Kc Lau

Pupillary constriction in response to light in rodents, which does not depend on central neural pathways.

We show here that the widely held belief that reflex constriction of the mammalian pupil in response to light depends exclusively upon neural pathways between eye and brain is in need of revision. We investigated the response of the pupil to light in dark-adapted rodents (golden hamsters; hooded rats; albino rats) subjected to a variety of surgical and pharmacological interventions designed to destroy or block all of the neural pathways and structures through which the reflex could be mediated. The interventions included bilateral intraorbital optic nerve section, or unilateral intracranial optic nerve section with enucleation of the contralateral eye, combined in some cases with bilateral removal of the superior cervical ganglia and/or pinealectomy; topical application of atropine; intraocular injection of tetrodotoxin (TTX). Golden hamsters and hooded rats, but not albino rats, retained an effective constriction of the pupil in response to light after all of these interventions, although the constriction was less and slower than in normal animals. These findings show that hamsters and hooded rats have both a neurally mediated fast light reflex that can be eliminated by severing connections between eye and brain, by blockade of cholinergic transmission to iris smooth muscle, and by blockade of action potentials by TTX; and a local, slower constriction in response to light, which remains after all these procedures. We have also confirmed previous observations of Bito and Turansky (1975) that pupillary constriction in response to light occurs in isolated in vitro anterior chamber preparations of hamster and hooded rat eyes.(ABSTRACT TRUNCATED AT 250 WORDS)

Microarrays for personalized genomic medicine

The combination of single nucleotide polymorphisms (SNPs) database and high-density SNP array allows the use of SNPs as informative polymorphic markers for Mendelian diseases with complex traits efficiently. With the high-density and high-resolution SNP arrays, we can detect even the smallest structural changes that would have been missed with conventional low-density cytogenetic techniques for prognostic and diagnostic utilities. Accurate mapping may be useful for genotype-phenotype correlation in individual basis and for prenatal investigations. Here, we review some applications of genome-wide SNP genotyping on detecting homozygous candidate region in consanguineous family priors to mutation analysis. In addition to personalized genomic medicine, studying the genetic heterogeneity in diverse ancestral population helps to implementing effective clinical management.

Non-invasive screening of HLA-DPA1 and HLA-DPB1 alleles for persistent hepatitis B virus infection: Susceptibility for vertical transmission and toward a personalized approach for vaccination and treatment

BACKGROUND Polymorphisms in the major histocompatibility complex (MHC) and non-MHC genes were recently reported to be associated with persistent hepatitis B virus (HBV) infection and host response to hepatitis B vaccine in Asian populations. We aimed to confirm the associations in Chinese population and develop a non-invasive screening method for the risk loci. METHODS We genotyped 2 risk alleles on the MHC loci, HLA-DPA1 (rs3077) and HLA-DPB1 (rs9277535), and 1 risk allele near a non-MHC gene, FOXP1 (rs6789153) using high-resolution melting curve analysis. With minimal processing steps and time, salivary DNA was extracted with a modified protocol of a blood kit. We compared the genotyping fidelity between peripheral blood DNA and salivary DNA. RESULTS Both rs3077 and rs9277535, but not rs6789153, are significantly associated with CHB in Chinese population (p-value<0.001). High genotype concordance between different sources of genomic DNA was obtained. CONCLUSIONS Genotyping salivary DNA using our modified methods provides a non-invasive fast screening for host susceptibility loci. The transmission mechanism of hepatitis B can now be modified by adding genetic susceptibility to the traditional vertical transmission model of hepatitis B.

Elimination of transient dendritic spines in ipsilaterally projecting retinal ganglion cells in rats with neonatal unilateral thalamotomy.

Using the DiI and intracellular Lucifer Yellow labeling techniques in the rat, we have demonstrated that the unilateral neonatal thalamotomy does not result in retention of transient dendritic spines of ipsilaterally projecting retinal ganglion cells (IPRGCs), although the thalamotomy is known to retain the normally transient IPRGCs (Chan et al., Dev. Brain Res., 49 (1989) 265-274). These results suggest that the process of elimination or retraction of transient dendritic spines occurs in retinal ganglion cells during development regardless of whether they make connections with appropriate or inappropriate loci in the visual targets, and/or a decrease in interactions with neighboring retinal ganglion cells.

