Positive epistasis between disease-causing missense mutations and silent polymorphism with effect on mRNA translation velocity.

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.

Genetic association and transcriptome integration identify contributing genes and tissues at cystic fibrosis modifier loci.

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.

LocusFocus: Web-based colocalization for the annotation and functional follow-up of GWAS.

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.

Correlating Cystic Fibrosis Transmembrane Conductance Regulator Function with Clinical Features to Inform Precision Treatment of Cystic Fibrosis.

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 FEV1% 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 FEV1% 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.

SBDS-Deficient Cells Have an Altered Homeostatic Equilibrium due to Translational Inefficiency Which Explains their Reduced Fitness and Provides a Logical Framework for Intervention.

Ribosomopathies are a family of inherited disorders caused by mutations in genes necessary for ribosomal function. Shwachman-Diamond Bodian Syndrome (SDS) is an autosomal recessive disease caused, in most patients, by mutations of the SBDS gene. SBDS is a protein required for the maturation of 60S ribosomes. SDS patients present exocrine pancreatic insufficiency, neutropenia, chronic infections, and skeletal abnormalities. Later in life, patients are prone to myelodisplastic syndrome and acute myeloid leukemia (AML). It is unknown why patients develop AML and which cellular alterations are directly due to the loss of the SBDS protein. Here we derived mouse embryonic fibroblast lines from an SbdsR126T/R126T mouse model. After their immortalization, we reconstituted them by adding wild type Sbds. We then performed a comprehensive analysis of cellular functions including colony formation, translational and transcriptional RNA-seq, stress and drug sensitivity. We show that: 1. Mutant Sbds causes a reduction in cellular clonogenic capability and oncogene-induced transformation. 2. Mutant Sbds causes a marked increase in immature 60S subunits, limited impact on mRNA specific initiation of translation, but reduced global protein synthesis capability. 3. Chronic loss of SBDS activity leads to a rewiring of gene expression with reduced ribosomal capability, but increased lysosomal and catabolic activity. 4. Consistently with the gene signature, we found that SBDS loss causes a reduction in ATP and lactate levels, and increased susceptibility to DNA damage. Combining our data, we conclude that a cell-specific fragile phenotype occurs when SBDS protein drops below a threshold level, and propose a new interpretation of the disease.

Cystic fibrosis gene modifier SLC26A9 modulates airway response to CFTR-directed therapeutics.

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.

A Joint Location-Scale Test Improves Power to Detect Associated SNPs, Gene Sets, and Pathways.

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.

Gene expression in transformed lymphocytes reveals variation in endomembrane and HLA pathways modifying cystic fibrosis pulmonary phenotypes.

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.

In Vivo Senescence in the Sbds-Deficient Murine Pancreas: Cell-Type Specific Consequences of Translation Insufficiency.

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 Tgfß, 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 Tgfß 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.

Sources of Variation in Sweat Chloride Measurements in Cystic Fibrosis.

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 (H2 = 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.

Prevalence of meconium ileus marks the severity of mutations of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene.

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.

Bone progenitor dysfunction induces myelodysplasia and secondary leukaemia.

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.

Variants in Solute Carrier SLC26A9 Modify Prenatal Exocrine Pancreatic Damage in Cystic Fibrosis.

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.

Deficiency of the ribosome biogenesis gene Sbds in hematopoietic stem and progenitor cells causes neutropenia in mice by attenuating lineage progression in myelocytes.

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.

Identification of the gene responsible for methylmalonic aciduria and homocystinuria, cblC type.

Methylmalonic aciduria and homocystinuria, cblC type (OMIM 277400), is the most common inborn error of vitamin B(12) (cobalamin) metabolism, with about 250 known cases. Affected individuals have developmental, hematological, neurological, metabolic, ophthalmologic and dermatologic clinical findings. Although considered a disease of infancy or childhood, some individuals develop symptoms in adulthood. The cblC locus was mapped to chromosome region 1p by linkage analysis. We refined the chromosomal interval using homozygosity mapping and haplotype analyses and identified the MMACHC gene. In 204 individuals, 42 different mutations were identified, many consistent with a loss of function of the protein product. One mutation, 271dupA, accounted for 40% of all disease alleles. Transduction of wild-type MMACHC into immortalized cblC fibroblast cell lines corrected the cellular phenotype. Molecular modeling predicts that the C-terminal region of the gene product folds similarly to TonB, a bacterial protein involved in energy transduction for cobalamin uptake.

Identification of a novel percent mammographic density locus at 12q24.

