Mutations in RIT1 cause Noonan syndrome - additional functional evidence and expanding the clinical phenotype.

RASopathies are a clinically heterogeneous group of conditions caused by mutations in 1 of 16 proteins in the RAS-mitogen activated protein kinase (RAS-MAPK) pathway. Recently, mutations in RIT1 were identified as a novel cause for Noonan syndrome. Here we provide additional functional evidence for a causal role of RIT1 mutations and expand the associated phenotypic spectrum. We identified two de novo missense variants p.Met90Ile and p.Ala57Gly. Both variants resulted in increased MEK-ERK signaling compared to wild-type, underscoring gain-of-function as the primary functional mechanism. Introduction of p.Met90Ile and p.Ala57Gly into zebrafish embryos reproduced not only aspects of the human phenotype but also revealed abnormalities of eye development, emphasizing the importance of RIT1 for spatial and temporal organization of the growing organism. In addition, we observed severe lymphedema of the lower extremity and genitalia in one patient. We provide additional evidence for a causal relationship between pathogenic mutations in RIT1, increased RAS-MAPK/MEK-ERK signaling and the clinical phenotype. The mutant RIT1 protein may possess reduced GTPase activity or a diminished ability to interact with cellular GTPase activating proteins; however the precise mechanism remains unknown. The phenotypic spectrum is likely to expand and includes lymphedema of the lower extremities in addition to nuchal hygroma.

Mutations in LOXHD1 gene can cause auditory neuropathy spectrum disorder.

Objectives: The aim of this paper was to study the auditory phenotype of three related children with sensorineural hearing loss (2 sisters and their cousin) following genetic analysis revealing mutations in LOXHD1. Methods: Genetic testing was conducted on three related children. They were assessed with a standard clinical test battery including distortion otoacoustic emissions, auditory brainstem responses and audiometry. Results: We identified heterozygous variants in LOXHD1 in a family of Irish/German and Italian/Irish ancestry with autosomal recessive auditory neuropathy spectrum disorder (ANSD). Mutations in LOXHD1 (MIM #613072) have been linked to an autosomal recessive nonsyndromic hearing loss (DFNB77), mapped to the locus 18q12-q21. All three subjects had evidence of some, albeit few, functioning cochlear hair cells as revealed by the presence of a cochlear microphonic and/or partial otoacoustic emissions early in life. Conclusion: To our knowledge, this is the first association between LOXHD1 mutations and ANSD in two patients who have been successfully managed with cochlear implants.

High resolution linkage and linkage disequilibrium analyses of chromosome 1p36 SNPs identify new positional candidate genes for low bone mineral density.

UNLABELLED: A quantitative trait locus (QTL) for BMD maps to chromosome 1p36. We have analyzed a high density SNP panel from this region for linkage and association to BMD in 39 osteoporosis pedigrees. Our results support the presence of genes controlling BMD on 1p36 and indicate new candidates for further analyses. INTRODUCTION: Low BMD is one of the major risk factors for osteoporosis. Following a genome scan in a sample of Caucasian families recruited through probands with low BMD, a region on 1p36 near marker D1S214 received support as a QTL for BMD from linkage (maximum lod-score = 2.87) and linkage disequilibrium (LD) analysis (p < 0.01). METHODS: To better characterize the genetic risk factors for low BMD located in this genomic region, we have genotyped the same group of families for 1095 SNPs located across 11 Mb on 1p36. Linkage and LD analyses have been performed using the variance component approach. RESULTS: Multivariate linkage analysis indicated two QTLs for femoral neck BMD, lumbar spine BMD and trochanter BMD simultaneously on 1p36, with maximum lod-scores of 4.37 at 12 cM and 3.59 at 22 cM. LD analysis identified several SNPs potentially associated with BMD, including the RERE gene SNP rs11121179 (p = 0.000005 for lumbar spine BMD). Other candidate genes include G1P2, SSU72 and CCDC27 (each containing 1 SNP with p < 0.001 for at least one BMD trait). CONCLUSIONS: This study supports the presence in 1p36 of QTLs affecting BMD at multiple skeletal sites. Replication of our results in other independent cohorts is warranted.

Meiosis in Drosophila melanogaster. II. The prometaphase-I kinetochore microtubule bundle and kinetochore orientation in males.

