Shuchao Pang

Altered expression of autophagic genes in the peripheral leukocytes of patients with sporadic Parkinson's disease.

Parkinsons disease (PD) is a progressive neurodegenerative disease caused by interaction of genetic and environmental factors. To date, genetic genes and variants causing PD remain largely unknown. Autophagy is a conserved cellular process including three subtypes, macroautophagy (hereafter referred to as autophagy), microautophagy and chaperone-mediated autophagy (CMA). Although reduced CMA and induced autophagy are observed in human PD brain samples, cell and animal PD models, CMA and autophagy have not been systemically studied in sporadic PD patients. In the peripheral leukocytes of sporadic PD patients, we examined gene expression levels of lysosome-associated membrane 2 (LAMP-2), a CMA receptor and a limiting step, and microtubule-associated protein 1 light chain 3 (LC3), product of which is sequentially cleaved and lipidated to form LC3-II as an autophagosome marker. Compared to age- and sex-matched healthy controls, LAMP-2 gene expression and protein levels in sporadic PD patients were significantly decreased, which may lead to reduced CMA activity and impaired fusion of autophagosome and lysosome. LC3 gene expression and LC3-II protein levels were significantly increased in sporadic PD patients, suggesting that autophagosomes are accumulated. Our findings, decreased LAMP-2 gene expression and increased LC3 gene expression, are consistent to the previous studies with dopaminergic neuronal cells in vitro and in vivo, which may contribute to the pathogenesis of sporadic PD by altering CMA and autophagy activities. The genetic causes leading to decreased LAMP-2 gene expression need further investigation and genetic or pharmacological restoration of LAMP-2 might be a novel strategy for treating PD patients.

Genetic analysis of SIRT1 gene promoter in sporadic Parkinson’s disease

Parkinsons disease (PD) is one of the most common neurodegenerative diseases. To date, genetic causes and underlying molecular mechanisms for sporadic PD remain largely unknown. Sirtuis are highly conserved NAD-dependent class III deacetylases. SIRT1, the closest to yeast Sir2, has deacetylase activity and ADP-ribosyltransferase activity. SIRT1 gene has been connected to many cellular processes and implicated in human diseases, such as obesity, type 2 diabetes, cancer and neurodegenerative diseases. Studies in animal model have also associated SIRT1 with aggregation of alpha-synuclein, a critical protein in the PD pathogenesis. We hypothesized that the genetic variants within the regulatory regions of SIRT1 gene that repress its gene expression, rather than mutations in its coding region that abolish SIRT1 function, may contribute to PD as a risk factor. In this study, we genetically analyzed the promoter region of SIRT1 gene in sporadic PD patients and ethic-matched healthy controls. Three novel heterozygous sequence variants, g.69644133C>G, g.69644213G>A and g.69644351G>A, were identified in PD patients, but in none of controls, which may alter the transcriptional activities of SIRT1 gene promoter, resulting in reduced SIRT1 levels. One novel heterozygous variant, g.69644219G>A, linked with single-nucleotide polymorphism - g.69644217A>C (rs932658), was only found in one control, which may have no functional activity. Therefore, our results suggested that genetic variants within the SIRT1 gene promoter may repress SIRT1 gene expression, contributing to PD as a risk factor.

Genetic analysis of the SIRT1 gene promoter in myocardial infarction.

Myocardial infarction (MI) is a restrictive phenotype of coronary artery disease. To date, a group of genes and genetic loci have been associated to MI. However, the genetic causes and underlying molecular mechanisms for MI remain largely unknown. SIRT1, one of highly conserved NAD-dependent class III deacetylases, has been involved in several cellular processes and implicated in human diseases. Autophagy is one of major cellular degradative pathways, which plays important roles in lipid metabolism. Recent studies have shown that SIRT1 deacetylates autophagy-related genes, and the expressions of autophagic genes are altered in MI patients. Accordingly, we hypothesized that SIRT1 may be linked to the MI pathogenesis. In this study, the SIRT1 gene promoter were genetically analyzed in large cohorts of MI patients (n = 327) and controls (n = 358). The results showed that six single-nucleotide polymorphisms and 14 sequence variants were identified. Among these, five novel heterozygous variants (g.69643743Ins, g.69643840Ins, g.69643903G > C, g.69644235G > C and g.69644353G > T) and one single-nucleotide polymorphism (rs35706870) were identified in MI patients, but in none of controls. Moreover, five novel heterozygous variants (g.69643672G > A, g.69644226C > T, g.69644278A > G, g.69644408G > A and g.69644408G > T) were only found in controls. The rest variants were found in MI patients and controls with similar frequencies. Taken together, the variants identified in MI patients may alter the transcriptional activities of SIRT1 gene promoter, which may change SIRT1 levels, contributing to the MI pathogenesis as a risk factor.

