Glutamate neurotransmission from leptin receptor cells is required for typical puberty and reproductive function in female mice.

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.

Glutamate neurotransmission from leptin receptor cells is required for typical puberty and reproductive function in female mice.

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.

Deletion of Androgen Receptor in LepRb Cells Improves Estrous Cycles in Prenatally Androgenized Mice.

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.

ERα Signaling in GHRH/Kiss1 Dual-Phenotype Neurons Plays Sex-Specific Roles in Growth and Puberty.

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, ∼30% 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.

Hypothalamic and Cell-Specific Transcriptomes Unravel a Dynamic Neuropil Remodeling in Leptin-Induced and Typical Pubertal Transition in Female Mice.

Epidemiological and genome-wide association studies (GWAS) have shown high correlation between childhood obesity and advance in puberty. Early age at menarche is associated with a series of morbidities, including breast cancer, cardiovascular diseases, type 2 diabetes, and obesity. The adipocyte hormone leptin signals the amount of fat stores to the neuroendocrine reproductive axis via direct actions in the brain. Using mouse genetics, we and others have identified the hypothalamic ventral premammillary nucleus (PMv) and the agouti-related protein (AgRP) neurons in the arcuate nucleus (Arc) as primary targets of leptin action in pubertal maturation. However, the molecular mechanisms underlying leptin's effects remain unknown. Here we assessed changes in the PMv and Arc transcriptional program during leptin-stimulated and typical pubertal development using overlapping analysis of bulk RNA sequecing, TRAP sequencing, and the published database. Our findings demonstrate that dynamic somatodendritic remodeling and extracellular space organization underlie leptin-induced and typical pubertal maturation in female mice.

Dissociated Pmch and Cre Expression in Lactating Pmch-Cre BAC Transgenic Mice.

The melanin-concentrating hormone (MCH) system plays a role in many physiological processes including reproduction and lactation. However, research regarding the function of MCH on different aspects of the reproductive function lags, due in part to a lack of validated genetic models with which to interrogate the system. This is particularly true in the case of female reproduction, as the anatomy and function of the MCH system is not well-characterized in the female mouse. We set out to determine whether the commercially available Pmch-Cre transgenic mouse line is a viable model to study the role of MCH neurons in distinct female reproductive states. We found that Pmch is transiently expressed in several nuclei of the rostral forebrain at the end of lactation. This includes the medial subdivision of the medial preoptic nucleus, the paraventricular nucleus of the hypothalamus, the ventral subdivision of the lateral septum, the anterodorsal preoptic nucleus and the anterodorsal nucleus of the thalamus. The Pmch expression in these sites, however, does not reliably induce Cre expression in the Pmch-Cre (BAC) transgenic mouse, making this line an inadequate model with which to study the role of MCH in behavioral and/or neuroendocrine adaptations of lactation. We also contribute to the general knowledge of the anatomy of the murine MCH system by showing that lactation-induced Pmch expression in the rostral forebrain is mostly observed in GABAergic (VGAT) neurons, in contrast to the typical MCH neurons of the tuberal and posterior hypothalamus which are glutamatergic (VGLUT2).

PI3K signalling in leptin receptor cells: Role in growth and reproduction.

Nutrition and growth are important signals for pubertal development, although how they are perceived and integrated in brain circuits has not been well defined. Growth hormones and metabolic cues both recruit phosphatidylinositol 3-kinase (PI3K) signalling in hypothalamic sites, although whether they converge into the same neuronal population(s) is also not known. In this review, we discuss recent findings from our laboratory showing the role of PI3K subunits in cells directly responsive to the adipocyte-derived hormone leptin in the coordination of growth, pubertal development and fertility. Mice with deletion of PI3K p110α and p110ß catalytic subunits in leptin receptor cells (LRΔα+ß ) have a lean phenotype associated with increased energy expenditure, locomotor activity and thermogenesis. The LRΔα+ß mice also show deficient growth and delayed puberty. Deletion of a single subunit (ie, p110α) in LR cells (LRΔα ) causes a similar phenotype of increased energy expenditure, deficient growth and delayed pubertal development, indicating that these functions are preferably controlled by p110α. The LRΔα mice show enhanced leptin sensitivity in metabolic regulation but, remarkably, these mice are unresponsive to the effects of leptin on growth and puberty. PI3K is also recruited by insulin and a subpopulation of LR neurones is responsive to i.c.v. insulin administration. Deletion of insulin receptor in LR cells causes no changes in body weight or linear growth and induces only a mild delay in pubertal completion. Our findings demonstrate that PI3K in LR cells plays an essential role in growth and reproduction. We will also discuss the potential neural pathways underlying these effects.

