ABSTRACT
Nephronophthisis-related ciliopathies (NPHP-RC) are degenerative recessive diseases that affect kidney, retina, and brain. Genetic defects in NPHP gene products that localize to cilia and centrosomes defined them as "ciliopathies." However, disease mechanisms remain poorly understood. Here, we identify by whole-exome resequencing, mutations of MRE11, ZNF423, and CEP164 as causing NPHP-RC. All three genes function within the DNA damage response (DDR) pathway. We demonstrate that, upon induced DNA damage, the NPHP-RC proteins ZNF423, CEP164, and NPHP10 colocalize to nuclear foci positive for TIP60, known to activate ATM at sites of DNA damage. We show that knockdown of CEP164 or ZNF423 causes sensitivity to DNA damaging agents and that cep164 knockdown in zebrafish results in dysregulated DDR and an NPHP-RC phenotype. Our findings link degenerative diseases of the kidney and retina, disorders of increasing prevalence, to mechanisms of DDR.
Subject(s)
DNA Damage , DNA-Binding Proteins/metabolism , Exome , Kidney Diseases, Cystic/genetics , Microtubule Proteins/metabolism , Animals , Cilia/metabolism , Gene Knockdown Techniques , Genes, Recessive , Humans , MRE11 Homologue Protein , Mice , Proteins , Signal Transduction , Zebrafish/embryology , Zebrafish/metabolismABSTRACT
Gonadal steroids modulate growth hormone (GH) secretion and the pubertal growth spurt via undefined central pathways. GH-releasing hormone (GHRH) neurons express estrogen receptor α (ERα) and androgen receptor (AR), suggesting changing levels of gonadal steroids during puberty directly modulate the somatotropic axis. We generated mice with deletion of ERα in GHRH cells (GHRHΔERα), which displayed reduced body length in both sexes. Timing of puberty onset was similar in both groups, but puberty completion was delayed in GHRHΔERα females. Lack of AR in GHRH cells (GHRHΔAR mice) induced no changes in body length, but puberty completion was also delayed in females. Using a mouse model with two reporter genes, we observed that, while GHRHtdTom neurons minimally colocalize with Kiss1hrGFP in prepubertal mice, â¼30% of GHRH neurons coexpressed both reporter genes in adult females, but not in males. Developmental analysis of Ghrh and Kiss1 expression suggested that a subpopulation of ERα neurons in the arcuate nucleus of female mice undergoes a shift in phenotype, from GHRH to Kiss1, during pubertal transition. Our findings demonstrate that direct actions of gonadal steroids in GHRH neurons modulate growth and puberty and indicate that GHRH/Kiss1 dual-phenotype neurons play a sex-specific role in the crosstalk between the somatotropic and gonadotropic axes during pubertal transition.SIGNIFICANCE STATEMENT Late maturing adolescents usually show delayed growth and bone age. At puberty, gonadal steroids have stimulatory effects on the activation of growth and reproductive axes, but the existence of gonadal steroid-sensitive neuronal crosstalk remains undefined. Moreover, the neural basis for the sex differences observed in the clinical arena is unknown. Lack of ERα in GHRH neurons disrupts growth in both sexes and causes pubertal delay in females. Deletion of androgen receptor in GHRH neurons only delayed female puberty. In adult females, not males, a subset of GHRH neurons shift phenotype to start producing Kiss1. Thus, direct estrogen action in GHRH/Kiss1 dual-phenotype neurons modulates growth and puberty and may orchestrate the sex differences in endocrine function observed during pubertal transition.
