Inactivation of menin, a Smad3-interacting protein, blocks transforming growth factor type β signaling

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by endocrine tumors of parathyroids, pancreatic islets, and anterior pituitary. The MEN1 gene encodes a nuclear protein called menin. In MEN1 carriers inactivating mutations give rise to a truncated product consistent with menin acting as a tumor suppressor gene. However, the role of menin in tumorigenesis and its physiological functions are not known. Here, we show that menin inactivation by antisense RNA antagonizes transforming growth factor type β-mediated cell growth inhibition. Menin interacts with Smad3, and antisense menin suppresses transforming growth factor type β-induced and Smad3-induced transcriptional activity by inhibiting Smad3/4-DNA binding at specific transcriptional regulatory sites. These results implicate a mechanism of tumorigenesis by menin inactivation.

Cloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia

Tumor-induced osteomalacia (TIO) is one of the paraneoplastic diseases characterized by hypophosphatemia caused by renal phosphate wasting. Because removal of responsible tumors normalizes phosphate metabolism, an unidentified humoral phosphaturic factor is believed to be responsible for this syndrome. To identify the causative factor of TIO, we obtained cDNA clones that were abundantly expressed only in a tumor causing TIO and constructed tumor-specific cDNA contigs. Based on the sequence of one major contig, we cloned 2,270-bp cDNA, which turned out to encode fibroblast growth factor 23 (FGF23). Administration of recombinant FGF23 decreased serum phosphate in mice within 12 h. When Chinese hamster ovary cells stably expressing FGF23 were s.c. implanted into nude mice, hypophosphatemia with increased renal phosphate clearance was observed. In addition, a high level of serum alkaline phosphatase, low 1,25-dihydroxyvitamin D, deformity of bone, and impairment of body weight gain became evident. Histological examination showed marked increase of osteoid and widening of growth plate. Thus, continuous production of FGF23 reproduced clinical, biochemical, and histological features of TIO in vivo. Analyses for recombinant FGF23 products produced by Chinese hamster ovary cells indicated proteolytic cleavage of FGF23 at the RXXR motif. Recent genetic study indicates that missense mutations in this RXXR motif of FGF23 are responsible for autosomal dominant hypophosphatemic rickets, another hypophosphatemic disease with similar features to TIO. We conclude that overproduction of FGF23 causes TIO, whereas mutations in the FGF23 gene result in autosomal dominant hypophosphatemic rickets possibly by preventing proteolytic cleavage and enhancing biological activity of FGF23.

The genetic basis of epidermolysis bullosa simplex with mottled pigmentation.

Epidermolysis bullosa simplex (EBS) is a group of autosomal dominant skin diseases characterized by blistering, due to mechanical stress-induced degeneration of basal epidermal cells. It is now well-established that the three major subtypes of EBS are genetic disorders of the basal epidermal keratins, keratin 5 (K5) and keratin 14 (K14). Here we show that a rare subtype, referred to as EBS with mottled pigmentation (MP), is also a disorder of these keratins. Affected members of two seemingly unrelated families with EBS-MP had a C to T point mutation in the second base position of codon 24 of one of two K5 alleles, leading to a Pro: Leu mutation. This mutation was not present in unaffected members nor in 100 alleles from normal individuals. Linkage analyses mapped the defect to this type II keratin gene (peak logarithm of odds score at phi = 0 of 3.9), which is located on chromosome 12q11-q13. This provides strong evidence that this mutation is responsible for the EBS-MP phenotype. Only conserved between K5 and K6, and not among any of the other type II keratins, Pro-24 is in the nonhelical head domain of K5, and only mildly perturbs the length of 10-nm keratin filaments assembled in vitro. However, this part of the K5 head domain is likely to protrude on the filament surface, perhaps leading to additional aberrations in intermediate filament architecture and/or in melanosome distribution that are seen ultrastructurally in patients with the mutation.

Constitutive receptor activation by Crouzon syndrome mutations in fibroblast growth factor receptor (FGFR)2 and FGFR2/Neu chimeras.

