Detection and characterization of male sex chromosome abnormalities in the UK Biobank study.

PURPOSE: The study aimed to systematically ascertain male sex chromosome abnormalities, 47,XXY (Klinefelter syndrome [KS]) and 47,XYY, and characterize their risks of adverse health outcomes. METHODS: We analyzed genotyping array or exome sequence data in 207,067 men of European ancestry aged 40 to 70 years from the UK Biobank and related these to extensive routine health record data. RESULTS: Only 49 of 213 (23%) of men whom we identified with KS and only 1 of 143 (0.7%) with 47,XYY had a diagnosis of abnormal karyotype on their medical records or self-report. We observed expected associations for KS with reproductive dysfunction (late puberty: risk ratio [RR] = 2.7; childlessness: RR = 4.2; testosterone concentration: RR = -3.8 nmol/L, all P < 2 × 10-8), whereas XYY men appeared to have normal reproductive function. Despite this difference, we identified several higher disease risks shared across both KS and 47,XYY, including type 2 diabetes (RR = 3.0 and 2.6, respectively), venous thrombosis (RR = 6.4 and 7.4, respectively), pulmonary embolism (RR = 3.3 and 3.7, respectively), and chronic obstructive pulmonary disease (RR = 4.4 and 4.6, respectively) (all P < 7 × 10-6). CONCLUSION: KS and 47,XYY were mostly unrecognized but conferred substantially higher risks for metabolic, vascular, and respiratory diseases, which were only partially explained by higher levels of body mass index, deprivation, and smoking.

GIGYF1 loss of function is associated with clonal mosaicism and adverse metabolic health.

Mosaic loss of chromosome Y (LOY) in leukocytes is the most common form of clonal mosaicism, caused by dysregulation in cell-cycle and DNA damage response pathways. Previous genetic studies have focussed on identifying common variants associated with LOY, which we now extend to rarer, protein-coding variation using exome sequences from 82,277 male UK Biobank participants. We find that loss of function of two genes-CHEK2 and GIGYF1-reach exome-wide significance. Rare alleles in GIGYF1 have not previously been implicated in any complex trait, but here loss-of-function carriers exhibit six-fold higher susceptibility to LOY (OR = 5.99 [3.04-11.81], p = 1.3 × 10-10). These same alleles are also associated with adverse metabolic health, including higher susceptibility to Type 2 Diabetes (OR = 6.10 [3.51-10.61], p = 1.8 × 10-12), 4 kg higher fat mass (p = 1.3 × 10-4), 2.32 nmol/L lower serum IGF1 levels (p = 1.5 × 10-4) and 4.5 kg lower handgrip strength (p = 4.7 × 10-7) consistent with proposed GIGYF1 enhancement of insulin and IGF-1 receptor signalling. These associations are mirrored by a common variant nearby associated with the expression of GIGYF1. Our observations highlight a potential direct connection between clonal mosaicism and metabolic health.

Differential IRF8 Transcription Factor Requirement Defines Two Pathways of Dendritic Cell Development in Humans.

The formation of mammalian dendritic cells (DCs) is controlled by multiple hematopoietic transcription factors, including IRF8. Loss of IRF8 exerts a differential effect on DC subsets, including plasmacytoid DCs (pDCs) and the classical DC lineages cDC1 and cDC2. In humans, cDC2-related subsets have been described including AXL+SIGLEC6+ pre-DC, DC2 and DC3. The origin of this heterogeneity is unknown. Using high-dimensional analysis, in vitro differentiation, and an allelic series of human IRF8 deficiency, we demonstrated that cDC2 (CD1c+DC) heterogeneity originates from two distinct pathways of development. The lymphoid-primed IRF8hi pathway, marked by CD123 and BTLA, carried pDC, cDC1, and DC2 trajectories, while the common myeloid IRF8lo pathway, expressing SIRPA, formed DC3s and monocytes. We traced distinct trajectories through the granulocyte-macrophage progenitor (GMP) compartment showing that AXL+SIGLEC6+ pre-DCs mapped exclusively to the DC2 pathway. In keeping with their lower requirement for IRF8, DC3s expand to replace DC2s in human partial IRF8 deficiency.

