Bone resorption in syndromes of the Ras/MAPK pathway.

Disorders of the Ras/mitogen-activated protein kinase (MAPK) pathway have an overlapping skeletal phenotype (e.g. scoliosis, osteopenia). The Ras proteins regulate cell proliferation and differentiation and neurofibromatosis type 1 (NF1) individuals have osteoclast hyperactivity and increased bone resorption as measured by urine pyridinium crosslinks [pyridinoline (Pyd) and deoxypyridinoline (Dpd)]. Pyd and Dpd are hydroxylysine-derived crosslinks of collagen found in bone and cartilage and excreted in the urine. Dpd is most abundant in bone. The aim of this study was to evaluate if other syndromes of the Ras/MAPK pathway have increased bone resorption, which may impact the skeletal phenotype. Participants were individuals with Noonan syndrome (n = 14), Costello syndrome (n = 21), and cardiofaciocutaneous (CFC) syndrome (n = 14). Pyridinium crosslinks from two consecutive first morning urines were extracted after acid hydrolysis and analyzed by high performance liquid chromatography. Three separate analyses of covariance were performed to compare Pyd, Dpd, and Dpd/Pyd ratio of each group to controls after controlling for age. Data were compared to 99 healthy controls. The Dpd and the Dpd/Pyd ratio were elevated (p < 0.0001) in all three conditions compared to controls suggesting that collagen degradation was predominantly from bone. The data suggest that the Ras/MAPK signal transduction pathway is important in bone homeostasis.

Molecular diagnosis in hereditary hemorrhagic telangiectasia: findings in a series tested simultaneously by sequencing and deletion/duplication analysis.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular disorder characterized by a unique pattern of telangiectasia and arteriovenous malformations (AVMs). Mutations in one of two genes (ENG and ACVRL1) cause approximately 85% of cases. Genetic testing impacts clinical management because genotype/phenotype correlations exist, and early preventive screening for internal AVMs is recommended in affected individuals prior to the age at which a diagnosis can typically be made based on clinical criteria. We report 383 consecutive cases in which sequencing and large deletion/duplication analysis were performed simultaneously for endoglin (ENG) and activin-like receptor kinase 1 (ACVRL1). We report the first case of mosaicism in an affected individual and 61 novel mutations. We discuss the potential benefits of a diagnostic testing approach for HHT whereby ENG and ACVRL1 are analyzed simultaneously by sequencing and a method which detects large deletion/duplications, rather than by a sequential or reflex testing protocol. We report a case in which a deletion would probably have been missed if large deletion/duplication analysis was performed only if a suspected pathogenic mutation was not first identified by sequencing.

A new distal arthrogryposis syndrome characterized by plantar flexion contractures.

The distal arthrogryposis (DA) syndromes are a distinct group of disorders characterized by contractures of two or more different body areas. More than a decade ago, we revised the classification of DAs and distinguished several new syndromes. This revision has facilitated the identification of five genes (i.e., TNNI2, TNNT3, MYH3, MYH8, and TPM2) that encode components of the contractile apparatus of fast-twitch myofibers and cause DA syndromes. We now report on the phenotypic features of a novel DA disorder characterized primarily by plantar flexion contractures in a large five-generation Utah family. Contractures of hips, elbows, wrists, and fingers were much milder though they varied in severity among affected individuals. All affected individuals had normal neurological examinations; electromyography and creatinine kinase levels were normal on selected individuals. We have tentatively labeled this condition distal arthrogryposis type 10 (DA10).

Clinical and molecular aspects of an informative family with neurofibromatosis type 1 and Noonan phenotype.

Neurofibromatosis-Noonan syndrome (NFNS) has been described as a unique phenotype, combining manifestations of neurofibromatosis type 1 (NF1) and Noonan syndrome, which are separate syndromes. Potential etiologies of NFNS include a discrete syndrome of distinct etiology, co-segregation of two mutated common genes, variable clinical expressivity of NF1, and/or allelic heterogeneity. We present an informative family with an unusual NF1 mutation with variable features of NF1 and Noonan syndrome. We hypothesize that an NF1 mutant allele can lead to diagnostic manifestations of Noonan syndrome, supporting the hypothesis that NF1 allelic heterogeneity causes NFNS.

