Down syndrome and the molecular pathogenesis resulting from trisomy of human chromosome 21

Elizabeth Fisher and Victor collaboratively for many years on Tybulewicz have worked the Down syndrome mouse model project. Elizabeth Fisher＇s background is in molecular genetics and mouse models, with an interest in anueploidy. Victor Tybulewicz is an immunologist whose primary interest is in signal transduction from the antigen receptors of B and T cells.

Studies on the integration of hepatitis B virus DNA sequence in human sperm chromosomes

Aim: To study the integration of hepatitis B virus (HBV) DNA into sperm chromosomes in hepatitis B patients and the features of its integration. Methods: Sperm chromosomes of 14 subjects (5 healthy controls and 9 HB patients, including 1 acute hepatitis B, 2 chronic active hepatitis B, 4 chronic persistent hepatitis B, 2 HBsAg chronic carriers with no clinical symptoms) were prepared using interspecific in vitro fertilization between zona-free hamster oocytes and human spermatozoa. Fluorescence in situ hybridization (FISH) to sperm chromosome spreads was carried out with biotin-labeled full length HBV DNA probe to detect the specific HBV DNA sequences in the sperm chromosomes. Results: Specific fluorescent signal spots for HBV DNA were seen in sperm chromosomes of one patient with chronic persistent hepatitis B. In 9(9/42) sperm chromosome complements containing fluorescent signal spots, one presented 5 obvious FISH spots and the others 2 to 4 signals. The fluorescence intensity showed significant difference among the signal spots. The distribution of signal sites among chromosomes seems to be random. Conclusion: HBV could integrate into human sperm chromosomes. Results suggest that the possibility of vertical transmission of HBV via the germ line to the next generation is present.

Repeat Sequences and Base Correlations in Human Y Chromosome Palindromes

On the basis of information theory and statistical methods, we use mutual information, n- tuple entropy and conditional entropy, combined with biological characteristics, to analyze the long range correlation and short range correlation in human Y chromosome palindromes. The magnitude distribution of the long range correlation which can be reflected by the mutual information is PS〉PSa〉PSb （P5a and P5b are the sequences that replace solely Alu repeats and all interspersed repeats with random uneorrelated sequences in human Y chromosome palindrome 5, respectively）; and the magnitude distribution of the short range correlation which can be reflected by the n-tuple entropy and the conditional entropy is PS〉P5a〉PSb〉random uncorrelated sequence. In other words, when the Alu repeats and all interspersed repeats replace with random uneorrelated sequence, the long range and short range correlation decrease gradually. However, the random nncorrelated sequence has no correlation. This research indicates that more repeat sequences result in stronger correlation between bases in human Y chromosome. The analyses may be helpful to understand the special structures of human Y chromosome palindromes profoundly.

Exploring Codon Usage Patterns of Alternatively Spliced Genes in Human Chromosome 1

In this study, 414 whole protein-coding sequences (238 004 codons) of alternatively spliced genes of human chromosome 1 have been employed to explore the patterns of codon usage bias among genes. Overall codon usage data analysis indicates that G- and C-ending codons are predominant in the genes. The base usage in all three codon positions suggests a selection-mutation balance. Multivariate statistical analysis reveals that the codon usage variation has a strong positive correlation with the expressivities of the genes (r=0.5790, P<0.0001). All 27 codons identified as optimal are G- and C-ending codons. Correlation analysis shows a strong negative correlation between the gene length and codon adaptation index value (r=0.2252, P<0.0001), and a significantly positive correlation between the gene length and Nc values (r=0.1876, P<0.0001). These results suggest that the comparatively shorter genes in the genes have higher codon usage bias to maximize translational efficiency, and selection may also contribute to the reduction of highly expressed proteins.

