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Background: Drug addiction is influenced by genetic factors., Aim: To determine if genetic variants in the serotonergic and adrenergic pathways are associated with heroin and/or cocaine addiction., Subjects & Methods: The study examined 140 polymorphisms in 19 genes in 1855 subjects with predominantly European or African ancestries., Results: A total of 38 polymorphisms (13 genes) showed nominal associations, including novel associations in S100A10 (p11) and SLC18A2 (VMAT2). The association of HTR3B SNP rs11606194 with heroin addiction in the European ancestry subgroup remained significant after correction for multiple testing (p(corrected) = 0.04)., Conclusion: The study strengthens our previous findings of association of polymorphisms in HTR3A, HTR3B and ADRA1A. The study suggests partial overlap in genetic susceptibility between populations of different ancestry and between heroin and cocaine addiction.

Background & Aim: The dopaminergic pathways have been implicated in the etiology of drug addictions. The aim of this study was to determine if variants in dopaminergic genes are associated with heroin addiction., Materials & Methods: The study includes 828 former heroin addicts and 232 healthy controls, of predominantly European ancestry. Ninety seven SNPs (13 genes) were analyzed., Results: Nine nominally significant associations were observed at CSNK1E, ANKK1, DRD2 and DRD3., Conclusion: The results support our previous report of association of CSNK1E SNP rs1534891 with protection from heroin addiction. CSNK1E interacts with circadian rhythms and DARPP-32 and has been implicated in negative regulation of sensitivity to opioids in rodents. It may be a target for drug addiction treatment. Original submitted 8 August 2014; Revision submitted 8 October 2014.

Stress is a significant risk factor in the development of drug addictions and in addiction relapse susceptibility. This hypothesis-driven study was designed to determine if specific SNPs in genes related to stress response are associated with heroin and/or cocaine addiction in African Americans. The analysis included 27 genes (124 SNPs) and was performed independently for each addiction. The sample consisted of former heroin addicts in methadone maintenance treatment (n = 314), cocaine addicts (n = 281), and controls (n = 208). Fourteen SNPs showed nominally significant association with heroin addiction (p < 0.05), including the African-specific, missense SNP rs5376 (Asn334Ser) in the galanin receptor type 1 gene (GALR1) and the functional FKBP5 intronic SNP rs1360780. Thirteen SNPs showed association with cocaine addiction, including the synonymous SNPs rs237902, in the oxytocin receptor gene (OXTR), and rs5374 in GALR1. No signal remained significant after correction for multiple testing. Four additional SNPs (GALR1 rs2717162, AVP rs2282018, CRHBP rs1875999, and NR3C2 rs1040288) were associated with both addictions and may indicate common liability. The study provides preliminary evidence for novel association of variants in several stress-related genes with heroin and/or cocaine addictions and may enhance the understanding of the interaction between stress and addictions., (© 2014 John Wiley & Sons Ltd/University College London.)

Background: Chemokine receptors CCR2 and CCR5 play a key role in immune and inflammatory responses and have been associated with several diseases, including AIDS. In order to comprehend health disparities it is important to understand the nature of genetic variation in specific genes of interest in different populations. Current studies of the CCR2 and CCR5 receptor genes are primarily focused on the CCR5-Δ32, and CCR2-V64I SNPs., Methods: Sanger sequencing was used to sequence the regions containing 16 SNPs in the adjacent CCR2 and CCR5 genes (including CCR5-Δ32, and CCR2-V64I) in 249 subjects of African, European and Hispanic ancestry. Linkage disequilibrium (LD) and haplotypes were determined using Haploview., Results: The data revealed large differences in allele frequencies of several SNPs and LD patterns among the ethnic groups, including SNPs that were restricted to Africans or Europeans. Seven known CCR5 haplotypes and six novel CCR2 haplotypes were identified. A rare case of an HIV+ subject with the CCR5-Δ32/Δ32 was identified., Conclusions: These data demonstrate a LD between CCR2 and CCR5 at several loci and provide new information about CCR2 that contributes to our understanding of its population-specific genetic variability. The data indicate that in addition to CCR5-Δ32 and CCR2-V64I, other SNPs and haplotypes may be important genetic determinants of disease and should be investigated., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Adequate methadone dosing in methadone maintenance treatment (MMT) for opioid addiction is critical for therapeutic success. One of the challenges in dose determination is the inter-individual variability in dose-response. Methadone metabolism is attributed primarily to cytochrome P450 enzymes CYP3A4, CYP2B6 and CYP2D6. The CYP2B6*6 allele [single nucleotide polymorphisms (SNPs) 785A>G (rs2279343) and 516G>T (rs3745274)] was associated with slow methadone metabolism. To explore the effects of CYP2B6*6 allele on methadone dose requirement, it was genotyped in a well-characterized sample of 74 Israeli former heroin addicts in MMT. The sample is primarily of Middle Eastern/European ancestry, based on ancestry informative markers (AIMs). Only patients with no major co-medication that may affect methadone metabolism were included. The stabilizing daily methadone dose in this sample ranges between 13 and 260mg (mean 140±52mg). The mean methadone doses required by subjects homozygous for the variant alleles of the CYP2B6 SNPs 785A>G and 516G>T (88, 96mg, respectively) were significantly lower than those of the heterozygotes (133, 129mg, respectively) and the non-carriers (150, 151mg, respectively) (nominal P=0.012, 0.048, respectively). The results remain significant after controlling for age, sex and the ABCB1 SNP 1236C>T (rs1128503), which was previously shown to be associated with high methadone dose requirement in this population (P=0.006, 0.030, respectively). An additional 77 CYP2B6, CYP3A4 and CYP2D6 SNPs were genotyped. Of these, 24 SNPs were polymorphic and none showed significant association with methadone dose. Further studies are necessary to replicate these preliminary findings in additional subjects and other populations., (© 2011 The Authors, Addiction Biology © 2011 Society for the Study of Addiction.)

