Genetic analysis of a pedigree with MECP2 duplication syndrome in China.

BACKGROUND: MECP2 duplication syndrome (MDS) is a rare X-linked genomic disorder that primarily affects males. It is characterized by delayed or absent speech development, severe motor and cognitive impairment, and recurrent respiratory infections. MDS is caused by the duplication of a chromosomal region located on chromosome Xq28, which contains the methyl CpG binding protein-2 (MECP2) gene. MECP2 functions as a transcriptional repressor or activator, regulating genes associated with nervous system development. The objective of this study is to provide a clinical description of MDS, including imaging changes observed from the fetal period to the neonatal period. METHODS: Conventional G-banding was employed to analyze the chromosome karyotypes of all pedigrees under investigation. Subsequently, whole exome sequencing (WES), advanced biological information analysis, and pedigree validation were conducted, which were further confirmed by copy number variation sequencing (CNV-seq). RESULTS: Chromosome karyotype analysis revealed that a male patient had a chromosome karyotype of 46,Y,dup(X)(q27.2q28). Whole-exon duplication in the MECP2 gene was revealed through WES results. CNV-seq validation confirmed the presence of Xq27.1q28 duplicates spanning 14.45 Mb, which was inherited from a mild phenotype mother. Neither the father nor the mother's younger brother carried this duplication. CONCLUSION: In this study, we examined a male child in a family who exhibited developmental delay and recurrent respiratory tract infections as the main symptoms. We conducted thorough family investigations and genetic testing to determine the underlying causes of the disease. Our findings will aid in early diagnosis, genetic counseling for male patients in this family, as well as providing prenatal diagnosis and reproductive guidance for female carriers.

[Study of GCN repeats of PHOX2B gene among individuals from southwest China and diagnosis of two patients with Congenital central hypoventilation syndrome].

OBJECTIVE: To study the trinucleotide repeats of GCN (GCA, GCT, GCC, GCG) encoding Alanine in exon 3 of the PHOX2B gene among healthy individuals from southwest China and two patients with Congenital central hypoventilation syndrome (CCHS). METHODS: The number and sequence of the GCN repeats of the PHOX2B gene were analyzed by capillary electrophoresis, Sanger sequencing and cloning sequencing of 518 healthy individuals and two newborns with CCHS, respectively. RESULTS: Among the 1036 alleles of the 518 healthy individuals, five alleles were identified, including (GCN)7, (GCN)13, (GCN)14, (GCN)15 and (GCN)20. The frequency of the (GCN)20 allele was the highest (94.79%). And five genotypes were identified, which included (GCN)7/(GCN)20, (GCN)13/(GCN)20, (GCN)14/(GCN)20, (GCN)15/(GCN)20, (GCN)20/(GCN)20. The homozygous genotypes were all (GCN)20/(GCN)20, and the carrier rate was 89.58%. Four GCN sequences of the (GCN)20 homozygous genotypes were identified among the 464 healthy individuals. The GCN repeat numbers in the exon 3 of the PHOX2B gene showed no significant difference between the expected and observed values, and had fulfilled the,Hardy-Weinberg equilibrium. The genotypes of the two CCHS patients were (GCN)20/(GCN)25 and (GCN)20/(GCN)30, respectively. CONCLUSION: It is important to determine the GCN repeats and genotypic data of the exon 3 of the PHOX2B gene among the healthy individuals. The number of GCN repeats in 518 healthy individuals was all below 20. The selection of appropriate methods can accurately detect the polyalanine repeat mutations (PARMs) of the PHOX2B gene, which is conducive to the early diagnosis, intervention and treatment of CCHS.

[Clinical application of non-invasive prenatal testing for twin pregnancies].

