Highly efficient gene release in spatiotemporal precision approached by light and pH dual responsive copolymers.

Triblock copolymer of poly(ethylene glycol)-b-poly(2-dimethylaminoethyl methacrylate)-b-poly(pyrenylmethyl methacrylate) (PEG-b-PDMAEMA-b-PPy) has been developed for use as an ideal gene delivery system, which showed both high stability under physiological conditions and efficient gene release in a mimetic cancer environment. The siRNA release from this system without external stimulation was 16% in 1 h and then remained steady. However, the photo-triggered siRNA release was 78% within 1 h and was higher than 91% after 24 h. The remarkable contrast between the stability and release efficiency of these siRNA-condensed micelleplexes before and after photo-irradiation has been rationalized by the light- and pH-induced structural transitions of the triblock copolymer micelles. The negligible cytotoxicity, high cellular uptake efficiency, and remarkable knockdown efficiency shown in in vitro tests further revealed the promising potential of these triblock copolymer micelleplexes for use in stimuli-responsive gene therapy.

THE EFFECTS ON DRIVING SPEED OF A HEAD-UP DISPLAY OF ROAD WARNINGS (1).

This study investigated the superimposition of the projected markings on the road with head-up display, as well as their effects on the driver's speed. Two experiments were conducted. In Exp. 1, driving operations were simulated with a desktop computer to assess 18 deceleration markings (from the factors position, shape, and color) and determined the factors and levels influencing driving speeds. Based on the results of Exp. 1, six deceleration markings (from the factors shape and color) were selected in the driving simulator for conducting Exp. 2. The results of Exp. 1 showed that markings at the sides were better than the markings in the center. In Exp. 2, there was no significant difference between the effects of the arrangement of markings and the change of shape and color on driving stability. Yellow and white colors had no significant effect on speed; however, bar markings were better than zigzag markings. The results indicated that the projection of markings on a head-up display was helpful for indicating necessary deceleration.

Clinical observations and treatment of pediatric homozygous familial hypercholesterolemia due to a low-density lipoprotein receptor defect.

BACKGROUND: Clinical observation and treatment of children with homozygous familial hypercholesterolemia (HoFH) has rarely been reported. We report clinical observations and treatment of 10 ethnic Chinese children with HoFH due to low-density lipoprotein receptor (LDLR) defect. OBJECTIVES: In children with HoFH, we evaluated the response to conventional cholesterol-lowering drug therapy and performed LDLR gene analysis. METHODS: A retrospective review of lipid profile changes in pediatric patients diagnosed with HoFH seen in our pediatric endocrinology outpatient clinic was performed. HoFH was diagnosed by molecular study of these patients and their parents. RESULTS: One novel (c.64del G) and 12 known mutations were found in the LDLR gene. Mutation of p.C308Y was the most common and was found in 26% of the studied alleles.Seven patients had fair responses to conventional drug therapy (high-dose statin with ezetimibe) with a reduction of 50% or more of the total cholesterol levels. The low-density lipoprotein-cholesterol levels of three patients decreased to lower than 160 mg/dL. One who had a good response to conventional drug therapy developed significant atheromatous plaques (largest plaque: 7.4 × 2.7 cm) in the extracranial carotid arteries and myocardial ischemia changes at 11 years old. CONCLUSION: The results suggest that despite aggressive therapy, many patients are not well controlled; atherosclerosis may progress, and novel therapies are required.

Enzyme assay and clinical assessment in subjects with a Chinese hotspot late-onset Fabry mutation (IVS4 + 919G→A).

Newborn screening for Fabry disease in Taiwan Chinese has revealed a high incidence of the late-onset GLA mutation IVS4 + 919G→A (∼1 in 1,500-1,600 males). We studied 94 adults with this mutation [22 men, 72 women; mean age: men 57.8 ± 6.0 (range 42-68), women 39.1 ± 14.1 years (range 19-82)]. Plasma α-galactosidase A activity assay was 10.4 ± 11.2% of normal in the men and 48.6 ± 19.5% of normal in the women. Echocardiography in 90 of the adults revealed left ventricular hypertrophy (LVH) in 19 (21%), including 14 of 21 men (67%) and 5 of 69 women (7%). Microalbuminuria, based on the urine albumin-to-creatinine ratio measured on at least two occasions, was present in 17 of 86 subjects (20%) (men: 5/20, 25%; women 12/66, 18%). At least one ocular manifestation consistent with Fabry disease was present in 41 of 52 subjects (79%) who underwent ophthalmologic examination, including 8 (15%) with conjunctival vessel tortuosity, 15 (29%) with cornea verticillata, 10 (19%) with Fabry cataract, and 34 (65%) with retinal vessel tortuosity. Among subjects over 40 years of age, men were more likely than women to have LVH [14/21 (67%) vs 5/25 (20%), p < 0.001]. Cardiovascular, renal and ocular abnormalities are highly prevalent in adult Taiwan Chinese subjects with the Fabry mutation IVS4 + 919G→A. Our findings contribute to the limited understanding of the course of this late-onset disease variant and underscore the need for close follow up in such patients.