A novel and functional variant within the ATG5 gene promoter in sporadic Parkinson's disease

Parkinsons disease (PD) is one of the most common neurodegenerative diseases. Majority of PD are sporadic, for which genetic causes remain largely unknown. Alpha-synuclein, the main component of Lewy bodies, plays a central role in the PD pathogenesis. Macroautophagy is a highly conserved cellular process that digests dysfunctional macromolecules and damaged organelles. Accumulating evidence indicates that macroautophagy (hereafter referred to as autophagy) is involved in alpha-synuclein degradation. Dysregulation of autophagy has been observed in the brain tissues from PD patients and animal models. We hypothesized that change expression levels of autophagy-related genes (ATG), including ATG5, may contribute to PD. In this study, we genetically and functionally analyzed the ATG5 gene promoter in groups of sporadic PD patients and ethnic-matched healthy controls. A novel heterozygous variant, 106774459T>A, was identified in one female patient, but in none of controls, which significantly enhanced transcriptional activities of the ATG5 gene promoter. Furthermore, ATG5 gene expression level in the PD patient was significantly elevated than that in controls. Four novel heterozygous variants, 106774423C>A, 106774418C>A, 106774382C>A and 106774206G>A, were only found in controls. The variant, 106774464C>T, and SNP-106774030A>G (rs510432) were found in PD patients and controls with similar frequencies. Collectively, the variant identified in PD patient may change ATG5 protein levels and alter autophagy activities, contributing to PD onset as a risk factor.

Genetic analysis of the TBX20 gene promoter region in patients with ventricular septal defects

Congenital heart disease (CHD) is the most common human birth defect. The morbidity and mortality of CHD patients are significantly higher than normal population even after surgical correction of cardiac defects, which is likely caused by genetic defects. To date, genetic causes for CHD remain largely unknown. TBX20 gene encodes a T-box transcription factor that plays pivotal roles in cardiac morphogenesis and is required for maintaining adult heart function and maturation. Mutations in TBX20 gene have been reported in familiar and sporadic CHD patients. However, the promoter region of TBX20 gene has not been genetically analyzed in CHD patients. As TBX20 functions as a dosage-dependent regulator during the heart development, we hypothesized that the sequence variants within the promoter region of the TBX20 gene may contribute to CHD. In this study, we bi-directionally sequenced the promoter region of the TBX20 gene in 265 patients with ventricular septal defects (VSD) and 242 controls. Within the promoter region of the TBX20 gene, one single-nucleotide polymorphism (SNP), rs336284 (g.4740T>C), and one novel heterozygous variant g.4741 G>A, which was linked with rs336284 (g.4740 T>C), were found in both VSD patients and controls with similar frequencies. A novel heterozygous variant, g.4932 G>A, was found in one VSD patient, but in none of controls, which significantly inhibited the transcriptional activities of TBX20 gene promoter, suggesting that the variant may contribute to the VSD etiology. Therefore, our data provides new information with respect to TBX20 gene mutations in CHD patients.

Genetic analysis of the ATG16L1 gene promoter in sporadic Parkinson's disease

Parkinsons disease (PD) is a common and progressive neurodegenerative disease in which the majority of cases arise sporadically. Sporadic PD is caused by the interactions of genetic and environmental factors. To date, genetic causes for sporadic PD remain largely unknown. Autophagy, a highly conserved cellular process, has been implicated in PD pathogenesis. We speculated that genetic variants in autophagy-related genes (ATG) that regulate gene expression may contribute to PD development. In our previous studies, we have identified several functional DNA sequence variants (DSVs) in the ATG5, ATG7 and LC3 genes in sporadic PD patients. In this study, we further genetically and functionally analyzed the promoter of the ATG16L1 gene, a critical gene for autophagosome formation, in groups of sporadic PD patients and ethnic-matched healthy controls. One novel heterozygous DSV, 233251432C>T, was found in one PD patient. Functionally, this DSV did not affect the transcriptional activity of the ATG16L1 gene promoter in human dopaminergic SH-SY5Y cells. Two heterozygous DSVs including one SNP, 233251286G>A (rs539735288) and 233251582C>T, were found only in controls. In addition, five other SNPs were found in both PD patients and controls. Taken together, the data suggested that genetic variants within the ATG16L1 gene promoter were not a risk factor for sporadic PD development.

Genetic analysis of the LAMP-2 gene promoter in patients with sporadic Parkinson's disease.

