Structural and non-coding variants increase the diagnostic yield of clinical whole genome sequencing for rare diseases.

BACKGROUND: Whole genome sequencing is increasingly being used for the diagnosis of patients with rare diseases. However, the diagnostic yields of many studies, particularly those conducted in a healthcare setting, are often disappointingly low, at 25-30%. This is in part because although entire genomes are sequenced, analysis is often confined to in silico gene panels or coding regions of the genome. METHODS: We undertook WGS on a cohort of 122 unrelated rare disease patients and their relatives (300 genomes) who had been pre-screened by gene panels or arrays. Patients were recruited from a broad spectrum of clinical specialties. We applied a bioinformatics pipeline that would allow comprehensive analysis of all variant types. We combined established bioinformatics tools for phenotypic and genomic analysis with our novel algorithms (SVRare, ALTSPLICE and GREEN-DB) to detect and annotate structural, splice site and non-coding variants. RESULTS: Our diagnostic yield was 43/122 cases (35%), although 47/122 cases (39%) were considered solved when considering novel candidate genes with supporting functional data into account. Structural, splice site and deep intronic variants contributed to 20/47 (43%) of our solved cases. Five genes that are novel, or were novel at the time of discovery, were identified, whilst a further three genes are putative novel disease genes with evidence of causality. We identified variants of uncertain significance in a further fourteen candidate genes. The phenotypic spectrum associated with RMND1 was expanded to include polymicrogyria. Two patients with secondary findings in FBN1 and KCNQ1 were confirmed to have previously unidentified Marfan and long QT syndromes, respectively, and were referred for further clinical interventions. Clinical diagnoses were changed in six patients and treatment adjustments made for eight individuals, which for five patients was considered life-saving. CONCLUSIONS: Genome sequencing is increasingly being considered as a first-line genetic test in routine clinical settings and can make a substantial contribution to rapidly identifying a causal aetiology for many patients, shortening their diagnostic odyssey. We have demonstrated that structural, splice site and intronic variants make a significant contribution to diagnostic yield and that comprehensive analysis of the entire genome is essential to maximise the value of clinical genome sequencing.

Prolidase Deficiency Causes Spontaneous T Cell Activation and Lupus-like Autoimmunity.

Prolidase deficiency (PD) is a multisystem disorder caused by mutations in the PEPD gene, which encodes a ubiquitously expressed metallopeptidase essential for the hydrolysis of dipeptides containing C-terminal proline or hydroxyproline. PD typically presents in childhood with developmental delay, skin ulcers, recurrent infections, and, in some patients, autoimmune features that can mimic systemic lupus erythematosus. The basis for the autoimmune association is uncertain, but might be due to self-antigen exposure with tissue damage, or indirectly driven by chronic infection and microbial burden. In this study, we address the question of causation and show that Pepd-null mice have increased antinuclear autoantibodies and raised serum IgA, accompanied by kidney immune complex deposition, consistent with a systemic lupus erythematosus-like disease. These features are associated with an accumulation of CD4 and CD8 effector T cells in the spleen and liver. Pepd deficiency leads to spontaneous T cell activation and proliferation into the effector subset, which is cell intrinsic and independent of Ag receptor specificity or antigenic stimulation. However, an increase in KLRG1+ effector CD8 cells is not observed in mixed chimeras, in which the autoimmune phenotype is also absent. Our findings link autoimmune susceptibility in PD to spontaneous T cell dysfunction, likely to be acting in combination with immune activators that lie outside the hemopoietic system but result from the abnormal metabolism or loss of nonenzymatic prolidase function. This knowledge provides insight into the role of prolidase in the maintenance of self-tolerance and highlights the importance of treatment to control T cell activation.

NDRG1 is induced by antigen-receptor signaling but dispensable for B and T cell self-tolerance.

Peripheral tolerance prevents the initiation of damaging immune responses by autoreactive lymphocytes. While tolerogenic mechanisms are tightly regulated by antigen-dependent and independent signals, downstream pathways are incompletely understood. N-myc downstream-regulated gene 1 (NDRG1), an anti-cancer therapeutic target, has previously been implicated as a CD4+ T cell clonal anergy factor. By RNA-sequencing, we identified Ndrg1 as the third most upregulated gene in anergic, compared to naïve follicular, B cells. Ndrg1 is upregulated by B cell receptor activation (signal one) and suppressed by co-stimulation (signal two), suggesting that NDRG1 may be important in B cell tolerance. However, though Ndrg1-/- mice have a neurological defect mimicking NDRG1-associated Charcot-Marie-Tooth (CMT4d) disease, primary and secondary immune responses were normal. We find that B cell tolerance is maintained, and NDRG1 does not play a role in downstream responses during re-stimulation of in vivo antigen-experienced CD4+ T cells, demonstrating that NDGR1 is functionally redundant for lymphocyte anergy.