Newborn screening for methylmalonic aciduria by tandem mass spectrometry: 7 years' experience from two centers in Taiwan.

BACKGROUND: The clinical course of methylmalonic aciduria (MMA) is fulminant in neonates and emergency management is necessary to save lives. It is therefore very important to differentiate affected from unaffected neonates immediately when there are abnormal results regarding MMA in newborn screening. METHODS: Between January 2002 and December 2008, 598,522 newborns were screened for MMA by 2 neonatal screening centers: the Chinese Foundation of Health and the Taipei Institute of Pathology. A total of 22 newborns were referred to confirmatory medical centers, and 7 were confirmed as having MMA. The initial propionylcarnitine (C3) level, C3/acetylcarnitine (C2) ratio, plasma ammonia, liver function tests, blood pH and bicarbonate were compared between the true-positive and false-positive groups. RESULTS: The C3/C2 ratio and plasma ammonia were markedly higher in the true-positive MMA group (p < 0.0001). Blood gas pH (p = 0.029), bicarbonate (p = 0.019), and aspartate aminotransferase (p = 0.005) also significantly differed between these 2 groups. CONCLUSION: Referred newborns with elevated plasma C3/C2 ratios > 0.4 or ammonia levels > 200 mg/dL should be highly suspected of having MMA.

Clinical observations, molecular genetic analysis, and treatment of sitosterolemia in infants and children.

The clinical observation and treatment of young children with sitosterolemia has rarely been reported. We report clinical, biochemical, and molecular genetic observations and treatment outcomes for five Chinese children from four separate families presenting with sitosterolemia in whom we identified two new (Y329X, G269R) and three known (R446X, N437K, R389H) mutations in the ABCG5 gene. The R389H mutation was found in 50% of alleles. Three of these five patients received cholestyramine therapy with a very good response. However, all patients discontinued this therapy because of poor compliance. Finally, all patients were on ezetimibe therapy and had satisfactory total serum cholesterol levels, though their plant sterol levels were still higher than normal. Another noteworthy finding is that a female infant had a serum cholesterol level of 654 mg/dl at 7 months of age, despite being breast fed (with very tiny amounts of plant sterols) since birth and undergoing 4 months of ezetimibe administration. Although she failed to respond to ezetimibe during this period, she did show improvement when the therapy was started again at 2 years of age. It is possible that another 23-month-old female patient also responded more slowly to ezetimibe treatment than older patients.

Severe hyponatremia due to ACTH insufficiency in a 14 year-old girl with growth hormone deficiency.

SIADH-like hyponatremia as the presenting manifestation of ACTH deficiency is rare in childhood. Here we report a 14 year-old girl who, after 8 years of GH replacement and subsequent treatment for subclinical secondary hypothyroidism, presented with confusion and disorientation due to severe hyponatremia. When her pituitary axis was re-assessed, she was diagnosed as having ACTH deficiency associated with multiple pituitary hormone deficiencies (MPHD) (including GH, FSH, LH, and subclinical TSH deficiencies). She responded poorly to treatment with only hypertonic fluid, but improved after addition of hydrocortisone replacement. The purpose of this paper is to emphasize the importance of suspecting ACTH insufficiency in children with GH deficiency if hyponatremia develops.

High incidence of the cardiac variant of Fabry disease revealed by newborn screening in the Taiwan Chinese population.

BACKGROUND: Fabry disease is a treatable lysosomal storage disorder, which is often misdiagnosed or belatedly diagnosed. METHODS AND RESULTS: To determine the disease incidence in the Taiwan Chinese population, a Fabry disease newborn screening study was initiated. A total of 110 027 newborns were screened by assaying the alpha-galactosidase A (alpha-Gal A) activity using dry blood spots. Low plasma alpha-Gal A activity and presence of a Fabry mutation was demonstrated in 45 neonates (3 females). Eight different mutations were identified, including 3 known missense mutations (R112H, A143T, and R356W), 4 novel missense mutations (G104V, M296L, G360C, and K391T), and one known intronic mutation (IVS4+919G-->A). The IVS4+919G-->A mutation was most common (82% of patients). A total of 20 maternal grandparents of infants harboring this intronic mutation were evaluated by echocardiography, mutation analysis and alpha-Gal A activity assay. The intronic mutation was found in 9 grandfathers and 11 grandmothers. Of these grandparents, 3 grandfathers (33%) but none of the grandmothers had hypertrophic cardiomyopathy. Additionally, 16 males who had been diagnosed with idiopathic hypertrophic cardiomyopathy were screened by mutation analysis and alpha-Gal A activity; 4 (25%) showed deficient plasma alpha-Gal A activity in combination with the intronic mutation. CONCLUSIONS: We found an unexpected high prevalence of the cardiac variant Fabry mutation IVS4+919G-->A among both newborns (approximately 1 in 1600 males) and patients with idiopathic hypertrophic cardiomyopathy in the Taiwan Chinese population. The early identification of undiagnosed patients allows timely therapeutic intervention providing a better clinical outcome.

