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1.  Genome-wide copy number variation study associates metabotropic glutamate receptor gene networks with attention deficit hyperactivity disorder 
Nature genetics  2011;44(1):78-84.
Attention deficit hyperactivity disorder (ADHD) is a common, heritable neuropsychiatric disorder of unknown etiology. We performed a whole-genome copy number variation (CNV) study on 1,013 cases with ADHD and 4,105 healthy children of European ancestry using 550,000 SNPs. We evaluated statistically significant findings in multiple independent cohorts, with a total of 2,493 cases with ADHD and 9,222 controls of European ancestry, using matched platforms. CNVs affecting metabotropic glutamate receptor genes were enriched across all cohorts (P = 2.1 × 10−9). We saw GRM5 (encoding glutamate receptor, metabotropic 5) deletions in ten cases and one control (P = 1.36 × 10−6). We saw GRM7 deletions in six cases, and we saw GRM8 deletions in eight cases and no controls. GRM1 was duplicated in eight cases. We experimentally validated the observed variants using quantitative RT-PCR. A gene network analysis showed that genes interacting with the genes in the GRM family are enriched for CNVs in ~10% of the cases (P = 4.38 × 10−10) after correction for occurrence in the controls. We identified rare recurrent CNVs affecting glutamatergic neurotransmission genes that were overrepresented in multiple ADHD cohorts.
PMCID: PMC4310555  PMID: 22138692
2.  Association of variants of the interleukin-23 receptor (IL23R) gene with susceptibility to pediatric Crohn’s disease 
Background & Aims
Recently an association was demonstrated between the single nucleotide polymorphism (SNP), rs11209026, within the interleukin-23 receptor (IL23R) locus and Crohn’s disease (CD) as a consequence of a genome wide association study of this disease in adults. We examined the effects of this and other previously reported SNPs at this locus with respect to CD in children.
Utilizing data from our ongoing genome-wide association study in our cohort of 142 pediatric CD cases and 281 matched controls, we investigated the association of the previously reported SNPs at the IL23R locus with the childhood form of this disease.
Using a Fisher’s exact test, the minor allele frequency (MAF) of rs1120902 in the cases was 1.75% while it was 6.61% in controls, yielding a protective odds ratio (OR) of 0.25 (95% CI 0.10 – 0.65; one-sided P = 9.2×10−4). Furthermore, of all the SNPs previously reported, rs11209026 was the most strongly associated. A subsequent family-based association test (which is more resistant to population stratification) with 65 sets of trios derived from our initial patient cohort yielded significant association with rs11209026 in a transmission disequilibrium test (one-sided P=0.0017). In contrast, no association was detected to the CARD15 gene for the IBD phenotype.
The OR of the IL23R variant in our pediatric study is highly comparable with that reported previously in a non-Jewish adult IBD case-control cohort (OR=0.26). As such, variants in IL23R gene confer a similar magnitude of risk of CD to children as for their adult counterparts.
PMCID: PMC4287202  PMID: 17618837
IL23R; gene; association; Crohn’s Disease
3.  A candidate gene approach to identify modifiers of the palatal phenotype in 22q11.2 deletion syndrome patients 
Palatal anomalies are one of the identifying features of 22q11.2 deletion syndrome (22q11.2DS) affecting about one third of patients. To identify genetic variants that increase the risk of cleft or palatal anomalies in 22q11.2DS patients, we performed a candidate gene association study in 101 patients with 22q11.2DS genotyped with the Affymetrix genome-wide human SNP array 6.0.
Patients from Children's Hospital of Philadelphia, USA and Wilhelmina Children's Hospital Utrecht, The Netherlands were stratified based on palatal phenotype (overt cleft, submucosal cleft, bifid uvula). SNPs in 21 candidate genes for cleft palate were analyzed for genotype-phenotype association. In addition, TBX1 sequencing was carried out. Quality control and association analyses were conducted using the software package PLINK.
Genotype and phenotype data of 101 unrelated patients (63 non-cleft subjects (62.4%), 38 cleft subjects (37.6%)) were analyzed. A Total of 39 SNPs on 10 genes demonstrated a p-value ≤0.05 prior to correction. The most significant SNPs were found on FGF10. However none of the SNPs remained significant after correcting for multiple testing.
Although these results are promising, analysis of additional samples will be required to confirm that variants in these regions influence risk for cleft palate or palatal anomalies in 22q11.2DS patients.
PMCID: PMC4098838  PMID: 23121717
22q11.2 deletion syndrome; Cleft palate; Candidate gene
4.  Meta-Analysis of Genome-Wide Scans Provides Evidence for Sex- and Site-Specific Regulation of Bone Mass 
Several genome-wide scans have been performed to detect loci that regulate BMD, but these have yielded inconsistent results, with limited replication of linkage peaks in different studies. In an effort to improve statistical power for detection of these loci, we performed a meta-analysis of genome-wide scans in which spine or hip BMD were studied. Evidence was gained to suggest that several chromosomal loci regulate BMD in a site-specific and sex-specific manner.
