Randomised trial of genetic testing and targeted intervention to prevent the development and progression of Paget's disease of bone.

INTRODUCTION: Paget's disease of bone (PDB) frequently presents at an advanced stage with irreversible skeletal damage. Clinical outcomes might be improved by earlier diagnosis and prophylactic treatment. METHODS: We randomised 222 individuals at increased risk of PDB because of pathogenic SQSTM1 variants to receive 5 mg zoledronic acid (ZA) or placebo. The primary outcome was new bone lesions assessed by radionuclide bone scan. Secondary outcomes included change in existing lesions, biochemical markers of bone turnover and skeletal events related to PDB. RESULTS: The median duration of follow-up was 84 months (range 0-127) and 180 participants (81%) completed the study. At baseline, 9 (8.1%) of the ZA group had PDB lesions vs 12 (10.8%) of the placebo group. Two of the placebo group developed new lesions versus none in the ZA group (OR 0.41, 95% CI 0.00 to 3.43, p=0.25). Eight of the placebo group had a poor outcome (lesions which were new, unchanged or progressing) compared with none of the ZA group (OR 0.08, 95% CI 0.00 to 0.42, p=0.003). At the study end, 1 participant in the ZA group had lesions compared with 11 in the placebo group. Biochemical markers of bone turnover were significantly reduced in the ZA group. One participant allocated to placebo required rescue therapy with ZA because of symptomatic disease. The number and severity of adverse events did not differ between groups. CONCLUSIONS: Genetic testing for pathogenic SQSTM1 variants coupled with intervention with ZA is well tolerated and has favourable effects on the progression of early PDB. TRIAL REGISTRATION NUMBER: ISRCTN11616770.

Idiopathic Pulmonary Fibrosis Is Associated with Common Genetic Variants and Limited Rare Variants.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a rare, irreversible, and progressive disease of the lungs. Common genetic variants, in addition to nongenetic factors, have been consistently associated with IPF. Rare variants identified by candidate gene, family-based, and exome studies have also been reported to associate with IPF. However, the extent to which rare variants, genome-wide, may contribute to the risk of IPF remains unknown. Objectives: We used whole-genome sequencing to investigate the role of rare variants, genome-wide, on IPF risk. Methods: As part of the Trans-Omics for Precision Medicine Program, we sequenced 2,180 cases of IPF. Association testing focused on the aggregated effect of rare variants (minor allele frequency ⩽0.01) within genes or regions. We also identified individual rare variants that are influential within genes and estimated the heritability of IPF on the basis of rare and common variants. Measurements and Main Results: Rare variants in both TERT and RTEL1 were significantly associated with IPF. A single rare variant in each of the TERT and RTEL1 genes was found to consistently influence the aggregated test statistics. There was no significant evidence of association with other previously reported rare variants. The SNP heritability of IPF was estimated to be 32% (SE = 3%). Conclusions: Rare variants within the TERT and RTEL1 genes and well-established common variants have the largest contribution to IPF risk overall. Efforts in risk profiling or the development of therapies for IPF that focus on TERT, RTEL1, common variants, and environmental risk factors are likely to have the largest impact on this complex disease.

