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1.  Genome-wide analysis of rare copy number variations reveals PARK2 as a candidate gene for attention-deficit/hyperactivity disorder 
Molecular Psychiatry  2012;19(1):115-121.
Attention-deficit/hyperactivity disorder (ADHD) is a common, highly heritable neurodevelopmental disorder. Genetic loci have not yet been identified by genome-wide association studies. Rare copy number variations (CNVs), such as chromosomal deletions or duplications, have been implicated in ADHD and other neurodevelopmental disorders. To identify rare (frequency ⩽1%) CNVs that increase the risk of ADHD, we performed a whole-genome CNV analysis based on 489 young ADHD patients and 1285 adult population-based controls and identified one significantly associated CNV region. In tests for a global burden of large (>500 kb) rare CNVs, we observed a nonsignificant (P=0.271) 1.126-fold enriched rate of subjects carrying at least one such CNV in the group of ADHD cases. Locus-specific tests of association were used to assess if there were more rare CNVs in cases compared with controls. Detected CNVs, which were significantly enriched in the ADHD group, were validated by quantitative (q)PCR. Findings were replicated in an independent sample of 386 young patients with ADHD and 781 young population-based healthy controls. We identified rare CNVs within the parkinson protein 2 gene (PARK2) with a significantly higher prevalence in ADHD patients than in controls (P=2.8 × 10−4 after empirical correction for genome-wide testing). In total, the PARK2 locus (chr 6: 162 659 756–162 767 019) harboured three deletions and nine duplications in the ADHD patients and two deletions and two duplications in the controls. By qPCR analysis, we validated 11 of the 12 CNVs in ADHD patients (P=1.2 × 10−3 after empirical correction for genome-wide testing). In the replication sample, CNVs at the PARK2 locus were found in four additional ADHD patients and one additional control (P=4.3 × 10−2). Our results suggest that copy number variants at the PARK2 locus contribute to the genetic susceptibility of ADHD. Mutations and CNVs in PARK2 are known to be associated with Parkinson disease.
PMCID: PMC3873032  PMID: 23164820
ADHD; children; CNVs; GWAS; PARK2
2.  Copy Number Variation in Familial Parkinson Disease 
PLoS ONE  2011;6(8):e20988.
Copy number variants (CNVs) are known to cause Mendelian forms of Parkinson disease (PD), most notably in SNCA and PARK2. PARK2 has a recessive mode of inheritance; however, recent evidence demonstrates that a single CNV in PARK2 (but not a single missense mutation) may increase risk for PD. We recently performed a genome-wide association study for PD that excluded individuals known to have either a LRRK2 mutation or two PARK2 mutations. Data from the Illumina370Duo arrays were re-clustered using only white individuals with high quality intensity data, and CNV calls were made using two algorithms, PennCNV and QuantiSNP. After quality assessment, the final sample included 816 cases and 856 controls. Results varied between the two CNV calling algorithms for many regions, including the PARK2 locus (genome-wide p = 0.04 for PennCNV and p = 0.13 for QuantiSNP). However, there was consistent evidence with both algorithms for two novel genes, USP32 and DOCK5 (empirical, genome-wide p-values<0.001). PARK2 CNVs tended to be larger, and all instances that were molecularly tested were validated. In contrast, the CNVs in both novel loci were smaller and failed to replicate using real-time PCR, MLPA, and gel electrophoresis. The DOCK5 variation is more akin to a VNTR than a typical CNV and the association is likely caused by artifact due to DNA source. DNA for all the cases was derived from whole blood, while the DNA for all controls was derived from lymphoblast cell lines. The USP32 locus contains many SNPs with low minor allele frequency leading to a loss of heterozygosity that may have been spuriously interpreted by the CNV calling algorithms as support for a deletion. Thus, only the CNVs within the PARK2 locus could be molecularly validated and associated with PD susceptibility.
PMCID: PMC3149037  PMID: 21829596
3.  Analysis of Dosage Mutation in PARK2 among Korean Patients with Early-Onset or Familial Parkinson's Disease 
Background and Purpose
There is some controversy regarding heterozygous mutations of the gene encoding parkin (PARK2) as risk factors for Parkinson's disease (PD), and all previous studies have been performed in non-Asian populations. Dosage mutation of PARK2, rather than a point mutation or small insertion/deletion mutation, was reported to be a risk factor for familial PD; dosage mutation of PARK2 is common in Asian populations.
We performed a gene-dosage analysis of PARK2 using real-time polymerase chain reaction for 189 patients with early-onset PD or familial PD, and 191 control individuals. In the case of PD patients with heterozygous gene-dosage mutation, we performed a sequencing analysis to exclude compound heterozygous mutations. The association between heterozygous mutation of PARK2 and PD was tested.
We identified 22 PD patients with PARK2 mutations (11.6%). Five patients (2.6%) had compound heterozygous mutations, and 13 patients (6.9%) had a heterozygous mutation. The phase could not be determined in one patient. Three small sequence variations were found in 30 mutated alleles (10.0%). Gene-dosage mutation accounted for 90% of all of the mutations found. The frequency of a heterozygous PARK2 gene-dosage mutation was higher in PD patients than in the controls.
Heterozygous gene-dosage mutation of PARK2 is a genetic risk factor for patients with early-onset or familial PD in Koreans.
PMCID: PMC4101102  PMID: 25045378
Parkinson's disease; PARK2; gene-dosage change; risk factor
4.  Hereditary breast and ovarian cancer: assessment of point mutations and copy number variations in Brazilian patients 
BMC Medical Genetics  2014;15:55.
Germ line mutations in BRCA1 and BRCA2 (BRCA1/2) and other susceptibility genes have been identified as genetic causes of hereditary breast and ovarian cancer (HBOC). To identify the disease-causing mutations in a cohort of 120 Brazilian women fulfilling criteria for HBOC, we carried out a comprehensive screening of BRCA1/2, TP53 R337H, CHEK2 1100delC, followed by an analysis of copy number variations in 14 additional breast cancer susceptibility genes (PTEN, ATM, NBN, RAD50, RAD51, BRIP1, PALB2, MLH1, MSH2, MSH6, TP53, CDKN2A, CDH1 and CTNNB1).
