[Identification of novel KIT gene mutations in two Chinese families with piebaldism].

OBJECTIVE: To screen for potential mutations of KIT gene for two Chinese families affected with piebaldism in order to facilitate genetic counseling and assisted reproduction. METHODS: Peripheral blood samples were collected from 2 patients of family 1 and the proband and 3 unaffected members of family 2 for the extraction of DNA and RNA. PCR-sequencing and reverse transcription PCR-sequencing were used to screen KIT mutations. RESULTS: All of the patients from family 1 were found to carry heterozygous IVS12+2-+7delinsACATCTTTA, a splicing mutation undocumented in the human gene mutation data base (HGMD) database. This mutation has resulted in c.1765-1779del in cDNA and p.Gly592Ala/del:E12, which has led to skipping of exon 12 and no expression of cDNA. The proband from family 2 has carried a heterozygous c.2401A>C mutation in KIT gene. The same mutation was not found in unaffected members. CONCLUSION: We have attained definite diagnosis for both families, which has facilitated genetic counseling and assisted reproduction for our patients and their family members.

[Mutation screening and prenatal diagnosis of tuberous sclerosis complex].

OBJECTIVE: To screen mutations of tuberous sclerosis complex (TSC) patients to confirm a clinical diagnosis of TSC, and to perform prenatal diagnosis for families with mutations. METHODS: In this study, PCR-denaturing high-performance liquid chromatography(DHPLC), supplemented with sequencing when necessary, was used to screen TSC1 and TSC2 mutations in 21 patients from 19 pedigrees visited author's hospital in the last five years. For novel mutations, one hundred unrelated healthy individuals were screened to exclude the possibility of polymorphism. RESULTS: Seventeen different mutations were found in 21 patients of 19 pedigrees with 13 being novel mutations, including c. 2672delA, c. 2672insA of TSC1 gene and c.4918insCGCC, c.1143delG, Intron27+1 G>A, c.1957-1958delAG, Intron5+1 G>A, c.910insCT, c.2753 C>G, c.4078dupAGCAAGTCCAGCTCCTC, Intron 11 -1 G>A, Intron 14+1 G>A, c.684 C>A of TSC2 gene, indicating a high frequency of de novo mutations in TSC. Three of these mutations were in the TSC1 gene (N762S, c.2672insA and c. 2672delA), while all remaining 14 were in the TSC2 gene. Prenatal diagnosis for TSC was performed for 7 fetuses from these pedigrees. The six fetuses that tested negative for TSC mutations were carried to term and, to date, none of these children has shown symptoms of TSC. CONCLUSION: Author's data showed that a mutation detection rate of tuberous sclerosis was 89.5%(17/19) among patients in author's hospital. The ratio of TSC2 and TSC1 mutations was about 1:1 in the familial cases, but TSC2 mutation was more common than TSC1 mutation in sporadic cases. Author's data demonstrated that birth of TSC children for those with familial history of TSC could be prevented through prenatal diagnosis.

Crypt Y chromosome fragment resulting from an X;Y translocation in a patient with premature ovarian failure.

OBJECTIVE: To identify a cryptic Y chromosome fragment that resulted from a X;Y translocation in a patient with premature ovarian failure (POF) and analyze the karyotype-phenotype correlation. DESIGN: Case report. SETTING: A university-based reproductive medicine center. PATIENT(S): A 33-year-old woman with POF. INTERVENTION(S): Karyotyping analysis, comparative genomic hybridization, fluorescence in situ hybridization, and polymerase chain reaction (PCR) analysis for the patient. MAIN OUTCOME MEASURE(S): Karyotype determination of the patient. RESULT(S): The patient was suspected to carry an abnormal X chromosome by traditional cytogenetic analysis. A Y chromosome hybridization signal was found in the patient's genome by comparative genomic hybridization analysis. The fluorescence in situ hybridization result showed that the Y chromosome material resulted from a translocation between Xq and Yq. Using the specific sequence-tagged sites, the breakpoints on the X and Y chromosomes were located at Xq26.3 and Yq11.223, respectively. Combined with chromosome G banding and C banding, the karyotype of the patient was determined as 46,X,der(X)t(X;Y) (q26.3;q11.223). CONCLUSION(S): The advanced molecular cytogenetic techniques are helpful to detect cryptic chromosome aberrancies in patients with POF. This rare case supports that Xq26-q28 is the critical region of POF, and is helpful to analyze the risk of gonadoblastoma in patients with POF with Y chromosomal material.