Integrative multi-omics analysis reveals novel idiopathic pulmonary fibrosis endotypes associated with disease progression.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is characterized by the accumulation of extracellular matrix in the pulmonary interstitium and progressive functional decline. We hypothesized that integration of multi-omics data would identify clinically meaningful molecular endotypes of IPF. METHODS: The IPF-PRO Registry is a prospective registry of patients with IPF. Proteomic and transcriptomic (including total RNA [toRNA] and microRNA [miRNA]) analyses were performed using blood collected at enrollment. Molecular data were integrated using Similarity Network Fusion, followed by unsupervised spectral clustering to identify molecular subtypes. Cox proportional hazards models tested the relationship between these subtypes and progression-free and transplant-free survival. The molecular subtypes were compared to risk groups based on a previously described 52-gene (toRNA expression) signature. Biological characteristics of the molecular subtypes were evaluated via linear regression differential expression and canonical pathways (Ingenuity Pathway Analysis [IPA]) over-representation analyses. RESULTS: Among 232 subjects, two molecular subtypes were identified. Subtype 1 (n = 105, 45.3%) and Subtype 2 (n = 127, 54.7%) had similar distributions of age (70.1 +/- 8.1 vs. 69.3 +/- 7.6 years; p = 0.31) and sex (79.1% vs. 70.1% males, p = 0.16). Subtype 1 had more severe disease based on composite physiologic index (CPI) (55.8 vs. 51.2; p = 0.002). After adjusting for CPI and antifibrotic treatment at enrollment, subtype 1 experienced shorter progression-free survival (HR 1.79, 95% CI 1.28,2.56; p = 0.0008) and similar transplant-free survival (HR 1.30, 95% CI 0.87,1.96; p = 0.20) as subtype 2. There was little agreement in the distribution of subjects to the molecular subtypes and the risk groups based on 52-gene signature (kappa = 0.04, 95% CI= -0.08, 0.17), and the 52-gene signature risk groups were associated with differences in transplant-free but not progression-free survival. Based on heatmaps and differential expression analyses, proteins and miRNAs (but not toRNA) contributed to classification of subjects to the molecular subtypes. The IPA showed enrichment in pulmonary fibrosis-relevant pathways, including mTOR, VEGF, PDGF, and B-cell receptor signaling. CONCLUSIONS: Integration of transcriptomic and proteomic data from blood enabled identification of clinically meaningful molecular endotypes of IPF. If validated, these endotypes could facilitate identification of individuals likely to experience disease progression and enrichment of clinical trials. TRIAL REGISTRATION: NCT01915511.

SDHC phaeochromocytoma and paraganglioma: A UK-wide case series.

OBJECTIVE: Phaeochromocytomas and paragangliomas (PPGL) are rare, but strongly heritable tumours. Variants in succinate dehydrogenase (SDH) subunits are identified in approximately 25% of cases. However, clinical and genetic information of patients with SDHC variants are underreported. DESIGN: This retrospective case series collated data from 18 UK Genetics and Endocrinology departments. PATIENTS: Both asymptomatic and disease-affected patients with confirmed SDHC germline variants are included. MEASUREMENTS: Clinical data including tumour type and location, surveillance outcomes and interventions, SDHC genetic variant assessment, interpretation, and tumour risk calculation. RESULTS: We report 91 SDHC cases, 46 probands and 45 non-probands. Fifty-one cases were disease-affected. Median age at genetic diagnosis was 43 years (range: 11-79). Twenty-four SDHC germline variants were identified including six novel variants. Head and neck paraganglioma (HNPGL, n = 30, 65.2%), extra-adrenal paraganglioma (EAPGL, n = 13, 28.2%) and phaeochromocytomas (PCC) (n = 3, 6.5%) were present. One case had multiple PPGLs. Malignant disease was reported in 19.6% (9/46). Eight cases had non-PPGL SDHC-associated tumours, six gastrointestinal stromal tumours (GIST) and two renal cell cancers (RCC). Cumulative tumour risk (95% CI) at age 60 years was 0.94 (CI: 0.79-0.99) in probands, and 0.16 (CI: 0-0.31) in non-probands, respectively. CONCLUSIONS: This study describes the largest cohort of 91 SDHC patients worldwide. We confirm disease-affected SDHC variant cases develop isolated HNPGL disease in nearly 2/3 of patients, EAPGL and PCC in 1/3, with an increased risk of GIST and RCC. One fifth developed malignant disease, requiring comprehensive lifelong tumour screening and surveillance.

Identification and Prognosis of Patients With Interstitial Pneumonia With Autoimmune Features.

