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The amount of radiologically dense breast-tissue appearing on a mammogram varies between women because of differences in the composition of breast tissue, and is referred to here as mammographic density. This review presents evidence that mammographic density is a strong risk factor for breast cancer, and that risk of breast cancer is four to five times greater in women with density in more than 75% of the breast than in women with little or no density in the breast. Density in more than 50% of the breast could account for about a third of breast cancers. The epidemiology of mammographic density is consistent with its being a marker of susceptibility to breast cancer. Twin studies have shown that the proportion of the breast occupied by density, at a given age, is highly heritable, and inherited factors explain 63% of the variance. Mammographic breast density has the characteristics of a quantitative trait and might be determined by genes that are easier to identify than those for breast cancer itself. The genes that determine breast density might also be associated with risk of breast cancer, and their identification is also likely to provide insights into the biology of the breast and identify potential targets for preventive strategies. [ABSTRACT FROM AUTHOR]

Introduction: Mammographic breast density is a highly heritable (h2 > 0.6) and strong risk factor for breast cancer. We conducted a genome-wide linkage study to identify loci influencing mammographic breast density (MD).Methods: Epidemiological data were assembled on 1,415 families from the Australia, Northern California and Ontario sites of the Breast Cancer Family Registry, and additional families recruited in Australia and Ontario. Families consisted of sister pairs with age-matched mammograms and data on factors known to influence MD. Single nucleotide polymorphism (SNP) genotyping was performed on 3,952 individuals using the Illumina Infinium 6K linkage panel.Results: Using a variance components method, genome-wide linkage analysis was performed using quantitative traits obtained by adjusting MD measurements for known covariates. Our primary trait was formed by fitting a linear model to the square root of the percentage of the breast area that was dense (PMD), adjusting for age at mammogram, number of live births, menopausal status, weight, height, weight squared, and menopausal hormone therapy. The maximum logarithm of odds (LOD) score from the genome-wide scan was on chromosome 7p14.1-p13 (LOD = 2.69; 63.5 cM) for covariate-adjusted PMD, with a 1-LOD interval spanning 8.6 cM. A similar signal was seen for the covariate adjusted area of the breast that was dense (DA) phenotype. Simulations showed that the complete sample had adequate power to detect LOD scores of 3 or 3.5 for a locus accounting for 20% of phenotypic variance. A modest peak initially seen on chromosome 7q32.3-q34 increased in strength when only the 513 families with at least two sisters below 50 years of age were included in the analysis (LOD 3.2; 140.7 cM, 1-LOD interval spanning 9.6 cM). In a subgroup analysis, we also found a LOD score of 3.3 for DA phenotype on chromosome 12.11.22-q13.11 (60.8 cM, 1-LOD interval spanning 9.3 cM), overlapping a region identified in a previous study.Conclusions: The suggestive peaks and the larger linkage signal seen in the subset of pedigrees with younger participants highlight regions of interest for further study to identify genes that determine MD, with the goal of understanding mammographic density and its involvement in susceptibility to breast cancer. [ABSTRACT FROM AUTHOR]

In December 2020, the U.S. Food and Drug Administration (FDA) expanded the list of CFTR variants approved for treatment with CFTR modulators drugs from 39 to 183. Clinicians should be aware that individuals harboring certain variants approved for treatment may not respond to or benefit from this therapy. After review, the expert panel leading the CFTR2 project identified four categories of variants that may not result in a clinical response to modulator treatment: 15 variants assigned as non CF-causing; 45 variants of unknown significance; six variants known or suspected to cause mis-splicing as their primary defect rather than an amino acid substitution; and eight variants known to occur together in cis with another deleterious variant not expected to lead to CFTR protein (nonsense or frameshift). The potential risks and benefits of CFTR modulator therapy should be considered carefully for individuals harboring these variants., Competing Interests: Declaration of Competing Interest CC serves on advisory boards for Vertex Pharmaceuticals, Mylan, Chiesi Farmaceutici, and Actelion. GRC served as a consultant for Vertex Pharmaceuticals and has a relative employed by Vertex Pharmaceuticals. All other authors report none., (Copyright © 2022. Published by Elsevier B.V.)

Background: Variation in respiratory response to cystic fibrosis (CF) small molecule therapies is due in part to the contribution of CF lung disease modifier genes. Cultured human bronchial epithelia (HBE) is the gold standard respiratory model for assessing CF therapeutic efficacy but it is hard to access. Cultured human nasal epithelia (HNE) is proposed as a more accessible surrogate model but it is unknown whether the expression profile of the modifier genes are comparable between HNE and HBE which we assess here., Methods: RNA-sequencing was conducted on paired cultured and fresh HNE and HBE (n = 71 samples) collected from 21 individuals with CF. Genome-wide gene expression was first compared between cultured and fresh cells and then between cultured HNE and HBE based on an equivalence testing procedure we implemented. The co-expression relationships of CFTR and CF lung disease modifier genes were compared between cultured HNE and HBE to determine equivalent interactions., Results: The culturing process had little impact on the expression level of CF lung disease modifier genes. Over 90% of expressed genes showed significant equivalent expression level across cultured HNE and HBE (expression fold-change<2, FDR<0.1), including CFTR and CF lung disease modifier genes. The difference in co-expression relationships among these genes was not significant (p-value=0.99), suggesting their functional interactions are likely to be consistent in the two models., Conclusions: Cultured HNE recapitulates the expression profile of CF lung disease modifier genes in cultured HBE, suggesting the biological processes involving these genes are likely to be consistent across the two models., Competing Interests: Declaration of Competing Interest There is no conflict of interest to be declared., (Copyright © 2022. Published by Elsevier B.V.)