Subject(s)
Estrogen Receptor alpha/physiology , Growth Hormone-Releasing Hormone/physiology , Growth/physiology , Kisspeptins/physiology , Sexual Maturation/physiology , Signal Transduction/physiology , Animals , Estrogen Receptor alpha/genetics , Female , Gonadal Steroid Hormones/blood , Gonadal Steroid Hormones/physiology , Growth/genetics , Growth Hormone-Releasing Hormone/genetics , Hypothalamus/metabolism , Kisspeptins/genetics , Male , Mice , Mice, Knockout , Receptors, Androgen/physiology , Sex Characteristics , Sexual Maturation/genetics , Signal Transduction/geneticsABSTRACT
Hypothalamic neurons detect changes in circulating hormones such as leptin and insulin and put forward outputs to sustain energy and glucose homeostasis. Because leptin and insulin receptors colocalize in ~40-60% of neurons in the hypothalamus, we characterized the metabolic phenotype of mice with selective deletion of the insulin receptor (InsR) in LepR cells. LRΔInsR mice presented no difference in body weight and insulin levels but increased fat mass. In the light phase, LRΔInsR mice exhibited increased food intake, locomotor activity, carbon dioxide production, and respiratory exchange rate. These mice showed reduced fat oxidation and reduced expression of cluster of differentiation 36 and AMP-activated protein kinase-α1 in the liver, increased glucose oxidation in the light phase, and overall reduced basal glucose levels. To verify the impact of InsR deletion in LepR cells in obesity, we generated ob/ ob InsRfl, ob/ ob LRcre, and ob/ ob LRΔInsR mice. The ob/ ob LRΔInsR mice had higher body weight, fat mass, and expression of genes related to fat metabolism in the liver. No difference in food intake despite increased neuropeptide Y and agouti-related peptide expression, and no difference in energy expenditure, fat, or glucose oxidation was found in ob/ ob LRΔInsR compared with LRcre or LRΔInsR controls. Remarkably, basal glucose levels were reduced, and the expression of genes associated with glucose metabolism in the liver was higher. Insulin signaling in LepR cells is required for the proper fat and glucose oxidation. These effects are independent of leptin given that the leptin-deficient ob/ ob LRΔInsR mice also presented reduced glycemia and higher adiposity. The mechanisms underlying these responses remain to be unveiled.
Subject(s)
Diabetes Mellitus, Type 2/metabolism , Glucose/metabolism , Insulin/metabolism , Leptin/metabolism , Neurons/metabolism , Obesity/metabolism , Receptor, Insulin/genetics , Receptors, Leptin/metabolism , AMP-Activated Protein Kinases/metabolism , Animals , CD36 Antigens/metabolism , Energy Metabolism , Female , Gene Deletion , Homeostasis , Hyperinsulinism/metabolism , Hypothalamus/cytology , Hypothalamus/metabolism , Liver/metabolism , Male , Mice , Mice, ObeseABSTRACT
The anaphylactoid reaction described follows cessation of ranitidine in a 19-year-old female with the disease cluster: mast cell activation syndrome, hypermobile Ehlers-Danlos syndrome and postural tachycardia syndrome. Anaphylaxis can give wide-ranging symptoms from rhinorrhoea and urticaria to tachycardia and system-wide, life-threatening, anaphylactic shock. Individuals with a disorder of mast cell activation can experience many such symptoms. H2 receptor antagonists, such as ranitidine, are commonly prescribed in this population. A mechanism for the reaction is proposed in the context of ranitidine, as an inverse agonist, causing upregulation of H2 histamine receptors and raised histamine levels due to enzyme induction. This effect, following extended and/or high antihistamine dosing, may have implications for other individuals with a disorder of mast cell activation, such as mastocytosis or mast cell activation syndrome. There are potential policy and patient guidance implications for primary and secondary care with respect to cessation of H2 antagonists.
Subject(s)
Anaphylaxis/immunology , Histamine/blood , Receptors, Histamine H2/metabolism , Withholding Treatment , Adult , Anaphylaxis/blood , Anaphylaxis/diagnosis , Anaphylaxis/drug therapy , Chlorpheniramine/therapeutic use , Epinephrine/administration & dosage , Female , Histamine/immunology , Histamine H1 Antagonists/therapeutic use , Histamine H2 Antagonists/administration & dosage , Humans , Ranitidine/administration & dosage , Receptors, Histamine H2/immunology , Treatment Outcome , Up-Regulation , Young AdultABSTRACT
Nephrotic syndrome (NS) is a genetically heterogeneous group of diseases that are divided into steroid-sensitive NS (SSNS) and steroid-resistant NS (SRNS). SRNS inevitably leads to end-stage kidney disease, and no curative treatment is available. To date, mutations in more than 24 genes have been described in Mendelian forms of SRNS; however, no Mendelian form of SSNS has been described. To identify a genetic form of SSNS, we performed homozygosity mapping, whole-exome sequencing, and multiplex PCR followed by next-generation sequencing. We thereby detected biallelic mutations in EMP2 (epithelial membrane protein 2) in four individuals from three unrelated families affected by SRNS or SSNS. We showed that EMP2 exclusively localized to glomeruli in the kidney. Knockdown of emp2 in zebrafish resulted in pericardial effusion, supporting the pathogenic role of mutated EMP2 in human NS. At the cellular level, we showed that knockdown of EMP2 in podocytes and endothelial cells resulted in an increased amount of CAVEOLIN-1 and decreased cell proliferation. Our data therefore identify EMP2 mutations as causing a recessive Mendelian form of SSNS.
