A mutation in the nucleoporin-107 gene causes XX gonadal dysgenesis

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Abstract

Ovarian development and maintenance are poorly understood; however, diseases that affect these processes can offer insights into the underlying mechanisms. XX female gonadal dysgenesis (XX-GD) is a rare, genetically heterogeneous disorder that is characterized by underdeveloped, dysfunctional ovaries, with subsequent lack of spontaneous pubertal development, primary amenorrhea, uterine hypoplasia, and hypergonadotropic hypogonadism. Here, we report an extended consanguineous family of Palestinian origin, in which 4 females exhibited XX-GD. Using homozygosity mapping and whole-exome sequencing, we identified a recessive missense mutation in nucleoporin-107 (NUP107, c.1339G>A, p.D447N). This mutation segregated with the XX-GD phenotype and was not present in available databases or in 150 healthy ethnically matched controls. NUP107 is a component of the nuclear pore complex, and the NUP107-associated protein SEH1 is required for oogenesis in Drosophila. In Drosophila, Nup107 knockdown in somatic gonadal cells resulted in female sterility, whereas males were fully fertile. Transgenic rescue of Drosophila females bearing the Nup107^D364N mutation, which corresponds to the human NUP107 (p.D447N), resulted in almost complete sterility, with a marked reduction in progeny, morphologically aberrant eggshells, and disintegrating egg chambers, indicating defective oogenesis. These results indicate a pivotal role for NUP107 in ovarian development and suggest that nucleoporin defects may play a role in milder and more common conditions such as premature ovarian failure.

Authors

Ariella Weinberg-Shukron, Paul Renbaum, Rachel Kalifa, Sharon Zeligson, Ziva Ben-Neriah, Amatzia Dreifuss, Amal Abu-Rayyan, Noa Maatuk, Nilly Fardian, Dina Rekler, Moien Kanaan, Abraham O. Samson, Ephrat Levy-Lahad, Offer Gerlitz, David Zangen

Reengineering a transmembrane protein to treat muscular dystrophy using exon skipping

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Abstract

Exon skipping uses antisense oligonucleotides as a treatment for genetic diseases. The antisense oligonucleotides used for exon skipping are designed to bypass premature stop codons in the target RNA and restore reading frame disruption. Exon skipping is currently being tested in humans with dystrophin gene mutations who have Duchenne muscular dystrophy. For Duchenne muscular dystrophy, the rationale for exon skipping derived from observations in patients with naturally occurring dystrophin gene mutations that generated internally deleted but partially functional dystrophin proteins. We have now expanded the potential for exon skipping by testing whether an internal, in-frame truncation of a transmembrane protein γ-sarcoglycan is functional. We generated an internally truncated γ-sarcoglycan protein that we have termed Mini-Gamma by deleting a large portion of the extracellular domain. Mini-Gamma provided functional and pathological benefits to correct the loss of γ-sarcoglycan in a Drosophila model, in heterologous cell expression studies, and in transgenic mice lacking γ-sarcoglycan. We generated a cellular model of human muscle disease and showed that multiple exon skipping could be induced in RNA that encodes a mutant human γ-sarcoglycan. Since Mini-Gamma represents removal of 4 of the 7 coding exons in γ-sarcoglycan, this approach provides a viable strategy to treat the majority of patients with γ-sarcoglycan gene mutations.

Authors

Quan Q. Gao, Eugene Wyatt, Jeff A. Goldstein, Peter LoPresti, Lisa M. Castillo, Alec Gazda, Natalie Petrossian, Judy U. Earley, Michele Hadhazy, David Y. Barefield, Alexis R. Demonbreun, Carsten Bönnemann, Matthew Wolf, Elizabeth M. McNally

Proof-of-principle rapid noninvasive prenatal diagnosis of autosomal recessive founder mutations

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Abstract

BACKGROUND. Noninvasive prenatal testing can be used to accurately detect chromosomal aneuploidies in circulating fetal DNA; however, the necessity of parental haplotype construction is a primary drawback to noninvasive prenatal diagnosis (NIPD) of monogenic disease. Family-specific haplotype assembly is essential for accurate diagnosis of minuscule amounts of circulating cell-free fetal DNA; however, current haplotyping techniques are too time-consuming and laborious to be carried out within the limited time constraints of prenatal testing, hampering practical application of NIPD in the clinic. Here, we have addressed this pitfall and devised a universal strategy for rapid NIPD of a prevalent mutation in the Ashkenazi Jewish (AJ) population.