[Mutation screening of the TYR and P gene in three patients with oculocutaneous albinism].

OBJECTIVE: To identify the mutations of the tyrosinase gene (TYR) and P gene in patients with oculocutaneous albinism (OCA). METHODS: Polymerase chain reaction (PCR) and denaturing high performance liquid chromatography (DHPLC) were applied to detect the mutations in all exons of TYR gene and P gene. Then DNA sequencing and restriction endonuclease analysis were used to confirm the mutations detected by DHPLC. Novel mutations were screened in 100 unrelated persons with normal phenotypes to exclude the possibility of polymorphism. RESULTS: Two mutations were detected in the P gene of the three patients and none in TYR gene. Heterozygous mutation of T450M in exon 13 of the P gene was detected in patient 1. Patient 2 had a heterozygous mutation of T450M in exon 13 and a heterozygous mutation of G775R in exon 23 of the P gene. Patient 3 had a heterozygous mutation of G775R as well. Restriction endonuclease analysis of the P gene exon 13 showed that the Oli I site had partly disappeared resulting from the heterozygous mutation T450M in patient 1 and patient 2, but not in 100 unrelated individuals. The heterozygous mutation T450M is a novel mutation. CONCLUSION: Gene diagnosis of OCA can be carried out effectively by combining PCR, DHPLC, DNA sequencing and restriction endonuclease analysis.

[Expression of a novel spermatogenesis gene SRG4 in postpartum normal and cryptorchid mouse testis].

To understand the function of SRG4, a novel spermatogenesis gene, we studied its expression pattern during normal mouse development and in experimental cryptorchidism. Testis tissues were collected from 1-, 3-, and 12-week-old normal mice and immunohistochemistry was used to detect the expression of SRG4. We performed surgery on mice to cre- ate unilateral cryptorchidism and monitored SRG4 mRNA levels by semi-quantitative RT-PCR in the cryptorchid testis from day 0-18. At post-operative day 18, the cryptorchid testis and the contralateral control testis were harvested and assayed for SRG4 expression by in situ hybridization. Immunohistochemistry results showed that SRG4 protein was hardly detected in 1-week-old mouse testis, but the expression was present in 3-week-old mouse and abundant in 12-week-old mouse testis. SRG4 immunostaining was mainly localized to the cytoplasm and membrane of spermatocytes and round spermatids. Moreover, semi-quantitative RT-PCR result showed the expression of SRG4 mRNA did not decrease until 9 d after cryptorchid surgery, and continued to decline thereafter. In situ hybridization revealed that in contrast to the abundant SRG4 expression in the control side, few remaining germ cells in the crytorchid testis were positive for SRG4 at d 18 after surgery. The results indicated that the expression of SRG4 was regulated by development, and SRG4 was mainly expressed in the cytoplasm of spermatocytes and round spermatids. However, in cryptorchid testis, in which most germ cells undergo apoptosis, only a few of SRG4 is observed, suggesting that SRG4 may be as a specific marker to evaluate the process of spermatogenesis.

[Mutation screening and prenatal diagnosis of hidrotic ectodermal dysplasia in a Chinese family].

