Loss-of-function CFTR p.G970D missense mutation might cause congenital bilateral absence of the vas deferens and be associated with impaired spermatogenesis / 亚洲男科学杂志(英文版)

Congenital bilateral absence of the vas deferens (CBAVD) is observed in 1%-2% of males presenting with infertility and is clearly associated with cystic fibrosis transmembrane conductance regulator (CFTR) mutations. CFTR is one of the most well-known genes related to male fertility. The frequency of CFTR mutations or impaired CFTR expression is increased in men with nonobstructive azoospermia (NOA). CFTR mutations are highly polymorphic and have established ethnic specificity. Compared with F508Del in Caucasians, the p.G970D mutation is reported to be the most frequent CFTR mutation in Chinese patients with cystic fibrosis. However, whether p.G970D participates in male infertility remains unknown. Herein, a loss-of-function CFTR p.G970D missense mutation was identified in a patient with CBAVD and NOA. Subsequent retrospective analysis of 122 Chinese patients with CBAVD showed that the mutation is a common pathogenic mutation (4.1%, 5/122), excluding polymorphic sites. Furthermore, we generated model cell lines derived from mouse testes harboring the homozygous Cftr p.G965D mutation equivalent to the CFTR variant in patients. The Cftr p.G965D mutation may be lethal in spermatogonial stem cells and spermatogonia and affect the proliferation of spermatocytes and Sertoli cells. In spermatocyte GC-2(spd)ts (GC2) Cftr p.G965D cells, RNA splicing variants were detected and CFTR expression decreased, which may contribute to the phenotypes associated with impaired spermatogenesis. Thus, this study indicated that the CFTR p.G970D missense mutation might be a pathogenic mutation for CBAVD in Chinese males and associated with impaired spermatogenesis by affecting the proliferation of germ cells.

A novel homozygous frameshift mutation in / 亚洲男科学杂志(英文版)

Oligoasthenoteratozoospermia (OAT) refers to the combination of various sperm abnormalities, including a decreased sperm count, reduced motility, and abnormal sperm morphology. Only a few genetic causes have been shown to be associated with OAT. Herein, we identified a novel homozygous frameshift mutation in meiosis-specific nuclear structural 1 (MNS1; NM_018365: c.603_604insG: p.Lys202Glufs*6) by whole-exome sequencing in an OAT proband from a consanguineous Chinese family. Subsequent variant screening identified four additional heterozygous MNS1 variants in 6/219 infertile individuals with oligoasthenospermia, but no MNS1 variants were observed among 223 fertile controls. Immunostaining analysis showed MNS1 to be normally located in the whole-sperm flagella, but was absent in the proband's sperm. Expression analysis by Western blot also confirmed that MNS1 was absent in the proband's sperm. Abnormal flagellum morphology and ultrastructural disturbances in outer doublet microtubules were observed in the proband's sperm. A total of three intracytoplasmic sperm injection cycles were carried out for the proband's wife, but they all failed to lead to a successful pregnancy. Overall, this is the first study to report a loss-of-function mutation in MNS1 causing OAT in a Han Chinese patient.

Improving native human sperm freezing protection by using a modified vitrification method / 亚洲男科学杂志(英文版)

Slow freezing is the most commonly used technique for the cryopreservation of spermatozoa in clinical practice. However, it has been shown to have a negative impact on sperm function and structure. Vitrification as a successful alternative method has been proved to have better protective effects on human embryos, but vitrification of spermatozoa is still subject to low recovery rates. In this study, a modified vitrification method for native spermatozoa was developed. A total of 28 semen samples were included; each sample was divided into three equal parts and assigned to fresh, slow freezing, and vitrification groups. Sperm vitality, motility, morphology, DNA integrity, and acrosome reaction were assessed for each of the groups. The results showed that vitrification achieves better results for several sperm protection parameters than slow freezing; vitrification achieves a higher recovery rate (P < 0.05), motility (P <0.05), morphology (P <0.05), and curve line velocity (P <0.05) than slow freezing. Furthermore, DNA fragmentation was decreased (P <0.05) and better acrosome protection (P <0.05) was exhibited in the spermatozoa after vitrification. Principal component analysis of all sperm parameters revealed that the vitrification cluster was closer to the fresh cluster, indicating that spermatozoa are better preserved through vitrification. In conclusion, while both slow freezing and vitrification have negative effects on sperm function and structure, the vitrification protocol described here had a relatively better recovery rate (65.8%) and showed improved preservation of several sperm quality parameters compared with slow freezing.