Determining the Relationship Between Blood Pressure, Kidney Function, and Chronic Kidney Disease: Insights From Genetic Epidemiology.

BACKGROUND: It is well established that decreased kidney function can increase blood pressure (BP), but it is unproven whether moderately elevated BP causes chronic kidney disease (CKD) or glomerular hyperfiltration. METHODS: 311 119 White British UK Biobank participants were included in logistic regression analyses to estimate the odds of CKD (defined as long-term kidney replacement therapy, estimated glomerular filtration rate [eGFR]< 60mL/min/1.73m2, or urinary albumin:creatinine ratio ≥3 mg/mmol) associated with higher genetically predicted BP using genetic risk scores comprising 219 systolic and 223 diastolic BP loci. Analyses estimating associations with clinical categories of eGFR and urinary albumin:creatinine ratio were also conducted, with an eGFR ≥120 mL (min·1.73m2) considered evidence of glomerular hyperfiltration. RESULTS: 21 623 participants had CKD: 7781 with reduced eGFR and 15 500 with albuminuria. 1828 participants had an eGFR ≥120 mL/min/1.73m2. Each genetically predicted 10 mmHg higher systolic BP and 5 mmHg higher diastolic BP were associated with a 37% (95% CI, 1.29-1.45) and 19% (1.14-1.25) higher odds of CKD, respectively. Associations were evident for both the reduced eGFR and albuminuria components of the CKD outcome. The odds of hyperfiltration (versus an eGFR ≥60 and <90 mL/min/1.73m2 were 49% higher (95% CI, 1.21-1.84) for each genetically predicted 10 mmHg higher systolic BP. Associations with CKD and hyperfiltration were similar irrespective of preexisting diabetes, vascular disease, or different levels of adiposity. CONCLUSIONS: In this general population, genetic epidemiological evidence supports a causal role of life-long differences in BP for decreased kidney function, glomerular hyperfiltration, and albuminuria. Physiological autoregulation may not afford complete renal protection against the moderate BP elevations.

An ontogenetic switch drives the positive and negative selection of B cells.

Developing B cells can be positively or negatively selected by self-antigens, but the mechanisms that determine these outcomes are incompletely understood. Here, we show that a B cell intrinsic switch between positive and negative selection during ontogeny is determined by a change from Lin28b to let-7 gene expression. Ectopic expression of a Lin28b transgene in murine B cells restored the positive selection of autoreactive B-1 B cells by self-antigen in adult bone marrow. Analysis of antigen-specific immature B cells in early and late ontogeny identified Lin28b-dependent genes associated with B-1 B cell development, including Arid3a and Bhleh41, and Lin28b-independent effects are associated with the presence or absence of self-antigen. These findings identify cell intrinsic and extrinsic determinants of B cell fate during ontogeny and reconcile lineage and selection theories of B cell development. They explain how changes in the balance of positive and negative selection may be able to adapt to meet the immunological needs of an individual during its lifetime.

High-throughput phenotyping reveals expansive genetic and structural underpinnings of immune variation.

By developing a high-density murine immunophenotyping platform compatible with high-throughput genetic screening, we have established profound contributions of genetics and structure to immune variation (http://www.immunophenotype.org). Specifically, high-throughput phenotyping of 530 unique mouse gene knockouts identified 140 monogenic 'hits', of which most had no previous immunologic association. Furthermore, hits were collectively enriched in genes for which humans show poor tolerance to loss of function. The immunophenotyping platform also exposed dense correlation networks linking immune parameters with each other and with specific physiologic traits. Such linkages limit freedom of movement for individual immune parameters, thereby imposing genetically regulated 'immunologic structures', the integrity of which was associated with immunocompetence. Hence, we provide an expanded genetic resource and structural perspective for understanding and monitoring immune variation in health and disease.

An essential role for the Zn2+ transporter ZIP7 in B cell development.