Fourteen prometaphase kinetochore microtubule bundles have been examined in electron micrographs of serial sections. The majority (54%) of the microtubules extended from the polar region towards the kinetochore but do not end in the kinetochore proper. Rather, they stop short of the kinetochore (21%), graze the kinetochore (19%), or pass through the kinetochore (9%), displaying a free end distal to the pole. Other microtubules that make up the kinetochore bundle include: kinetochore-to-pole microtubules (24%), chromosome-to-pole microtubules (5%), pieces with two free ends (14%), and those microtubules with one end in the kinetochore and a free end distal to the kinetochore (9%). We conclude that the majority of the microtubules in the kinetochore bundle are most likely of polar origin rather than having been nucleated at the kinetochore. Prometaphase-I kinetochores can display any one of four patterns of microtubule connections with the poles, but the pattern of microtubule connections is not always correlated with kinetochore position. For instance, a kinetochore directly facing one pole may have microtubule connections with both poles while a kinetochore positioned 90 degrees to the spindle axis may have microtubules running towards one pole only.

Micromanipulated bivalents can trigger mini-spindle formation in Drosophila melanogaster spermatocyte cytoplasm.

Single (individual) bivalents in cultured Drosophila melanogaster primary spermatocytes were detached from the spindle with a micromanipulation needle and placed in the cytoplasm. Such bivalents are prevented from rejoining the spindle by a natural membrane barrier that surrounds the spindle, but they quickly orient as if on a spindle of their own and the half-bivalents separate in anaphase. Serial section electron microscopy shows that a mini-spindle forms around the cytoplasmic bivalent, i.e., the microtubule density in the vicinity of the bivalent is much greater than in other cytoplasmic regions. This microtubule population cannot be accounted for solely by kinetochore nucleation and/or capture of microtubules. Furthermore, the mini-spindles frequently form at odd angles to the main spindle, so that at least one pole has no relationship to the poles of the main spindle. We conclude that a bivalent, or factors that become associated with the bivalent as a result of the manipulation, can either stabilize microtubules or promote their assembly. The bivalent activates latent microtubule organizing centers, or alternatively, polar organizing material has been passively transported from the main spindle to the cytoplasm by the micromanipulation procedure.

Mouse cathepsin M, a placenta-specific lysosomal cysteine protease related to cathepsins L and P.

The complete nucleotide sequence of a novel cathepsin cDNA derived from mouse placenta was determined and is termed cathepsin M. The predicted protein of 333 amino acid is a member of the family C1A proteases and is related to mouse cathepsins L and P. Mouse cathepsin M is highly expressed in placenta, whereas no detectable levels were found in lung, spleen, heart, brain, kidney, thymus, testicle, liver, or embryo. Phylogenic analyses of the sequences of human and mouse cathepsins show that cathepsin M is most closely related to cathepsins P and L. However, the differences are sufficiently large to indicate that the enzymes will be found in other species. This is in contrast to human cathepsins L and V, which probably resulted from a gene duplication after divergence of mammalian species.

Characterization of mouse cathepsin R, a new member of a family of placentally expressed cysteine proteases.

A new mouse cysteine protease, termed cathepsin R, has been identified. The complete nucleotide sequence of this gene was derived from a set of cDNAs generated from 15.5-day mouse placenta. Sequence analysis revealed an open reading frame encoding a 334 amino acid long polypeptide closely related to placentally expressed cathepsins P, Q, and M. RT-PCR analysis indicated that cathepsin R is only expressed in placenta and thus is a new member of the emerging family of cathepsins whose expression is regulated during mouse embryonic development. Modeling and structural analysis suggests that cathepsin R will have a restricted substrate specificity when compared to that of cathepsin L.

Meiosis in Drosophila melanogaster, III. The effect of orientation disruptor (ord) on gonial mitotic and the meiotic divisions in males.

Orientation disruptor (ord), a meiotic mutant that is recombination defective in females and disjunction defective in males and females, has been analyzed using serial section electron and light microscopy. From analysis of primary spermatocytes we have confirmed that ord males are defective in some aspect of the mechanism(s) that holds sister chromatids together during meiosis. In addition, we have determined that ord causes high frequencies of nondisjunction during spermatogonial mitotic divisions, as well as during the meiotic divisions. Mitotic nondisjunction involves the large autosomes more frequently than the sex chromosomes or chromosome 4 and results in high frequencies of primary spermatocytes that are either monosomic or trisomic for chromosome 2 or 3. Abnormalities in spermatocyte cyst formation are also observed in males homozygous for ord. These abnormalities include loss of regulation of meiotic synchrony and the number of gonial cell divisions.

