Direct measurement of pregnanediol 3-glucuronide (PDG) in dried urine spots by liquid chromatography-mass spectrometry to detect ovulation.

BACKGROUND: Non-invasive self-testing using an objective chemical method to detect ovulation is valuable for women planning conception, practising contraception or undergoing infertility investigations or treatment. METHODS: Based on luteal phase secretion of progesterone (P4) and excretion of its major metabolite, pregnanediol glucuronide (PDG), we developed a novel direct liquid chromatography-mass spectrometry (LCMS) method to measure PDG and other steroid glucuronides in urine and in dried urine spots (DUS) without deconjugation or derivatization. Urine PDG by LCMS and immunoassay (P3G) and P4 by immunoassay with and without adjustment for creatinine were evaluated in daily first void urine samples from 10 women through a single menstrual cycle in which ovulation was confirmed by serial transvaginal ultrasound. RESULTS: Urine PDG with and without creatinine adjustment was stable during the follicular phase with the expected striking rise in the luteal phase peaking at 5 days after ovulation. Using a single spot urine sample (100 µL) or a DUS (<20 µL urine) and an optimal threshold to distinguish pre- from post-ovulatory samples, in ROC analysis urine PDG adjusted for creatinine accurately identified ovulation in 92 % of samples was comparable with P3G immunoassay and superior to urine P4 with or without adjustment for creatinine. Extending the analysis to two or three consecutive daily samples reduced the false negative rate from 8% to 2.6 % for two and 1.9 % for three urine samples. CONCLUSIONS: This method holds promise as a non-invasive self-test method for women to determine by an objective chemical method their ovulatory status.

Denisovan, modern human and mouse TNFAIP3 alleles tune A20 phosphorylation and immunity.

Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.

Elongator mutation in mice induces neurodegeneration and ataxia-like behavior.

Cerebellar ataxias are severe neurodegenerative disorders with an early onset and progressive and inexorable course of the disease. Here, we report a single point mutation in the gene encoding Elongator complex subunit 6 causing Purkinje neuron degeneration and an ataxia-like phenotype in the mutant wobbly mouse. This mutation destabilizes the complex and compromises its function in translation regulation, leading to protein misfolding, proteotoxic stress, and eventual neuronal death. In addition, we show that substantial microgliosis is triggered by the NLRP3 inflammasome pathway in the cerebellum and that blocking NLRP3 function in vivo significantly delays neuronal degeneration and the onset of ataxia in mutant animals. Our data provide a mechanistic insight into the pathophysiology of a cerebellar ataxia caused by an Elongator mutation, substantiating the increasing body of evidence that alterations of this complex are broadly implicated in the onset of a number of diverse neurological disorders.

A mutation in the viral sensor 2'-5'-oligoadenylate synthetase 2 causes failure of lactation.

We identified a non-synonymous mutation in Oas2 (I405N), a sensor of viral double-stranded RNA, from an ENU-mutagenesis screen designed to discover new genes involved in mammary development. The mutation caused post-partum failure of lactation in healthy mice with otherwise normally developed mammary glands, characterized by greatly reduced milk protein synthesis coupled with epithelial cell death, inhibition of proliferation and a robust interferon response. Expression of mutant but not wild type Oas2 in cultured HC-11 or T47D mammary cells recapitulated the phenotypic and transcriptional effects observed in the mouse. The mutation activates the OAS2 pathway, demonstrated by a 34-fold increase in RNase L activity, and its effects were dependent on expression of RNase L and IRF7, proximal and distal pathway members. This is the first report of a viral recognition pathway regulating lactation.

Mutations in DZIP1L, which encodes a ciliary-transition-zone protein, cause autosomal recessive polycystic kidney disease.