Insulin signaling in LepR cells modulates fat and glucose homeostasis independent of leptin.

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.

Can H2 -receptor upregulation and raised histamine explain an anaphylactoid reaction on cessation of ranitidine in a 19-year-old female? A case report.

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.

Obesity and High-Fat Diet Induce Distinct Changes in Placental Gene Expression and Pregnancy Outcome.

Obese women are at high risk of pregnancy complications, including preeclampsia, miscarriage, preterm birth, stillbirth, and neonatal death. In the current study, we aimed to determine the effects of obesity on pregnancy outcome and placental gene expression in preclinical mouse models of genetic and nutritional obesity. The leptin receptor (LepR) null-reactivatable (LepRloxTB), LepR-deficient (Leprdb/+), and high-fat diet (HFD)-fed mice were assessed for fertility, pregnancy outcome, placental morphology, and placental transcriptome using standard quantitative polymerase chain reaction (qPCR) and qPCR arrays. The restoration of fertility of LepRloxTB was performed by stereotaxic delivery of adeno-associated virus-Cre into the hypothalamic ventral premammillary nucleus. Fertile LepRloxTB females were morbidly obese, whereas the wild-type mice-fed HFD showed only a mild increase in body weight. Approximately 80% of the LepRloxTB females had embryo resorptions (∼40% of the embryos). In HFD mice, the number of resorptions was not different from controls fed a regular diet. Placentas of resorbed embryos from obese mice displayed necrosis and inflammatory infiltrate in the labyrinth and changes in the expression of genes associated with angiogenesis and inflammation (e.g., Vegfa, Hif1a, Nfkbia, Tlr3, Tlr4). In contrast, placentas from embryos of females on HFD showed changes in a different set of genes, mostly associated with cellular growth and response to stress (e.g., Plg, Ang, Igf1, Igfbp1, Fgf2, Tgfb2, Serpinf1). Sexual dimorphism in gene expression was only apparent in placentas from obese LepRloxTB mice. Our findings indicate that an obese environment and HFD have distinct effects on pregnancy outcome and the placental transcriptome.

PI3Kα inactivation in leptin receptor cells increases leptin sensitivity but disrupts growth and reproduction.

The role of PI3K in leptin physiology has been difficult to determine due to its actions downstream of several metabolic cues, including insulin. Here, we used a series of mouse models to dissociate the roles of specific PI3K catalytic subunits and of insulin receptor (InsR) downstream of leptin signaling. We show that disruption of p110α and p110ß subunits in leptin receptor cells (LRΔα+ß) produces a lean phenotype associated with increased energy expenditure, locomotor activity, and thermogenesis. LRΔα+ß mice have deficient growth and delayed puberty. Single subunit deletion (i.e., p110α in LRΔα) resulted in similarly increased energy expenditure, deficient growth, and pubertal development, but LRΔα mice have normal locomotor activity and thermogenesis. Blunted PI3K in leptin receptor (LR) cells enhanced leptin sensitivity in metabolic regulation due to increased basal hypothalamic pAKT, leptin-induced pSTAT3, and decreased PTEN levels. However, these mice are unresponsive to leptin's effects on growth and puberty. We further assessed if these phenotypes were associated with disruption of insulin signaling. LRΔInsR mice have no metabolic or growth deficit and show only mild delay in pubertal completion. Our findings demonstrate that PI3K in LR cells plays an essential role in energy expenditure, growth, and reproduction. These actions are independent from insulin signaling.

AMPKα2 in Kiss1 Neurons Is Required for Reproductive Adaptations to Acute Metabolic Challenges in Adult Female Mice.