Subject(s)
Membrane Glycoproteins/genetics , Mutation , Nephrotic Syndrome/genetics , Alleles , Animals , Caveolin 1/metabolism , Cell Proliferation , Child, Preschool , Chromosome Mapping , Endothelial Cells/pathology , Gene Expression Regulation , Genetic Loci , Homozygote , Humans , Infant , Kidney/pathology , Kidney Failure, Chronic/etiology , Kidney Failure, Chronic/genetics , Membrane Glycoproteins/metabolism , Nephrotic Syndrome/complications , Zebrafish/embryology , Zebrafish/geneticsABSTRACT
Chronically increased echogenicity on renal ultrasound is a sensitive early finding of chronic kidney disease that can be detected before manifestation of other symptoms. Increased echogenicity, however, is not specific for a certain etiology of chronic kidney disease. Here, we performed whole exome sequencing in 79 consanguineous or familial cases of suspected nephronophthisis in order to determine the underlying molecular disease cause. In 50 cases, there was a causative mutation in a known monogenic disease gene. In 32 of these cases whole exome sequencing confirmed the diagnosis of a nephronophthisis-related ciliopathy. In 8 cases it revealed the diagnosis of a renal tubulopathy. The remaining 10 cases were identified as Alport syndrome (4), autosomal-recessive polycystic kidney disease (2), congenital anomalies of the kidney and urinary tract (3), and APECED syndrome (1). In 5 families, in whom mutations in known monogenic genes were excluded, we applied homozygosity mapping for variant filtering and identified 5 novel candidate genes (RBM48, FAM186B, PIAS1, INCENP, and RCOR1) for renal ciliopathies. Thus, whole exome sequencing allows the detection of the causative mutation in 2/3 of affected individuals, thereby presenting the etiologic diagnosis, and allows identification of novel candidate genes.
Subject(s)
Renal Insufficiency, Chronic/genetics , Age of Onset , Cohort Studies , DNA Mutational Analysis , Exome , Humans , Kidney Diseases, Cystic/congenital , Kidney Diseases, Cystic/genetics , Renal Insufficiency, Chronic/diagnostic imagingABSTRACT
Defects of motile cilia cause primary ciliary dyskinesia (PCD), characterized by recurrent respiratory infections and male infertility. Using whole-exome resequencing and high-throughput mutation analysis, we identified recessive biallelic mutations in ZMYND10 in 14 families and mutations in the recently identified LRRC6 in 13 families. We show that ZMYND10 and LRRC6 interact and that certain ZMYND10 and LRRC6 mutations abrogate the interaction between the LRRC6 CS domain and the ZMYND10 C-terminal domain. Additionally, ZMYND10 and LRRC6 colocalize with the centriole markers SAS6 and PCM1. Mutations in ZMYND10 result in the absence of the axonemal protein components DNAH5 and DNALI1 from respiratory cilia. Animal models support the association between ZMYND10 and human PCD, given that zmynd10 knockdown in zebrafish caused ciliary paralysis leading to cystic kidneys and otolith defects and that knockdown in Xenopus interfered with ciliogenesis. Our findings suggest that a cytoplasmic protein complex containing ZMYND10 and LRRC6 is necessary for motile ciliary function.
Subject(s)
Cilia/genetics , Kartagener Syndrome/genetics , Proteins/genetics , Respiratory System/metabolism , Tumor Suppressor Proteins/genetics , Animals , Autoantigens/genetics , Autoantigens/metabolism , Axonemal Dyneins/genetics , Axonemal Dyneins/metabolism , Biomarkers/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cilia/metabolism , Cilia/pathology , Cytoskeletal Proteins , Exome , Gene Expression Regulation , High-Throughput Nucleotide Sequencing , Humans , Kartagener Syndrome/metabolism , Kartagener Syndrome/pathology , Male , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Mutation , Pedigree , Protein Binding , Protein Structure, Tertiary , Proteins/metabolism , Rats , Respiratory System/pathology , Tumor Suppressor Proteins/metabolism , Xenopus laevis/genetics , Xenopus laevis/metabolism , Zebrafish/genetics , Zebrafish/metabolismABSTRACT
Leptin signals energy sufficiency to the reproductive hypothalamic-pituitary-gonadal (HPG) axis. Studies using genetic models have demonstrated that hypothalamic neurons are major players mediating these effects. Leptin receptor (LepR) is also expressed in the pituitary gland and in the gonads, but the physiological effects of leptin in these sites are still unclear. Female mice with selective deletion of LepR in a subset of gonadotropes show normal pubertal development but impaired fertility. Conditional deletion approaches, however, often result in redundancy or developmental adaptations, which may compromise the assessment of leptin's action in gonadotropes for pubertal maturation. To circumvent these issues, we adopted a complementary genetic approach and assessed if selective reexpression of LepR only in gonadotropes is sufficient to enable puberty and improve fertility of LepR null female mice. We initially assessed the colocalization of gonadotropin-releasing hormone receptor (GnRHR) and LepR in the HPG axis using GnRHR-IRES-Cre (GRIC) and LepR-Cre reporter (tdTomato or enhanced green fluorescent protein) mice. We found that GRIC and leptin-induced phosphorylation of STAT3 are expressed in distinct hypothalamic neurons. Whereas LepR-Cre was observed in theca cells, GRIC expression was rarely found in the ovarian parenchyma. In contrast, a subpopulation of gonadotropes expressed the LepR-Cre reporter gene (tdTomato). We then crossed the GRIC mice with the LepR null reactivable (LepR(loxTB)) mice. These mice showed an increase in FSH levels, but they remained in a prepubertal state. Together with previous findings, our data indicate that leptin-selective action in gonadotropes serves a role in adult reproductive physiology but is not sufficient to allow pubertal maturation in mice.