BMD is a heritable trait and an important predictor of osteoporotic fracture risk. Several genome-wide scans have been performed in an attempt to detect loci that regulate BMD, but there has been limited replication of linkage peaks between studies. In an attempt to resolve these inconsistencies, we conducted a collaborative meta-analysis of genome-wide linkage scans in which femoral neck BMD (FN-BMD) or lumbar spine BMD (LS-BMD) had been studied.
Materials and Methods
Data were accumulated from nine genome-wide scans involving 11,842 subjects. Data were analyzed separately for LS-BMD and FN-BMD and by sex. For each study, genomic bins of 30 cM were defined and ranked according to the maximum LOD score they contained. While various densitometers were used in different studies, the ranking approach that we used means that the results are not confounded by the fact that different measurement devices were used. Significance for high average rank and heterogeneity was obtained through Monte Carlo testing.
For LS-BMD, the quantitative trait locus (QTL) with greatest significance was on chromosome 1p13.3-q23.3 (p = 0.004), but this exhibited high heterogeneity and the effect was specific for women. Other significant LS-BMD QTLs were on chromosomes 12q24.31-qter, 3p25.3-p22.1, 11p12-q13.3, and 1q32-q42.3, including one on 18p11-q12.3 that had not been detected by individual studies. For FN-BMD, the strongest QTL was on chromosome 9q31.1-q33.3 (p = 0.002). Other significant QTLs were identified on chromosomes 17p12-q21.33, 14q13.1-q24.1, 9q21.32-q31.1, and 5q14.3-q23.2. There was no correlation in average ranks of bins between men and women and the loci that regulated BMD in men and women and at different sites were largely distinct.
This large-scale meta-analysis provided evidence for replication of several QTLs identified in previous studies and also identified a QTL on chromosome 18p11-q12.3, which had not been detected by individual studies. However, despite the large sample size, none of the individual loci identified reached genome-wide significance.
PMCID: PMC4016811  PMID: 17228994
osteoporosis; BMD; linkage; meta-analysis; genome search; genome scan
5.  Evidence From Human and Zebrafish That GPC1 Is a Biliary Atresia Susceptibility Gene 
Gastroenterology  2013;144(5):1107-1115.e3.
Biliary atresia (BA) is a progressive fibroinflammatory disorder of infants involving the extrahepatic and intrahepatic biliary tree. Its etiology is unclear but is believed to involve exposure of a genetically susceptible individual to certain environmental factors. BA occurs exclusively in the neonatal liver, so variants of genes expressed during hepatobiliary development could affect susceptibility. Genome-wide association studies previously identified a potential region of interest at 2q37. We continued these studies to narrow the region and identify BA susceptibility genes.
We searched for copy number variants that were increased among patients with BA (n = 61) compared with healthy individuals (controls; n = 5088). After identifying a candidate gene, we investigated expression patterns of orthologues in zebrafish liver and the effects of reducing expression, with morpholino antisense oligonucleotides, on biliary development, gene expression, and signal transduction.
We observed a statistically significant increase in deletions at 2q37.3 in patients with BA that resulted in deletion of one copy of GPC1, which encodes glypican 1, a heparan sulfate proteoglycan that regulates Hedgehog signaling and inflammation. Knockdown of gpc1 in zebrafish led to developmental biliary defects. Exposure of the gpc1 morphants to cyclopamine, a Hedgehog antagonist, partially rescued the gpc1-knockdown phenotype. Injection of zebrafish with recombinant Sonic Hedgehog led to biliary defects similar to those of the gpc1 morphants. Liver samples from patients with BA had reduced levels of apical GPC1 in cholangiocytes compared with samples from controls.
Based on genetic analysis of patients with BA and zebrafish, GPC1 appears to be a BA susceptibility gene. These findings also support a role for Hedgehog signaling in the pathogenesis of BA.
PMCID: PMC3736559  PMID: 23336978
GWA; Susceptibility Loci; Animal Model; Bile Duct Growth and Development
6.  Replication of GWAS-identified neuroblastoma risk loci strengthens the role of BARD1 and affirms the cumulative effect of genetic variations on disease susceptibility 
Carcinogenesis  2012;34(3):605-611.