Crouzon syndrome is an autosomal dominant condition primarily characterized by craniosynostosis. This syndrome has been associated with a variety of amino acid point mutations in the extracellular domain of fibroblast growth factor receptor 2 (FGFR2). FGFR2/Neu chimeras were generated by substituting the extracellular domain of Neu with that of FGFR2 containing the following Crouzon mutations: Tyr-340-->His; Cys-342-->Tyr; Cys-342-->Arg; Cys-342-->Ser; Ser-354-->Cys: and delta17 (deletion of amino acids 345-361). Each of the mutant chimeric FGFR2/Neu constructs stimulated focus formation in NIH 3T3 cells, indicating that Crouzon mutations can stimulate signal transduction through a heterologous receptor tyrosine kinase. In vitro kinase assay results indicate that FGFR2 receptors containing Crouzon mutations have increased tyrosine kinase activity and, when analyzed under nonreducing conditions, exhibited disulfide-bonded dimers. Thus the human developmental abnormality Crouzon syndrome arises from constitutive activation of FGFR2 due to aberrant intermolecular disulfide-bonding. These results together with our earlier observation that achondroplasia results from constitutive activation of the related receptor FGFR3, leads to the prediction that other malformation syndromes attributed to FGFRs, such as Pfeiffer syndrome and Thanatophoric dysplasia, also arise from constitutive receptor activation.

Structure and function in rhodopsin: correct folding and misfolding in two point mutants in the intradiscal domain of rhodopsin identified in retinitis pigmentosa.

The rhodopsin mutants P23H and G188R, identified in autosomal dominant retinitis pigmentosa (ADRP), and the site-specific mutants D190A and DeltaY191-Y192 were expressed in COS cells from synthetic mutant opsin genes containing these mutations. The proteins expressed from P23H and D190A partially regenerated the rhodopsin chromophore with 11-cis-retinal and were mixtures of the correctly folded (retinal-binding) and misfolded (non-retinal-binding) opsins. The mixtures were separated into pure, correctly folded mutant rhodopsins and misfolded opsins. The proteins expressed from the ADRP mutant G188R and the mutant DeltaY191-Y192 were composed of totally misfolded non-retinal-binding opsins. Far-UV CD spectra showed that the correctly folded mutant rhodopsins had helical content similar to that of the wild-type rhodopsin, whereas the misfolded opsins had helical content 50-70% of the wild type. The near-UV CD spectra of the misfolded mutant proteins lack the characteristic band pattern seen in the wild-type opsin, indicative of a different tertiary structure. Further, whereas the folded mutant rhodopsins were essentially resistant to trypsin digestion, the misfolded opsins were degraded to small fragments under the same conditions. Therefore, the misfolded opsins appear to be less compact in their structures than the correctly folded forms. We suggest that most, if not all, of the point mutations in the intradiscal domain identified in ADRP cause partial or complete misfolding of rhodopsin.

Structure and function in rhodopsin: correct folding and misfolding in point mutants at and in proximity to the site of the retinitis pigmentosa mutation Leu-125-->Arg in the transmembrane helix C.

L125R is a mutation in the transmembrane helix C of rhodopsin that is associated with autosomal dominant retinitis pigmentosa. To probe the orientation of the helix and its packing in the transmembrane domain, we have prepared and studied the mutations E122R, I123R, A124R, S127R, L125F, and L125A at, and in proximity to, the above mutation site. Like L125R, the opsin expressed in COS-1 cells from E122R did not bind 11-cis-retinal, whereas those from I123R and S127R formed the rhodopsin chromophore partially. A124R opsin formed the rhodopsin chromophore (lambda max 495 nm) in the dark, but the metarhodopsin II formed on illumination decayed about 6.5 times faster than that of the wild type and was defective in transducin activation. The mutant opsins from L125F and L125A bound 11-cis-retinal only partially, and in both cases, the mixtures of the proteins produced were separated into retinal-binding and non-retinal-binding (misfolded) fractions. The purified mutant rhodopsin from L125F showed lambda max at 500 nm, whereas that from L125A showed lambda max at 503 nm. The mutant rhodopsin L125F showed abnormal bleaching behavior and both mutants on illumination showed destabilized metarhodopsin II species and reduced transducin activation. Because previous results have indicated that misfolding in rhodopsin is due to the formation of a disulfide bond other than the normal disulfide bond between Cys-110 and Cys-187 in the intradiscal domain, we conclude from the misfolding in mutants L125F and L125A that the folding in vivo in the transmembrane domain is coupled to that in the intradiscal domain.

Spectrum of HERG K+-channel dysfunction in an inherited cardiac arrhythmia.