Subject(s)
Fertility/physiology , Follicle Stimulating Hormone/biosynthesis , Gene Expression Regulation, Developmental/physiology , Puberty/physiology , Receptors, LHRH/metabolism , Receptors, Leptin/metabolism , Animals , Cells, Cultured , Mice , Mice, Knockout , Receptors, Leptin/geneticsABSTRACT
The molecular basis of nephronophthisis, the most frequent genetic cause of renal failure in children and young adults, and its association with retinal degeneration and cerebellar vermis aplasia in Joubert syndrome are poorly understood. Using positional cloning, we here identify mutations in the gene CEP290 as causing nephronophthisis. It encodes a protein with several domains also present in CENPF, a protein involved in chromosome segregation. CEP290 (also known as NPHP6) interacts with and modulates the activity of ATF4, a transcription factor implicated in cAMP-dependent renal cyst formation. NPHP6 is found at centrosomes and in the nucleus of renal epithelial cells in a cell cycle-dependent manner and in connecting cilia of photoreceptors. Abrogation of its function in zebrafish recapitulates the renal, retinal and cerebellar phenotypes of Joubert syndrome. Our findings help establish the link between centrosome function, tissue architecture and transcriptional control in the pathogenesis of cystic kidney disease, retinal degeneration, and central nervous system development.
Subject(s)
Activating Transcription Factor 4/genetics , Antigens, Neoplasm/genetics , Mutation , Neoplasm Proteins/genetics , Animals , Cell Cycle Proteins , Cytoskeletal Proteins , Female , Genetic Linkage , Humans , In Situ Hybridization , Male , Pedigree , Syndrome , ZebrafishABSTRACT
Rare single-gene disorders cause chronic disease. However, half of the 6000 recessive single gene causes of disease are still unknown. Because recessive disease genes can illuminate, at least in part, disease pathomechanism, their identification offers direct opportunities for improved clinical management and potentially treatment. Rare diseases comprise the majority of chronic kidney disease (CKD) in children but are notoriously difficult to diagnose. Whole-exome resequencing facilitates identification of recessive disease genes. However, its utility is impeded by the large number of genetic variants detected. We here overcome this limitation by combining homozygosity mapping with whole-exome resequencing in 10 sib pairs with a nephronophthisis-related ciliopathy, which represents the most frequent genetic cause of CKD in the first three decades of life. In 7 of 10 sibships with a histologic or ultrasonographic diagnosis of nephronophthisis-related ciliopathy, we detect the causative gene. In six sibships, we identify mutations of known nephronophthisis-related ciliopathy genes, while in two additional sibships we found mutations in the known CKD-causing genes SLC4A1 and AGXT as phenocopies of nephronophthisis-related ciliopathy. Thus, whole-exome resequencing establishes an efficient, noninvasive approach towards early detection and causation-based diagnosis of rare kidney diseases. This approach can be extended to other rare recessive disorders, thereby providing accurate diagnosis and facilitating the study of disease mechanisms.