Autosomal recessive polycystic kidney disease (ARPKD), usually considered to be a genetically homogeneous disease caused by mutations in PKHD1, has been associated with ciliary dysfunction. Here, we describe mutations in DZIP1L, which encodes DAZ interacting protein 1-like, in patients with ARPKD. We further validated these findings through loss-of-function studies in mice and zebrafish. DZIP1L localizes to centrioles and to the distal ends of basal bodies, and interacts with septin2, a protein implicated in maintenance of the periciliary diffusion barrier at the ciliary transition zone. In agreement with a defect in the diffusion barrier, we found that the ciliary-membrane translocation of the PKD proteins polycystin-1 and polycystin-2 is compromised in DZIP1L-mutant cells. Together, these data provide what is, to our knowledge, the first conclusive evidence that ARPKD is not a homogeneous disorder and further establish DZIP1L as a second gene involved in ARPKD pathogenesis.

Heterogeneity of Human Neutrophil CD177 Expression Results from CD177P1 Pseudogene Conversion.

Most humans harbor both CD177neg and CD177pos neutrophils but 1-10% of people are CD177null, placing them at risk for formation of anti-neutrophil antibodies that can cause transfusion-related acute lung injury and neonatal alloimmune neutropenia. By deep sequencing the CD177 locus, we catalogued CD177 single nucleotide variants and identified a novel stop codon in CD177null individuals arising from a single base substitution in exon 7. This is not a mutation in CD177 itself, rather the CD177null phenotype arises when exon 7 of CD177 is supplied entirely by the CD177 pseudogene (CD177P1), which appears to have resulted from allelic gene conversion. In CD177 expressing individuals the CD177 locus contains both CD177P1 and CD177 sequences. The proportion of CD177hi neutrophils in the blood is a heritable trait. Abundance of CD177hi neutrophils correlates with homozygosity for CD177 reference allele, while heterozygosity for ectopic CD177P1 gene conversion correlates with increased CD177neg neutrophils, in which both CD177P1 partially incorporated allele and paired intact CD177 allele are transcribed. Human neutrophil heterogeneity for CD177 expression arises by ectopic allelic conversion. Resolution of the genetic basis of CD177null phenotype identifies a method for screening for individuals at risk of CD177 isoimmunisation.

Arrested Hematopoiesis and Vascular Relaxation Defects in Mice with a Mutation in Dhfr.

Dihydrofolate reductase (DHFR) is a critical enzyme in the folate metabolism pathway and also plays a role in regulating nitric oxide (NO) signaling in endothelial cells. Although both coding and noncoding mutations with phenotypic effects have been identified in the human DHFR gene, no mouse model is currently available to study the consequences of perturbing DHFR in vivo In order to identify genes involved in definitive hematopoiesis, we performed a forward genetic screen and produced a mouse line, here referred to as Orana, with a point mutation in the Dhfr locus leading to a Thr136Ala substitution in the DHFR protein. Homozygote Orana mice initiate definitive hematopoiesis, but expansion of progenitors in the fetal liver is compromised, and the animals die between embryonic day 13.5 (E13.5) and E14.5. Heterozygote Orana mice survive to adulthood but have tissue-specific alterations in folate abundance and distribution, perturbed stress erythropoiesis, and impaired endothelium-dependent relaxation of the aorta consistent with the role of DHFR in regulating NO signaling. Orana mice provide insight into the dual roles of DHFR and are a useful model for investigating the role of environmental and dietary factors in the context of vascular defects caused by altered NO signaling.

Comparison of predicted and actual consequences of missense mutations.

Each person's genome sequence has thousands of missense variants. Practical interpretation of their functional significance must rely on computational inferences in the absence of exhaustive experimental measurements. Here we analyzed the efficacy of these inferences in 33 de novo missense mutations revealed by sequencing in first-generation progeny of N-ethyl-N-nitrosourea-treated mice, involving 23 essential immune system genes. PolyPhen2, SIFT, MutationAssessor, Panther, CADD, and Condel were used to predict each mutation's functional importance, whereas the actual effect was measured by breeding and testing homozygotes for the expected in vivo loss-of-function phenotype. Only 20% of mutations predicted to be deleterious by PolyPhen2 (and 15% by CADD) showed a discernible phenotype in individual homozygotes. Half of all possible missense mutations in the same 23 immune genes were predicted to be deleterious, and most of these appear to become subject to purifying selection because few persist between separate mouse substrains, rodents, or primates. Because defects in immune genes could be phenotypically masked in vivo by compensation and environment, we compared inferences by the same tools with the in vitro phenotype of all 2,314 possible missense variants in TP53; 42% of mutations predicted by PolyPhen2 to be deleterious (and 45% by CADD) had little measurable consequence for TP53-promoted transcription. We conclude that for de novo or low-frequency missense mutations found by genome sequencing, half those inferred as deleterious correspond to nearly neutral mutations that have little impact on the clinical phenotype of individual cases but will nevertheless become subject to purifying selection.