METHODS. Pregnant AJ couples, carrying mutation(s) in GBA, which encodes acid β-glucosidase, were recruited at the SZMC Gaucher Clinic. Targeted next-generation sequencing of GBA-flanking SNPs was performed on peripheral blood samples from each couple, relevant mutation carrier family members, and unrelated individuals who are homozygotes for an AJ founder mutation. Allele-specific haplotypes were constructed based on linkage, and a consensus Gaucher disease–associated founder mutation–flanking haplotype was fine mapped. Together, these haplotypes were used for NIPD. All test results were validated by conventional prenatal or postnatal diagnostic methods.

RESULTS. Ten parental alleles in eight unrelated fetuses were diagnosed successfully based on the noninvasive method developed in this study. The consensus mutation–flanking haplotype aided diagnosis for 6 of 9 founder mutation alleles.

CONCLUSIONS. The founder NIPD method developed and described here is rapid, economical, and readily adaptable for prenatal testing of prevalent autosomal recessive disease-causing mutations in an assortment of worldwide populations.

FUNDING. SZMC, Protalix Biotherapeutics Inc., and Centogene AG.

Authors

David A. Zeevi, Gheona Altarescu, Ariella Weinberg-Shukron, Fouad Zahdeh, Tama Dinur, Gaya Chicco, Yair Herskovitz, Paul Renbaum, Deborah Elstein, Ephrat Levy-Lahad, Arndt Rolfs, Ari Zimran

RAP1-mediated MEK/ERK pathway defects in Kabuki syndrome

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Abstract

The genetic disorder Kabuki syndrome (KS) is characterized by developmental delay and congenital anomalies. Dominant mutations in the chromatin regulators lysine (K)–specific methyltransferase 2D (KMT2D) (also known as MLL2) and lysine (K)–specific demethylase 6A (KDM6A) underlie the majority of cases. Although the functions of these chromatin-modifying proteins have been studied extensively, the physiological systems regulated by them are largely unknown. Using whole-exome sequencing, we identified a mutation in RAP1A that was converted to homozygosity as the result of uniparental isodisomy (UPD) in a patient with KS and a de novo, dominant mutation in RAP1B in a second individual with a KS-like phenotype. We elucidated a genetic and functional interaction between the respective KS-associated genes and their products in zebrafish models and patient cell lines. Specifically, we determined that dysfunction of known KS genes and the genes identified in this study results in aberrant MEK/ERK signaling as well as disruption of F-actin polymerization and cell intercalation. Moreover, these phenotypes could be rescued in zebrafish models by rebalancing MEK/ERK signaling via administration of small molecule inhibitors of MEK. Taken together, our studies suggest that the KS pathophysiology overlaps with the RASopathies and provide a potential direction for treatment design.

Authors

Nina Bögershausen, I-Chun Tsai, Esther Pohl, Pelin Özlem Simsek Kiper, Filippo Beleggia, E. Ferda Percin, Katharina Keupp, Angela Matchan, Esther Milz, Yasemin Alanay, Hülya Kayserili, Yicheng Liu, Siddharth Banka, Andrea Kranz, Martin Zenker, Dagmar Wieczorek, Nursel Elcioglu, Paolo Prontera, Stanislas Lyonnet, Thomas Meitinger, A. Francis Stewart, Dian Donnai, Tim M. Strom, Koray Boduroglu, Gökhan Yigit, Yun Li, Nicholas Katsanis, Bernd Wollnik

Rapamycin improves TIE2-mutated venous malformation in murine model and human subjects