A de novo partial 5p deletion and cryptic 18p duplication detected by SNP-Array in a boy featuring Cri du Chat syndrome / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To determine the karyotype of a boy suspected to have Cri du Chat syndrome with severe clinical manifestations, and to assess the recurrence risk for his family.</p><p><b>METHODS</b>High-resolution GTG banding was performed to analyze the patient and his parents. Fluorescence in situ hybridization (FISH) with Cri du Chat syndrome region probe as well as subregional probes mapped to 5pter, 5qter, 18pter, 18qter, and whole chromosome painting probe 18 was performed to analyze the patient and his parents. In addition, single nucleotide polymorphism-based arrays (SNP-Array) analysis with Affymetrix GeneChip Genome-wide Human SNP Nsp/Sty 6.0 were also performed to analyze the patient.</p><p><b>RESULTS</b>Karyotype analysis indicated that the patient has carried a terminal deletion in 5p. FISH with Cri du Chat syndrome region probe confirmed that D5S23 and D5S721 loci are deleted. SNP-Array has detected a 15 Mb deletion at 5p and a 2 Mb duplication at 18p. FISH with 5p subtelomeric probes and 18p subtelomeric probe further confirmed that the derivative chromosome 5 has derived from a translocation between 5p and 18p, which has given rise to a 46,XY,der(5)t(5;18)(p15.1;p11.31)dn karyotype.</p><p><b>CONCLUSION</b>A de novo 5p partial deletion in conjunction with a cryptic 18p duplication has been detected in a boy featuring Cri-du-Chat syndrome. His parents, both with negative findings, have a low recurrence risk. For its ability to detect chromosomal imbalance, SNP-Array has a great value for counseling of similar patients and assessment of recurrence risks.</p>

Karyotype analysis of qualified sperm donors on preliminary screening / 中华男科学杂志

<p><b>OBJECTIVE</b>To explore the significance of karyotype analysis in screening sperm donors.</p><p><b>METHODS</b>From January 1, 2004 to December 31, 2008, a total of 2537 potential sperm donors passed our preliminary screening, and all were routinely karyo-typed via peripheral blood. Follow-ups were conducted on the pregnancy outcome and congenital malformation after artificial insemination with the sperm from the qualified donors.</p><p><b>RESULTS</b>Among the 2537 qualified sperm donors, 2362 were of the normal karyotype 46, XY and 135 showed polymorphism. Abnormal karyotype was found in 6 cases, and controversial abnormal karyotype in 34.</p><p><b>CONCLUSION</b>Karyotype analysis can reduce the risk of chromosomal disease in neonates from artificial insemination, and genetic counseling for abnormal karyotype sperm donors may help them solve their future reproductive problems.</p>

Chromosome copy analysis by single-cell comparative genomic hybridization technique based on primer extension preamplification and degenerate oligonucleotide primed-PCR / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a single-cell whole genome amplification (WGA) technique, in combination with comparative genomic hybridization (CGH), for analyzing chromosomal copy number changes, and to explore its clinical application in preimplantation genetic diagnosis (PGD).</p><p><b>METHODS</b>Twelve single-cell samples with known karyotypes, including 5 chorionic villus samples, 4 human embryonic stem cell (hESC) samples and 3 peripheral lymphocyte samples, and 4 single blastomere samples carrying chromosomal abnormalities detected by PGD, were collected for whole genome amplification by combining primer extension preamplification (PEP) with degenerate oligonucleotide primed-PCR (DOP-PCR) amplification. The amplified products labeled by red fluorescence were mixed with control DNA labeled by green fluorescence, and then the mixture was analyzed by CGH. As a comparison, 10 single cell samples were amplified by DOP-PCR only and then CGH analysis was performed.</p><p><b>RESULTS</b>The amplification using PEP-DOP-PCR was more stable than traditional DOP-PCR. The products of PEP-DOP-PCR range from 100 bp to 1000 bp, with the mean size being about 400 bp. The CGH results were consistent with analyses by other methods. However, only 6 out of 10 single cell samples were successfully amplified by DOP-PCR, and CGH analysis showed a high background and 2 samples showed inconsistent results from other methods.</p><p><b>CONCLUSION</b>PEP-DOP-PCR can effectively amplify the whole genome DNA of single cell. Combined with CGH, this WGA method can successfully detect single-cell chromosomal copy number changes, while DOP-PCR was easy to fail to amplify and amplify inhomogeneously, and CGH analysis using this PCR product usually showed high background. These results suggest that PEP-DOP-CGH is a promising method for preimplantation genetic diagnosis.</p>