A temporary and reversible inhibition of the hypothalamo-pituitary-gonadal axis is adaptive when energy reserves are diminished, allowing individual survival and energy accumulation for eventual reproduction. The AMP-activated protein kinase (AMPK) works as a cellular sensor of the AMP to ATP ratio and ultimately of energy availability. Activation of AMPK suppresses ATP-consuming processes and stimulates ATP-producing pathways. The AMPK α2 catalytic subunit is expressed in multiple hypothalamic nuclei including those associated with reproductive control, ie, the anteroventral periventricular nucleus and the arcuate nucleus. Subsets of kisspeptin neurons in the anteroventral periventricular nucleus (20% in females) and arcuate nucleus (45% in males and 65% in females) coexpress AMPKα2 mRNA. Using the Cre-loxP approach, we assessed whether AMPKα2 in Kiss1 cells is required for body weight and reproductive function. The AMPKα2-deleted mice show no difference in body weight and time for sexual maturation compared with controls. Males and females are fertile and have normal litter size. The AMPKα2-deleted and control females have similar estradiol feedback responses and show no difference in Kiss1 mRNA expression after ovariectomy or ovariectomy plus estradiol replacement. In males, acute fasting decreased Kiss1 mRNA expression in both groups, but no effect was observed in females. However, after an acute fasting, control mice displayed prolonged diestrous phase, but AMPKα2-deleted females showed no disruption of estrous cycles. Our findings demonstrate that the AMPKα2 catalytic subunit in Kiss1 cells is dispensable for body weight and reproductive function in mice but is necessary for the reproductive adaptations to conditions of acute metabolic distress.

Leptin receptor null mice with reexpression of LepR in GnRHR expressing cells display elevated FSH levels but remain in a prepubertal state.

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.

Whole exome sequencing identifies causative mutations in the majority of consanguineous or familial cases with childhood-onset increased renal echogenicity.

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.

Role of the adipocyte-derived hormone leptin in reproductive control.

Achievement of sexual maturation and maintenance of fertility in adulthood are functions that are sensitive to the metabolic status of the organism, particularly the magnitude of fat reserves. In this sense, the adipocyte-derived hormone, leptin, plays a major role in linking metabolic cues and the control of multiple neuroendocrine axes. The hypothalamus is a key site mediating leptin actions, including those involved in the modulation of the hypothalamus-pituitary-gonads (HPG) axis at different stages of development and in different environmental conditions. In the present review, we provide an update of the role of leptin in reproduction and discuss its interactions with neurons, neurotransmitters and downstream targets of the reproductive axis, with a special emphasis on the actions of leptin in the central nervous system. We hope this review will contribute to the understanding of the mechanisms whereby metabolic signals, especially leptin, influence the reproductive neuroendocrine axis modulating its activity in different nutritional states. Special attention will be given to recent advances in the identification of key hypothalamic sites and signaling pathways relevant to leptin's action in reproductive control.

Mutations in EMP2 cause childhood-onset nephrotic syndrome.

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.

Whole-exome resequencing distinguishes cystic kidney diseases from phenocopies in renal ciliopathies.

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.

ADCK4 mutations promote steroid-resistant nephrotic syndrome through CoQ10 biosynthesis disruption.

Identification of single-gene causes of steroid-resistant nephrotic syndrome (SRNS) has furthered the understanding of the pathogenesis of this disease. Here, using a combination of homozygosity mapping and whole human exome resequencing, we identified mutations in the aarF domain containing kinase 4 (ADCK4) gene in 15 individuals with SRNS from 8 unrelated families. ADCK4 was highly similar to ADCK3, which has been shown to participate in coenzyme Q10 (CoQ10) biosynthesis. Mutations in ADCK4 resulted in reduced CoQ10 levels and reduced mitochondrial respiratory enzyme activity in cells isolated from individuals with SRNS and transformed lymphoblasts. Knockdown of adck4 in zebrafish and Drosophila recapitulated nephrotic syndrome-associated phenotypes. Furthermore, ADCK4 was expressed in glomerular podocytes and partially localized to podocyte mitochondria and foot processes in rat kidneys and cultured human podocytes. In human podocytes, ADCK4 interacted with members of the CoQ10 biosynthesis pathway, including COQ6, which has been linked with SRNS and COQ7. Knockdown of ADCK4 in podocytes resulted in decreased migration, which was reversed by CoQ10 addition. Interestingly, a patient with SRNS with a homozygous ADCK4 frameshift mutation had partial remission following CoQ10 treatment. These data indicate that individuals with SRNS with mutations in ADCK4 or other genes that participate in CoQ10 biosynthesis may be treatable with CoQ10.

ZMYND10 is mutated in primary ciliary dyskinesia and interacts with LRRC6.

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.

Identification of 99 novel mutations in a worldwide cohort of 1,056 patients with a nephronophthisis-related ciliopathy.

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.