Long QT syndrome (LQT) is an autosomal dominant disorder that can cause sudden death from cardiac arrhythmias. We recently discovered that mutations in HERG, a K+-channel gene, cause chromosome 7-linked LQT. Heterologous expression of HERG in Xenopus oocytes revealed that HERG current was similar to a well-characterized cardiac delayed rectifier K+ current, IKr, and led to the hypothesis that mutations in HERG reduced IKr, causing prolonged myocellular action potentials. To define the mechanism of LQT, we injected oocytes with mutant HERG complementary RNAs, either singly or in combination with wild-type complementary RNA. Some mutations caused loss of function, whereas others caused dominant negative suppression of HERG function. These mutations are predicted to cause a spectrum of diminished IKr and delayed ventricular repolarization, consistent with the prolonged QT interval observed in individuals with LQT.

Genetic dissection of Alzheimer disease, a heterogeneous disorder.

The genetics of Alzheimer disease (AD) are complex and not completely understood. Mutations in the amyloid precursor protein gene (APP) can cause early-onset autosomal dominant AD. In vitro studies indicate that cells expressing mutant APPs overproduce pathogenic forms of the A beta peptide, the major component of AD amyloid. However, mutations in the APP gene are responsible for 5% or less of all early-onset familial AD. A locus on chromosome 14 is responsible for AD in other early-onset AD families and represents the most severe form of the disease in terms of age of onset and rate of decline. Attempts to identify the AD3 gene by positional cloning methods are underway. At least one additional early-onset AD locus remains to be located. In late-onset AD, the apolipoprotein E gene allele epsilon 4 is a risk factor for AD. This allele appears to act as a dose-dependent age-of-onset modifier. The epsilon 2 allele of this gene may be protective. Other late-onset susceptibility factors remain to be identified.

Mapping a gene causing cerebral cavernous malformation to 7q11.2-q21.

Cerebral cavernous malformation is a common disease of the brain vasculature of unknown cause characterized by dilated thin-walled sinusoidal vessels (caverns); these lesions cause varying clinical presentations which include headache, seizure, and hemorrhagic stroke. This disorder is frequently familial, with autosomal dominant inheritance. Using a general linkage approach in two extended cavernous malformation kindreds, we have identified linkage of this trait to chromosome 7q11.2-q21. Multipoint linkage analysis yields a peak logarithm of odds (lod) score of 6.88 with zero recombination with locus D7S669 and localizes the gene to a 7-cM region in the interval between loci ELN and D7S802.

Premature suture closure and ectopic cranial bone in mice expressing Msx2 transgenes in the developing skull.

The coordinate growth of the brain and skull is achieved through a series of interactions between the developing brain, the growing bones of the skull, and the fibrous joints, or sutures, that unite the bones. These interactions couple the expansion of the brain to the growth of the bony plates at the sutures. Craniosynostosis, the premature fusion of the bones of the skull, is a common birth defect (1 in 3000 live births) that disrupts coordinate growth and often results in profoundly abnormal skull shape. Individuals affected with Boston-type craniosynostosis, an autosomal dominant disorder, bear a mutated copy of MSX2, a homeobox gene thought to function in tissue interactions. Here we show that expression of the mouse counterpart of this mutant gene in the developing skulls of transgenic mice causes craniosynostosis and ectopic cranial bone. These mice provide a transgenic model of craniosynostosis as well as a point of entry into the molecular mechanisms that coordinate the growth of the brain and skull.

Mutações inativadoras no gene MKRN3 são causa de puberdade precoce central familial

