBmtDNA in two distinct regions: nt 3600-4400 including ND1-3 tRNAs - ND2 and nt 8600-9400 encompassing COX III.A related analysis revealed recombinant molecules in 1/10 heart, 1/14 brain, 5/12 kidney, 2/12 muscle, 0/6 testis, and4/14 ovarian clones. Somatic mutations were also observed. Therefore, recombinant mtDNAs can be prevalent inheteroplasmic mammals. These results are disturbing. In assisted reproduction, the cytoplasm from younger oocytes isbeing injected into older oocytes resulting in heteroplasmic children (Barritt, J., et al., 2001, Hum Repro 16:513) and intherapeutic cloning heteroplasmy can be expected. Consequently, both practices could result in recombinant mtDNAs,something that evolution has gone to considerable lengths to avoid. Supported from NIH grant NS21328 (DCW) andDept. Neurological Sciences, University of Milan, fellowship (MC) .

Copyright © 2005 The American Society of Human Genetics. All rights reserved.

Development of a novel orally administered macrophage mediated gene therapy for Gaucher Disease. E.I. Ginns,D.M. Faryna, M. Galdzicka, C. Chrzanowski, G.R. Ostroff. Program in Medical Genetics, Pediatric Neurology, UMassMedical School, Worcester, MA 01605.

A novel orally administered macrophage delivered gene therapy is being developed for treatment of Gaucher disease.Current enzyme replacement therapy (ERT) improves blood counts and reverses hepatosplenomegaly. However, ERT isintravenously administered, costly, and has not significantly addressed bone or neurological complications. To evaluatethe efficacy of our orally administered gene therapy method, DNA encoding human glucocerebrosidase (huGBA)wasformulated inside micron-sized yeast cell wall particles (YCWP). YCWP-huGBA formulations were used to introducehuGBA DNA into J774 murine macrophages in culture and into long-lived Gaucher mice generated by gene targeting.As observed in human patients, the reduced GBA activity in these Gaucher mice results in glucocerebroside storage andGaucher cells in tissues. The clinical manifestations in these mice can be accelerated by short courses of conduritol--epoxide. Following oral intake, YCWP-huGBA formulations are taken up through intestinal Peyers Patches where theyare phagocytosed by macrophages that then migrate to organs of the reticuloendothelial system. Within the macrophageendosome the huGBA DNA is released at acid pH, migrates to the nucleus, and is expressed to produce normal huGBA.This macrophage mediated treatment results in huGBA expression in J774 murine macrophages in-vitro, and in tissuesof Gaucher mice. Preliminary findings suggest that this therapy sufficiently corrects tissue GBA activity to amelioratesymptoms in treated, compared to untreated, severely affected Gaucher mice. As a consequence of improved delivery ofhuGBA, we expect that this approach will achieve significant reversal of tissue pathology, including bone. Ifmacrophages containing huGBA migrate into brain, then resulting increased GBA levels could also provide clinicalbenefit for neurologic manifestations of the disease. The successful development of this therapy should provide a safer,more efficient and cost effective treatment for patients with Gaucher disease, as well as other lysosomal diseases.


Adiponectin: An Inherited Survival Factor in Families with Exceptional Longevity. A.R. Shuldiner1, J. Crandall2,T.I. Pollin1, K. Tanner1, M. Rincon2, R. Lipton2, N. Barzilai2, G. Atzmon2. 1) University of Maryland School ofMedicine,Baltimore; 2) Institute for Aging Research AECOM, New York.

Adiponectin (ADIPOQ), a serum protein expressed by adipose tissue, is lower with obesity and has a protective roleagainst insulin resistance and atherosclerosis. To test the hypothesis that ADIPOQ influences survival, we examinedADIPOQ levels and genetic variation in the ADIPOQ gene in subjects with exceptional longevity. We studied unrelatedAshkenazi Jews between the ages of 60 and 108 years (n=366), of which 169 where over the age of 95 years. To testheritability, ADIPOQ levels were also measured in 222 of their offspring. Homozygosity for the ADIPOQ +2019del/del increased from 12% at age 65 to 31% at age 105, (=0.35, p=0.05) and was associated with significantly higherserum ADIPOQ levels, independent of BMI (p = 0.01). Serum ADIPOQ levels were greater in subjects with exceptionallongevity (13.00.5 ug/mL vs. 17.1 0.6 ug/mL in age 60-94 years and 95 years, respectively, p=0.0001), an effect thatwas independent of gender or BMI. Subjects with high serum ADIPOQ levels had a greater percentage of large HDLand LDL particle sizes(by NMR spectroscopy), higher HDL-cholesterol levels, lower insulin resistance (by HOMA) andlower prevalence of the metabolic syndrome (by NCEP III guidlines)compared to those with low ADIPOQ levels. Thedistribution of adiponectin levels in the offspring was bimodal, and ADIPOQ levels were significantly heritable(h2=0.36, p=0.05) in offspring, suggesting inheritance of a high (protective) ADIPOQ phenotype in families withexceptional longevity. Exceptional longevity is associated with high levels of ADIPOQ and with favorable lipoproteinprofile and protection from the metabolic syndrome. This phenotype is inherited in part by a novel ADIPOQ genotype.We suggest that ADIPOQ may be an inherited survival factor in exceptional longevity acting by increasing insulinsensitivity and providing protection from the metabolic syndrome and cardiovascular disease.


Homozygous HOXA1 mutations disrupt human brainstem, inner ear, cardiovascular, and cognitivedevelopment. M.A. Tischfield1,3, T.M. Bosley4,5, M.A.M Salih6, I.A. Alorainy7, E.C. Sener8, M.J. Nester5, D.T.Oystreck4, W. Chan1, C. Andrews1, R.P. Erickson9, E.C. Engle1,2,3. 1) Dept of Medicine, Program in Genomics,Children's Hosp Boston; 2) Dept of Neurology, Children's Hosp Boston; 3) Program in Neuroscience, Div Med Sci,Harvard Med School, Boston; 4) Neuro-ophthalmology Div, King Khaled Eye Specialist Hospital, Riyadh, SA; 5)Neuroscience Dept, King Faisal Specialist Hospital and Research Centre, Riyadh, SA; 6) Division of Peds Neuro,Depart of Peds, College of Medicine, King Khalid Univ Hosp, Riyadh, SA; 7) Depart of Radiology & DiagnosticImaging, College of Med, King Saud University, Riyadh, SA; 8) Dept of Ophthal, Hacettepe Univ Hospital, Ankara,Turkey; 9) Dept of Pediatrics, Mol. & Cell Biology, Univ. of Arizona College of Med, Tucson, AZ.

Using a positional cloning approach, we have identified homozygous coding mutations in HOXA1 in three geneticallyisolated populations in the Middle East and American southwest. Affected individuals have a pleiotropic spectrum ofphenotypes including horizontal gaze abnormalities, facial weakness, deafness, hypoventilation, skull deformities,autism, mental retardation, internal carotid artery malformations, and conotruncal heart defects. Although horizontalgaze abnormalities and sensorineural deafness are the most penetrant aspects of the syndrome, the remainingphenotypes demonstrate considerable variable expressivity that can be dependent upon genetic background. This is thefirst report of mutations in a HOX gene critical for CNS development and the first description of viable homozygousmutations in any human HOX gene. The identified mutations, two non-sense and one frameshift, introduce prematurestop codons in exon 1. Truncated mutant proteins should lack all known functional domains resulting in a complete lossof HOXA1 function. Our results demonstrate a new function for HOXA1 in vascular patterning that had not beenreported in Hoxa1-/- mice. We also demonstrate that HOXA1 is necessary for proper cognitive and behavioraldevelopment, intriguing considering the canonical CNS expression domain of Hoxa1 has a rostal boundary in thebrainstem.


Ancient Hybridization in the History of Homo sapiens. M.F. Hammer, D. Garrigan, Z. Mobasher, S.B. Kingan, J.A.Wilder. Div Biotechnology, Univ Arizona, Tucson, AZ.

Fossil evidence links human ancestry with populations that evolved modern gracile morphology in Africa 130,000 -160,000 years ago. Yet fossils alone do not provide clear answers to the question of whether the ancestors of all modernHomo sapiens comprised a single African population or an amalgamation of distinct archaic populations. DNAsequence data have consistently supported a single origin model in which anatomically modern Africans expanded andcompletely replaced all other archaic hominin populations. Here we present novel sequence data from a 17.5-kb X-linked locus, Xp21.1, that exhibits ancient divergence among lineages. We analyze levels of haplotype divergence andlinkage disequilibrium (LD) in the framework of models predicting patterns of nucleotide variation expected as aconsequence of admixture between historically isolated subpopulations. No previous human polymorphism study hasbeen specifically designed to utilize these measures to assess the probability of a single population origin foranatomically modern humans. The Xp21.1 locus was selected because it is located in a non-coding region of the Xchromosome with moderately high levels of recombination and very low gene density. This serves to minimize thepotenital impact of natural selection acting on linked functional sites. Monte Carlo computer simulations show that it ishighly improbable that the pattern of nucleotide variation observed at the Xp21.1 locus could arise in a single, panmicticpopulation, as predicted by the recent African replacement model. We consider several plausible alternative hypothesesand conclude that ancient population structure in the evolutionary lineage leading to AMH is the most likely explanationfor the Xp21.1 data. This inference supports human evolution models that incorporate admixture between divergentAfrican branches of the genus Homo.


The International HapMap Project: A haplotype map of the human genome with 4 million SNPs. P. Donnelly, onbehalf of The International HapMap Consortium. University of Oxford, Oxford, UK.

