Siu Yuen Chan

MANNOSE-BINDING LECTIN AND RHEUMATOID ARTHRITIS IN SOUTHERN CHINESE

OBJECTIVE Insufficiency of mannose-binding lectin (MBL) is associated with recurrent infections. Rheumatoid arthritis (RA) may represent an aberrant immune response to infections. This study examined the phenotypic expression and variant alleles of the MBL gene and its etiologic role in Chinese with RA. METHODS We studied 211 RA patients and 196 healthy subjects. Serum MBL concentrations and codon-54 mutation of the MBL gene and its promoter polymorphisms were analyzed. Clinical characteristics and disease activity were also examined. RESULTS Patients with RA had significantly lower serum MBL levels and higher frequency of codon-54 mutation of the MBL gene compared with controls. Additionally, there was a significant difference in the distribution of promoter polymorphisms, H/L, between RA patients and controls, although the frequencies of Y/X and those of nonstructural polymorphisms, P/Q, did not differ between the 2 groups. Furthermore, patients with RA had a lower incidence of the highest-producing haplotype HY and a higher incidence of the lowest-producing haplotype LX compared with controls. Serum MBL levels did not correlate with drug treatment or with disease activity. However, patients with erosive and serious extraarticular disease had significantly lower serum MBL levels than those without these disease manifestations at the time of study. Also, significantly more patients with erosive disease had a codon-54 mutation of the MBL gene compared with those with nonerosive disease. CONCLUSION The codon-54 mutation and low-producing promoter polymorphisms of the MBL gene are associated with RA. A low serum level of MBL predisposes to the development of RA and is a risk factor for severe disease in southern Chinese.

Characterization of additional genetic events in childhood acute lymphoblastic leukemia with TEL/AML1 gene fusion : a molecular cytogenetics study

TEL/AML1 gene fusion that results from a cryptic t(12;21) is the most common genetic aberration in childhood B-lineage acute lymphoblastic leukemia (ALL). While the translocation may initiate the leukemic process, critical secondary genetic events are currently believed to be pivotal for leukemogenesis. We investigated 12 cases of childhood ALL with TEL/AML1 gene fusion by fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) and documented additional or secondary genetic changes in seven patients (58%). Three patients showed extra copies of chromosome 21 including a case in which the trisomy 21 (+21) clone was distinct from the one harboring TEL/AML1 gene fusion. Interestingly, one patient without +21 showed amplification of the AML1 gene on chromosome 21q, supporting the contention that AML1 amplification may be an important additional genetic event. Gene expression study by semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) in two of these four patients showed an increase in AML1 transcripts that paralleled the increase in gene copy number. Deletion of the normal TEL allele was detected in two patients, with one of them showing loss of chromosome 12 together with duplication of the der(12)t(12;21). Finally, one patient showed duplication of the fusion signal. Our findings confirm that additional or secondary genetic changes including AML1 amplification are commonly encountered in childhood ALL with TEL/AML1 gene fusion, which are envisaged to play significant roles in disease progression.

Mannose-binding protein in preterm infants: developmental profile and clinical significance.