Over 400 variants in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) are CF-causing. CFTR modulators target variants to improve lung function, but marked variability in response exists and current therapies do not address all CF-causing variants highlighting unmet needs. Alternative epithelial ion channel/transporters such as SLC26A9 could compensate for CFTR dysfunction, providing therapeutic targets that may benefit all individuals with CF. We investigate the relationship between rs7512462, a marker of SLC26A9 activity, and lung function pre- and post-treatment with CFTR modulators in Canadian and US CF cohorts, in the general population, and in those with chronic obstructive pulmonary disease (COPD). Rs7512462 CC genotype is associated with greater lung function in CF individuals with minimal function variants (for which there are currently no approved therapies; p = 0.008); and for gating (p = 0.033) and p.Phe508del/ p.Phe508del (p = 0.006) genotypes upon treatment with CFTR modulators. In parallel, human nasal epithelia with CC and p.Phe508del/p.Phe508del after Ussing chamber analysis of a combination of approved and experimental modulator treatments show greater CFTR function (p = 0.0022). Beyond CF, rs7512462 is associated with peak expiratory flow in a meta-analysis of the UK Biobank and Spirometa Consortium (p = 2.74 × 10 -44 ) and provides p = 0.0891 in an analysis of COPD case-control status in the UK Biobank defined by spirometry. These findings support SLC26A9 as a therapeutic target to improve lung function for all people with CF and in individuals with other obstructive lung diseases., (© 2022. The Author(s).)

Purpose: Cystic fibrosis (CF), caused by pathogenic variants in the CF transmembrane conductance regulator (CFTR), affects multiple organs including the exocrine pancreas, which is a causal contributor to cystic fibrosis-related diabetes (CFRD). Untreated CFRD causes increased CF-related mortality whereas early detection can improve outcomes., Methods: Using genetic and easily accessible clinical measures available at birth, we constructed a CFRD prediction model using the Canadian CF Gene Modifier Study (CGS; n = 1,958) and validated it in the French CF Gene Modifier Study (FGMS; n = 1,003). We investigated genetic variants shown to associate with CF disease severity across multiple organs in genome-wide association studies., Results: The strongest predictors included sex, CFTR severity score, and several genetic variants including one annotated to PRSS1, which encodes cationic trypsinogen. The final model defined in the CGS shows excellent agreement when validated on the FGMS, and the risk classifier shows slightly better performance at predicting CFRD risk later in life in both studies., Conclusion: We demonstrated clinical utility by comparing CFRD prevalence rates between the top 10% of individuals with the highest risk and the bottom 10% with the lowest risk. A web-based application was developed to provide practitioners with patient-specific CFRD risk to guide CFRD monitoring and treatment.

Epistasis refers to the dependence of a mutation on other mutation(s) and the genetic context in general. In the context of human disorders, epistasis complicates the spectrum of disease symptoms and has been proposed as a major contributor to variations in disease outcome. The nonadditive relationship between mutations and the lack of complete understanding of the underlying physiological effects limit our ability to predict phenotypic outcome. Here, we report positive epistasis between intragenic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR)-the gene responsible for cystic fibrosis (CF) pathology. We identified a synonymous single-nucleotide polymorphism (sSNP) that is invariant for the CFTR amino acid sequence but inverts translation speed at the affected codon. This sSNP in cis exhibits positive epistatic effects on some CF disease-causing missense mutations. Individually, both mutations alter CFTR structure and function, yet when combined, they lead to enhanced protein expression and activity. The most robust effect was observed when the sSNP was present in combination with missense mutations that, along with the primary amino acid change, also alter the speed of translation at the affected codon. Functional studies revealed that synergistic alteration in ribosomal velocity is the underlying mechanism; alteration of translation speed likely increases the time window for establishing crucial domain-domain interactions that are otherwise perturbed by each individual mutation., Competing Interests: The authors declare no competing interest.

Genome-wide association studies (GWAS) have primarily identified trait-associated loci in the non-coding genome. Colocalization analyses of SNP associations from GWAS with expression quantitative trait loci (eQTL) evidence enable the generation of hypotheses about responsible mechanism, genes and tissues of origin to guide functional characterization. Here, we present a web-based colocalization browsing and testing tool named LocusFocus (https://locusfocus.research.sickkids.ca). LocusFocus formally tests colocalization using our established Simple Sum method to identify the most relevant genes and tissues for a particular GWAS locus in the presence of high linkage disequilibrium and/or allelic heterogeneity. We demonstrate the utility of LocusFocus, following up on a genome-wide significant locus from a GWAS of meconium ileus (an intestinal obstruction in cystic fibrosis). Using LocusFocus for colocalization analysis with eQTL data suggests variation in ATP12A gene expression in the pancreas rather than intestine is responsible for the GWAS locus. LocusFocus has no operating system dependencies and may be installed in a local web server. LocusFocus is available under the MIT license, with full documentation and source code accessible on GitHub at https://github.com/naim-panjwani/LocusFocus., Competing Interests: The authors have declared that no competing interests exist.