Case-control study of the muscular compartments and osseous strength in neurofibromatosis type 1 using peripheral quantitative computed tomography.

Skeletal anomalies are observed in neurofibromatosis type 1 (NF1), but the pathogenesis is unknown. Given that muscle mass is important in the development of the strength of bone, peripheral quantitative computed tomography (pQCT) was utilized to compare measurements of muscle compartments between NF1 individuals and controls. Forty individuals with NF1 (age 5-18 years) were evaluated. Cross-sectional measurements, at the 66% tibial site, were obtained using pQCT (XCT-2000, Stratec) and variables were compared to controls without NF1 ((age 5-18 years, N=380) using analysis-of-covariance controlling for age, height, Tanner stage, and gender. The NF1 cohort showed decreased total cross-sectional area [p<0.001], decreased muscle plus bone cross-sectional area [p<0.001], decreased muscle cross-sectional area [p<0.001], and decreased Stress Strain Index [p=0.010]. These data indicate that NF1 individuals have decreased muscle cross-sectional area and decreased bone strength than individuals without NF1.

Hydranencephaly in an infant with vascular malformations.

Hydranencephaly is a condition in which cerebral hemispheres are absent and reduced to fluid-filled sacs in a normal skull. Numerous causes have been proposed. We report a male infant with hydranencephaly and congenital vascular malformations (port wine stains, generalized nevus flammeus, anomalous retinal vessels, and absent internal carotid flow). Magnetic resonance imaging of the brain showed absence of most of the cerebrum except for small portions of the occipital cortex and thalami. Magnetic resonance angiography showed flow within the vertebral and basilar arteries without internal carotid intracranial flow above the internal carotid petrous and cavernous portion. This is a report of cutaneous and retinal malformations associated with hydranencephaly. Vascular malformations of larger vessels (e.g., webbing of the carotid arteries and an absent internal carotid arterial system) have been observed in other infants with hydranencephaly, and are proposed to lead to brain destruction. The case reported herein supports the role of primary vascular malformations in the development of some cases of hydranencephaly.

Colorectal cancer genomics: evidence for multiple genotypes which influence survival.

Colorectal cancer (CRC) is a leading cause of cancer death and the mechanism for variable outcome in this disease is not yet fully understood. It is hypothesized that differences in the genetic make-up of tumours may be partially responsible for the differences observed in survival among same staged individuals for this disease. In this study the tumour genomes of 29 consecutive patients undergoing surgery for Dukes' C CRC were assessed by comparative genomic hybridization (CGH). In addition, the CGH profiles from the tumours were compared with those from eight colorectal cell lines. Great variation in genetic grade (all detectable aberrations i.e., loss + gain) was observed in 29 Dukes' C colorectal tumours by CGH (median four aberrations per tumour, range 0-20). Gain was found in 76% and loss in 41% of tumours. The most frequently observed regions of gain were 13q (27.6%), 20q (27.6%), 7p (24.1%), 8q (24.1%), and 1q (20.7%) and loss were 18q (31%), 4q (20.7%), 17p (20.7%), 18p (20.7%), and 15q (20.1%). None of these specific genomic aberrations were associated with patient survival. However, patients with more than two aberrations had a better survival than patients with fewer regions of loss and gain (P = 0.02). CRC cell lines had similar regions of loss or gain as the tumours. However, the frequency of genomic aberrations was much greater in the CRC cell lines. Although genomic change in CRC is relevant to the survival of patients with Dukes' C CRC, careful analysis is required to identify cell lines which are representative models of CRC genomics.

Characterization of the topoisomerase I locus in human colorectal cancer.

DNA topoisomerase I (topo I) is the principle target for Camptothecin and its analogues. The topo I gene is located on chromosome 20q11.2-q13.1 and variation in topo I gene copy number has been shown to have impact on the in vitro sensitivity to topoisomerase I inhibitor chemotherapy. Fluorescence in situ hybridization (FISH) was used to detect and compare the TOPO I gene copy number between metaphase and interphase nuclei in a panel of 7 colorectal cancer cell lines. TOPO I gene copy number varied from 2 to 8 between cell lines, and signal in interphase nuclei demonstrated a linear relationship with that detected in metaphase nuclei. The structure of gene amplification included isochromosome formation, amplicon extension, and marker chromosome generation. Comparative genomic hybridization (CGH) was then used to further define the region of gain on chromosome 20. The region of gain contained the topo I gene and involved nearly all of 20q in most cases. This demonstrates a high degree of intrinsic variation in topo I gene copy number and the involvement of a 20q amplicon in colorectal cancer, which may have important implications for colorectal tumorigenesis and the use of chemotherapy.

