High-quality read-based phasing of cystic fibrosis cohort informs genetic understanding of disease modification.

Phasing of heterozygous alleles is critical for interpretation of cis-effects of disease-relevant variation. We sequenced 477 individuals with cystic fibrosis (CF) using linked-read sequencing, which display an average phase block N50 of 4.39 Mb. We use these samples to construct a graph representation of CFTR haplotypes, demonstrating its utility for understanding complex CF alleles. These are visualized in a Web app, CFTbaRcodes, that enables interactive exploration of CFTR haplotypes present in this cohort. We perform fine-mapping and phasing of the chr7q35 trypsinogen locus associated with CF meconium ileus, an intestinal obstruction at birth associated with more severe CF outcomes and pancreatic disease. A 20-kb deletion polymorphism and a PRSS2 missense variant p.Thr8Ile (rs62473563) are shown to independently contribute to meconium ileus risk (p = 0.0028, p = 0.011, respectively) and are PRSS2 pancreas eQTLs (p = 9.5 × 10-7 and p = 1.4 × 10-4, respectively), suggesting the mechanism by which these polymorphisms contribute to CF. The phase information from linked reads provides a putative causal explanation for variation at a CF-relevant locus, which also has implications for the genetic basis of non-CF pancreatitis, to which this locus has been reported to contribute.

Genetic evidence supports the development of SLC26A9 targeting therapies for the treatment of lung disease.

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.

Cystic fibrosis-related diabetes onset can be predicted using biomarkers measured at birth.

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.

Therapeutic benefit after intracranial gene therapy delivered during the symptomatic stage in a feline model of Sandhoff disease.

Sandhoff disease (SD) is an autosomal recessive lysosomal storage disease caused by defects in the ß-subunit of ß-N-acetylhexosaminidase (Hex), the enzyme that catabolizes GM2 ganglioside. Hex deficiency causes neuronal storage of GM2 and related glycoconjugates, resulting in progressive neurodegeneration and death, typically in infancy. No effective treatment exists for human patients. Adeno-associated virus (AAV) gene therapy led to improved clinical outcome and survival of SD cats treated before the onset of disease symptoms. Most human patients are diagnosed after clinical disease onset, so it is imperative to test AAV-gene therapy in symptomatic SD cats to provide a realistic indication of therapeutic benefits that can be expected in humans. In this study, AAVrh8 vectors injected into the thalamus and deep cerebellar nuclei of symptomatic SD cats resulted in widespread central nervous system enzyme distribution, although a substantial burden of storage material remained. Cats treated in the early symptomatic phase showed delayed disease progression and a significant survival increase versus untreated cats. Treatment was less effective when administered later in the disease course, although therapeutic benefit was still possible. Results are encouraging for the treatment of human patients and provide support for the development AAV-gene therapy for human SD.

Sustained normalization of neurological disease after intracranial gene therapy in a feline model.

Progressive debilitating neurological defects characterize feline G(M1) gangliosidosis, a lysosomal storage disease caused by deficiency of lysosomal ß-galactosidase. No effective therapy exists for affected children, who often die before age 5 years. An adeno-associated viral vector carrying the therapeutic gene was injected bilaterally into two brain targets (thalamus and deep cerebellar nuclei) of a feline model of G(M1) gangliosidosis. Gene therapy normalized ß-galactosidase activity and storage throughout the brain and spinal cord. The mean survival of 12 treated G(M1) animals was >38 months, compared to 8 months for untreated animals. Seven of the eight treated animals remaining alive demonstrated normalization of disease, with abrogation of many symptoms including gait deficits and postural imbalance. Sustained correction of the G(M1) gangliosidosis disease phenotype after limited intracranial targeting by gene therapy in a large animal model suggests that this approach may be useful for treating the human version of this lysosomal storage disorder.

Therapeutic response in feline sandhoff disease despite immunity to intracranial gene therapy.