Publisher Correction: Whole-genome sequencing of a sporadic primary immunodeficiency cohort.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Whole-genome sequencing of a sporadic primary immunodeficiency cohort.

Primary immunodeficiency (PID) is characterized by recurrent and often life-threatening infections, autoimmunity and cancer, and it poses major diagnostic and therapeutic challenges. Although the most severe forms of PID are identified in early childhood, most patients present in adulthood, typically with no apparent family history and a variable clinical phenotype of widespread immune dysregulation: about 25% of patients have autoimmune disease, allergy is prevalent and up to 10% develop lymphoid malignancies1-3. Consequently, in sporadic (or non-familial) PID genetic diagnosis is difficult and the role of genetics is not well defined. Here we address these challenges by performing whole-genome sequencing in a large PID cohort of 1,318 participants. An analysis of the coding regions of the genome in 886 index cases of PID found that disease-causing mutations in known genes that are implicated in monogenic PID occurred in 10.3% of these patients, and a Bayesian approach (BeviMed4) identified multiple new candidate PID-associated genes, including IVNS1ABP. We also examined the noncoding genome, and found deletions in regulatory regions that contribute to disease causation. In addition, we used a genome-wide association study to identify loci that are associated with PID, and found evidence for the colocalization of-and interplay between-novel high-penetrance monogenic variants and common variants (at the PTPN2 and SOCS1 loci). This begins to explain the contribution of common variants to the variable penetrance and phenotypic complexity that are observed in PID. Thus, using a cohort-based whole-genome-sequencing approach in the diagnosis of PID can increase diagnostic yield and further our understanding of the key pathways that influence immune responsiveness in humans.

Thousands of missing variants in the UK Biobank are recoverable by genome realignment.

The UK Biobank is an unprecedented resource for human disease research. In March 2019, 49,997 exomes were made publicly available to investigators. Here we note that thousands of variant calls are unexpectedly absent from this dataset, with 641 genes showing zero variation. We show that the reason for this was an erroneous read alignment to the GRCh38 reference. The missing variants can be recovered by modifying read alignment parameters to correctly handle the expanded set of contigs available in the human genome reference. Given the size and complexity of such population scale datasets, we propose a simple heuristic that can uncover systematic errors using summary data accessible to most investigators.

Predicting the Occurrence of Variants in RAG1 and RAG2.

While widespread genome sequencing ushers in a new era of preventive medicine, the tools for predictive genomics are still lacking. Time and resource limitations mean that human diseases remain uncharacterized because of an inability to predict clinically relevant genetic variants. A strategy of targeting highly conserved protein regions is used commonly in functional studies. However, this benefit is lost for rare diseases where the attributable genes are mostly conserved. An immunological disorder exemplifying this challenge occurs through damaging mutations in RAG1 and RAG2 which presents at an early age with a distinct phenotype of life-threatening immunodeficiency or autoimmunity. Many tools exist for variant pathogenicity prediction, but these cannot account for the probability of variant occurrence. Here, we present a method that predicts the likelihood of mutation for every amino acid residue in the RAG1 and RAG2 proteins. Population genetics data from approximately 146,000 individuals was used for rare variant analysis. Forty-four known pathogenic variants reported in patients and recombination activity measurements from 110 RAG1/2 mutants were used to validate calculated scores. Probabilities were compared with 98 currently known human cases of disease. A genome sequence dataset of 558 patients who have primary immunodeficiency but that are negative for RAG deficiency were also used as validation controls. We compared the difference between mutation likelihood and pathogenicity prediction. Our method builds a map of most probable mutations allowing pre-emptive functional analysis. This method may be applied to other diseases with hopes of improving preparedness for clinical diagnosis.