Several neuroblastoma (NB) susceptibility loci have been identified within LINC00340, BARD1, LMO1, DUSP12, HSD17B12, DDX4, IL31RA, HACE1 and LIN28B by genome-wide association (GWA) studies including European American individuals. To validate and comprehensively evaluate the impact of the identified NB variants on disease risk and phenotype, we analyzed 16 single nucleotide polymorphisms (SNPs) in an Italian population (370 cases and 809 controls). We assessed their regulatory activity on gene expression in lymphoblastoid (LCLs) and NB cell lines. We evaluated the cumulative effect of the independent loci on NB risk and high-risk phenotype development in Italian and European American (1627 cases and 2575 controls) populations. All NB susceptibility genes replicated in the Italian dataset except for DDX4 and IL31RA, and the most significant SNP was rs6435862 in BARD1 (P = 8.4×10–15). BARD1 showed an additional and independent SNP association (rs7585356). This variant influenced BARD1 mRNA expression in LCLs and NB cell lines. No evidence of epistasis among the NB-associated variants was detected, whereas a cumulative effect of risk variants on NB risk (European Americans: P trend = 6.9×10–30, Italians: P trend = 8.55×1013) and development of high-risk phenotype (European Americans: P trend = 6.9×10–13, Italians: P trend = 2.2×10–1) was observed in a dose-dependent manner. These results provide further evidence that the risk loci identified in GWA studies contribute to NB susceptibility in distinct populations and strengthen the role of BARD1 as major genetic contributor to NB risk. This study shows that even in the absence of interaction the combination of several low-penetrance alleles has potential to distinguish subgroups of patients at different risks of developing NB.
PMCID: PMC3716226  PMID: 23222812
7.  Overt Cleft Palate Phenotype and TBX1 Genotype Correlations in Velo-cardio-facial/DiGeorge/22q11.2 Deletion Syndrome Patients 
Velo-cardio-facial syndrome/DiGeorge syndrome, also known as 22q11.2 deletion syndrome (22q11DS) is the most common microdeletion syndrome, with an estimated incidence of 1/2,000 – 1/4,000 live births. Approximately 9–11% of patients with this disorder have an overt cleft palate (CP), but the genetic factors responsible for CP in the 22q11DS subset are unknown. The TBX1 gene, a member of the T-box transcription factor gene family, lies within the 22q11.2 region that is hemizygous in patients with 22q11DS. Inactivation of one allele of Tbx1 in the mouse does not result in CP, but inactivation of both alleles does. Based on these data, we hypothesized that DNA variants in the remaining allele of TBX1 may confer risk to CP in patients with 22q11DS. To test the hypothesis, we evaluated TBX1 exon sequencing (n = 360) and genotyping data (n = 737) with respect to presence (n = 54) or absence (n = 683) of CP in patients with 22q11DS. Two upstream SNPs (rs4819835 and rs5748410) showed individual evidence for association but they were not significant after correction for multiple testing. Associations were not identified between DNA variants and haplotypes in 22q11DS patients with CP. Overall, this study indicates that common DNA variants in TBX1 may be nominally causative for CP in patients with 22q11DS. This raises the possibility that genes elsewhere on the remaining allele of 22q11.2 or in the genome could be relevant.
PMCID: PMC3477281  PMID: 23034814
22q11.2 deletion syndrome; TBX1 sequencing; cleft palate; genomic disorder
8.  Replication of neuroblastoma SNP association at the BARD1 locus in African-Americans 
Neuroblastoma (NBL) is an often-fatal pediatric cancer more frequent in European-American than African-American children. African-American children, however, are at higher risk for the more severe form of NBL, and have worse overall survival than European-American children. Genome wide association studies (GWAS) have identified several SNPs associated to NBL in children of European descent. Knowledge of their association to NBL in African-American children is still lacking.
We genotyped and imputed SNPs located in three gene regions reported to be associated to NBL in children of European descent, and tested them for association in 390 African-American NBL patients compared to 2500 healthy, ethnically matched controls.
SNPs in the BARD1 gene region show a similar pattern of association to NBL in African-American and European-American children. The more restricted extent of linkage disequilibrium in the African-American population suggests a smaller candidate region for the putative causal variants than previously reported. Limited association was observed at the other two gene regions tested, including LMO1 in 11p15 and FLJ22536 in 6p22.
Common BARD1 SNPs affect risk of NBL in African-Americans. The role of other SNPs associated to NBL in children of European descent could not be confirmed, possibly due to different patterns of linkage disequilibrium or limited statistical power to detect association to variants with small effect on disease risk. Extension of GWAS to populations of African descent is important to confirm their results and validity beyond the European populations, and can help to refine the location of the putative causal variants.
PMCID: PMC3319325  PMID: 22328350
Neuroblastoma; BARD1; African-Americans; SNPs; genetic association
9.  Common variation at 6q16 within HACE1 and LIN28B influences susceptibility to neuroblastoma 
Nature genetics  2012;44(10):1126-1130.
Neuroblastoma is a cancer of the sympathetic nervous system that accounts for approximately 10% of all pediatric oncology deaths1. Here we report on a genome-wide association study of 2,817 neuroblastoma cases and 7,473 controls. We identified two new associations at 6q16, the first within HACE1 (rs4336470; combined P = 2.7 × 10−11, odds ratio 1.26, 95% CI: 1.18–1.35) and the second within LIN28B (rs17065417; combined P = 1.2 × 10−8, odds ratio 1.38, 95% CI: 1.23–1.54). Expression of LIN28B and let-7 miRNA correlated with rs17065417 genotype in neuroblastoma cell lines, and we observed significant growth inhibition upon depletion of LIN28B specifically in neuroblastoma cells homozygous for the risk allele. Low HACE1 and high LIN28B expression in diagnostic primary neuroblastomas were associated with worse overall survival (P = 0.008 and 0.014, respectively). Taken together, we show that common variants in HACE1 and LIN28B influence neuroblastoma susceptibility and that both genes likely play a role in disease progression.