The International HapMap Consortium, which includes scientists from Canada, China, Japan, Nigeria, the UK, and theUS, has developed a human haplotype map describing the common properties and patterns of DNA sequence variationacross the human genome. The primary data are the genotypes of about four million SNPs, their allele frequencies, andthe degree of association among them, in a set of 270 DNA samples. In addition, ten 500 kb ENCODE regions wereresequenced in 48 samples and the HapMap samples were genotyped for all known and newly discovered SNPs. SNPswere genotyped using technologies developed by Perlegen, Illumina, Third Wave, Sequenom, ParAllele, and Perkin-Elmer. Four quality assessment exercises found that the data are > 99% complete and > 99.5% accurate. The HapMapdata are freely and publicly available at the HapMap Data Coordination Center (www.hapmap.org and hapmap.jst.go.jp)and dbSNP (www.ncbi.nlm.nih.gov/SNP), and are incorporated into the UCSC Genome Browser and Ensembl. TheDNA samples were collected through processes of community engagement and public consultation with individualinformed consent. The 270 samples included 30 Yoruba trios from Ibadan, Nigeria, 45 Japanese from Tokyo, Japan, 45Han Chinese from Beijing, China, and 30 Utah CEPH trios (northern and western European ancestry). Analyses of thedata were performed on both the ENCODE genotypes and the genome-wide HapMap genotypes to assess how sensitivethe inferences of patterns of LD and choice of markers for tagging haplotypes are to SNP density. Specifically, weanalyzed the types and patterns of LD and common haplotypes, estimated a fine-structure recombination map, searchedfor associations with genomic features, and sought evidence for natural selection as an explanation of specific patterns.All analyses were conducted genome-wide, for each population sampled.


Effect of mutation type and location on clinical outcome in 1081 Marfan syndrome patients or relatedphenotypes with FBN1 mutations : an international study. L. Faivre1, G. Beroud2, A. Child3, B. Callewaert4, C.Binquet1, E. Gautier1, E. Arbustini5, A. Kiotsekoglou3, P. Comeglio3, C. Beroud2, C. Bonithon1, D. Halliday6, C. Muti7,L. Ades8, J. De Baecker4, P. Coucke4, U. Francke9, A. De Paepe4, G. Jondeau7, C. Boileau7. 1) Centre dépidémiologie- investigation clinique et centre de Génétique, CHU Dijon, France; 2) IURC, Montpellier, France; 3) St. GeorgesHospital, London, UK; 4) Ghent University Hospital, Belgium; 5) Policlinico San Matteo, Pavia, Italy; 6) University ofOxford, UK; 7) Hôpital Ambroise Paré, Boulogne, France; 8) Royal Alexandra Hospital, Sydney, Australia; 9) StanfordUniversity Medical Center, USA.

Mutations in the FBN1 gene cause Marfan syndrome (MFS) and have been associated with a wide range of milderoverlapping phenotypes. A large collaborative study based on the international FBN1 mutation database including 1081patients (820 probands) allowed us to investigate the relationship between the FBN1 genotype and the nature andseverity of the clinical phenotype. A set of qualitative and quantitative clinical parameters was compared for differentclasses of mutation. Patients with a FBN1 null mutation had a more severe skeletal, aortic and lung phenotype thanpatients with a mutation producing an altered protein (p=0.005, p=0.0004 and p=0.007), whereas the frequency ofectopia lentis was lower (p0.0001). A higher risk of ectopia lentis was found in patients with a missense mutationsubstituting or producing a cysteine when compared to patients with other missense mutations (p0.0001), as well as witha mutation within the 5 region versus 3 (p=0.0003). Patients with mutations within exons 24-32 had a more severephenotype (younger age at diagnosis, at aortic dilatation or aortic surgery and shorter survival) than patients withmutations located elsewhere (p0.0001), even when neonatal MFS were excluded. No significant differences were foundfor any clinical parameter in patients with a mutation located in EGF domains versus TGFBP domains. This study is thelargest ever reported and indicates high-risk groups for cardiac and ophthalmologic manifestations, and can be helpful inmonitoring patients with a FBN1 mutation.


A meta-analysis of FXTAS patients with and without family history of fragile X syndrome: a probable thresholdmodel for the toxicity of CGG repeats. S. Jacquemont1, L. Beckett2, M. Leehey3, F. Tassone4, R. Hagerman5, P.Hagerman3. 1) Département de Génétique, C H U, Nantes, France; 2) Division of Biostatistics, Department of PublicHealth Sciences, UC Davis, CA; 3) Department of Neurology, University of Colorado Health Sciences Center Denver,CO; 4) Department of Biochemistry and Molecular Medicine, School of Medicine, University of California Davis, CA;5) MIND Institute, University of California Davis Medical Center Sacramento, CA.

Results of genetic screening for the FMR1 premutation (55-200 CGG repeats) have been published by 12 independentgroups. 2820 patients were ascertained through movement disorder clinics independent of their family history. Weconducted a meta analysis in order to determine the prevalence of FXTAS for the main movement disorder diagnosesand established guidelines for fragile X premutation screening in neurology clinics. The overall prevalence of the FMR1premutation regardless of the movement disorder diagnosis is 1%, 7 times greater than expected based on the prevalenceof the premutation in males of the general population (OR = 6.9 ; 95% CI : 1.8 , 65.5). This figure has limited relevancesince the excess of premutation allele was significant only in the group of males presenting cerebellar ataxia after theage of 50 years. In that group, the prevalence of the premutation rises to 2.7% ; (21/778), 22 times greater than expectedbased on the prevalence of the premutation in males of the general population (OR = 22.4 ; 95% CI : 5.8, 86.7). Theclinical descriptions of these patients were compared to the cases identified through fragile X families. The metaanalysis of the molecular data from patients recruited with or without a family history of fragile X supports thehypothesis of a threshold model of the toxicity of CGG repeats. We conclude that the toxicity of CGG repeats below 70may not have functional repercussions or may be associated with FXTAS related disorders. This meta-analysis hasconsequences for the projected prevalence of FXTAS in the general population, which is lower than the initial estimateof 1/ 3000.


FXTAS: a descriptive study of premutation carriers from fragile X families. E.G. Allen, J. Juncos, M. Rusin, G.Novak, L. Shubeck, S.L. Sherman, R. Letz. Emory Univ, Atlanta, GA.

We are conducting a study to further examine the symptoms, penetrance, and risk factors associated with thetremor/ataxia syndrome (FXTAS) among carriers of premutation alleles of the FMR1 gene. Our study populationincludes all siblings of premutation carrier males over the age of 50 identified through a survey of families with fragileX syndrome. We conducted a comprehensive battery of tests including a medical history, a neuropsychological testbattery, and quantitative neurological assessment. Within the neurological assessment, we use a series of tests to obtainobjective, quantitative measures of key features observed in FXTAS cases to date: 1) resting, postural, and intentionupper-limb tremor, 2) reduced vibration sensation, a surrogate of possible neuropathy, 3) decreased postural stability,and 4) dysdiadokinesis, or upper limb uncoordination. We have obtained these measures on 49 males (mean age=63.8)and 20 females (mean age=65.3). Subjects were scored as expressing the above phenotypes if they scored above the95th percentile of age-adjusted standards. Among premutation males, we have seen a significantly increased incidenceof tremor (45%), peripheral neuropathy (14%), decreased postural stability (57%), and dysdiadokinesis (78%). Incontrast, these phenotypes were not markedly increased in females or non-carrier male relatives. These findings as wellas neuropsychological measures will be examined with molecular correlates of FMR1.


Spectrum of CHD7 mutations in 113 patients with CHARGE Syndrome. S.R. Lalani1, A.M. Safiullah1, S.D.Fernbach1, L.M. Molinari1, C. Bacino1, S.L. Davenport4, M.A. Hefner2, J.M. Graham Jr.3, J.W. Belmont1. 1)Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 2) Department of Pediatrics,St. Louis University School of Medicine St. Louis, MO; 3) Medical Genetics Institute, Cedars-Sinai Medical Center,Los Angeles, CA; 4) Sensory Genetics/Neuro-Development, St. Paul, Minnesota.

CHARGE is a complex birth defect, characterized by non-random occurrence of coloboma, choanal atresia, cranialnerve defects, distinctive inner ear abnormalities, heart and urogenital anomalies and growth retardation. Recently,intragenic mutation of CHD7, the chromodomain-helicase DNA binding protein was reported to be a major cause ofCHARGE syndrome. Chromatin remodeling is a well-recognized mechanism of gene expression regulation and thegene is likely to play a significant role in embryonic development and cell cycle regulation. We report the spectrum ofCHD7 mutations in 113 individuals with sporadic and familial CHARGE. Mutations were found in 62 patients (55%)distributed throughout the gene. About 68% were truncating mutations, most likely leading to haploinsufficiency. Wehave obtained phenotypic information on all patients and have performed multivariate analysis conditioned on themutation status. The analysis shows that congenital heart defects (50/55 compared to mutation negative group, p=0.014)and growth retardation (42/48 compared to mutation negative group, p=0.002) are more frequent in patients with CHD7mutation. Mouse embryo in situ hybridization shows expression of this gene in the oto-acoustic complex, brain, ear,pharyngeal endoderm, and heart tube. We have also performed microarray analysis of gene expression usinglymphoblastoid cell lines of seven CHD7 mutation positive patients and compared the expression pattern in these to fivecontrol subjects and four affected patients with no abnormality of CHD7 gene by sequence analysis. Preliminaryanalysis shows significant gene expression differences among these three groups. The differential gene expressionpattern in the CHD7 mutation negative group may further assist in understanding the molecular basis of disorder in thisgroup of affected children.