Despite the known importance of zinc for human immunity, molecular insights into its roles have remained limited. Here we report a novel autosomal recessive disease characterized by absent B cells, agammaglobulinemia and early onset infections in five unrelated families. The immunodeficiency results from hypomorphic mutations of SLC39A7, which encodes the endoplasmic reticulum-to-cytoplasm zinc transporter ZIP7. Using CRISPR-Cas9 mutagenesis we have precisely modeled ZIP7 deficiency in mice. Homozygosity for a null allele caused embryonic death, but hypomorphic alleles reproduced the block in B cell development seen in patients. B cells from mutant mice exhibited a diminished concentration of cytoplasmic free zinc, increased phosphatase activity and decreased phosphorylation of signaling molecules downstream of the pre-B cell and B cell receptors. Our findings highlight a specific role for cytosolic Zn2+ in modulating B cell receptor signal strength and positive selection.

Interventions for chronic kidney disease in people with sickle cell disease.

BACKGROUND: Sickle cell disease (SCD) is one of the commonest severe monogenic disorders in the world, due to the inheritance of two abnormal haemoglobin (beta-globin) genes. SCD can cause severe pain, significant end-organ damage, pulmonary complications, and premature death. Kidney disease is a frequent and potentially severe complication in people with SCD.Chronic kidney disease is defined as abnormalities of kidney structure or function, present for more than three months. Sickle cell nephropathy refers to the spectrum of kidney complications in SCD.Glomerular damage is a cause of microalbuminuria and can develop at an early age in children with SCD, and increases in prevalence in adulthood. In people with sickle cell nephropathy, outcomes are poor as a result of the progression to proteinuria and chronic kidney insufficiency. Up to 12% of people who develop sickle cell nephropathy will develop end-stage renal disease. OBJECTIVES: To assess the effectiveness of any intervention in preventing or reducing kidney complications or chronic kidney disease in people with SCD (including red blood cell transfusions, hydroxyurea and angiotensin-converting enzyme inhibitor (ACEI)), either alone or in combination with each other. SEARCH METHODS: We searched for relevant trials in the Cochrane Library, MEDLINE (from 1946), Embase (from 1974), the Transfusion Evidence Library (from 1980), and ongoing trial databases; all searches current to 05 April 2016. We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group Trials Register: 13 April 2017. SELECTION CRITERIA: Randomised controlled trials comparing interventions to prevent or reduce kidney complications or chronic kidney disease in people with SCD. There were no restrictions by outcomes examined, language or publication status. DATA COLLECTION AND ANALYSIS: Two authors independently assessed trial eligibility, extracted data and assessed the risk of bias. MAIN RESULTS: We included two trials with 215 participants. One trial was published in 2011 and included 193 children aged 9 months to 18 months, and compared treatment with hydroxyurea to placebo. The second trial was published in 1998 and included 22 adults with normal blood pressure and microalbuminuria and compared ACEI to placebo.We rated the quality of evidence as low to very low across different outcomes according to GRADE methodology. This was due to trials having: a high or unclear risk of bias including attrition and detection bias; indirectness (the available evidence was for children aged 9 months to 18 months in one trial and a small and select adult sample size in a second trial); and imprecise outcome effect estimates of significant benefit or harm. Hydroxyurea versus placebo We are very uncertain if hydroxyurea reduces or prevents progression of kidney disease (assessed by change in glomerular filtration rate), or reduces hyperfiltration in children aged 9 to 18 months, mean difference (MD) 0.58 (95% confidence interval (CI) -14.60 to 15.76 (mL/min per 1.73 m²)) (one study; 142 participants; very low-quality evidence).In children aged 9 to 18 months, hydroxyurea may improve the ability to concentrate urine, MD 42.23 (95% CI 12.14 to 72.32 (mOsm/kg)) (one study; 178 participants; low-quality evidence).Hydroxyurea may make little or no difference to SCD-related serious adverse events including: incidence of acute chest syndrome, risk ratio (RR) 0.39 (99% CI 0.13 to 1.16); painful crisis, RR 0.68 (99% CI 0.45 to 1.02); and hospitalisations, RR 0.83 (99% CI 0.68 to 1.01) (one study, 193 participants; low-quality evidence).No deaths occurred in the trial. Quality of life was not reported. ACEI versus placeboWe are very uncertain if ACEI reduces proteinuria in adults with SCD who have normal blood pressure and microalbuminuria, MD -49.00 (95% CI -124.10 to 26.10 (mg per day)) (one study; 22 participants; very low-quality evidence). We are very uncertain if ACEI reduce or prevent kidney disease as measured by creatinine clearance. The authors state that creatinine clearance remained constant over six months in both groups, but no comparative data were provided (very low-quality evidence).All-cause mortality, serious adverse events and quality of life were not reported. AUTHORS' CONCLUSIONS: In young children aged 9 months to 18 months, we are very uncertain if hydroxyurea improves glomerular filtration rate or reduces hyperfiltration, but it may improve young children's ability to concentrate urine and may make little or no difference on the incidence of acute chest syndrome, painful crises and hospitalisations.We are very uncertain if giving ACEI to adults with normal blood pressure and microalbuminuria has any effect on preventing or reducing kidney complications.This review identified no trials that looked at red cell transfusions nor any combinations of interventions to prevent or reduce kidney complications.Due to lack of evidence this review cannot comment on the management of either children aged over 18 months or adults with any known genotype of SCD.We have identified a lack of adequately-designed and powered studies, and no ongoing trials which address this critical question. Trials of hydroxyurea, ACEI or red blood cell transfusion in older children and adults are urgently needed to determine any effect on prevention or reduction kidney complications in people with SCD.