Context: Individuals with cystic fibrosis (CF) develop a distinct form of diabetes characterized by β-cell dysfunction and islet amyloid accumulation similar to type 2 diabetes (T2D), but generally have normal insulin sensitivity. CF-related diabetes (CFRD) risk is determined by both CFTR, the gene responsible for CF, and other genetic variants., Objective: To identify genetic modifiers of CFRD and determine the genetic overlap with other types of diabetes., Design and Patients: A genome-wide association study was conducted for CFRD onset on 5740 individuals with CF. Weighted polygenic risk scores (PRSs) for type 1 diabetes (T1D), T2D, and diabetes endophenotypes were tested for association with CFRD., Results: Genome-wide significance was obtained for variants at a novel locus (PTMA) and 2 known CFRD genetic modifiers (TCF7L2 and SLC26A9). PTMA and SLC26A9 variants were CF-specific; TCF7L2 variants also associated with T2D. CFRD was strongly associated with PRSs for T2D, insulin secretion, postchallenge glucose concentration, and fasting plasma glucose, and less strongly with T1D PRSs. CFRD was inconsistently associated with PRSs for insulin sensitivity and was not associated with a PRS for islet autoimmunity. A CFRD PRS comprising variants selected from these PRSs (with a false discovery rate < 0.1) and the genome-wide significant variants was associated with CFRD in a replication population., Conclusions: CFRD and T2D have more etiologic and mechanistic overlap than previously known, aligning along pathways involving β-cell function rather than insulin sensitivity. Two CFRD risk loci are unrelated to T2D and may affect multiple aspects of CF. An 18-variant PRS stratifies risk of CFRD in an independent population., (© Endocrine Society 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Rationale: The advent of precision treatment for cystic fibrosis using small-molecule therapeutics has created a need to estimate potential clinical improvements attributable to increases in cystic fibrosis transmembrane conductance regulator (CFTR) function. Objectives: To derive CFTR function of a variety of CFTR genotypes and correlate with key clinical features (sweat chloride concentration, pancreatic exocrine status, and lung function) to develop benchmarks for assessing response to CFTR modulators. Methods: CFTR function assigned to 226 unique CFTR genotypes was correlated with the clinical data of 54,671 individuals enrolled in the Clinical and Functional Translation of CFTR (CFTR2) project. Cross-sectional FEV 1 % predicted measurements were plotted by age at which measurement was obtained. Shifts in sweat chloride concentration and lung function reported in CFTR modulator trials were compared with function-phenotype correlations to assess potential efficacy of therapies. Measurements and Main Results: CFTR genotype function exhibited a logarithmic relationship with each clinical feature. Modest increases in CFTR function related to differing genotypes were associated with clinically relevant improvements in cross-sectional FEV 1 % predicted over a range of ages (6-82 yr). Therapeutic responses to modulators corresponded closely to predictions from the CFTR2-derived relationship between CFTR genotype function and phenotype. Conclusions: Increasing CFTR function in individuals with severe disease will have a proportionally greater effect on outcomes than similar increases in CFTR function in individuals with mild disease and should reverse a substantial fraction of the disease process. This study provides reference standards for clinical outcomes that may be achieved by increasing CFTR function.

Cystic Fibrosis (CF) exhibits morbidity in several organs, including progressive lung disease in all patients and intestinal obstruction at birth (meconium ileus) in ~15%. Individuals with the same causal CFTR mutations show variable disease presentation which is partly attributed to modifier genes. With >6,500 participants from the International CF Gene Modifier Consortium, genome-wide association investigation identified a new modifier locus for meconium ileus encompassing ATP12A on chromosome 13 (min p = 3.83x10(-10)); replicated loci encompassing SLC6A14 on chromosome X and SLC26A9 on chromosome 1, (min p<2.2x10(-16), 2.81x10(-11), respectively); and replicated a suggestive locus on chromosome 7 near PRSS1 (min p = 2.55x10(-7)). PRSS1 is exclusively expressed in the exocrine pancreas and was previously associated with non-CF pancreatitis with functional characterization demonstrating impact on PRSS1 gene expression. We thus asked whether the other meconium ileus modifier loci impact gene expression and in which organ. We developed and applied a colocalization framework called the Simple Sum (SS) that integrates regulatory and genetic association information, and also contrasts colocalization evidence across tissues or genes. The associated modifier loci colocalized with expression quantitative trait loci (eQTLs) for ATP12A (p = 3.35x10(-8)), SLC6A14 (p = 1.12x10(-10)) and SLC26A9 (p = 4.48x10(-5)) in the pancreas, even though meconium ileus manifests in the intestine. The meconium ileus susceptibility locus on chromosome X appeared shifted in location from a previously identified locus for CF lung disease severity. Using the SS we integrated the lung disease association locus with eQTLs from nasal epithelia of 63 CF participants and demonstrated evidence of colocalization with airway-specific regulation of SLC6A14 (p = 2.3x10(-4)). Cystic Fibrosis is realizing the promise of personalized medicine, and identification of the contributing organ and understanding of tissue specificity for a gene modifier is essential for the next phase of personalizing therapeutic strategies., Competing Interests: The authors have declared that no competing interests exist.