Parkinsons disease (PD) is a common neurodegenerative disease in the people of over 65. Majority of PD is sporadic, which is caused by interaction of genetic and environmental factors. To date, genetic causes and underlying molecular mechanisms for sporadic PD remain largely unknown. Autophagy is a conserved cellular degradative process, consisting of macroautophagy, microautophagy and chaperone-mediated autophagy (CMA). Macroautophagy (hereafter referred to as autophagy) and CMA are involved in the degradation of alpha-synuclein, a critical protein in the PD pathogenesis. Previous studies with brain tissues and leukocytes have shown that the expression levels of lysosome-associated membrane-2 (LAMP-2) gene are significantly decreased in PD patients. In this study, we genetically and functionally analyze the promoter region of LAMP-2 gene in sporadic PD patients. Two novel sequence variants and two single nucleotide polymorphisms (SNPs) were identified. The heterozygous variant, g.4127A>C, which was only found in one female PD patient, significantly reduced the transcriptional activities of LAMP-2 gene promoter. The hemizygous variant, g.5038G>A, which was only found in one male control, enhanced the transcriptional activities of LAMP-2 gene promoter. No significant difference in frequencies of the SNPs, rs42900 (g.4569A>C) and rs28603270 (g.4760T>G), was observed between PD patients and controls. Collectively, the sequence variants within the LAMP-2 gene promoter may be linked to the PD onset by changing LAMP-2 protein levels and impairing autophagy and CMA activities.

Two novel and functional DNA sequence variants within an upstream enhancer of the human NKX2-5 gene in ventricular septal defects.

Mortality in patients with congenital heart disease (CHD) is significantly increased even with successful surgeries. The main causes are late cardiac complications, such as heart failure and arrhythmia, probably due to genetic defects. To date, genetic causes for CHD remain largely unknown. NKX2-5 gene encodes a highly conserved homeobox transcription factor, which is essential to the heart development in embryos and cardiac function in adults. Mutations in NKX2-5 gene have been implicated in diverse types of CHD, including ventricular septal defect (VSD). As NKX2-5 is a dosage-sensitive regulator, we have speculated that changed NKX2-5 levels may mediate CHD development by influencing cardiac gene regulatory network. In previous studies, we have analyzed the NKX2-5 gene promoter and a proximal enhancer in VSD patients. In the present study, we further genetically and functionally analyzed an upstream enhancer of the NKX2-5 gene in large cohorts of VSD patients (n=340) and controls (n=347). Two novel heterozygous DNA sequence variants (DSVs), g.17483576C>G and g.17483564C>T, were identified in three VSD patients, but none in controls. Functionally, these two DSVs significantly decreased the activity of the enhancer (P<0.01). Another novel heterozygous DSV, g.17483557Ins, was found in both VSD patients and controls with similar frequencies (P>0.05). Taken together, our data suggested that the DSVs within the upstream enhancer of the NKX2-5 gene may contribute to a small number of VSD. Therefore, genetic studies of CHD may provide insight into designing novel therapies for adult CHD patients.

Genetic and functional analysis of the NKX2-5 gene promoter in patients with ventricular septal defects.

The ventricular septal defect (VSD) is the most common type of congenital heart disease (CHD). The morbidity and mortality of CHD patients are significantly higher due to late cardiac complications, likely caused by genetic defects. Mutations in cardiac transcription factor genes such as GATA-4, TBX5, and NKX2-5 have been implicated in CHD cases. The NKX2-5 gene, a homeobox gene, is expressed in the developing heart and the adult heart. Because NKX2-5 is a dosage-sensitive regulator during embryonic development, the authors hypothesized that the expression levels of the NKX2-5 gene rather than the mutant protein may play important roles in CHD. In this study, the promoter regions and exon regions of the NKX2-5 gene were bidirectionally sequenced in large cohorts of VSD patients and healthy control subjects. The results showed that a novel sequence variant (g.4574c>deletion), found only in one VSD patient, and a single nucleotide polymorphism (rs118026695), the frequency of which was significantly higher in VSD patients, were identified within the promoter region. Functional analysis confirmed that these sequence variants significantly enhanced the transcriptional activities of the NKX2-5 gene promoter, altering the expression of the NKX2-5 gene and the cardiac gene regulatory network. In addition, a synonymous mutation in the second exon of the NKX2-5 gene was identified in one VSD patient, which may affect the translation process. Therefore, the authors’ data provide supportive evidence that mutations in the coding region of the NKX2-5 gene and sequence variants within its promoter region may be among the contributors to the CHD etiology.

Decreased gene expression of LC3 in peripheral leucocytes of patients with coronary artery disease.

Eur J Clin Invest 2011; 41 (9): 958–963