Molecular investigations of a novel iduronate-2-sulfatase mutant in a Chinese patient

BACKGROUND Molecular investigations of iduronate-2-sulfatase (IDS) mutants for the X-linked lysosomal storage disease mucopolysaccharidosis type II (MPS II, Hunter disease), commonly depends on transient expression studies to verify a single nucleotide change to be pathogenic. In 2 severely affected patients, IDS missense mutations, c.1016T>C (novel) and c.1016T>G (known) were identified predicting the substitution of an ambivalent cyclic proline and a hydrophilic arginine respectively for the hydrophobic leucine at residue 339. We hypothesized that residue Leu339 may be functionally critical. METHODS We performed a study for the 2 mutations by in-situ mutagenesis, in vitro expression, and functional analysis. RESULTS Transient expression revealed that both the missense variants had stable mRNA but their residual enzyme activities remained <2.5% of normal level. The effect of the missense mutations on protein expression was detected by Western blot analysis. Both the missense mutations synthesized the precursor form but had reduced mature form of IDS. CONCLUSION The novel mutation p.L339P is a disease-causing mutation affecting maturation of the protein.

A fast modified protocol for random-access ultra-high density whole-genome scan: a tool for personalized genomic medicine, positional mapping, and cytogenetic analysis.

BACKGROUND High density single nucleotide polymorphism (SNP) genotyping array is widely applied on genome-wide association study of common diseases. In these studies, a fixed batch-size of 48 or 96 samples allows high-throughput analysis. To enhance the clinical application of microarray analysis on personalized medicine, we describe a modified PCR purification protocol without batch-size limitation for whole-genome scan using ultra-high density SNP microarray. METHODS Enzyme-digested PCR products were purified with the use of magnetic beads. Separation of the magnetic particles applies magnetic stand devices instead of vacuum pumps. With no batch-size limitation, we genotyped 17 genomic samples and 3 whole genome amplified samples in order to examine the performance of the modified protocol. RESULTS Our method is simple and fast, provides sufficient amount and high quality PCR products for subsequent fragmentation and labeling procedures prior to GeneChip hybridization. We show that the purified DNA can be genotyped with good QC call rate of >93% in average similar to standard protocol. With the use of the short protocol, we successfully identified the breakpoint localization of a ring chromosome in a female and located the disease gene in a consanguineous family affected by limb-girdle muscular dystrophy. CONCLUSION By modifying a single step in the original protocol, we are able to speed up the overall genotyping analysis and change the batch-wise analysis to random-access analysis for ultra-high density whole-genome scan for personalized medicine, positional mapping, and cytogenetic analysis.

Microarray analysis unmasked paternal uniparental disomy of chromosome 12 in a patient with isolated sulfite oxidase deficiency

BACKGROUND In the investigation of a proband with a biochemical diagnosis of isolated sulfite oxidase deficiency, we identified a homozygous nonsense mutation of the SUOX gene in the proband. However, the mutation was only detected in the father and not the mother. Deletion of the SUOX gene of the mother and paternal disomy of chromosome 12, where the SUOX gene is located, were suspected in view that allele dropout of the mother non-amplified wild-type allele is unlikely. METHODS To distinguish the two possible causes, we performed a genome wide microarray analysis in the patient and parents using high-density single-nucleotide microarrays. Whole genome allele sharing of the genomes of the patient and parents were performed by dChip. RESULTS In the proband, the whole genome scan showed loss of heterozygosity (LOH) of the entire chromosome 12. However, the LOH is copy neutral and deletion of the SUOX gene of the mother was thus excluded. On whole genome allele sharing analysis, the proband showed a high degree of allele sharing with the father and a very low allele sharing with the mother only in chromosome 12. The cause of the homozygosity of the mutation of the patient is UPD (12) pat. CONCLUSIONS To the best of our knowledge, this study is the first UPD (12) pat causing isolated sulfite oxidase deficiency in humans. Even with one parent being a carrier of an autosomal recessive disease, a fetus with the autosomal recessive disease is still possible. This will have clinical impact on genetic counseling.

DNA-based diagnosis of erythropoietic protoporphyria in two families and the frequency of a low-expression FECH allele in a Chinese population