BACKGROUND/OBJECTIVE: Patients classified as interstitial pneumonia with autoimmune features (IPAF) have interstitial lung disease (ILD) and features of autoimmunity but do not fulfill criteria for connective tissue diseases (CTDs). Our goal was to identify patients classifiable as IPAF, CTD-ILD, and idiopathic pulmonary fibrosis (IPF) from a preexisting pulmonary cohort and evaluate the prognosis of patients with IPAF. METHODS: We reviewed the medical records of 456 patients from a single-center pulmonary ILD cohort whose diagnoses were previously established by a multidisciplinary panel that did not include rheumatologists. We reclassified patients as IPAF, CTD-ILD, or IPF. We compared transplant-free survival using Kaplan-Meier methods and identified prognostic factors using Cox models. RESULTS: We identified 60 patients with IPAF, 113 with CTD-ILD, and 126 with IPF. Transplant-free survival of IPAF was not statistically significantly different from that of CTD-ILD or IPF. Among IPAF patients, male sex (hazard ratio, 4.58 [1.77-11.87]) was independently associated with worse transplant-free survival. During follow-up, only 10% of IPAF patients were diagnosed with CTD-ILD, most commonly antisynthetase syndrome. CONCLUSION: Despite similar clinical characteristics, most patients with IPAF did not progress to CTD-ILD; those who did often developed antisynthetase syndrome, highlighting the critical importance of comprehensive myositis autoantibody testing in this population. As in other types of ILD, male sex may portend a worse prognosis in IPAF. The routine engagement of rheumatologists in the multidisciplinary evaluation of ILD will help ensure the accurate classification of these patients and help clarify prognostic factors.

Integrated plasma proteomics and lung transcriptomics reveal novel biomarkers in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease with a significant unmet medical need. Development of transformational therapies for IPF is challenging in part to due to lack of robust predictive biomarkers of prognosis and treatment response. Importantly, circulating biomarkers of IPF are limited and none are in clinical use. METHODS: We previously reported dysregulated pathways and new disease biomarkers in advanced IPF through RNA sequencing of lung tissues from a cohort of transplant-stage IPF patients (n = 36) in comparison to normal healthy donors (n = 19) and patients with acute lung injury (n = 11). Here we performed proteomic profiling of matching plasma samples from these cohorts through the Somascan-1300 SomaLogics platform. RESULTS: Comparative analyses of lung transcriptomic and plasma proteomic signatures identified a set of 34 differentially expressed analytes (fold change (FC) ≥ ± 1.5, false discovery ratio (FDR) ≤ 0.1) in IPF samples compared to healthy controls. IPF samples showed strong enrichment of chemotaxis, tumor infiltration and mast cell migration pathways and downregulated extracellular matrix (ECM) degradation. Mucosal (CCL25 and CCL28) and Th2 (CCL17 and CCL22) chemokines were markedly upregulated in IPF and highly correlated within the subjects. The mast cell maturation chemokine, CXCL12, was also upregulated in IPF plasma (fold change 1.92, FDR 0.006) and significantly correlated (Pearson r = - 0.38, p = 0.022) to lung function (%predicted FVC), with a concomitant increase in the mast cell Tryptase, TPSB2. Markers of collagen III and VI degradation (C3M and C6M) were significantly downregulated (C3M p < 0.001 and C6M p < 0.0001 IPF vs control) and correlated, Pearson r = 0.77) in advanced IPF consistent with altered ECM homeostasis. CONCLUSIONS: Our study identifies a panel of tissue and circulating biomarkers with clinical utility in IPF that can be validated in future studies across larger cohorts.

Lung Innate Lymphoid Cell Composition Is Altered in Primary Graft Dysfunction.