Aim: The interindividual variability in the dose required for effective methadone maintenance treatment (MMT) for opioid addiction may be influenced in part by genetic variations in genes encoding pharmacodynamic factors of methadone. This study was conducted to identify some of these variants., Materials & Methods: This study focused on 11 genes encoding components of the opioidergic (OPRM1, POMC and ARRB2), the dopaminergic (ANKK1 and DRD2) and the glutamatergic pathways (GRIN1 and GRIN2A), as well as the neurotrophin system (NGFB, BDNF, NTRK1 and NTRK2). The study includes 227 Israeli patients undergoing stable MMT., Results: Out of the 110 variants analyzed, 12 SNPs (in BDNF, NTRK2, OPRM1, DRD2 and ANKK1) were associated with methadone dose (nominal p < 0.05). Of these SNPs, ANKK1 rs7118900 and DRD2 rs2283265 are known to affect gene expression. Logistic regression of five representative SNPs discriminated between individuals requiring a methadone dose of >120 mg/day and <120 mg/day (p = 0.019), and showed moderate sensitivity and specificity (AUC of 0.63 in receiver operating characteristic analysis)., Conclusion: This data should stimulate further research on the potential influence and clinical significance of these variants on MMT.

Objective: Studies indicate cross-desensitization between opioid receptors (eg, kappa opioid receptor, OPRK1) and chemokine receptors (eg, CXCR4) involved in HIV infection. Whether gene variants of OPRK1 and its ligand, prodynorphin (PDYN), influence the outcome of HIV therapy was tested., Methods: Three study points, admission to the Women's Interagency HIV Study, initiation of highly active antiretroviral therapy (HAART), and the most recent visit, were chosen for analysis as crucial events in the clinical history of the HIV patients. Regression analyses of 17 variants of OPRK1 and 11 variants of PDYN with change of viral load (VL) and CD4 count between admission and initiation of HAART and initiation of HAART to the most recent visit to Women's Interagency HIV Study were performed in 598 HIV+ subjects, including African Americans, Hispanics, and Whites. Association with HIV status was done in 1009 subjects., Results: Before HAART, greater VL decline (improvement) in carriers of PDYN IVS3+189C>T and greater increase of CD4 count (improvement) in carriers of OPRK -72C>T were found in African Americans. Also, greater increase of CD4 count in carriers of OPRK1 IVS2+7886A>G and greater decline of CD4 count (deterioration) in carriers of OPRK1 -1205G>A were found in Whites. After HAART, greater decline of VL in carriers of OPRK1 IVS2+2225G>A and greater increase of VL in carriers of OPRK1 IVS2+10658G>T and IVS2+10963A>G were found in Whites. Also, a lesser increase of CD4 count was found in Hispanic carriers of OPRK1 IVS2+2225G>A., Conclusions: OPRK1 and PDYN polymorphisms may alter severity of HIV infection and response to treatment.

Addictive diseases, including addiction to heroin, prescription opioids, or cocaine, pose massive personal and public health costs. Addictions are chronic relapsing diseases of the brain caused by drug-induced direct effects and persisting neuroadaptations at the epigenetic, mRNA, neuropeptide, neurotransmitter, or protein levels. These neuroadaptations, which can be specific to drug type, and their resultant behaviors are modified by various internal and external environmental factors, including stress responsivity, addict mindset, and social setting. Specific gene variants, including variants encoding pharmacological target proteins or genes mediating neuroadaptations, also modify vulnerability at particular stages of addiction. Greater understanding of these interacting factors through laboratory-based and translational studies have the potential to optimize early interventions for the therapy of chronic addictive diseases and to reduce the burden of relapse. Here, we review the molecular neurobiology and genetics of opiate addiction, including heroin and prescription opioids, and cocaine addiction.