Does genotype imputation with public reference panels identify variants contributing to disease? Genotype imputation using the 1000 Genomes Project (1KG; 2504 individuals) displayed poor coverage at the causal cystic fibrosis (CF) transmembrane conductance regulator ( CFTR ) locus for the International CF Gene Modifier Consortium. Imputation with the larger Haplotype Reference Consortium (HRC; 32,470 individuals) displayed improved coverage but low sensitivity of variants clinically relevant for CF. A hybrid reference that combined whole genome sequencing (WGS) from 101 CF individuals with the 1KG imputed a greater number of single-nucleotide variants (SNVs) that would be analyzed in a genetic association study ( r 2  ≥ 0.3 and MAF ≥ 0.5%) than imputation with the HRC, while the HRC excelled in the lower frequency spectrum. Using the 1KG or HRC as reference panels missed the most common CF-causing variants or displayed low imputation accuracy. Designs that incorporate population-specific WGS can improve imputation accuracy at disease-specific loci, while imputation using public data sets can omit disease-relevant genotypes., Competing Interests: The authors declare no competing financial interests.

Background: Mammographic density defined by the conventional pixel brightness threshold, and adjusted for age and body mass index (BMI), is a well-established risk factor for breast cancer. We asked if higher thresholds better separate women with and without breast cancer., Methods: We studied Australian women, 354 with breast cancer over-sampled for early-onset and family history, and 944 unaffected controls frequency-matched for age at mammogram. We measured mammographic dense area and percent density using the CUMULUS software at the conventional threshold, which we call Cumulus , and at two increasingly higher thresholds, which we call Altocumulus and Cirrocumulus , respectively. All measures were Box-Cox transformed and adjusted for age and BMI. We estimated the odds per adjusted standard deviation (OPERA) using logistic regression and the area under the receiver operating characteristic curve (AUC)., Results: Altocumulus and Cirrocumulus were correlated with Cumulus (r ∼ 0.8 and 0.6 , respectively) . For dense area, the OPERA was 1.62, 1.74 and 1.73 for Cumulus, Altocumulus and Cirrocumulus , respectively (all P  < 0.001). After adjusting for Altocumulus and Cirrocumulus , Cumulus was not significant ( P  > 0.6). The OPERAs for percent density were less but gave similar findings. The mean of the standardized adjusted Altocumulus and Cirrocumulus dense area measures was the best predictor; OPERA = 1.87 [95% confidence interval (CI): 1.64-2.14] and AUC = 0.68 (0.65-0.71)., Conclusions: The areas of higher mammographically dense regions are associated with almost 30% stronger breast cancer risk gradient, explain the risk association of the conventional measure and might be more aetiologically important. This has substantial implications for clinical translation and molecular, genetic and epidemiological research., (© The Author 2016. Published by Oxford University Press on behalf of the International Epidemiological Association)

Rationale: Expanding the use of cystic fibrosis transmembrane conductance regulator (CFTR) potentiators and correctors for the treatment of cystic fibrosis (CF) requires precise and accurate biomarkers. Sweat chloride concentration provides an in vivo assessment of CFTR function, but it is unknown the degree to which CFTR mutations account for sweat chloride variation., Objectives: To estimate potential sources of variation for sweat chloride measurements, including demographic factors, testing variability, recording biases, and CFTR genotype itself., Methods: A total of 2,639 sweat chloride measurements were obtained in 1,761 twins/siblings from the CF Twin-Sibling Study, French CF Modifier Gene Study, and Canadian Consortium for Genetic Studies. Variance component estimation was performed by nested mixed modeling., Measurements and Main Results: Across the tested CF population as a whole, CFTR gene mutations were found to be the primary determinant of sweat chloride variability (56.1% of variation) with contributions from variation over time (e.g., factors related to testing on different days; 13.8%), environmental factors (e.g., climate, family diet; 13.5%), other residual factors (e.g., test variability; 9.9%), and unique individual factors (e.g., modifier genes, unique exposures; 6.8%) (likelihood ratio test, P < 0.001). Twin analysis suggested that modifier genes did not play a significant role because the heritability estimate was negligible (H 2  = 0; 95% confidence interval, 0.0-0.35). For an individual with CF, variation in sweat chloride was primarily caused by variation over time (58.1%) with the remainder attributable to residual/random factors (41.9%)., Conclusions: Variation in the CFTR gene is the predominant cause of sweat chloride variation; most of the non-CFTR variation is caused by testing variability and unique environmental factors. If test precision and accuracy can be improved, sweat chloride measurement could be a valuable biomarker for assessing response to therapies directed at mutant CFTR.