Rationale: Primary graft dysfunction (PGD) is the leading cause of early morbidity and mortality after lung transplantation, but the immunologic mechanisms are poorly understood. Innate lymphoid cells (ILC) are a heterogeneous family of immune cells regulating pathologic inflammation and beneficial tissue repair. However, whether changes in donor-derived lung ILC populations are associated with PGD development has never been examined.Objectives: To determine whether PGD in chronic obstructive pulmonary disease or interstitial lung disease transplant recipients is associated with alterations in ILC subset composition within the allograft.Methods: We performed a single-center cohort study of lung transplantation patients with surgical biopsies of donor tissue taken before, and immediately after, allograft reperfusion. Donor immune cells from 18 patients were characterized phenotypically by flow cytometry for single-cell resolution of distinct ILC subsets. Changes in the percentage of ILC subsets with reperfusion or PGD (grade 3 within 72 h) were assessed.Measurements and Main Results: Allograft reperfusion resulted in significantly decreased frequencies of natural killer cells and a trend toward reduced ILC populations, regardless of diagnosis (interstitial lung disease or chronic obstructive pulmonary disease). Seven patients developed PGD (38.9%), and PGD development was associated with selective reduction of the ILC2 subset after reperfusion. Conversely, patients without PGD exhibited significantly higher ILC1 frequencies before reperfusion, accompanied by elevated ILC2 frequencies after allograft reperfusion.Conclusions: The composition of donor ILC subsets is altered after allograft reperfusion and is associated with PGD development, suggesting that ILCs may be involved in regulating lung injury in lung transplant recipients.

Second International Guidelines for the Diagnosis and Management of Hereditary Hemorrhagic Telangiectasia.

DESCRIPTION: Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disease with an estimated prevalence of 1 in 5000 that is characterized by the presence of vascular malformations (VMs). These result in chronic bleeding, acute hemorrhage, and complications from shunting through VMs. The goal of the Second International HHT Guidelines process was to develop evidence-based consensus guidelines for the management and prevention of HHT-related symptoms and complications. METHODS: The guidelines were developed using the AGREE II (Appraisal of Guidelines for Research and Evaluation II) framework and GRADE (Grading of Recommendations Assessment, Development and Evaluation) methodology. The guidelines expert panel included expert physicians (clinical and genetic) in HHT from 15 countries, guidelines methodologists, health care workers, health care administrators, patient advocacy representatives, and persons with HHT. During the preconference process, the expert panel generated clinically relevant questions in 6 priority topic areas. A systematic literature search was done in June 2019, and articles meeting a priori criteria were included to generate evidence tables, which were used as the basis for recommendation development. The expert panel subsequently convened during a guidelines conference to conduct a structured consensus process, during which recommendations reaching at least 80% consensus were discussed and approved. RECOMMENDATIONS: The expert panel generated and approved 6 new recommendations for each of the following 6 priority topic areas: epistaxis, gastrointestinal bleeding, anemia and iron deficiency, liver VMs, pediatric care, and pregnancy and delivery (36 total). The recommendations highlight new evidence in existing topics from the first International HHT Guidelines and provide guidance in 3 new areas: anemia, pediatrics, and pregnancy and delivery. These recommendations should facilitate implementation of key components of HHT care into clinical practice.

Skeletal muscle adiposity and outcomes in candidates for lung transplantation: a lung transplant body composition cohort study.

CT measurement of body composition may improve lung transplant candidate selection. We assessed whether skeletal muscle adipose deposition on abdominal and thigh CT scans was associated with 6 min walk distance (6MWD) and wait-list survival in lung transplant candidates. Each ½-SD decrease in abdominal muscle attenuation (indicating greater lipid content) was associated with 14 m decrease in 6MWD (95% CI -20 to -8) and 20% increased risk of death or delisting (95% CI 10% to 40%). Each ½-standard deviation decrease in thigh muscle attenuation was associated with 15 m decrease in 6MWD (95% CI -21 to -10). CT imaging may improve candidate risk stratification.

Relationship between neuropsychiatric disorders and cognitive and behavioural change in MND.