PMCID: PMC3459292  PMID: 22941191
10.  Computerized Neurocognitive Profile in Young People with 22q11.2 Deletion Syndrome Compared to Youths with Schizophrenia and At-risk for Psychosis 
Adults with 22q11.2 Deletion Syndrome (22q11DS) have increased prevalence of schizophrenia features. Our goal is to compare the neurocognitive profile in 22q11DS, schizophrenia and individuals at risk for schizophrenia.
Twenty-one 22q11DS patients (8y-32y, mean 14.9y, 15M 6F) were matched to four comparison groups on age: low risk (n=21), first-degree family members of schizophrenia patients (genetic risk, n=20), individuals exhibiting putatively prodromal symptoms (clinical risk, n=19), and patients with schizophrenia (n=21). All participants received semi-structured interviews [Diagnostic Interview for Genetic Studies (DIGS) and the Structured Interview for Prodromal Syndromes (SIPS)], and a computerized neurocognitive battery (CNB) measuring the following domains: Abstraction and Mental Flexibility, Attention, Working Memory, Verbal Memory, Face Memory, Spatial Memory, Language, Spatial Processing, Sensorimotor Dexterity, and Emotion Processing.
60% of 22q11DS participants met SIPS criteria for prodromal symptoms and one participant met criteria for paranoid schizophrenia. 38% met criteria for Depressive Disorders. All 22q11DS participants successfully completed the CNB. 22q11DS participants were significantly less accurate in nearly all domains, but had similar speed of response compared to the other groups. Their profile resembled that of the psychosis groups in accuracy and speed, except for more pronounced deficits in accuracy for face memory and emotion processing.
Subthreshold psychotic symptoms are present in a high proportion of 22q11DS participants. Deficits shown in the CNB are more pronounced for accuracy than speed relative to the psychosis groups with similar profiles. Similar deficits have been described in the 22q11DS population using non-computerized measures, which require increased testing time.
PMCID: PMC3272485  PMID: 22170773
22q11.2 Deletion Syndrome; VCFS; schizophrenia risk; neuropsychology
11.  Genotype and cardiovascular phenotype correlations with TBX1 in 1,022 velo-cardio- facial/DiGeorge/22q11.2 deletion syndrome patients 
Human mutation  2011;32(11):1278-1289.
Haploinsufficiency of TBX1, encoding a T-box transcription factor, is largely responsible for the physical malformations in velo-cardio-facial/DiGeorge/22q11.2 deletion syndrome (22q11DS) patients. Cardiovascular malformations in these patients are highly variable, raising the question as to whether DNA variations in the TBX1 locus on the remaining allele of 22q11.2, could be responsible. To test this, a large sample size is needed. The TBX1 gene was sequenced in 360 consecutive 22q11DS patients. Rare and common variations were identified. We did not detect enrichment in rare SNP number in those with or without a congenital heart defect. One exception was that there was increased number of very rare SNPs between those with normal heart anatomy compared to those with right-sided aortic arch or persistent truncus arteriosus, suggesting potentially protective roles in the SNPs for these phenotype enrichment groups. Nine common SNPs (MAF >0.05) were chosen and used to genotype the entire cohort of 1,022 22q11DS subjects. We did not find a correlation between common SNPs or haplotypes and cardiovascular phenotype. This work demonstrates that common DNA variations in TBX1 do not explain variable cardiovascular expression in 22q11DS patients, implicating existence of modifiers in other genes on 22q11.2 or elsewhere in the genome.
PMCID: PMC3196824  PMID: 21796729
22q11.2 deletion syndrome; TBX1 sequencing; cardiovascular defects; genomic disorder
12.  Common Variation at BARD1 Results in the Expression of an Oncogenic Isoform that Influences Neuroblastoma Susceptibility and Oncogenicity 
Cancer Research  2012;72(8):2068-2078.
The mechanisms underlying genetic susceptibility at loci discovered by genome-wide association study (GWAS) approaches in human cancer remain largely undefined. In this study we characterized the high-risk neuroblastoma association at the BRCA1-related locus, BARD1, showing that disease-associated variations correlate with increased expression of the oncogenically activated isoform, BARD1β. In neuroblastoma cells, silencing of BARD1β showed genotype-specific cytotoxic effects, including decreased substrate-adherent, anchorage-independent, and foci growth. In established murine fibroblasts, overexpression of BARD1β was sufficient for neoplastic transformation. BARD1β stabilized the Aurora family of kinases in neuroblastoma cells, suggesting both a mechanism for the observed effect and a potential therapeutic strategy. Together, our findings identify BARD1β as an oncogenic driver of high-risk neuroblastoma tumorigenesis, and more generally, they illustrate how robust GWAS signals offer genomic landmarks to identify molecular mechanisms involved in both tumor initiation and malignant progression. The interaction of BARD1β with the Aurora family of kinases lends strong support to the ongoing work to develop Aurora kinase inhibitors for clinically aggressive neuroblastoma.