Subject(s)
Genetic Testing/methods , Kidney Diseases, Cystic/diagnosis , Kidney Diseases, Cystic/genetics , Adolescent , Adult , DNA Mutational Analysis , Early Diagnosis , Exome , Genes, Recessive , Humans , Infant , Male , Mutation , Phenotype , Young AdultABSTRACT
The hypothalamic ventral premammillary nucleus (PMv) is a glutamatergic nucleus essential for the metabolic control of reproduction. However, conditional deletion of leptin receptor (LepRb) in vesicular glutamate transporter 2 (Vglut2) expressing neurons results in virtually no reproductive deficits. In this study, we determine the role of glutamatergic signaling from leptin responsive PMv neurons on puberty and fertility. We first assessed if stimulation of PMv neurons induces LH release in fed adult females. We used the stimulatory form of designer receptor exclusively activated by designer drugs (DREADDs) in LepRb-Cre mice. We collected blood sequentially before and for 1h after iv. clozapine-N-oxide injection. LH level increased in animals correctly targeted to the PMv, and LH level was correlated to the number of cFos immunoreactive neurons in the PMv. Next, females with deletion of Vglut2 in LepRb neurons (LepR∆VGlut2) showed delayed age of puberty, disrupted estrous cycles, increased GnRH concentration in the axon terminals and disrupted LH responses, suggesting impaired GnRH release. To assess if glutamate is required for PMv actions in pubertal development, we generated a Cre-induced reexpression of endogenous LepRb (LepRloxTB) with concomitant deletion of Vglut2 (Vglut2-floxed) mice. Rescue of Lepr and deletion of Vglut2 in the PMv was obtained by stereotaxic injection of an adeno-associated virus vector expressing Cre recombinase. Control LepRloxTB mice with PMv LepRb rescue showed vaginal opening, follicle maturation and became pregnant, while LepRloxTB;Vglut2flox mice showed no pubertal development. Our results indicate that glutamatergic signaling from leptin sensitive neurons regulates the reproductive axis, and that leptin action on pubertal development via PMv neurons requires Vglut2.
ABSTRACT
The hypothalamic ventral premammillary nucleus (PMv) is a glutamatergic nucleus essential for the metabolic control of reproduction. However, conditional deletion of leptin receptor long form (LepRb) in vesicular glutamate transporter 2 (Vglut2) expressing neurons results in virtually no reproductive deficits. In this study, we determined the role of glutamatergic neurotransmission from leptin responsive PMv neurons on puberty and fertility. We first assessed if stimulation of PMv neurons induces luteinizing hormone (LH) release in fed adult females. We used the stimulatory form of designer receptor exclusively activated by designer drugs (DREADDs) in LeprCre (LepRb-Cre) mice. We collected blood sequentially before and for 1 hr after intravenous clozapine-N-oxide injection. LH level increased in animals correctly targeted to the PMv, and LH level was correlated to the number of Fos immunoreactive neurons in the PMv. Next, females with deletion of Slc17a6 (Vglut2) in LepRb neurons (LeprΔVGlut2) showed delayed age of puberty, disrupted estrous cycles, increased gonadotropin-releasing hormone (GnRH) concentration in the axon terminals, and disrupted LH secretion, suggesting impaired GnRH release. To assess if glutamate is required for PMv actions in pubertal development, we generated a Cre-induced reexpression of endogenous LepRb (LeprloxTB) with concomitant deletion of Slc17a6 (Vglut2flox) mice. Rescue of Lepr and deletion of Slc17a6 in the PMv was obtained by stereotaxic injection of an adeno-associated virus vector expressing Cre recombinase. Control LeprloxTB mice with PMv LepRb rescue showed vaginal opening, follicle maturation, and became pregnant, while LeprloxTB;Vglut2flox mice showed no pubertal development. Our results indicate that glutamatergic neurotransmission from leptin sensitive neurons regulates the reproductive axis, and that leptin action on pubertal development via PMv neurons requires Vglut2.
Subject(s)
Glutamic Acid , Receptors, Leptin , Sexual Maturation , Synaptic Transmission , Animals , Female , Receptors, Leptin/metabolism , Receptors, Leptin/genetics , Mice , Glutamic Acid/metabolism , Neurons/metabolism , Neurons/physiology , Reproduction , Vesicular Glutamate Transport Protein 2/metabolism , Vesicular Glutamate Transport Protein 2/genetics , Luteinizing Hormone/blood , Luteinizing Hormone/metabolismABSTRACT
Nephronophthisis-related ciliopathies (NPHP-RC) are autosomal-recessive cystic kidney diseases. More than 13 genes are implicated in its pathogenesis to date, accounting for only 40 % of all cases. High-throughput mutation screenings of large patient cohorts represent a powerful tool for diagnostics and identification of novel NPHP genes. We here performed a new high-throughput mutation analysis method to study 13 established NPHP genes (NPHP1-NPHP13) in a worldwide cohort of 1,056 patients diagnosed with NPHP-RC. We first applied multiplexed PCR-based amplification using Fluidigm Access-Array™ technology followed by barcoding and next-generation resequencing on an Illumina platform. As a result, we established the molecular diagnosis in 127/1,056 independent individuals (12.0 %) and identified a single heterozygous truncating mutation in an additional 31 individuals (2.9 %). Altogether, we detected 159 different mutations in 11 out of 13 different NPHP genes, 99 of which were novel. Phenotypically most remarkable were two patients with truncating mutations in INVS/NPHP2 who did not present as infants and did not exhibit extrarenal manifestations. In addition, we present the first case of Caroli disease due to mutations in WDR19/NPHP13 and the second case ever with a recessive mutation in GLIS2/NPHP7. This study represents the most comprehensive mutation analysis in NPHP-RC patients, identifying the largest number of novel mutations in a single study worldwide.