Capillary sequencing and multiplex ligation-dependent probe amplification (MLPA) were used for detecting point mutations and copy number variations (CNVs), respectively, for the BRCA1 and BRCA2 genes; capillary sequencing was used for point mutation for both variants TP53 R337H and CHEK2 1100delC, and finally array comparative genomic hybridization (array-CGH) was used for identifying CNVs in the 14 additional genes.
The positive detection rate in our series was 26%. BRCA1 pathogenic mutations were found in 20 cases, including two cases with CNVs, whereas BRCA2 mutations were found in 7 cases. We also found three patients with the TP53 R337H mutation and one patient with the CHEK2 1100delC mutation. Seven (25%) pathogenic mutations in BRCA1/2 were firstly described, including a splice-site BRCA1 mutation for which pathogenicity was confirmed by the presence of an aberrant transcript showing the loss of the last 62 bp of exon 7. Microdeletions of exon 4 in ATM and exon 2 in PTEN were identified in BRCA2-mutated and BRCA1/2-negative patients, respectively.
In summary, our results showed a high frequency of BRCA1/2 mutations and a higher prevalence of BRCA1 (64.5%) gene. Moreover, the detection of the TP53 R337H variant in our series and the fact that this variant has a founder effect in our population prompted us to suggest that all female breast cancer patients with clinical criteria for HBOC and negative for BRCA1/2 genes should be tested for the TP53 R337H variant. Furthermore, the presence of genomic structural rearrangement resulting in CNVs in other genes that predispose breast cancer in conjunction with BRCA2 point mutations demonstrated a highly complex genetic etiology in Brazilian breast cancer families.
PMCID: PMC4038072  PMID: 24884479
Breast cancer; Mutation; BRCA1; BRCA2; HBOC; CHEK 1100delC; TP53 R337H
5.  Identification of Rare Recurrent Copy Number Variants in High-Risk Autism Families and Their Prevalence in a Large ASD Population 
PLoS ONE  2013;8(1):e52239.
Structural variation is thought to play a major etiological role in the development of autism spectrum disorders (ASDs), and numerous studies documenting the relevance of copy number variants (CNVs) in ASD have been published since 2006. To determine if large ASD families harbor high-impact CNVs that may have broader impact in the general ASD population, we used the Affymetrix genome-wide human SNP array 6.0 to identify 153 putative autism-specific CNVs present in 55 individuals with ASD from 9 multiplex ASD pedigrees. To evaluate the actual prevalence of these CNVs as well as 185 CNVs reportedly associated with ASD from published studies many of which are insufficiently powered, we designed a custom Illumina array and used it to interrogate these CNVs in 3,000 ASD cases and 6,000 controls. Additional single nucleotide variants (SNVs) on the array identified 25 CNVs that we did not detect in our family studies at the standard SNP array resolution. After molecular validation, our results demonstrated that 15 CNVs identified in high-risk ASD families also were found in two or more ASD cases with odds ratios greater than 2.0, strengthening their support as ASD risk variants. In addition, of the 25 CNVs identified using SNV probes on our custom array, 9 also had odds ratios greater than 2.0, suggesting that these CNVs also are ASD risk variants. Eighteen of the validated CNVs have not been reported previously in individuals with ASD and three have only been observed once. Finally, we confirmed the association of 31 of 185 published ASD-associated CNVs in our dataset with odds ratios greater than 2.0, suggesting they may be of clinical relevance in the evaluation of children with ASDs. Taken together, these data provide strong support for the existence and application of high-impact CNVs in the clinical genetic evaluation of children with ASD.
PMCID: PMC3544904  PMID: 23341896
6.  Genetic Variation of the Fc Gamma Receptor 3B Gene and Association with Rheumatoid Arthritis 
PLoS ONE  2010;5(10):e13173.
Fc gamma receptors (FcγRs) play a crucial role in immunity by linking IgG antibody-mediated responses with cellular effector and regulatory functions. Genetic variants in these receptors have been previously identified as risk factors for several chronic inflammatory conditions. The present study aimed to investigate the presence of copy number variations (CNVs) in the FCGR3B gene and its potential association with the autoimmune disease rheumatoid arthritis (RA).
Methodology/Principal Findings
CNV of the FCGR3B gene was studied using Multiplex Ligation Dependent Probe Amplification (MLPA) in 518 Dutch RA patients and 304 healthy controls. Surprisingly, three independent MLPA probes targeting the FCGR3B promoter measured different CNV frequencies, with probe#1 and #2 measuring 0 to 5 gene copies and probe#3 showing little evidence of CNV. Quantitative-PCR correlated with the copy number results from MLPA probe#2, which detected low copy number (1 copy) in 6.7% and high copy number (≥3 copies) in 9.4% of the control population. No significant difference was observed between RA patients and the healthy controls, neither in the low copy nor the high copy number groups (p-values = 0.36 and 0.71, respectively). Sequencing of the FCGR3B promoter region revealed an insertion/deletion (indel) that explained the disparate CNV results of MLPA probe#1. Finally, a non-significant trend was found between the novel -256A>TG indel and RA (40.7% in healthy controls versus 35.9% in RA patients; P = 0.08).
The current study highlights the complexity and poor characterization of the FCGR3B gene sequence, indicating that the design and interpretation of genotyping assays based on specific probe sequences must be performed with caution. Nonetheless, we confirmed the presence of CNV and identified novel polymorphisms in the FCGR3B gene in the Dutch population. Although no association was found between RA and FCGR3B CNV, the possible protective effect of the -256A>TG indel polymorphism must be addressed in larger studies.