Addiction to drugs is a chronic, relapsing brain disease that has major medical, social, and economic complications. It has been established that genetic factors contribute to the vulnerability to develop drug addiction and to the effectiveness of its treatment. Identification of these factors may increase our understanding of the disorders, help in the development of new treatments and advance personalized medicine. In this review, we will describe the genetics of the major genes of the opioid system (opioid receptors and their endogenous ligands) in connection to addiction to opioids, cocaine, alcohol and methamphetamines. Particular emphasis is given to association and functional studies of specific variants. We will provide information on the sample populations and the size of each study, as well as a list of the variants implicated in association with addiction-related phenotypes, and with the effectiveness of pharmacotherapy for addiction.

Background: Mu opioid receptor (OPRM1) ligands may alter expression of chemokines and chemokine receptors involved in penetration of human immunodeficiency virus (HIV) type 1 into the cell. We suggest that OPRM1 variants may affect the pathophysiology of HIV infection., Methods: DNA samples from 1031 eligible African Americans, Hispanics, and whites from the Women's Interagency HIV Study (WIHS) who were alive as of April 2006 were analyzed. We performed regression analysis of association of 18 OPRM1 variants with a change of viral load and CD4 cell count during 2 periods: between admission to WIHS and the start of highly active antiretroviral therapy (HAART) (interval X) and between the start of HAART and the most recent WIHS visit (interval Y), and examined the association of these variants with HIV status., Results: Regardless of genotype, a significant decrease in viral load during interval X was found for each ethnicity. Whites with allele G of the functional polymorphism 118A > G (reference sequence rs1799971) showed a smaller decrease in viral load; those bearing minor alleles IVS1 + 1050A, IVS1 + 14123A, and IVS2 + 31A showed a larger decrease in viral load over interval X (0.01 < P < .05). Hispanics with the same alleles showed a greater increase in CD4 cell count over interval Y (0.01 < P < .05). We found an association between OPRM1 variants and HIV status in African Americans and whites., Conclusions: OPRM1 polymorphisms may alter the severity of HIV infection before and after HAART.

Objective: Dynorphins, the endogenous ligands for the κ opioid receptor, are implicated in neuropsychiatric disorders through modulation of basal and stimuli-induced dopaminergic, glutamatergic, and serotonergic tones. Expression of the prodynorphin gene (PDYN) is critical for rewarding properties of drugs of abuse and stress-induced responses. Epigenetic factors, such as DNA methylation, play an important role in modulation of gene expression., Methods: We analyzed DNA methylation patterns of three CpG-rich regions of PDYN, a CpG island, and cluster A in the proximal promoter, and cluster B in coding exon 4, by bisulfite sequencing of DNA from the caudate and anterior cingulate cortex from post-mortem brain of 35 individuals (22 HIV seropositive), and in peripheral blood mononuclear cells from 21 of these individuals., Results: We found remarkably similar patterns of methylation across CpG sites in these tissues. However, there were tissue-specific differences in methylation levels (P=0.000001) of the CpG island: higher levels in peripheral blood mononuclear cells (82%) than in the brain tissues, the caudate (62%), and the anterior cingulate cortex (44%). But there was higher PDYN expression in the caudate than in the anterior cingulate cortex. In contrast, cluster A near the transcription start site is hypomethylated., Conclusion: This DNA methylation profile of the PDYN gene is typical for primary responsive genes with regulatory elements for both basal and tissue-specific transcription. Our findings provide a rationale for further studies of the role of other epigenetic factors in the regulation of PDYN expression in individuals with psychiatric and neurological disorders.

The opioid system is involved in the action of opiate drugs, opioid addiction, pain experience and analgesia. Individual differences in opioid effect may be attributed in part to genetic variations. Long-range cis regulatory elements and intronic variants are potential sources of functional diversity. Recently, we have detected association of two intronic OPRM1 variants with heroin addiction in European Americans. In this study, we analyzed the genetic variations in the OPRM1 100 kb 5'-flanking region and intron 1 in the HapMap Caucasian population. Four major linkage disequilibrium blocks were identified, consisting of 28, 22, 15 and 42 single-nucleotide polymorphisms (SNPs), respectively. The locations of these blocks are (-100 to -90), (-90 to -67), (-20 to -1) and (+1 to +44) kb, respectively. The two intronic variants, indicated in our recent study, are part of a distinct haplogroup that includes SNPs from intron 1, and the proximal 5' region. The 118G (rs1799971) allele is part of a different haplogroup that includes several variants in the distal 5' region that may have a regulatory potential. These findings were corroborated by genotyping eight SNPs in a sample of European Americans and suggest an extended OPRM1 locus with potential new regulatory regions.

