Identification of novel KIT gene mutations in two Chinese families with piebaldism / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To screen for potential mutations of KIT gene for two Chinese families affected with piebaldism in order to facilitate genetic counseling and assisted reproduction.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>We have attained definite diagnosis for both families, which has facilitated genetic counseling and assisted reproduction for our patients and their family members.</p>

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>

Mutation screening of the F VIII gene in 10 hemophilia A families / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify the F VIII gene mutations of patients and suspected female carriers in 10 Hemophilia A (HA) families, and to guide the prenatal diagnosis.</p><p><b>METHODS</b>PCR, denaturinghigh performance liquid chromatogramphy (DHPLC) and DNA sequencing technologies were applied to screen the F VIII gene of 8 HA patients and 12 suspected female carriers in the 10 families. Linkage analysis was performed by using St 14(DXS 52), intron 13 (CA)n and EX18/Bcl I of the F VIII gene in the HA families. In prenatal diagnosis, we screened the same mutation found in the patients. PCR-restriction fragment length polymorphism was applied to detect the new missense mutations of F VIII gene in 100 unrelated healthy individuals to exclude the possibility of polymorphism.</p><p><b>RESULTS</b>Five missense mutations, 3 frameshift mutations, 2 nonsense mutations and 2 single nucleotide polymorphism (SNP) were identified in 10 the HA families. Among them, c.878A to G, c.1015A to G, c.6870G to T, c.1282delA, c.3072_3073insT, c.4880_4881insA and c.5000G to A were novel mutations or polymorphism. No missense mutations c.878A G, c.1015A to G and c.6870G to T, were found in the 100 healthy unrelated controls. (2) Nine suspected female carriers were confirmed at the gene level. (3) X risk chromosome could be determined to in 4 HA families by genetic linkage analysis. (4) Among the four fetuses for prenatal diagnosis, 2 were normal, 1 was carrier and the remaining 1 was a patient.</p><p><b>CONCLUSION</b>Six novel mutations, i.e., c.878A to G, c.1015A to G, c.6870G to T, c.1282delA, c.3072_3073insT and c.4880_4881insA, were identified in this study. PCR, DHPLC and DNA sequencing could be used to screen the gene mutations of HA patients, to carry out carrier detection and prenatal diagnosis of HA families efficiently, by combining with restriction endonuclease analysis and genetic linkage analysis.</p>

Mutation screening and prenatal diagnosis of a pedigree with Glanzmann's thrombasthenia / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>Mutation screening was performed in a pedigree of Glanzmann's thrombasthenia (GT) and prenatal diagnosis was performed.</p><p><b>METHODS</b>In this study, reverse transcription-PCR-sequencing and PCR-sequencing, as well as restriction fragment length polymorphism(RFLP) and A/T-cloned-sequencing, were used to screen the ITGA2B and ITGB3 mutation in a pedigree with Glanzmann's thrombasthenia in the RNA and DNA level. Prenatal diagnosis was performed for this pedigree.</p><p><b>RESULTS</b>Deletion of 99 bps was found in the cDNA of the patient in the pedigree, leading to deletion of 33 codons (from codon 160 to 192). After genomic analysis, the patient was found to be a compound heterozygote of c.374C to G mutation and intron 4(IVS-4) + 5 G to C mutation. The two mutations were inherited from the parents. IVS-4 + 5 G to C mutation was a point mutation in the splice site, while c.374C to G mutation was out of the splice site. But both of them resulted in the same splice pattern in RNA. The two mutations were novel mutations which have not been reported in Human Gene Mutation Database (HGMD) and the mutation data base of Glanzmann's thrombasthenia. The results of ITGB3 gene screening is normal in the proband and his parents.</p><p><b>CONCLUSION</b>Two novel mutation, c.374C to G and IVS-4 + 5 G to C were found in this study, which might be the cause of GT in the pedigree.</p>

Mutation screening and prenatal diagnosis of tuberous sclerosis complex / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</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>

Mutation screening of the TYR and P gene in three patients with oculocutaneous albinism / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify the mutations of the tyrosinase gene (TYR) and P gene in patients with oculocutaneous albinism (OCA).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>Gene diagnosis of OCA can be carried out effectively by combining PCR, DHPLC, DNA sequencing and restriction endonuclease analysis.</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>

Mutation detection of PKD1 gene in patients with autosomal dominant polycystic kidney diseases / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To detect gene mutation in the patients with autosomal dominant polycystic kidney disease (PKD).</p><p><b>METHODS</b>Polymerase chain reaction (PCR)-denaturing high-performance liquid chromatography (DHPLC) analyses were performed in 3o single copy region of PKD 1 gene (PKD1). DNA sequencing were carried out on PCR products with abnormal peak shape afterwards.</p><p><b>RESULTS</b>A new nonsense mutation (C11901A in exon 42 of PKD1 was identified to cause serine in position 3897 turning to a stop codon. A missense mutation, C10737T, was detected in exon 35 which caused threonine in position 3509 turn to methionine. Two kinds of samesense mutation, G11824A and C11860T in exon 42, were found in normal control.</p><p><b>CONCLUSION</b>PKD1 mutation were detected successfully by PCR-DHPLC. A new nonsense mutation, a missense mutation and two polymorphisms are identified in this study.</p>

