Pharmacogenetic study of methadone treatment for heroin addiction: associations between drug-metabolizing gene polymorphisms and treatment efficacy.

OBJECTIVES: Opioid dependence is currently one of the most serious problems affecting the social norms and public health system. Methadone maintenance therapy (MMT) is being widely used in treating heroin-dependent patients. The mechanism of methadone metabolism and disposition has been shown to involve cytochrome P450 (CYP450) and P-glycoprotein. The aim of this study was to explore the relationships among genetic polymorphisms, BMI and effective dose of methadone used in MMT within a northern Taiwan cohort. METHODS: One hundred heroin-dependent patients were enrolled in the study. The clinical data gathered included methadone dose, sex and BMI. DNA was collected from the oral swab of the participants to analyze the relevant alleles. RESULTS: An effective methadone dose correlated with sex, BMI and the presence of ABCB1 2677GG (rs2032582) and CYP2B6 516GG (rs374527). Furthermore, the CYP2B6 516GG homozygote was related to a higher average dose of methadone (GG: 68.50 ± 32.43; GT: 52.28 ± 25.75; TT: 44.44 ± 29.64; P < 0.02), whereas the ABCB1 2677GG homozygote was related to a lower dose (GG: 51.09 ± 20.83; GT: 69.65 ± 37.51; TT: 62.52 ± 30.44; P < 0.05). We examined the predictive effect of polymorphisms combined with sex and BMI on methadone dose by conducting multiple linear regressions. Our data predicted the average dose of methadone in approximately 30% of heroin-dependent patients. CONCLUSION: The interactions between genetic polymorphisms and clinical features proved useful in identifying the effective dose of MMT for heroin-dependent patients in Taiwan more precisely.

Effect of cell culture biomaterials for completely xeno-free generation of human induced pluripotent stem cells.

Human induced pluripotent stem cells (hiPSCs) were generated on several biomaterials from human amniotic fluid in completely xeno-free and feeder-free conditions via the transfection of pluripotent genes using a nonintegrating RNA Sendai virus vector. The effect of xeno-free culture medium on the efficiency of the establishment of human amniotic fluid stem cells from amniotic fluid was evaluated. Subsequently, the effect of cell culture biomaterials on the reprogramming efficiency was investigated during the reprogramming of human amniotic fluid stem cells into hiPSCs. Cells cultured in laminin-511, laminin-521, and Synthemax II-coated dishes and hydrogels having optimal elasticity that were engrafted with specific oligopeptides derived from vitronectin could be reprogrammed into hiPSCs with high efficiency. The reprogrammed cells expressed pluripotency proteins and had the capability to differentiate into cells derived from all three germ layers in vitro and in vivo. Human iPSCs could be generated successfully and at high efficiency (0.15-0.25%) in completely xeno-free conditions from the selection of optimal cell culture biomaterials.

Xeno-free and feeder-free culture and differentiation of human embryonic stem cells on recombinant vitronectin-grafted hydrogels.

Recombinant vitronectin-grafted hydrogels were developed by adjusting surface charge of the hydrogels with grafting of poly-l-lysine for optimal culture of human embryonic stem cells (hESCs) under xeno- and feeder-free culture conditions, with elasticity regulated by crosslinking time (10-30 kPa), in contrast to conventional recombinant vitronectin coating dishes, which have a fixed stiff surface (3 GPa). hESCs proliferated on the hydrogels for over 10 passages and differentiated into the cells derived from three germ layers indicating the maintenance of pluripotency. hESCs on the hydrogels differentiated into cardiomyocytes under xeno-free culture conditions with much higher efficiency (80% of cTnT+ cells) than those on conventional recombinant vitronectin or Matrigel-coating dishes just only after 12 days of induction. It is important to have an optimal design of cell culture biomaterials where biological cues (recombinant vitronectin) and physical cues (optimal elasticity) are combined for high differentiation of hESCs into specific cell lineages, such as cardiomyocytes, under xeno-free and feeder-free culture conditions.