OBJECTIVE: To evaluate the feasibility of non-invasive prenatal testing (NIPT) for the screening of fetal chromosome aneuploidies in twin pregnancies. METHODS: A total of 2 745 women with twin-pregnancies were subjected for NIPT screening. Chromosomal karyotyping and chromosomal microarray analysis (CMA) were carried out on amniotic fluid samples from those with a high risk for fetal chromosome aneuploidies, and the diagnosis and pregnancy outcome were followed up. The sensitivity, specificity, positive predictive value and false positive rate of the NIPT were calculated. RESULTS: Compared with other chromosomal abnormalities, NIPT had a higher efficacy for trisomy 21 and sex chromosomal aneuploidy (SCA) in twin pregnancies (with sensitivity being 100%, 100%, and specificity being 99.93%, 99.9%, respectively). It is difficult to evaluate the efficacy for trisomies 18 and 13 due to the limited data. For chromosome microdeletions and microduplications spanning 15 ~ 21 Mb, NIPT also had a certain detection rate. Compared with women with natural conception, NIPT had a higher detection rate for those with twin pregnancies by assisted reproduction (P < 0.05). CONCLUSION: It is feasible to use NIPT for the detection of chromosome aneuploidies in women with twin pregnancies.

Prenatal diagnosis of mosaic chromosomal aneuploidy and uniparental disomy and clinical outcomes evaluation of four fetuses.

BACKGROUND: Few co-occurrence cases of mosaic aneuploidy and uniparental disomy (UPD) chromosomes have been reported in prenatal periods. It is a big challenge for us to predict fetal clinical outcomes with these chromosome abnormalities because of their highly heterogeneous clinical manifestations and limited phenotype attainable by ultrasound. METHODS: Amniotic fluid samples were collected from four cases. Karyotype, chromosome microarray analysis, short tandem repeats, and whole exome sequencing were adopted to analyze fetal chromosomal aneuploidy, UPD, and gene variation. Meanwhile, CNVseq analysis proceeded for cultured and uncultured amniocytes in case 2 and case 4 and MS-MLPA for chr11 and chr15 in case 3. RESULTS: All four fetuses showed mosaic chromosomal aneuploidy and UPD simultaneously. The results were: Case 1: T2(7%) and UPD(2)mat(12%). Case 2: T15(60%) and UPD(15)mat(40%). Case 3: 45,X(13%) and genome-wide paternal UPD(20%). Case 4: <10% of T20 and > 90% UPD(20)mat in uncultured amniocyte. By analyzing their formation mechanism of mosaic chromosomal aneuploidy and UPD, at least two adverse genetic events happened during their meiosis and mitosis. The fetus of case 1 presented a benign with a normal intrauterine phenotype, consistent with a low proportion of trisomy cells. However, the other three fetuses had adverse pregnancy outcomes, resulting from the UPD chromosomes with imprinted regions involved or a higher level of mosaic aneuploidy. CONCLUSION: UPD is often present with mosaic aneuploidy. It is necessary to analyze them simultaneously using a whole battery of analyses for these cases when their chromosomes with imprinted regions are involved or known carriers of a recessive allele. Fetal clinical outcomes were related to the affected chromosomes aneuploidy and UPD, mosaic levels and tissues, methylation status, and homozygous variation of recessive genes on the UPD chromosome. Genetic counseling for pregnant women with such fetuses is crucial to make informed choices.

Delineation of an inverted tandem Xq23-26.3 duplication in a female featuring extremely short stature and mild mental deficiency.

BACKGROUND: Partial duplications involving the long arm of the X chromosome are associated with mental retardation, short stature, microcephaly, and a wide range of physical findings. Female carriers usually have no clinical phenotype. Occasionally, they may also have heterogeneous features due to non-random inactivation of the X chromosome. METHODS: The peripheral blood sample was collected from the patient and subjected to a few genetic testing, including chromosomal karyotyping, Chromosomal microarray analysis (CMA), Optical genome mapping, short tandem repeat (STR) analysis for Determination of parental origin, and X chromosome inactivation (XCI) analysis. RESULTS: We have identified a de novo Xq23-Xq26.3 duplication in an adult female featuring extremely short stature and mild mental deficiency. Chromosome analysis detected a duplication on Xq23-q26.3 with a size of approximately 20 Mb. The duplication region has encompassed a number of genes, among which ARHGEF6, PHF6, HPRT1 and SLC9A6 are associated with X-linked mental retardation. Further analysis suggested that the duplication has derived from her father, was of the inversion duplication type and involved various degrees of skewed X chromosome inactivation. CONCLUSION: Correlation with her phenotypes might indicate new mechanisms by which the X chromosome may lead to short stature and mental retardation. Our findings thereby may shed more light on the phenotypic implication of functional disomy of X-chromosome genes.