OBJECTIVE: To analyze the mutations in Cx30 gene in a Chinese family with hidrotic ectodermal dysplasia (HED) and to make prenatal diagnosis on the embryo which has been pregnant for 5 months. METHODS: A family including 2 affected and 4 unaffected individuals was collected, and their informed consents were obtained. The affected woman had a five-month pregnancy. An 884 bp fragment containing the whole GJB6 coding sequence was amplified by PCR and the products were bi-direction sequenced directly. The mutation was further confirmed with restriction endoenzyme digesting. On the base of successful gene diagnosis, the following detection procedure on the pregnant baby was performed. First the whole coding region of Cx30 was amplified using primers Cx30-F and Cx30-R and the PCR products were digested by Hae II. Then the PCR products were cloned into pUCm-T vector. Blue-white blot screening method and PCR-restriction endoenzyme digesting technique were used to identify the correct clones. The mutant allele clone was sequenced to confirmed the mutation. RESULTS: A heterozygous missense mutation 263C --> T in the Cx30 gene was detected in the affected little girl and her affected mother, which led to an amino acid substitution (A88V) in the second transmembrane domain of GJB6. The mutation was confirmed by Hae II digestion. A88V mutant allele cannot be cut while the wild normal allele can be cut into two fragments, 520 and 278 bp. The result of analyse on the five-month pregnancy show the embryo carried the A88V mutation too. So the embryo will be a patient. CONCLUSION: An A88V missense mutation in the Cx30 gene can also cause HED in Chinese Han population. Based on the gene diagnosis, prenatal diagnosis can be played using bi-direction sequencing and confirmed with restriction endoenzyme digesting.

[A mutation in TGF beta1 gene encoding the latency-associated peptide in a Chinese patient with Camurati-Engelmann disease].

OBJECTIVE: To identify the mutation in transforming growth factor-beta1 gene (TGF beta1) in a Chinese patient with Camurati-Engelmann disease(CED). METHODS: Denaturing high-performance liquid chromatography (DHPLC) analysis was performed on the whole seven coding exons and exon-intron boundaries, then the mutation was identified by direct sequencing. RESULTS: Mutation screening of TGF beta1 in this patient revealed a heterozygous missense mutation R218H in exon 4. CONCLUSION: The identification of the mutation could provide essential data for subsequent therapy and genetic counseling.

Molecular cloning and preliminary function study of a novel human gene, TSARG7, related to spermatogenesis.

A novel human gene TSARG7 (GenBank accession No. AY513610) was identified from a human testis cDNA library by using the mTSARG7 gene (GenBank accession No. AY489184) as an electronic probe. The gene whose full cDNA length is 2,463 bp containing 12 exons and 11 introns is located in the human chromosome 8p11.21. The predicted protein encoded by this gene contains 456 amino acids with a theoretical molecular weight of 56,295 dalton and isoelectric point of 9.13. It is a new member of the acyltransferase family since its sequence possesses the highly conserved PlsC domain existing in all acyltransferase-like proteins. Two groups, the TSARG7 and mTSARG7, the TSARG7 and Au041707, share 97% identity in the 456 amino acids. Expression of the TSARG7 gene is restricted to the testis. Subcellular localization studies show that the EGFP-tagged TSARG7 protein was localized in the cytoplasm of GC-1 cells. The TSARG7 mRNA expression was initiated in the testis of a 13-year-old boy, and its level increased steadily along with spermatogenesis and sexual maturation of the human. The results of heat stress experiment demonstrate that TSARG7 expression has a relation with temperature. In conclusion, our study suggests that we have cloned a novel human gene and this gene may play an important role in human spermatogenesis and sexual maturation.

Molecular cloning of a novel mouse testis-specific spermatogenic cell apoptosis inhibitor gene mTSARG7 as a candidate oncogene.

A novel mouse gene, mTSARG7 (GenBank accession No. AY489184), with a full cDNA length of 2279 bp and containing 12 exons and 11 introns, was cloned from a mouse expressed sequence tag (GenBank accession No. BE644543) that was significantly up-regulated in cryptorchidism. The gene was located in mouse chromosome 8A1.3 and encoded a protein containing 403 amino acid residues that was a new member of the acyltransferase family because the sequence contained the highly conserved phosphate acyltransferase (PlsC) domain existing in all acyltransferase-like proteins. The mTSARG7 protein and AU041707 protein shared 83.9% identity in 402 amino acid residues. Expression of the mTSARG7 gene was restricted to the mouse testis. The results of the in situ hybridization analysis revealed that the mTSARG7 mRNA was expressed in mouse spermatogonia and spermatocytes. Subcellular localization studies showed that the EGFP-tagged mTSARG7 protein was localized in the cytoplasm of GC-1 spg cells. The mTSARG7 mRNA expression was initiated in the mouse testis in the second week after birth, and the expression level increased steadily with spermatogenesis and sexual maturation of the mouse. The results of the heat stress experiment showed that the mTSARG7 mRNA expression gradually decreased as the heating duration increased. The pcDNA3.1 Hygro(-)/mTSARG7 plasmid was constructed and introduced into GC-1 spg cells by liposome transfection. The mTSARG7 can accelerate GC-1 spg cells, causing them to traverse the S-phase and enter the G2-phase, compared with the control group where this did not occur as there was no transfection of mTSARG7. In conclusion, our results suggest that this gene may play an important role in spermatogenesis and the development of cryptorchid testes, and is a testis-specific apoptosis candidate oncogene.