The use of dried blood spot sampling for the measurement of HbA1c: a cross-sectional study.

BACKGROUND: The use of dried blood spot (DBS) sampling is an alternative to traditional venous blood collection, and particularly useful for people living in rural and remote areas, and for those who are infirm, house-bound or time-poor. The objective of this study was to assess whether the measurement of glycated haemoglobin A1c (HbA1c) in DBS samples provided comparative and acceptably precise results. METHODS: Venous and capillary blood samples were collected from 115 adult participants. After proper instruction, each participant punctured his/her own finger and collected capillary blood samples on pieces of a proprietary cellulose filter paper. Each filter paper was subsequently placed inside a breathable envelope, stored at room temperature, and processed on the same day (D0), four (D4), seven (D7) and fourteen (D14) days after collection. HbA1c was measured in duplicates/triplicates in whole venous blood (WB), capillary blood (capDBS) and venous blood placed on the matrix paper (venDBS), by turbidimetric inhibition immunoassay. Intra-assay coefficients of variation (CV) were calculated. DBS values were compared to WB results using linear regression, Bland-Altman plots and cross-validation models. RESULTS: Eleven and 56 patients had type 1 and type 2 diabetes mellitus, respectively. Mean HbA1c levels were 6.22 ± 1.11 % for WB samples (n = 115). The median intra-assay CV was lower than 3 % for WB and capDBS on all days. Results from capDBS and venDBS showed high correlation and agreement to WB results, with narrow 95 % limits of agreement (except for results from D14 samples), as observed in Bland-Altman plots. When capDBS values were applied to equations derived from regression analyses, results approached those of WB values. A cross-validation model showed that capDBS results on D0, D4 and D7 were close to the WB results, with prediction intervals that were narrow enough to be clinically acceptable. CONCLUSIONS: The measurement of HbA1c from DBS samples provided results that were comparable to results from WB samples, if measured up to seven days after collection. Intra-assay coefficients of variation were low, results were in agreement with the gold-standard, and prediction intervals were clinically acceptable. The measurement of HbA1c through DBS sampling may be considered in situations where traditional venipuncture is not available. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ID ACTRN12613000769785.

Identification of phenotypically and functionally heterogeneous mouse mucosal-associated invariant T cells using MR1 tetramers.

Studies on the biology of mucosal-associated invariant T cells (MAIT cells) in mice have been hampered by a lack of specific reagents. Using MR1-antigen (Ag) tetramers that specifically bind to the MR1-restricted MAIT T cell receptors (TCRs), we demonstrate that MAIT cells are detectable in a broad range of tissues in C57BL/6 and BALB/c mice. These cells include CD4(-)CD8(-), CD4(-)CD8(+), and CD4(+)CD8(-) subsets, and their frequency varies in a tissue- and strain-specific manner. Mouse MAIT cells have a CD44(hi)CD62L(lo) memory phenotype and produce high levels of IL-17A, whereas other cytokines, including IFN-γ, IL-4, IL-10, IL-13, and GM-CSF, are produced at low to moderate levels. Consistent with high IL-17A production, most MAIT cells express high levels of retinoic acid-related orphan receptor γt (RORγt), whereas RORγt(lo) MAIT cells predominantly express T-bet and produce IFN-γ. Most MAIT cells express the promyelocytic leukemia zinc finger (PLZF) transcription factor, and their development is largely PLZF dependent. These observations contrast with previous reports that MAIT cells from Vα19 TCR transgenic mice are PLZF(-) and express a naive CD44(lo) phenotype. Accordingly, MAIT cells from normal mice more closely resemble human MAIT cells than previously appreciated, and this provides the foundation for further investigations of these cells in health and disease.