Verification of a cryptic t(Y;15) translocation in a male with an apparent 45,X karyotype.

BACKGROUND: A rare disease is that an individual with a non-chimeric karyotype of 45,X develops into a male. We explored the genetic aetiology of an infertile male with an apparent 45,X karyotype, which was subsequently verified as cryptic translocation between chromosomes Y and 15. METHODS: DNA was extracted from the patient's peripheral blood. A range of genetic testing was performed, including conventional chromosomal karyotyping, short tandem repeat (STR) analysis for azoospermia factor (AZF) region, fluorescence in situ hybridization (FISH) with specific probes groups of DXZ1/DYZ3, DYZ3/D15Z1/PML and SRY/D15Z1/PML, and chromosomal microarray analysis (CMA) for genomic copy number variations (CNVs). RESULTS: The patient was found to have an apparent 45,X karyotype. STR analysis showed that he possessed a short arm of the Y chromosome, including the SRY gene; however, he was missing the long arm of the Y chromosome, including AZFa + b + c and Yqter. A FISH assay of DXZ1 and DYZ3 probes showed a green signal of the X centromere and a red of the Y centromeric signal on a D-group-sized chromosome. By FISH assaying with D15Z1 and DYZ3 probes, chromosomes 15 and Y centromeric signals appeared closely on a single chromosome, as the PML control probe ascertained. A further FISH assay with D15Z1 and SRY probes revealed a signal of the SRY gene at the end of one arm of chromosome 15. The result of the CMA indicated a deletion with an approximate size of 45.31 Mb spanning from Yq11 to Yter. CONCLUSION: Our study enriched the karyotype-phenotype correlation of Y and 15 chromosomes translocation. It strengthened the critical roles of molecular genetic techniques in identifying the chromosomal breakpoints and regions involved. Genetic aetiology can guide early intervention in childhood and assisted reproduction in adulthood.

Identification of an SRY-negative 46,XX infertility male with a heterozygous deletion downstream of SOX3 gene.

BACKGROUND: A male individual with a karyotype of 46,XX is very rare. We explored the genetic aetiology of an infertility male with a kayrotype of 46,XX and SRY negative. METHODS: The peripheral blood sample was collected from the patient and subjected to a few genetic testing, including chromosomal karyotyping, azoospermia factor (AZF) deletion, short tandem repeat (STR) analysis for AMELX, AMELY and SRY, fluorescence in situ hybridization (FISH) with specific probes for CSP 18/CSP X/CSP Y/SRY, chromosomal microarray analysis (CMA) for genomic copy number variations(CNVs), whole-genome analysis(WGA) for genomic SNV&InDel mutation, and X chromosome inactivation (XCI) analysis. RESULTS: The patient had a karyotype of 46,XX. AZF analysis showed that he missed the AZF region (including a, b and c) and SRY gene. STR assay revealed he possessed the AMELX in the X chromosome, but he had no the AMELY and SRY in the Y chromosome. FISH analysis with CSP X/CSP Y/SRY showed only two X centromeric signals, but none Y chromosome and SRY. The above results of the karyotype, FISH and STR analysis did not suggest a Y chromosome chimerism existed in the patient's peripheral blood. The result of the CMA indicated a heterozygous deletion with an approximate size of 867 kb in Xq27.1 (hg19: chrX: 138,612,879-139,480,163 bp), located at 104 kb downstream of SOX3 gene, including F9, CXorf66, MCF2 and ATP11C. WGA also displayed the above deletion fragment but did not present known pathogenic or likely pathogenic SNV&InDel mutation responsible for sex determination and development. XCI assay showed that he had about 75% of the X chromosome inactivated. CONCLUSIONS: Although the pathogenicity of 46,XX male patients with SRY negative remains unclear, SOX3 expression of the acquired function may be associated with partial testis differentiation of these patients. Therefore, the CNVs analysis of the SOX3 gene and its regulatory region should be performed routinely for these patients.