Loss of the interleukin-6 receptor causes immunodeficiency, atopy, and abnormal inflammatory responses.

IL-6 excess is central to the pathogenesis of multiple inflammatory conditions and is targeted in clinical practice by immunotherapy that blocks the IL-6 receptor encoded by IL6R We describe two patients with homozygous mutations in IL6R who presented with recurrent infections, abnormal acute-phase responses, elevated IgE, eczema, and eosinophilia. This study identifies a novel primary immunodeficiency, clarifying the contribution of IL-6 to the phenotype of patients with mutations in IL6ST, STAT3, and ZNF341, genes encoding different components of the IL-6 signaling pathway, and alerts us to the potential toxicity of drugs targeting the IL-6R.

Pathogenic NFKB2 variant in the ankyrin repeat domain (R635X) causes a variable antibody deficiency.

Genetic studies are identifying an increasing number of monogenic causes of Common Variable Immunodeficiency (CVID). Pathogenic variants in the C-terminus of NFKB2 have been identified in the subset of CVID patients whose immunodeficiency is associated with ectodermal dysplasia and central adrenal insufficiency. We describe 2 unrelated CVID pedigrees with 4 cases of pathogenic stop gain variants (c.1903C > T) in the ankyrin repeat domain (ARD) of NF-κB2, leading to a premature truncation of the protein at p.Arg635Term (R635X). By immunophenotyping and functional ex vivo B- and T-cell experiments we characterized the variant by reduced class-switched memory B-cell counts and immature plasmablasts, unable to produce IgG and IgA. Features of a poor proliferative T-cell response and reduced expansion of CD4+CXCR5+ T cells was only observed in the two clinically affected index cases without any clear clinical correlate. In conclusion, pathogenic stop variants in the ARD of NFKB2 can cause 'infection-only' CVID with an abnormal B-cell phenotype and a variable clinical penetrance.

A Specific CNOT1 Mutation Results in a Novel Syndrome of Pancreatic Agenesis and Holoprosencephaly through Impaired Pancreatic and Neurological Development.

We report a recurrent CNOT1 de novo missense mutation, GenBank: NM_016284.4; c.1603C>T (p.Arg535Cys), resulting in a syndrome of pancreatic agenesis and abnormal forebrain development in three individuals and a similar phenotype in mice. CNOT1 is a transcriptional repressor that has been suggested as being critical for maintaining embryonic stem cells in a pluripotent state. These findings suggest that CNOT1 plays a critical role in pancreatic and neurological development and describe a novel genetic syndrome of pancreatic agenesis and holoprosencephaly.

Copy number variation of LINGO1 in familial dystonic tremor.

OBJECTIVE: To elucidate the genetic cause of a large 5 generation South Indian family with multiple individuals with predominantly an upper limb postural tremor and posturing in keeping with another form of tremor, namely, dystonic tremor. METHODS: Whole-genome single nucleotide polymorphism (SNP) microarray analysis was undertaken to look for copy number variants in the affected individuals. RESULTS: Whole-genome SNP microarray studies identified a tandem duplicated genomic segment of chromosome 15q24 present in all affected family members. Whole-genome sequencing demonstrated that it comprised a ∼550-kb tandem duplication encompassing the entire LINGO1 gene. CONCLUSIONS: The identification of a genomic duplication as the likely molecular cause of this condition, resulting in an additional LINGO1 gene copy in affected cases, adds further support for a causal role of this gene in tremor disorders and implicates increased expression levels of LINGO1 as a potential pathogenic mechanism.

Loss-of-function nuclear factor κB subunit 1 (NFKB1) variants are the most common monogenic cause of common variable immunodeficiency in Europeans.

