Cloning and characterization of the mouse Mcoln1 gene reveals an alternatively spliced transcript not seen in humans

BACKGROUND: Mucolipidosis type IV (MLIV) is an autosomal recessive lysosomal storage disorder characterized by severe neurologic and ophthalmologic abnormalities. Recently the MLIV gene, MCOLN1, has been identified as a new member of the transient receptor potential (TRP) cation channel superfamily. Here we report the cloning and characterization of the mouse homologue, Mcoln1, and report a novel splice variant that is not seen in humans. RESULTS: The human and mouse genes display a high degree of synteny. Mcoln1 shows 91% amino acid and 86% nucleotide identity to MCOLN1. Also, Mcoln1 maps to chromosome 8 and contains an open reading frame of 580 amino acids, with a transcript length of approximately 2 kb encoded by 14 exons, similar to its human counterpart. The transcript that results from murine specific alternative splicing encodes a 611 amino acid protein that differs at the c-terminus. CONCLUSIONS: Mcoln1 is highly similar to MCOLN1, especially in the transmembrane domains and ion pore region. Also, the late endosomal/lysosomal targeting signal is conserved, supporting the hypothesis that the protein is localized to these vesicle membranes. To date, there are very few reports describing species-specific splice variants. While identification of Mcoln1 is crucial to the development of mouse models for MLIV, the fact that there are two transcripts in mice suggests an additional or alternate function of the gene that may complicate phenotypic assessment

Clinical Management of Congenital Hypogonadotropic Hypogonadism

The initiation and maintenance of reproductive capacity in humans is dependent on pulsatile secretion of the hypothalamic hormone GnRH. Congenital hypogonadotropic hypogonadism (CHH) is a rare disorder that results from the failure of the normal episodic GnRH secretion, leading to delayed puberty and infertility. CHH can be associated with an absent sense of smell, also termed Kallmann syndrome, or with other anomalies. CHH is characterized by rich genetic heterogeneity, with mutations in >30 genes identified to date acting either alone or in combination. CHH can be challenging to diagnose, particularly in early adolescence where the clinical picture mirrors that of constitutional delay of growth and puberty. Timely diagnosis and treatment will induce puberty, leading to improved sexual, bone, metabolic, and psychological health. In most cases, patients require lifelong treatment, yet a notable portion of male patients (approximate to 10% to 20%) exhibit a spontaneous recovery of their reproductive function. Finally, fertility can be induced with pulsatile GnRH treatment or gonadotropin regimens in most patients. In summary, this review is a comprehensive synthesis of the current literature available regarding the diagnosis, patient management, and genetic foundations of CHH relative to normal reproductive development.Peer reviewe

Fat-to-glucose interconversion by hydrodynamic transfer of two glyoxylate cycle enzyme genes

The glyoxylate cycle, which is well characterized in higher plants and some microorganisms but not in vertebrates, is able to bypass the citric acid cycle to achieve fat-to-carbohydrate interconversion. In this context, the hydrodynamic transfer of two glyoxylate cycle enzymes, such as isocytrate lyase (ICL) and malate synthase (MS), could accomplish the shift of using fat for the synthesis of glucose. Therefore, 20 mice weighing 23.37 ± 0.96 g were hydrodinamically gene transferred by administering into the tail vein a bolus with ICL and MS. After 36 hours, body weight, plasma glucose, respiratory quotient and energy expenditure were measured. The respiratory quotient was increased by gene transfer, which suggests that a higher carbohydrate/lipid ratio is oxidized in such animals. This application could help, if adequate protocols are designed, to induce fat utilization for glucose synthesis, which might be eventually useful to reduce body fat depots in situations of obesity and diabetes