PMCID: PMC3328617  PMID: 22350409
genome-wide association; neuroblastoma; BARD1; cancer susceptibility genes; functional genomics; oncogenes; genotype-phenotype correlations
13.  Genome-Wide Linkage Analysis to Identify Genetic Modifiers of ALK Mutation Penetrance in Familial Neuroblastoma 
Human Heredity  2011;71(2):135-139.
Neuroblastoma (NB) is an important childhood cancer with a strong genetic component related to disease susceptibility. Approximately 1% of NB cases have a positive family history. Following a genome-wide linkage analysis and sequencing of candidate genes in the critical region, we identified ALK as the major familial NB gene. Dominant mutations in ALK are found in more than 50% of familial NB cases. However, in the families used for the linkage study, only about 50% of carriers of ALK mutations are affected by NB.
To test whether genetic variation may explain the reduced penetrance of the disease phenotype, we analyzed genome-wide genotype data in ALK mutation-positive families using a model-based linkage approach with different liability classes for carriers and non-carriers of ALK mutations.
The region with the highest LOD score was located at chromosome 2p23–p24 and included the ALK locus under models of dominant and recessive inheritance.
This finding suggests that variants in the non-mutated ALK gene or another gene linked to it may affect penetrance of the ALK mutations and risk of developing NB in familial cases.
PMCID: PMC3136385  PMID: 21734404
Neuroblastoma; Penetrance; Linkage analysis; Genetic modifier
14.  Integrative genomics identifies LMO1 as a neuroblastoma oncogene 
Nature  2010;469(7329):216-220.
Neuroblastoma is a childhood cancer of the sympathetic nervous system that accounts for approximately 10% of all paediatric oncology deaths1,2. To identify genetic risk factors for neuroblastoma, we performed a genome-wide association study (GWAS) on 2,251 patients and 6,097 control subjects of European ancestry from four case series. Here we report a significant association within LIM domain only 1 (LMO1) at 11p15.4 (rs110419, combined P = 5.2 × 10−16, odds ratio of risk allele = 1.34 (95% confidence interval 1.25–1.44)). The signal was enriched in the subset of patients with the most aggressive form of the disease. LMO1 encodes a cysteine-rich transcriptional regulator, and its paralogues (LMO2, LMO3 and LMO4) have each been previously implicated in cancer. In parallel, we analysed genome-wide DNA copy number alterations in 701 primary tumours. We found that the LMO1 locus was aberrant in 12.4% through a duplication event, and that this event was associated with more advanced disease (P < 0.0001) and survival (P = 0.041). The germline single nucleotide polymorphism (SNP) risk alleles and somatic copy number gains were associated with increased LMO1 expression in neuroblastoma cell lines and primary tumours, consistent with a gain-of-function role in tumorigenesis. Short hairpin RNA (shRNA)-mediated depletion of LMO1 inhibited growth of neuroblastoma cells with high LMO1 expression, whereas forced expression of LMO1 in neuroblastoma cells with low LMO1 expression enhanced proliferation. These data show that common polymorphisms at the LMO1 locus are strongly associated with susceptibility to developing neuroblastoma, but also may influence the likelihood of further somatic alterations at this locus, leading to malignant progression.
PMCID: PMC3320515  PMID: 21124317
15.  Phenotype Restricted Genome-Wide Association Study Using a Gene-Centric Approach Identifies Three Low-Risk Neuroblastoma Susceptibility Loci 
PLoS Genetics  2011;7(3):e1002026.
Neuroblastoma is a malignant neoplasm of the developing sympathetic nervous system that is notable for its phenotypic diversity. High-risk patients typically have widely disseminated disease at diagnosis and a poor survival probability, but low-risk patients frequently have localized tumors that are almost always cured with little or no chemotherapy. Our genome-wide association study (GWAS) has identified common variants within FLJ22536, BARD1, and LMO1 as significantly associated with neuroblastoma and more robustly associated with high-risk disease. Here we show that a GWAS focused on low-risk cases identified SNPs within DUSP12 at 1q23.3 (P = 2.07×10−6), DDX4 and IL31RA both at 5q11.2 (P = 2.94×10−6 and 6.54×10−7 respectively), and HSD17B12 at 11p11.2 (P = 4.20×10−7) as being associated with the less aggressive form of the disease. These data demonstrate the importance of robust phenotypic data in GWAS analyses and identify additional susceptibility variants for neuroblastoma.