Subject(s)
Caroli Disease/genetics , Cilia/genetics , Cilia/pathology , Genes, Recessive/genetics , Kidney Diseases, Cystic/genetics , Membrane Proteins/genetics , Mutation/genetics , Adaptor Proteins, Signal Transducing/genetics , Caroli Disease/pathology , Cohort Studies , Cytoskeletal Proteins , DNA Mutational Analysis , Female , Global Health , High-Throughput Nucleotide Sequencing , Humans , Kidney Diseases, Cystic/pathology , Male , Multiplex Polymerase Chain Reaction , Pedigree , Pilot ProjectsABSTRACT
OBJECTIVE: To identify disease-causing mutations within coding regions of 11 known NPHP genes (NPHP1-NPHP11) in a cohort of 192 patients diagnosed with a nephronophthisis-associated ciliopathy, at low cost. METHODS: Mutation analysis was carried out using PCR-based 48.48 Access Array microfluidic technology (Fluidigm) with consecutive next-generation sequencing. We applied a 10-fold primer multiplexing approach allowing PCR-based amplification of 475 amplicons (251 exons) for 48 DNA samples simultaneously. After four rounds of amplification followed by indexing all of 192 patient-derived products with different barcodes in a subsequent PCR, 2 × 100 paired-end sequencing was performed on one lane of a HiSeq2000 instrument (Illumina). Bioinformatics analysis was performed using 'CLC Genomics Workbench' software. Potential mutations were confirmed by Sanger sequencing and shown to segregate. RESULTS: Bioinformatics analysis revealed sufficient coverage of 30 × for 168/192 (87.5%) DNA samples (median 449 ×) and of 234 out of 251 targeted coding exons (sensitivity: 93.2%). For proof-of-principle, we analysed 20 known mutations and identified 18 of them in the correct zygosity state (90%). Likewise, we identified pathogenic mutations in 34/192 patients (18%) and discovered 23 novel mutations in the genes NPHP3 (7), NPHP4 (3), IQCB1 (4), CEP290 (7), RPGRIP1L (1), and TMEM67 (1). Additionally, we found 40 different single heterozygous missense variants of unknown significance. CONCLUSIONS: We conclude that the combined approach of array-based multiplexed PCR-amplification on a Fluidigm Access Array platform followed by next-generation sequencing is highly cost-efficient and strongly facilitates diagnostic mutation analysis in broadly heterogeneous Mendelian disorders.
Subject(s)
DNA Mutational Analysis , High-Throughput Nucleotide Sequencing , Kidney Diseases, Cystic/congenital , Multiplex Polymerase Chain Reaction , Base Sequence , Cilia/pathology , Computational Biology/methods , Exons , Genotype , Humans , Kidney Diseases, Cystic/genetics , Kidney Diseases, Cystic/pathology , Mutation , Reproducibility of ResultsABSTRACT
Androgens are steroid hormones crucial for sexual differentiation of the brain and reproductive function. In excess, however, androgens may decrease fertility as observed in polycystic ovary syndrome, a common endocrine disorder characterized by oligo/anovulation and/or polycystic ovaries. Hyperandrogenism may also disrupt energy homeostasis, inducing higher central adiposity, insulin resistance, and glucose intolerance, which may exacerbate reproductive dysfunction. Androgens bind to androgen receptors (ARs), which are expressed in many reproductive and metabolic tissues, including brain sites that regulate the hypothalamo-pituitary-gonadal axis and energy homeostasis. The neuronal populations affected by androgen excess, however, have not been defined. We and others have shown that, in mice, AR is highly expressed in leptin receptor (LepRb) neurons, particularly in the arcuate (ARH) and the ventral premammillary nuclei (PMv). Here, we assessed if LepRb neurons, which are critical in the central regulation of energy homeostasis and exert permissive actions on puberty and fertility, have a role in the pathogenesis of female hyperandrogenism. Prenatally androgenized (PNA) mice lacking AR in LepRb cells (LepRbΔAR) show no changes in body mass, body composition, glucose homeostasis, or sexual maturation. They do show, however, a remarkable improvement of estrous cycles combined with normalization of ovary morphology compared to PNA controls. Our findings indicate that the prenatal androgenization effects on adult reproductive physiology (ie, anestrus and anovulation) are mediated by a subpopulation of LepRb neurons directly sensitive to androgens. They also suggest that the effects of hyperandrogenism on sexual maturation and reproductive function in adult females are controlled by distinct neural circuits.