Phenotypic spectrum of CHARGE syndrome with CHD7 mutations. K. Kosaki1, M. Aramaki1, T. Udaka1, R.Koaki2, Y. Makita3, N. Okamoto4, H. Yoshihashi5, H. Oki5, Y. Fukushima6, H. Kawame7. 1) Dept Pediatrics, Div MedGen, Keio Univ, Tokyo, Japan; 2) Dept of Clin Genet & Mol Med, Nat'l Children'fs Med Ctr, Tokyo, Japan; 3) Dept ofPediatrics, Asahikawa Med Col, Asahikawa, Japan; 4) Department of Planning and Research, Osaka Medical Centerand Res Inst for Maternal and Child Health, Osaka, Japan; 5) Tokyo Metropolitan Kiyose Children'fs Hosp,Tokyo; 6)Dept of Med Genet, Shinshu Univ Sch of Med, Matsumoto, Japan; 7) Division of Clinical Genetics, Nagano Children'sHosp, Nagano, Japan.

CHARGE syndrome is characterized by a constellation of non-randomly associated malformations: C - coloboma ofthe iris or retina, H - heart defects, A - atresia of the choanae, R - retardation of growth and/or development, G - genitalanomalies, and E - ear abnormalities. Recently, Vissers et al. identified the gene Chromodomain helicase DNA-bindingprotein-7 (CHD7) at chromosome 8q12.1 as a causative gene of CHARGE syndrome. We further delineated thephenotypic spectrum of CHARGE patients with mutations in CHD7. Twenty-three patients who fulfilled Blake'fscriteria were screened for CHD7 mutations by using DHPLC. PCR products corresponding to all variant elution profileswere sequenced bidirectionally using the dideoxy sequencing method. We identified heterozygous CHD7 mutations in17 (71%) of the 24 patients enrolled in the study: 7 frameshift mutations, 6 nonsense mutations, 3 splice-site mutations,and 1 intragenic deletion from exon 8 to 12. These mutation classes are likely to result in a prematurely truncatedprotein or the loss of protein expression. Altogether, 15 of the 17 cases had coloboma of the eyes. However, only 2patients had iridal coloboma and 5 patients had only disc coloboma, without iridal or retinal coloboma. Thus, the retinaand optic disc must be thoroughly examined when CHARGE syndrome is suspected, even in the absence of iridalcoloboma. Choanal atresia/stenosis was not common (5 patients) and was less frequent than oral clefts (8 patients).Severe hearing-loss, laryngomalacia, and developmental delays were prevalent. Genetic testing of CHD7 will be helpfulin confirming the diagnosis and in providing accurate genetic counseling to the families.


Genotype-phenotype correlations in Rubinstein-Taybi syndrome. E.K. Schorry1, M. Keddache1, B. Abiramikumar1,N. Lanphear2, J. Rubinstein2, G.A. Grabowski1. 1) Div. of Human Genetics; 2) Div. of Developmental Disabilities,Cincinnati Children's Hospital.

Rubinstein-Taybi syndrome (RTS) is a well-described multiple congenital anomaly/mental retardation syndromewhich usually occurs sporadically. Loss of function in CREBBP or EP300 genes, which encode histone acetyltransferases, has been found in about 50% of patients with RTS, with the majority of mutations in CREBBP. Weperformed mutation analysis of CREBBP in 98 patients who met diagnostic criteria for RTS during 2 international RTSfamily conferences. DNA was extracted from peripheral blood, and mutation analysis of CREBBP was performed on all31 coding exons and exon-intron junctions by bidirectional comparative PCR sequencing. A total of 59 differentvariations were observed in the DNA sequence. All mutations involving a change in the protein were unique, and wereequally distributed throughout the 31 coding exons. Twenty-five of the mutations created a truncated protein product orclearly altered the splice-donor/splice-acceptor consensus sequence; 11 mutations resulted in single amino acid changes.Forty-five patients did not have an identifiable mutation in CREBBP. Extensive phenotypic data was also collected on the patients during the conference. All patients had the characteristicfacies, broad thumbs, and mental retardation. We analyzed phenotypic data to determine correlations with specificmutation types, i.e., truncating, splice site, single amino acid substitutions, or no identifiable mutation. There were nodifferences in the facial and broad thumb phenotype between the 4 groups, with all groups displaying the characteristicdysmorphology. Degree of mental retardation and growth retardation were similar in all groups. Congenital heartdisease was seen in 40% of the group overall, but was less frequent (18%) in patients with single amino acidsubstitutions. Interestingly, duplication of the great toe was seen in 8 patients with no identifiable mutation, but in noneof the patients with CREBBP mutations. Further research is needed to determine the additional genes which cause thisphenotype.


Phenotypic characterization of familial oculo-auriculo-vertebral (OAV) spectrum: assessment of 19 additionalfamilies. A.E. Beck1, L. Hudgins2, A.W. Grix4, N.H. Robin5, E. Chen6, L.C. Lazzeroni3, H.E. Hoyme2, U. Francke1,2. 1)Dept Genetics and; 2) Dept Pediatrics, Div Medical Genetics and; 3) Dept Health Research & Policy, Div Biostatistics,Stanford Univ, Stanford, CA; 4) Permanente Medical Group, Sacramento, CA; 5) Dept Genetics, Univ Alabama atBirmingham, Birmingham, AL; 6) Permanente Medical Group, Oakland, CA.

Although the OAV spectrum is considered a sporadic condition, a few large studies suggest that up to 45% ofprobands with hemifacial microsomia or microtia have affected relatives. In the present study, we evaluated 19 familiesin which probands with OAV had additional family members with OAV features. We ascertained 54 affected familymembers (including probands) and 18 normal relatives who were apparently transmitting carriers. Of thosephenotypically affected, 57% were female and 43% were male. 63% had unilateral and 37% had bilateral findings. 93%exhibited auricular anomalies including preauricular tags (23 cases) and microtia (30 cases, often with canalnarrowing/atresia and conductive deafness). Eye malformations in 20% included epibulbar dermoids (8/11),microphthalmia, and iris/retinal colobomas. Mild facial asymmetry was seen in 30%, but the marked hemifacialmicrosomia often seen in sporadic OAV was uncommon. Renal (4%), vertebral (11%) and cardiac (7 %) anomaliesoccurred less frequently than in sporadic cases. Only 2 affected subjects had intellectual impairment. Chromosomalstudies in probands were normal. Although mouse models and candidate regions in the human genome are underinvestigation, no human gene mutations for OAV are known. Our new data suggest that there are genetic subtypeswithin the OAV spectrum consistent with autosomal dominant and possibly X-linked inheritance of a single genedisplaying variable expressivity and incomplete penetrance. Careful evaluation of relatives for minor manifestations ofOAV is essential before quoting recurrence risks. Since microtia can result from aberrant migration of neural crest cellsinto the 1st/2nd branchial arches during embryonic weeks 4-5, it is tempting to postulate that the altered gene(s) in ourfamilial cases are involved with neural crest regulation.


Array CGH identifies chromosome abnormalities with unexpected clinical variability in contiguous genesyndromes. B.A. Bejjani, B.C. Ballif, C.D. Kashork, E. Rorem, K. Sundin, L.G. Shaffer. Signature GenomicLaboratories, LLC, Spokane, WA.

Array CGH provides distinct advantages over conventional cytogenetics by detecting aneuploidy, microdeletions,microduplications, and subtelomeric rearrangements in a single, simultaneous assay. The SignatureChip was designed todetect the common microdeletions, reciprocal microduplications, subtelomeric and pericentromeric alterations,unbalanced translocations, and aneuploidy, while avoiding most common population variants. The design also allowsfor distinguishing the common-sized microdeletions that are flanked by low copy repeats from larger deletions throughthe use of flanking control loci. The array uses 831 BACs covering 126 clinical and 104 control loci in 3-6 clonecontigs. Of the 1,300 clinical cases submitted to our laboratory for diagnostic testing, we identified 73 cases (5.6%) withclinically relevant abnormalities. The majority of these cases have had at least one previous cytogenetic study. Amongthese, 22 telomeric deletions or unbalanced rearrangements and 21 cases with syndromic deletions were identified. Wealso identified 3 cases of interstitial deletions of 1p36 and three cases of interstitial duplications on 3q, 4q and 16q, noneof which would be detected using available sets of subtelomere probes. The finding of 21 cases with syndromicmicrodeletions most of which were submitted by clinical geneticists after at least one previous cytogenetic studysuggests that these well-characterized syndromes have unrecognized variability in clinical presentation that may preventthe clinician from readily reaching a diagnosis. The clinical variability of these syndromes will be reviewed. In addition,microduplications of 15q12, 17p11.2, and 22q11.2 have been identified, confirming that reciprocal products of commonmicrodeletion syndromes will be detected. These microduplications are unlikely to be identified by conventionalmetaphase FISH assays. Thus, array CGH is a powerful approach for uncovering subtelomeric rearrangements,microdeletions and microduplications, even in patients who are not suggestive of a particular syndrome.


9q34.3 microdeletion syndrome: clinical and genetic insights. S.A. Yatsenko1, H. Firth4, S. Tomkins5, O. Rittinger6,E. Lammer7, K.S. Lewis8, S.W. Cheung1, P. Stankiewicz1, J.R. Lupski1,2,3. 1) Dept Molec & Human Genetics, BaylorCol Medicine, Houston, TX; 2) Pediatrics, Baylor College of Medicine; 3) Texas Children Hospital, Houston, TX; 4)Addenbrookes Hospital, Cambridge, UK; 5) St. Michael's Hospital, Bristol, UK; 6) Klinische Genetik,Landeskinderklinik Salzburg, Salzburg, Austria; 7) Children's Hospital and Research Center at Oakland, Oakland, CA;8) St. Joseph's Hospital And Medical Center, Phoenix, AZ.