Using real time qPCR, we examined the expression of mRNAs for the five somatostatin receptors (SSTRs) in the caudate putamen of male C57BL/6J and 129P3/J mice. Animals were exposed to multiple injections of heroin, or saline, in the setting of a conditioned place preference study. The relative expression levels of the five SSTR mRNAs differed between the two strains. In both strains, SSTR-1 mRNA was expressed at the highest levels and SSTR-5 at the lowest. Interestingly, in 129P3/J mice SSTR-3 mRNA was not detected in the caudate putamen. We confirmed this finding in the frontal cortex, hypothalamus, nucleus accumbens and a region containing the substantia nigra and ventral tegmental area. We also found strain differences in the mRNA levels of SSTR-2 and -4. Intermittent heroin administration had a dose-dependent effect on the levels of SSTR-1 and -3 mRNAs. These results demonstrate strain differences in the expression of specific mRNAs and a heroin-induced dose-dependent elevation of SSTR-1 and -3 mRNAs in the mouse caudate putamen., (Copyright 2010 Elsevier B.V. All rights reserved.)

Addiction to opiates and illicit use of psychostimulants is a chronic, relapsing brain disease that, if left untreated, can cause major medical, social, and economic problems. This article reviews recent progress in studies of association of gene variants with vulnerability to develop opiate and cocaine addictions, focusing primarily on genes of the opioid and monoaminergic systems. In addition, we provide the first evidence of a cis-acting polymorphism and a functional haplotype in the PDYN gene, of significantly higher DNA methylation rate of the OPRM1 gene in the lymphocytes of heroin addicts, and significant differences in genotype frequencies of three single-nucleotide polymorphisms of the P-glycoprotein gene (ABCB1) between "higher" and "lower" methadone doses in methadone-maintained patients. In genomewide and multigene association studies, we found association of several new genes and new variants of known genes with heroin addiction. Finally, we describe the development and application of a novel technique: molecular haplotyping for studies in genetics of drug addiction.

Dynorphin peptides and the kappa-opioid receptor are important in the rewarding properties of cocaine, heroin, and alcohol. We tested polymorphisms of the prodynorphin gene (PDYN) for association with cocaine dependence and cocaine/alcohol codependence. We genotyped six single nucleotide polymorphisms (SNPs), located in the promoter region, exon 4 coding, and 3' untranslated region, in 106 Caucasians and 204 African Americans who were cocaine dependent, cocaine/alcohol codependent, or controls. In Caucasians, we found point-wise significant associations of 3'UTR SNPs (rs910080, rs910079, and rs2235749) with cocaine dependence and cocaine/alcohol codependence. These SNPs are in high linkage disequilibrium, comprising a haplotype block. The haplotype CCT was significantly experiment-wise associated with cocaine dependence and with combined cocaine dependence and cocaine/alcohol codependence (false discovery rate, q=0.04 and 0.03, respectively). We investigated allele-specific gene expression of PDYN, using SNP rs910079 as a reporter, in postmortem human brains from eight heterozygous subjects, using SNaPshot assay. There was significantly lower expression for C allele (rs910079), with ratios ranging from 0.48 to 0.78, indicating lower expression of the CCT haplotype of PDYN in both the caudate and nucleus accumbens. Analysis of total PDYN expression in 43 postmortem brains also showed significantly lower levels of preprodynorphin mRNA in subjects having the risk CCT haplotype. This study provides evidence that a 3'UTR PDYN haplotype, implicated in vulnerability to develop cocaine addiction and/or cocaine/alcohol codependence, is related to lower mRNA expression of the PDYN gene in human dorsal and ventral striatum.

This review primarily focuses on our recent findings in bidirectional translational research on opiate and cocaine addictions. First, we present neurobiological and molecular studies on endogenous opioid systems (e.g. proopiomelanocortin, mu opioid receptor, dynorphin, and kappa opioid receptor), brain stress-responsive systems (e.g. orexin, arginine vasopressin, V1b receptor, and corticotropin-releasing factor), hypothalamic-pituitary-adrenal axis, and neurotransmitters (especially dopamine), in response to both chronic cocaine or opiate exposure and to drug withdrawal, using several newly developed animal models and molecular approaches. The second aspect is human molecular genetic association investigations including hypothesis-driven studies and genome-wide array studies, to define particular systems involved in vulnerability to develop specific addictions, and response to pharmacotherapy.

Methadone is a mu-opioid receptor agonist used for treating opiate dependence. The range of effective methadone doses is broad. Part of the large inter-individual variability in efficacy may be accounted for by genetic factors. Methadone is a substrate of the transporter P-glycoprotein (P-gp) 170 that is encoded by the ABCB1 (MDR1) gene. Thus, P-gp variants may play a role in methadone absorption and distribution. We assessed the association between ABCB1 polymorphisms and methadone dose requirements in 98 methadone-maintained patients. The stabilizing methadone doses were normally distributed with a mean and median dose of 160 mg/day (range 30-280 mg/day). Statistical analysis showed significant difference in genotype frequencies between the 'higher' (>150 mg/day) and 'lower' (< or =150 mg/day) methadone dose groups for single nucleotide polymorphism (SNP) 1236C>T (rs1128503) (experiment-wise P = 0.0325). Furthermore, individuals bearing the 3-locus genotype pattern TT-TT-TT (rs1045642, rs2032582 and rs1128503) have an approximately 5-fold chance of requiring the 'higher' methadone dose, while individuals heterozygous for these three SNPs have an approximately 3-fold chance of stabilizing at the 'lower' methadone dose (point-wise P-value = 0.026). These data suggest that specific ABCB1 variants may have clinical relevance by influencing the methadone dose required to prevent withdrawal symptoms and relapse in this population.