PMCID: PMC2950138  PMID: 20957197
7.  PARK2 variability in Polish Parkinson’s disease patients - interaction with mitochondrial haplogroups 
Parkinsonism & related disorders  2012;18(5):520-524.
Aims and objectives
A new pathomechanism of Parkinson’s disease (PD) involving regulation of mitochondrial functions was recently proposed. Parkin complexed with mitochondrial transcription factor A (TFAM) binds mtDNA and promotes mitochondrial biogenesis, which is abolished by PARK2 gene mutations. We have previously shown that mitochondrial haplogroups/clusters and TFAM common variation influenced PD risk. We investigate the role of PARK2 polymorphisms on PD risk and their interactions with mitochondrial haplogroups/clusters as well as with TFAM variability.
104 early-onset PD patients (EOPD, age at onset ≤ 50 years) were screened for PARK2 coding sequence changes including gene dosage alterations. Three selected PARK2 polymorphisms (S167N, V380L, D394N) were genotyped in 326 PD patients and 315 controls using TaqMan allelic discrimination assay.
PARK2 screen revealed two heterozygous changes in two EOPD patients: exon 2 deletion and one novel synonymous variation (c.999C>A, P333P).
In association study no differences in genotype/allele frequencies of S167N, V380L, D394N were found between analyzed groups. Stratification by mitochondrial clusters revealed higher frequency of V380L G/G genotype and allele G in PD patients, within HV cluster (p=0.040; p=0.022, respectively). Moreover, interaction between genotypes G/G V380L of PARK2 and G/G rs2306604 of TFAM, within HV cluster was significant (OR 2.05; CI 1.04 – 4.04; p=0.038).
Our results indicate that co-occurence of G/G V380L PARK2 and G/G rs2306604 TFAM on the prooxidative HV cluster background can contribute to PD risk. We confirm low PARK2 mutation frequency in Polish EOPD patients.
PMCID: PMC3358581  PMID: 22361577
Parkinson’s disease risk factors; PARK2; mitochondrial clusters; mitochondrial transcription factor A (TFAM)
8.  Combined examination of sequence and copy number variations in human deafness genes improves diagnosis for cases of genetic deafness 
Copy number variations (CNVs) are the major type of structural variation in the human genome, and are more common than DNA sequence variations in populations. CNVs are important factors for human genetic and phenotypic diversity. Many CNVs have been associated with either resistance to diseases or identified as the cause of diseases. Currently little is known about the role of CNVs in causing deafness. CNVs are currently not analyzed by conventional genetic analysis methods to study deafness. Here we detected both DNA sequence variations and CNVs affecting 80 genes known to be required for normal hearing.
Coding regions of the deafness genes were captured by a hybridization-based method and processed through the standard next-generation sequencing (NGS) protocol using the Illumina platform. Samples hybridized together in the same reaction were analyzed to obtain CNVs. A read depth based method was used to measure CNVs at the resolution of a single exon. Results were validated by the quantitative PCR (qPCR) based method.
Among 79 sporadic cases clinically diagnosed with sensorineural hearing loss, we identified previously-reported disease-causing sequence mutations in 16 cases. In addition, we identified a total of 97 CNVs (72 CNV gains and 25 CNV losses) in 27 deafness genes. The CNVs included homozygous deletions which may directly give rise to deleterious effects on protein functions known to be essential for hearing, as well as heterozygous deletions and CNV gains compounded with sequence mutations in deafness genes that could potentially harm gene functions.
We studied how CNVs in known deafness genes may result in deafness. Data provided here served as a basis to explain how CNVs disrupt normal functions of deafness genes. These results may significantly expand our understanding about how various types of genetic mutations cause deafness in humans.
PMCID: PMC4194081  PMID: 25342930
Genetic deafness; Copy number variations; Sequence mutations; Next-generation sequencing; Deafness gene panel; Hearing
9.  Structural genomic variation in ischemic stroke 
Neurogenetics  2008;9(2):101-108.
Technological advances in molecular genetics allow rapid and sensitive identification of genomic copy number variants (CNVs). This, in turn, has sparked interest in the function such variation may play in disease. While a role for copy number mutations as a cause of Mendelian disorders is well established, it is unclear whether CNVs may affect risk for common complex disorders. We sought to investigate whether CNVs may modulate risk for ischemic stroke (IS) and to provide a catalog of CNVs in patients with this disorder by analyzing copy number metrics produced as a part of our previous genome-wide single-nucleotide polymorphism (SNP)-based association study of ischemic stroke in a North American white population. We examined CNVs in 263 patients with ischemic stroke (IS). Each identified CNV was compared with changes identified in 275 neurologically normal controls. Our analysis identified 247 CNVs, corresponding to 187 insertions (76%; 135 heterozygous; 25 homozygous duplications or triplications; 2 heterosomic) and 60 deletions (24%; 40 heterozygous deletions;3 homozygous deletions; 14 heterosomic deletions). Most alterations (81%) were the same as, or overlapped with, previously reported CNVs. We report here the first genome-wide analysis of CNVs in IS patients. In summary, our study did not detect any common genomic structural variation unequivocally linked to IS, although we cannot exclude that smaller CNVs or CNVs in genomic regions poorly covered by this methodology may confer risk for IS. The application of genome-wide SNP arrays now facilitates the evaluation of structural changes through the entire genome as part of a genome-wide genetic association study.
PMCID: PMC2730937  PMID: 18288507
Stroke; Cerebral ischemia; Genetics; Copy number variants
10.  Exon Dosage Variations in Brazilian Patients with Parkinson’s Disease: Analysis of SNCA, PARKIN, PINK1 and DJ-1 Genes 
Disease markers  2012;32(3):173-178.