OBJECTIVE: In this population-based study, we aimed to determine whether neuropsychiatric history, medication or family history of neuropsychiatric disorders predicted cognitive and/or behavioural impairment in motor neuron disease (MND). METHODS: People with MND (pwMND) on the Scottish Clinical, Audit, Research and Evaluation of MND (CARE-MND) register, diagnosed from January 2015 to January 2018, with cognitive and/or behavioural data measured using the Edinburgh Cognitive and Behavioural ALS Screen were included. Data were extracted on patient neuropsychiatric, medication and family history of neuropsychiatric disorders. We identified patients with cognitive impairment (motor neuron disease with cognitive impairment (MNDci)), behavioural impairment (motor neuron disease with behavioural impairment (MNDbi), both (motor neuron disease with cognitive and behavioural impairment (MNDcbi)) or motor neuron disease-frontotemporal dementia (MND-FTD). RESULTS: Data were available for 305 pwMND (mean age at diagnosis=62.26 years, SD=11.40), of which 60 (19.7%) had a neuropsychiatric disorder. A family history of neuropsychiatric disorders was present in 36/231 (15.58%) of patients. Patient premorbid mood disorders were associated with increased apathy (OR=2.78, 95% CI 1.083 to 7.169). A family history of any neuropsychiatric disorder was associated with poorer visuospatial scores, MNDbi (OR=3.14, 95% CI 1.09 to 8.99) and MND-FTD (OR=5.08, 95% CI 1.26 to 20.40). A family history of mood disorders was associated with poorer overall cognition (exp(b)=0.725, p=0.026), language, verbal fluency and visuospatial scores, and MND-FTD (OR=7.57, 95% CI 1.55 to 46.87). A family history of neurotic disorders was associated with poorer language (exp(b)=0.362, p<0.001), visuospatial scores (exp(b)=0.625, p<0.009) and MND-FTD (OR=13.75, 95% CI 1.71 to 110.86). CONCLUSION: Neuropsychiatric disorders in patients and their families are associated with cognitive and behavioural changes post-MND diagnosis, with many occurring independently of MND-FTD and C9orf72 status. These findings support an overlap between MND, frontotemporal dementia and neuropsychiatric disorders, particularly mood disorders.

Postreperfusion plasma endothelial activation markers are associated with acute kidney injury after lung transplantation.

Acute kidney injury (AKI) is common after lung transplantation, but molecular markers remain poorly studied. The endothelial activation markers soluble thrombomodulin (sTM), protein C, and plasminogen activator inhibitor-1 (PAI-1) are implicated in kidney microcirculatory injury in animal models of AKI. We tested the association of 6-hour postreperfusion plasma levels of these markers with posttransplant AKI severity in patients enrolled in the Lung Transplant Outcomes Group prospective cohort study at the University of Pennsylvania during two eras: 2004-06 (n = 61) and 2013-15 (n = 67). We defined AKI stage through postoperative day 5 using Kidney Disease Improving Global Outcomes creatinine criteria. We used multivariable ordinal logistic regression to determine the association of each biomarker with AKI, adjusted for primary graft dysfunction and extracorporeal life support. AKI occurred in 57 (45%) patients across both eras: 28 (22%) stage 1, 29 (23%) stage 2-3. Higher sTM and lower protein C plasma levels were associated with AKI stage in each era and remained so in multivariable models utilizing both eras (sTM: OR 1.76 [95% CI 1.19-2.60] per standard deviation, P = .005; protein C: OR 0.54 [1.19-2.60], P = .003). We conclude that 6-hour postreperfusion plasma sTM and protein C levels are associated with early postlung transplant AKI severity.

The association of post-lung transplant acute kidney injury with mortality is independent of primary graft dysfunction: A cohort study.

BACKGROUND: Prior studies of post-lung transplant acute kidney injury (AKI) have not accounted for confounding effects of primary graft dysfunction (PGD). We sought to test the impact of PGD on AKI risk factors and on the association of AKI with mortality. METHODS: We included patients transplanted at the University of Pennsylvania from 2005-12, defined AKI using consensus criteria during transplant hospitalization, and defined PGD as grade 3 at 48-72 hours. We used multivariable logistic regression to test the impact of PGD on AKI risk factors and Cox models to test association of AKI with one-year mortality adjusting for PGD and other confounders. RESULTS: Of 299 patients, 188 (62.9%) developed AKI with 142 (75%) cases occurring by postoperative day 4. In multivariable models, PGD was strongly associated with AKI (OR 3.76, 95% CI 1.72-8.19, P = .001) but minimally changed associations of other risk factors with AKI. Both AKI (HR 3.64, 95% CI 1.68-7.88, P = .001) and PGD (HR 2.55, 95% CI 1.40-4.64, P = .002) were independently associated with one-year mortality. CONCLUSIONS: Post-lung transplant AKI risk factors and association of AKI with mortality were independent of PGD. AKI may therefore be a target for improving lung transplant mortality rather than simply an epiphenomenon of PGD.