A maioria dos casos de puberdade precoce central (PPC) em meninas permanece idiopática. A hipótese de uma causa genética vem se fortalecendo após a descoberta de alguns genes associados a este fenótipo, sobretudo aqueles implicados com o sistema kisspeptina (KISS1 e KISS1R). Entretanto, apenas casos isolados de PPC foram relacionados à mutação na kisspeptina ou em seu receptor. Até recentemente, a maioria dos estudos genéticos em PPC buscava genes candidatos selecionados com base em modelos animais, análise genética de pacientes com hipogonadismo hipogonadotrófico, ou ainda, nos estudos de associação ampla do genoma. Neste trabalho, foi utilizado o sequenciamento exômico global, uma metodologia mais moderna de sequenciamento, para identificar variantes associadas ao fenótipo de PPC. Trinta e seis indivíduos com a forma de PPC familial (19 famílias) e 213 casos aparentemente esporádicos foram inicialmente selecionados. A forma familial foi definida pela presença de mais de um membro afetado na família. DNA genômico foi extraído dos leucócitos do sangue periférico de todos os pacientes. O estudo de sequenciamento exômico global realizado pela técnica ILLUMINA, em 40 membros de 15 famílias com PPC, identificou mutações inativadoras em um único gene, MKRN3, em cinco dessas famílias. Pesquisa de mutação no MKRN3 realizada por sequenciamento direto em duas famílias adicionais (quatro pacientes) identificou duas novas variantes nesse gene. O MKRN3 é um gene de um único éxon, localizado no cromossomo 15 em uma região crítica para a síndrome de Prader Willi. O gene MKRN3 sofre imprinting materno, sendo expresso apenas pelo alelo paterno. A descoberta de mutações em pacientes com PPC familial despertou o interesse para a pesquisa de mutações nesse gene em 213 pacientes com PPC aparentemente esporádica por meio de reação em cadeia de polimerase seguida de purificação enzimática e sequenciamento automático direto (Sanger). Três novas mutações e duas já anteriormente identificadas, incluindo quatro frameshifts e uma variante missense, foram encontradas, em heterozigose, em seis meninas não relacionadas. Todas as novas variantes identificadas estavam ausentes nos bancos de dados (1000 Genomes e Exome Variant Server). O estudo de segregação familial em três dessas meninas com PPC aparentemente esporádica e mutação no MKRN3 confirmou o padrão de herança autossômica dominante com penetrância completa e transmissão exclusiva pelo alelo paterno, demonstrando que esses casos eram, na verdade, também familiares. A maioria das mutações encontradas no MKRN3 era do tipo frameshift ou nonsense, levando a stop códons prematuros e proteínas truncadas e, portanto, confirmando a associação com o fenótipo. As duas mutações missenses (p.Arg365Ser e p.Phe417Ile) identificadas estavam localizadas em regiões de dedo ou anel de zinco, importantes para a função da proteína. Além disso, os estudos in silico dessas duas variantes demonstraram patogenicidade. Todos os pacientes com mutação no MKRN3 apresentavam características clínicas e hormonais típicas de ativação prematura do eixo reprodutivo. A mediana de idade de início da puberdade foi de 6 anos nas meninas (variando de 3 a 6,5) e 8 anos nos meninos (variando de 5,9 a 8,5). Tendo em vista o fenômeno de imprinting, análise de metilação foi também realizada em um subgrupo de 52 pacientes com PPC pela técnica de MS-MLPA, mas não foram encontradas alterações no padrão de metilação. Em conclusão, este trabalho identificou um novo gene associado ao fenótipo de PPC. Atualmente, mutações inativadoras no MKRN3 representam a causa genética mais comum de PPC familial (33%). O MKRN3 é o primeiro gene imprintado associado a distúrbios puberais em humanos. O mecanismo preciso de ação desse gene na regulação da secreção de GnRH necessita de estudos adicionais

Tauroursodeoxycholic acid prevents retinal degeneration in transgenic P23H rats

Purpose. To evaluate the preventive effect of tauroursodeoxycholic acid (TUDCA) on photoreceptor degeneration, synaptic connectivity and functional activity of the retina in the transgenic P23H rat, an animal model of autosomal dominant retinitis pigmentosa (RP). Methods. P23H line-3 rats were injected with TUDCA once a week from postnatal day (P)21 to P120, in parallel with vehicle-administered controls. At P120, functional activity of the retina was evaluated by electroretinographic (ERG) recording. The effects of TUDCA on the number, morphology, integrity, and synaptic connectivity of retinal cells were characterized by immunofluorescence confocal microscopy. Results. The amplitude of ERG a- and b-waves was significantly higher in TUDCA-treated animals under both scotopic and photopic conditions than in control animals. In the central area of the retina, TUDCA-treated P23H rats showed threefold more photoreceptors than control animals. The number of TUNEL-positive cells was significantly smaller in TUDCA-treated rats, in which photoreceptor morphology was preserved. Presynaptic and postsynaptic elements, as well as the synaptic contacts between photoreceptors and bipolar or horizontal cells, were preserved in TUDCA-treated P23H rats. Furthermore, in TUDCA-treated rat retinas, the number of both rod bipolar and horizontal cell bodies, as well as the density of their synaptic terminals in the outer plexiform layer, was greater than in control rats. Conclusions. TUDCA treatment was capable of preserving cone and rod structure and function, together with their contacts with their postsynaptic neurons. The neuroprotective effects of TUDCA make this compound potentially useful for delaying retinal degeneration in RP.