Fanconi anemia (FA) is a rare autosomal recessive disease characterized by a greatly increased risk of cancer among those diagnosed with the syndrome. The question as to whether FA heterozygotes are at increased risk for cancer is of great importance to those at risk for being a carrier. To address this question, we formed a cohort of grandparents of probands identified through the International Fanconi Anemia Registry. We obtained informed consent, a short questionnaire, and either blood or buccal swab DNA. After diagnosis of the proband was confirmed and complementation studies or DNA sequencing on the proband were completed, mutation analyses of the putative carriers and noncarriers was carried out. Standardized incidence ratios (SIR) were calculated to compare the observed cancer incidence of the grandparents and other relatives with the expected rates of cancer, using the Surveillance, Epidemiology, and End Results registries and the Connecticut Cancer registry. In the 944 study subjects who participated (784 grandparents and 160 other relatives), there was no suggestion of an increase in overall cancer incidence. On the other hand, a significantly higher rate of breast cancer than expected was observed among carrier grandmothers [SIR, 1.7; 95% confidence interval (95% CI), 1.1-2.7]. Among the grandmothers, those who were carriers of FANCC mutations were found to be at highest risk (SIR, 2.4; 95% CI, 1.1-5.2). Overall, there was no increased risk for cancer among FA heterozygotes in this study of Fanconi relatives, although there is some evidence that FANCC mutations are possibly breast cancer susceptibility alleles.

Fanconi anemia (FA) is a rare autosomal recessive disorder that results from mutations in at least 11 different genes. Recent studies have demonstrated that clinical progression of the disease may be influenced by inter- and intragenic variations, emphasizing the importance of identifying the complementation groups. In the present study we have employed bicistronic retrovirus vectors that coexpress FA-specific cDNAs for complementation groups A, C, F, and G, together with the enhanced green fluorescence protein (EGFP), allowing for specific analysis of transduced EGFP+ cells within bulk cultures by flow cytometry. In addition, the assay relies on the correction of the characteristic FA-associated G2/M arrest after treatment of cells with DNA-damaging agents, which is analyzed by flow cytometry. Results obtained with this assay matched the complementation groups known for 12 control lymphoblast cell lines tested. We report here the results obtained for 48 FA patients with unknown complementation groups using this new assay. Complementation groups were identified for 24 patients. We have identified mutations in the genes corresponding to the assigned complementation group in 23 samples. This assay has now been established in a standardized fashion for complementation assignments in FA patients and the subsequent directing of rapid mutation analysis in those patients.

In the search for genetic modifiers of the Fanconi anaemia (FA) phenotype, we examined the role of polymorphism in three glutathione s-transferase genes (GSTT1, GSTM1 and GSTP1) in 356 FA patients enrolled in the International Fanconi Anaemia Registry (IFAR). Cellular sensitivity to 1,2:3,4 diepoxybutane was significantly increased in GSTT1 deleted compared with GSTT1 positive cases (median chromosomal breaks 11.1 vs. 8.3, P < 0.01) but there was no effect on clinical manifestations of FA. GSTM1 genotype significantly influenced time to bone marrow failure in complementation group FA-C, (median age 3.0 years vs. 7.0 years, P < 0.01). GSTP1 genotype did not influence FA phenotype.

Seven Fanconi anemia-associated proteins (FANCA, FANCB, FANCC, FANCE, FANCF, FANCG and FANCL) form a nuclear Fanconi anemia core complex that activates the monoubiquitination of FANCD2, targeting FANCD2 to BRCA1-containing nuclear foci. Cells from individuals with Fanconi anemia of complementation groups D1 and J (FA-D1 and FA-J) have normal FANCD2 ubiquitination. Using genetic mapping, mutation identification and western-blot data, we identify the defective protein in FA-J cells as BRIP1 (also called BACH1), a DNA helicase that is a binding partner of the breast cancer tumor suppressor BRCA1.

