Candidate Modifiers Research Articles

Disease severity in a mouse model of ataxia telangiectasia is modulated by the DNA damage checkpoint gene Hus1

The human genomic instability syndrome ataxia telangiectasia (A-T), caused by mutations in the gene encoding the DNA damage checkpoint kinase ATM, is characterized by multisystem defects including neurodegeneration, immunodeficiency and increased cancer predisposition. ATM is central to a pathway that responds to double-strand DNA breaks, whereas the related kinase ATR leads a parallel signaling cascade that is activated by replication stress. To dissect the physiological relationship between the ATM and ATR pathways, we generated mice defective for both. Because complete ATR pathway inactivation causes embryonic lethality, we weakened the ATR mechanism to different degrees by impairing HUS1, a member of the 911 complex that is required for efficient ATR signaling. Notably, simultaneous ATM and HUS1 defects caused synthetic lethality. Atm/Hus1 double-mutant embryos showed widespread apoptosis and died mid-gestationally. Despite the underlying DNA damage checkpoint defects, increased DNA damage signaling was observed, as evidenced by H2AX phosphorylation and p53 accumulation. A less severe Hus1 defect together with Atm loss resulted in partial embryonic lethality, with the surviving double-mutant mice showing synergistic increases in genomic instability and specific developmental defects, including dwarfism, craniofacial abnormalities and brachymesophalangy, phenotypes that are observed in several human genomic instability disorders. In addition to identifying tissue-specific consequences of checkpoint dysfunction, these data highlight a robust, cooperative configuration for the mammalian DNA damage response network and further suggest HUS1 and related genes in the ATR pathway as candidate modifiers of disease severity in A-T patients.

Phenotype variations in Lafora progressive myoclonus epilepsy: possible involvement of genetic modifiers?

Lafora progressive myoclonus epilepsy, also known as Lafora disease (LD), is the most severe and fatal form of progressive myoclonus epilepsy with its typical onset during the late childhood or early adolescence. LD is characterized by recurrent epileptic seizures and progressive decline in intellectual function. LD can be caused by defects in any of the two known genes and the clinical features of these two genetic groups are almost identical. The past one decade has witnessed considerable success in identifying the LD genes, their mutations, the cellular functions of gene products and on molecular basis of LD. Here, we briefly review the current literature on the phenotype variations, on possible presence of genetic modifiers, and candidate modifiers as targets for therapeutic interventions in LD.

Investigation of modifier genes within copy number variations in Rett syndrome

MECP2 mutations are responsible for two different phenotypes in females, classical Rett syndrome and the milder Zappella variant (Z-RTT). We investigated whether Copy Number Variants (CNVs) may modulate the phenotype by comparison of array-CGH data from two discordant pairs of sisters and four additional discordant pairs of unrelated girls matched by mutation type. We also searched for potential MeCP2 targets within CNVs by ChIP-chip analysis. We did not identify one major common gene/region, suggesting that modifiers may be complex and variable between cases. However, we detected CNVs correlating with disease severity that contain candidate modifiers. CROCC (1p36.13) is a potential MeCP2 target in which a duplication in a Z-RTT and a deletion in a classic patient were observed. CROCC encodes a structural component of ciliary motility that is required for correct brain development. CFHR1 and CFHR3, on 1q31.3, may be involved in the regulation of complement during synapse elimination and were found to be deleted in a Z-RTT but duplicated in two classic patients. The duplication of 10q11.22, present in two Z-RTT patients, includes GPRIN2, a regulator of neurite outgrowth and PPYR1, involved in energy homeostasis. Functional analyses are necessary to confirm candidates and to define targets for future therapies.

C05 TP53 R72P polymorphism as a modifier of age at onset of Huntington's disease

BackgroundThe size of the CAG repeat tract is the major determinant of age of onset (AO) in Huntington's disease (HD). However, the number of the CAG repeats does not allow...