Safranal, a saffron constituent, attenuates retinal degeneration in P23H rats

Saffron, an extract from Crocus sativus, has been largely used in traditional medicine for its antiapoptotic and anticarcinogenic properties. In this work, we investigate the effects of safranal, a component of saffron stigmas, in attenuating retinal degeneration in the P23H rat model of autosomal dominant retinitis pigmentosa. We demonstrate that administration of safranal to homozygous P23H line-3 rats preserves both photoreceptor morphology and number. Electroretinographic recordings showed higher a- and b-wave amplitudes under both photopic and scotopic conditions in safranal-treated versus non-treated animals. Furthermore, the capillary network in safranal-treated animals was preserved, unlike that found in untreated animals. Our findings indicate that dietary supplementation with safranal slows photoreceptor cell degeneration and ameliorates the loss of retinal function and vascular network disruption in P23H rats. This work also suggests that safranal could be potentially useful to retard retinal degeneration in patients with retinitis pigmentosa.

Proinsulin slows retinal degeneration and vision loss in the P23H rat model of retinitis pigmentosa

Proinsulin has been characterized as a neuroprotective molecule. In this work we assess the therapeutic potential of proinsulin on photoreceptor degeneration, synaptic connectivity, and functional activity of the retina in the transgenic P23H rat, an animal model of autosomal dominant retinitis pigmentosa (RP). P23H homozygous rats received an intramuscular injection of an adeno-associated viral vector serotype 1 (AAV1) expressing human proinsulin (hPi+) or AAV1-null vector (hPi−) at P20. Levels of hPi in serum were determined by enzyme-linked immunosorbent assay (ELISA), and visual function was evaluated by electroretinographic (ERG) recording at P30, P60, P90, and P120. Preservation of retinal structure was assessed by immunohistochemistry at P120. Human proinsulin was detected in serum from rats injected with hPi+ at all times tested, with average hPi levels ranging from 1.1 nM (P30) to 1.4 nM (P120). ERG recordings showed an amelioration of vision loss in hPi+ animals. The scotopic b-waves were significantly higher in hPi+ animals than in control rats at P90 and P120. This attenuation of visual deterioration correlated with a delay in photoreceptor degeneration and the preservation of retinal cytoarchitecture. hPi+ animals had 48.7% more photoreceptors than control animals. Presynaptic and postsynaptic elements, as well as the synaptic contacts between photoreceptors and bipolar or horizontal cells, were preserved in hPi+ P23H rats. Furthermore, in hPi+ rat retinas the number of rod bipolar cell bodies was greater than in control rats. Our data demonstrate that hPi expression preserves cone and rod structure and function, together with their contacts with postsynaptic neurons, in the P23H rat. These data strongly support the further development of proinsulin-based therapy to counteract retinitis pigmentosa.

Neuroprotective effects of the cannabinoid agonist HU210 on retinal degeneration

Cannabinoids have been demonstrated to exert neuroprotective effects on different types of neuronal insults. Here we have addressed the therapeutic potential of the synthetic cannabinoid HU210 on photoreceptor degeneration, synaptic connectivity and functional activity of the retina in the transgenic P23H rat, an animal model for autosomal dominant retinitis pigmentosa (RP). In P23H rats administered with HU210 (100 μg/kg, i.p.) from P24 to P90, ERG recordings showed an amelioration of vision loss, as compared to vehicle-administered animals. Under scotopic conditions, the maximum a-wave amplitudes recorded at P60 and P90 were higher in HU210-treated animals, as compared to the values obtained in untreated animals. The scotopic b-waves were significantly higher in treated animals than in untreated rats at P30, P60 and P90. This attenuation of visual deterioration correlated with a delay in photoreceptor degeneration and the preservation of retinal cytoarchitecture. HU210-treated animals had 40% more photoreceptors than untreated animals. Presynaptic and postsynaptic elements, as well as the synaptic contacts between photoreceptors and bipolar or horizontal cells, were also preserved in HU210-treated P23H rats. These results indicate that HU210 preserves cone and rod structure and function, together with their contacts with postsynaptic neurons, in P23H rats. These data suggest that cannabinoids are potentially useful to delay retinal degeneration in RP patients.