BACKGROUND: The genetic cause of primary immunodeficiency disease (PID) carries prognostic information. OBJECTIVE: We conducted a whole-genome sequencing study assessing a large proportion of the NIHR BioResource-Rare Diseases cohort. METHODS: In the predominantly European study population of principally sporadic unrelated PID cases (n = 846), a novel Bayesian method identified nuclear factor κB subunit 1 (NFKB1) as one of the genes most strongly associated with PID, and the association was explained by 16 novel heterozygous truncating, missense, and gene deletion variants. This accounted for 4% of common variable immunodeficiency (CVID) cases (n = 390) in the cohort. Amino acid substitutions predicted to be pathogenic were assessed by means of analysis of structural protein data. Immunophenotyping, immunoblotting, and ex vivo stimulation of lymphocytes determined the functional effects of these variants. Detailed clinical and pedigree information was collected for genotype-phenotype cosegregation analyses. RESULTS: Both sporadic and familial cases demonstrated evidence of the noninfective complications of CVID, including massive lymphadenopathy (24%), unexplained splenomegaly (48%), and autoimmune disease (48%), features prior studies correlated with worse clinical prognosis. Although partial penetrance of clinical symptoms was noted in certain pedigrees, all carriers have a deficiency in B-lymphocyte differentiation. Detailed assessment of B-lymphocyte numbers, phenotype, and function identifies the presence of an increased CD21low B-cell population. Combined with identification of the disease-causing variant, this distinguishes between healthy subjects, asymptomatic carriers, and clinically affected cases. CONCLUSION: We show that heterozygous loss-of-function variants in NFKB1 are the most common known monogenic cause of CVID, which results in a temporally progressive defect in the formation of immunoglobulin-producing B cells.

Diagnosis of lethal or prenatal-onset autosomal recessive disorders by parental exome sequencing.

OBJECTIVE: Rare genetic disorders resulting in prenatal or neonatal death are genetically heterogeneous, but testing is often limited by the availability of fetal DNA, leaving couples without a potential prenatal test for future pregnancies. We describe our novel strategy of exome sequencing parental DNA samples to diagnose recessive monogenic disorders in an audit of the first 50 couples referred. METHOD: Exome sequencing was carried out in a consecutive series of 50 couples who had 1 or more pregnancies affected with a lethal or prenatal-onset disorder. In all cases, there was insufficient DNA for exome sequencing of the affected fetus. Heterozygous rare variants (MAF < 0.001) in the same gene in both parents were selected for analysis. Likely, disease-causing variants were tested in fetal DNA to confirm co-segregation. RESULTS: Parental exome analysis identified heterozygous pathogenic (or likely pathogenic) variants in 24 different genes in 26/50 couples (52%). Where 2 or more fetuses were affected, a genetic diagnosis was obtained in 18/29 cases (62%). In most cases, the clinical features were typical of the disorder, but in others, they result from a hypomorphic variant or represent the most severe form of a variable phenotypic spectrum. CONCLUSION: We conclude that exome sequencing of parental samples is a powerful strategy with high clinical utility for the genetic diagnosis of lethal or prenatal-onset recessive disorders. © 2017 The Authors Prenatal Diagnosis published by John Wiley & Sons Ltd.

Recessively Inherited LRBA Mutations Cause Autoimmunity Presenting as Neonatal Diabetes.

Young-onset autoimmune diabetes associated with additional autoimmunity usually reflects a polygenic predisposition, but rare cases result from monogenic autoimmunity. Diagnosing monogenic autoimmunity is crucial for patients' prognosis and clinical management. We sought to identify novel genetic causes of autoimmunity presenting with neonatal diabetes (NDM) (diagnosis <6 months). We performed exome sequencing in a patient with NDM and autoimmune lymphoproliferative syndrome and his unrelated, unaffected parents and identified compound heterozygous null mutations in LRBA Biallelic LRBA mutations cause common variable immunodeficiency-8; however, NDM has not been confirmed in this disorder. We sequenced LRBA in 169 additional patients with diabetes diagnosed <1 year without mutations in the 24 known NDM genes. We identified recessive null mutations in 8 additional probands, of which, 3 had NDM (<6 months). Diabetes was the presenting feature in 6 of 9 probands. Six of 17 (35%) patients born to consanguineous parents and with additional early-onset autoimmunity had recessive LRBA mutations. LRBA testing should be considered in patients with diabetes diagnosed <12 months, particularly if they have additional autoimmunity or are born to consanguineous parents. A genetic diagnosis is important as it can enable personalized therapy with abatacept, a CTLA-4 mimetic, and inform genetic counseling.