Modulation and Detection of Tau Aggregation with Small-Molecule Ligands

Recent results from high-throughput and other screening approaches reveal that small molecules can directly interact with recombinant full-length tau monomers and fibrillar tau aggregates in three distinct modes. First, in the high concentration regime (>10 micromolar), certain anionic molecules such as Congo red efficiently promote tau filament formation through a nucleation-elongation mechanism involving a dimeric nucleus and monomer-mediated elongation. These compounds are useful for modeling tau aggregation in vitro and in biological models. Second, in the low concentration regime (<1 micromolar), other ligands, including cyanine dyes, display aggregation antagonist activity. Compounds that can prevent or reverse fibrillization are candidate modifiers of disease pathology. Finally, certain compounds bind mature tau fibrils with varying affinities at multiple binding sites without modulating the aggregation reaction. For some ligands, >10-fold selectivity for tau aggregates relative to filaments composed of beta-amyloid or alpha-synuclein can be demonstrated at the level of binding affinity. Together these observations suggest that small-molecules have utility for interrogating the tau aggregation pathway, for inhibiting neuritic lesion formation, and for selective pre-mortem detection of neurofibrillary lesions through whole brain imaging.

Genetic modifiers of degeneration in the cathepsin D deficient Drosophila model for neuronal ceroid lipofuscinosis

Neuronal ceroid lipofuscinoses (NCLs) are pediatric, neurodegenerative, lysosomal storage disorders. Mutations in cathepsin D result in the most severe, congenital form of NCLs. We have previously generated a cathepsin D deficient Drosophila model, which exhibits the key features of NCLs: progressive intracellular accumulation of autofluorescent storage material and modest neurodegeneration in the brain areas related to visual functions. Here we extend the phenotypic characterization of cathepsin D deficient Drosophila and report that modest degenerative changes are also present in their retinae. Furthermore, by utilizing this phenotype, we examined the possible effect of 17 candidate modifiers, selected based on the results from other cathepsin D deficiency models. We found enhancers of this phenotype that support the involvement of endocytosis-, lipid metabolism- and oxidation-related factors in the cathepsin D deficiency induced degeneration. Our results warrant further investigation of these mechanisms in the pathogenesis of cathepsin D deficiency.

Expression analysis of genes lying in the NF1 microdeletion interval points to four candidate modifiers for neurofibroma formation

The hallmark of neurofibromatosis type 1 (NF1) are multiple dermal neurofibromas. They show high inter- and intrafamilial variability for which the influence of modifying genes is discussed. NF1 patients presenting microdeletions spanning NF1 and several contiguous genes have an earlier onset and higher number of dermal neurofibromas than classical NF1 patients, pointing to one of the deleted genes as modifier. Expression analysis of 13 genes of the microdeletion region in dermal neurofibromas and other tissues revealed four candidates for the modification of neurofibroma formation: CENTA2, RAB11FIP4, C17orf79, and UTP6.

Induction of the Phase II Detoxification Pathway Suppresses Neuron Loss inDrosophilaModels of Parkinson's Disease

Alpha-synuclein aggregates are a common feature of sporadic Parkinson's disease (PD), and mutations that increase alpha-synuclein abundance confer rare heritable forms of PD. Although these findings suggest that alpha-synuclein plays a central role in the pathogenesis of this disorder, little is known of the mechanism by which alpha-synuclein promotes neuron loss or the factors that regulate alpha-synuclein toxicity. To address these matters, we tested candidate modifiers of alpha-synuclein toxicity using a Drosophila model of PD. In the current work, we focused on phase II detoxification enzymes involved in glutathione metabolism. We find that the neuronal death accompanying alpha-synuclein expression in Drosophila is enhanced by loss-of-function mutations in genes that promote glutathione synthesis and glutathione conjugation. This neuronal loss can be overcome by genetic or pharmacological interventions that increase glutathione synthesis or glutathione conjugation activity. Moreover, these same pharmacological agents suppress neuron loss in Drosophila parkin mutants, a loss-of-function model of PD. Our results suggest that oxidative stress is a feature of alpha-synuclein toxicity and that induction of the phase II detoxification pathway represents a potential preventative therapy for PD.