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Abstract

Venous malformations (VMs) are composed of ectatic veins with scarce smooth muscle cell coverage. Activating mutations in the endothelial cell tyrosine kinase receptor TIE2 are a common cause of these lesions. VMs cause deformity, pain, and local intravascular coagulopathy, and they expand with time. Targeted pharmacological therapies are not available for this condition. Here, we generated a model of VMs by injecting HUVECs expressing the most frequent VM-causing TIE2 mutation, TIE2-L914F, into immune-deficient mice. TIE2-L914F–expressing HUVECs formed VMs with ectatic blood-filled channels that enlarged over time. We tested both rapamycin and a TIE2 tyrosine kinase inhibitor (TIE2-TKI) for their effects on murine VM expansion and for their ability to inhibit mutant TIE2 signaling. Rapamycin prevented VM growth, while TIE2-TKI had no effect. In cultured TIE2-L914F–expressing HUVECs, rapamycin effectively reduced mutant TIE2-induced AKT signaling and, though TIE2-TKI did target the WT receptor, it only weakly suppressed mutant-induced AKT signaling. In a prospective clinical pilot study, we analyzed the effects of rapamycin in 6 patients with difficult–to-treat venous anomalies. Rapamycin reduced pain, bleeding, lesion size, functional and esthetic impairment, and intravascular coagulopathy. This study provides a VM model that allows evaluation of potential therapeutic strategies and demonstrates that rapamycin provides clinical improvement in patients with venous malformation.

Authors

Elisa Boscolo, Nisha Limaye, Lan Huang, Kyu-Tae Kang, Julie Soblet, Melanie Uebelhoer, Antonella Mendola, Marjut Natynki, Emmanuel Seront, Sophie Dupont, Jennifer Hammer, Catherine Legrand, Carlo Brugnara, Lauri Eklund, Miikka Vikkula, Joyce Bischoff, Laurence M. Boon

Med12 gain-of-function mutation causes leiomyomas and genomic instability

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Abstract

Uterine leiomyomas are benign tumors that can cause pain, bleeding, and infertility in some women. Mediator complex subunit 12 (MED12) exon 2 variants are associated with uterine leiomyomas; however, the causality of MED12 variants, their genetic mode of action, and their role in genomic instability have not been established. Here, we generated a mouse model that conditionally expresses a Med12 missense variant (c.131G>A) in the uterus and demonstrated that this alteration alone promotes uterine leiomyoma formation and hyperplasia in both WT mice and animals harboring a uterine mesenchymal cell–specific Med12 deletion. Compared with WT animals, expression of Med12 c.131G>A in conditional Med12–KO mice resulted in earlier onset of leiomyoma lesions that were also greater in size. Moreover, leiomyomatous, Med12 c.131G>A variant–expressing uteri developed chromosomal rearrangements. Together, our results show that the common human leiomyoma–associated MED12 variant can cause leiomyomas in mice via a gain of function that drives genomic instability, which is frequently observed in human leiomyomas.

Authors

Priya Mittal, Yong-hyun Shin, Svetlana A. Yatsenko, Carlos A. Castro, Urvashi Surti, Aleksandar Rajkovic

DNA methylation directs functional maturation of pancreatic β cells

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Abstract

Pancreatic β cells secrete insulin in response to postprandial increases in glucose levels to prevent hyperglycemia and inhibit insulin secretion under fasting conditions to protect against hypoglycemia. β cells lack this functional capability at birth and acquire glucose-stimulated insulin secretion (GSIS) during neonatal life. Here, we have shown that during postnatal life, the de novo DNA methyltransferase DNMT3A initiates a metabolic program by repressing key genes, thereby enabling the coupling of insulin secretion to glucose levels. In a murine model, β cell–specific deletion of Dnmt3a prevented the metabolic switch, resulting in loss of GSIS. DNMT3A bound to the promoters of the genes encoding hexokinase 1 (HK1) and lactate dehydrogenase A (LDHA) — both of which regulate the metabolic switch — and knockdown of these two key DNMT3A targets restored the GSIS response in islets from animals with β cell–specific Dnmt3a deletion. Furthermore, DNA methylation–mediated repression of glucose-secretion decoupling genes to modulate GSIS was conserved in human β cells. Together, our results reveal a role for DNA methylation to direct the acquisition of pancreatic β cell function.

Authors

Sangeeta Dhawan, Shuen-ing Tschen, Chun Zeng, Tingxia Guo, Matthias Hebrok, Aleksey Matveyenko, Anil Bhushan