Restoration of pulsatile luteinizing hormone secretion after fasting in rhesus monkeys (Macaca mulatta): dependence on size of the refeed meal.

In men and adult male rhesus monkeys brief periods of fasting (i.e. 1-2 days) can often lead to a suppression of hypothalamic-pituitary-testicular axis activity, marked by decreased circulating concentrations of LH, FSH, and testosterone. Refeeding a normal meal can rapidly restore normal secretory patterns of LH and testosterone. To learn more about the nature of the signal that links reproductive hormone secretion to nutrient intake, we examined whether the degree of restoration of pulsatile LH and testosterone secretion after a brief period of fasting is influenced by the size of the refeed meal. Adult male rhesus monkeys had indwelling venous catheters surgically implanted and were maintained on swivel/tether systems for remote collection of blood samples. Monkeys were fasted for 1 day and then on the following day they were refed one of five meals which varied in size and calorie content (i.e. 0 Cal/0 pellets Purina monkey chow; 100 Cal/5 pellets; 200 Cal/10 pellets; 300 Cal/15 pellets; or 600 Cal/30 pellets). Blood samples were collected from 0800-2400 h at 20-min intervals on the days of refeeding and assayed for LH and testosterone content. After a day of fasting, the frequency of pulsatile LH secretion the following morning was very low (0.56 +/- 0.1 pulses/3 h, measured from 0800-1100 h). When monkeys were not refed (i.e. were subjected to a second day of fasting; 0 pellet group) LH pulse frequency remained low [2.88 +/- 0.72 pulses/13 h, measured from 1100-2400 h (equivalent to 0.66 pulses/3 h)] throughout the day and evening hours. Refeeding monkeys 5, 10, 15, and 30 pellets caused a progressive stimulation of pulsatile LH secretion (3.17 +/- 0.54, 3.67 +/- 0.56, 5.5 +/- 0.67, and 6.38 +/- 0.53 pulses/13 h, respectively). Like LH pulse frequency, mean circulating LH and testosterone concentrations progressively increased as the size of the refeed meal was increased. For all three parameters (i.e. LH pulse frequency, mean LH concentration, and mean testosterone concentration) there was a significant, P less than or equal to 0.001, linear trend with increasing meal size. These findings indicate a very strong relation between the level of nutritional intake and the level of central drive to the reproductive axis during nutritional recovery from fasting, and suggest a physiological parameter that responds in a progressive fashion to increasing food intake serves as the signal to link central drive to the reproductive axis to changes in nutrient intake.(ABSTRACT TRUNCATED AT 400 WORDS)

Lexical judgments after right- or left-hemisphere injury.

Patients with unilateral left- or right-hemisphere lesions were asked to make similarity judgments to visually presented words on the basis of rhyme, meaning or visual similarity. The left-hemisphere-injured group was significantly impaired, relative to controls, for all types of lexical judgments, with the greatest impairment in the rhyme condition. Patients with right-hemisphere injury were also impaired, but only when lexical judgments were based on meaning. The results are generally consistent with normal and split-brain lateralization findings, and provide evidence supportive of a right-hemisphere contribution to some aspects of lexical semantic processing.

Mononucleotide gas-phase proton affinities as determined by the kinetic method.

The goal of this work is to determine the proton affinities of (deoxy)nucleoside 5'- and 3'-monophosphates (mononucleotides) using the kinetic method with fast atom bombardment mass spectrometry. The proton affinities of the (deoxy)nucleoside 5'- and 3'-monophosphates yielded the following trend: (deoxy)adenosine monophosphates > (deoxy)guanosine monophosphates > (deoxy)cytidine monophosphates >> deoxythymidine/uridine monophosphates. In all cases the proton affinity decreases or remains the same with the addition of the phosphate group from those values reported for nucleosides. The proton affinity is dependent on the location of the phosphate backbone (5'-vs. 3'-phosphates): the 3'-monophosphates have lower proton affinities than the 5'-monophosphates except for the thymidine/uridine monophosphates where the trend is reversed. Molecular modeling was utilized to determine if multiple protonation sites and intramolecular hydrogen bond formation would influence the proton affinity measurements. Semiempirical calculations of the proton affinities at various locations on each mononucleotide were performed and compared to the experimental results. The possible influence of intramolecular hydrogen bonding between the nucleobases and the phosphate group on the measured and calculated proton affinities is discussed.