Salutary responses to adeno-associated viral (AAV) gene therapy have been reported in the mouse model of Sandhoff disease (SD), a neurodegenerative lysosomal storage disease caused by deficiency of ß-N-acetylhexosaminidase (Hex). While untreated mice reach the humane endpoint by 4.1 months of age, mice treated by a single intracranial injection of vectors expressing human hexosaminidase may live a normal life span of 2 years. When treated with the same therapeutic vectors used in mice, two cats with SD lived to 7.0 and 8.2 months of age, compared with an untreated life span of 4.5 ± 0.5 months (n = 11). Because a pronounced humoral immune response to both the AAV1 vectors and human hexosaminidase was documented, feline cDNAs for the hexosaminidase α- and ß-subunits were cloned into AAVrh8 vectors. Cats treated with vectors expressing feline hexosaminidase produced enzymatic activity >75-fold normal at the brain injection site with little evidence of an immune infiltrate. Affected cats treated with feline-specific vectors by bilateral injection of the thalamus lived to 10.4 ± 3.7 months of age (n = 3), or 2.3 times as long as untreated cats. These studies support the therapeutic potential of AAV vectors for SD and underscore the importance of species-specific cDNAs for translational research.

Treatment of Aspergillus fumigatus in patients with cystic fibrosis: a randomized, placebo-controlled pilot study.

BACKGROUND: Many patients with cystic fibrosis develop persistent airway infection/colonization with Aspergillus fumigatus, however the impact of A. fumigatus on clinical outcomes remains unclear. The objective of this study was to determine whether treatment directed against Aspergillus fumigatus improves pulmonary function and clinical outcomes in patients with cystic fibrosis (CF). METHODS: We performed a double-blind randomized placebo-controlled pilot clinical trial involving 35 patients with CF whose sputum cultures were chronically positive for A. fumigatus. Participants were centrally randomized to receive either oral itraconazole 5 mg/kg/d (N = 18) or placebo (N = 17) for 24 weeks. The primary outcome was the proportion of patients who experienced a respiratory exacerbation requiring intravenous antibiotics over the 24 week treatment period. Secondary outcomes included changes in FEV(1) and quality of life. RESULTS: Over the 24 week treatment period, 4 of 18 (22%) patients randomized to itraconazole experienced a respiratory exacerbation requiring intravenous antibiotics, compared to 5 of 16 (31%) placebo treated patients, P = 0.70. FEV(1) declined by 4.62% over 24 weeks in the patients randomized to itraconazole, compared to a 0.32% improvement in the placebo group (between group difference = -4.94%, 95% CI: -15.33 to 5.45, P = 0.34). Quality of life did not differ between the 2 treatment groups throughout the study. Therapeutic itraconazole blood levels were not achieved in 43% of patients randomized to itraconazole. CONCLUSION: We did not identify clinical benefit from itraconazole treatment for CF patients whose sputum was chronically colonized with A. fumigatus. Limitations of this pilot study were its small sample size, and failure to achieve therapeutic levels of itraconazole in many patients. TRIAL REGISTRATION: ClinicalTrials.gov NCT00528190.

Acute suppression of bone turnover with calcium infusion in persons with spinal cord injury.

BACKGROUND: Some people with chronic spinal cord injury (SCI) have low vitamin D levels and secondary hyperparathyroidism. OBJECTIVE: To determine whether, and to what extent, an acute calcium infusion decreased levels of N-telopeptide (NTx), a marker of osteoclastic activity, in individuals with chronic SCI. STUDY DESIGN: Case series. SUBJECTS: Eight men with chronic SCI. A relatively low serum 25 hydroxyvitamin D concentration (25[OH]D < or =20 ng/mL) and/or a high parathyroid hormone (PTH) (>55 pg/mL) was a prerequisite for study inclusion. METHODS: Calcium gluconate bolus 0.025 mmol elemental calcium/kg over 20 minutes followed by a constant infusion of 0.025 mmol/kg per hour for 6 hours was infused; blood samples were collected every 2 hours for measurement of serum total calcium, creatinine, NTx, and PTH. RESULTS: All results are expressed as means (+/- SDs). Baseline serum 25-hydroxyvitamin D level was 14.5 +/- 3.5 ng/mL (range: 10.2-19.6 ng/mL); PTH, 70 +/- 25 pg/mL (range: 37-100 pg/mL); and NTx, 21 +/- 7 nM bone collagen equivalents (BCE) (range: 14-34 nM). At 2, 4, and 6 hours after the calcium infusion, serum calcium rose from 9.3 +/- 0.2 to 10.8 +/- 0.9, 10.5 +/- 0.8, and 10.6 +/- 0.6 mg/d; PTH was suppressed from 70 +/- 25 pg/mL to 18 +/- 12, 16 +/- 9, and 15 +/- 9 pg/mL, respectively; NTx fell from 21 +/- 8 nM BCE to 17 +/- 5, 12 +/- 4, and 12 +/- 3 nM BCE, respectively. CONCLUSIONS: Serum NTx is a marker for bone collagen catabolism, and its reduction suggests that bone turnover was decreased. A relative deficiency of vitamin D associated with chronically elevated levels of PTH would be expected to increase bone turnover and to worsen the bone loss associated with immobilization.