9q34.3 microdeletion syndrome is a contiguous gene syndrome characterized by craniofacial dysmorphism, neonatalhypotonia, childhood obesity, microcephaly, mental retardation and absence of expressive speech. Depending on thedeletion size, additional clinical features can include congenital heart defects, seizures, abnormal male genitalia, limband brain anomalies. Recently we identified an ~700 kb critical region at the most distal portion of 9q34.3encompassing genes EHMT1 (euchromatic histone methyltransferase) and CACNA1B in association with a minimalphenotype (Yatsenko et al. 2005). Haploinsufficiency of EHMT1 in all our patients and disruption of EHMT1 identifiedin a child with features of 9q deletion syndrome (Kleefstra et al. 2005) provide the first evidence that dysregulation ofhistone modifications and chromatin structure can be a pathophysiological mechanism underlying mental retardationand neurobehavioral disorders. Here we present the FISH mapping of the breakpoints in six new patients with del(9)(q34.3) using BAC and fosmidclones, and compare the deletion sizes and clinical manifestations. We constructed a deletion map of 9q34.3chromosome region using cell lines from eleven affected individuals. In addition molecular cytogenetic analysisrevealed one patient with der(9)del(9)(q34.3)dup(9)(q34.3q34.2). Our investigation showed that the 9q34.3 deletionsvaried from ~700 kb to 3.5 Mb in size and complexity of the rearrangement can be detected only by FISH or microarrayCGH analyses. These observations provide new insights for genetic counseling and for the search of new candidategenes. We discuss genotype-phenotype correlation, and the possible molecular mechanism of deletion.


Overgrowth in association with chromosomal anomalies. G.S. Ball, R.K. Aldrich, J. Lee, S. Li, K.A. Casas.Department of Pediatrics, University of Oklahoma, Rare Chromosomal Anomalies Registry, Oklahoma City, OK.

Overgrowth is a feature of very few chromosomal syndromes. We report two new patients with overgrowth. Patient 1presented at birth with distal joint contractures and failed newborn hearing screen. Imaging studies revealed agenesis ofthe corpus callosum and left cystic kidney. Routine chromosome analysis performed shortly after birth showed extramaterial on chromosome 6q. Subtelomeric fluorescent in situ hybridization (FISH) using a 10p subtelomeric probeconfirmed that the extra material on 6q was derived from 10p, resulting in terminal deletion of 6q and trisomy 10p. At 5months of age, her length was 4 standard deviations above the mean for age. Weight and occipitofrontal circumferencewere at the 75th centile. Palm length was greater than the 97th centile for age, and there was an advanced bone age of 2years. Patient 2 presented at 15 years of age with mild mental retardation and recent onset of seizures and hypertension.Her height was over 3 standard deviations above the mean for an adult female, weight was 4 standard deviations abovethe mean for an adult female, and occipitofrontal circumference was 3 standard deviations above the mean for an adultfemale. Palm length was at the 97th centile for an adult female. Routine chromosome analysis had been performed inearly childhood with normal results. Subtelomeric FISH testing at 15 years of age revealed an unbalancedrearrangement with deletion of 8p and trisomy of 12p. Overgrowth is not a reported feature of the 6q deletion or 10pduplication syndromes, suggesting a fusion gene or other mechanism unique to the (6;10) translocation in Patient 1.Overgrowth is not a reported feature of the 8p deletion syndrome, but increased birth length and birth weight havepreviously been associated with duplication 12p and tetrasomy 12p, suggesting a dosage effect of a growth factor gene.Results in Patient 2 narrow the candidate region for this gene(s) to the 12p subtelomere.


Identification of Segmental Chromosomal Duplications and Deletions in Prostate Cancer. A. Pearlman, M.Salman, H. Ostrer. Human Genetics Program, New York University School of Medicine, New York, NY.

Recurrent somatic genetic alterations contribute to the development and progression of prostate cancer. These eventscan be identified using arrayCGH, a high-resolution technique that maps the duplicated or deleted segments onto arraysof well-characterized bacterial artificial chromosomes (BACs) or oligonucleotides. As part of our efforts to identifysomatic alterations that are predictive of racial differences or prognosis, and to aid the identification of tumor suppressorgenes, we compared the performance of Affymetrix 10K SNP chips and 19K BAC arrays using three Gleason 7 prostatecancers and their matched normal tissue. We analyzed the Affymetrix 10K SNP chip arrays using GDAS 2.0,dChipSNP and the Affymetrix copy number tool and the BAC arrays using the CGH Explorer, DNAcopy, vMAP, andGLAD segmentation tools. SNP chips provided calls for 93-98% of the probes. The gender calls were as expected for allsamples and the concordance between replicates was 97%. No duplication events were identified with confidence. LOHevents were observed on chromosomes 1, 13, and 21 for tumor 3 and in an overlapping region on 13 for tumor 1.Among the 60,000 events sampled, 4 small, LOH events occurred in single samples, but not in their replicates,suggesting that the frequency of false positives was

Finding prostate cancer susceptibility genes using linkage-based and candidate gene approaches. E.A. Ostrander,on behalf of The International Consortium of Prostate Cancer Genetics. Cancer Genetics Branch, NHGRI/NIH,Bethesda, MD.

Identification of prostate cancer (PC) susceptibility genes has been hampered by the phenotypic heterogeneityassociated with the disease, the large number of contributing loci, and the associated difficulties in accurately stratifyingfamilies based on features of disease, family history, or clinical presentation. Although genome-wide screens have beenperformed in over a dozen independent studies, few chromosomal regions have been consistently identified as regionsof interest. The International Consortium for Prostate Cancer Genetics (ICPCG) was formed to facilitate theidentification of PC susceptibility genes. The ICPCG has undertaken analyses aimed at utilizing the large familyresources available (over 1800 PC families). These include: 1) combining genome-wide screen linkage data from a totalof 1,233 PC families collected by members of the ICPCG. Using parametric and non parametric analyses five regionswere identified with suggestive linkage (LOD score >1.86) on 5q12, 8p21, 15q11, 17q21 and 22q12, as well as asignificant linkage at 22q12 (LOD 3.57) in a subset of families with 5 or more affected members. These findings areconsistent with the hypothesis of multiple PC susceptibility genes with modest effects, or several major genessegregating in small subsets of families; 2) multi-locus analysis of genome wide scan data in all families using orderedsubset analysis and pedigree covariate analysis using recursive partitioning; 3) a genome wide scan for linkage in 189families that have three or more first degree relatives with aggressive prostate cancer; 4) genome wide scans for linkagein families with both prostate and other cancers (e.g. gastric cancer) and prostate cancer families of Ashkenazi Jewishdescent. Finally, we have undertaken replication studies for previously described loci on the X chromosome, and testingof provocative candidate genes such as CHEK2 and NBS1. In addition, novel methodologic approaches are beingdeveloped and implemented to analyze this large and informative data set.


Genome-wide linkage scan for prostate cancer susceptibility loci in men with aggressive disease: The Universityof Michigan Prostate Cancer Genetics Project. L.A. Ho1, J.B. Dimmer2, Y. Wang1, E.M. Gillanders3, K.A. Cooney2,E.M. Lange1. 1) University of North Carolina, Chapel Hill, NC; 2) The University of Michigan, Ann Arbor, MI; 3)NHGRI, NIH, Bethesda, MD.

Identifying prostate cancer susceptibility genes using linkage analysis has proven to be a difficult challenge. It is clearthat there is considerable genetic heterogeneity which results in reduced power to detect linkage signals. Another likelycomplication in mapping prostate cancer susceptibility genes is that there is considerable heterogeneity in the phenotypeof prostate cancer, with a considerable range in the aggressiveness of the disease even among family members. Weperformed a genome-wide nonparametric linkage scan for genes that predispose to aggressive forms of the diseasebased on 405 genetic markers and 79 informative pedigrees with two or more cases of aggressive disease. Onlyindividuals defined to have aggressive disease were coded as "affected" in our analyses. An indicator variable foraggressive disease was created using the following criteria: 1) Regional or distant stage (based on pathology if radicalprostatectomy done, otherwise clinical stage, T3, T4, N1, M1) or 2) Gleason score of 7 or higher or poorly differentiatedgrade (if no Gleason score is available) or 3) PSA at diagnosis 20 or higher or 4) prostate cancer listed as primary causeof death on death certificate. We found strong evidence for linkage on chromosome 15q (LOD = 3.43) near markerD15S1002. Suggestive linkage was found on chromosome 6p (LOD = 2.21) near marker D6S422. Additional LODscores greater than 1 were found on chromosomes 3q (1.43), 18q (1.17), 20q (1.05) and Xp (1.02). Using a more rigiddefinition of prostate cancer will result in a severe reduction in the effective sample sizes available for linkage analysis.However, the reduction in heterogeneity or "noise" in the phenotype may ultimately prove to increase the statisticalpower to detect susceptibility genes for this complex trait.


Single locus gene mutations and prostate cancer: Can mutations in the androgen receptor gene be directly linkedto the occurrence of prostate cancer? B. Gottlieb1,3,5, C. Alvarado1, L.K. Beitel1,2, K. Sircar4, A. Aprikian4, M.Trifiro1,2,3. 1) Dept Cell Genetics, Lady Davis Inst Medical Res, Montreal, PQ, Canada; 2) Dept Medicine, McGillUniversity, Montreal, PQ, Canada; 3) Dept Human Genetics, McGill University, Montreal, PQ, Canada; 4) DeptUrology, McGill University, Montreal, PQ, Canada; 5) Dept Biology. John Abbott College, Ste Anne De Bellvue, PQ,Canada.

One of the Holy Grails of cancer genetics has been to find mutations in a specific gene linked directly to a specificcancer. A possible candidate has been the androgen receptor gene (AR), which recent evidence has shown is importantin all prostate cancer (CaP) stages. However, numerous studies that have examined the possible association between ARmutations and CaP have produced very inconclusive results. To resolve this issue we have considered the fact that tumortissue heterogeneity has rarely been considered an indicator of genetic heterogeneity. To see if AR genetic heterogeneitydoes indeed exist we have conducted a micro-genetic examination of tumors from CaP patients using laser capturemicrodissection (LCM) to isolate specific pathologically-identified tissues and then sequence their AR. To help correlatemutations with tissue type, we have prepared cancerous and benign tissue micro-arrays from prostates from men withCaP. To date we have found a number of different mutations, in distinct areas isolated from a single prostate, all ofwhich appear to be unique. Mutations were found in both cancerous and benign tissue from the central zone of theprostate, but not from benign peripheral and transitional zones. Even more intriguing is that different mutations werefound in cancerous and benign tissues. Thus, it appears that somatic mosaicism of the AR occurs within CaP tumors,although the exact relationship between AR mutations and CaP must await an analysis of many more prostate tumors.We believe that this study, which is the first to conduct a micro-genetic analysis of AR mutations in CaP by utilizingLCM, points the way to an approach that can much more accurately monitor genetic events that lead to the initiation andprogression of cancer.