Mosaic PPM1D mutations are associated with predisposition to breast and ovarian cancer.

Improved sequencing technologies offer unprecedented opportunities for investigating the role of rare genetic variation in common disease. However, there are considerable challenges with respect to study design, data analysis and replication. Using pooled next-generation sequencing of 507 genes implicated in the repair of DNA in 1,150 samples, an analytical strategy focused on protein-truncating variants (PTVs) and a large-scale sequencing case-control replication experiment in 13,642 individuals, here we show that rare PTVs in the p53-inducible protein phosphatase PPM1D are associated with predisposition to breast cancer and ovarian cancer. PPM1D PTV mutations were present in 25 out of 7,781 cases versus 1 out of 5,861 controls (P = 1.12 × 10(-5)), including 18 mutations in 6,912 individuals with breast cancer (P = 2.42 × 10(-4)) and 12 mutations in 1,121 individuals with ovarian cancer (P = 3.10 × 10(-9)). Notably, all of the identified PPM1D PTVs were mosaic in lymphocyte DNA and clustered within a 370-base-pair region in the final exon of the gene, carboxy-terminal to the phosphatase catalytic domain. Functional studies demonstrate that the mutations result in enhanced suppression of p53 in response to ionizing radiation exposure, suggesting that the mutant alleles encode hyperactive PPM1D isoforms. Thus, although the mutations cause premature protein truncation, they do not result in the simple loss-of-function effect typically associated with this class of variant, but instead probably have a gain-of-function effect. Our results have implications for the detection and management of breast and ovarian cancer risk. More generally, these data provide new insights into the role of rare and of mosaic genetic variants in common conditions, and the use of sequencing in their identification.

Quantitative Evidence for Revising the Definition of Primary Graft Dysfunction after Lung Transplant.

RATIONALE: Primary graft dysfunction (PGD) is a form of acute lung injury that occurs after lung transplantation. The definition of PGD was standardized in 2005. Since that time, clinical practice has evolved, and this definition is increasingly used as a primary endpoint for clinical trials; therefore, validation is warranted. OBJECTIVES: We sought to determine whether refinements to the 2005 consensus definition could further improve construct validity. METHODS: Data from the Lung Transplant Outcomes Group multicenter cohort were used to compare variations on the PGD definition, including alternate oxygenation thresholds, inclusion of additional severity groups, and effects of procedure type and mechanical ventilation. Convergent and divergent validity were compared for mortality prediction and concurrent lung injury biomarker discrimination. MEASUREMENTS AND MAIN RESULTS: A total of 1,179 subjects from 10 centers were enrolled from 2007 to 2012. Median length of follow-up was 4 years (interquartile range = 2.4-5.9). No mortality differences were noted between no PGD (grade 0) and mild PGD (grade 1). Significantly better mortality discrimination was evident for all definitions using later time points (48, 72, or 48-72 hours; P < 0.001). Biomarker divergent discrimination was superior when collapsing grades 0 and 1. Additional severity grades, use of mechanical ventilation, and transplant procedure type had minimal or no effect on mortality or biomarker discrimination. CONCLUSIONS: The PGD consensus definition can be simplified by combining lower PGD grades. Construct validity of grading was present regardless of transplant procedure type or use of mechanical ventilation. Additional severity categories had minimal impact on mortality or biomarker discrimination.

Risks of breast or ovarian cancer in BRCA1 or BRCA2 predictive test negatives: findings from the EMBRACE study.