Effects of hepatitis B virus infection on human sperm chromosomes

AIM: To evaluate the level of sperm chromosome aberrations in male patients with hepatitis B, and to directly detect whether there are HBV DNA integrations in sperm chromosomes of hepatitis B patients.METHODS: Sperm chromosomes of 14 tested subjects (5healthy controls, 9 patients with HBV infection, including 1with acute hepatitis B, 2 with chronic active hepatitis B, 4with chronic persistent hepatitis B, 2 chronic HBsAg carriers with no clinical symptoms) were prepared using interspecific in vitro fertilization between zona-free golden hamster ova and human spermatozoa, and the frequencies of aberration spermatozoa were compared between subjects of HBV infection and controls. Fluorescence in situ hybridization (FISH) to sperm chromosome spreads was carried out with biotin-labeled full length HBV DNA probe to detect the specific HBV DNA sequences in the sperm chromosomes.RESULTS: The total frequency of sperm chromosome aberrations in HBV infection group (14.8%, 33/223) was significantly higher than that in the control group (4.3%,5/116). Moreover, the sperm chromosomes in HBV infection patients commonly presented stickiness, clumping, failure to staining, etc, which would affect the analysis of sperm chromosomes. Specific fluorescent signal spots for HBV DNA were seen in sperm chromosomes of one patient with chronic persistent hepatitis. In 9 (9/42) sperm chromosome complements containing fluorescent signal spots, one presented 5 obvious FISH spots, others presented 2 to 4signals. There was significant difference of fluorescence intensity among the signal spots. The distribution of signal sites among chromosomes was random.CONCLUSION: HBV infection can bring about mutagenic effects on sperm chromosomes. Integrations of viral DNA into sperm chromosomes which are multisites and nonspecific, can further increase the instability of sperm chromosomes. This study suggested that HBV infection can create extensively hereditary effects by alteration genetic constituent and/or induction chromosome aberrations, as well as the possibility of vertical transmission of HBV via the germ line to the next generation.

Relationship between chromosome behaviour and stability of human-mouse and human-(human-mouse)hvbridomas

Chromosomes in human-mouse and human-(human-mouse)hybridomas wereanalysed by G-banding methods.It was found that most human chromosomes,exceptNo.13 and X,Y,were retained.The frequencies of chromosomes No.1,3,4,5,6,17,19,21 and 22 were higher than those of other chromosomes in each hybridoma clone.The myeloma cell lines X63-Ag8.653 and SHM-D33 were also analysed.The morphologyof marker chromosomes was apparently different between hybridomas.There were 7 kindsof marker chromosomes in human-mouse hybridomas and 16 kinds of markerchromosomes in human-(human-mouse)hybridomas.Clones that retained humanchromosome No.1 were more stable and clones that did not retain human chromosomeNo.14 were still capable of secreting human immunoglobulin.Clones that retained humanchromosome No.2 did not secret human k light chain McAb while clones that retainedhuman chromosomes No.2 and No.22 only secreted λ light chain.

LOSS OF HETEROZYGOSITY ON CHROMOSOME 13 IN SQUAMOUS CELL CARCINOMAS OF THE LARYNX

Objective: To locate lost region of tumor suppressor gene on chromosome 13q in squamous cell carcinoma of the larynx (LSCC) and to provide clues and evidence for discovering and locating new suppressor gene. Methods: Loss of heterozygosity (LOH) on chromosome 13q was analyzed in 58 LSCC patients by microsatellite polymorphic sequences in loci D13S765 (13q13), RB120 (13q142), D13S133 (13q143) and D13S318 (13q21) on chromosome 13 by PCR. Results: There weren't any LOH on chromosome 13q in 3 cases with preinvasive LSCC. Fortyfive percentage (24/53) of the 53 invasive LSCC cases showed LOH at one or more loci on chromosome 13q region. The highest percentage of LOH on chromosome 13q was 52% (22/53) at D13S765 locus. Conclusion: The deletion region on chromosome 13q was located near by D13S765 locus which is centromeric to RB1. In this region there is suppressor gene, which is related to the genesis and development of LSCC, possibly including RB1. The inactivation of these suppressor genes may be related to the genesis and development of invasive LSCC.