Translocation (X;5)(q13;q33) in essential thrombocythemia.

We report a novel chromosomal translocation (X;5)(q13;q33) in a woman with no history of prior chemotherapy or radiotherapy, found to have essential thrombocythemia. Aberrations in chromosome 5, mostly deletions of 5q, have been described in essential thrombocythemia; however, a t(X;5) translocation has not been reported.

Descriptive analysis of tibial pseudarthrosis in patients with neurofibromatosis 1.

Five percent of individuals with neurofibromatosis type 1 (NF1) present with congenital long bone pseudarthrosis (PA). In large series, 50-80% of patients with congenital long bone PA also have NF1. Very little information exists on the natural history and pathogenesis of PA in NF1. This report is a descriptive analysis of a large series of patients with NF1 and tibial bowing or PA. Study A is a case-control study using the National Neurofibromatosis Foundation International Database (NNFFID). Eighty-five patients with PA were compared to a control group from the same database. There was a statistically significant male predominance of NF1 cases with PA (54 males to 31 females), compared to controls (85 males to 87 females) (chi2 = 4.0, P = 0.046, using a two-tailed test with Yates' correction). There was no significant difference in the clinical presentation of NF1 manifestations in NF1 patients with PA than in NF1 patients without PA. Of the affected individuals with PA, there were 24 de novo cases and 21 familial cases (9 through maternal and 12 through paternal inheritance). Questions that could not be answered by Study A were addressed by a partially overlapping case-series report, Study B, in which data on 75 cases ascertained through questionnaires completed by NF center directors were collected. From Study B we determined that half of the patients who had a fracture sustained it before age 2, and approximately 16% of the pseudarthrosis patients had an amputation. Our data indicate a male predominance and no parent-of-origin effect. Male gender may be a susceptibility factor for pseudarthrosis in NF1.

Comparative genomic hybridization analysis of chromosomal alterations induced by the development of resistance to thymidylate synthase inhibitors.

Acquired resistance to chemotherapy is a major obstacle to the successful treatment of cancer. In the past, technical limitations prevented the detection of genetic alterations associated with such resistance on a genome-wide scale. This study evaluated comparative genomic hybridization (CGH) as a tool to detect candidate regions of the genome associated with chemoresistance. Using a variation of conventional CGH, DNA from cell lines that were resistant to thymidylate synthase inhibitors (raltitrexed and 5-fluorouracil) and their sensitive parent cells were evaluated. In MCF-7 and H630 cells that were resistant to raltitrexed, only a single region of change (18p gain) was apparent. The third cell line, H630R10, which was resistant to 5-fluorouracil, had changes in several genomic regions following the acquisition of resistance, including 18p gain. Gain in the chromosomal region containing the thymidylate synthase gene (18p11.32) was detected by CGH in all three resistant cell lines. However, additional novel regions of interest were identified in the cells that were resistant to 5-fluorouracil. These results suggest that CGH is of potential use in the detection of regions of the genome involved in chemoresistance.

Specificity of diastereomers of [99mTc]TRODAT-1 as dopamine transporter imaging agents.