Huntingtin Interacting Proteins Are Genetic Modifiers of Neurodegeneration

Huntington's disease (HD) is a fatal neurodegenerative condition caused by expansion of the polyglutamine tract in the huntingtin (Htt) protein. Neuronal toxicity in HD is thought to be, at least in part, a consequence of protein interactions involving mutant Htt. We therefore hypothesized that genetic modifiers of HD neurodegeneration should be enriched among Htt protein interactors. To test this idea, we identified a comprehensive set of Htt interactors using two complementary approaches: high-throughput yeast two-hybrid screening and affinity pull down followed by mass spectrometry. This effort led to the identification of 234 high-confidence Htt-associated proteins, 104 of which were found with the yeast method and 130 with the pull downs. We then tested an arbitrary set of 60 genes encoding interacting proteins for their ability to behave as genetic modifiers of neurodegeneration in a Drosophila model of HD. This high-content validation assay showed that 27 of 60 orthologs tested were high-confidence genetic modifiers, as modification was observed with more than one allele. The 45% hit rate for genetic modifiers seen among the interactors is an order of magnitude higher than the 1%–4% typically observed in unbiased genetic screens. Genetic modifiers were similarly represented among proteins discovered using yeast two-hybrid and pull-down/mass spectrometry methods, supporting the notion that these complementary technologies are equally useful in identifying biologically relevant proteins. Interacting proteins confirmed as modifiers of the neurodegeneration phenotype represent a diverse array of biological functions, including synaptic transmission, cytoskeletal organization, signal transduction, and transcription. Among the modifiers were 17 loss-of-function suppressors of neurodegeneration, which can be considered potential targets for therapeutic intervention. Finally, we show that seven interacting proteins from among 11 tested were able to co-immunoprecipitate with full-length Htt from mouse brain. These studies demonstrate that high-throughput screening for protein interactions combined with genetic validation in a model organism is a powerful approach for identifying novel candidate modifiers of polyglutamine toxicity.

Modifiers of risk in familial adenomatous polyposis

Familial adenomatous polyposis (FAP) is an autosomal dominant condition caused by mutations in the adenomatous polyposis coli (APC) gene. The phenotypic expression of FAP is extremely variable, and only part of this variability reflects the influence of different germline APC mutations. The remaining differences are likely attributable to the action of modifier genes and environmental (including pharmacologic) factors. In this review, we discuss recent investigations of candidate modifiers of FAP risk, including studies examining the roles of the MutY homolog, insulin-like growth factor-2, EphB receptors, detoxification enzymes, cyclooxygenase-2, and lipoprotein lipase. Identification of FAP modifier genes will provide new insight into the mechanisms of colorectal cancer development and may suggest novel therapeutic targets as well as candidate genes for colorectal cancer susceptibility in the general population.

Respiratory Epithelial Gene Expression in Patients with Mild and Severe Cystic Fibrosis Lung Disease

Despite having identical cystic fibrosis transmembrane conductance regulator genotypes, individuals with DeltaF508 homozygous cystic fibrosis (CF) demonstrate significant variability in severity of pulmonary disease. This investigation used high-density oligonucleotide microarray analysis of nasal respiratory epithelium to investigate the molecular basis of phenotypic differences in CF by (1) identifying differences in gene expression between DeltaF508 homozygotes in the most severe 20th percentile of lung disease by forced expiratory volume in 1 s and those in the most mild 20th percentile of lung disease and (2) identifying differences in gene expression between DeltaF508 homozygotes and age-matched non-CF control subjects. Microarray results from 23 participants (12 CF, 11 non-CF) met the strict quality control guidelines and were used for final data analysis. A total of 652 of the 11,867 genes identified as present in 75% of the samples were significantly differentially expressed in one of the three disease phenotypes: 30 in non-CF, 53 in mild CF, and 569 in severe CF. An analysis of genes differentially expressed by severity of CF lung disease demonstrated significant upregulation in severe CF of genes involved in protein ubiquination (P < 0.04), mitochondrial oxidoreductase activity (P < 0.01), and lipid metabolism (P < 0.03). Analysis of genes with decreased expression in patients with CF compared with control subjects demonstrated significant downregulation of genes involved in airway defense (P < 0.047) and protein metabolism (P < 0.048). This study suggests that differences in CF lung phenotype are associated with differences in expression of genes involving airway defense, protein ubiquination, and mitochondrial oxidoreductase activity and identifies specific new candidate modifiers of the CF phenotype.