Parkinson’s disease is one of the most common neurodegenerative disorders associated with aging, reaching ∼ 2% of individuals over 65 years. Knowledge achieved in the last decade about the genetic basis of Parkinson’s disease clearly shows that genetic factors play an important role in the etiology of this disorder. Exon dosage variations account for a high proportion of Parkinson’s disease mutations, mainly for PARKIN gene. In the present study, we screened genomic rearrangements in SNCA, PARKIN, PINK1 and DJ-1 genes in 102 Brazilian Parkinson’s disease patients with early onset (age of onset ≤ 50 years), using the multiplex ligation-dependent probe amplification method. Family history was reported by 24 patients, while 78 were sporadic cases. Screening of exon dosage revealed PARKIN and PINK1 copy number variations, but no dosage alteration was found in SNCA and DJ-1 genes. Most of the carriers harbor heterozygous deletions or duplications in the PARKIN gene and only one patient was found to have a deletion in PINK1 exon 1. Data about dosage changes are scarce in the Brazilian population, which stresses the importance of including exon dosage analysis in Parkinson’s disease genetic studies.
PMCID: PMC3826582  PMID: 22377733
Copy number variation; early-onset; MLPA; Deletion; Duplication
11.  Genetic variability at the PARK16 locus 
European Journal of Human Genetics  2010;18(12):1356-1359.
Parkinson's disease (PD) is a complex neurodegenerative disease which is clinically heterogeneous and pathologically consists of loss of dopaminergic neurons in the substantia nigra and intracytoplasmic neuronal inclusions containing alpha-synuclein aggregations known as Lewy bodies. Although the majority of PD is idiopathic, pathogenic mutations in several mendelian genes have been successfully identified through linkage analyses. To identify susceptibility loci for idiopathic PD, several genome-wide association studies (GWAS) within different populations have recently been conducted in both idiopathic and familial forms of PD. These analyses have confirmed SNCA and MAPT as loci harboring PD susceptibility. In addition, the GWAS identified several other genetic loci suggestively associated with the risk of PD; among these, only one was replicated by two different studies of European and Asian ancestries. Hence, we investigated this novel locus known as PARK16 for coding mutations in a large series of idiopathic pathologically proven PD cases, and also conducted an association study in a case–control cohort from the United Kingdom. An association between a novel RAB7L1 mutation, c.379-12insT, and disease (P-value=0.0325) was identified. Two novel coding variants present only in the PD cohort were also identified within the RAB7L1 (p.K157R) and SLC41A1 (p.A350V) genes. No copy number variation analyses have yet been performed within this recently identified locus. We concluded that, although both coding variants and risk alleles within the PARK16 locus seem to be rare, further molecular analyses within the PARK16 locus and within different populations are required in order to examine its biochemical role in the disease process.
PMCID: PMC3002857  PMID: 20683486
PARK16 locus; genetics; association studies
12.  Androgen Receptor Copy Number Variation and Androgenetic Alopecia: A Case-Control Study 
PLoS ONE  2009;4(4):e5081.
The functional polymorphism that explains the established association of the androgen receptor (AR) with androgenetic alopecia (AGA) remains unidentified, but Copy Number Variation (CNV) might be relevant. CNV involves changes in copy number of large segments of DNA, leading to the altered dosage of gene regulators or genes themselves. Two recent reports indicate regions of CNV in and around AR, and these have not been studied in relation to AGA. The aim of this preliminary case-control study was to determine if AR CNV is associated with AGA, with the hypothesis that CNV is the functional AR variant contributing to this condition.
Methodology/Principal Findings
Multiplex Ligation-dependent Probe Amplification was used to screen for CNV in five AR exons and a conserved, non-coding region upstream of AR in 85 men carefully selected as cases and controls for maximal phenotypic contrast. There was no evidence of CNV in AR in any of the cases or controls, and thus no evidence of significant association between AGA and AR CNV.
The results suggest this form of genomic variation at the AR locus is unlikely to predispose to AGA.
PMCID: PMC2659771  PMID: 19340294
13.  Predictors of Parkin Mutations in Early Onset Parkinson disease: the CORE-PD Study 
Archives of Neurology  2010;67(6):731-738.
Mutations in the parkin gene are the most common genetic cause of early-onset Parkinson’s disease (EOPD). Results from a multi-center study of cases with PD systematically sampled by age at onset (AAO) have not been reported.
To determine risk factors associated with carrying mutations in the parkin gene.
Cross-sectional observational study
13 movement disorders centers
956 EOPD cases defined as AAO <51.
Main Outcome Measures
Presence of heterozygous, homozygous or compound heterozygous parkin mutations.
14.7% of cases reported a family history of PD in a first-degree relative using a previously validated interview. Sixty-four cases (6.7%) had parkin mutations (3.9% heterozygous, 0.6% homozygotes, 2.2% compound heterozgyotes). Copy Number Variation (CNV) was present in 52.3% (31.6% of heterozygotes, 83.3% of homozygotes, 81.0% of compound heterozygotes). Deletions in exons 3–4 and 255delA, were common in Hispanics, and specifically, in the Puerto Rican population. Earlier AAO, Hispanic ethnicity (OR compared to White non-Hispanic 2.7 95% CI 1.3–5.7, p<0.009) and family history of PD in a first-degree relative (OR 2.8 95%CI 1.5–5.3, p<0.002) were associated with carrying any mutation in the parkin gene (heterozygous, homozygous, compound heterozygous). Hispanic ethnicity was associated with carrying a heterozygous mutation (OR compared to non-Hispanic Caucasian 2.8 95%CI 1.1–7.2, p<0.03) after adjustment for covariates.
AAO, Hispanic ethnicity and family history of PD are associated with carrying any parkin mutation (heterozygous, homozygous, compound heterozygous) and heterozygous mutations alone. The increased odds of carrying a parkin mutation in Hispanics warrants further study.
PMCID: PMC3329757  PMID: 20558392
14.  Copy number variation and cytidine analogue cytotoxicity: A genome-wide association approach 
BMC Genomics  2010;11:357.