The Potential Effect of Different Doses of Ionizing Radiation on Genes and Disease.

AIM: The chromosomal aberrations induced by radiation appear about nonrandomly distributed across the whole genome. Previous studies have shown that chromosomes with high DNA content are less frequently involved in the formation of symmetrical translocations and dicentric chromosomes than expected, whereas smaller chromosomes are more frequently involved. We hypothesized that these translocation regions are linked to radiation sensitivity. MATERIALS AND METHODS: We investigated the frequencies of chromosome translocations induced by radiation exposure and adjusted the results according to chromosome length. We specifically analyzed whole blood samples from 3 participants. The samples were irradiated using 60Co at doses of 0.5, 1, 2.5, and 5 Gy. Traditional Giemsa-trypsin-Wright band staining was performed to identify the translocations in the chromosomes, and results were compared with microarray data generated in our previous study. RESULTS: Our analysis indicated that chromosomes 9q were the most sensitive to translocations after various doses of radiation, and such translocations occurred in the euchromatin region. Chromosomes 1, 9, 15, and 17 were more sensitive to radiation doses of 0.5 Gy. This observation could be useful when selecting sensitive reference chromosomes in the low-dose region. The results of expression profiling analysis for radiation-sensitive regions were similar to those of chromosome translocation analysis. CONCLUSION: This study shows that some chromosomes or genomic regions are more sensitive to alteration by radiation exposure.

Clinical and molecular characterization of three genomic rearrangements at chromosome 22q13.3 associated with autism spectrum disorder.

OBJECTIVES: Chromosome 22q13 is a hot region of genomic rearrangements that may result in deletion, duplication, and translocation, and that may lead to neurodevelopmental disorders in affected patients. MATERIALS AND METHODS: We carried out an array-based comparative genomic hybridization analysis to detect copy number variations (CNVs) of genomic DNA in patients with autism spectrum disorders (ASD) who were consecutively recruited into our molecular genetic study of ASD. Karyotyping, fluorescent in-situ hybridization analysis, and real time-quantitative PCR were used for validation tests. RESULTS: We completed a genome-wide CNV analysis of 335 patients with ASD from Taiwan. Three unrelated male patients were found to carry three different CNVs at 22q13.3, respectively, including a de novo terminal deletion of ∼106 kb at 22q13.33, a de novo interstitial duplication of ∼1.8 Mb at 22q13.32-q13.33, and a microdeletion of ∼147 kb at 22q13.33. These three CNVs all involved the dosage change of the SHANK3 gene. The last patient also carried a genomic duplication of ∼3.86 Mb at 19q13.42-q13.4 in addition to a microdeletion of ∼147 kb at 22q13.33. His younger sister also carried these two CNVs, but she had developmental delay and other neurological deficits without ASD. These two CNVs were transmitted from their unaffected father, who carried a balanced translocation between chromosome 22q and 19q. CONCLUSION: Our data support that recurrent genomic rearrangements at 22q13.3 are part of the genetic landscape of ASD in our patients and changes in SHANK3 dosage are associated with neurodevelopmental disorders. However, the clinical symptoms of patients with 22q13.3 rearrangements can vary depending on other genetic and nongenetic factors, not limited to genes involved in CNVs in this region.

Complex genomic variants contribute toward the genetic architecture of autism spectrum disorder.

Genetic factor plays a critical role in the etiology of autism spectrum disorder (ASD). Both common variants with a small effect and rare mutations with a large effect contribute toward the genetic basis of ASD, showing the high genetic heterogeneity of ASD. Genomic rearrangements account for around 10-15% of its genetic landscape. However, they are highly individualized and each of them has a very rare frequency.

Interferon gamma polymorphisms associated with susceptibility to tuberculosis in a Han Taiwanese population.