Themis2 lowers the threshold for B cell activation during positive selection.

The positive and negative selection of lymphocytes by antigen is central to adaptive immunity and self-tolerance, yet how this is determined by different antigens is not completely understood. We found that thymocyte-selection-associated family member 2 (Themis2) increased the positive selection of B1 cells and germinal center B cells by self and foreign antigens. Themis2 lowered the threshold for B-cell activation by low-avidity, but not high-avidity, antigens. Themis2 constitutively bound the adaptor protein Grb2, src-kinase Lyn and signal transducer phospholipase γ2 (PLC-γ2), and increased activation of PLC-γ2 and its downstream pathways following B cell receptor stimulation. Our findings identify a unique function for Themis2 in differential signaling and provide insight into how B cells discriminate between antigens of different quantity and quality.

VEGF regulates local inhibitory complement proteins in the eye and kidney.

Outer retinal and renal glomerular functions rely on specialized vasculature maintained by VEGF that is produced by neighboring epithelial cells, the retinal pigment epithelium (RPE) and podocytes, respectively. Dysregulation of RPE- and podocyte-derived VEGF is associated with neovascularization in wet age-related macular degeneration (ARMD), choriocapillaris degeneration, and glomerular thrombotic microangiopathy (TMA). Since complement activation and genetic variants in inhibitory complement factor H (CFH) are also features of both ARMD and TMA, we hypothesized that VEGF and CFH interact. Here, we demonstrated that VEGF inhibition decreases local CFH and other complement regulators in the eye and kidney through reduced VEGFR2/PKC-α/CREB signaling. Patient podocytes and RPE cells carrying disease-associated CFH genetic variants had more alternative complement pathway deposits than controls. These deposits were increased by VEGF antagonism, a common wet ARMD treatment, suggesting that VEGF inhibition could reduce cellular complement regulatory capacity. VEGF antagonism also increased markers of endothelial cell activation, which was partially reduced by genetic complement inhibition. Together, these results suggest that VEGF protects the retinal and glomerular microvasculature, not only through VEGFR2-mediated vasculotrophism, but also through modulation of local complement proteins that could protect against complement-mediated damage. Though further study is warranted, these findings could be relevant for patients receiving VEGF antagonists.

Mutation of Fnip1 is associated with B-cell deficiency, cardiomyopathy, and elevated AMPK activity.

Folliculin (FLCN) is a tumor-suppressor protein mutated in the Birt-Hogg-Dubé (BHD) syndrome, which associates with two paralogous proteins, folliculin-interacting protein (FNIP)1 and FNIP2, forming a complex that interacts with the AMP-activated protein kinase (AMPK). Although it is clear that this complex influences AMPK and other metabolic regulators, reports of its effects have been inconsistent. To address this issue, we created a recessive loss-of-function variant of Fnip1 Homozygous FNIP1 deficiency resulted in profound B-cell deficiency, partially restored by overexpression of the antiapoptotic protein BCL2, whereas heterozygous deficiency caused a loss of marginal zone B cells. FNIP1-deficient mice developed cardiomyopathy characterized by left ventricular hypertrophy and glycogen accumulation, with close parallels to mice and humans bearing gain-of-function mutations in the γ2 subunit of AMPK. Concordantly, γ2-specific AMPK activity was elevated in neonatal FNIP1-deficient myocardium, whereas AMPK-dependent unc-51-like autophagy activating kinase 1 (ULK1) phosphorylation and autophagy were increased in FNIP1-deficient B-cell progenitors. These data support a role for FNIP1 as a negative regulator of AMPK.

Interventions for chronic kidney disease in people with sickle cell disease.

This is the protocol for a review and there is no abstract. The objectives are as follows: To assess the effectiveness of any intervention in preventing or reducing kidney complications or CKD in people with SCD (including red blood cell transfusions, hydroxyurea and ACEI (either alone or in combination with each other)).