The human genome displays extensive copy-number variation (CNV). Recent discoveries have shown that large segments of DNA, ranging in size from hundreds to thousands of nucleotides, are either deleted or duplicated. This CNV may encompass genes, leading to a change in phenotype, including drug response phenotypes. Gemcitabine and 1-β-D-arabinofuranosylcytosine (AraC) are cytidine analogues used to treat a variety of cancers. Previous studies have shown that genetic variation may influence response to these drugs. In the present study, we set out to test the hypothesis that variation in copy number might contribute to variation in cytidine analogue response phenotypes.
We used a cell-based model system consisting of 197 ethnically-defined lymphoblastoid cell lines for which genome-wide SNP data were obtained using Illumina 550 and 650 K SNP arrays to study cytidine analogue cytotoxicity. 775 CNVs with allele frequencies > 1% were identified in 102 regions across the genome. 87/102 of these loci overlapped with previously identified regions of CNV. Association of CNVs with gemcitabine and AraC IC50 values identified 11 regions with permutation p-values < 0.05. Multiplex ligation-dependent probe amplification assays were performed to verify the 11 CNV regions that were associated with this phenotype; with false positive and false negative rates for the in-silico findings of 1.3% and 0.04%, respectively. We also had basal mRNA expression array data for these same 197 cell lines, which allowed us to quantify mRNA expression for 41 probesets in or near the CNV regions identified. We found that 7 of those 41 genes were highly expressed in our lymphoblastoid cell lines, and one of the seven genes (SMYD3) that was significant in the CNV association study was selected for further functional experiments. Those studies showed that knockdown of SMYD3, in pancreatic cancer cell lines increased gemcitabine and AraC resistance during cytotoxicity assay, consistent with the results of the association analysis.
These results suggest that CNVs may play a role in variation in cytidine analogue effect. Therefore, association studies of CNVs with drug response phenotypes in cell-based model systems, when paired with functional characterization, might help to identify CNV that contributes to variation in drug response.
PMCID: PMC2894803  PMID: 20525348
15.  Characteristic Face: A Key Indicator for Direct Diagnosis of 22q11.2 Deletions in Chinese Velocardiofacial Syndrome Patients 
PLoS ONE  2013;8(1):e54404.
Velocardiofacial syndrome (VCFS) is a disease in human with an expansive phenotypic spectrum and diverse genetic mechanisms mainly associated with copy number variations (CNVs) on 22q11.2 or other chromosomes. However, the correlations between CNVs and phenotypes remain ambiguous. This study aims to analyze the types and sizes of CNVs in VCFS patients, to define whether correlations exist between CNVs and clinical manifestations in Chinese VCFS patients. In total, 55 clinically suspected Chinese VCFS patients and 100 normal controls were detected by multiplex ligation-dependent probe amplification (MLPA). The data from MLPA and all the detailed clinical features of the objects were documented and analyzed. A total of 44 patients (80.0%) were diagnosed with CNVs on 22q11.2. Among them, 43 (78.2%) presented with 22q11.2 heterozygous deletions, of whom 40 (93.0%) had typical 3-Mb deletion, and 3 (7.0%) exhibited proximal 1.5-Mb deletion; no patient was found with atypical deletion on 22q11.2. One patient (1.8%) presented with a 3-Mb duplication mapping to the typical 3-Mb region on 22q11.2, while none of the chromosomal abnormalities in the MLPA kit were found in the other 11 patients and 100 normal controls. All the 43 patients with 22q11.2 deletions displayed characteristic face and palatal anomalies; 37 of them (86.0%) had cognitive or behavioral disorders, and 23 (53.5%) suffered from immune deficiencies; 10 patients (23.3%) manifested congenital heart diseases. Interestingly, all patients with the characteristic face had 22q11.2 heterozygous deletions, but no difference in phenotypic spectrum was observed between 3-Mb and 1.5-Mb deletions. Our data suggest that the characteristic face can be used as a key indicator for direct diagnosis of 22q11.2 deletions in Chinese VCFS patients.
PMCID: PMC3547028  PMID: 23342150
16.  Novel ATP13A2 (PARK9) homozygous mutation in a family with marked phenotype variability 
Neurogenetics  2010;12(1):33-39.
Mutations in the ATP13A2 (PARK9) and FBXO7 (PARK15) genes are linked to different forms of autosomal recessive juvenile-onset neurodegenerative diseases with overlapping phenotypes, including levodopa-responsive parkinsonism, pyramidal disturbances, cognitive decline, and supranuclear gaze disturbance. However, the associated genotypes and phenotypes are poorly characterized due to the small number of patients described. Here, we report clinical, instrumental, and genetic findings in an Italian family with novel PARK9 and PARK15 mutations. The proband developed a severe progressive phenotype including juvenile-onset parkinsonism, pyramidal disturbances, cognitive decline, and oculomotor abnormalities. On the contrary, his brother only shows mild abnormalities (pyramidal, cognitive, and oculomotor) on the neurological examination at the age of 31 years. These two brothers both carry a novel homozygous PARK9 missense (p.G877R) and a novel heterozygous PARK15 mutation (p.R481C). The PARK9 mutation replaces a crucial residue for the ATPase activity, and is therefore most likely a loss-of-function mutation and disease-causing in homozygous state. The pathogenic significance of the PARK15 single heterozygous mutation remains unclear. In both sibs, DaTSCAN single photon emission computed tomography showed marked nigrostriatal dopaminergic defects, and transcranial magnetic stimulation detected prolonged central motor conduction time. MRI, including T2*-weighted imaging, detected no evidence of brain iron accumulation. This family, the third reported with homozygous PARK9 mutations and the first with mutations in two genes for atypical juvenile parkinsonism, illustrates that PARK9-linked disease might display wide intra-familial clinical variability and milder phenotypes, suggesting the existence of strong, still unknown, modifiers.
PMCID: PMC3029807  PMID: 20853184
Parkinsonism; Movement disorders; PARK9; PARK15; ATP13A2; FBXO7
17.  Both Rare and De Novo Copy Number Variants Are Prevalent in Agenesis of the Corpus Callosum but Not in Cerebellar Hypoplasia or Polymicrogyria 
PLoS Genetics  2013;9(10):e1003823.