The aim of this study was to determine the developmental profile of mannose‐binding protein (MBP) in preterm infants. MBP was measured in 885 longitudinally collected serum samples from 168 preterm infants, and 63 were genotyped with respect to the codon 54 mutation in the MBP gene. MBP level/codon 54 genotyping were also determined on the cord blood of 146/123 term infants and 138/123 adults, respectively. The best cut‐off values of MBP for dividing preterm, term infants and adults into ‘low’ and ‘high’ M BP groups were 400 ng/ml (55 low, 113 high), 700 ng/ml (35 low, 111 high) and 750 ng/ml (33 low. 105 high), respectively, by achieving the least number of misclassifications according to the codon 54 mutation. The relative risk of the ‘low’ groups for presence of the codon 54 mutation compared with ‘high’ groups were 42 4, 67–9 and 22–9 for preterm, term infants and adults, respectively (P KO 00001). The gestational age and birth weight of the ‘low’(n = 55) and ‘high’(n= 113) MBP groups of the 168 preterm infants were 29.5 ± 2.8 weeks, 30.5±2.8 weeks (p=0.03) and 1230±317g, 1277±289g (p = 0.35). respectively. The mean MBP levels of these two groups of preterm infants were different (P<0001) at all ages measured. As a whole group, the MBP level rose from a mean of 500 ng/ml at 25 weeks gestation to 1700ng/ml at 20 weeks post full‐term. The mortality rates of ‘low’ and ‘high’ MBP groups of preterm infants were 22% and 12%, respectively (p‐0.113). This difference in mortality was due to gestational age and birth weight standard deviation score (SDS) after adjusting for length of gestation and gender (p = 0.0001) rather than to low MBP levels (p = 0 65). MBP levels were not related to birthweight SDS score (P = 0 26). The mean ± sd. MBP levels for preterm, term infants and adults without the codon 54 mutation were 1225 ±701 ng/ml (n = 45), 2064 ± 829 ng/ml (n= 88) and 2473 ± 1395ng/ml (n = 95), respectively; the corresponding values for those with the codon 54 mutation were 130 ±275 ng/ml (n= 18), 533 ±665 ng/ml (n = 35)and 330±225ng/ ml (n= 28), respectively. Intra‐uterine growth retardation in preterm infants does not influence MBP levels. For those without the codon 54 mutation, there is a significant difference in MBP level between the three age groups. For those with the codon 54 mutation, there is a significant difference between preterm and term infants, but not between term infants and adults. We conclude that there is a maturation in MBP levels for preterm infants, and that a moderately low MBP phenotype does not affect survival. We cannot exclude an effect of profoundly reduced MBP levels (characteristic of individuals homozygous for the codon 54 mutation), since no such preterm infant was identified in this study.

RET receptor tyrosine kinase isoforms in kidney function and disease

The RET proto-oncogene encodes two major isoforms, RET9 and RET51, which differ at the carboxyl-terminal. Loss-of-function mutations in RET result in gut aganglionosis while gain of function mutations result in cancer syndromes. From studies on transgenic mice, RET9 is important for early development of the kidney and the enteric nervous system. Little is known about the function of RET isoforms in later life. Here we report the expression of RET isoforms and its signalling complex, GDNF and GFRα1, in foetal and adult human kidneys. We found their expression in both the developing and the adult renal collecting system. We further show that only RET51 but not RET9 could promote the survival and tubulogenesis of mIMCD3 (mouse inner medullary collecting duct) cells in collagen gel. Our results agree with the hypothesis that RET51 signalling is related to differentiation events in later kidney organogenesis. In addition, it may also have a function in the adult kidney. We further extend our study by showing increased RET and GDNF expression in collecting duct cysts of polycystic kidney patients. This suggests that GDNF/RET signalling may contribute to proliferation of the collecting duct epithelium in an autocrine/paracrine manner.

EXPRESSION OF TRANSFORMING GROWTH FACTOR ALPHA AND EPIDERMAL GROWTH FACTOR RECEPTOR IN ADULT POLYCYSTIC KIDNEY DISEASE

Adult polycystic kidney disease (APKD) is a common genetic disease with a frequency of 1:1000. Evidence suggests that transforming growth factor alpha (TGF alpha) signaling may contribute to the hyperproliferation of the cystic epithelia in APKD. TGF alpha and epidermal growth factor (EGF) are well known mitogens expressed in the kidney and both exert their biological activities through binding to the same EGF receptor. A transgenic mouse that over-expressed TGF alpha developed renal cysts; raised levels of TGF alpha and EGF receptor mRNA were found in kidneys from two autosomal dominant APKD patients. To study the role of TGF alpha in cyst formation, we analyzed nine anatomically diagnosed adult polycystic kidneys and four normal kidneys using immunohistochemistry. We also traced the possible origins of the cysts by staining with the proximal convoluted tubule (PCT) marker, gp330, and the distal convoluted tubule (DCT) and collecting tubule (CT) marker, peanut agglutinin (PNA). In normal kidneys, TGF alpha protein was concentrated in the DCT and CT and EGF receptor protein in all three tubule types. In the early cysts of APKD, the cystic epithelia showed strong positive staining with TGF alpha, EGF receptor and gp330 but negative with PNA. Strong TGF alpha and EGF receptor staining was also found in the mixture of advanced cysts in the end-stage cystic kidneys although the cysts are likely to be derived from different segment of the renal tubules. This increased TGF alpha and EGF receptor expression in all cases and all types of cysts suggests that autocrine/paracrine stimulation by TGF alpha may be a common mechanism in cyst development in APKD.