Fanconi anemia (FA) is an autosomal recessive disorder that is defined by cellular hypersensitivity to DNA cross-linking agents, and is characterized clinically by developmental abnormalities, progressive bone-marrow failure, and predisposition to leukemia and solid tumors. There is extensive genetic heterogeneity, with at least 11 different FA complementation groups. FA-A is the most common group, accounting for approximately 65% of all affected individuals. The mutation spectrum of the FANCA gene, located on chromosome 16q24.3, is highly heterogeneous. Here we summarize all sequence variations (mutations and polymorphisms) in FANCA described in the literature and listed in the Fanconi Anemia Mutation Database as of March 2004, and report 61 novel FANCA mutations identified in FA patients registered in the International Fanconi Anemia Registry (IFAR). Thirty-eight novel SNPs, previously unreported in the literature or in dbSNP, were also identified. We studied the segregation of common FANCA SNPs in FA families to generate haplotypes. We found that FANCA SNP data are highly useful for carrier testing, prenatal diagnosis, and preimplantation genetic diagnosis, particularly when the disease-causing mutations are unknown. Twenty-two large genomic deletions were identified by detection of apparent homozygosity for rare SNPs. In addition, a conserved SNP haplotype block spanning at least 60 kb of the FANCA gene was identified in individuals from various ethnic groups., ((c) 2005 Wiley-Liss, Inc.)

The breast cancer susceptibility gene BRCA2 has recently been identified as identical to the Fanconi anemia (FA) gene FANCD1. Here we expand the clinical implications of this discovery. Notably, we identified 6 children in 5 kindreds exhibiting the co-occurrence of BRCA2 mutations, FA, and early onset acute leukemia. Leukemia occurred at a median of 2.2 years of age in the BRCA2 patients in contrast to a median onset of 13.4 years in all other FA patients in the International Fanconi Anemia Registry (IFAR; P <.0001). Breast cancer was noted in 4 of the 5 kindreds. Of the 6 children with leukemia, 4 were treated with bone marrow transplantation and 2 are alive at 3 and 9 months after treatment. Our results suggest that BRCA2 testing should be considered in all patients with FA in whom the complementation group cannot be defined or in whom leukemia is diagnosed at or before 5 years of age.

Fanconi anemia is an autosomal recessive disorder characterized by congenital malformations, bone marrow failure, and the development of squamous cell carcinomas (SCCs) and other cancers. Recent clinicopathologic evidence has raised the possibility that an environmental factor such as human papillomavirus (HPV) may be involved in the pathogenesis of SCCs in Fanconi anemia patients. Given the high prevalence of p53 mutations in SCCs among the general population and the lack of p53 mutations in HPV-related carcinogenesis, we evaluated the role of HPV and p53 mutations and polymorphisms in SCC from Fanconi anemia patients. We used polymerase chain reaction (PCR) screening and real-time PCR to detect and quantify HPV DNA in DNA extracted from microdissected SCCs obtained from 24 Fanconi anemia patients (n = 25 SCCs; case subjects) and 50 age-, sex-, and tumor site-matched SCC patients without Fanconi anemia (n = 50 SCCs; control subjects). We PCR-amplified and sequenced exons 4-9 of the p53 gene from SCC DNA. We detected HPV DNA in 84% of the SCC specimens from the case subjects and in 36% of the SCC specimens from the control subjects (P<.001). The prevalence of p53 mutations in SCCs from the case subjects (0%, 0/25) was statistically significantly lower than that of SCCs from the control subjects (36%, 12/33; P<.001). A greater proportion of patients with Fanconi anemia and SCC were homozygous for Arg72, a p53 polymorphism that may be associated with increased risk for HPV-associated human malignancies, than an ethnically-matched cohort of Fanconi anemia patients without SCC (75% versus 51%; P =.05). These data suggest that Fanconi anemia is associated with increased susceptibility to HPV-induced carcinogenesis.

Fanconi anemia is an inherited disease characterized by bone marrow failure, congenital malformations, and predisposition to cancer. The breast cancer susceptibility gene BRCA2 was recently found to be associated with Fanconi anemia complementation group D1 (FA-D1). We examined four kindreds afflicted with Fanconi anemia for the presence of germline BRCA2 mutations. One kindred, of Ashkenazi Jewish ancestry, had five members who were diagnosed with breast cancer and two cousins who were BRCA2*6174delT/C3069X compound heterozygotes and had Fanconi anemia and brain tumors. In another kindred of Ashkenazi Jewish and Lithuanian Catholic ancestry, a child with Fanconi anemia and a medulloblastoma was a BRCA2*6174delT/886delGT compound heterozygote. Two other kindreds each contained a Fanconi anemia-afflicted child who developed medulloblastoma; one child was of Latin American ancestry and a compound heterozygote for BRCA2*I2490T/ 5301insA and the other was African American and a compound heterozygote for BRCA2*Q3066X/E1308X. Median age of the Fanconi anemia-afflicted children at brain tumor diagnosis was 3.5 years. The co-occurrence of brain tumors, Fanconi anemia, and breast cancer observed in one of these kindreds constitutes a new syndromic association. Individuals who carry a germline BRCA2 mutation and who plan to have children with a partner of Ashkenazi Jewish descent should consider undergoing genetic counseling.