Agenesis of the corpus callosum (ACC), cerebellar hypoplasia (CBLH), and polymicrogyria (PMG) are severe congenital brain malformations with largely undiscovered causes. We conducted a large-scale chromosomal copy number variation (CNV) discovery effort in 255 ACC, 220 CBLH, and 147 PMG patients, and 2,349 controls. Compared to controls, significantly more ACC, but unexpectedly not CBLH or PMG patients, had rare genic CNVs over one megabase (p = 1.48×10−3; odds ratio [OR] = 3.19; 95% confidence interval [CI] = 1.89–5.39). Rare genic CNVs were those that impacted at least one gene in less than 1% of the combined population of patients and controls. Compared to controls, significantly more ACC but not CBLH or PMG patients had rare CNVs impacting over 20 genes (p = 0.01; OR = 2.95; 95% CI = 1.69–5.18). Independent qPCR confirmation showed that 9.4% of ACC patients had de novo CNVs. These, in comparison to inherited CNVs, preferentially overlapped de novo CNVs previously observed in patients with autism spectrum disorders (p = 3.06×10−4; OR = 7.55; 95% CI = 2.40–23.72). Interestingly, numerous reports have shown a reduced corpus callosum area in autistic patients, and diminished social and executive function in many ACC patients. We also confirmed and refined previously known CNVs, including significantly narrowing the 8p23.1-p11.1 duplication present in 2% of our current ACC cohort. We found six novel CNVs, each in a single patient, that are likely deleterious: deletions of 1p31.3-p31.1, 1q31.2-q31.3, 5q23.1, and 15q11.2-q13.1; and duplications of 2q11.2-q13 and 11p14.3-p14.2. One ACC patient with microcephaly had a paternally inherited deletion of 16p13.11 that included NDE1. Exome sequencing identified a recessive maternally inherited nonsense mutation in the non-deleted allele of NDE1, revealing the complexity of ACC genetics. This is the first systematic study of CNVs in congenital brain malformations, and shows a much higher prevalence of large gene-rich CNVs in ACC than in CBLH and PMG.
Author Summary
Here, we systematically test the genetic etiology of three common developmental brain malformations: agenesis of the corpus callosum (ACC), cerebellar hypoplasia (CBLH), and polymicrogyria (PMG) by copy number variation (CNV) analysis in a large cohort of brain malformation patients and controls. We found significantly more ACC but not CBLH or PMG patients with rare genic CNVs over one megabase and with rare CNVs impacting over 20 genes when compared with controls. De novo CNVs were found in 9.4% of ACC patients, and interestingly many such CNVs overlapped with de novo CNVs observed in autism. Notably, numerous studies have demonstrated a reduction in the corpus callosum area in autistic brains. Our analysis also refined previously known large CNVs that cause these malformations, and identified six novel CNVs that are likely deleterious. One ACC patient had inherited a deletion from the father which, through exome sequencing, was found to uncover a recessive nonsense mutation in NDE1 on the non-deleted allele inherited from the mother. Our study is the first to systematically evaluate the burden of rare genic CNVs in congenital brain malformations and shows that large gene-rich CNVs are more common in ACC than in CBLH and PMG.
PMCID: PMC3789824  PMID: 24098143
18.  Common Variants in PARK Loci and Related Genes and Parkinson’s Disease 
Rare mutations in PARK loci genes cause Parkinson’s disease (PD) in some families and isolated populations. We investigated the association of common variants in PARK loci and related genes with PD susceptibility and age at onset in an outbred population. 1,103 PD cases from the upper Midwest, USA were individually matched to unaffected siblings (n = 654) or unrelated controls (n = 449) from the same region. Using a sequencing approach in 25 cases and 25 controls, single nucleotide polymorphisms (SNPs) in species-conserved regions of PARK loci and related genes were detected. We selected additional tag SNPs from the HapMap. We genotyped a total of 235 SNPs and two variable number tandem repeats (VNTRs) in the ATP13A2, DJ1, LRRK1, LRRK2, MAPT, Omi/HtrA2, PARK2, PINK1, SNCA, SNCB, SNCG, SPR, and UCHL1 genes in all 2,206 subjects. Case-control analyses were performed to study association with PD susceptibility, while cases-only analyses were used to study association with age at onset. Only MAPT SNP rs2435200 was associated with PD susceptibility after correction for multiple testing (OR = 0.74, 95% CI = 0.64 – 0.86, uncorrected P < 0.0001, log additive model); however, 16 additional MAPT variants, seven SNCA variants, and one LRRK2, PARK2, and UCHL1 variants each had significant uncorrected P-values. There were no significant associations for age at onset after correction for multiple testing. Our results confirm the association of MAPT and SNCA genes with PD susceptibility, but show limited association of other PARK loci and related genes with PD.
PMCID: PMC3606822  PMID: 21412835
19.  Autism genome-wide copy number variation reveals ubiquitin and neuronal genes 
Nature  2009;459(7246):569-573.
Autism spectrum disorders (ASDs) are childhood neurodevelopmental disorders with complex genetic origins1–4. Previous studies focusing on candidate genes or genomic regions have identified several copy number variations (CNVs) that are associated with an increased risk of ASDs5–9. Here we present the results from a whole-genome CNV study on a cohort of 859 ASD cases and 1,409 healthy children of European ancestry who were genotyped with ~550,000 single nucleotide polymorphism markers, in an attempt to comprehensively identify CNVs conferring susceptibility to ASDs. Positive findings were evaluated in an independent cohort of 1,336 ASD cases and 1,110 controls of European ancestry. Besides previously reported ASD candidate genes, such as NRXN1 (ref. 10) and CNTN4 (refs 11, 12), several new susceptibility genes encoding neuronal cell-adhesion molecules, including NLGN1 and ASTN2, were enriched with CNVs in ASD cases compared to controls (P = 9.5 × 10−3). Furthermore, CNVs within or surrounding genes involved in the ubiquitin pathways, including UBE3A, PARK2, RFWD2 and FBXO40, were affected by CNVs not observed in controls (P = 3.3 × 10−3). We also identified duplications 55 kilobases upstream of complementary DNA AK123120 (P = 3.6 × 10−6). Although these variants may be individually rare, they target genes involved in neuronal cell-adhesion or ubiquitin degradation, indicating that these two important gene networks expressed within the central nervous system may contribute to the genetic susceptibility of ASD.