Allele-specific expression in familial pancreatic cancer. J. Fan1, A. Maitra2, M. Bibikova1, E. Chudin1, E.W.Garcia1, D. Barker1, P. Chen2, C. Karikari2, M.G. Goggins2, R.H. Hruban2, A. Chakravarti2. 1) Illumina, Inc, SanDiego, CA; 2) Johns Hopkins University, Baltimore, MD.

Pancreatic cancer afflicts over 30,000 individuals each year in the United States, and is mostly lethal within months ofdiagnosis. About 10% of pancreatic cancer is familial due to an inherited predisposition. Despite considerable effort,germline mutations have been identified in less than 20% of families, in a few genes such as BRCA1/2. To enhancegene discovery, we analyzed germline allele-specific expression (ASE) patterns in familial pancreatic cancer (FPC). Ourcentral hypothesis is that the gene responsible for FPC would be hemizygously mutated in the germline of affectedindividuals, and hence, would only or predominantly be expressed hemi-allelically from the wild-type allele (at least fora subset of mutations such as nonsense mutations or deletions). A microarray-based ASE assay was developed for 2,140coding SNPs (cSNPs) derived from 680 cancer-associated genes, such as cell cycle checkpoint and apoptosis-inducinggenes. Lymphoblastoid cell lines from 48 age-matched individuals (16 CEPH family trios) were analyzed for genomicheterozygosity and allele-specific differences in expression, if any, at heterozygous cSNPs. Genes demonstratingsignificant ASE in these normal individuals were catalogued for reference. Subsequently, lymphoblastoid cell lines from109 individuals with FPC were examined for ASE variations. Overall, 331 genes demonstrated potential ASE in at leastone of the 157 samples, of which 201 demonstrated ASE in both CEPH and FPC samples. These include knownimprinted genes as well as multiple X-chromosome transcripts, confirming the overall validity of our platform. Theremaining 130 genes demonstrated ASE in one or more of the 109 FPC patient samples only, and include both knowngenes implicated in FPC (e.g., BRCA1/2) as well as novel candidate genes (e.g., XRCC5, BPAG1, AGO, etc.). We arecurrently validating array-based ASE on a gene-by-gene basis using quantitative RT-PCR. Mutational screening will beperformed for genes that display confirmed ASE variation in FPC patients but not (or at a significantly lower frequency)in CEPH controls.


PRKAR1A mutations leading to Carney complex, PPNAD and related disorders: Analysis of the largest databaseto date, identification of large gene rearrangments and other novel disease-causing alterations, and functionalcharacterization of expressed mutants. A. Horvath, S. Boikos, F. Weinberg, E. Meoli, S. Stergiopoulos, T. Bei, L.Matyakhina, I. Bossis, C. Stratakis. Section on Endocrinology & Genetics, DEB, NICHD, NIH, Bethesda, MD.

Carney complex (CNC) and primary pigmented nodular adrenocortical disease (PPNAD) are caused by inactivatingmutations in PRKAR1A, the main regulatory subunit of cAMP-dependent protein kinase (PKA). We have screened forPRKAR1A sequence changes a total of 655 individuals from 171 non-related affacted kindreds, by DHPLC and genomicsequencing; negative samples were then screened by Southern blotting; 161 patients have been found to have 55 (33novel) different disease-causing single base substitutions or small insertions/deletions scattered over the codingsequence and the splice junctions. Six index cases have shown alternative patterns in heterozygote state by Southernanalysis using EcoRI; further analysis confirmed two distinct deletions, one ~5 kb from the exon3/intron3 region, andanother deleting ~4 kb from between exons 4 and 8. These data were confirmed by long distance PCR; FISH did notshow any abnormalities. Four specimens were sharing common EcoRI altered restriction pattern, suggesting mutationsof EcoRI site. In contrast to previous studies, where the vast majority of the mutations were proven to lead to NMD, andsubsequently, not to be expressed, we identified 10 different expressed mutations, which allowed us to assess thegenotype-phenotype correlations. Two of the expressed mutations were associated with particularly aggressive clinicalphenotype, two other were associated almost exclusively with PPNAD only. We conclude that (1) there is a largenumber of new PRKAR1A mutations associated with CNC and PPNAD, (2) the existence of large gene rearrangementsconfirms the complex PRKAR1A disease-mutation spectrum (3) approximately 20% of the mutations do not undergoNMD and lead to the expression of various mutant PRKAR1A proteins, and (4) there is genotype-phenotypecorrelation. This study has important implications for counseling, and for the molecular pathophysiology of PKA-associated tumorigenesis.


A mouse model employing the tet-off system in an antisense construct to Prkar1a confirms tTA-independentexpression of the transgene and develops tumors consistent with Carney complex (MIM160980). K.J. Griffin, F.Weinberg, S. Stergiopoulos, L. Matyakhina, L. Kirschner, C. Stratakis. SEGEN, DEB, NICHD,NIH, Bethesda, MD.

Carney complex (CNC) is a multiple neoplasia syndrome associated with adrenal, pituitary, thyroid, and gonadalneoplasms, caused by PRKAR1A mutations inactivating the type IA regulatory subunit of protein kinase A (PKA). Atransgenic line carrying an antisense transgene for exon 2 (X2as) of Prkar1a was crossed with a tTA-transgene-expressing line. The resulting tTA/X2as mouse mimicked CNC in many respects (Cancer Res 2004;64:8811, J MedGenet 2004;41:923), and developed lymphoid hyperplasia. The mice with only the X2as transgene also developedabnormalities that were not present in mice carrying tTA alone or in wild type mice of the same background. Previousreports have suggested that the tet-off system is leaky, allowing for the occasional expression of the target constructwithout the tTA factor. This study reports 136 mice: wild type (16), those carrying only the X2as transgene (60), thoseexpressing tTA only (15), and the tTA/X2as (45) line published recently. The X2as mice developed a variety of tumorsthat were not present in controls, and not different from those in tTA/X2as mice. These include follicular adenomas ofthe thyroid, pigmentation and x-zone vacuolization of the adrenal cortex, uterine cysts, and abnormalities of the testis.The X2as mice did not develop lymphoid hyperplasia or lymphoma as frequently as tTA/X2as mice. PKA activities inX2as tissues were intermediate between tTA/X2as and controls. Genetic analysis of X2as-derived sarcomas showedinvolvement of the mouse chromosome 11 Prkar1a locus. Expression of the X2as transgene was confirmed by proteinand mRNA studies; proliferation assays of X2as and tTA/X2as mouse-embryonic fibroblasts were similar. We concludethat the X2as mice developed tumors similar to those seen in CNC, tTA/X2as, and Prkar1a+/-mice. These studiesconfirm the leakiness of the tet-off system but also provide the opportunity to test which features are related to Prkar1a-down regulation vs. expression of the tTA transgene.


Mutations and Interstitial Deletions Involving Exon 3 of the Beta-catenin Gene are Detected in SporadicAdrenocortical Tumors. I. Bourdeau, A. Lampron, M. Tadjine. Division of Endocrinology, Department of Medicine,Centre hospitalier de lUniversité de Montréal (CHUM)-Hôtel-Dieu, Montréal, Québec, Canada H2W 1T8.

Adrenocortical lesions are diagnosed frequently as benign adenomas or nodular hyperplasias, and more rarely asmalignant adrenocortical carcinomas. The genetic background of sporadic adrenocortical lesions is poorly characterized.In our previous work, involving large-scale cDNA microarray analysis, we demonstrated aberrant differentialexpression of a number of Wnt/-catenin signaling-related genes implicated in adrenocortical hyperplasias. To furtherexplore the role of Wnt/-catenin signaling in adrenocortical tumorigenesis, we conducted a search for mutations of exon3 of the -catenin gene. DNA was extracted from 48 human adrenocortical samples and the human adrenocortical cancercell line NCI-H295R. The adrenal tissue samples included 28 patients with adenomas, 4 with carcinomas, 13 withACTH-independent macronodular adrenal hyperplasia (AIMAH) and 3 with ACTH-dependent adrenal hyperplasia. Thesamples were screened for somatic mutations in exon 3 of the -catenin gene using deletion screening by polymerasechain reaction (PCR) and direct sequencing. We found genetic alterations in 6 out of 28 adenomas (21,4%). There wereno mutations detected in adrenocortical carcinomas, AIMAH, ACTH-dependent hyperplasia and the NCI-H295R cellline. Three point mutations occurred at potential serine/threonine phosphorylation residues of codons 37 or 45 [S37C(n=2) and S45F n=1)]. The remaining 3 tumors contained deletions of 6, 55 and 271 bp, each including part of exon 3.Reverse transcription-PCR experiments with RNAs isolated from the adenoma with the 271bp deletion detectedtranscripts that lacked exon 3, in addition to the normal transcript. These mutations, as previously reported in severaltypes of tumors, but never in adrenocortical tumors, abrogate the phosphorylation-dependent interaction of -cateninknown to lead to transcription activation of Wnt target genes. Our results suggest, for the first time, that -catenin genemutations are frequent in adrenocortical adenomas. Possible involvement of -catenin activation could contribute toadrenocortical tumorigenesis.