Cystic fibrosis is realizing the promise of personalized medicine. Recent advances in drug development that target the causal CFTR directly result in lung function improvement, but variability in response is demanding better prediction of outcomes to improve management decisions. The genetic modifier SLC26A9 contributes to disease severity in the CF pancreas and intestine at birth and here we assess its relationship with disease severity and therapeutic response in the airways. SLC26A9 association with lung disease was assessed in individuals from the Canadian and French CF Gene Modifier consortia with CFTR-gating mutations and in those homozygous for the common Phe508del mutation. Variability in response to a CFTR-directed therapy attributed to SLC26A9 genotype was assessed in Canadian patients with gating mutations. A primary airway model system determined if SLC26A9 shows modification of Phe508del CFTR function upon treatment with a CFTR corrector. In those with gating mutations that retain cell surface-localized CFTR we show that SLC26A9 modifies lung function while this is not the case in individuals homozygous for Phe508del where cell surface expression is lacking. Treatment response to ivacaftor, which aims to improve CFTR-channel opening probability in patients with gating mutations, shows substantial variability in response, 28% of which can be explained by rs7512462 in SLC26A9 (P = 0.0006). When homozygous Phe508del primary bronchial cells are treated to restore surface CFTR, SLC26A9 likewise modifies treatment response (P = 0.02). Our findings indicate that SLC26A9 airway modification requires CFTR at the cell surface, and that a common variant in SLC26A9 may predict response to CFTR-directed therapeutics.

Rationale: Meconium ileus (MI) is a perinatal complication in cystic fibrosis (CF), which is only minimally influenced by environmental factors. We derived and examined MI prevalence (MIP) scores to assess CFTR phenotype-phenotype correlation for severe mutations., Method: MIP scores were established using a Canadian CF population (n = 2,492) as estimates of the proportion of patients with MI among all patients carrying the same CFTR mutation, focusing on patients with p.F508del as the second allele. Comparisons were made to the registries from the US CF Foundation (n = 43,432), Italy (Veneto/Trentino/Alto Adige regions) (n = 1,788), and Germany (n = 3,596)., Results: The prevalence of MI varied among the different registries (13-21%). MI was predominantly prevalent in patients with pancreatic insufficiency carrying "severe" CFTR mutations. In this severe spectrum MIP scores further distinguished between mutation types, for example, G542X (0.31) with a high, F508del (0.22) with a moderate, and G551D (0.08) with a low MIP score. Higher MIP scores were associated with more severe clinical phenotypes, such as a lower forced expiratory volume in 1 second (P = 0.01) and body mass index z score (P = 0.04)., Conclusions: MIP scores can be used to rank CFTR mutations according to their clinical severity and provide a means to expand delineation of CF phenotypes.Genet Med 18 4, 333-340.

Shwachman-Diamond syndrome is a congenital bone marrow failure disorder characterized by debilitating neutropenia. The disease is associated with loss-of-function mutations in the SBDS gene, implicated in ribosome biogenesis, but the cellular and molecular events driving cell specific phenotypes in ribosomopathies remain poorly defined. Here, we established what is to our knowledge the first mammalian model of neutropenia in Shwachman-Diamond syndrome through targeted downregulation of Sbds in hematopoietic stem and progenitor cells expressing the myeloid transcription factor CCAAT/enhancer binding protein α (Cebpa). Sbds deficiency in the myeloid lineage specifically affected myelocytes and their downstream progeny while, unexpectedly, it was well tolerated by rapidly cycling hematopoietic progenitor cells. Molecular insights provided by massive parallel sequencing supported cellular observations of impaired cell cycle exit and formation of secondary granules associated with the defect of myeloid lineage progression in myelocytes. Mechanistically, Sbds deficiency activated the p53 tumor suppressor pathway and induced apoptosis in these cells. Collectively, the data reveal a previously unanticipated, selective dependency of myelocytes and downstream progeny, but not rapidly cycling progenitors, on this ubiquitous ribosome biogenesis protein, thus providing a cellular basis for the understanding of myeloid lineage biased defects in Shwachman-Diamond syndrome., (Copyright© Ferrata Storti Foundation.)

The identification of small molecules that target specific CFTR variants has ushered in a new era of treatment for cystic fibrosis (CF), yet optimal, individualized treatment of CF will require identification and targeting of disease modifiers. Here we use genome-wide association analysis to identify genetic modifiers of CF lung disease, the primary cause of mortality. Meta-analysis of 6,365 CF patients identifies five loci that display significant association with variation in lung disease. Regions on chr3q29 (MUC4/MUC20; P=3.3 × 10(-11)), chr5p15.3 (SLC9A3; P=6.8 × 10(-12)), chr6p21.3 (HLA Class II; P=1.2 × 10(-8)) and chrXq22-q23 (AGTR2/SLC6A14; P=1.8 × 10(-9)) contain genes of high biological relevance to CF pathophysiology. The fifth locus, on chr11p12-p13 (EHF/APIP; P=1.9 × 10(-10)), was previously shown to be associated with lung disease. These results provide new insights into potential targets for modulating lung disease severity in CF.