PMCID: PMC2925224  PMID: 19404257
20.  Genetic Etiology of Parkinson Disease Associated with Mutations in the SNCA, PARK2, PINK1, PARK7, and LRRK2 Genes: A Mutation Update 
Human Mutation  2010;31(7):763-780.
To date, molecular genetic analyses have identified over 500 distinct DNA variants in five disease genes associated with familial Parkinson disease; α-synuclein (SNCA), parkin (PARK2), PTEN-induced putative kinase 1 (PINK1), DJ-1 (PARK7), and Leucine-rich repeat kinase 2 (LRRK2). These genetic variants include ∼82% simple mutations and ∼18% copy number variations. Some mutation subtypes are likely underestimated because only few studies reported extensive mutation analyses of all five genes, by both exonic sequencing and dosage analyses. Here we present an update of all mutations published to date in the literature, systematically organized in a novel mutation database ( In addition, we address the biological relevance of putative pathogenic mutations. This review emphasizes the need for comprehensive genetic screening of Parkinson patients followed by an insightful study of the functional relevance of observed genetic variants. Moreover, while capturing existing data from the literature it became apparent that several of the five Parkinson genes were also contributing to the genetic etiology of other Lewy Body Diseases and Parkinson-plus syndromes, indicating that mutation screening is recommendable in these patient groups. Hum Mutat 31:763–780, 2010. © 2010 Wiley-Liss, Inc.
PMCID: PMC3056147  PMID: 20506312
Parkinson disease; genetic etiology; database; SNCA; PARK2; PINK1; PARK7; LRRK2
21.  The Effect of Algorithms on Copy Number Variant Detection 
PLoS ONE  2010;5(12):e14456.
The detection of copy number variants (CNVs) and the results of CNV-disease association studies rely on how CNVs are defined, and because array-based technologies can only infer CNVs, CNV-calling algorithms can produce vastly different findings. Several authors have noted the large-scale variability between CNV-detection methods, as well as the substantial false positive and false negative rates associated with those methods. In this study, we use variations of four common algorithms for CNV detection (PennCNV, QuantiSNP, HMMSeg, and cnvPartition) and two definitions of overlap (any overlap and an overlap of at least 40% of the smaller CNV) to illustrate the effects of varying algorithms and definitions of overlap on CNV discovery.
Methodology and Principal Findings
We used a 56 K Illumina genotyping array enriched for CNV regions to generate hybridization intensities and allele frequencies for 48 Caucasian schizophrenia cases and 48 age-, ethnicity-, and gender-matched control subjects. No algorithm found a difference in CNV burden between the two groups. However, the total number of CNVs called ranged from 102 to 3,765 across algorithms. The mean CNV size ranged from 46 kb to 787 kb, and the average number of CNVs per subject ranged from 1 to 39. The number of novel CNVs not previously reported in normal subjects ranged from 0 to 212.
Conclusions and Significance
Motivated by the availability of multiple publicly available genome-wide SNP arrays, investigators are conducting numerous analyses to identify putative additional CNVs in complex genetic disorders. However, the number of CNVs identified in array-based studies, and whether these CNVs are novel or valid, will depend on the algorithm(s) used. Thus, given the variety of methods used, there will be many false positives and false negatives. Both guidelines for the identification of CNVs inferred from high-density arrays and the establishment of a gold standard for validation of CNVs are needed.
PMCID: PMC3012691  PMID: 21209939
22.  Copy Number Variations in a Population-Based Study of Charcot-Marie-Tooth Disease 
BioMed Research International  2015;2015:960404.
Copy number variations (CNVs) are important in relation to diversity and evolution but can sometimes cause disease. The most common genetic cause of the inherited peripheral neuropathy Charcot-Marie-Tooth disease is the PMP22 duplication; otherwise, CNVs have been considered rare. We investigated CNVs in a population-based sample of Charcot-Marie-Tooth (CMT) families. The 81 CMT families had previously been screened for the PMP22 duplication and point mutations in 51 peripheral neuropathy genes, and a genetic cause was identified in 37 CMT families (46%). Index patients from the 44 CMT families with an unknown genetic diagnosis were analysed by whole-genome array comparative genomic hybridization to investigate the entire genome for larger CNVs and multiplex ligation-dependent probe amplification to detect smaller intragenomic CNVs in MFN2 and MPZ. One patient had the pathogenic PMP22 duplication not detected by previous methods. Three patients had potentially pathogenic CNVs in the CNTNAP2, LAMA2, or SEMA5A, that is, genes related to neuromuscular or neurodevelopmental disease. Genotype and phenotype correlation indicated likely pathogenicity for the LAMA2 CNV, whereas the CNTNAP2 and SEMA5A CNVs remained potentially pathogenic. Except the PMP22 duplication, disease causing CNVs are rare but may cause CMT in about 1% (95% CI 0–7%) of the Norwegian CMT families.
PMCID: PMC4306395  PMID: 25648254
23.  Copy number variation of the APC gene is associated with regulation of bone mineral density☆ 
Bone  2012;51(5):939-943.
Genetic studies of osteoporosis have commonly examined SNPs in candidate genes or whole genome analyses, but insertions and deletions of DNA, collectively called copy number variations (CNVs), also comprise a large amount of the genetic variability between individuals. Previously, SNPs in the APC gene have been strongly associated with femoral neck and lumbar spine volumetric bone mineral density in older men. In addition, familial adenomatous polyposis patients carrying heterozygous mutations in the APC gene have been shown to have significantly higher mean bone mineral density than age- and sex-matched controls suggesting the importance of this gene in regulating bone mineral density. We examined CNV within the APC gene region to test for association with bone mineral density.