Identification of a cryptic 1p36.3 microdeletion in a patient with Prader-Willi-like syndrome features / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To determine the karyotype of a patient with Prader-Willi-like syndrome features.</p><p><b>METHODS</b>Chromosomal high resolution banding was carried out to analyze the karyotype of the patient, and methylation-specific PCR was used to analyze the imprinting region of chromosome 15. Subtelomeric region was screened by multiplex ligation-dependent probe amplification (MLPA), and fluorescent in situ hybridization (FISH) and real-time quantitative PCR were further performed to identify the deleted region.</p><p><b>RESULTS</b>No abnormality was discovered by high resolution karyotype analysis and methylation-specific PCR studies. MLPA analysis showed that the patient had a deletion of 1p subtelomeric area, which was confirmed by FISH analysis. The deleted region was shown within a 4.2 Mb in the distal 1p by 3 BAC FISH probes of 1p36 combined with real-time PCR technique. Family pedigree investigation showed the chromosome abnormality was de novo. Therefore, partial monosomy 1p36 was likely responsible for the mental retardation of the patient.</p><p><b>CONCLUSION</b>Molecular cytogenetic techniques should be performed to those patients with Prader-Willi-like syndrome features, to determine their karyotypes.</p>

Delineating a supernumerary marker chromosome by combining several cytogenetic and molecular cytogenetic techniques / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To characterize a supernumerary marker chromosome (SMC) by comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH) and traditional cytogenetic techniques, and to explore the clinical application of these techniques in delineating de novo marker chromosomes.</p><p><b>METHODS</b>A mental retardation patient received chromosome test by ordinary G banding. CGH and FISH techniques were used to analyze the origin of the de novo SMC, and N banding technique and C banding techniques were used to analyze the SMC structure. The phenotypic effects of the SMC were analyzed after the karyotype was determined.</p><p><b>RESULTS</b>By G banding technique, the patient was showed to have a mosaic karyotype with SMC: mos.47, XX, +mar [31]/48, XX, +2mar[29]. CGH analysis showed a gain of 15q11 --> q14, and the result was confirmed by FISH with chromosome 15 painting probe. The further FISH analysis showed the SMC had two signals with UBE3A probe for detecting Prader-willi syndrome/Angelman syndrome (PWS/AS). N banding and C banding analysis showed the SMC had a double satellite and double centromere, respectively. Combined with the above results, the karyotype of the patient was: mos.47, XX, +der (15) (pter --> q14::q14 --> pter) [31]/48, XX, +2der (15) (pter --> q14::q14 --> pter) [29]. ish der(15)(WCP15+, UBE3A++, PML-).</p><p><b>CONCLUSION</b>CGH is a valuable method to detect imbalanced chromosomal rearrangement. Combined with FISH and the traditional cytogenetic technique, it provides a valuable technique platform for characterizing the structure of the de novo SMC, and a basis for exploring the relation between karyotype and phenotype, prognosis and recurrent risk.</p>

Cytogenetic analysis and phenotype location analysis on the karyotype of a ring chromosome 21 syndrome / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To search the forming cause and the correlation between the clinical phenotype and chromosome band by the cytogenetic analysis on a case of ring chromosome 21 syndrome.</p><p><b>METHODS</b>Identification and location of 21 ring chromosome were performed with the G-banding, C-banding, N-banding, high-resolution banding and fluorescence in situ hybridization (FISH) techniques.</p><p><b>RESULTS</b>It was found that the karyotypes of the patient's parents are normal. The patient's karyotype is 46,XY, r(21)[91]/46,XY,r(21;21)(p11q22.3;p11q22.3) [5]/45,XY,-21[4].</p><p><b>CONCLUSION</b>The clinical phenotype of ring chromosome 21 syndrome is related to the deletion of distal segment of 21q, and the abnormal sexual development of male is related with the deletion of 21q22.3.</p>

Molecular cytogenetic analysis for a familial complex chromosomal rearrangement / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To determine a complex chromosomal rearrangement by advanced molecular cytogenetic techniques and analyze its clinical effect.</p><p><b>METHODS</b>A complex chromosomal rearrangement (CCR) involved in chromosomes 5, 16 and 20 in a 29-year-old male carrier was determined by chromosomal microdissection and multicolor fluorescence in situ hybridization (M-FISH), and family degree investigation was further performed.</p><p><b>RESULTS</b>The karyotype of the case was a complex chromosomal translocation among chromosomes 5, 20 and 16, and accompanied with a band of chromosome 20 inserted into chromosome 5. His mother and sister both had the same abnormal karyotype by familial investigation.</p><p><b>CONCLUSION</b>The combined use of M-FISH and chromosome microdissection is a powerful tool to determine CCR. The complex chromosomal rearrangement could be transmitted stably in the family, but still the carriers could give birth to a healthy baby by chance.</p>