Genome-wide localization of menin and assocated histone methytransferase complex members. P.C. Scacheri1, S.Davis1, D.T. Odom2, S. Perkins1, A.M. Spiegel3, P.S. Meltzer1, F.S. Collins1. 1) NHGRI, Bethesda, MD; 2) WhiteheadInstitute, Cambridge, MA; 3) NIDCD, Bethesda, MD.

Multiple Endocrine Neoplasia, type I (MEN1) is a familial cancer syndrome characterized primarily by multipletumors of the endocrine glands. The gene for MEN1 encodes a ubiquitously expressed tumor suppressor protein calledmenin. Menin was recently shown to interact with several components of a trithorax family histone methyltransferasecomplex including ASH2, Rbbp5, WDR5, and the leukemia proto-oncoprotein MLL. These findings suggested a rolefor menin in transcriptional regulation, potentially mediated through covalent modification of histone H3 at lysine 4. Togain insights into menin's role as a tumor suppressor, we determined the genomic occupancy of menin, MLL, andRbbp5, using a strategy that couples chromatin immunoprecipitation with DNA microarray technology (ChIP-chip). Themicroarrays used in this approach were custom designed to contain high density oligonucleotide tiling paths across20,000 human promoter segments. Given that MLL is reported to regulate homeobox (HOX) transcription, these arraysalso harbored tiling paths across all four HOX clusters. Our data in HeLa cells indicate that menin, MLL, and RBBP5co-localize with RNA polymerase II to transcriptional start sites, where histone H3 lysine 4 trimethylation occurs.Binding to the HOX clusters is strikingly different, with broad footprints that extend across inter- and intragenic regionsof the HOX A and C clusters. To gain insights to the endocrine-specific tumor phenotype in MEN1, we compared thesebinding sites to those we mapped in primary human pancreatic islets, a common site of tumor formation in MEN1patients. Menin was found to bind to a similar set of target genes in pancreatic islets, but with notable exceptions at theHOX clusters and elsewhere. Targets bound by menin exclusively in islet cells include key transcription factorsinvolved in pancreatic endocrine development and insulin regulation. We suggest that absence of menin initiatestumorigenesis by mediating epigenetic changes in chromatin structure, potentially at the promoters of these keyendocrine-specific genes.


Expression profiling identifies similar expression pattern of uveal melanomas with chromosome 3 isodiosmy tothose with monosomy 3. M. Abdel-Rahman1,2, Y. Yang2, F. Davidorf2, C. Eng1. 1) Clinical Cancer Genetics Prog,Ohio State Univ, Columbus, OH; 2) Department of Ophthalmology, Ohio Sate Univ, Columbus, OH.

Purpose: Monosomy of chromosome 3 (M3) is the most frequent somatic alteration in uveal melanomas (UMs). M3has been suggested as a useful marker for detection of aggressive UMs. Contrary to earlier reports, recent studiesindicate a high frequency of partial chromosome 3 deletion in UMs. Moreover, chromosome 3 isodisomy has beenreported in up to 16% of UMs without M3. The aim of the study is to identify the expression pattern associated withchromosome 3 isodisomy and partial chromosome 3 alterations compared to M3 in UMs. Methods: We used acombination of comparative genomic hybridization (CGH), high resolution genotyping utilizing 38 microsatellitemarkers on chromosome 3 and gene expression profiling to study 13 primary UMs. The frequency of partialchromosome 3 alterations was studied in a total of 47 UMs. Results: 7/13 UMs with available CGH data and expressionprofiling showed M3. In addition 2/13 showed isodiomy of chromosome 3 indicated by minimal or no alteration ofchromosome 3 by CGH and LOH of most of the markers on both chromosome 3 arms. The remaining 4/13 UMsshowed partial gains and/or loss of chromosome 3. All samples with partial chromosome 3 alterations were confirmedby genotyping. Expression profiling identified similar expression patterns of samples with chromosome 3 isodisomy tothose with M3. Expression profiling identified several regions of common decreased expression on both chromosomearms 3p and 3q. Finally we identified a high frequency (22/47) of UMs with partial chromosome 3 alterations.Conclusions: Our data have revealed similar expression patterns of UMs with chromosome 3 isodisomy to those withM3, suggesting that isodisomy 3 may portend similar poor prognosis as M3. These results also indicate that partialchromosome 3 alterations are under-diagnosed in UMs. The clinical significance of partial chromosome 3 alterationswill need to be further explored. Taken together, our results are important in designing clinical diagnostic strategies forthe detection of chromosome 3 alterations in UMs patients.


Advanced genetic analyses on migraine: Trait component analysis and mouse model expression study. M.Wessman1,2, G. Oswell3, M. Kallela4, V. Anttila1, M. Kaunisto1, P. Tikka1, E. Hämäläinen1, D. Nyholt5, J. Terwilliger6,J. Kaprio7, M. Färkkilä4, L. Peltonen8, A. Palotie1. 1) Finnish Genome Center and Dept of Clin Chem, Univ Helsinki,Finland; 2) Folkhälsan Research Center, Helsinki, Finland; 3) Dept of Pathology, UCLA, USA; 4) Dept of Neurology,Helsinki University Central Hospital, Finland; 5) Queensland Inst of Med Research, Brisbane, Australia; 6) ColumbiaGenome Center, Columbia University, NYC, USA; 7) Dept of Public Health, Univ Helsinki, Finland; 8) Dept of HumanMolecular Genetics, National Public Health Institute, Helsinki, Finland.

Migraine is a common chronic severe headache disorder with a strong genetic component and a prevalence of ~ 15%in the Caucasian population. We have collected nearly 700 multiple case families and based the diagnosis on theInternational Headache Society (IHS) criteria. This enables us to use medium-to-large size families for genome-widelinkage analysis and to perform case control association studies. Our previous genome-wide linkage study identified alocus on 4q24 linked to the endpoint diagnosis migraine with aura. We subsequently performed a trait componentlinkage analysis to test which IHS trait components provide best evidence of linkage to the 4q24 locus and whetheradditional susceptibility regions could be localized. Strongest evidence of linkage to 4q24 was achieved withunilaterality (parametric two-point LOD score 4.20), photophobia (3.73) and phonophobia (3.52). Pulsation was linkedto 17p13 (4.65) and the IHS C-criteria to 18q12 (3.27). To identify positional candidate genes we performed wholetranscriptome expression analysis with the Affymetrix 430 2.0 GeneChip on primary cultures of neurons and glial cellsfrom the tottering mouse with a mutation in Cacna1a. Using a novel pathway identifying algorithm we identified 238genes comprising 108 GeneOntology pathways as being differentially regulated including Ca2+ channel genes Cacna1band Cacng7, Na+/K+ pumps Atp1a1 and 2, Atp1b1, 2 and 3. We also identified several genes in our Chr 4 and Chr 17restricted regions. Candidate genes identified in the mouse expression study are to be tested in our case control sampleof 1800 subjects.


A common non-synonymous SNP in the BRI3 (ITM2C) gene accounts for a significant portion of the linkageevidence for Parkinson disease on chromosome 2q. N. Pankratz1, L. Byder2, C.A. Halter1, A. Rudolph3, C.W.Shults4,5, P.M. Conneally1, T. Foroud1, W.C. Nichols2, Parkinson Study Group-PROGENI Investigators. 1) Medical &Molec Genetics, Indiana Univ Sch Medicine, Indianapolis, IN; 2) Children's Hospital Research Foundation, Cincinnati,OH; 3) Univ Rochester, Rochester, NY; 4) Univ California, San Diego, CA; 5) VA San Diego Healthcare System, SanDiego, CA.

Previously, we identified significant linkage to chromosome 2q35 using a sample of individuals with familialParkinson disease (PD). The linkage was strongest among those families with verified PD and those with a strongerfamily history (four or more affected or an affected sibpair with an affected parent). The disease allele appeared to beinherited in an autosomal dominant fashion, and penetrance appeared to be high in those individuals with a strongfamily history. Subsequently, we have genotyped six additional markers in the linkage region. With these additionalmarkers, the autosomal dominant LOD score for the subset remained significant, and the non-parametric linkageevidence of the verified PD sample increased from a LOD score of 2.5 to 4.0. One candidate gene in this region is BRI3(ITM2C), which belongs to a family of integral membrane proteins. One of its homologs (BRI2) is associated withfamilial dementia. Little is known about BRI3, except that it is highly enriched in human brain and that it interacts withBACE1, an enzyme responsible for converting beta-amyloid, a protein involved in Alzheimer disease pathogenesis.When we screened our PD sample for mutations in the BRI3 gene, we identified a rare non-synonymous point mutation(Arg236Trp) in one of our patients, as well as a non-synonymous polymorphism (Gly53Ser) that appears to be commonin the general population (allele frequency = 0.40). Using the Genotype-IBD Sharing Test (GIST) under the samehypothesis of autosomal dominant inheritance, we have shown that this common SNP accounts for a significant portionof the linkage to chromosome 2q (p=0.03). It is therefore possible that this polymorphism is a common PDsusceptibility gene with low penetrance (similar to APOE for Alzheimer disease).


High-density linkage screen in multiple sclerosis. J. Haines, for the International Multiple Sclerosis GeneticsConsortium. Ctr Human Genetics, Vanderbilt Univ Medical Ctr, Nashville, TN.