Percent mammographic density adjusted for age and body mass index (BMI) is one of the strongest risk factors for breast cancer and has a heritable component that remains largely unidentified. We performed a three-stage genome-wide association study (GWAS) of percent mammographic density to identify novel genetic loci associated with this trait. In stage 1, we combined three GWASs of percent density comprised of 1241 women from studies at the Mayo Clinic and identified the top 48 loci (99 single nucleotide polymorphisms). We attempted replication of these loci in 7018 women from seven additional studies (stage 2). The meta-analysis of stage 1 and 2 data identified a novel locus, rs1265507 on 12q24, associated with percent density, adjusting for age and BMI (P = 4.43 × 10(-8)). We refined the 12q24 locus with 459 additional variants (stage 3) in a combined analysis of all three stages (n = 10 377) and confirmed that rs1265507 has the strongest association in the 12q24 region (P = 1.03 × 10(-8)). Rs1265507 is located between the genes TBX5 and TBX3, which are members of the phylogenetically conserved T-box gene family and encode transcription factors involved in developmental regulation. Understanding the mechanism underlying this association will provide insight into the genetics of breast tissue composition.

Unraveling the complex genetic model for cystic fibrosis: pleiotropic effects of modifier genes on early cystic fibrosis-related morbidities.

The existence of pleiotropy in disorders with multi-organ involvement can suggest therapeutic targets that could ameliorate overall disease severity. Here we assessed pleiotropy of modifier genes in cystic fibrosis (CF). CF, caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, affects the lungs, liver, pancreas and intestines. However, modifier genes contribute to variable disease severity across affected organs, even in individuals with the same CFTR genotype. We sought to determine whether SLC26A9, SLC9A3 and SLC6A14, that contribute to meconium ileus in CF, are pleiotropic for other early-affecting CF co-morbidities. In the Canadian CF population, we assessed evidence for pleiotropic effects on (1) pediatric lung disease severity (n = 815), (2) age at first acquisition of Pseudomonas aeruginosa (P. aeruginosa) (n = 730), and (3) prenatal pancreatic damage measured by immunoreactive trypsinogen (n = 126). A multiple-phenotype analytic strategy assessed evidence for pleiotropy in the presence of phenotypic correlation. We required the same alleles to be associated with detrimental effects. SLC26A9 was pleiotropic for meconium ileus and pancreatic damage (p = 0.002 at rs7512462), SLC9A3 for meconium ileus and lung disease (p = 1.5 × 10(-6) at rs17563161), and SLC6A14 for meconium ileus and both lung disease and age at first P. aeruginosa infection (p = 0.0002 and p = 0.006 at rs3788766, respectively). The meconium ileus risk alleles in SLC26A9, SLC9A3 and SLC6A14 are pleiotropic, increasing risk for other early CF co-morbidities. Furthermore, co-morbidities affecting the same organ tended to associate with the same genes. The existence of pleiotropy within this single disorder suggests that complementary therapeutic strategies to augment solute transport will benefit multiple CF-associated tissues.

Mammographic density and breast cancer: a comparison of related and unrelated controls in the Breast Cancer Family Registry.

INTRODUCTION: Percent mammographic density (PMD) is a strong and highly heritable risk factor for breast cancer. Studies of the role of PMD in familial breast cancer may require controls, such as the sisters of cases, selected from the same 'risk set' as the cases. The use of sister controls would allow control for factors that have been shown to influence risk of breast cancer such as race/ethnicity, socioeconomic status and a family history of breast cancer, but may introduce 'overmatching' and attenuate case-control differences in PMD. METHODS: To examine the potential effects of using sister controls rather than unrelated controls in a case-control study, we examined PMD in triplets, each comprised of a case with invasive breast cancer, an unaffected full sister control, and an unaffected unrelated control. Both controls were matched to cases on age at mammogram. Total breast area and dense area in the mammogram were measured in the unaffected breast of cases and a randomly selected breast in controls, and the non-dense area and PMD calculated from these measurements. RESULTS: The mean difference in PMD between cases and controls, and the standard deviation (SD) of the difference, were slightly less for sister controls (4.2% (SD = 20.0)) than for unrelated controls (4.9% (SD = 25.7)). We found statistically significant correlations in PMD between cases (n = 228) and sister controls (n = 228) (r = 0.39 (95% CI: 0.28, 0.50; P <0.0001)), but not between cases and unrelated controls (n = 228) (r = 0.04 (95% CI: -0.09, 0.17; P = 0.51)). After adjusting for other risk factors, square root transformed PMD was associated with an increased risk of breast cancer when comparing cases to sister controls (adjusted odds ratio (inter-quintile odds ratio (IQOR) = 2.19, 95% CI = 1.20, 4.00) or to unrelated controls (adjusted IQOR = 2.62, 95% CI = 1.62, 4.25). CONCLUSIONS: The use of sister controls in case-control studies of PMD resulted in a modest attenuation of case-control differences and risk estimates, but showed a statistically significant association with risk and allowed control for race/ethnicity, socioeconomic status and family history.

Deficiency of Sbds in the mouse pancreas leads to features of Shwachman-Diamond syndrome, with loss of zymogen granules.

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.