A de novo Mutation in KMT2A (MLL) in monozygotic twins with Wiedemann-Steiner syndrome.

Growth deficiency, psychomotor delay, and facial dysmorphism was originally described in a male patient in 1989 by Wiedemann et al. and later in 2000 by Steiner et al. Wiedemann-Steiner syndrome (WSS) has since been described only a few times in the literature, with the phenotypic spectrum both expanding and becoming more delineated with each patient reported. We report on the clinical and molecular features of monozygotic twins with a de novo mutation in KMT2A. Single nucleotide polymorphism (SNP) microarray was done on both twins and whole-exome sequencing was done using both parents and one of the affected twins. SNP microarray confirmed that they were monozygotic twins. A de novo heterozygous variant (p. Arg1083*) in the KMT2A gene was identified through whole-exome sequencing, confirming the diagnosis of WSS. In this study, we have identified a de novo mutation in KMT2A associated with psychomotor developmental delay, facial dysmorphism, short stature, hypertrichosis cubiti, and small kidneys. This finding in monozygotic twins gives specificity to the WSS. The description of more cases of WSS is needed for further delineation of this condition. Small kidneys with normal function have not been described in this condition in the medical literature before.

LRGUK-1 is required for basal body and manchette function during spermatogenesis and male fertility.

Male infertility affects at least 5% of reproductive age males. The most common pathology is a complex presentation of decreased sperm output and abnormal sperm shape and motility referred to as oligoasthenoteratospermia (OAT). For the majority of OAT men a precise diagnosis cannot be provided. Here we demonstrate that leucine-rich repeats and guanylate kinase-domain containing isoform 1 (LRGUK-1) is required for multiple aspects of sperm assembly, including acrosome attachment, sperm head shaping and the initiation of the axoneme growth to form the core of the sperm tail. Specifically, LRGUK-1 is required for basal body attachment to the plasma membrane, the appropriate formation of the sub-distal appendages, the extension of axoneme microtubules and for microtubule movement and organisation within the manchette. Manchette dysfunction leads to abnormal sperm head shaping. Several of these functions may be achieved in association with the LRGUK-1 binding partner HOOK2. Collectively, these data establish LRGUK-1 as a major determinant of microtubule structure within the male germ line.

Reducing the search space for causal genetic variants with VASP.

MOTIVATION: Increasingly, cost-effective high-throughput DNA sequencing technologies are being utilized to sequence human pedigrees to elucidate the genetic cause of a wide variety of human diseases. While numerous tools exist for variant prioritization within a single genome, the ability to concurrently analyze variants within pedigrees remains a challenge, especially should there be no prior indication of the underlying genetic cause of the disease. Here, we present a tool, variant analysis of sequenced pedigrees (VASP), a flexible data integration environment capable of producing a summary of pedigree variation, providing relevant information such as compound heterozygosity, genome phasing and disease inheritance patterns. Designed to aggregate data across a sequenced pedigree, VASP allows both powerful filtering and custom prioritization of both single nucleotide variants (SNVs) and small indels. Hence, clinical and research users with prior knowledge of a disease are able to dramatically reduce the variant search space based on a wide variety of custom prioritization criteria. AVAILABILITY AND IMPLEMENTATION: Source code available for academic non-commercial research purposes at https://github.com/mattmattmattmatt/VASP.

A deleterious RNF43 germline mutation in a severely affected serrated polyposis kindred.

We report a germline nonsense mutation within the extracellular domain of the RING finger ubiquitin ligase RNF43, segregating with a severe form of serrated polyposis within a kindred. The finding provides evidence that inherited RNF43 mutations define a familial cancer syndrome.