Assessment of Combined Karyotype Analysis and Chromosome Microarray Analysis in Prenatal Diagnosis: A Cohort Study of 3710 Pregnancies.

Objective: The current study aimed to compare the characteristics of chromosome abnormalities detected by conventional G-banding karyotyping, chromosome microarray analysis (CMA), or fluorescence in situ hybridization (FISH)/CNVplex analysis and further explore the application value of combined karyotype analysis and CMA in prenatal diagnosis with a larger sample size. Methods: From March 2019 to March 2021, 3710 amniocentesis samples were retrospectively collected from women who accepted prenatal diagnosis at 16 to 22 + 6 weeks of pregnancy. The pregnant women underwent karyotype analysis and CMA. In the case of fetal chromosomal mosaicism, FISH or CNVplex analysis was utilized for validation. Results: In total, 3710 G-banding karyotype results and CMA results from invasive prenatal diagnosis were collected. Of these, 201 (5.41%) fetuses with an abnormal karyotype were observed. The CMA analysis showed that the abnormality rate was 9.14% (340/3710). The detection rate of CMA combined with karyotype analysis was 0.35% higher than that of CMA alone and 4.08% higher than that of karyotyping alone. Additionally, 12 cases had abnormal karyotype analysis, despite normal CMA results. To further detect the chromosome mosaicism, we used FISH analysis to correct the karyotype results of case 1. Correspondingly, a total of 157 cases showed abnormal CMA results but normal karyotype analysis. We also found chromosomal mosaicism in 4 cases using CMA. Moreover, CNVplex and CMA demonstrated that representative case 15 was mosaicism for trisomy 2. Conclusions: Conventional G-banding karyotyping and CMA have their own advantages and limitations. A combination of karyotype analysis and CMA can increase the detection rate of chromosome abnormalities and make up for the limitation of signal detection.

A novel SRY pathogenic variant from a 46,XY female harboring a nonsense point mutation (G to A) in position 293.

46,XY female is a genetic disorder characterized by gonad gender not consistent with chromosomal sex. The SRY gene mutation is a common cause of 46,XY reversal type 1 (OMIM: 400044). Peripheral blood was collected from a 46,XY female patient and her father. Sex chromosomes were confirmed by karyotype analysis and fluorescence in situ hybridization (FISH) detection of the specific probe of sex chromosomes with cultured lymphocytes. After extracting blood genomic DNA, SRY characteristic fluorescence peak was detected by quantitative fluorescence PCR (QF-PCR) method. Whole exome was sequenced with NGS, and SRY gene was sequenced by Sanger sequencing, respectively. The chromosomes X and Y of the patient were confirmed by karyotype of 46,XY, and FISH specific probe of chromosome X and Y. SRY specific fluorescence peak was observed by QF-PCR. The whole-exome sequencing results showed chrY: 2655352(GRCh37): c.293G>A hemizygote mutation, confirmed by Sanger sequencing. The de novo mutation resulted in the mRNA encoding the tryptophan codon of 98 (UGG) change into a termination codon (UAG) (P.Trp98ter), and the translation process was terminated prematurely. The discovery of this novel mutation in the SRY gene helps elucidate the molecular mechanism of 46,XY female sex reversal and enriches such patients' genetic mutation spectrum.

2+0 CYP21A2 deletion carrier - a limitation of the genetic testing and counseling: A case report.

BACKGROUND: CYP21A2 gene mutations may all cause reduction or loss of 21-hydroxylase activity, leading to development of congenital adrenal hyperplasia (CAH) with different clinical phenotypes. For families with CAH children, genetic testing of the parents and genetic counseling are recommended to assess the risk of recurrence. CASE SUMMARY: We report a case of CAH with a high suspicion before delivery. The risk of the child suffering from CAH during the pregnancy had been underestimated due to the deviation of genetic counseling and genetic testing results. Our report confirmed a CYP21A2 homozygous deletion in this case, CYP21A2 heterozygous deletion in the mother, and a rare 2+0 CYP21A2 deletion in the father. CONCLUSION: It is important to analyze the distribution of CYP21A2 gene in the two alleles of parents of children with CAH.