Ten centimorgan microsatellite map have been the standard tool used for whole genome linkage screening since themid 1990s and to date 11 screens employing this methodology have been published in multiple sclerosis. Inspection ofthese data shows that average genotyping success and information extraction rates are disappointing (80% and 44%respectively). Moreover available evidence suggests that genotyping errors rates in these studies (particularly the olderones) are likely to be > 1% for many if not most of the markers typed. The advent of high throughput cost effective SNPgenotyping has provided new whole genome linkage screening tools promising significantly improved genotypingsuccess rates and accuracy, which at high density should substantially enhance information extraction. Prompted by thepotential benefits of these new tools we have typed the Illumina BeadArray linkage mapping panel (version 4) in a setof 730 multiplex families from Australia (97), Scandinavia (165), the United Kingdom (298) and the United States(170), which together provide 945 affected relative pairs (830 sib pairs, 14 half sib pairs, 54 cousin pairs and 47avunclular pairs). After excluding 474 markers with genotyping success rates of 0.16) data is available from 4506 markers spanning the genome.A total of 2709 samples have been typed. The mean information extraction is 79.3% (range 42.4 - 91.3%) and theobserved Mendelian inconsistencies suggest that within this data the genotyping error rate is just 0.002%. Highlysignificant linkage is observed in the region of the MHC (MLS 11.7) while suggestive linkage is seen on chromosomes17q23 and 5q33. This screen provides the most definitive linkage map for multiple sclerosis currently available andillustrates the substantial increase in power that can be achieved using high density SNP linkage mapping sets. It is clearthat future attempts to identify non-MHC susceptibility genes in multiple sclerosis will have to involve large samplesizes and association based methodology.


Homozygosity mapping with high-density SNP arrays identifies a novel gene for Bardet-Biedl Syndrome. A.P.Chiang1, J.S. Beck1,2, A.L. Ferguson1, K. Elbedour3, R. Carmi3, H.-J. Yen1,2, M.K. Tayeh1,2, J. Wei1,2, D.Y.Nishimura1, T.A. Braun1, E.M. Stone1,2, T.L. Casavant1, V.C. Sheffield1,2. 1) University Iowa, Iowa City, IA, USA; 2)Howard Hughes Medical Institute, Iowa City, IA, USA; 3) Genetic Institute, Ben Gurion University of the Negev, Beer-Sheva, Israel.

The identification of mutations in genes that cause human diseases has largely been accomplished through the use ofpositional cloning, which relies on linkage mapping. In studies of rare diseases, the resolution of linkage mapping islimited by the number of meioses and marker density. One recent technological advance is the development of highdensity Single Nucleotide Polymorphism (SNP) microarrays for genotyping. The SNP arrays overcome low markerinformativity by using a large number of markers to achieve greater coverage at finer resolution. To determine the utilityof this technology for homozygosity mapping using small pedigrees, we genotyped a small consanguineous IsraeliBedouin (AT) family with autosomal recessive Bardet-Biedl Syndrome (BBS; obesity, pigmentary retinopathy,polydactyly, hypognoadism, renal and cardiac abnormalities, and cognitive impairment). While eight genes have beenidentified to cause BBS, over half of BBS patients have unknown genetic defects, leaving the possibility of additionalgenes, that when mutated, cause BBS. The AT family was a good candidate for homozygosity mapping with highdensity SNP arrays because it was not linked to any of the known BBS loci and a genome-wide scan with microsatellitemarkers at ~10 cM density did not reveal a linked locus. DNA from all four individuals affected with BBS from thefamily was hybridized to the HindIII chip of the Affymetrix 100K SNP chip set and the resulting data analyzed forregions of homozygosity. Regions of homozygosity were prioritized based on the physical distance, the number ofconsecutive homozygous SNPs, and marker density. Mutation analysis in the best candidate homozygosity regionidentified a gene with a conserved homozygous missense mutation. Functional analysis of this gene in a zebrafishsystem provides additional evidence that this is a BBS gene (BBS9).


Genomeutwin-European twin cohorts: Combined analysis of genome scans guide to two QTL:s for body statureincluding one common Caucasoid X-chromosomal locus. L. Peltonen1, S. Sammalisto1, T. Hiekkalinna1, N. Martin2,J. Harris3, D. Boomsma4, K. Christensen5, K. Ohm Kyvik5, N. Pedersen6, T. Andrew7, T. Spector7, E. Widen8, A.Palotie8, M. Perola1, GenomEUtwin. 1) Dept Med Genetics & Molec Med, Univ Helsinki & Nat Pub Hth In, Helsinki,Finland; 2) Queensland Institute of Medical Research, Brisbane, Australia; 3) Norwegian Institute of Public Health,Oslo; 4) Free University, Amsterdam, Netherlands; 5) University of Southern Denmark, Odense, Denmark; 6)Karolinska Institutet, Stockholm, Sweden; 7) St Thomas Hospital, London, United Kingdom; 8) University of Helsinki,Finnish Genome Center, Finland.

Twin cohorts provide a unique advantage for investigations of the role of genetics and environment in the etiology ofcommon traits. Co-twins share environment throughout critical fetal period and early years of life and twin designharmonizes this component of complex traits in a unique manner. The EU-funded GenomEUtwin(www.genomeutwin.org) consortium consists of eight twin cohorts (Australian, Danish, Dutch, Finnish, Italian,Norwegian, UK and Swedish) with the total number of 600000 twin pairs. Federated database with open source codehas been created to share the data across the cohorts. We performed QTL analysis of stature (body height) usinggenome-wide scans performed for 8775 individuals: Australia (n=3730), Denmark (628), Finland (772), Netherlands(1313), Sweden (102) and United Kingdom (2230). The marker maps were combined and related to the sequencepositions using computer program developed by us which uses DeCode genetic map markers as an anchoring set(www.bioinfo.helsinki.fi/cartographer). We used the program Merlin for variance components analysis with age, sexand country-of-origin as covariates. The covariate adjusted heritability was 82% for stature in the pooled data set. Wefound evidence for two major QTLs for human stature on 15q24 (LOD=3.75, 1-lod drop 11cm) and Xq25 (LOD=2.73,15cM). Especially the evidence for linkage for the X-chromosomal locus is contributed by most of the cohorts, thussuggesting an evolutionally old genetic variant having effect on the growth in European-based populations.


Evidence for association between hepatic nuclear factor 4, alpha and triglycerides in Finnish familial combinedhyperlipidemia families. D. Weissglas1, E. Suviolahti1,2, J. Lee1, A. Jokiaho1, M-R. Taskinen3, P. Pajukanta1. 1)Department of Human Genetics, UCLA, Los Angeles, CA; 2) Department of Molecular Medicine, National PublicHealth Institute, Biomedicum, Helsinki, Finland; 3) Department of Medicine, University of Helsinki, Finland.

Familial combined hyperlipidemia (FCHL), characterized by increased levels of serum total cholesterol, triglycerides(TGs) or both, is observed in up to 20% of patients with premature coronary heart disease. Previously we identified alocus linked to elevated TGs on chromosome 20q13.11 in Finnish FCHL families. Numerous linkage studies of type 2diabetes mellitus (T2DM) have also found evidence of linkage to this region. Recently, several independent groupsidentified associations between single nucleotide polymorphisms (SNPs) in the hepatic nuclear factor 4, alpha (HNF4A)gene on 20q12-q13.1 and T2DM in Finnish and Ashkenazi Jews. Because there is a clear phenotypic overlap betweenFCHL and T2DM, we tested this gene region for association with FCHL in Finnish families. To date no associationbetween plasma lipid levels and HNF4A has been identified in FCHL. We constructed the linkage disequilibrium (LD)structure of the HNF4A region by using both the HapMap data and recently published Finnish LD data. Based on thatinformation, we genotyped the seven SNPs, previously associated with T2DM, and three additional tag-SNPs fromdistinct haplotype blocks to capture most of the genetic variation within the HNF4A gene, including promoters 1 and 2.A SNP in promoter 2 of the HNF4A gene was significantly associated with high TGs (P=0.007) in 720 family membersof 60 FCHL families. Haplotype analyses also showed significant associations with TGs as well as with plasmaapolipoprotein B and high-density lipoprotein levels for several HNF4A haplotypes. The most significant haplotype forhigh TGs (P=0.008) has a frequency of 0.289 in the Finnish FCHL families. In conclusion, this study is the first toprovide significant evidence for association between the intragenic variants of the HNF4A gene and plasma lipid levelsin FCHL.


A new method to correct for population stratification in genetic case-control association studies: centralizing thenon-central chi-square. P. Gorroochurn1, G.A. Heiman2, S.E. Hodge1,3, D.A. Greenberg1,3. 1) Division of StatisticalGenetics,Dept Biostatistics, Columbia Univ, New York, NY; 2) Department of Epidemilogy, Columbia Univ, NewYork, NY; 3) Clinical-Genetic Epidemiology Unit, New York State Psychiatric Institute, New York, NY.

Recently, several authors have investigated the use of genomic information in an attempt to eliminate bias due topopulation stratification (PS) in case-control association studies. We here present a new method, the delta-centralization(DC) method, to correct for PS. DC works well even when there is a lot of bias due to PS. In the presence of PS, theusual chi-square statistics used to test for association have non-central chi-square distributions. Other methods approachthe non-centrality indirectly, but we deal with it directly, by estimating the non-centrality parameter itself. Specifically:(1) We define a quantity delta, a function of the relevant subpopulation parameters, that exactly predicts (in relativelylarge samples) the elevation of the false positive rate due to PS, when there is no true association between markergenotype and disease. (Delta is quite different from F_ST and can be large even when F_ST is small.) (2) We show howto estimate delta, using a panel of unlinked neutral loci. (3) We show that the square of delta corresponds to the non-centrality parameter of the chi-square distribution. Thus, we can centralize the chi-square using our estimate of delta;this is the DC method. (4) We demonstrate, via computer simulations, that DC works well with as few as 25-30unlinked markers, where the markers are chosen to have allele frequencies reasonably close (within 0.1) to those at thetest locus. (5) We compare DC with genomic control and show that whereas the latter becomes over-conservative whenthere is considerable bias due to PS (i.e. when delta is large), DC performs well for all values of delta.


A Spatial Clustering Approach to the Analysis of Genetic Association Studies. E.R.B. Waldron1, J.C. Whittaker1,2,D.J. Balding1. 1) Epidemiology and Public Health, Imperial College London, London, United Kingdom; 2)Epidemiology and Population Health, London School of Hygiene and Tropical Medicine, London.