Author Summary
Neuroblastoma is the most common solid tumor outside the central nervous system and is accountable for 10% of the mortality rate of all children's cancers. It has distinctive clinical behaviors and is categorized into different risk groups: high-risk, intermediate-risk, and low-risk. Genome-wide association studies have reported a number of genetic variations predisposing to high-risk neuroblastoma. This study focuses on the low-risk neuroblastoma group and identifies four novel genes (DUSP12, DDX4, IL31RA, and HSD17B12) at three distinct genomic positions that harbor disease-causing variants. This study also reports several gene sets that are enriched in overall neuroblastoma as well as in both high-risk and low-risk groups. Also of importance is that this study adopts a new computational method that identifies genes, instead of only one single nucleotide polymorphism, as disease-causing variants. Shown to have superior power of detection genome-wide association signals for neuroblastoma, the methodology presented in this study has great potential applications in case-control association studies in other diseases.
PMCID: PMC3060064  PMID: 21436895
16.  The Otto Aufranc Award: Identification of a 4 Mb Region on Chromosome 17q21 Linked to Developmental Dysplasia of the Hip in One 18-member, Multigeneration Family 
Developmental dysplasia of the hip (DDH) is a disabling condition that, depending on geography, can afflict between 20% and 80% of patients with end-stage arthritis of the hip. Despite its prevalence, the etiology of this disease remains unknown. DDH is a complex disorder with both environmental and genetic causes. Based on the literature the candidate genes for the disease are HOXB9, collagen type I α1, and DLX 3. The purpose of our study was to map and characterize the gene or genes responsible for this disorder by family linkage analysis. We recruited one 18-member, multigeneration affected family to provide cheek swabs and blood samples for isolation of DNA. Amplified DNA underwent a total genome scan using GeneChip Mapping 250 K Assay (Affymetrix, Santa Clara, CA). We observed only one region with a LOD score greater than 1.5: a 4 Mb region on chromosome 17q21.32, yielding a LOD score of 1.82. While a LOD score of 1.82 does not meet the accepted standard for linkage we interpret these data as suggesting the responsible gene could be linked to this region, which includes a cluster of homeobox genes (HOX genes) that are part of the developmental regulatory system providing cells with specific positional identities along the developing joint and spine. Discovering the genetic basis of the disease would be an important step in understanding the etiology of this disabling condition.
PMCID: PMC2807013  PMID: 19756907
18.  Comparative analysis of different approaches for dealing with candidate regions in the context of a genome-wide association study 
BMC Proceedings  2009;3(Suppl 7):S93.
Genome-wide association studies (GWAS) test hundreds of thousands of single-nucleotide polymorphisms (SNPs) for association to a trait, treating each marker equally and ignoring prior evidence of association to specific regions. Typically, promising regions are selected for further investigation based on p-values obtained from simple tests of association. However, loci that exert only a weak, low-penetrant role on the trait, producing modest evidence of association, are not detectable in the context of a GWAS. Implementing prior knowledge of association in GWAS could increase power, help distinguish between false and true positives, and identify better sets of SNPs for follow-up studies.
Here we performed a GWAS on rheumatoid arthritis (RA) patients and controls (Problem 1, Genetic Analysis Workshop 16). In order to include prior information in the analysis, we applied four methods that distinctively deal with markers in candidate genes in the context of GWAS. SNPs were divided into a random and a candidate subset, then we applied empirical correction by permutation, false-discovery rate, false-positive report probability, and posterior odds of association using different prior probabilities. We repeated the same analyses on two different sets of candidate markers defined on the basis of previously reported association to RA following two different approaches. The four methods showed similar relative behavior when applied to the two sets, with the proportion of candidate SNPs ranked among the top 2,000 varying from 0 to 100%. The use of different prior probabilities changed the stringency of the methods, but not their relative performance.
PMCID: PMC2795997  PMID: 20018090
19.  Common variations in BARD1 influence susceptibility to high-risk neuroblastoma 
Nature genetics  2009;41(6):718-723.
We conducted a SNP-based genome-wide association study (GWAS) focused on the high-risk subset of neuroblastoma1. As our previous unbiased GWAS showed strong association of common 6p22 SNP alleles with aggressive neuroblastoma2, we now restricted our analysis to 397 high-risk cases compared to 2,043 controls. We detected new significant association of six SNPs at 2q35 within the BARD1 gene locus (Pallelic = 2.35×10−9 − 2.25×10−8). Each SNP association was confirmed in a second series of 189 high-risk cases and 1,178 controls (Pallelic = 7.90×10−7 − 2.77×10−4). The two most significant SNPs (rs6435862, rs3768716) were also tested in two additional independent high-risk neuroblastoma case series, yielding combined allelic odds-ratios of 1.68 each (P = 8.65×10−18 and 2.74×10−16, respectively). Significant association was also found with known BARD1 nsSNPs. These data show that common variation in BARD1 contributes to the etiology of the aggressive and most clinically relevant subset of human neuroblastoma.