Subject(s)
Anovulation , Hyperandrogenism , Polycystic Ovary Syndrome , Pregnancy , Humans , Mice , Female , Animals , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Hyperandrogenism/genetics , Hyperandrogenism/complications , Receptors, Leptin/genetics , Sexual Maturation , Androgens/pharmacology , Polycystic Ovary Syndrome/metabolism , Virilism , Estrous CycleABSTRACT
BACKGROUND: Nephronophthisis associated ciliopathies (NPHP-AC) comprise a group of autosomal recessive cystic kidney diseases that includes nephronophthisis (NPHP), Senior-Loken syndrome (SLS), Joubert syndrome (JBTS), and Meckel-Gruber syndrome (MKS). To date, causative mutations in NPHP-AC have been described for 18 different genes, rendering mutation analysis tedious and expensive. To overcome the broad genetic locus heterogeneity, a strategy of DNA pooling with consecutive massively parallel resequencing (MPR) was devised. METHODS: In 120 patients with severe NPHP-AC phenotypes, five pools of genomic DNA with 24 patients each were prepared which were used as templates in order to PCR amplify all 376 exons of 18 NPHP-AC genes (NPHP1, INVS, NPHP3, NPHP4, IQCB1, CEP290, GLIS2, RPGRIP1L, NEK8, TMEM67, INPP5E, TMEM216, AHI1, ARL13B, CC2D2A, TTC21B, MKS1, and XPNPEP3). PCR products were then subjected to MPR on an Illumina Genome-Analyser and mutations were subsequently assigned to their respective mutation carrier via CEL I endonuclease based heteroduplex screening and confirmed by Sanger sequencing. RESULTS: For proof of principle, DNA from patients with known mutations was used and detection of 22 out of 24 different alleles (92% sensitivity) was demonstrated. MPR led to the molecular diagnosis in 30/120 patients (25%) and 54 pathogenic mutations (27 novel) were identified in seven different NPHP-AC genes. Additionally, in 24 patients only single heterozygous variants of unknown significance were found. CONCLUSIONS: The combined approach of DNA pooling followed by MPR strongly facilitates mutation analysis in broadly heterogeneous single gene disorders. The lack of mutations in 75% of patients in this cohort indicates further extensive heterogeneity in NPHP-AC.
Subject(s)
Cilia/genetics , DNA Mutational Analysis/methods , Heteroduplex Analysis/methods , Kidney Diseases, Cystic/genetics , Cilia/pathology , Humans , Nucleic Acid Amplification Techniques , Polymerase Chain ReactionABSTRACT
The identification of recessive disease-causing genes by homozygosity mapping is often restricted by lack of suitable consanguineous families. To overcome these limitations, we apply homozygosity mapping to single affected individuals from outbred populations. In 72 individuals of 54 kindred ascertained worldwide with known homozygous mutations in 13 different recessive disease genes, we performed total genome homozygosity mapping using 250,000 SNP arrays. Likelihood ratio Z-scores (ZLR) were plotted across the genome to detect ZLR peaks that reflect segments of homozygosity by descent, which may harbor the mutated gene. In 93% of cases, the causative gene was positioned within a consistent ZLR peak of homozygosity. The number of peaks reflected the degree of inbreeding. We demonstrate that disease-causing homozygous mutations can be detected in single cases from outbred populations within a single ZLR peak of homozygosity as short as 2 Mb, containing an average of only 16 candidate genes. As many specialty clinics have access to cohorts of individuals from outbred populations, and as our approach will result in smaller genetic candidate regions, the new strategy of homozygosity mapping in single outbred individuals will strongly accelerate the discovery of novel recessive disease genes.