Lack of evidence for leukocyte maternal microchimerism in primary biliary cirrhosis

AIM:It is reasonable to assume that microchimerism could also be involved in the induction of primary biliary cirrhosis (PBC).However,previous reports investigated only fetus-microchimerism in women patients.Maternal microchimerism has not been investigated until now. The current study aimed to clear either maternal microchimerism was involved in the pathogenesis of PBC or not. METHODS:We used fluorescence in situ hybridization on paraffin-embedded tissue (We called“Tissue-FiSH”.) to determine whether maternal cells infiltrated in male patients who were diagnosed as having PBC.Tissue-FiSH was performed by using both X and Y specific probes on the biopsy liver sample of 3 male PBC patients. RESULTS:Infiltrating lymphocytes demonstrated both X and Y signals in all 3 male patients. CONCLUSION:Maternal microchimerism dose not play a significant role in PBC.PBC may not relate to fetus and maternal microchimerism.

PCR analysis of Yq microdeletions in infertile males, a study from South India

AIM: To estimate the frequency of microdeletions in the long arm of Y-chromosome of 20 infertile males from South India. METHODS: Polymerase chain reaction (PCR) amplification using Y-specific STS of azoospermia factor (AZF) regions i.e., SY 84 for AZFa, SY 127 for AZFb and SY 254 for AZFc. RESULTS: Of the 20 infertile subjects 3 (15 %), one azoospermic and two oligozoospermic, showed microdeletions in the AZF region of Y-chromosome. CONCLUSION: The frequency of deletions involving AZF region of the Y-chromosome is 15 % in azoospermic and severely oligozoospermic infertile men. PCR amplification of AZF locus is useful for the diagnosis of microdeletions in the Y-chromosome.

Natural Selection on Human Y Chromosomes

In the field of anthropology, the uniparerttally inherited Y chromosome has long been used to trace the paternal lineage of the populations and to understand differences in migration and population genetics between males and females, with additional advantages of small effective population size, suf- ficient markers, and population-specific haplotype distribution （Jobling and Tyler-Smith, 1995; Jin and Su, 2000; Underhill et al., 2000）. Many such population studies have rested on the assumption that all the Y chromosome markers in the non- recombination regions are selectively neutral （Jobling and Tyler-Smith, 2003）.

Studies on in situ PCR Detected by the BrdU Antibody Technique

Using the BrdU antibody technique followed by an immuno-chemical staining(BAT),the amplification o f DNA fragments specific to human Y chromosome on cell specimen slides was efficiently detected. Whether direct BrdU incorporation into PCR products or in situ hybridization with PCR products on slides, the amplified targetDNA fragments of specimen were visualized by BAT under the microscope. The availability of BAT and differencesin the sensitivity and efficiency between BAT and dig--if-dUTP labeling in cell in situ PCR were disCussed.

High frequency loss of heterozygosity on the long arms of chromosomes 13 and 14 in nasopharyngeal carcinoma in Southern China

OBJECTIVE: To investigate the loss of heterozygosity (LOH) on chromosomal arms 13q and 14q in nasopharyngeal carcinoma (NPC) using 21 microsatellite polymorphic markers and to study whether there is a correlation between LOH and clinicopathologic parameters and/or Epstein-Barr virus (EBV) infection in NPC. METHODS: Sixty cases of NPC were studied using polymerase chain reaction based microsatellite analysis with genescan and genotyping techniques. RESULTS: LOH was detected on 13q in 78% of NPC tumors, high frequency LOH loci (more than 30%) clustered to 13q12.3-q14.3 and 13q32. On chromosome 14q, LOH was detected in 80% of NPC tumors; high frequency LOH loci clustered to 14q11-q13, 14q21-q24 and 14q32. High frequency LOH at 13q31-q32 correlated with a lower level of EBV infection; LOH on chromosome 14q was closely associated with poor differentiation of NPC tumor cells. CONCLUSION: Our results suggest that in NPC, LOH on chromosome 13q and 14q are common genetic events, and putative tumor suppressor genes (TSG) residing in these regions may be involved in tumorigenesis.