Randomized trial of a decision aid for patients with cystic fibrosis considering lung transplantation.

RATIONALE: We developed an evidence-based decision aid for patients with advanced cystic fibrosis considering referral for lung transplantation. OBJECTIVES: To prospectively evaluate whether use of the decision aid increased knowledge about the options, improved realistic expectations, and decreased decisional conflict in adult patients. METHODS: We performed a single-blind randomized controlled trial involving 149 adult patients with cystic fibrosis with an FEV(1)

Effect of pamidronate administration on bone in patients with acute spinal cord injury.

Eleven subjects participated in a prospective placebo-controlled trial to address the efficacy of pamidronate in reducing bone loss in persons with acute spinal cord injury (SCI). We administered pamidronate (treatment) or normal saline (placebo) intravenously at baseline (22 to 65 days after injury) and sequentially over 12 months, with follow-up at 18 and 24 months. Regional bone mineral density (BMD) was lost over time, regardless of group. In the treatment group compared with the placebo group, we noted a mild early reduction in loss of total leg BMD. Significant bone loss from baseline occurred earlier in the placebo group at the regional sites than in the treatment group. However, by the end of the treatment and follow-up phases, both groups demonstrated a similar percent bone loss from baseline. Despite an early reduction in bone loss, pamidronate failed to prevent major, long-term bone loss in persons with acute neurologically complete SCI.

Novel metal clusters isolated from blood are lethal to cancer cells.

Unfolding and subsequent aggregation of proteins is a common phenomenon that is linked to many human disorders. Misfolded hemoglobin is generally manifested in various autoimmune, infectious and inherited diseases. We isolated micrometer and submicrometer particles, termed proteons, from human and animal blood. Proteons lack nucleic acids but contain two major polypeptide populations with homology to the hemoglobin alpha-chain. Proteons form by reversible seeded aggregation of proteins around proteon nucleating centers (PNCs). PNCs are comprised of 1- to 2-nm metallic nanoclusters containing 40-300 atoms. Each milliliter of human blood contained approximately 7 x 10(13) PNCs and approximately 3 x 10(8) proteons. Exposure of isolated blood plasma to elevated temperatures increased the number of proteons. When an aliquot of this heated plasma was introduced into untreated plasma that was subsequently heated, the number of proteons further increased, reaching a maximum after a total of three such iterations. Small concentrations of PNCs were lethal to cultured cancer cells, whereas noncancerous cells were much less affected.

Effect of a vitamin D analog on leg bone mineral density in patients with chronic spinal cord injury.

A randomized, placebo-controlled trial was performed to determine the effect of a vitamin D analog (1-alpha-hydroxyvitamin D(2) [1-alpha D(2)]) on the bone mineral density (BMD) in patients with chronic spinal cord injury (SCI). Forty subjects with chronic complete motor SCI were enrolled. The mean plus or minus standard deviation age and duration of injury were 42 plus or minus 12 yr and 11 plus or minus 10 yr, respectively. Either 4 micrograms 1-alpha D(2) (n = 19) or placebo (n = 21) was administered daily for 24 mo. Metabolic markers of bone resorption and formation were obtained. Regional lower-limb dual-energy x-ray absorptiometry was performed at baseline and at 6, 12, 18, and 24 mo. Leg BMD and percent change from baseline significantly increased at 6 (percent change only), 12, 18, and 24 mo in the treatment group, but not in the placebo group. Urinary N-telopeptide, a marker of bone resorption, was significantly reduced during treatment with 1-alpha D(2), but markers of bone formation were not changed.

Cell targeted phagemid rescued by preselected landscape phage.