Whole exome sequencing identifies c.963T > A and c.492 + 1G > A mutations in REN responsible for autosomal recessive renal tubular dysgenesis.

AIM: This study was aimed to identify the potentially pathogenic gene variants that contribute to the etiology of the autosomal recessive renal tubular dysgenesis (RTD) in the aborted fetus. METHODS: Illumina infinium global screening array was used to analyze chromosome karyotype of the aborted fetus. The exomes of the aborted fetus and his parents were sequenced using the whole exome sequencing technology. The resulting variants from whole exome sequencing were filtered by basic and advanced biological information analysis and the candidate mutation was verified by Sanger sequencing. RESULTS: Trisomy in chromosome 10 was found in the aborted fetus. The exon heterozygous variant of c.963T > A (p.Y321X) (nonsense mutation) and intron heterozygous variant of c.492 + 1G > A (splicing site mutation) in REN was first identified and validated by Sanger sequencing. Moreover, the exon heterozygous variant of c.963T > A (p.Y321X) and intron heterozygous variant of c.492 + 1G > A was from the mother and father, respectively. CONCLUSION: Our results reported the novel exon heterozygous variant of c.963T > A (p.Y321X) and intron heterozygous variant of c.492 + 1G > A in REN may contribute to autosomal recessive RTD, expanding our understanding of the causally relevant mutations for this disorder.

Growth hormone alleviates oxidative stress and improves the IVF outcomes of poor ovarian responders: a randomized controlled trial.

BACKGROUND: Oxidative stress (OS), defined as an imbalance between excessive reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) production and antioxidant insufficiency, has been suggested to be involved in the pathogenesis of poor ovarian response (POR). Growth hormone (GH) can reduce OS in some cell types. This study investigated whether GH can improve OS and the in vitro fertilization and embryo transfer (IVF-ET) outcomes of poor ovarian responders. METHODS: This study enrolled 105 patients with POR and 58 patients without POR (controls) who were diagnosed according to the Bologna criteria and underwent conventional IVF-ET. Poor ovarian responders were randomly assigned to two groups: the POR-GH group, which received pretreatment with GH 4 IU/d on day 2 of the previous menstrual cycle before IVF until the trigger day, and the POR-C group, which received no pretreatment. OS markers in follicular fluid (FF), ROS levels in granulosa cells (GCs), and the IVF outcomes of the groups were compared. RESULTS: Endometrial thickness on trigger day, the number of cleaved embryos, the number of higher-quality embryos, and the rates of embryo formation, higher-quality embryo formation, implantation and clinical pregnancy were significantly increased in the POR-GH group compared with the POR-C group (P < 0.05). Moreover, compared to those in the non-POR group, FF malondialdehyde (MDA), total oxidant status (TOS), oxidative stress index (OSI) and ROS levels in GCs were significantly higher, whereas superoxide dismutase (SOD) and the total antioxidant capacity (TAC) were significantly lower in the POR-C group (P < 0.05). Furthermore, compared with those in the POR-C group, the FF TAC was significantly increased in the POR-GH group, and TOS, OSI and intracellular ROS levels were significantly reduced (P < 0.05). CONCLUSIONS: Pretreatment with GH alleviates OS and improves oocyte quality and IVF outcomes of poor ovarian responders. TRIAL REGISTRATION: Chinese Clinical Trial Registry. ChiCTR1900021269 . Registered 8 February 2019, http://www.chictr.org.cn/edit.aspx?pid=35837&htm=4 .

[Prenatal diagnosis of a case of Pallister-Killian syndrome].

OBJECTIVE: To carry out G-banded chromosomal karyotyping and chromosomal microarray analysis (CMA) for a fetus featuring multiple malformations. METHODS: The fetus was found to have increased nuchal thickness, generalized edema, asymmetric lower limbs, tetralogy of Fallot, nasal bone anomaly and cleft palate. Following amniocentesis, G-band karyotyping and CMA were carried out. RESULTS: The fetus had a karyotype of 47,XX,+i(12)(p10) [14]/46,XX[6]. CMA has identified a 33.9 Mb duplication at 12p13.33-p11.1, which was suggestive of tetrasomy 12p. CONCLUSION: Combined chromosomal karyotyping and CMA can delineate the origin of abnormal chromosomal fragments during prenatal diagnosis. The fetus was diagnosed with Pallister-Killian syndrome.