BACKGROUND: Polymorphisms of the interferon gamma (IFN-γ) gene are associated with the risk of tuberculosis (TB) in different populations. However, the genetic susceptibility to TB in Han Chinese living in Taiwan is still unknown. The purpose of this study is to evaluate whether the polymorphisms of the IFN-γ gene are associated with TB in Han Taiwanese. METHODS: A total of 200 TB patients and 202 age-matched non-TB individuals were enrolled. Five tag single nucleotide polymorphisms (tSNPs) and rs2430561 (+874) of IFN-γ were selected from a public database. The genotypes were determined using polymerase chain reaction assays. RESULTS: Three IFN-γ polymorphisms in intron 3, rs1861494 and rs2069718, and rs2430561 in interon 1 were strongly associated with TB. The C carrier (CT+TT) of rs1861494, TT homozygous of rs2069718, and AA homozygous of rs2430561 were risk genotypes for susceptibility to TB. CONCLUSION: The IFN-γ polymorphisms, rs1861494, rs2069718, and rs2430561, may confer the risk of TB in Han Taiwanese.

MicroRNA expression profiling altered by variant dosage of radiation exposure.

Various biological effects are associated with radiation exposure. Irradiated cells may elevate the risk for genetic instability, mutation, and cancer under low levels of radiation exposure, in addition to being able to extend the postradiation side effects in normal tissues. Radiation-induced bystander effect (RIBE) is the focus of rigorous research as it may promote the development of cancer even at low radiation doses. Alterations in the DNA sequence could not explain these biological effects of radiation and it is thought that epigenetics factors may be involved. Indeed, some microRNAs (or miRNAs) have been found to correlate radiation-induced damages and may be potential biomarkers for the various biological effects caused by different levels of radiation exposure. However, the regulatory role that miRNA plays in this aspect remains elusive. In this study, we profiled the expression changes in miRNA under fractionated radiation exposure in human peripheral blood mononuclear cells. By utilizing publicly available microRNA knowledge bases and performing cross validations with our previous gene expression profiling under the same radiation condition, we identified various miRNA-gene interactions specific to different doses of radiation treatment, providing new insights for the molecular underpinnings of radiation injury.

Gene expression profiling of biological pathway alterations by radiation exposure.

Though damage caused by radiation has been the focus of rigorous research, the mechanisms through which radiation exerts harmful effects on cells are complex and not well-understood. In particular, the influence of low dose radiation exposure on the regulation of genes and pathways remains unclear. In an attempt to investigate the molecular alterations induced by varying doses of radiation, a genome-wide expression analysis was conducted. Peripheral blood mononuclear cells were collected from five participants and each sample was subjected to 0.5 Gy, 1 Gy, 2.5 Gy, and 5 Gy of cobalt 60 radiation, followed by array-based expression profiling. Gene set enrichment analysis indicated that the immune system and cancer development pathways appeared to be the major affected targets by radiation exposure. Therefore, 1 Gy radioactive exposure seemed to be a critical threshold dosage. In fact, after 1 Gy radiation exposure, expression levels of several genes including FADD, TNFRSF10B, TNFRSF8, TNFRSF10A, TNFSF10, TNFSF8, CASP1, and CASP4 that are associated with carcinogenesis and metabolic disorders showed significant alterations. Our results suggest that exposure to low-dose radiation may elicit changes in metabolic and immune pathways, potentially increasing the risk of immune dysfunctions and metabolic disorders.

The identification of a spontaneous 47, XX, +21/46, XY chimeric fetus with male genitalia.