Gene-based, pathway, and other multivariate association methods are motivated by the possibility of GxG and GxE interactions; however, accounting for such interactions is limited by the challenges associated with adequate modeling information. Here we propose an easy-to-implement joint location-scale (JLS) association testing framework for single-variant and multivariate analysis that accounts for interactions without explicitly modeling them. We apply the JLS method to a gene-set analysis of cystic fibrosis (CF) lung disease, which is influenced by multiple environmental and genetic factors. We identify and replicate an association between the constituents of the apical plasma membrane and CF lung disease (p = 0.0099 and p = 0.0180, respectively) and highlight a role for the SLC9A3-SLC9A3R1/2-EZR complex in contributing to CF lung disease. Many association studies could benefit from re-analysis with the JLS method that leverages complex genetic architecture for SNP, gene, and pathway identification. Analytical verification, simulation, and additional proof-of-principle applications support our approach., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Genetic models of ribosome dysfunction show selective organ failure, highlighting a gap in our understanding of cell-type specific responses to translation insufficiency. Translation defects underlie a growing list of inherited and acquired cancer-predisposition syndromes referred to as ribosomopathies. We sought to identify molecular mechanisms underlying organ failure in a recessive ribosomopathy, with particular emphasis on the pancreas, an organ with a high and reiterative requirement for protein synthesis. Biallelic loss of function mutations in SBDS are associated with the ribosomopathy Shwachman-Diamond syndrome, which is typified by pancreatic dysfunction, bone marrow failure, skeletal abnormalities and neurological phenotypes. Targeted disruption of Sbds in the murine pancreas resulted in p53 stabilization early in the postnatal period, specifically in acinar cells. Decreased Myc expression was observed and atrophy of the adult SDS pancreas could be explained by the senescence of acinar cells, characterized by induction of Tgfβ, p15(Ink4b) and components of the senescence-associated secretory program. This is the first report of senescence, a tumour suppression mechanism, in association with SDS or in response to a ribosomopathy. Genetic ablation of p53 largely resolved digestive enzyme synthesis and acinar compartment hypoplasia, but resulted in decreased cell size, a hallmark of decreased translation capacity. Moreover, p53 ablation resulted in expression of acinar dedifferentiation markers and extensive apoptosis. Our findings indicate a protective role for p53 and senescence in response to Sbds ablation in the pancreas. In contrast to the pancreas, the Tgfβ molecular signature was not detected in fetal bone marrow, liver or brain of mouse models with constitutive Sbds ablation. Nevertheless, as observed with the adult pancreas phenotype, disease phenotypes of embryonic tissues, including marked neuronal cell death due to apoptosis, were determined to be p53-dependent. Our findings therefore point to cell/tissue-specific responses to p53-activation that include distinction between apoptosis and senescence pathways, in the context of translation disruption.

Objectives: To test the hypothesis that multiple constituents of the apical plasma membrane residing alongside the causal cystic fibrosis (CF) transmembrane conductance regulator protein, including known CF modifiers SLC26A9, SLC6A14, and SLC9A3, would be associated with prenatal exocrine pancreatic damage as measured by newborn screened (NBS) immunoreactive trypsinogen (IRT) levels., Study Design: NBS IRT measures and genome-wide genotype data were available on 111 subjects from Colorado, 37 subjects from Wisconsin, and 80 subjects from France. Multiple linear regression was used to determine whether any of 8 single nucleotide polymorphisms (SNPs) in SLC26A9, SLC6A14, and SLC9A3 were associated with IRT and whether other constituents of the apical plasma membrane contributed to IRT., Results: In the Colorado sample, 3 SLC26A9 SNPs were associated with NBS IRT (min P=1.16×10(-3); rs7512462), but no SLC6A14 or SLC9A3 SNPs were associated (P>.05). The rs7512462 association replicated in the Wisconsin sample (P=.03) but not in the French sample (P=.76). Furthermore, rs7512462 was the top-ranked apical membrane constituent in the combined Colorado and Wisconsin sample., Conclusions: NBS IRT is a biomarker of prenatal exocrine pancreatic disease in patients with CF, and a SNP in SLC26A9 accounts for significant IRT variability. This work suggests SLC26A9 as a potential therapeutic target to ameliorate exocrine pancreatic disease., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Variation in cystic fibrosis (CF) phenotypes, including lung disease severity, age of onset of persistent Pseudomonas aeruginosa (P. aeruginosa) lung infection, and presence of meconium ileus (MI), has been partially explained by genome-wide association studies (GWASs). It is not expected that GWASs alone are sufficiently powered to uncover all heritable traits associated with CF phenotypic diversity. Therefore, we utilized gene expression association from lymphoblastoid cells lines from 754 p.Phe508del CF-affected homozygous individuals to identify genes and pathways. LPAR6, a G protein coupled receptor, associated with lung disease severity (false discovery rate q value = 0.0006). Additional pathway analyses, utilizing a stringent permutation-based approach, identified unique signals for all three phenotypes. Pathways associated with lung disease severity were annotated in three broad categories: (1) endomembrane function, containing p.Phe508del processing genes, providing evidence of the importance of p.Phe508del processing to explain lung phenotype variation; (2) HLA class I genes, extending previous GWAS findings in the HLA region; and (3) endoplasmic reticulum stress response genes. Expression pathways associated with lung disease were concordant for some endosome and HLA pathways, with pathways identified using GWAS associations from 1,978 CF-affected individuals. Pathways associated with age of onset of persistent P. aeruginosa infection were enriched for HLA class II genes, and those associated with MI were related to oxidative phosphorylation. Formal testing demonstrated that genes showing differential expression associated with lung disease severity were enriched for heritable genetic variation and expression quantitative traits. Gene expression provided a powerful tool to identify unrecognized heritable variation, complementing ongoing GWASs in this rare disease., (Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Circulating immunoreactive trypsinogen (IRT), a biomarker of exocrine pancreatic disease in cystic fibrosis (CF), is elevated in most CF newborns. In those with severe CF transmembrane conductance regulator (CFTR) genotypes, IRT declines rapidly in the first years of life, reflecting progressive pancreatic damage. Consistent with this progression, a less elevated newborn IRT measure would reflect more severe pancreatic disease, including compromised islet compartments, and potentially increased risk of CF-related diabetes (CFRD). We show in two independent CF populations that a lower newborn IRT estimate is associated with higher CFRD risk among individuals with severe CFTR genotypes, and we provide evidence to support a causal relationship. Increased loge(IRT) at birth was associated with decreased CFRD risk in Canadian and Colorado samples (hazard ratio 0.30 [95% CI 0.15-0.61] and 0.39 [0.18-0.81], respectively). Using Mendelian randomization with the SLC26A9 rs7512462 genotype as an instrumental variable since it is known to be associated with IRT birth levels in the CF population, we provide evidence to support a causal contribution of exocrine pancreatic status on CFRD risk. Our findings suggest CFRD risk could be predicted in early life and that maintained ductal fluid flow in the exocrine pancreas could delay the onset of CFRD., (© 2014 by the American Diabetes Association.)