[Genetic analysis of a 46,XY female with sex reversal due to duplication of NR0B1 gene].

OBJECTIVE: To explore the pathogenesis of a 46,XY female with sex reversal. METHODS: Peripheral blood lymphocytes of the patient were subjected to G-banding karyotype analysis. Sex chromosomes were analyzed with fluorescence in situ hybridization (FISH). SRY gene was analyzed by Sanger sequencing. The whole exome of the patient was subjected to next generation sequencing. Copy number variations (CNVs) of the NR0B1, SF1, SRY, SOX9 and WNT4 genes were validated by multiplex ligation-dependent probe amplification (MLPA). RESULTS: The patient had a 46,XY karyotype. FISH analysis showed that her sex chromosomes were X and Y. No mutation was found in the SRY gene, and no pathogenic mutation was detected in her exome. However, a duplication spanning approximately 67.31 kb encompassing the MAGEB1, MAGEB3, MAGEB4 and NR0B1 genes at Xp21, was predicted by software analysis. MLPA confirmed duplication of the NR0B1 gene in the patient and her mother. CONCLUSION: A duplication fragment of Xp21 encompassing the NR0B1 gene in the 46,XY female with sex reversal is transmitted from her asymptomatic carrier mother. Attention should be paid towards the insidious nature and high morbidity of this duplication.

[Application of quantitative fluorescencet-PCR in the prenatal diagnosis of chromosomale aneuploidies].

OBJECTIVE: To assess the accuracy of quantitative fluorescence PCR(QF-PCR) for the detection of fetal chromosomal aneuploidies and its values for prenatal diagnosis. METHODS: QF-PCR and chromosomal karyotyping were used to analyze 6066 amniotic fluid samples derived from 6034 pregnant women. RESULTS: Both QF-PCR and karyotyping analysis have detected 135 cases of fetal aneuploidies involving chromosomes 21, 18, 13, X, and Y. The QF-PCR assay was also successful in 67 cases for which amniotic fluid culture has failed. Furthermore, it has identified maternal cell contamination in 7 cases. By determining the consistency of short tandem repeat (STR) sites, the QF-PCR assay has identified 22 dizygotic twins among 32 twins with double chorions and double amniotic sacs. In 12 cases, it has signaled numerical chromosomal aberration by critical or partial abnormal values for the fluorescence peak area ratio, which were verified by karyotyping analysis as mosaicisms of chromosome aneuploidies. CONCLUSION: The QF-PCR can provide an useful supplement for chromosomal karyotyping and has an important role in rapid prenatal diagnosis.

[Analysis of genetics mechanism for the phenotypic diversity in a patient carrying a rare ring chromosome 9].

OBJECTIVE: To explore the genetics mechanism for the phenotypic variability in a patient carrying a rare ring chromosome 9. METHODS: The karyotype of the patient was analyzed with cytogenetics method. Presence of sex chromosome was confirmed with fluorescence in situ hybridization. The SRY gene was subjected to PCR amplification and direct sequencing. Potential deletion and duplication were detected with array-based comparative genomic hybridization (array-CGH). RESULTS: The karyotype of the patient has comprised 6 types of cell lines containing a ring chromosome 9. The SRY gene sequence was normal. By array-CGH, the patient has carried a hemizygous deletion at 9p24.3-p23 (174 201-9 721 761) encompassing 30 genes from Online Mendelian Inheritance in Man. CONCLUSION: The phenotypic variability of the 9p deletion syndrome in conjunct with ring chromosome 9 may be attributable to multiple factors including loss of chromosomal material, insufficient dosage of genes, instability of ring chromosome, and pattern of inheritance.

[Detection of chromosomal aneuploidies in spontaneous abortion samples by fluorescence in situ hybridization].