Cytogenetic study of 1780 cases of spontaneous abortion / 中南大学学报(医学版)

OBJECTIVE@#To explore the relationship between chromosome anomaly and spontaneous abortion, and to provide useful information for genetic counseling and prenatal diagnosis in reproductive clinic.@*METHODS@#A total of 1 780 patients who had a history of spontaneous abortion before 24 weeks of gestation were enrolled. The lymphocyte culture and harvest were performed according to standard methods. Karyotypes were analyzed by G-banding in all cases and C- banding in some cases in addition.@*RESULTS@#Altogether 57 abnormal karyotypes were found and the overall incidence of chromosomal abnormalities was 3.20% (women 3.32%; men 2.12%). Among them 23 cases were the balanced translocation; 14 cases were the Robertsonian translocation, 3 cases were the complex chromosomal rearrangement, and the other 17 cases were the other abnormalities. In women with 1, 2, 3 or more spontaneous abortion, the incidence of chromosomal abnormalities was 1.7%, 2.3%, and 5.8%, respectively.@*CONCLUSION@#Translocations are the major abnormal karyotpes associated with spontaneous abortions. The chance of finding chromosomal aberration increases with the number of abortions. Chromosomal abnormalities are more common in women with 3 or more spontaneous abortions.

Prenatal diagnosis of common chromosomal aneuploidies on uncultured amniotic fluid cells by fluorescence in situ hybridization / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To evaluate the feasibility of using fluorescence in situ hybridization(FISH) for the detection of a few common chromosome aneuploidies on interphase nuclei of uncultured amniotic fluid cells.</p><p><b>METHODS</b>Amniotic fluid samples were taken from 55 women at 16-32 weeks of pregnancy; interphase FISH was performed for diagnosing Down syndrome and aneuploidies of other four chromosomes 13, 18, X and Y. Then the karyotypes from standard cytogenetic analysis after percutaneous umbilical blood sampling(PUBS) were compared to the FISH results.</p><p><b>RESULTS</b>Each of the 55 uncultured amniotic fluid samples tested with FISH was enumerated 200 nuclei. Fifty-three samples were normal. Two samples were found to have trisomy 21(one is a case of standard trisomy 21 with three signals in all 200 nuclei, the other is a mosaic trisomy 21).</p><p><b>CONCLUSION</b>Interphase FISH analysis of uncultured amniotic fluid cells is a rapid, accurate and very sensitive method. It could be used in the prenatal cytogenetic laboratory.</p>

Molecular cytogenetic detection of partial chromosome 13q trisomy and its relation with the clinical features of tortilcollis / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To search for the possible relation between tortilcollis and partial chromosome 13q trisomy.</p><p><b>METHODS</b>Fluorescence in situ hybridization (FISH) technique combined with chromosome banding was performed to determine the karyotype of two patients with typical clinical features of partial 13q trisomy syndrome, then their manifestations were compared with those of the literatures published previously.</p><p><b>RESULTS</b>The two cases were partial trisomy of 13q14--> ter with a different second derivative chromosome, in spite of this difference, both of them had tortilcollis.</p><p><b>CONCLUSION</b>It is suggested that a potential site for tortilcollis may locate on the long arm of chromosome 13. With reference to a report previously published, the more precise candidate related region may be 13q32--> qter.</p>

Identification and characterization of marker chromosome in Turner syndrome / 中华妇产科杂志

Objective To analyze the karyotypes of 11 cases of Turner syndrome with marker chromosome,and study the phenotypic effects resulting from the abnormal karyotype.Methods Eleven Turner syndrome patients had a mosaic karyotype and carried a marker chromosome,and 6 marker chromosomes were ring chromosomes.Their karyotypes were showed as mos.45,X/46,X,+mar or mos. 45,X/46,X,+r.Fluorescence in situ hybridization(FISH)technique with X/Y centromere probes was performed to determine the origin of the marker chromosome.Reverse chromosome painting technique was used to identify the breakpoints of two largest markers.Phenotype effects with different chromosome breakpoints were compared.Results All the 11 marker chromosomes were ring X chromosomes.The breakpoints of the r(X)were involved in Xp22,Xq22,Xq24 and Xq26,etc.Conclusions The marker chromosomes in Turner syndrome mainly originate from X chromosome and form ring chromosome X.Each r (X)in our patients was mosaic,indicating it was originated from mitosis error during early embryo development.To analyze the origin of the marker chromosome and the breakpoint of r(X)will provide guidance for the therapy and prognosis of the Turner syndrome patient.