BACKGROUND: Approximately 30 sex-chromosome discordant chimera cases have been reported to date, of which only four cases carried trisomy 21. Here, we present an additional case, an aborted fetus with a karyotype of 47,XX, +21/46,XY. CASE PRESENTATION: Autopsy demonstrated that this fetus was normally developed and had male genitalia. Major characteristics of Down syndrome were not observed except an enlarged gap between the first and second toes. Karyotyping of tissues cultured from the fetus revealed the same chimeric chromosomal composition detected in the amniotic fluid but with a different ratio of [47,XX,+21] to [46,XY]. Further short tandem repeat analysis indicated a double paternal contribution and single maternal contribution to the fetus, with the additional chromosome 21 in the [47,XX,+21] cell lineage originating from the paternal side. CONCLUSION: We thus propose that this chimeric fetus was formed via the dispermic fertilization of a parthenogenetic ovum with one (Y) sperm and one (X,+21) sperm.

Identification and characterization of three inherited genomic copy number variations associated with familial schizophrenia.

Schizophrenia is a complex mental disorder with high degree of genetic influence in its etiology. Several recent studies revealed that copy number variations (CNVs) of genomic DNA contributed significantly to the genetic architecture of sporadic schizophrenia. This study aimed to investigate whether CNVs also contribute to the familial forms of schizophrenia. Using array-based comparative genomic hybridization technology, we searched for pathogenic CNV associated with schizophrenia in a sample of 60 index cases from multiplex schizophrenia families. We detected three inherited CNVs that were associated with schizophrenia in three families, including a microdeletion of ~4.4Mb at chromosome 6q12-q13, a microduplication of ~1Mb at chromosome 18q12.3, and an interstitial duplication of ~5Mb at chromosome 15q11.2-q13.1. Our data indicate that CNVs contribute to the genetic underpinnings of the familial forms of schizophrenia as well as of the sporadic form. As 15q11-13 duplication is a well-known recurrent CNV associated with autism in the literature, the detection of the 15q11.2-q13.1 duplication in our schizophrenia patients provides additional support to other studies reporting that schizophrenia is part of the clinical spectrum of 15q11-q13 duplication syndrome.

Clinical and molecular characterization of a transmitted reciprocal translocation t(1;12)(p32.1;q21.3) in a family co-segregating with mental retardation, language delay, and microcephaly.

BACKGROUND: Chromosome translocation associated with neurodevelopmental disorders provides an opportunity to identify new disease-associated genes and gain new insight into their function. During chromosome analysis, we identified a reciprocal translocation between chromosomes 1p and 12q, t(1; 12)(p32.1; q21.3), co-segregating with microcephaly, language delay, and severe psychomotor retardation in a mother and her two affected boys. METHODS: Fluorescence in situ hybridization (FISH), long-range PCR, and direct sequencing were used to map the breakpoints on chromosomes 1p and 12q. A reporter gene assay was conducted in human neuroblastoma (SKNSH) and Chinese hamster ovary (CHO) cell lines to assess the functional implication of the fusion sequences between chromosomes 12 and 1. RESULTS: We determined both breakpoints at the nucleotide level. Neither breakpoint disrupted any known gene directly. The breakpoint on chromosome 1p was located amid a gene-poor region of ~ 1.1 Mb, while the breakpoint on chromosome 12q was located ~ 3.4 kb downstream of the ALX1 gene, a homeobox gene. In the reporter gene assay, we discovered that the fusion sequences construct between chromosomes 12 and 1 had a ~ 1.5 to 2-fold increased reporter gene activity compared with the corresponding normal chromosome 12 sequences construct. CONCLUSION: Our findings imply that the translocation may enhance the expression of the ALX1 gene via the position effect and result in the clinical symptoms of this family. Our findings may also expand the clinical phenotype spectrum of ALX1-related human diseases as loss of the ALX1 function was recently reported to result in abnormal craniofacial development.

Molecular cytogenetic analysis and clinical manifestations of a case with de novo mosaic ring chromosome 7.