Current strategies for mutation detection in phenotype-driven screens utilising next generation sequencing.

Mutagenesis-based screens in mice are a powerful discovery platform to identify novel genes or gene functions associated with disease phenotypes. An N-ethyl-N-nitrosourea (ENU) mutagenesis screen induces single nucleotide variants randomly in the mouse genome. Subsequent phenotyping of mutant and wildtype mice enables the identification of mutated pathways resulting in phenotypes associated with a particular ENU lesion. This unbiased approach to gene discovery conducts the phenotyping with no prior knowledge of the functional mutations. Before the advent of affordable next generation sequencing (NGS), ENU variant identification was a limiting step in gene characterization, akin to 'finding a needle in a haystack'. The emergence of a reliable reference genome alongside advances in NGS has propelled ENU mutation discovery from an arduous, time-consuming exercise to an effective and rapid form of mutation discovery. This has permitted large mouse facilities worldwide to use ENU for novel mutation discovery in a high-throughput manner, helping to accelerate basic science at the mechanistic level. Here, we describe three different strategies used to identify ENU variants from NGS data and some of the subsequent steps for mutation characterisation.

Next-generation sequencing to dissect hereditary nephrotic syndrome in mice identifies a hypomorphic mutation in Lamb2 and models Pierson's syndrome.

The study of mutations causing the steroid-resistant nephrotic syndrome in children has greatly advanced our understanding of the kidney filtration barrier. In particular, these genetic variants have illuminated the roles of the podocyte, glomerular basement membrane and endothelial cell in glomerular filtration. However, in a significant number of familial and early onset cases, an underlying mutation cannot be identified, indicating that there are likely to be multiple unknown genes with roles in glomerular permeability. We now show how the combination of N-ethyl-N-nitrosourea mutagenesis and next-generation sequencing could be used to identify the range of mutations affecting these pathways. Using this approach, we isolated a novel mouse strain with a viable nephrotic phenotype and used whole-genome sequencing to isolate a causative hypomorphic mutation in Lamb2. This discovery generated a model for one part of the spectrum of human Pierson's syndrome and provides a powerful proof of principle for accelerating gene discovery and improving our understanding of inherited forms of renal disease.

Unlocking the bottleneck in forward genetics using whole-genome sequencing and identity by descent to isolate causative mutations.

Forward genetics screens with N-ethyl-N-nitrosourea (ENU) provide a powerful way to illuminate gene function and generate mouse models of human disease; however, the identification of causative mutations remains a limiting step. Current strategies depend on conventional mapping, so the propagation of affected mice requires non-lethal screens; accurate tracking of phenotypes through pedigrees is complex and uncertain; out-crossing can introduce unexpected modifiers; and Sanger sequencing of candidate genes is inefficient. Here we show how these problems can be efficiently overcome using whole-genome sequencing (WGS) to detect the ENU mutations and then identify regions that are identical by descent (IBD) in multiple affected mice. In this strategy, we use a modification of the Lander-Green algorithm to isolate causative recessive and dominant mutations, even at low coverage, on a pure strain background. Analysis of the IBD regions also allows us to calculate the ENU mutation rate (1.54 mutations per Mb) and to model future strategies for genetic screens in mice. The introduction of this approach will accelerate the discovery of causal variants, permit broader and more informative lethal screens to be used, reduce animal costs, and herald a new era for ENU mutagenesis.

B cell survival, surface BCR and BAFFR expression, CD74 metabolism, and CD8- dendritic cells require the intramembrane endopeptidase SPPL2A.

Druggable proteins required for B lymphocyte survival and immune responses are an emerging source of new treatments for autoimmunity and lymphoid malignancy. In this study, we show that mice with an inactivating mutation in the intramembrane protease signal peptide peptidase-like 2A (SPPL2A) unexpectedly exhibit profound humoral immunodeficiency and lack mature B cell subsets, mirroring deficiency of the cytokine B cell-activating factor (BAFF). Accumulation of Sppl2a-deficient B cells was rescued by overexpression of the BAFF-induced survival protein B cell lymphoma 2 (BCL2) but not BAFF and was distinguished by low surface BAFF receptor and IgM and IgD B cell receptors. CD8-negative dendritic cells were also greatly decreased. SPPL2A deficiency blocked the proteolytic processing of CD74 MHC II invariant chain in both cell types, causing dramatic build-up of the p8 product of Cathepsin S and interfering with earlier steps in CD74 endosomal retention and processing. The findings illuminate an important role for the final step in the CD74-MHC II pathway and a new target for protease inhibitor treatment of B cell diseases.