We have developed a gene delivery system that utilizes a cell-binding helper phage preselected from a landscape phage display library, and a phagemid harboring a marker gene and all regulatory elements (origins of replication and promoter-enhancer cassettes) necessary for replication of the phagemid and expression of the marker gene in the targeted cell. All the proteins required for encapsulation of the phagemid DNA and cell targeting are provided by the phage helper and are separate from the phagemid. Therefore, the resultant Phagemid Infective Particles (PIPs) are able to bind and infect target cells and express the marker gene from within the cell. Our approach, shown here for glioma cells, differs from others in that a phagemid expressing a model marker or particular therapeutic gene can be easily exchanged for a phagemid expressing a different therapeutic gene. Also, a different helper phage, selected from a phage display library, such as the f8-8-mer landscape library used here, can target any cell type and direct the encapsulation of any therapeutic gene encoding phagemid. Because of its versatility, the PIPs system may be readily used for optimization of the gene-delivery strategies applied to specific cell and tissue targets.

Phage matrix for isolation of glioma cell membrane proteins.

Cell-binding ligands for RG2 rat glioma were identified in our recent study from a library of peptides that are displayed as fusion molecules on phage particles. Here, one of the phage clones was used to affinity purify those cell membrane components to which the displayed peptides bind. This phage clone, displaying the ELRGDSLP peptide, was shown to recognize glioma cells specifically in comparison to control phage-expressing peptides of either similar or irrelevant sequences. Blocking experiments with synthetic RGDS peptide demonstrated that the phage-glioma cell recognition occurs via the RGD motif known to be present in many integrin-binding proteins. To form an affinity matrix that would bind to glioma cell membrane molecules, ELRGDSLP phage particles were cross-linked using dextran polymer. Whole cell lysate from RG2 rat glioma cells was passed through the matrix, resulting in the isolation of cell membrane components having strong affinity to the peptides on phage and molecules associated with those components. One of the isolated proteins was found to be CD44s, a cell surface adhesion molecule involved in glioma cell invasion and migration, which likely formed a complex with an RGD-binding integrin. Cell membrane proteins isolated with this innovative approach could be used for the design of cell-specific anticancer treatments.

Atypical presentation of lymphomatoid granulomatosis in a patient with longstanding sarcoidosis.

A patient was recently evaluated who had longstanding sarcoidosis with lymphadenopathy and multiple, small lung nodules, and who developed a new, 9 cm solitary pulmonary mass in the right lower lobe. After thoracotomy, this lesion was ultimately found to be lymphomatoid granulomatosis, a rare lymphoproliferative disorder. Radiographic evaluations of patients with this disorder characteristically show multiple, bilateral reticulonodular opacities that follow the bronchovascular bundles; however, presentation with a solitary, large pulmonary mass is rare. The present case illustrates the need for complete evaluation of new clinical and radiographic findings in the setting of chronic lung disease.

Phage probes for malignant glial cells.

Early diagnosis and effective treatment of malignant gliomas, which are heterogeneous brain tumors with variable expression of cell surface markers, are inhibited by the lack of means to characterize and target tumor-selective molecules. To create molecular profiles for RG2 rat glioma cells, we used phage display technology, an approach capable of producing valuable ligands to unknown cell surface targets. The ligands were selected from libraries of peptides displayed as fusion molecules on phage particles. Modifications of the selection conditions resulted in identification of three distinctive families of peptide ligands for malignant glioma cells. The first family with V (D)/(G) L P (E)/(T) H(3) binding motif appeared to target a marker that is common for glioma cells, normal brain cells, and cells of non-brain origin. The second group of peptide-presented phage displayed D (T)/S/(L) T K consensus sequence and contained peptides with pronounced glioma-selective properties. Phage clones expressing peptides with E (L)/V/(S) R G D S motif were found in cell lysates and represented the third family of glioma-specific ligands. All peptides within this family contain the RGD amino acid sequence, which is known to bind to a number of integrins. Phage clones that belong to this family were internalized by RG2 glioma cells about 63-fold more efficiently than by astrocytes. The approach described could be applicable for accurate detection of glioma expression patterns in individual tumors. Such patterns could be beneficial in the design of effective combinations of drugs for anti-glioma treatments.