PURPOSE: BRCA1/BRCA2 predictive test negatives are proven noncarriers of a BRCA1/BRCA2 mutation that is carried by their relatives. The risk of developing breast cancer (BC) or epithelial ovarian cancer (EOC) in these women is uncertain. The study aimed to estimate risks of invasive BC and EOC in a large cohort of BRCA1/BRCA2 predictive test negatives. METHODS: We used cohort analysis to estimate incidences, cumulative risks, and standardized incidence ratios (SIRs). RESULTS: A total of 1,895 unaffected women were eligible for inclusion in the BC risk analysis and 1,736 in the EOC risk analysis. There were 23 incident invasive BCs and 2 EOCs. The cumulative risk of invasive BC was 9.4% (95% confidence interval (CI) 5.9-15%) by age 85 years and the corresponding risk of EOC was 0.6% (95% CI 0.2-2.6%). The SIR for invasive BC was 0.93 (95% CI 0.62-1.40) in the overall cohort, 0.85 (95% CI 0.48-1.50) in noncarriers from BRCA1 families, and 1.03 (95% CI 0.57-1.87) in noncarriers from BRCA2 families. The SIR for EOC was 0.79 (95% CI 0.20-3.17) in the overall cohort. CONCLUSION: Our results did not provide evidence for elevated risks of invasive BC or EOC in BRCA1/BRCA2 predictive test negatives.

Patients' Views of Treatment-Focused Genetic Testing (TFGT): Some Lessons for the Mainstreaming of BRCA1 and BRCA2 Testing.

This paper explores patients' views and experiences of undergoing treatment-focused BRCA1 and BRCA2 genetic testing (TFGT), either offered following triaging to clinical genetics (breast cancer) or as part of a mainstreamed care pathway in oncology (ovarian cancer). Drawing on 26 in-depth interviews with patients with breast or ovarian cancer who had undergone TFGT, this retrospective study examines patients' views of genetic testing at this point in their care pathway, focusing on issues, such as initial response to the offer of testing, motivations for undergoing testing, and views on care pathways. Patients were amenable to the incorporation of TFGT at an early stage in their cancer care irrespective of (any) prior anticipation of having a genetic test or family history. While patients were glad to have been offered TFGT as part of their care, some questioned the logic of the test's timing in relation to their cancer treatment. Crucially, patients appeared unable to disentangle the treatment role of TFGT from its preventative function for self and other family members, suggesting that some may undergo TFGT to obtain information for others rather than for self.

Adaptor protein-2 sigma subunit mutations causing familial hypocalciuric hypercalcaemia type 3 (FHH3) demonstrate genotype-phenotype correlations, codon bias and dominant-negative effects.

The adaptor protein-2 sigma subunit (AP2σ2) is pivotal for clathrin-mediated endocytosis of plasma membrane constituents such as the calcium-sensing receptor (CaSR). Mutations of the AP2σ2 Arg15 residue result in familial hypocalciuric hypercalcaemia type 3 (FHH3), a disorder of extracellular calcium (Ca(2+) o) homeostasis. To elucidate the role of AP2σ2 in Ca(2+) o regulation, we investigated 65 FHH probands, without other FHH-associated mutations, for AP2σ2 mutations, characterized their functional consequences and investigated the genetic mechanisms leading to FHH3. AP2σ2 mutations were identified in 17 probands, comprising 5 Arg15Cys, 4 Arg15His and 8 Arg15Leu mutations. A genotype-phenotype correlation was observed with the Arg15Leu mutation leading to marked hypercalcaemia. FHH3 probands harboured additional phenotypes such as cognitive dysfunction. All three FHH3-causing AP2σ2 mutations impaired CaSR signal transduction in a dominant-negative manner. Mutational bias was observed at the AP2σ2 Arg15 residue as other predicted missense substitutions (Arg15Gly, Arg15Pro and Arg15Ser), which also caused CaSR loss-of-function, were not detected in FHH probands, and these mutations were found to reduce the numbers of CaSR-expressing cells. FHH3 probands had significantly greater serum calcium (sCa) and magnesium (sMg) concentrations with reduced urinary calcium to creatinine clearance ratios (CCCR) in comparison with FHH1 probands with CaSR mutations, and a calculated index of sCa × sMg/100 × CCCR, which was ≥ 5.0, had a diagnostic sensitivity and specificity of 83 and 86%, respectively, for FHH3. Thus, our studies demonstrate AP2σ2 mutations to result in a more severe FHH phenotype with genotype-phenotype correlations, and a dominant-negative mechanism of action with mutational bias at the Arg15 residue.