Examination of ALAS2, ABC7, Rag1, and IL6 Genes as Candidate Modifiers of Iron Overload in HFE C282Y Homozygotes with Severe Iron Overload.

The variable penetrance of HFE hemochromatosis led us to examine candidate modifier genes in 24 adult probands homozygous for the HFE C282Y mutation with severe iron overload. Mutations of the X-linked genes ALAS2 and ABC7 are associated with sideroblastic anemia and are such candidate genes. One C282Y homozygote had a P520L mutation in ALAS2 and another had an an ABC7 ivs2+1 G->A mutation that would result in the loss of a splice site and predict skipping of exon 2 with an in-frame deletion of 27 amino acids from the mature protein. Examination of respective family members demonstrated that inheritance of the ALAS2 P520L mutation or the ABC7 ivs2+1 mutation alone was not associated with excessive iron storage or sideroblastic anemia. Only subjects homozygous for HFE C282Y and hemizygous or heterozygous for these ALAS2 or ABC7 mutations had iron overload. Because the families were small, it was not possible to establish conclusively that these mutations affected the severity of iron overload. Nevertheless, these data suggest that if ALAS2 and ABC7 mutations are modifers of HFE hemochromatosis, they occur at a low frequency and do not account for the majority of cases of severe iron overload in C282Y homozygotes. Mice expressing double knockouts of rag1 and hfe or beta 2 microglobulin exhibit more severe iron overload than mice with knockouts of either gene alone. Thus, we sought to determine if mutations in RAG1 were associated with severe iron overload in 20 HFE C282Y homozygotes with severe iron overload. Four non-synonymous SNPs were identified in the HFE C282Y homozygotes: R249H, R449K, K820R, and M1006V. These mutations occurred at allele frequencies of 0.3500, 0.0238, 0.1190, and 0.0227, respectively. In 364 control subjects, the allele frequencies of R249H (0.3750) and K820R (0.1133) did not differ significantly from those in HFE C282Y homozygotes. A promoter polymorphism in IL6 at nt −174 has been shown to be associated with IL-6 expression: the G allele with high expression and the C allele with low expression. Lower levels of IL-6 expression might result in lower levels of hepcidin expression, further decreasing the already lower levels of hepcidin resulting from HFE C282Y homozygosity. Analysis of 340 control whites disclosed 62 IL6 -174C/C subjects had a mean transferrin saturation of 28% and 107 IL6 -174G/G subjects had a mean transferrin saturation of 25% (p <0.05). We found that the C allele frequency was 0.4800 in C282Y homozygotes with severe iron overload; this did not differ significantly from the frequency of 0.4338 in white control subjects. These data suggest that the IL6 -174 C allele is not significantly associated with severe iron overload in HFE C282Y homozygotes. We conclude that mutations of ALAS2 and ABC7 may be rare causes of severe iron loading in HFE C282Y homozygotes, but it is unlikely that RAG1 and the IL6 promoter polymorphisms contribute significantly to the iron loading of such subjects.

Modifier genes in cystic fibrosis

Although over 1,000 disease-causing mutations in the CFTR gene have been described, the highly variable disease phenotype in cystic fibrosis (CF) cannot be explained on the basis of this gene alone. Both the environment and other non-CFTR genes are likely to be important. The increased understanding of pathophysiological processes in the CF lung has led to several studies on genes in these pathways, including those involved in host defense, mucin production, and airway responsiveness. Additionally, candidate modifiers of the gastrointestinal manifestations of CF have been explored. One of the major aims of such studies is to produce targets for novel drug developments. This review will summarize the field to date and discuss some of the methodological issues important in the design and interpretation of such studies.