OBJECTIVE: To analyze 81 spontaneous abortion samples with fluorescence in situ hybridization (FISH). METHODS: Chromosome 13, 21, 16, 22, 18, X and Y probes were used to detect the samples. RESULTS: FISH was successful in 80 cases (98.77%). Among these, 35 (43.75%) had an abnormal karyotype, which included 19 autosomal aneuploidies, 6 sex chromosome aneuploidies, 9 triploidies and 1 tetraploidy. CONCLUSION: FISH is a rapid and easy method for detecting chromosomal aneuploidies in spontaneous abortion samples, and has a higher detection rate in early spontaneous abortion samples.

[Analysis of 89 amniotic samples using fluorescent in situ hybridization].

OBJECTIVE: To assess the value of fluorescent in situ hybridization (FISH) for detecting common chromosome aneuploidies in interphase nuclei of amniotic fluid cells. METHODS: Eighty two uncultured amniotic fluid samples and supernatants from 2 successfully and 5 unsuccessfully cultured amniotic fluid samples were analyzed with FISH. Results from standard cytogenetic analysis of 79 uncultured amniotic fluid samples and 2 successfully cultured amniotic fluid samples were compared with FISH results. RESULTS: All of the 89 samples were succeeded analyzed with FISH. Positive findings included 3 cases with trisomy 21, 1 case with 47, XYY and 1 case with 69, XXX, which were consistent with results of karyotype analysis. CONCLUSION: FISH is a rapid and accurate method for prenatal diagnosis, and can also provide a remedy to failed amniotic fluid cells culture.

Reference values of biochemical and hematological parameters for Guizhou minipigs.

The pig is not only an economically important livestock animal but is also a valuable model animal for biomedical research and xenotransplantation. Reference values for clinical biochemical and hematological parameters are required for accurate data interpretation while using a pig model. In this study, whole blood samples were collected from 54 healthy Chinese Guizhou minipigs. We analyzed routine biochemical and hematological parameters and special coagulation parameters, including thrombelastography and coagulation factor activities, and have presented the baseline values of these parameters. These data provide valuable information for investigators using minipigs as animal models in biomedical studies and useful physiological data for veterinarians and livestock producers. We also compared all the results for the minipigs with the corresponding data from healthy humans. The bilirubin, uric acid and cholesterol levels of minipigs were significantly lower than those of humans (14%, 0.086% and 48% of human levels, respectively), whereas the serum enzyme levels were much higher than those in humans (e.g. the hydroxybutyrate dehydrogenase and creatine kinase levels of the minipigs were 19- and 8.4-fold higher than the human reference values). The red blood cell counts, platelet counts and white blood cell counts of the minipigs were significantly higher than those of the humans. The coagulation activities of factor VII and factor X were higher in minipigs than in humans. The significant differences observed between minipigs and humans for many of these parameters suggest substantial interspecies disparities in organs and tissues. These differences merit greater attention in biomedical research involving minipigs, particularly in the area of pig-to-human transplantation.

Cloning and comparison of factor X from rhesus monkey (Macaca mulatta).

The reliability of the rhesus monkey as an important experimental animal depends on its genetic concordance with human. During our assessment of the rhesus monkey as a preclinical model for coagulation-related research, we cloned the full-length cDNA of rhesus monkey factor X (FX) and compared its genetic characteristics and coagulation activity with those of human FX. The full-length cDNA of rhesus monkey FX was 1683 bp in length, corresponding to 487 coding amino acids and sharing 94.71% nucleotide identity and 93.65% amino acid identity with human FX. When FX sequences from different animals were compared with that of human FX, rhesus monkey and baboon FX showed similar degrees of homology to human FX, which were less than that between human and chimpanzee FX sequences but remarkably higher than those of another 2 monkey species, bovine, pig, and rodents. Comparison of functional sites between human and rhesus monkey FX revealed high similarities between their amino acids sequences and 3-dimensional structures. The average coagulation activity of FX from 24 rhesus monkeys was in the normal range of that of healthy humans. The rhesus monkey therefore may be a suitable animal model for research addressing coagulation factor X.