PMCID: PMC2753610  PMID: 19412175
20.  Precocious Sister Chromatid Separation (PSCS) in Cornelia de Lange Syndrome 
The Cornelia de Lange syndrome (CdLS) (OMIM# 122470) is a dominantly inherited multisystem developmental disorder. The phenotype consists of characteristic facial features, hirsutism, abnormalities of the upper extremities ranging from subtle changes in the phalanges and metacarpal bones to oligodactyly and phocomelia, gastroesophageal dysfunction, growth retardation, and neurodevelopmental delay. Prevalence is estimated to be as high as 1 in 10,000. Recently, mutations in NIPBL were identified in sporadic and familial CdLS cases. To date, mutations in this gene have been identified in over 45% of individuals with CdLS. NIPBL is the human homolog of the Drosophila Nipped-B gene. Although its function in mammalian systems has not yet been elucidated, sequence homologs of Nipped-B in yeast (Scc2 and Mis4) are required for sister chromatid cohesion during mitosis, and a similar role was recently demonstrated for Nipped-B in Drosophila. In order to evaluate NIPBL role in sister chromatid cohesion in humans, metaphase spreads on 90 probands (40 NIPBL mutation positive and 50 NIPBL mutation negative) with CdLS were evaluated for evidence of precocious sister chromatid separation (PSCS). We screened 50 metaphases from each proband and found evidence of PSCS in 41% (compared to 9% in control samples). These studies indicate that NIPBL may play a role in sister chromatid cohesion in humans as has been reported for its homologs in Drosophila and yeast.
PMCID: PMC2766539  PMID: 16100726
cornelia de Lange syndrome; CdLS; NIPBL; Nipped-B; precocious sister chromatid separation; PSCS
21.  Copy number variation at 1q21.1 associated with neuroblastoma 
Nature  2009;459(7249):987-991.
Common copy number variations (CNVs) represent a significant source of genetic diversity, yet their influence on phenotypic variability, including disease susceptibility, remains poorly understood. To address this problem in cancer, we performed a genome-wide association study (GWAS) of CNVs in the childhood cancer neuroblastoma, a disease where SNP variations are known to influence susceptibility1,2. We first genotyped 846 Caucasian neuroblastoma patients and 803 healthy Caucasian controls at 550,000 single nucleotide polymorphisms, and performed a CNV-based test for association. We then replicated significant observations in two independent sample sets comprised of a total of 595 cases and 3,357 controls. We identified a common CNV at 1q21.1 associated with neuroblastoma in the discovery set, which was confirmed in both replication sets (Pcombined = 2.97 × 10−17; OR = 2.49, 95% CI: 2.02 to 3.05). This CNV was validated by quantitative PCR, fluorescent in situ hybridization, and analysis of matched tumor specimens, and was shown to be heritable in an independent set of 713 cancer-free trios. We identified a novel transcript within the CNV which showed high sequence similarity to several “Neuroblastoma breakpoint family” (NBPF) genes3,4 and represents a new member of this gene family (NBPFX). This transcript was preferentially expressed in fetal brain and fetal sympathetic nervous tissues, and expression level was strictly correlated with CNV state in neuroblastoma cells. These data demonstrate that inherited copy number variation at 1q21.1 is associated with neuroblastoma and implicate a novel NBPF gene in early tumorigenesis of this childhood cancer.
PMCID: PMC2755253  PMID: 19536264
22.  A genome-wide association study identifies a susceptibility locus to clinically aggressive neuroblastoma at 6p22 
The New England journal of medicine  2008;358(24):2585-2593.
Neuroblastoma is a malignancy of the developing sympathetic nervous system that most commonly affects young children and is often lethal. The etiology of this embryonal cancer is not known.
We performed a genome-wide association study by first genotyping 1,032 neuroblastoma patients and 2,043 controls of European descent using the Illumina HumanHap550 BeadChip. Three independent groups of neuroblastoma cases (N=720) and controls (N=2128) were then genotyped to replicate significant associations.
We observed highly significant association between neuroblastoma and the common minor alleles of three single nucleotide polymorphisms (SNPs) within a 94.2 kilobase (Kb) linkage disequilibrium block at chromosome band 6p22 containing the predicted genes FLJ22536 and FLJ44180 (P-value range = 1.71×10-9-7.01×10-10; allelic odds ratio range 1.39-1.40). Homozygosity for the at-risk G allele of the most significantly associated SNP, rs6939340, resulted in an increased likelihood of developing neuroblastoma of 1.97 (95% CI 1.58-2.44). Subsequent genotyping of these 6p22 SNPs in the three independent case series confirmed our observation of association (P=9.33×10-15 at rs6939340 for joint analysis). Furthermore, neuroblastoma patients homozygous for the risk alleles at 6p22 were more likely to develop metastatic (Stage 4) disease (P=0.02), show amplification of the MYCN oncogene in the tumor cells (P=0.006), and to have disease relapse (P=0.01).
Common genetic variation at chromosome band 6p22 is associated with susceptibility to neuroblastoma.