Polycystic Kidney Disease with Hyperinsulinemic Hypoglycemia Caused by a Promoter Mutation in Phosphomannomutase 2.

Hyperinsulinemic hypoglycemia (HI) and congenital polycystic kidney disease (PKD) are rare, genetically heterogeneous disorders. The co-occurrence of these disorders (HIPKD) in 17 children from 11 unrelated families suggested an unrecognized genetic disorder. Whole-genome linkage analysis in five informative families identified a single significant locus on chromosome 16p13.2 (logarithm of odds score 6.5). Sequencing of the coding regions of all linked genes failed to identify biallelic mutations. Instead, we found in all patients a promoter mutation (c.-167G>T) in the phosphomannomutase 2 gene (PMM2), either homozygous or in trans with PMM2 coding mutations. PMM2 encodes a key enzyme in N-glycosylation. Abnormal glycosylation has been associated with PKD, and we found that deglycosylation in cultured pancreatic ß cells altered insulin secretion. Recessive coding mutations in PMM2 cause congenital disorder of glycosylation type 1a (CDG1A), a devastating multisystem disorder with prominent neurologic involvement. Yet our patients did not exhibit the typical clinical or diagnostic features of CDG1A. In vitro, the PMM2 promoter mutation associated with decreased transcriptional activity in patient kidney cells and impaired binding of the transcription factor ZNF143. In silico analysis suggested an important role of ZNF143 for the formation of a chromatin loop including PMM2 We propose that the PMM2 promoter mutation alters tissue-specific chromatin loop formation, with consequent organ-specific deficiency of PMM2 leading to the restricted phenotype of HIPKD. Our findings extend the spectrum of genetic causes for both HI and PKD and provide insights into gene regulation and PMM2 pleiotropy.

SOS1 frameshift mutations cause pure mucosal neuroma syndrome, a clinical phenotype distinct from multiple endocrine neoplasia type 2B.

BACKGROUND: Mucosal neuromas, thickened corneal nerves and marfanoid body habitus are characteristic phenotypic features of multiple endocrine neoplasia type 2B (MEN2B) and often provide an early clue to the diagnosis of the syndrome. Rarely, patients present with typical physical features of MEN2B but without associated endocrinopathies (medullary thyroid carcinoma or pheochromocytoma) or a RET gene mutation; this clinical presentation is thought to represent a distinct condition termed 'pure mucosal neuroma syndrome'. METHODS: Exome sequencing was performed in two unrelated probands with mucosal neuromas, thickened corneal nerves and marfanoid body habitus, but no MEN2B-associated endocrinopathy or RET gene mutation. Sanger sequencing was performed to confirm mutations detected by exome sequencing and to test in family members and 3 additional unrelated index patients with mucosal neuromas or thickened corneal nerves. RESULTS: A heterozygous SOS1 gene frameshift mutation (c.3266dup or c.3248dup) was identified in each proband. Sanger sequencing showed that proband 1 inherited the c.3266dup mutation from his affected mother, while the c.3248dup mutation had arisen de novo in proband 2. Sanger sequencing also identified one further novel SOS1 mutation (c.3254dup) in one of the 3 additional index patients. CONCLUSION: Our results demonstrate the existence of pure mucosal neuroma syndrome as a clinical entity distinct from MEN2B that can now be diagnosed by genetic testing.