Diastolic Dysfunction Increases the Risk of Primary Graft Dysfunction after Lung Transplant.

RATIONALE: Primary graft dysfunction (PGD) is a significant cause of early morbidity and mortality after lung transplant and is characterized by severe hypoxemia and infiltrates in the allograft. The pathogenesis of PGD involves ischemia-reperfusion injury. However, subclinical increases in pulmonary venous pressure due to left ventricular diastolic dysfunction may contribute by exacerbating capillary leak. OBJECTIVES: To determine whether a higher ratio of early mitral inflow velocity (E) to early diastolic mitral annular velocity (é), indicative of worse left ventricular diastolic function, is associated with a higher risk of PGD. METHODS: We performed a retrospective cohort study of patients in the Lung Transplant Outcomes Group who underwent bilateral lung transplant at our institution between 2004 and 2014 for interstitial lung disease, chronic obstructive pulmonary disease, or pulmonary arterial hypertension. Transthoracic echocardiograms obtained during evaluation for transplant listing were analyzed for E/é and other measures of diastolic function. PGD was defined as PaO2/FiO2 less than or equal to 200 with allograft infiltrates at 48 or 72 hours after reperfusion. The association between E/é and PGD was assessed with multivariable logistic regression. MEASUREMENTS AND MAIN RESULTS: After adjustment for recipient age, body mass index, mean pulmonary arterial pressure, and pretransplant diagnosis, higher E/é and E/é greater than 8 were associated with an increased risk of PGD (E/é odds ratio, 1.93; 95% confidence interval, 1.02-3.64; P = 0.04; E/é >8 odds ratio, 5.29; 95% confidence interval, 1.40-20.01; P = 0.01). CONCLUSIONS: Differences in left ventricular diastolic function may contribute to the development of PGD. Future trials are needed to determine whether optimization of left ventricular diastolic function reduces the risk of PGD.

Genetic testing and reproductive choice in neurological disorders.

Genetic testing is increasingly important for investigating suspected inherited neurological conditions. A genetic diagnosis can have a huge impact on patients and also their families. It is important for neurologists to appreciate the presymptomatic and prenatal testing options available to patients and their at-risk relatives once a genetic disorder is diagnosed. Asymptomatic family members can experience considerable psychological distress from the knowledge that they might have inherited a neurodegenerative condition. They may also be concerned about the risk of their children inheriting the condition. Information on reproductive options can provide hope and reassurance. This paper reviews the principles of genetic testing in neurological practice, and how they can be applied in prenatal and preimplantation genetic diagnosis. We explain the basis for direct and exclusion testing, use case examples to illustrate the process by which families are counselled and discuss the ethical implications of reproductive technologies.

Improved PCR based methods for detecting C9orf72 hexanucleotide repeat expansions.

Due to the GC-rich, repetitive nature of C9orf72 hexanucleotide repeat expansions, PCR based detection methods are challenging. Several limitations of PCR have been reported and overcoming these could help to define the pathogenic range. There is also a need to develop improved repeat-primed PCR assays which allow detection even in the presence of genomic variation around the repeat region. We have optimised PCR conditions for the C9orf72 hexanucleotide repeat expansion, using betaine as a co-solvent and specific cycling conditions, including slow ramping and a high denaturation temperature. We have developed a flanking assay, and repeat-primed PCR assays for both 3' and 5' ends of the repeat expansion, which when used together provide a robust strategy for detecting the presence or absence of expansions greater than ∼100 repeats, even in the presence of genomic variability at the 3' end of the repeat. Using our assays, we have detected repeat expansions in 47/442 Scottish ALS patients. Furthermore, we recommend the combined use of these assays in a clinical diagnostic setting.