Zinc finger protein Zfp335 is required for the formation of the naïve T cell compartment.

The generation of naïve T lymphocytes is critical for immune function yet the mechanisms governing their maturation remain incompletely understood. We have identified a mouse mutant, bloto, that harbors a hypomorphic mutation in the zinc finger protein Zfp335. Zfp335(bloto/bloto) mice exhibit a naïve T cell deficiency due to an intrinsic developmental defect that begins to manifest in the thymus and continues into the periphery, affecting T cells that have recently undergone thymic egress. The effects of Zfp335(bloto) are multigenic and cannot be attributed to altered thymic selection, proliferation or Bcl2-dependent survival. Zfp335 binds to promoter regions via a consensus motif, and its target genes are enriched in categories related to protein metabolism, mitochondrial function, and transcriptional regulation. Restoring the expression of one target, Ankle2, partially rescues T cell maturation. These findings identify Zfp335 as a transcription factor and essential regulator of late-stage intrathymic and post-thymic T cell maturation.

Autosomal-dominant B-cell deficiency with alopecia due to a mutation in NFKB2 that results in nonprocessable p100.

Most genetic defects that arrest B-cell development in the bone marrow present early in life with agammaglobulinemia, whereas incomplete antibody deficiency is usually associated with circulating B cells. We report 3 related individuals with a novel form of severe B-cell deficiency associated with partial persistence of serum immunoglobulin arising from a missense mutation in NFKB2. Significantly, this point mutation results in a D865G substitution and causes a failure of p100 phosphorylation that blocks processing to p52. Severe B-cell deficiency affects mature and transitional cells, mimicking the action of rituximab. This phenotype appears to be due to disruption of canonical and noncanonical nuclear factor κB pathways by the mutant p100 molecule. These findings could be informative for therapeutics as well as immunodeficiency.

Zinc-finger protein ZFP318 is essential for expression of IgD, the alternatively spliced Igh product made by mature B lymphocytes.

IgD and IgM are produced by alternative splicing of long primary RNA transcripts from the Ig heavy chain (Igh) locus and serve as the receptors for antigen on naïve mature B lymphocytes. IgM is made selectively in immature B cells, whereas IgD is coexpressed with IgM when the cells mature into follicular or marginal zone B cells, but the transacting factors responsible for this regulated change in splicing have remained elusive. Here, we use a genetic screen in mice to identify ZFP318, a nuclear protein with two U1-type zinc fingers found in RNA-binding proteins and no known role in the immune system, as a critical factor for IgD expression. A point mutation in an evolutionarily conserved lysine-rich domain encoded by the alternatively spliced Zfp318 exon 10 abolished IgD expression on marginal zone B cells, decreased IgD on follicular B cells, and increased IgM, but only slightly decreased the percentage of B cells and did not decrease expression of other maturation markers CD21, CD23, or CD62L. A targeted Zfp318 null allele extinguished IgD expression on mature B cells and increased IgM. Zfp318 mRNA is developmentally regulated in parallel with IgD, with little in pro-B cells, moderate amounts in immature B cells, and high levels selectively in mature follicular B cells. These findings identify ZFP318 as a crucial factor regulating the expression of the two major antibody isotypes on the surface of most mature B cells.

Rasgrp1 mutation increases naive T-cell CD44 expression and drives mTOR-dependent accumulation of Helios⁺ T cells and autoantibodies.