Recently, we reported the first human study of [99mTc]TRODAT-1, technetium, 2-[[2-[[[3-(4-chlorophenyl)-8-methyl-8-azabicyclo[3.2.1]oct-2- yl]methyl](2-mercaptoethyl)amino]ethyl]amino]-ethanethiolato(3-)-o xo- [1R-(exo-exo)]-, as an imaging agent of central nervous system (CNS) dopamine transporters. Due to the existence of several chiral centers on this molecule, upon the formation of [99mTc]TRODAT-1 complex (2) several diastereomers could be created. Two major diastereomers of [99mTc]TRODAT-1 (2), designated as peak A (2A) and peak B (2B), were separated by HPLC. Biodistribution of the purified diastereomers 2A,B was evaluated in rats. It appears that 2A displayed a higher lipophilicity than 2B (PC = 305 and 229, respectively), and a similar trend was observed for the initial brain uptake at 2 min postinjection (0.50% and 0.28% dose/organ for 2A,B, respectively). At 60 min post-iv-injection, the specific uptakes, as measured by [striatum - cerebellum]/cerebellum ([ST-CB]/CB) ratio, were 1.72 and 2.79 for 2A,B, respectively. The higher [ST-CB]/CB ratio observed for 2B was corroborated by the results of an in vitro binding assay. Higher binding affinity for dopamine transporters was observed for 3B (Ki = 13.87 and 8.42 nM for the analogous rhenium complexes 3A,B, respectively). The structure of the [99mTc]TRODAT-1 complexes was deduced using nonradioactive rhenium as a surrogate for radioactive technetium complex. Reacting free TRODAT-1 ligand with [Bu4N][ReOCl4] yielded two major complexes: Re-TRODAT-1A (3A) and Re-TRODAT-1B (3B) (corresponding with peaks A and B of [99mTc]TRODAT-1, respectively), whose structures were determined by X-ray analysis. The X-ray structures show that both complexes have a pseudo-square-pyramidal structure of [RevO]3+N2S2 core with oxygen occupying the apical position and the N-alkyl substitution in syn-configuration to the oxo-rhenium bond. In conclusion, TRODAT-1 formed at least two diastereomers after complexing with a metal(V)-oxo (M = 99mTc, Re) center core. The two isomers display different binding affinities toward dopamine transporters and distinct properties of localization in the striatum area of the brain where the transporters are located.

Small and neutral Tc(v)O BAT, bisaminoethanethiol (N2S2) complexes for developing new brain imaging agents.

Bisaminoethanethiol (BAT) ligands with various gem-dimethyl and amide groups were prepared, and the corresponding neutral Tc-99m complexes were prepared and evaluated for their relative stabilities by ligand-exchange reactions. It was demonstrated that technetium complexes containing gem-dimethyl substituents have higher lipophilicities, whereas those with an amide group possess greater stability, which enhances ligand-exchange reaction. The most interesting observation was that the brain uptake in rats is not determined only by lipophilicity. Apparently, Tc-99m complexes with an amide functional group display lower brain uptakes in rats compared to those without an amide group. The brain uptake was strongly influenced by substituents on the BAT ligand. These factors are critically important and should be taken into consideration when designing Tc-99m-labeled agents for CNS receptor imaging.

[99mTc]TRODAT-1: a novel technetium-99m complex as a dopamine transporter imaging agent.

Technetium-99m is the most commonly used radionuclide in routine nuclear medicine imaging procedures. Development of 99mTc-labeled receptor-specific imaging agents for studying the central nervous system is potentially useful for evaluation of brain function in normal and disease states. A novel 99mTc-labeled tropane derivative, [99mTc]TRODAT-1, which is useful as a potential CNS dopamine transporter imaging agent, was evaluated and characterized. After i. v. injection into rats, [99mTc]TRODAT-1 displayed specific brain uptake in the rat striatal region (striatum-cerebellum/cerebellum ratio 1.8 at 60 min), where dopamine neurons are concentrated. The specific striatal uptake could be blocked by pretreating rats with a dose of competing dopamine transporter ligand, beta-CIT (or RTI-55, i.v., 1 mg/kg). However, the specific striatal uptake of [99mTc]TRODAT-1 was not affected by co-injection of excess free ligand (TRODAT-1, up to 200 microg per rat) or by pretreating the rats with haloperidol (i.v., 1 mg/kg). The specific uptake in striatal regions of rats that had prior 6-hydroxydopamine lesion in the substantia nigra area showed a dramatic reduction. The radioactive material recovered from the rat striatal homogenates at 60 min after i.v. injection of [99mTc]TRODAT-1 showed primarily the original compound (>95%), a good indication of in vivo stability in brain tissue. Similar and comparable organ distribution patterns and brain regional uptakes of [99mTc]TRODAT-1 were obtained for male and female rats. Ex vivo autoradiography results of rat brain sections further confirmed the high uptake and retention of [99mTc]TRODAT-1 in the striatal region. In vitro binding studies measuring the affinity to dopamine transporters for the free ligand, TRODAT-1, and a nonradioactive rhenium derivative, Re-TRODAT-1, showed Ki values of 9.7 nM and 14.1 nM, respectively. Behavioral studies in rats using the free ligand, TRODAT-1 and Re-TRODAT-1 indicated that, unlike other tropane derivatives, they displayed no effect on locomotor activity, suggesting low toxicity. These results strongly support the conclusions that this novel 99mTc radioligand binds selectively to dopamine transporters in the brain and that is is potentially useful for in vivo assessment of the loss of dopamine neurons in Parkinson's and other neurodegenerative diseases.