Subject(s)
Genes, Recessive , DNA Mutational Analysis , False Positive Reactions , Family Health , Female , Genetic Markers , Genetics, Population , Homozygote , Humans , Kidney Diseases, Cystic/genetics , Male , Models, Genetic , Nephrotic Syndrome/genetics , Pedigree , Steroids/pharmacologyABSTRACT
Nephronophthisis (NPHP), an autosomal recessive cystic kidney disease, is the most frequent genetic cause for end-stage renal failure in the first three decades of life. Mutations in 13 genes (NPHP1-NPHP11, AHI1, and CC2D2A) cause NPHP with ubiquitous expression of the corresponding proteins consistent with the multiorgan involvement of NPHP-related diseases. The genotype-phenotype correlation in these ciliopathies can be explained by gene locus heterogeneity, allelism, and the impact of modifier genes. In some NPHP-related ciliopathies, the nature of the recessive mutations determines disease severity. In order to define the genotype-phenotype correlation more clearly, we evaluated a worldwide cohort of 440 patients from 365 families with NPHP-related ciliopathies, in whom both disease-causing alleles were identified. The phenotypes were ranked in the order of severity from degenerative to degenerative/dysplastic to dysplastic. A genotype of two null alleles caused a range of phenotypes, with an increasing order of severity of NPHP1, NPHP3, NPHP4, NPHP5, NPHP2, NPHP10, NPHP6, to AHI1. Only NPHP6 showed allelic influences on the phenotypes; the presence of two null mutations caused dysplastic phenotypes, whereas at least one missense allele rescued it to a milder degenerative phenotype. We also found nine novel mutations in the NPHP genes. Thus, our studies have important implications for genetic counseling and planning of renal replacement therapy.
Subject(s)
Genetic Association Studies , Kidney Diseases, Cystic/congenital , Adaptor Proteins, Signal Transducing/genetics , Alleles , Cytoskeletal Proteins , Family , Humans , Kidney Diseases, Cystic/epidemiology , Kidney Diseases, Cystic/genetics , Kidney Failure, Chronic/epidemiology , Kidney Failure, Chronic/genetics , Membrane Proteins/genetics , MutationABSTRACT
Bardet-Biedl syndrome (BBS) is a rare, primarily autosomal-recessive ciliopathy. The phenotype of this pleiotropic disease includes retinitis pigmentosa, postaxial polydactyly, truncal obesity, learning disabilities, hypogonadism and renal anomalies, among others. To date, mutations in 15 genes (BBS1-BBS14, SDCCAG8) have been described to cause BBS. The broad genetic locus heterogeneity renders mutation screening time-consuming and expensive. We applied a strategy of DNA pooling and subsequent massively parallel resequencing (MPR) to screen individuals affected with BBS from 105 families for mutations in 12 known BBS genes. DNA was pooled in 5 pools of 21 individuals each. All 132 coding exons of BBS1-BBS12 were amplified by conventional PCR. Subsequent MPR was performed on an Illumina Genome Analyzer II™ platform. Following mutation identification, the mutation carrier was assigned by CEL I endonuclease heteroduplex screening and confirmed by Sanger sequencing. In 29 out of 105 individuals (28%), both mutated alleles were identified in 10 different BBS genes. A total of 35 different disease-causing mutations were confirmed, of which 18 mutations were novel. In 12 additional families, a total of 12 different single heterozygous changes of uncertain pathogenicity were found. Thus, DNA pooling combined with MPR offers a valuable strategy for mutation analysis of large patient cohorts, especially in genetically heterogeneous diseases such as BBS.
Subject(s)
Bardet-Biedl Syndrome/genetics , DNA Mutational Analysis/methods , Proteins/genetics , Amino Acid Sequence , Base Sequence , Chromosome Mapping , Cohort Studies , Consanguinity , Exons/genetics , Genetic Heterogeneity , Genetic Loci/genetics , Genetic Predisposition to Disease , Group II Chaperonins/genetics , Humans , Microtubule-Associated Proteins , Molecular Sequence Data , Mutation/geneticsABSTRACT
Nephronophthisis (NPHP) is an autosomal recessive kidney disease characterized by tubular basement membrane disruption, interstitial infiltration, and tubular cysts. NPHP leads to end-stage renal failure (ESRD) in the first three decades of life and is the most frequent genetic cause of chronic renal failure in children and young adults. Extrarenal manifestations are known, such as retinitis pigmentosa, brainstem and cerebellar anomalies, liver fibrosis, and ocular motor apraxia type Cogan. We report on a Turkish family with clinical signs of nephronophthisis. The phenotype occurred in two generations and therefore seemed to be inherited in an autosomal dominant pattern. Nevertheless, a deletion analysis of the NPHP1 gene on chromosome 2 was performed and showed a homozygous deletion. Analysis of the family pedigree indicated no obvious consanguinity in the last three generations. However, haplotype analysis demonstrated homozygosity on chromosome 2 indicating a common ancestor to the parents of all affected individuals. NPHP1 deletion analysis should always be considered in patients with apparently dominant nephronophthisis. Furthermore, three out of four patients developed ESRD between 27 and 43 years of age, which may be influenced by yet unknown modifier genes.