Linkage analysis of a region on chromosome 2 with essential hypertension in Chinese families

OBJECTIVE: To verify the linkage of the candidate regions identified in a previous study (markers D2S168, D2S151, D2S142 on chromosome 2) with hypertension in Chinese families. METHODS: A genetic linkage study focused on chromosome 2 was performed on 240 Chinese families containing 856 patients with essential hypertension. A total of 1080 individuals were genotyped using 9 highly polymorphic microsatellite markers around the candidate regions on chromosome 2 with an average spacing of 5 cM. Non-parametric linkage (NPL), parametric linkage analysis and transmission-disequilibrium test (TDT) with the GENEHUNTER software were used to assess evidence for linkage. RESULTS: Linkage of a region on chromosome 2 around D2S151 and D2S142 with hypertension was confirmed by different statistical methods (NPL, LOD score and TDT). However, the linkage of D2S168 could not be replicated in this extension study. CONCLUSIONS: The data suggest that a region on chromosome 2 at or near the loci of D2S142 and D2S151 may harbor genes responsible for the development of essential hypertension in Chinese.

Chromosome 14q may harbor multiple tumor suppressor genes in primary glioblastoma multiforme

OBJECTIVE: To evaluate whether deletion of chromosome 14q is involved in the carcinogenesis of primary glioblastoma multiforme and to identify possibly common deletion regions. METHJODS: Fourteen fluorescent dye-labeled polymorphic markers were used and polymerase chain reaction-based microsatellite analysis was employed to investigate loss of heterozygosity (LOH) on chromosome 14q in 20 primary glioblastoma multiforme (GBM). RESULTS: Ten of twenty (50%) GBM displayed LOH at one or more of the markers on chromosome 14q. Five tumors showed either LOH or non-informative on all markers tested. The most frequent LOH was observed at locus D14S65 (57.1%) on 14q32.1, and in the chromosomal region spanning from D14S63 (47.1%) to D14S74 (46.7%) on 14q23-31. None of the informative loci exhibited microsatellite instability. CONCLUSIONS: Allelic deletion on chromosome 14q plays an important role in the pathogenesis of GBM. Chromosomal regions at locus D14S65 on 14q32.1 and spanning from D14S63 to D14S74 on 14q23-31 may harbor multiple tumor suppressor genes associated with GBM.

Familial Wolff-Parkinson-White syndrome is linked to the loci on chromosome 7q3

OBJECTIVE: Wolff-Parkinson-White syndrome (WPW) is considered to be an autosomal dominant hereditary disease, but the gene is not identified. The objective of this study was to localize the genetic loci of Wolff-Parkinson-White syndrome. METHODS: Linkage analysis between the disease of Wolff-Parkinson-White syndrome and 3 STR (short tandem repeats) markers on 7q3 (D7S505, D7S688, and D7S483) was tested in 3 kindreds of the Wolff-Parkinson-White syndrome (101 numbers in total) by genotyping. RESULTS: Wolff-Parkinson-White syndrome was linked to the loci above. The maximum two-point Lod score detected at D7S505 was 6.4 at a recombination fraction (theta) of 0.1; the Lod score of D7S688, D7S483 was 5.3 vs 2.5. CONCLUSION: The gene of Wolff-Parkinson-White syndrome is located at 7q3.

How Often Does Human DNA Mutate?