Mulvihill-Smith progeria-like syndrome: a further report with delineation of phenotype, immunologic deficits, and novel observation of fibroblast abnormalities.

We report the seventh case of Mulvihill-Smith progeria-like syndrome in a 5-year-old boy with a thin, pinched face, failure to thrive, and cutaneous pigmented nevi. The patient's motor and intellectual development were normal. His immune function tests demonstrate evidence of lymphopenia with no selective loss of a major subpopulation, low immunoglobulin (Ig)G2 and IgG4 subclasses, and an absent in vitro proliferative response to pokeweed mitogen. Chromosomal mitomycin and radiation sensitivity were normal. The skin fibroblast growth in culture was slow, and the fibroblasts appeared morphologically different from normal controls in their size and large number of inclusions. In addition, primary cilia, which normally issue from the centrosome, were absent-a new finding in fibroblasts in this disorder. It remains to be seen if the relative absence of centrosomal cilia in cultured fibroblasts in early passages is a consistent finding in this progeria syndrome.

Synthesis and characterization of technetium-99m-labeled tropanes as dopamine transporter-imaging agents.

In the development of novel Tc-99m-labeled tropane derivatives as dopamine transporter (reuptake site)-imaging agents, a series of neutral and lipophilic complexes containing bis-(aminoethanethiol) as a neutral complexing moiety for a [99mTc]TcO3+ center core was successfully prepared. Biological evaluation of the Tc-99m-labeled complexes 13-16 as central nervous system (CNS) dopamine transporter-imaging agents was reported. Synthesis of the tropane derivatives was achieved by stepwise reactions adding two aminoethanethiol units. The final free thiol ligands were obtained by deblocking the 4-methoxybenzyl protecting group with Hg(OAc)2 to obtain trifluoroacetate salts. All of the Tc-99m complexes, with the exception of 16, displayed good initial brain uptake and selective uptake in the striatal area, where dopamine transporters are concentrated. One of the compounds, [2-[[2-[[[3-(4-chlorophenyl)-8-methyl-8-azabicyclo [3.2.1]oct-2-yl]methyl](2-mercaptoethyl)amino]ethy] amino]ethanethiolato-(3-)-N2,N2',S2,S2'] oxo-[1R-(exo-exo)]- [99mTc]technetium,[99mTc]TRODAT-1 (13), displayed the highest initial uptake in rat brain (0.4% at 2 min post iv injection); the striatal/cerebellar (ST/ CB) ratio reached 2.8 at 60 min after an iv injection. The specific uptake in rat brain can be blocked by pretreating rats with a competing dopamine transporter binding agent, beta-CIT (RTI-55, 2 beta-carbomethoxy-3 beta-(4-iodophenyl)tropane; iv, 1 mg/kg), which reduced the regional brain uptake ratio (ST/CB) to 1.2. In contrast, the specific striatal uptake was not affected by pretreating rats with a noncompeting ligand, haldol (iv, 1 mg/kg). After an iv injection of 9 mCi of [99mTc]TRODAT-1 (13), in vivo images of baboon brain using single-photon emission-computed tomography exhibited excellent localization in striatum (basal ganglia), where dopamine neurons are known to be concentrated. This series of compounds may provide a convenient source of short-lived imaging agents for routine diagnosis of CNS diseases (i.e., Parkinson's disease) in which changes in the dopamine transporter concentration are implicated.