Gas-phase hydrogen/deuterium exchange of positively charged mononucleotides by use of Fourier-transform ion cyclotron resonance mass spectrometry.

The gas-phase structures of protonated (deoxy)nucleoside-5'- and 3'-monophosphates (mononucleotides) have been examined by the use of gas-phase hydrogen/deuterium (H/D) exchange and high-field Fourier-transform ion cyclotron resonance mass spectrometry. These nucleotides were reacted with three different deuterating reagents: ND3, D2O, and D2S, of which ND3 was the most effective. All mononucleotides fully exchanged their labile hydrogen for deuterium with ND3 with the exception of deoxycytidine-3'-monophosphate, deoxyadenosine-5'-monophosphate, adenosine-5'-monophosphate, and adenosine-3'-monophosphate. Semiempirical calculations demonstrate the presence of hydrogen bonding upon protonation of the purine mononucleotides which may lead to incomplete H/D exchange. H/D exchange rates differed between the deoxymononucleotides and the ribomononucleotides, suggesting that the 2'-OH group plays an important role in the exchange process. Reactions of nucleosides and mononucleotides with D2O demonstrate that a structure-specific long-lived ion-molecule complex between D2O and the mononucleotide involving the phosphate group is necessary for exchange to overcome the high-energy activation barrier. In contrast, a structure-specific long-lived ion-molecule complex between the mononucleotides and ND3 is not required for exchange to occur.

Temporal summation functions for detection of sine-wave gratings in young and older adults.

Temporal summation functions for 0.416 and 7.5 c/deg sinusoidal gratings were measured in young and old observers in order to test the hypothesis of a shift in sensitivity from "transient" to "sustained" channels in the aging visual system. Results failed to support the transient-shift hypothesis. Additional tests showed no age-related changes in temporal summation even within a single channel. When all observers were refracted for the test distance and matched for retinal illuminance, no age-related differences in contrast sensitivity were found.

Maillard reaction induced lactose attachment to bovine beta-lactoglobulin: electrospray ionization and matrix-assisted laser desorption/ionization examination.

Nonenzymatic attachment of lactose to beta-lactoglobulin (beta-Lg) was investigated under different conditions. Solubilized conditions, dry environment, and a combination of dry and solubilized environments, were examined for their effects on lactosylation. Temperatures ranging from 50 to 65 degrees C and time intervals between 1 h and 4 days were used. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry were implemented to examine the reaction products. Maximum attachment efficiency occurred at 65 degrees C held for 3 h in dry-way conditions. Incubations held for long periods of time under dry-way conditions suggest possible denaturation. Both ESI and MALDI data suggest beta-Lg removal in the solubilized samples held for long periods of time. A combination of solubilized and dry environments led to very similar mass spectrogram results over time.

Empowerment and persons with cancer: politics in cancer medicine.

Persons with cancer are likely to encounter a loss of personal control as a result of their illness experience. An empowerment perspective, which emphasizes the possibility of patients "owning their own lives", is useful for understanding the interpersonal and social dynamics of patients' loss of control and for guiding the development of strategies aimed at maximizing control. Because the factors influencing an individual's sense of control are multi-leveled, optimal empowerment occurs when strategies are employed at several levels of social organization. In this paper, a number of barriers to the empowerment of cancer patients are identified and strategies recommended to enhance patient empowerment in cancer care. The empowerment needs and strategies discussed here include: (a) the provision of optimal care, (b) the enhancement of individual patient power, (c) the development of a strong consumer voice in policy decision-making processes, and (d) societal attitude change.

Beyond "bad manners": the power relations of "consumer participation" in Ontario's community mental health system.

This article describes the work of the legislation subcommittee of the steering committee responsible for the implementation of the Graham Report, Ontario's current blueprint for community mental health. It describes barriers to psychiatric survivor participation in the subcommittee's 1990 provincial consultation, including professional/bureaucratic characterization of survivor actions during the event as "bad manners." I argue that this naming is an act of power. Conflicts arose because the two groups operate from different behavioural codes in which the pivotal issue, acted out indirectly in all kinds of interactions, was whether and how deeply to include personal experience and emotions as forms of knowledge. The cultural dimensions of "consumer participation" must be more broadly recognized and more consciously considered if this policy is to remain viable, particularly in a time of major economic restructuring.