Pathogenic mosaic variants in congenital hypogonadotropic hypogonadism

PURPOSE Congenital hypogonadotropic hypogonadism (CHH) is a rare disorder resulting in absent puberty and infertility. The genetic architecture is complex with multiple loci involved, variable expressivity, and incomplete penetrance. The majority of cases are sporadic, consistent with a disease affecting fertility. The current study aims to investigate mosaicism as a genetic mechanism for CHH, focusing on de novo rare variants in CHH genes. METHODS We evaluated 60 trios for de novo rare sequencing variants (RSV) in known CHH genes using exome sequencing. Potential mosaicism was suspected among RSVs with altered allelic ratios and confirmed using customized ultradeep sequencing (UDS) in multiple tissues. RESULTS Among the 60 trios, 10 probands harbored de novo pathogenic variants in CHH genes. Custom UDS demonstrated that three of these de novo variants were in fact postzygotic mosaicism-two in FGFR1 (p.Leu630Pro and p.Gly348Arg), and one in CHD7 (p.Arg2428*). Statistically significant variation across multiple tissues (DNA from blood, buccal, hair follicle, urine) confirmed their mosaic nature. CONCLUSIONS We identified a significant number of de novo pathogenic variants in CHH of which a notable number (3/10) exhibited mosaicism. This report of postzygotic mosaicism in CHH patients provides valuable information for accurate genetic counseling

Neuron-Derived Neurotrophic Factor Is Mutated in Congenital Hypogonadotropic Hypogonadism

Congenital hypogonadotropic hypogonadism (CHH) is a rare genetic disorder characterized by infertility and the absence of puberty. Defects in GnRH neuron migration or altered GnRH secretion and/or action lead to a severe gonadotropin-releasing hormone (GnRH) deficiency. Given the close developmental association of GnRH neurons with the olfactory primary axons, CHH is often associated with anosmia or hyposmia, in which case it is defined as Kallmann syndrome (KS). The genetics of CHH are heterogeneous, and >40 genes are involved either alone or in combination. Several CHH-related genes controlling GnRH ontogeny encode proteins containing fibronectin-3 (FN3) domains, which are important for brain and neural development. Therefore, we hypothesized that defects in other FN3-superfamily genes would underlie CHH. Next-generation sequencing was performed for 240 CHH unrelated probands and filtered for rare, protein-truncating variants (PTVs) in FN3-superfamily genes. Compared to gnomAD controls the CHH cohort was statistically enriched for PTVs in neuron-derived neurotrophic factor (NDNF) (p = 1.40 x 10(-6)). Three heterozygous PTVs (p.Lys62*, p.Tyr128Thrfs*55, and p.Trp469*, all absent from the gnomAD database) and an additional heterozygous missense mutation (p.Thr201Ser) were found in four KS probands. Notably, NDNF is expressed along the GnRH neuron migratory route in both mouse embryos and human fetuses and enhances GnRH neuron migration. Further, knock down of the zebrafish ortholog of NDNF resulted in altered GnRH migration. Finally, mice lacking Ndnf showed delayed GnRH neuron migration and altered olfactory axonal projections to the olfactory bulb; both results are consistent with a role of NDNF in GnRH neuron development. Altogether, our results highlight NDNF as a gene involved in the GnRH neuron migration implicated in KS.Peer reviewe

KLB , encoding β‐Klotho, is mutated in patients with congenital hypogonadotropic hypogonadism

Congenital hypogonadotropic hypogonadism (CHH) is a rare genetic form of isolated gonadotropin‐releasing hormone (GnRH) deficiency caused by mutations in > 30 genes. Fibroblast growth factor receptor 1 (FGFR1) is the most frequently mutated gene in CHH and is implicated in GnRH neuron development and maintenance. We note that a CHH FGFR1 mutation (p.L342S) decreases signaling of the metabolic regulator FGF21 by impairing the association of FGFR1 with β‐Klotho (KLB), the obligate co‐receptor for FGF21. We thus hypothesized that the metabolic FGF21/KLB/FGFR1 pathway is involved in CHH. Genetic screening of 334 CHH patients identified seven heterozygous loss‐of‐function KLB mutations in 13 patients (4%). Most patients with KLB mutations (9/13) exhibited metabolic defects. In mice, lack of Klb led to delayed puberty, altered estrous cyclicity, and subfertility due to a hypothalamic defect associated with inability of GnRH neurons to release GnRH in response to FGF21. Peripheral FGF21 administration could indeed reach GnRH neurons through circumventricular organs in the hypothalamus. We conclude that FGF21/KLB/FGFR1 signaling plays an essential role in GnRH biology, potentially linking metabolism with reproduction