Editor＇s comments The human mutation rate how often new changes appear in the DNA--is fundamental to understanding many aspects of medical genetics and human evolutionary genetics. But it is low, and has therefore been difficult to measure. In the past, scientists could only estimate it approximately, either by observing how often mutant phenotypes appeared, or by comparison of humans and closely related species, such as chimpanzee, where many mutations could accumulate but the time period was uncertain. Now, a new study supported by the NSFC in China and The Royal Society in the UK reports the first direct measurement of the human mutation rate at the individual letters （ nucleotides or bases） of DNA. This was possible because new （ next ）-generation sequencing technology is much more powerful than the methods available previously. The work was published in the lead- ing journal Currerzt Biology on 15th September 2009. The results were reported in the news by Nature, Science and the BBC , as well as in more than 20 Chinese newspapers and radio stations after the work first appeared online on 27th August. It was also one of the research highlights in Nature on 3rd September, which commented ＂ This direct measurement of the human mutation rate should help researchers to refine evolutionary dating and better understand the source of genetic disease＇. From the work, researchers could estimate that everyone has around 200 new mutations in their genome ; as the authors said, ＂we are all mutants＂. The ability to reliably measure rates of DNA mutation means we can begin to ask how mutation rates vary between different regions of the genome and perhaps also between different individuals.

The predictive value of chromosome 8p deletion for metastasis of hepatocellular carcinoma:a summary of works in 10 years

Hepatocellular carcinoma(HCC)represents an extremely poor prognostic cancer,which is mainly due to the high frequency of metastasis/recurrence after surgical operation.Exploring the molecular mechanisms involved in HCC metastasis could be helpful in the pre-diction and early diagnosis of HCC recurrence and could also provide new therapeutic targets for HCC metastasis.In the recent decade,we analyzed the genomic aberrations of the clinical specimens,as well as the metastatic models and cell lines of human HCC to identify the genetic mar-kers related to HCC metastasis and to verify their clinical values in the prediction and control of metastasis of HCC.Using the comparative genomic hybridization(CGH)technique,we compared the differences of chromosomal aberrations between primary HCC tumors and their matched metastatic lesions,and found that chromosome 8p deletions might contribute to HCC metastasis.This novel finding was further confirmed by comparison between nude mice models of HCC with different meta-static potentials.By the more sensitive genome-wide microsatellite analysis,8p deletion was defined to 8p23.3 and 8p11.2,which are two likely regions harboring meta-stasis-related genes of HCC.Using‘8p-specific’microar-rays,two novel metastatic suppressors(HTPAP and MRSA)were identified,and were proven to suppress in vitro invasion and in vivo metastasis of HCC.Clinical studies indicate that 8p deletion detected in HCC or cir-culating plasma DNA of patients is a useful predictor for metastatic recurrence and prognosis,even for patients with early stage HCC.These novel findings are regarded as important advances in the study of the molecular mechanisms of HCC metastasis,which provide not only a holistic view on the molecular cytogenetic bases of HCC metastasis,but also candidate regions for further study to identify metastatic suppressor genes.

Expression of recombinant human butyrylcholinesterase in the milk of transgenic mice

Butyrylcholinesterase(BCHE)is a natural bioscavenger that protects humans against organophosphate toxicity.Due to the limited yield of human BCHE(hBCHE)when purifying from human plasma,it is necessary to find an alternative method to produce this protein.One potential method is to produce transgenic livestock that make modified milk containing high concentration of hBCHE.In this study,we cloned the hBCHEgene into a human lactoferrin(hLF)bacterial artificial chromosome(BAC)construct to make a hLFhBCHE BAC construct.Subsequently,we injected the BAC construct into pronuclei of mouse fertilized embryos and generated transgenic mice.Expression analysis showed that recombinant hBCHE(rhBCHE)was expressed efficiently in the mammary gland of the transgenic mice and the concentration of rhBCHE in the milk of individual mice ranged from 76
12 to 159
28 mg·L^(–1).Protein function tests showed that rhBCHE has the same enzymatic activity as the native hBCHE.Our results pave the way for making transgenic livestock to produce large quantities of rhBCHE.