Diabetes is a common age-dependent complication of cystic fibrosis (CF) that is strongly influenced by modifier genes. We conducted a genome-wide association study in 3,059 individuals with CF (644 with CF-related diabetes [CFRD]) and identified single nucleotide polymorphisms (SNPs) within and 5' to the SLC26A9 gene that associated with CFRD (hazard ratio [HR] 1.38; P = 3.6 × 10(-8)). Replication was demonstrated in 694 individuals (124 with CFRD) (HR, 1.47; P = 0.007), with combined analysis significant at P = 9.8 × 10(-10). SLC26A9 is an epithelial chloride/bicarbonate channel that can interact with the CF transmembrane regulator (CFTR), the protein mutated in CF. We also hypothesized that common SNPs associated with type 2 diabetes also might affect risk for CFRD. A previous association of CFRD with SNPs in TCF7L2 was replicated in this study (P = 0.004; combined analysis P = 3.8 × 10(-6)), and type 2 diabetes SNPs at or near CDKAL1, CDKN2A/B, and IGF2BP2 were associated with CFRD (P < 0.004). These five loci accounted for 8.3% of the phenotypic variance in CFRD onset and had a combined population-attributable risk of 68%. Diabetes is a highly prevalent complication of CF, for which susceptibility is determined in part by variants at SLC26A9 (which mediates processes proximate to the CF disease-causing gene) and at four susceptibility loci for type 2 diabetes in the general population.

Allelic heterogeneity in disease-causing genes presents a substantial challenge to the translation of genomic variation into clinical practice. Few of the almost 2,000 variants in the cystic fibrosis transmembrane conductance regulator gene CFTR have empirical evidence that they cause cystic fibrosis. To address this gap, we collected both genotype and phenotype data for 39,696 individuals with cystic fibrosis in registries and clinics in North America and Europe. In these individuals, 159 CFTR variants had an allele frequency of ł0.01%. These variants were evaluated for both clinical severity and functional consequence, with 127 (80%) meeting both clinical and functional criteria consistent with disease. Assessment of disease penetrance in 2,188 fathers of individuals with cystic fibrosis enabled assignment of 12 of the remaining 32 variants as neutral, whereas the other 20 variants remained of indeterminate effect. This study illustrates that sourcing data directly from well-phenotyped subjects can address the gap in our ability to interpret clinically relevant genomic variation.

Shwachman Diamond syndrome (SDS) is a rare inherited bone marrow failure syndrome (IBMFS) characterized by neutropenia, exocrine pancreatic dysfunction, and cancer predisposition. Patients are at risk for myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML) but, unlike other IBMFS, there have been no reported cases of solid tumors. We report a novel case of a solid tumor in a patient with SDS and biallelic mutations in the Shwachman Bodian Diamond Syndrome gene (SBDS). Whether the development of breast cancer in this patient is due to SDS or an isolated case due to unknown factors requires further study., (Copyright © 2011 Wiley Periodicals, Inc.)

Background & Aims: Shwachman-Diamond syndrome (SDS) is the second leading cause of hereditary exocrine pancreatic dysfunction. More than 90% of patients with SDS have biallelic loss-of-function mutations in the Shwachman-Bodian Diamond syndrome (SBDS) gene, which encodes a factor involved in ribosome function. We investigated whether mutations in Sbds lead to similar pancreatic defects in mice., Methods: Pancreas-specific knock-out mice were generated using a floxed Sbds allele and bred with mice carrying a null or disease-associated missense Sbds allele. Cre recombinase, regulated by the pancreatic transcription factor 1a promoter, was used to disrupt Sbds specifically in the pancreas. Models were assessed for pancreatic dysfunction and growth impairment., Results: Disruption of Sbds in the mouse pancreas was sufficient to recapitulate SDS phenotypes. Pancreata of mice with Sbds mutations had decreased mass, fat infiltration, but general preservation of ductal and endocrine compartments. Pancreatic extracts from mutant mice had defects in formation of the 80S ribosomal complex. The exocrine compartment of mutant mice was hypoplastic and individual acini produced few zymogen granules. The null Sbds allele resulted in an earlier onset of phenotypes as well as endocrine impairment. Mutant mice had reduced serum levels of digestive enzymes and overall growth impairment., Conclusions: We developed a mouse model of SDS with pancreatic phenotypes similar to those of the human disease. This model could be used to investigate organ-specific consequences of Sbds-associated ribosomopathy. Sbds genotypes correlated with phenotypes. Defects developed specifically in the pancreata of mice, reducing growth of mice and production of digestive enzymes. SBDS therefore appears to be required for normal pancreatic development and function., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Variants associated with meconium ileus in cystic fibrosis were identified in 3,763 affected individuals by genome-wide association study (GWAS). Five SNPs at two loci near SLC6A14 at Xq23-24 (minimum P = 1.28 × 10(-12) at rs3788766) and SLC26A9 at 1q32.1 (minimum P = 9.88 × 10(-9) at rs4077468) accounted for ~5% of phenotypic variability and were replicated in an independent sample of affected individuals (n = 2,372; P = 0.001 and 0.0001, respectively). By incorporating the knowledge that disease-causing mutations in CFTR alter electrolyte and fluid flux across surface epithelium into a hypothesis-driven GWAS (GWAS-HD), we identified associations with the same SNPs in SLC6A14 and SLC26A9 and established evidence for the involvement of SNPs in a third solute carrier gene, SLC9A3. In addition, GWAS-HD provided evidence of association between meconium ileus and multiple genes encoding constituents of the apical plasma membrane where CFTR resides (P = 0.0002; testing of 155 apical membrane genes jointly and in replication, P = 0.022). These findings suggest that modulating activities of apical membrane constituents could complement current therapeutic paradigms for cystic fibrosis.