PMCID: PMC2742373  PMID: 18463370
23.  Identification of ALK as the Major Familial Neuroblastoma Predisposition Gene 
Nature  2008;455(7215):930-935.
Survival rates for the childhood cancer neuroblastoma have not substantively improved despite dramatic escalation in chemotherapy intensity. Like most human cancers, this embryonal malignancy can be inherited, but the genetic etiology of familial and sporadically occurring neuroblastoma was largely unknown. Here we show that germline mutations in the anaplastic lymphoma kinase gene (ALK) explain the majority of hereditary neuroblastomas, and that activating mutations can also be somatically acquired. We first identified a significant linkage signal at the short arm of chromosome 2 (maximum nonparametric LOD=4.23 at rs1344063) using a whole-genome scan in neuroblastoma pedigrees. Resequencing of regional candidate genes identified three separate missense mutations in the tyrosine kinase domain of ALK (G1128A, R1192P and R1275Q) that segregated with the disease in eight separate families. Examination of 491 sporadically occurring human neuroblastoma samples showed that the ALK locus was gained in 22.8%, and highly amplified in an additional 3.3%, and that these aberrations were highly associated with death from disease (P=0.0003). Resequencing of 194 high-risk neuroblastoma samples showed somatically acquired mutations within the tyrosine kinase domain in 12.4%. Nine of the ten mutations map to critical regions of the kinase domain and were predicted to be oncogenic drivers with high probability. Mutations resulted in constitutive phosphorylation consistent with activation, and targeted knockdown of ALK mRNA resulted in profound growth inhibition of 4 of 4 cell lines harboring mutant or amplified ALK, as well as 2 of 6 wild type for ALK. Our results demonstrate that heritable mutations of ALK are the major cause of familial neuroblastoma, and that germline or acquired activation of this cell surface kinase is a tractable therapeutic target for this lethal pediatric malignancy.
PMCID: PMC2672043  PMID: 18724359
24.  Variants of ST8SIA1 Are Associated with Risk of Developing Multiple Sclerosis 
PLoS ONE  2008;3(7):e2653.
Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system of unknown etiology with both genetic and environmental factors playing a role in susceptibility. To date, the HLA DR15/DQ6 haplotype within the major histocompatibility complex on chromosome 6p, is the strongest genetic risk factor associated with MS susceptibility. Additional alleles of IL7 and IL2 have been identified as risk factors for MS with small effect. Here we present two independent studies supporting an allelic association of MS with polymorphisms in the ST8SIA1 gene, located on chromosome 12p12 and encoding ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 1. The initial association was made in a single three-generation family where a single-nucleotide polymorphism (SNP) rs4762896, was segregating together with HLA DR15/DQ6 in MS patients. A study of 274 family trios ( affected child and both unaffected parents) from Australia validated the association of ST8SIA1 in individuals with MS, showing transmission disequilibrium of the paternal alleles for three additional SNPs, namely rs704219, rs2041906, and rs1558793, with p = 0.001, p = 0.01 and p = 0.01 respectively. These findings implicate ST8SIA1 as a possible novel susceptibility gene for MS.
PMCID: PMC2440423  PMID: 18612409
25.  Genetic loci linked to Type 1 Diabetes and Multiple Sclerosis families in Sardinia 
The Mediterranean island of Sardinia has a strikingly high incidence of the autoimmune disorders Type 1 Diabetes (T1D) and Multiple Sclerosis (MS). Furthermore, the two diseases tend to be co-inherited in the same individuals and in the same families. These observations suggest that some unknown autoimmunity variant with relevant effect size could be fairly common in this founder population and could be detected using linkage analysis.
To search for T1D and MS loci as well as any that predispose to both diseases, we performed a whole genome linkage scan, sequentially genotyping 593 microsatellite marker loci in 954 individuals distributed in 175 Sardinian families. In total, 413 patients were studied; 285 with T1D, 116 with MS and 12 with both disorders. Model-free linkage analysis was performed on the genotyped samples using the Kong and Cox logarithm of odds (LOD) score statistic.
In T1D, aside from the HLA locus, we found four regions showing a lod-score ≥1; 1p31.1, 6q26, 10q21.2 and 22q11.22. In MS we found three regions showing a lod-score ≥1; 1q42.2, 18p11.21 and 20p12.3. In the combined T1D-MS scan for shared autoimmunity loci, four regions showed a LOD >1, including 6q26, 10q21.2, 20p12.3 and 22q11.22. When we typed more markers in these intervals we obtained suggestive evidence of linkage in the T1D scan at 10q21.2 (LOD = 2.1), in the MS scan at 1q42.2 (LOD = 2.5) and at 18p11.22 (LOD = 2.6). When all T1D and MS families were analysed jointly we obtained suggestive evidence in two regions: at 10q21.1 (LOD score = 2.3) and at 20p12.3 (LOD score = 2.5).
This suggestive evidence of linkage with T1D, MS and both diseases indicates critical chromosome intervals to be followed up in downstream association studies.
PMCID: PMC2259316  PMID: 18205952

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