Haplotype-based approaches to the analysis of genetic association studies have important advantages over single-marker analyses, but they nevertheless suffer from limitations. For instance, rare haplotypes may not analysedeffectively. Furthermore, the relationships between distinct but similar haplotypes that may share recent commonancestry are often not accounted for. We have developed an algorithm based on spatial statistics techniques, employinga simple distance in haplotype space that reflects evolutionary processes. The algorithm searches for case-rich clustersof similar haplotypes. Membership of this cluster corresponds to predicting the allele at an unobserved causal SNP. Thealgorithm can be applied to fine-mapping if the distance metric depends on the putative location of a causal allele. Usingthis algorithm to analyse data for the CYP2D6 gene, for which the true causal polymorphism is fully characterised, wecorrectly predicted nearly 98% of the genotypes at the major causal polymorphism and the functional mutation wasmapped accurately. Simulation studies also revealed consistently better performance than alternative fine-mappingalgorithms and allowed us to identify situations in which multi-point approaches offer a substantial improvement oversingle-point analyses.


Admixture-Matched Case-Control Study: A Practical Approach for Genetic Association Studies in AdmixedPopulations. J. Kho1,2, H.J. Tsai1,2, N. Shaikh1,2, S. Choudry1,2, M. Navqi1,2, D. Navarro1,2, H. Matallana1,2, R.Castro1,2, C. Lilly3, H.G. Watson4, K. Meade5, M.L. Noir6, S. Thyne1, E. Ziv1, E.G. Burchard1,2. 1) UCSF, SanFrancisco, CA; 2) Lung Biology Center, San Francisco General Hospital, SF, CA; 3) Brigham and Womens Hospital,Boston, MA; 4) The James A. Watson Wellness Center, Oakland, CA; 5) Children's Hospital and Research Institute,Oakland, CA; 6) Bay Area Pediatrics, Oakland, CA.

Case-control genetic association studies in admixed populations are known to be susceptible to genetic confoundingdue to population stratification. The transmission/disequilibrium test (TDT) approach can avoid this problem. But, theTDT is expensive and impractical for late-onset diseases. Case-control study designs, in which cases and controls arematched by admixture, can be an appealing alternative for genetic association studies in admixed populations. Weapplied this matching strategy when recruiting our African American participants in the Study of African American,Asthma, Genes and Environments (SAGE). Group admixture in this cohort consists of 83% African ancestry and 17%European ancestry. By carrying out several complementary analyses, our results show that there is substructure, but thatthe admixture distributions are almost identical in cases and controls, and also in cases only. We performed associationtests for asthma-related traits with ancestry, and only found that FEV1, a measure of asthma severity, was associatedwith ancestry after adjusting for socio-economic and environmental factors (p=0.01). We did not observe an excess oftype I error rate in our association tests for ancestry informative markers and asthma-related traits when ancestry wasnot adjusted in the analyses. Furthermore, using the association tests between genetic variants in a known asthmacandidate gene, 2 adrenergic receptor (2AR) and FEF25-75, a measure of bronchodilator drug responsiveness to theasthma medication, albuterol, as an example, we showed population stratification was not a confounder in our study.Our work demonstrates that admixture-matched case-control strategies can efficiently control for populationstratification confounding in admixed populations.


Analysis of fine structure recombination patterns in a human chromosomal region not previously known toharbor a recombination hot spot. I. Tiemann-Boege1, P. Calabrese1, D.M. Cochran1, R. Sokol2, N. Arnheim1. 1)Molecular and Computational Biology Program, University of Southern California, Los Angeles, CA; 2) Health ScienceCenter, University of Southern California, Los Angeles, CA.

For decades, evidence has suggested that crossing over is not homogenous across the human genome but can behighly localized in hot spots. Recent molecular studies of regions known to contain a hot spot based on classicalmethods have revealed that hot spots can be present where strong linkage disequilibrium (LD) is interrupted by patternsof very low LD. Whether recombination is generally restricted to hot-spots and whether hot spots can be found in largerchromosomal regions without exceptional frequencies of crossing over is yet to be addressed. Using sperm typing thatselectively amplifies only recombinants in a pool of sperm genomes, we analyzed the recombination fractions in smallcontiguous intervals (~5 kb) of a 104 kb region of human chromosome 21 that showed no exceptional crossoveractivity. The observed recombination intensity ranges from 0.10 (95% confidence interval: 0.01, 0.29) to 12.21 (9.67,15.05) times the human genome average rate. We identified two hot-spots with a recombination activity of 11.17 (9.98,12.42) and 12.21 (9.67, 15.05) times the human average and four regions with a frequency ranging between 0.93 (0.59,1.33) and 3.62 (3.09, 4.19) fold human average. The remaining regions all have an estimated frequency below 0.55times the human average (all have a 95% upper bound below 0.84). The recombination patterns observed by spermtyping were compared with the patterns predicted by LDHat, Hotspotter and a third algorithm developed by one of us(P.C.). Patterns are roughly congruent with the recombination inferred from the population estimators with someexceptions. Our data suggests that it is likely that hotspots are usually found in larger chromosomal regions withoutexceptional frequencies of crossing over. Clearly, studies on other chromosomal intervals are needed to get a bettergenome-wide picture of the extent to which recombination is generally restricted to hot-spots and the degree to whichLD analyses can predict recombination intensities.


Improved Association Analyses of Disease Subtypes in Case-Parent Triads. M.P. Epstein1, I.D. Waldman2, G.A.Satten3. 1) Dept Human Genetics, Emory Univ, Atlanta, GA; 2) Dept Psychology, Emory Univ, Atlanta, GA; 3) Centersfor Disease Control and Prevention, Atlanta, GA.

The sampling of case-parent triads is an appealing strategy for conducting association analyses of complex diseases.In certain situations, one may have interest in using the triads to identify genetic variants that are associated with aspecific subtype of disease, perhaps related to severity of symptoms or sensitivity to medication. A straightforwardstrategy for conducting such a subtype analysis would be to analyze only those triads with the subtype of interest. Whilesuch a strategy is valid, we show that triads without the subtype provide additional genetic information that increasespower to detect association with the subtype. We incorporate this additional information using a likelihood-basedframework that permits flexible modeling and estimation of allelic effects on disease subtypes and also allows formissing parental data. Using simulated data under a variety of genetic models, we show that our proposed associationtest consistently outperforms association tests that only analyze subtype triads. We also apply our method to a triadstudy of attention-deficit hyperactivity disorder and identify a genetic variant in the dopamine transporter gene that isassociated with a hyperactive-impulsive subtype.


Estimating genetic effects from genomewide association scan data. S. Zollner, J. Pritchard. Dept Human Genetics,Univ Chicago, Chicago, IL.

Genomewide association studies are a powerful method to detect loci harboring variants that affect complex traits.After a successful scan for association it is of great interest to estimate the impact of a detected variant on the phenotypeof a carrier (penetrance) and on the population as a whole (frequency). These estimates allow to assess the importanceof a mutation, they may provide information about its biological effect and facilitate planning replication studies. Asthese estimations are usually performed based on the same data that were used to identify a variant, the results areaffected by ascertainment bias, causing the genetic effects of a variant to be overestimated if not corrected for. Thisoverestimation is considered the main reason that many replication studies fail as the sample size needed isunderestimated. The magnitude of this bias depends on the power of the initial association study. Here, we present anapproach that corrects for the ascertainment effect and generates an approximate maximum-likelihood estimate of thefrequency of a variant and its penetrance parameters. The method produces both a point-estimate and a confidenceregion for all parameters. We apply this estimator to simulated datasets and study its performance and its ability todistinguish between different models of penetrance. We show that by taking epidemiological data into account, it isoften possible to obtain fairly precise estimates of all parameters, even if the power of the genome scan is low. We alsoshow that the error of the estimate decreases with sample size, independent of the power of the original test forassociation. Finally, we demonstrate that the same algorithm can be used to assess interactions between a genetic variantand environmental risk factors.


Regression-Based QTL analysis method incorporating parent-of-origin effect. O. Gorlova, L. Lei, D. Zhu, Y.Zhang, W. Li, S. Shete, R.A. Price, C. Amos. Dept Epidemiology, Univ Texas MD Anderson CA Ctr, Houston, TX.

We present an extension of Sham et al.s (2002) regression-based quantitative-trait linkage analysis method toincorporate parent-of-origin effects. We separately regressed total, paternal, and maternal IBD sharing on traits squaredsums and differences. We also developed a test for imprinting that indicates whether there is any difference betweenpaternal and maternal regression. We use a panel of statistics to detect imprinting, which includes an overall T statistic(a test for total linkage), both parental T statistics (tests for parental-specific linkages), and the D index (a test forimprinting). We performed an extensive simulation to examine the performance of the panel. We found that when usingempirical percentiles the method is very powerful in detecting parent-specific linkage with correct type I error rate forthe non-linked parental component. Missing parental genotypes increase the type I error rate of both the linkage andimprinting tests and decreases the power of the imprinting test. When the major gene has low heritability, the power ofthe method decreases dramatically but the panel still performs well. We also used a permutation algorithm, which canensure the appropriate type I error rate. We applied the method to a data from a study of 6 body size related measuresand 23 loci on chromosome 7 for 255 nuclear families. Multivariate identities-by-descent were obtain using amodification of SIMWALK program. A parent-of-origin effect consistent with maternal imprinting was suggested at99.67-111.26 Mb for body mass index, bioelectrical impedance analysis, waist circumference, and leptin concentration.

Copyright © 2005 The American Soci

Documents

HIGH ABUNDANCE mtDNA RECOMBINANTS IN HETEROPLASMIC … · 2019-10-22 · of HOXA1 function. Our results demonstrate a new function for HOXA1 in vascular patterning that had not been