Haplotype analysis of carnitine transporters and left ventricular mass in human essential hypertension

The carnitine-associated alteration of myocardial fatty acid metabolism may be one of the molecular mechanisms underlying left ventricular hypertrophy (LVH) in essential hypertension. We tested the hypothesis that polymorphisms of the genes involved in carnitine transport, OCTN2, CPT1A, CPT1B, and CPT2, might be associated with LVH. Haplotype-based association analysis in an observational study. Outpatients from the Nephrology Division of the University Hospital. A total of 215 never-treated, middle-aged patients with mild essential hypertension. Relationships between left ventricular mass index (LVMI) (measured with m-mode echocardiography) and haplotype combinations for 13 common genetic variants selected from single nucleotide polymorphism database (dbSNPs). The SNPs were selected to cover the genomic region of the four loci, and a total of 23 haplotypes were identified: 8 for OCTN2 (H1 to H8), 8 for CPT1A (H9 to H16), 3 for CPT1B (H17 to H19), and 4 for CPT2 (H20 to H23). In a multilocus haplotype analysis, after adjusting for sex, age, systolic blood pressure, diastolic blood pressure, body mass index, and duration of hypertension, a significant effect on LVMI was seen for H13 (+8.9, P = .05), H14 (-5.63, P = .05), H15 (-18.79, P = .0006), H18 (-1.66, P = .03), and H22 (-3.42, P = .004). These significant haplotypes were respectively 3.7%, 1.6%, 1.6%, 39.3%, and 29.7% of the total population. These results identify the carnitine-transporter gene family as candidate modifiers of LVMI in human hypertension. The use of common SNPs to define informative haplotypes associated with the phenotype of interest is the starting point for progress toward identification of the trapped contributing SNP(s).

First Study of NAT1 and NAT2 Polymorphisms in Bulgarian Patients with Balkan Endemic Nephropathy (Ben) and Healthy Controls

ABSTRACTN-acetyltransferases (NAT) are involved in the metabolic activation and deactivation of environmental carcinogens, such as arylamines. Several epidemiological studies evaluated the role of NAT1 and NAT2 polymorphisms in a number of cancers. The suspected role of polycyclic aromatic hydrocarbons in the etiology of Balkan Endemic Nephropathy (BEN), as well as the high risk of uroepithelial tumor development in BEN patients necessitates clarification of the role of xenobiotic metabolizing enzymes for BEN susceptibility. The aim of this study was to determine the frequency of different polymorphisms in N- acetyltransferases in patients with BEN and to define the role NAT1 and NAT2 as candidate modifiers of BEN susceptibility.96 Bulgarian BEN patients and 112 healthy Bulgarians as controls were genotyped for NAT1 (C559T, G560A, T640G, T1088A, C1095A) and NAT2 (C282T, T341C). Rapid cycle PCR followed by melting curve analysis was used for genotyping.The frequencies of NAT1 and NAT2 polymorphisms were similar in BEN patients and controls. The data did not reveal a significantly elevated risk for BEN with NAT1 alleles *3, *10 and *11. NAT2 slow acetylators, predicted by C282T and T341C, also did not show an increased risk for BEN.For the first time the frequencies of different polymorphisms in NAT1 and NAT2 in patients with BEN and in the healthy Bulgarian population were established. No evidence for a predisposing role of N- acetyltransferases in the development of BEN was revealed. This study needs conformation in other endemic regions of Balkan countries.

Modifier genes in cystic fibrosis lung disease

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene and is characterized by progressive bronchiectatic lung disease and pancreatic exocrine insufficiency. A broad spectrum of disease severity exists; some individuals with CF die early in childhood, whereas others live well into adulthood with only mild lung disease. It is now clear that CFTR genotype alone does not account for the wide diversity in CF pulmonary phenotype. Evidence is accumulating that secondary genetic factors separate from the CFTR locus significantly influence the severity of CF lung disease. The general classes of these potential modifier genes include inflammatory and antiinflammatory mediators, antioxidants, mediators of airway reactivity, molecules involved in CFTR trafficking, and alternative ion channels. The best-studied CF candidate modifiers include mannose-binding lectin, glutathione-S-transferase, transforming growth factor-β1, tumor necrosis factor-α, β2-adrenegic receptor, and HLA class II antigens. Ongoing studies designed to identify genetic modifiers of CF pulmonary phenotype may offer new insights into the pathophysiology of CF lung disease and provide leads for new CF therapeutic interventions. (J Lab Clin Med 2003;141:237-41)

Identification of MEFV-Independent Modifying Genetic Factors for Familial Mediterranean Fever