Health impact of US military service in a large population-based military cohort: findings of the Millennium Cohort Study, 2001-2008

<p>Abstract</p> <p>Background</p> <p>Combat-intense, lengthy, and multiple deployments in Iraq and Afghanistan have characterized the new millennium. The US military's all-volunteer force has never been better trained and technologically equipped to engage enemy combatants in multiple theaters of operations. Nonetheless, concerns over potential lasting effects of deployment on long-term health continue to mount and are yet to be elucidated. This report outlines how findings from the first 7 years of the Millennium Cohort Study have helped to address health concerns related to military service including deployments.</p> <p>Methods</p> <p>The Millennium Cohort Study was designed in the late 1990s to address veteran and public concerns for the first time using prospectively collected health and behavioral data.</p> <p>Results</p> <p>Over 150 000 active-duty, reserve, and National Guard personnel from all service branches have enrolled, and more than 70% of the first 2 enrollment panels submitted at least 1 follow-up survey. Approximately half of the Cohort has deployed in support of operations in Iraq and Afghanistan.</p> <p>Conclusion</p> <p>The Millennium Cohort Study is providing prospective data that will guide public health policymakers for years to come by exploring associations between military exposures and important health outcomes. Strategic studies aim to identify, reduce, and prevent adverse health outcomes that may be associated with military service, including those related to deployment.</p