Missense variants are a major source of human genetic variation. Here we analyze a new mouse missense variant, Rasgrp1(Anaef), with an ENU-mutated EF hand in the Rasgrp1 Ras guanine nucleotide exchange factor. Rasgrp1(Anaef) mice exhibit anti-nuclear autoantibodies and gradually accumulate a CD44(hi) Helios(+) PD-1(+) CD4(+) T cell population that is dependent on B cells. Despite reduced Rasgrp1-Ras-ERK activation in vitro, thymocyte selection in Rasgrp1(Anaef) is mostly normal in vivo, although CD44 is overexpressed on naïve thymocytes and T cells in a T-cell-autonomous manner. We identify CD44 expression as a sensitive reporter of tonic mTOR-S6 kinase signaling through a novel mouse strain, chino, with a reduction-of-function mutation in Mtor. Elevated tonic mTOR-S6 signaling occurs in Rasgrp1(Anaef) naïve CD4(+) T cells. CD44 expression, CD4(+) T cell subset ratios and serum autoantibodies all returned to normal in Rasgrp1(Anaef)Mtor(chino) double-mutant mice, demonstrating that increased mTOR activity is essential for the Rasgrp1(Anaef) T cell dysregulation. DOI: http://dx.doi.org/10.7554/eLife.01020.001.

A missense mutation in the transcription factor ETV5 leads to sterility, increased embryonic and perinatal death, postnatal growth restriction, renal asymmetry and polydactyly in the mouse.

ETV5 (Ets variant gene 5) is a transcription factor that is required for fertility. In this study, we demonstrate that ETV5 plays additional roles in embryonic and postnatal developmental processes in the mouse. Through a genome-wide mouse mutagenesis approach, we generated a sterile mouse line that carried a nonsense mutation in exon 12 of the Etv5 gene. The mutation led to the conversion of lysine at position 412 into a premature termination codon (PTC) within the ETS DNA binding domain of the protein. We showed that the PTC-containing allele produced a highly unstable mRNA, which in turn resulted in an undetectable level of ETV5 protein. The Etv5 mutation resulted in male and female sterility as determined by breeding experiments. Mutant males were sterile due to a progressive loss of spermatogonia, which ultimately resulted in a Sertoli cell only phenotype by 8 week-of-age. Further, the ETV5 target genes Cxcr4 and Ccl9 were significantly down-regulated in mutant neonate testes. CXCR4 and CCL9 have been implicated in the maintenance and migration of spermatogonia, respectively. Moreover, the Etv5 mutation resulted in several developmental abnormalities including an increased incidence of embryonic and perinatal lethality, postnatal growth restriction, polydactyly and renal asymmetry. Thus, our data define a physiological role for ETV5 in many aspects of development including embryonic and perinatal survival, postnatal growth, limb patterning, kidney development and fertility.

RBM5 is a male germ cell splicing factor and is required for spermatid differentiation and male fertility.

Alternative splicing of precursor messenger RNA (pre-mRNA) is common in mammalian cells and enables the production of multiple gene products from a single gene, thus increasing transcriptome and proteome diversity. Disturbance of splicing regulation is associated with many human diseases; however, key splicing factors that control tissue-specific alternative splicing remain largely undefined. In an unbiased genetic screen for essential male fertility genes in the mouse, we identified the RNA binding protein RBM5 (RNA binding motif 5) as an essential regulator of haploid male germ cell pre-mRNA splicing and fertility. Mice carrying a missense mutation (R263P) in the second RNA recognition motif (RRM) of RBM5 exhibited spermatid differentiation arrest, germ cell sloughing and apoptosis, which ultimately led to azoospermia (no sperm in the ejaculate) and male sterility. Molecular modelling suggested that the R263P mutation resulted in compromised mRNA binding. Within the adult mouse testis, RBM5 localises to somatic and germ cells including spermatogonia, spermatocytes and round spermatids. Through the use of RNA pull down coupled with microarrays, we identified 11 round spermatid-expressed mRNAs as putative RBM5 targets. Importantly, the R263P mutation affected pre-mRNA splicing and resulted in a shift in the isoform ratios, or the production of novel spliced transcripts, of most targets. Microarray analysis of isolated round spermatids suggests that altered splicing of RBM5 target pre-mRNAs affected expression of genes in several pathways, including those implicated in germ cell adhesion, spermatid head shaping, and acrosome and tail formation. In summary, our findings reveal a critical role for RBM5 as a pre-mRNA splicing regulator in round spermatids and male fertility. Our findings also suggest that the second RRM of RBM5 is pivotal for appropriate pre-mRNA splicing.