Six new mutations of the thyroglobulin gene discovered in taiwanese children presenting with thyroid dyshormonogenesis.

BACKGROUND: Thyroglobulin (TG) defect is a rare cause of congenital hypothyroidism. Although only 44 mutations of the human TG gene have been identified, we have suspected a TG defect in 38% of Taiwan Chinese children/adolescents presenting with moderate or severe thyroidal dyshormonogenesis. STUDY OBJECTIVE: The aim of the study is to report the discovery of new TG gene mutations and associated clinical manifestations of the defective TG protein. PATIENTS AND RESULTS: In seven patients from six families, we detected six new TG gene mutations, including c.1348delT, p.R432X (c.1351C>T), g.IVS3 + 2T>G, c.1712delT, p.Q1765X (c.5350C>T), and c.6047delA. The c.1348delT and p.R432X mutations were the most common, detected in 33 and 25%, respectively, of alleles studied. Haplotype analysis suggested that the c.1348delT and g.IVS3 + 2T>G mutations are due to founder effects, whereas p.R432X is probably due to independently recurrent de novo mutations. mRNA transcript of the g.IVS3 + 2T>G mutant, detected in whole blood by reverse transcription-nested PCR, showed skipping of exon 3 (98-bp deletion) and a frameshift, with a terminal signal after 17 altered amino acid residues. CONCLUSIONS: TG defects have an important role in severe thyroidal dyshormonogenesis (pretreatment, or after a 3-wk T(4) withdrawal, plasma T(4) < or = 30 nmol/liter) in Taiwanese. Its genetic characteristics are markedly different from those described in other populations presenting with mutations of the TG gene.

Growth hormone therapy in neonatal patients with methylmalonic acidemia.

BACKGROUND: Information regarding growth hormone (GH) therapy in neonatal patients with methylmalonic academia (MMA) is lacking. We present our experience with GH therapy in neonatal patients with MMA. METHODS: Four neonatal patients with mut 0 type MMA were identified through newborn screening for elevated propionylcarnitine (C3) levels. GH therapy (0.6 IU/kg/week, subcutaneously) was prescribed for patient 1 after 1 month of admission, and was prescribed for patients 2, 3 and 4 on the 1st day of admission. We evaluated weight, skin erosion, hospital stay, and serum levels of C3 after GH therapy. RESULTS: All of the neonatal patients with MMA displayed obvious weight gain and distinct improvement in skin erosions after GH therapy. The duration of hospital stay for patients 2, 3 and 4 was reduced compared to that of patient 1. However, the metabolic effects of GH therapy on reducing serum levels of C3 seem to be indeterminate. CONCLUSION: Our clinical findings suggest that GH therapy has potentially beneficial effects on neonatal patients with MMA.

Unambiguous molecular detections with multiple genetic approach for the complicated chromosome 22q11 deletion syndrome.

BACKGROUND: Chromosome 22q11 deletion syndrome (22q11DS) causes a developmental disorder during the embryonic stage, usually because of hemizygous deletions. The clinical pictures of patients with 22q11DS vary because of polymorphisms: on average, approximately 93% of affected individuals have a de novo deletion of 22q11, and the rest have inherited the same deletion from a parent. Methods using multiple genetic markers are thus important for the accurate detection of these microdeletions. METHODS: We studied 12 babies suspected to carry 22q11DS and 18 age-matched healthy controls from unrelated Taiwanese families. We determined genomic variance using microarray-based comparative genomic hybridization (array-CGH), quantitative real-time polymerase chain reaction (qPCR) and multiplex ligation-dependent probe amplification (MLPA). RESULTS: Changes in genomic copy number were significantly associated with clinical manifestations for the classical criteria of 22q11DS using MPLA and qPCR (p < 0.01). An identical deletion was shown in three affected infants by MLPA. These reduced DNA dosages were also obtained partially using array-CGH and confirmed by qPCR but with some differences in deletion size. CONCLUSION: Both MLPA and qPCR could produce a clearly defined range of deleted genomic DNA, whereas there must be a deleted genome that is not distinguishable using MLPA. These data demonstrate that such multiple genetic approaches are necessary for the unambiguous molecular detection of these types of complicated genomic syndromes.