A mutation in TGF beta1 gene encoding the latency-associated peptide in a Chinese patient with Camurati-Engelmann disease / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify the mutation in transforming growth factor-beta1 gene (TGF beta1) in a Chinese patient with Camurati-Engelmann disease(CED).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>Mutation screening of TGF beta1 in this patient revealed a heterozygous missense mutation R218H in exon 4.</p><p><b>CONCLUSION</b>The identification of the mutation could provide essential data for subsequent therapy and genetic counseling.</p>

Mutation screening and prenatal diagnosis of hidrotic ectodermal dysplasia in a Chinese family / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Diagnosis of hemophilia A by a combination of St14(DXS52) VNTR polymorphism and (CA)n repeat polymorphism within FVIII gene / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To improve the accuracy and the diagnostic rate of gene diagnosis and prenatal gene diagnosis for hemophilia A (HA) families.</p><p><b>METHODS</b>Linkage analysis was performed by using St14(DXS52) VNTR polymorphism and intron 13 (CA)n repeat polymorphism of the factor VIII gene among HA families for indirect diagnosis.</p><p><b>RESULTS</b>The diagnostic rates using linkage analysis based upon one of the above mentioned two polymorphic loci among 9 HA families were 66.7% and 66.7%, respectively. The diagnostic rate rose to 88.9% by using a combination of the two polymorphic loci. Prenatal gene diagnoses were performed for 4 HA families. A wrong prenatal diagnosis which may happen when linkage analysis was performed by using only St14 VNTR was monitored.</p><p><b>CONCLUSION</b>The rapid and accurate gene diagnosis and prenatal gene diagnosis could be performed by a combination of the two polymorphic loci for about 90% HA families.</p>

Detection of a new mutation (G1253T) of iduronate-2-sulfatase gene for the patient with mucopolysaccharidosis type II / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify the mutations of iduronate-2-sulfatase (IDS) gene in mucopolysaccharidosis type II patients.</p><p><b>METHODS</b>PCR-SSCP analysis was applied to detect the common mutations in the exons 2, 3, 5, 7, 8, 9 in IDS-gene of the patient. DNA sequencing and PCR-RFLP were applied to analyze the mutation detected by PCR-SSCP.</p><p><b>RESULTS</b>A new mutation(1253G-->T) of exon 7 of the IDS gene was found by PCR-SSCP and DNA sequencing in the patient, The PCR-restriction enzyme digestion showed that enzyme digestion location appeared in the patient and his mother, which verified the results of sequencing analysis.</p><p><b>CONCLUSION</b>The mutation of patient with MPSII could be detected effectively and quickly by the applications of PCR-SSCP, DNA sequencing and PCR-restriction enzyme digestion analysis, and the new mutation thus detected is necessary for the prenatal diagnosis of the pedigree.</p>

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>

Diagnosing achondroplasia by single cell nested-PCR / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To research on the reliability of diagnosing achondroplasia (ACH) on single cell level and to provide a basis for preimplantation genetic diagnosis(PGD).</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>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%.</p><p><b>CONCLUSION</b>The diagnosis of ACH by single cell nested-PCR is comparatively stable and reliable.</p>

46,XY female sex reversal patient with a novel point mutation in the coding sequence of the SRY gene / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the molecular mechanism of a Chinese patient with 46, XY sex reversal.</p><p><b>METHODS</b>DNA fragments of the SRY gene from the typical XY female sex reversal patient and her father were amplified by polymerase chain reaction (PCR). The amplified PCR fragments were cloned into the pUCm-T vector, and direct sequencing was carried out on an ABI 377-3 automated DNA sequencer to detect the mutation. PCR-restriction enzyme digestion was applied to detect the results of DNA sequencing.</p><p><b>RESULTS</b>A novel mutation of the SRY gene was identified in the XY sex reversal patient of this study. A T is replaced by an A in codon 129 at position +387, resulting in the replacement of the polar amino acid tyrosine (TAT) by the stop code (TAA) in the HMG-box, whereas her father was proved to have the wild-type sequence. Because the mutation introduced an enzyme site of MaeIII, the PCR-restrict enzyme digestion showed that there were three bands (131 bp,231 bp and 247 bp) in the patient, whereas two bands (131 bp and 478 bp) in normal man. It verified the results of sequencing analysis. The results after searching the Human Gene Mutation Database showed that this mutation was not described before and should be a new mutation.</p><p><b>CONCLUSION</b>The novel mutation in SRY gene has provided valuable information for the understanding of molecular mechanism of the patient with 46,XY female sex reversal.</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.