Characterization of a novel iodinated ligand, IPMPP, for human dopamine D4 receptors expressed in CHO cells.

A novel radioiodinated ligand with a high specific activity (2,200 Ci/mmol), 3-[4-(4-iodophenyl)piperazin-1-yl]methyl-1H-pyrrolo(2,3-b)pyridine ([125I]IPMPP), was successfully prepared. Binding characteristics of [125I]IPMPP were evaluated using human dopamine D4 (D4.2 variant) receptors expressed in Chinese hamster ovary (CHO) cells. Saturation analysis revealed high-affinity binding sites for [125I]IPMPP (Kd = 0.39 +/- 0.18 nM). The number of D4 receptors labeled with [125I]IPMPP at room temperature was four times higher than that labeled with [125I]S(-)5-OH-PIPAT, a radioiodinated agonist ligand (572 fmol/mg protein vs. 125 fmol/mg protein). A significant decrease in the number of binding sites was observed with [125I]S(-)5-OH-PIPAT when assays were carried out at a higher temperature (37 degrees C vs. 25 degrees C). In contrast to [125I]S(-)5-OH-PIPAT, [125I]IPMPP labeled more D4 sites at 37 degrees C. Neither magnesium ion nor guanylimidodiphosphate (Gpp(NH)p) affected [125I]IPMPP binding. These data support the conclusion that [125I]IPMPP is an antagonist ligand. The potency of various compounds, including clozapine, to inhibit [125I]IPMPP binding is consistent with the rank order measured with other radioligands for D4 receptors. In addition, measuring D4 receptor stimulation of [35S]GTPgammaS binding further demonstrated the antagonist property of IPMPP.

Insights from a new analytical electrophoresis apparatus.

Charge is a fundamental property of macromolecules that is inextricably linked to their structure, solubility, stability, and interactions. Progress has been made on the theoretical and structural aspects of these relationships. However, for several reasons, charge is difficult to measure in solution. Consequently, there is a lack of experimental data that, independent of other macro-ion properties, determines the effective charge. To overcome this problem, novel instrumentation and methods are being developed in our laboratory. Described here is an analytical electrophoresis apparatus that permits both the measurement of electrophoretic mobilities and the determination of steady-state electrophoresis concentration distributions. The latter provides a different-perspective on the processes that influence macro-ion behavior in an electric field. In addition, the apparatus permits the determination of diffusion coefficients either from boundary spreading during transport or from the decay of a concentration gradient. All of these determinations can be made with a single, 8-microL sample in a variety of solvents, thus providing unique insights into the charge properties of a macro-ion. Presented here is a progress report about this emerging technology, including the description of a prototype apparatus and examples of its use with a DNA oligonucleotide.

In vivo SPECT imaging of 5-HT1A receptors with [123I]p-MPPI in nonhuman primates.

The in vivo imaging of a novel iodinated phenylpiperazine derivative for 5-HT1A receptors, [123I]p-MPPI (4-(2'-methoxy-)phenyl-1-[2'-(n-2"-pyridinyl)-p-iodobenzamido-] ethyl-piperazine), using single photon emission computed tomography (SPECT), was evaluated in nonhuman primates. After an i.v. injection, [123I]p-MPPI penetrated the blood-brain barrier quickly and localized in brain regions where 5-HT1A receptor density is high (hippocampus, frontal cortex, cingulate gyrus, entorhinal cortex). Maximum ratio of hippocampus to cerebellum was 3 to 1 at 50 min postinjection. The specific binding of the radioligand in the hippocampal region, an area rich in 5-HT1A receptor density, was blocked by a chasing dose of (+/-) 8-OH-DPAT (2 mg/kg, i.v.) or non-radioactive p-MPPI (1 mg/kg, i.v.), whereas the regional distribution of [123I]p-MPPI was unaffected by treatment with non 5-HT1A agents, such as ketanserin. Ex vivo and in vitro autoradiographic studies using monkey brain further confirmed that the specific binding of [123I]p-MPPI is associated with 5-HT1A receptor sites. However, the initial attempt at [123I]p-MPPI human imaging studies did not display specific localization of 5-HT1A receptors. This discrepancy observed for [123I]p-MPPI may be due to a dramatic difference in metabolic pathways between humans and monkeys.