Ferrochelatase (FECH; E.C. 4.99.1.1) is the terminal enzyme which catalyzes ferrous iron insertion into protoporphyrin IX in the haem biosynthetic pathway [1]. Partial deficiency of FECH activity causes excess accumulation of cellular protoporphyrin and results in erythropoietic protoporphyria (EPP; MIM 177000). Upon irradiation, the photodamage of the binding sites between protoporphyrin and hemoglobin may lead to the leakage of erythrocyte protoporphyrin into the plasma [2]. The phototoxicity reaction of protoporphyrin is responsible for the typical history of skin photosensitivity developed in the patients. Definitive diagnosis relies upon an image-based approach detecting fluorescent protoporphyrins [3]. The demonstration of fluorocytes/fluorescent red cells in a fresh blood film viewed under ultraviolet light is straightforward and reliable. In addition, examination of plasma fluorescence emission spectrum demonstrating a characteristic peak of plasma protoporphyrin fluorescence at 632 nm simply makes the diagnosis [4]. The human FECH gene is located on chromosome18q21.3 containing 11 exons [5]. The open reading frame extends for 1269 base pairs, coding a 423 amino acid proteinwhich acts in situ as an 82–84 kDa homodimer. The mode of inheritance of EPP is mainly autosomal dominant with incomplete penetrance. There is a low-expression allele, IVS3-48C, shown to have trans co-inheritance with an additional FECH genetic mutation in most overt EPP cases [6–8]. Identifying mutations in FECH gene provides additional information of the inheritance of the low expression allele and facilitates family screening. Recent studies only reported 2 FECH mutations (c.67+1GNC and p.R115X) in Chinese patients [9,10]. The proband in Family A (IIA-3) is a 12-y boy presentedwith hepatic dysfunction, severe abdominal pain, electrolyte disturbances and bizarre behaviour [11]. He complained of dark-color urine for one monthwith a history of skinphotosensitivity. Biochemical examinations revealed marked increase in erythrocyte protoporphyrin (8213 μg/dl; normal=20–80 μg/dl; 1 μg/dl=27.6 nmol/l) predominantly with free protoporphyrin and marked increase in plasma total porphyrins (87.2 μg/dl; normal=0–0.9 μg/dl) with a fluorescence emission spectrum consistent with EPP [4]. His symptomatic sister (IIA-1) also had greatly elevated erythrocyte protoporphyrin (3252 μg/dl). Fluorescence microscopy of red blood cells showed presence of fluorocytes in both IIA-1 and IIA-3. The proband in Family B (IIB-3) is 21-y presented with acute abdominal pain and complained of dark colored urine for 2 weeks. He has skin photosensitivity since early childhood. His liver function data were within normal limits but physical examination

Automated imaging of circulating fluorocytes for the diagnosis of erythropoietic protoporphyria: a pilot study for population screening.

Objectives To improve the traditional fresh blood film method to a high-throughput analysis of the presence of circulating fluorescent red cells (fluorocytes) in erythropoietic protoporphyria (EPP) using an automated imaging system. Methods Based on the autofluorescence of protoporphyrin, we used an automatic image acquisition platform for examining fluorocytes in peripheral blood with minimal sample preparation. The image acquisition is easy-to-use under automated operations of excitation, focusing, detection and data analysis. Quality image and semi-quantitative fluorescence measurement of fluorocytes can be generated in a single step. For high-throughput analysis, the platform can image more than 200 96-well micro-plates, i.e. 19200 samples, in approximately 10 hours. Importantly, the reagent cost of analysis is negligible. Results In this pilot study, three EPP patients were diagnosed and 4000 normal individuals were screened for EPP by this method. Our results showed that the method can distinguish the overt case and asymptomatic carriers. It gives reliable evidence for rapid EPP screening. Conclusion This automated imaging system provides multiple advantages that improve the traditional fresh blood film method as a more effective diagnostic tool and facilitates population screening for EPP. As fluorocytes are present in the umbilical cord blood of EPP patients, this high-throughput method can be potentially used for newborn screening of EPP.

Circulating fluorocytes at the first attack of acute intermittent porphyria: a missing link in the pathogenesis.

BACKGROUND Acute intermittent porphyria (AIP) is an autosomal dominant disorder of the haem biosynthesis resulting from a partial deficiency of hydroxymethylbilane synthase (HMBS) with incomplete penetrance. By conventional means, it is able to identify asymptomatic mutation carrier by molecular diagnosis, but one cannot reliably predict an acute porphyric attack. The presence of fluorescent red cells (fluorocytes) in AIP is probably under-recognized since AIP is a hepatic porphyria and not associated with photosensitivity. METHODS We used an automatic image acquisition platform to detect the circulating fluorocytes at 700 nm emission in a diabetic AIP patient during acute attack. We screened the patient and her family members for the mutation on HMBS, urine porphobilinogen and circulating fluorocytes. RESULTS The patient was heterozygous for a disease-causing mutation on HMBS and several bright circulating fluorocytes were detected. We showed evidence that protoporphyrin contributed to the erythrocyte auto-fluorescence. Interestingly, asymptomatic mutation carriers with increased urine porphobilinogen did not have circulating fluorocytes. All mutation-negative family members revealed no circulating fluorocytes. CONCLUSION Sudden decrease in plasma glucose concentration might invoke acute attack of AIP and appearance of circulatory fluorocytes. Potential of detecting fluorocytes as screening test or for predicting an acute attack of AIP in diabetes is worth investigating.