DNA was extracted from venous blood, genotyped using the Human Hap610 arrays and CNV determined from the fluorescence intensity data in 2070 Caucasian men and women aged 47.0 ± 13.0 (mean ± SD) years, to assess the effects of the CNV on bone mineral density at the forearm, spine and total hip sites.
Data for covariate adjusted bone mineral density from subjects grouped by APC CNV genotype showed significant difference (P = 0.02–0.002). Subjects with a single copy loss of APC had a 7.95%, 13.10% and 13.36% increase in bone mineral density at the forearm, spine and total hip sites respectively, compared to subjects with two copies of the APC gene.
These data support previous findings of APC regulating bone mineral density and demonstrate that a novel CNV of the APC gene is significantly associated with bone mineral density in Caucasian men and women.
► Previously, SNPs in APC gene have been associated with volumetric BMD in older men. ► We examined CNV in the APC gene and found significant association with BMD in the TwinsUK cohort. ► The maximum difference between subjects with APC CNV and wild type was 13.4% at the total hip site. ► APC plays an important role in bone regulation via the Wnt signaling pathway.
PMCID: PMC3918860  PMID: 22884971
Bone mineral density; Osteoporosis; Copy number variation; APC; Association
24.  De Novo CNV Formation in Mouse Embryonic Stem Cells Occurs in the Absence of Xrcc4-Dependent Nonhomologous End Joining 
PLoS Genetics  2012;8(9):e1002981.
Spontaneous copy number variant (CNV) mutations are an important factor in genomic structural variation, genomic disorders, and cancer. A major class of CNVs, termed nonrecurrent CNVs, is thought to arise by nonhomologous DNA repair mechanisms due to the presence of short microhomologies, blunt ends, or short insertions at junctions of normal and de novo pathogenic CNVs, features recapitulated in experimental systems in which CNVs are induced by exogenous replication stress. To test whether the canonical nonhomologous end joining (NHEJ) pathway of double-strand break (DSB) repair is involved in the formation of this class of CNVs, chromosome integrity was monitored in NHEJ–deficient Xrcc4−/− mouse embryonic stem (ES) cells following treatment with low doses of aphidicolin, a DNA replicative polymerase inhibitor. Mouse ES cells exhibited replication stress-induced CNV formation in the same manner as human fibroblasts, including the existence of syntenic hotspot regions, such as in the Auts2 and Wwox loci. The frequency and location of spontaneous and aphidicolin-induced CNV formation were not altered by loss of Xrcc4, as would be expected if canonical NHEJ were the predominant pathway of CNV formation. Moreover, de novo CNV junctions displayed a typical pattern of microhomology and blunt end use that did not change in the absence of Xrcc4. A number of complex CNVs were detected in both wild-type and Xrcc4−/− cells, including an example of a catastrophic, chromothripsis event. These results establish that nonrecurrent CNVs can be, and frequently are, formed by mechanisms other than Xrcc4-dependent NHEJ.
Author Summary
Copy number variants (CNVs) are a major factor in genetic variation and are a common and important class of mutation in genomic disorders, yet there is limited understanding of how many CNVs arise and the risk factors involved. One DNA damage response pathway implicated in CNV formation is nonhomologous end joining (NHEJ), which repairs broken DNA ends by Xrcc4-dependent direct ligation. We examined the effects of loss of Xrcc4 and NHEJ on CNV formation following replication stress in mouse cells. Cells lacking NHEJ displayed unaltered CNV frequencies, locations, and breakpoint structures compared to normal cells. These results establish that CNV mutations in a cell model system, and likely in vivo, arise by a mutagenic mechanism other than canonical NHEJ, a pattern similar to that reported for model translocation events. Potential roles of alternative end joining and template switching are discussed.
PMCID: PMC3447954  PMID: 23028374
25.  Molecular screening of the CYP4V2 gene in Bietti crystalline dystrophy that is associated with choroidal neovascularization 
Molecular Vision  2011;17:1970-1977.
Bietti crystalline dystrophy (BCD) is an autosomal recessive disease characterized by intraretinal deposits of multiple small crystals, with or without associated crystal deposits in the cornea. The disease is caused by mutation in the cytochrome p450, family 4, subfamily v, polypeptide 2 (CYP4V2) gene. Choroidal neovascularization (CNV) is a rare event in BCD. We report two cases of BCD associated with CNV. CYP4V2 and exon 5 of tissue inhibitor of metalloproteinase 3 (TIMP3) were screened in both cases. A patient with BCD, but without CNV, was also screened to identify pathogenic variations.
Three BCD families of Asian Indian origin were recruited after a comprehensive ophthalmic examination. Genomic DNA was isolated from blood leukocytes, and coding exons and flanking introns of CYP4V2 and exon 5 of TIMP3 were amplified via polymerase chain reaction (PCR) and were sequenced. Family segregation, control screening, and bioinformatics tools were used to assess the pathogenicity of the novel variations.
Of the three BCD patients, two had parafoveal CNV. The patient with BCD, but without CNV had novel single base-pair duplication (c.1062_1063dupA). This mutation results in a structurally defective and unstable protein with impaired protein function. Four novel benign variations (three in exons and one in an intron) were observed in the cohort. Screening of exon 5 of TIMP3 did not reveal any variation in these families.
A novel mutation was found in a patient with BCD but without CNV, while patients with BCD and CNV did not show any pathogenic variation. The modifier role of TIMP3 in the pathogenesis of CNV in BCD was partly ruled out, as no variation was observed in exon 5 of the gene. A larger BCD cohort with CNV needs to be studied and screened to understand the genetics of CNV in BCD.
PMCID: PMC3154135  PMID: 21850171

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