Fanconi anemia (FA) is a genetically heterogeneous autosomal recessive syndrome associated with chromosomal instability, hypersensitivity to DNA cross-linking agents, and predisposition to malignancy. The gene for FA complementation group G (FANCG) was the third FA gene to be cloned, and was found to be identical with human XRCC9, which maps to 9p13. The cDNA is predicted to encode a polypeptide of 622 amino acids, with no sequence similarities to any other known protein or motifs that could point to a molecular function for FANCG/XRCC9. We used single strand conformational polymorphism analysis (SSCP) to screen genomic DNA from a panel of 307 racially and ethnically diverse unrelated FA patients from the International Fanconi Anemia Registry (IFAR) for variants in FANCG. Twenty-seven abnormal SSCP patterns were found; 18 of these variants appear to be pathogenic mutations while nine are likely to be nonpathogenic polymorphisms. Direct sequencing of genomic DNA from seven FA-G probands with one mutant allele not detected in the SSCP study and three additional probands assigned to the FA-G complementation group by retroviral correction with FANCG resulted in the detection of nine additional pathogenic mutations and two common SNPs. Conditions for rapid screening for these mutations by DHPLC for use in a clinical laboratory setting were established. The most common FANCG mutations in the IFAR population were: IVS8-2A>G (seven Portuguese-Brazilian probands), IVS11+1G>C (seven French-Acadian probands), 1794&#95;1803del10 (seven European probands), and IVS3+1G>C (five Korean or Japanese probands). Our data suggest that the Portuguese-Brazilian, French-Acadian, and Korean/Japanese mutations were likely to have been present in a founding member of each of these populations., (Copyright 2003 Wiley-Liss, Inc.)

Somatic mosaicism has been observed previously in the lymphocyte population of patients with Fanconi anemia (FA). To identify the cellular origin of the genotypic reversion, we examined each lymphohematopoietic and stromal cell lineage in an FA patient with a 2815-2816ins19 mutation in FANCA and known lymphocyte somatic mosaicism. DNA extracted from individually plucked peripheral blood T cell colonies and marrow colony-forming unit granulocyte-macrophage and burst-forming unit erythroid cells revealed absence of the maternal FANCA exon 29 mutation in 74.0%, 80.3%, and 86.2% of colonies, respectively. These data, together with the absence of the FANCA exon 29 mutation in Epstein-Barr virus-transformed B cells and its presence in fibroblasts, indicate that genotypic reversion, most likely because of back mutation, originated in a lymphohematopoietic stem cell and not solely in a lymphocyte population. Contrary to a predicted increase in marrow cellularity resulting from reversion in a hematopoietic stem cell, pancytopenia was progressive. Additional evaluations revealed a partial deletion of 11q in 3 of 20 bone marrow metaphase cells. By using interphase fluorescence in situ hybridization with an MLL gene probe mapped to band 11q23 to identify colony-forming unit granulocyte-macrophage and burst-forming unit erythroid cells with the 11q deletion, the abnormal clone was exclusive to colonies with the FANCA exon 29 mutation. Thus, we demonstrate the spontaneous genotypic reversion in a lymphohematopoietic stem cell. The subsequent development of a clonal cytogenetic abnormality in nonrevertant cells suggests that ex vivo correction of hematopoietic stem cells by gene transfer may not be sufficient for providing life-long stable hematopoiesis in patients with FA.

Fanconi anemia (FA) is an autosomal recessive syndrome associated with hypersensitivity to DNA cross-linking agents and predisposition to neoplasia. Eight complementation groups (A-H) have been described, but the only FA genes cloned so far are FAC and FAA. We have recently identified 40 different germline mutations, including microdeletions, microinsertions, and point mutations in genomic DNA from 97 FA patients from the International Fanconi Anemia Registry (IFAR) by single-strand conformational polymorphism (SSCP) analysis. Interestingly, only one mutant allele was identified in many of these patients. Haplotype analysis with intragenic polymorphisms, as well as cDNA analysis of some patients suggested the presence of large deletions that would not be detected by SSCP analysis. In this study, we report the occurrence of Alu-mediated genomic deletions in FAA. Two different deletions of 1.2 kb and 1.9 kb were found. Both deletions include exons 16 and 17 and remove a 156-bp segment from the transcript causing a shorter in-frame message. Sequence analysis revealed that introns 15 and 17 are rich in partial and complete Alu repeats. There are at least four head-to-tail arranged Alu elements in intron 17 and one in intron 15, all oriented in the 3'-->5' direction. Sequence analysis of the deletions showed that the 5' breakpoints occurred at different sites in the same Alu element in intron 15, while the 3' breakpoints were located in different Alu repeats in intron 17. Numerous Alu repeats are present in FAA, suggesting that Alu-mediated recombination might be an important mechanism for the generation of FAA mutations.