AIM: Clinical and molecular cytogenetic investigations of a newborn girl exhibiting facial dysmorphism with developmental delay. METHODS: Phenotypic evaluation was first applied to examine the proband's developmental status. Computed tomography and colour transcranial Doppler were used then to investigate her brain structure and function. Subsequently, chromosomal abnormalities were examined by karyotyping and fluorescent in situ hybridization was performed to investigate size of fragments lost at the two distal ends of the ring chromosome 7. In addition, multicolour banding was applied to rule out structural rearrangement occurs in between the ring chromosome 7. RESULTS: The proband was born with mosaic supernumerary ring chromosome 7, without a normal karyotype detected in the peripheral blood lymphocytes. The distal arm of chromosome 7p (at least 255 kb from the telomere) was part of an extra ring chromosome 7. In addition, the distal arm of 7q, at least 8 kb from the telomere, was missing. There was no other chromosomal rearrangement detected by multicolour banding. INTERPRETATION: This is the 19th reported case of complete ring chromosome 7 mosaicism and the first survived case with mosaic supernumerary ring 7 without a normal karyotype detected in the peripheral lymphocytes.

Widespread expression of prostate apoptosis response-4 in nasopharyngeal carcinoma.

BACKGROUND: Prostate apoptosis response-4 (Par-4) augments apoptosis in various tumors, either during apoptotic insult or by ectopic overexpression. However, investigation of Par-4 expression in nasopharyngeal carcinoma (NPC) is lacking. METHODS: Specimens from patients with NPC, hypopharyngeal carcinoma (HPC), or oral cavity cancer were examined for Par-4 expression using immunohistochemistry. NPC cell proliferation and apoptosis were analyzed using immunohistochemical staining for Ki67, B-cell lymphoma 2 (Bcl-2), and in situ terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-16-dUTP nick end-labeling (TUNEL) assay, respectively. RESULTS: Par-4 was ubiquitously expressed in NPC biopsies (96.2%, 25/26) and was significantly higher than in HPC (47.6%, 50/105, p < .0001) and oral cavity cancers (38.7%, 12/31, p < .0001). Remarkably, apoptosis of NPC cells was absent and Par-4 expression was associated with obvious expression of Bcl-2 and Ki67 in all patients tested with NPC. CONCLUSIONS: Immunohistochemistry results showed widespread expression of Par-4 in NPC and revealed sustainable proliferation of NPC cells regardless of Par-4 expression.

Protein expression and intracellular localization of prostate apoptosis response-4 (Par-4) are associated with apoptosis induction in nasopharyngeal carcinoma cell lines.

Prostate apoptosis response-4 (Par-4) is a proapoptotic gene that selectively induces cell death in most cancer cells. In addition to the increased percentage of apoptotic cells, caspase-3 activity, and poly (ADP-ribose) polymerase (PARP) cleavage, we demonstrate that elevated expression of Par-4 and nuclear entry resulted in apoptosis of nasopharyngeal carcinoma (NPC) cell lines either in serum deprivation or by ectopic overexpression of Par-4. Moreover, disassociation from the Par-4/Akt complex was correlated with the induced proapoptotic ability of Par-4. Therefore, our data suggest that the cytoplasmic localization and expression level of endogenous Par-4 in NPC cells are not sufficient to augment apoptosis.

Prenatal diagnosis of occipital encephalocele, mega-cisterna magna, mesomelic shortening, and clubfeet associated with pure tetrasomy 20p.

We present the first case of a fetus with pure tetrasomy 20p proven by cord-blood sampling at 24 weeks of gestation. This case was diagnosed in utero with multiple congenital anomalies including occipital encephalocele, mega-cisterna magna, mesomelic shortening, and clubfeet. An analysis of GTG-banded chromosomes of 20 metaphase cells was performed. Female karyotype [47,XX, +i(20)(p10)] was revealed in all cells. Pure tetrasomy 20p was confirmed using fluorescent in situ hybridization (FISH) with a telomere probe for chromosome 20p in all seven metaphase cells. The pregnancy was terminated because of associated multiple anomalies and severe oligohydramnios. The postmortem examination confirmed the prenatal diagnosis.