A combined genome-wide association and linkage study was used to identify loci causing variation in cystic fibrosis lung disease severity. We identified a significant association (P = 3.34 × 10(-8)) near EHF and APIP (chr11p13) in p.Phe508del homozygotes (n = 1,978). The association replicated in p.Phe508del homozygotes (P = 0.006) from a separate family based study (n = 557), with P = 1.49 × 10(-9) for the three-study joint meta-analysis. Linkage analysis of 486 sibling pairs from the family based study identified a significant quantitative trait locus on chromosome 20q13.2 (log(10) odds = 5.03). Our findings provide insight into the causes of variation in lung disease severity in cystic fibrosis and suggest new therapeutic targets for this life-limiting disorder.

Mesenchymal cells contribute to the 'stroma' of most normal and malignant tissues, with specific mesenchymal cells participating in the regulatory niches of stem cells. By examining how mesenchymal osteolineage cells modulate haematopoiesis, here we show that deletion of Dicer1 specifically in mouse osteoprogenitors, but not in mature osteoblasts, disrupts the integrity of haematopoiesis. Myelodysplasia resulted and acute myelogenous leukaemia emerged that had acquired several genetic abnormalities while having intact Dicer1. Examining gene expression altered in osteoprogenitors as a result of Dicer1 deletion showed reduced expression of Sbds, the gene mutated in Schwachman-Bodian-Diamond syndrome-a human bone marrow failure and leukaemia pre-disposition condition. Deletion of Sbds in mouse osteoprogenitors induced bone marrow dysfunction with myelodysplasia. Therefore, perturbation of specific mesenchymal subsets of stromal cells can disorder differentiation, proliferation and apoptosis of heterologous cells, and disrupt tissue homeostasis. Furthermore, primary stromal dysfunction can result in secondary neoplastic disease, supporting the concept of niche-induced oncogenesis.

Objective: To investigate the cognitive, behavioral and adaptive functioning of children with Shwachman-Diamond syndrome (SDS)., Study Design: Thirty-two children with SDS (6-17 years) were evaluated by use of standardized neuropsychological tests. Results were compared with normative data, unaffected siblings (n = 13), and age-and sex-matched children with cystic fibrosis (CF; n = 20)., Results: Although intragroup variability in performance was evident, children with SDS displayed weaker overall intellectual reasoning, higher-order language skills, perceptual reasoning, visual-motor processing speed, visual motor- integration, visual executive problem-solving, attention, and aspects of academic achievement, as well as lower functional level of independence relative to the general population. Significant issues with behavior were also identified, including prior formal diagnoses and social problems. Lower abilities were found relative to sibling and CF control subjects and were not associated with secondary complications of SDS, age, or sex., Conclusion: Neurocognitive deficits in subjects with SDS are largely independent of family environment and having a chronic illness and are likely the consequences of Shwachman-Bodian-Diamond syndrome gene dysfunction. There is a need for a broad-based approach to the assessment of cognitive function and appropriate remediation of individuals with SDS., (Copyright 2010 Mosby, Inc. All rights reserved.)

Quebec platelet disorder (QPD) is an autosomal dominant bleeding disorder linked to a region on chromosome 10 that includes PLAU, the urokinase plasminogen activator gene. QPD increases urokinase plasminogen activator mRNA levels, particularly during megakaryocyte differentiation, without altering expression of flanking genes. Because PLAU sequence changes were excluded as the cause of this bleeding disorder, we investigated whether the QPD mutation involved PLAU copy number variation. All 38 subjects with QPD had a direct tandem duplication of a 78-kb genomic segment that includes PLAU. This mutation was specific to QPD as it was not present in any unaffected family members (n = 114), unrelated French Canadians (n = 221), or other persons tested (n = 90). This new information on the genetic mutation will facilitate diagnostic testing for QPD and studies of its pathogenesis and prevalence. QPD is the first bleeding disorder to be associated with a gene duplication event and a PLAU mutation.