Improving the genetic diagnosis of congenital hypogonadotropic hypogonadism

Congénital hypogonadotropic hypogonadism (CHH) is a rare endocrine disorder in which patients fail to go through puberty and are infertile as a result of gonadotropin- releasing hormone (GnRH) deficiency. Although mutations in &gt;30 genes have been shown to cause CHH, only 35% of patients have a mutation in these known genes. Oligogenicity, reduced penetrance, and variable expressivity complicates the genetic picture of the disorder as well. The overall goal of this thesis is to improve the genetic diagnosis of CHH for patients and their families. Project 1 focused on elucidating the genetic architecture and mutational spectrum in CHH, with a specific focus on differentiating CHH from constitutional delay of growth and puberty (CDGP)—a condition of transient GnRH deficiency that clinically overlaps with CHH. Both CHH and CDGP patients present in adolescence with a failure to initiate puberty, and differentiating these two disorders is challenging for clinicians. Distinct differences were found between CHH and CDGP patients—specifically that CHH patients have a higher burden of mutations in the known CHH genes, as well as a higher degree of oligogenicity (mutations in more than one gene). This work provides a foundation to further explore genetic tools to differentiate between these two conditions. Additional collaborative works describing the pre-pubertal diagnosis of CHH in a patient and refining the diagnosis of CHH patients with mutations in CHD7 are presented. In Project 2, 60 CHH trios (affected proband and unaffected parents) were used to evaluate the frequency of de novo mutations in the known CHH genes. A total of 10 probands had de novo mutations, most commonly in FGFR1 and CHD7. Additionally, 3 CHH patients were found to harbor post-zygotic mutations resulting in mosaicism. This was confirmed by demonstrating variable levels of the mutations across different tissue types. The results of this project provide valuable information for improving the accuracy and content of genetic testing and subsequent counseling of patients. In Project 3, a variety of methods were used to discover new genes related to CHH. Through our own work and European collaborations, we participated in the discovery of mutations in KLB, DCC, NTN1,AMH and PTCH1 underlying CHH. In these studies, genes were initially identified using biological methods, candidates from animal models, and genes involved in overlapping syndromes which were subsequently confirmed in CHH patients. -- L'hypogonadisme hypogonadotrope congénital (CHH) est un trouble endocrinien rare pour lequel les patients ne développent pas de puberté et sont infertiles. Ce trouble est provoqué par une déficience en hormone de libération des gonadotrophines hypophysaires (GnRH). Bien qu'il ait été démontré que des mutations dans plus de 30 gènes causent le CHH, seul 35% des patients ont une mutation dans un de ces gènes. Le phénomène d'oligogénicité, la pénétrance réduite et l'expressivité variable compliquent la compréhension génétique de la maladie. L’objectif global de cette thèse est d'améliorer le diagnostic génétique des patients CHH et de leur famille. Le projet 1 s'est concentré sur la résolution de l'architecture génétique et du spectre mutationnel dans le CHH, avec un accent particulier sur la différenciation entre le CHH et le retard constitutionnel de croissance et de la puberté (CDGP) - une condition de déficit transitoire en GnRH qui cliniquement chevauche le CHH. Les patients CHH et CDGP présentent à l'adolescence une incapacité à amorcer la puberté, et différencier ces deux troubles est difficile pour les cliniciens. Des différences distinctes ont été constatées entre les patients CHH et CDGP - en particulier, les patients CHH ont un fardeau plus élevé de mutations dans les gènes CHH connus, ainsi qu'un degré plus élevé d'oligogénicité (mutations dans plus d'un gène). Ce travail fournit une base pour explorer davantage les outils génétiques afin de différencier ces deux conditions. Des travaux collaboratifs supplémentaires sont présentés ; un diagnostic prépubère CHH chez un patient ainsi qu'un diagnostic affiné chez des patients CHH présentant des mutations dans le gène CHD7. Dans le projet 2, 60 trios CHH (proband affectés et parents non affectés) ont été utilisés pour évaluer la fréquence des mutations de novo dans les gènes CHH connus. Au total, 10 probands présentaient des mutations de novo, le plus souvent dans les gènes FGFR1 et CHD7. En outre, 3 patients CHH ont été trouvés pour héberger des mutations post-zygotiques résultant en un mosaïcisme qui a été confirmé en démontrant des niveaux variables des mutations à travers différents types de tissus. Les résultats de ce projet fournissent des informations précieuses qui permettent d'améliorer la précision et la teneur des tests génétiques ainsi que la qualité des conseils ultérieurs aux patients. Dans le projet 3, diverses méthodes ont été utilisées pour découvrir de nouveaux gènes liés à CHH. Grâce à notre propre travail et à nos collaborations européennes, nous avons participé à la découverte de mutations dans les gènes KLB, DCC, NTN1, AMH et PTCH1 qui causent un CHH. Dans ces études, les gènes ont été initialement identifiés à l'aide de méthodes biologiques, de candidats issus de modèles animaux et de gènes impliqués dans des syndromes se chevauchant, qui ont ensuite été confirmés chez des patients CHH

Biotin-Responsive Basal Ganglia Disease Maps to 2q36.3 and Is Due to Mutations in SLC19A3

Biotin-responsive basal ganglia disease (BBGD) is a recessive disorder with childhood onset that presents as a subacute encephalopathy, with confusion, dysarthria, and dysphagia, and that progresses to severe cogwheel rigidity, dystonia, quadriparesis, and eventual death, if left untreated. BBGD symptoms disappear within a few days with the administration of high doses of biotin (5–10 mg/kg/d). On brain magnetic resonance imaging examination, patients display central bilateral necrosis in the head of the caudate, with complete or partial involvement of the putamen. All patients diagnosed to date are of Saudi, Syrian, or Yemeni ancestry, and all have consanguineous parents. Using linkage analysis in four families, we mapped the genetic defect near marker D2S2158 in 2q36.3 (LOD=5.9; θ=0.0) to a minimum candidate region (∼2 Mb) between D2S2354 and D2S1256, on the basis of complete homozygosity. In this segment, each family displayed one of two different missense mutations that altered the coding sequence of SLC19A3, the gene for a transporter related to the reduced-folate (encoded by SLC19A1) and thiamin (encoded by SLC19A2) transporters