Cysteine (C)-X-C Receptor 4 Undergoes Transportin 1-Dependent Nuclear Localization and Remains Functional at the Nucleus of Metastatic Prostate Cancer Cells

The G-protein coupled receptor (GPCR), Cysteine (C)-X-C Receptor 4 (CXCR4), plays an important role in prostate cancer metastasis. CXCR4 is generally regarded as a plasma membrane receptor where it transmits signals that support transformation, progression and eventual metastasis. Due to the central role of CXCR4 in tumorigenesis, therapeutics approaches such as antagonist and monoclonal antibodies have focused on receptors that exist on the plasma membrane. An emerging concept for G-protein coupled receptors is that they may localize to and associate with the nucleus where they retain function and mediate nuclear signaling. Herein, we demonstrate that CXCR4 associated with the nucleus of malignant prostate cancer tissues. Likewise, expression of CXCR4 was detected in nuclear fractions among several prostate cancer cell lines, compared to normal prostate epithelial cells. Our studies identified a nuclear pool of CXCR4 and we defined a nuclear transport pathway for CXCR4. We reveal a putative nuclear localization sequence (NLS), ‘RPRK’, within CXCR4 that contributed to nuclear localization. Additionally, nuclear CXCR4 interacted with Transportinβ1 and Transportinβ1-binding to CXCR4 promoted its nuclear translocation. Importantly, Gαi immunoprecipitation and calcium mobilization studies indicated that nuclear CXCR4 was functional and participated in G-protein signaling, revealing that the nuclear pool of CXCR4 retained function. Given the suggestion that functional, nuclear CXCR4 may be a mechanism underlying prostate cancer recurrence, increased metastatic ability and poorer prognosis after tumors have been treated with therapy that targets plasma membrane CXCR4, these studies addresses a novel mechanism of nuclear signaling for CXCR4, a novel mechanism of clinical targeting, and demonstrate an active nuclear pool that provides important new information to illuminate what has been primarily clinical reports of nuclear CXCR4.

Identification of SCN1A and PCDH19 Mutations in Chinese Children with Dravet Syndrome

Background Dravet syndrome is a severe form of epilepsy. Majority of patients have a mutation in SCN1A gene, which encodes a voltage-gated sodium channel. A recent study has demonstrated that 16% of SCN1A-negative patients have a mutation in PCDH19, the gene encoding protocadherin-19. Mutations in other genes account for only a very small proportion of families. TSPYL4 is a novel candidate gene within the locus 6q16.3-q22.31 identified by linkage study. Objective The present study examined the mutations in epileptic Chinese children with emphasis on Dravet syndrome. Methods A hundred children with severe epilepsy were divided into Dravet syndrome and non-Dravet syndrome groups and screened for SCN1A mutations by direct sequencing. SCN1A-negative Dravet syndrome patients and patients with phenotypes resembling Dravet syndrome were checked for PCDH19 and TSPYL4 mutations. Results Eighteen patients (9 males, 9 females) were diagnosed to have Dravet syndrome. Among them, 83% (15/18) had SCN1A mutations including truncating (7), splice site (2) and missense mutations (6). The truncating/splice site mutations were associated with moderate to severe degree of intellectual disability (p<0.05). During the progression of disease, 73% (11/15) had features fitting into the diagnostic criteria of autism spectrum disorder and 53% (8/15) had history of vaccination-induced seizures. A novel PCDH19 p.D377N mutation was identified in one SCN1A-negative female patient with Dravet syndrome and a known PCDH19 p.N340S mutation in a female non-Dravet syndrome patient. The former also inherited a TSPYL4 p.G60R variant. Conclusion A high percentage of SCN1A mutations was identified in our Chinese cohort of Dravet syndrome patients but none in the rest of patients. We demonstrated that truncating/splice site mutations were linked to moderate to severe intellectual disability in these patients. A de novo PCDH19 missense mutation together with an inherited TSPYL4 missense variant were identified in a patient with Dravet syndrome.