Fanconi anemia (FA) is a genetically heterogeneous autosomal recessive syndrome associated with chromosomal instability, hypersensitivity to DNA crosslinking agents, and predisposition to malignancy. The gene for FA complementation group A (FAA) recently has been cloned. The cDNA is predicted to encode a polypeptide of 1,455 amino acids, with no homologies to any known protein that might suggest a function for FAA. We have used single-strand conformational polymorphism analysis to screen genomic DNA from a panel of 97 racially and ethnically diverse FA patients from the International Fanconi Anemia Registry for mutations in the FAA gene. A total of 85 variant bands were detected. Forty-five of the variants are probably benign polymorphisms, of which nine are common and can be used for various applications, including mapping studies for other genes in this region of chromosome 16q. Amplification refractory mutation system assays were developed to simplify their detection. Forty variants are likely to be pathogenic mutations. Seventeen of these are microdeletions/microinsertions associated with short direct repeats or homonucleotide tracts, a type of mutation thought to be generated by a mechanism of slipped-strand mispairing during DNA replication. A screening of 350 FA probands from the International Fanconi Anemia Registry for two of these deletions (1115-1118del and 3788-3790del) revealed that they are carried on about 2% and 5% of the FA alleles, respectively. 3788-3790del appears in a variety of ethnic groups and is found on at least two different haplotypes. We suggest that FAA is hypermutable, and that slipped-strand mispairing, a mutational mechanism recognized as important for the generation of germ-line and somatic mutations in a variety of cancer-related genes, including p53, APC, RB1, WT1, and BRCA1, may be a major mechanism for FAA mutagenesis.

Fanconi anemia (FA) is a genetically heterogenous disease involving at least five genes on the basis of complementation analysis (FAA to FAE). The FAA gene has been recently isolated by two independent approaches, positional and functional cloning. In the present study we describe the genomic structure of the FAA gene. The gene contains 43 exons spanning approximately 80 kb as determined by the alignment of four cosmids and the fine localization of the first and the last exons in restriction fragments of these clones. Exons range from 34 to 188 bp. All but three of the splice sites were consistent with the ag-gt rule. We also describe three alternative splicing events in cDNA clones that result in the loss of exon 37, a 23-bp deletion at the 5' end of exon 41, and a GCAG insertion at the 3' portion also in exon 41. Sequence analysis of the 5' region upstream of the putative transcription start site showed no obvious TATA and CAAT boxes, but did show a GC-rich region, typical of housekeeping genes. Knowledge of the structure of the FAA gene will provide an invaluable resource for the discovery of mutations in the gene that accounts for about 60-66% of FA patients.

We report the results of a genomewide scan using homozygosity mapping to identify genes causing Fanconi anemia, a genetically heterogeneous recessive disorder. By studying 23 inbred families, we detected linkage to a locus causing Fanconi anemia near marker D16S520 (16q24.3). Although -65% of our families displayed clear linkage to D16S520, we found strong evidence (P = .0013) of genetic heterogeneity. This result independently confirms the recent mapping of the FAA gene to chromosome 16 by Pronk et al. Family ascertainment was biased against a previously identified FAC gene on chromosome 9, and no linkage was observed to this locus. Simultaneous search analysis suggested several additional chromosomal regions that could account for a small fraction of Fanconi anemia in our families, but the sample size is insufficient to provide statistical significance. We also demonstrate the strong effect of marker allele frequencies on LOD scores obtained in homozygosity mapping and discuss ways to avoid false positives arising from this effect.

Acid sphingomyelinase (ASM; HGMW-approved symbol, SMPD1) is the lysosomal phosphodiesterase that hydrolyzes sphingomyelin to ceramide and phosphocholine. The deficient activity of this enzyme results in Types A and B Niemann-Pick disease (NPD). The full-length cDNA encoding human ASM has been isolated and characterized (E. H. Schuchman, M. Suchi, T. Takahashi, K. Sandhoff, and R. J. Desnick (1991) J. Biol. Chem. 66:8531-8539), and the ASM gene has been localized to chromosomal region 11p15.1-p15.4 (L. V. Pereira, R. J. Desnick, D. Adler, C. M. Disteche, and E. H. Schuchman (1991) Genomics 9:229-234). Using the cDNA as a probe, a genomic clone containing the ASM genomic region was isolated and the complete nucleotide sequence of the human ASM gene, including 1116 and 468 nucleotides upstream and downstream from the ASM coding region, respectively, was determined. This housekeeping gene contained six exons ranging in size from 77 to 773 bp and five introns ranging in size from 153 to 1059 bp. Exon 2 was unusually large and encoded 258 amino acids, or about 44% of the mature ASM polypeptide. The alternatively spliced 172-bp type 1-specific sequence was encoded by exon 3, whereas the type 2-specific sequence was located at the 5' end of intron 2. An analysis of the intron/exon junctions revealed that there was a weak donor splice site (AAA gtgagg) at the exon 3/intron 3 junction which occasionally leads to alternative splicing of exon 3 and the occurrence of the type 2 and 3 ASM transcripts. A single Alu1 element in the reverse orientation was in intron 2, immediately downstream from the type 2-specific sequence.(ABSTRACT TRUNCATED AT 250 WORDS)