Familial Mediterranean fever (FMF) is a recessively inherited disorder predisposing to renal amyloidosis and associated with mutations in MEFV, a gene encoding a protein of unknown function. Differences in clinical expression have been attributed to MEFV-allelic heterogeneity, with the M694V/M694V genotype associated with a high prevalence of renal amyloidosis. However, the variable risk for patients with identical MEFV mutations to develop this severe complication, prevented by lifelong administration of colchicine, strongly suggests a role for other genetic and/or environmental factors. To overcome the well-known difficulties in the identification of modifying genetic factors, we investigated a relatively homogeneous population sample consisting of 137 Armenian patients with FMF from 127 independent families living in Armenia. We selected the SAA1, SAA2, and APOE genes-encoding serum amyloid proteins and apolipoprotein E, respectively-as well as the patients' sex, as candidate modifiers for renal amyloidosis. A stepwise logistic-regression analysis showed that the SAA1alpha/alpha genotype was associated with a sevenfold increased risk for renal amyloidosis, compared with other SAA1 genotypes (odds ratio [OR] 6. 9; 95% confidence interval [CI] 2.5-19.0). This association, which was present whatever the MEFV genotype, was extremely marked in patients homozygous for M694V (11/11). The risk for male patients of developing renal amyloidosis was fourfold higher than that for female patients (OR=4.0; 95% CI=1.5-10.8). This association, particularly marked in patients who were not homozygous for M694V (34.0% vs. 11.6%), was independent of SAA1-allelic variations. Polymorphisms in the SAA2 or APOE gene did not appear to influence susceptibility to renal amyloidosis. Overall, these data, which provide new insights into the pathophysiology of FMF, demonstrate that susceptibility to renal amyloidosis in this Mendelian disorder is influenced by at least two MEFV-independent factors of genetic origin-SAA1 and sex-that act independently of each other.

Isolation and characterisation of cosmids to intervals within a 4.5Mb region at 6p21.3.

The gene responsible for hereditary haemochromatosis (HH) has recently been identified. One mutation in this gene, termed HFE, has been found in all Australian HH patients. We previously identified a predominant HH ancestral haplotype covering 4.5Mb at 6p21.3, and showed that patients with two copies of this haplotype express a more severe form of the disorder. One key question to now be resolved is why haplotype related variation in phenotypic expression of HH is present if all patients tested have the same HFE mutation. A cosmid resource covering the 4.5Mb HH ancestral haplo type region was obtained. These cosmids provide the material for the completion of a transcript map of this region, and will assist the identification of candidate modifiers of HFE expression.

Reply

<h3>Background</h3> Studies have identified many common genetic associations that influence renal function and all-cause CKD, but these explain only a small fraction of variance in these traits. The contribution of rare variants has not been systematically examined. <h3>Methods</h3> We performed exome sequencing of 3150 individuals, who collectively encompassed diverse CKD subtypes, and 9563 controls. To detect causal genes and evaluate the contribution of rare variants we used collapsing analysis, in which we compared the proportion of cases and controls carrying rare variants per gene. <h3>Results</h3> The analyses captured five established monogenic causes of CKD: variants in <i>PKD1</i>, <i>PKD2</i>, and <i>COL4A5</i> achieved study-wide significance, and we observed suggestive case enrichment for <i>COL4A4</i> and <i>COL4A3</i>. Beyond known disease-associated genes, collapsing analyses incorporating regional variant intolerance identified suggestive dominant signals in <i>CPT2</i> and several other candidate genes. Biallelic mutations in <i>CPT2</i> cause carnitine palmitoyltransferase II deficiency, sometimes associated with rhabdomyolysis and acute renal injury. Genetic modifier analysis among cases with <i>APOL1</i> risk genotypes identified a suggestive signal in <i>AHDC1</i>, implicated in Xia–Gibbs syndrome, which involves intellectual disability and other features. On the basis of the observed distribution of rare variants, we estimate that a two- to three-fold larger cohort would provide 80% power to implicate new genes for all-cause CKD. <h3>Conclusions</h3> This study demonstrates that rare-variant collapsing analyses can validate known genes and identify candidate genes and modifiers for kidney disease. In so doing, these findings provide a motivation for larger-scale investigation of rare-variant risk contributions across major clinical CKD categories.