Mediation of opioid analgesia by a truncated 6-transmembrane GPCR

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Abstract

The generation of potent opioid analgesics that lack the side effects of traditional opioids may be possible by targeting truncated splice variants of the μ-opioid receptor. μ-Opioids act through GPCRs that are generated from the Oprm1 gene, which undergoes extensive alternative splicing. The most abundant set of Oprm1 variants encode classical full-length 7 transmembrane domain (7TM) μ-opioid receptors that mediate the actions of the traditional μ-opioid drugs morphine and methadone. In contrast, 3-iodobenzoyl-6β-naltrexamide (IBNtxA) is a potent analgesic against thermal, inflammatory, and neuropathic pain that acts independently of 7TM μ-opioid receptors but has no activity in mice lacking a set of 6TM truncated μ-opioid receptor splice variants. Unlike traditional opioids, IBNtxA does not depress respiration or result in physical dependence or reward behavior, suggesting it acts through an alternative μ-opioid receptor target. Here we demonstrated that a truncated 6TM splice variant, mMOR-1G, can rescue IBNtxA analgesia in a μ-opioid receptor–deficient mouse that lacks all Oprm1 splice variants, ablating μ-opioid activity in these animals. Intrathecal administration of lentivirus containing the 6TM variant mMOR-1G restored IBNtxA, but not morphine, analgesia in Oprm1-deficient animals. Together, these results confirm that a truncated 6TM GPCR is both necessary and sufficient for IBNtxA analgesia.

Authors

Zhigang Lu, Jin Xu, Grace C. Rossi, Susruta Majumdar, Gavril W. Pasternak, Ying-Xian Pan

Dysfunctional SEMA3E signaling underlies gonadotropin-releasing hormone neuron deficiency in Kallmann syndrome

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Abstract

Individuals with an inherited deficiency in gonadotropin-releasing hormone (GnRH) have impaired sexual reproduction. Previous genetic linkage studies and sequencing of plausible gene candidates have identified mutations associated with inherited GnRH deficiency, but the small number of affected families and limited success in validating candidates have impeded genetic diagnoses for most patients. Using a combination of exome sequencing and computational modeling, we have identified a shared point mutation in semaphorin 3E (SEMA3E) in 2 brothers with Kallmann syndrome (KS), which causes inherited GnRH deficiency. Recombinant wild-type SEMA3E protected maturing GnRH neurons from cell death by triggering a plexin D1–dependent (PLXND1-dependent) activation of PI3K-mediated survival signaling. In contrast, recombinant SEMA3E carrying the KS-associated mutation did not protect GnRH neurons from death. In murine models, lack of either SEMA3E or PLXND1 increased apoptosis of GnRH neurons in the developing brain, reducing innervation of the adult median eminence by GnRH-positive neurites. GnRH neuron deficiency in male mice was accompanied by impaired testes growth, a characteristic feature of KS. Together, these results identify SEMA3E as an essential gene for GnRH neuron development, uncover a neurotrophic function for SEMA3E in the developing brain, and elucidate SEMA3E/PLXND1/PI3K signaling as a mechanism that prevents GnRH neuron deficiency.

Authors

Anna Cariboni, Valentina André, Sophie Chauvet, Daniele Cassatella, Kathryn Davidson, Alessia Caramello, Alessandro Fantin, Pierre Bouloux, Fanny Mann, Christiana Ruhrberg

Poly(A)-specific ribonuclease deficiency impacts telomere biology and causes dyskeratosis congenita

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Abstract

Dyskeratosis congenita (DC) and related syndromes are inherited, life-threatening bone marrow (BM) failure disorders, and approximately 40% of cases are currently uncharacterized at the genetic level. Here, using whole exome sequencing (WES), we have identified biallelic mutations in the gene encoding poly(A)-specific ribonuclease (PARN) in 3 families with individuals exhibiting severe DC. PARN is an extensively characterized exonuclease with deadenylation activity that controls mRNA stability in part and therefore regulates expression of a large number of genes. The DC-associated mutations identified affect key domains within the protein, and evaluation of patient cells revealed reduced deadenylation activity. This deadenylation deficiency caused an early DNA damage response in terms of nuclear p53 regulation, cell-cycle arrest, and reduced cell viability upon UV treatment. Individuals with biallelic PARN mutations and PARN-depleted cells exhibited reduced RNA levels for several key genes that are associated with telomere biology, specifically TERC, DKC1, RTEL1, and TERF1. Moreover, PARN-deficient cells also possessed critically short telomeres. Collectively, these results identify a role for PARN in telomere maintenance and demonstrate that it is a disease-causing gene in a subset of patients with severe DC.

Authors

Hemanth Tummala, Amanda Walne, Laura Collopy, Shirleny Cardoso, Josu de la Fuente, Sarah Lawson, James Powell, Nicola Cooper, Alison Foster, Shehla Mohammed, Vincent Plagnol, Thomas Vulliamy, Inderjeet Dokal