Subcellular Localization of SUN2 Is Regulated by Lamin A and Rab5

SUN2 is an inner nuclear membrane protein with a conserved Sad1/UNC-84 homology SUN-domain at the C-terminus. Intriguingly, SUN2 has also been reported to interact with Rab5, which localizes in early endosomes. To clarify the dual subcellular localization of SUN2, we investigated its localization in lamin A/C deficient cells rescued with lamin A or lamin C isoform, and in HeLa cells transfected with Rab5 or its mutants. We found that expression of lamin A but not lamin C partly restored the nuclear envelope localization of SUN2. SUN2 was redistributed to endosomes upon overexpression of Rab5, but remained on the nuclear envelope when the SUN domain was deleted. To explore the physiological function of SUN2 in vesicle trafficking and endocytosis, we demonstrated the colocalization of endogenous SUN2 and Rab5. Moreover, overexpression of SUN2 stimulated the uptake of transferrin while suppression of SUN2 expression attenuated the process. These findings support a role of SUN2 in endocytosis.

Isolation of differentially expressed genes in human heart tissues

We applied RNA arbitrarily primed-PCR (RAP-PCR) to screen the genes differentially expressed between common congenital heart defects (CHD) [atrial septal defect, ventricular septal defect, Tetrology of Fallot (TOF)] and normal human heart samples. Three of these differentially amplified fragments matched cDNA sequences coding for proteins of unknown function in humans: hCALO (human homologue of calossin), NP79 (coding for a nuclear protein of 79KD) and SUN2 (Sad-1 unc-84 domain protein 2). The other four fragments were from known human genes: apolipoprotein J, titin, dystrophin and protein kinase C-delta. Northern blot analysis confirmed that all of these genes are expressed in the human heart. The results of RAP-PCR were reconfirmed by quantitative RT-PCR in TOF and control heart samples. Both techniques showed the levels of expression of hCALO, NP79 and SUN2 to be comparable in TOF and control samples and the level of expression of dystrophin and titin, both coding for cytoskeletal proteins, to be significantly upregulated in TOF samples. In summary, we have shown that the RAP-PCR technique is useful in the identification of differentially expressed gene from biopsy samples of human CHD tissues. In this manner, we have identified three novel genes implicated in the normal function of the human heart and two known genes upregulated in TOF samples.

Neurobehavioral deficits of epidermal growth factor-overexpressing transgenic mice: impact on dopamine metabolism.

Epidermal growth factor (EGF) and its family member neuregulin-1 are implicated in the etiology of schizophrenia. Our recent pharmacological studies indicate that EGF injections to neonatal and adult rats both induce neurobehavioral deficits relevant to schizophrenia. We, however, did not evaluate the genetic impact of EGF transgene on neurobehavioral traits. Here we analyzed transgenic mice carrying the transgene of mouse EGF cDNA. As compared to control littermates, heterozygous EGF transgenic mice had an increase in EGF mRNA levels and showed significant decreases in prepulse inhibition and context-dependent fear learning, but there were no changes in locomotor behaviors and sound startle responses. In addition, these transgenic mice exhibited higher behavioral sensitivity to the repeated cocaine injections. There were neurochemical alterations in metabolic enzymes of dopamine (i.e., tyrosine hydroxylase, dopa decarboxylase, catechol-O-methyl transferase) and monoamine contents in various brain regions of the EGF transgenic mice, but there were no apparent neuropathological signs in the brain. The present findings rule out the indirect influence of anti-EGF antibody production on the reported behavioral deficits of EGF-injected mice. These results support the argument that aberrant hyper-signals of EGF have significant impact on mouse behavioral traits and dopamine metabolism.