Cloning of a full-length cDNA of human testis-specific spermatogenic cell apoptosis inhibitor TSARG2 as a candidate oncogene.

A novel human gene full-length cDNA sequence-TSARG2 was identified from a human testis cDNA library using the SRG2 gene (GenBank Accession No. ), which was significantly up-regulated in cryptorchidism, as an electronic probe. TSARG2 was 1223 bp in length. The putative protein encoded by this gene was 305 amino acids with a theoretical molecular weight of 34,751 and isoelectric point of 9.85. The sequence shared no significant homology with any known protein in databases except SRG2. Northern blot analysis revealed that 1.7 kb TSARG2 transcript was detected selectively in human testis. Furthermore, results of in situ hybridization assay confirmed that TSARG2 was expressed in seminiferous tubules, more precisely in spermatogonia and spermatocyte. No mutation was found by PCR-SSCP in 122 cases of azoospermia, severe oligzoospermia, and cryptorchidism. The green fluorescence produced by pEGFP-C1/TSARG2 was detected on the nucleus of COS7 cells after 24h post-transfection. The pcDNA3.1(-)/TSARG2 plasmid was constructed and introduced into MCF7 cells by liposome transfection. TSARG2 can accelerate MCF7 cells to traverse the S-phase and enter the G2-phase compared with the control without transfection of TSARG2, which suggested that this gene plays an important role in the development of cryptorchid testis and is a testis-specific apoptosis candidate oncogene.

Identification of a novel gene SRG4 expressed at specific stages of mouse spermatogenesis.

Spermatogenesis is a complex process. Two spermatocytes expression sequence tags (ESTs) BG101130 and BG100990 were found. Their putative amino acid sequences have high homology with rat Spag4 (sperm antigen 4). By electrical hybridization, a novel cDNA encoding polypeptide of 348 amino acid residues was identified from a mouse testis cDNA library. The new gene was designated as SRG4 (Spermatogenesis related gene 4) (GenBank accession No. AY307077). Results of Northern blot and RTPCR revealed that SRG4 expressed specifically in mouse testis. Changes of SRG4 expression in mouse different development stages were observed by RT-PCR. The SRG4 mRNA was hardly detected in 2 weeks postpartum, and expressed abundantly from 3 weeks later, reaching top lever at 4-5 weeks, while slightly down in aging mouse testis. Results of in situ hybridization showed that SRG4 gene expressed abundantly in spermatocytes, round spermatids. This indicated SRG4 gene may play an important role in mouse meiotic divisions of spermatocytes.

[Diagnosing achondroplasia by single cell nested-PCR].

OBJECTIVE: To research on the reliability of diagnosing achondroplasia (ACH) on single cell level and to provide a basis for preimplantation genetic diagnosis(PGD). METHODS: The high-frequency mutation region G380R of fibroblast growth factor receptor 3(FGFR3) gene was amplified by nested-PCR with single lymphocyte and single blastomere. The products of PCR were digested by restriction enzyme Bfm I, then the digested products were detected by 10% polyacrylamida gel electrophoresis(PAGE). RESULTS: The amplification success rate, allele dropout rate and correct diagnosis rate of single lymphocyte's PCR were 90.4%, 8.2% and 91.8%,respectively. The amplification success rate of single blastomere was 75.4%. CONCLUSION: The diagnosis of ACH by single cell nested-PCR is comparatively stable and reliable.