Analysis of clinical data and genetic mutations in three Chinese patients with tyrosinemia type I / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze clinical data and gene mutations in 3 Chinese patients with tyrosinemia type I, and to explore the correlation between genotypes and phenotypes.</p><p><b>METHODS</b>Three patients suspected with tyrosinemia I were tested by tandem mass spectrometry for the level of tyrosine, phenylalanine and succinylacetone in the blood, and by gas chromatography-mass spectrometry to determine the level of succinylacetone and organic acid in their urine. With the diagnosis established, the FAH gene was analyzed with polymerase chain reaction (PCR) and direct sequencing.</p><p><b>RESULTS</b>Two patients had acute onset of the disease, while another had subacute onset of the disease, with features including hepatomegaly and remarkably increased tyrosine and succinylacetone in the blood. Five mutations were detected in the FAH gene, which included c.455G>A (W152X), c.520C>T (R174X), c.974_976delCGAinsGC, c.1027 G>A (G343R) and c.1100 G>A (W367X), among which c.455G>A (W152X), c.974_976delCGAinsGC and c.1100 G>A (W367X) were not reported previously.</p><p><b>CONCLUSION</b>Tyrosinemia type I may be effectively diagnosed with the level of tyrosine and succinylacetone by tandem mass spectrometry and succinylacetone in the urine by gas chromatography mass spectrometry. Detection of underlying mutations mutations will be helpful for genetic counseling and further research.</p>

Clinical diagnosis and WISP3 gene mutation analysis for progressive pseudorheumatoid dysplasia / 中华儿科杂志

<p><b>OBJECTIVE</b>Progressive pseudorheumatoid dysplasia (PPD) (MIM#208230) is a rare autosomal recessive disease of cartilage homeostasis characterized by axial and peripheral skeletal dysplasia. Analysis of WISP3 (Wnt1-inducible signaling pathway protein 3, MIM#603400) gene mutation can confirm the clinical and radiographic diagnosis for PPD. This study aimed to recognize PPD based on clinical manifestations and imaging characteristics of bones, and to investigate the mutations of WISP3 gene in three patients with PPD.</p><p><b>METHOD</b>Three male patients (9 - 16 years old) from three unrelated Chinese families, who presented with joint pain, swelling, deformities and motion limitation, were referred to this study. PPD was diagnosed on the basis of the clinical manifestations, imaging characteristics of bones and laboratory evaluation. All five exons and their exon/intron boundaries of the WISP3 gene were amplified by polymerase chain reaction (PCR) from the peripheral blood DNA of three PPD family members, and mutation analysis was performed by bidirectional DNA sequencing.</p><p><b>RESULT</b>(1) Three patients were diagnosed as PPD by characteristic evidences: all patients presented with non-inflammatory multiple joints swelling and stiffness including joints in hand and feet as they age. Radiographs showed platyspondyly, ovoid or wedged anterior end-plate of vertebral bodies, coxa vara, widened epiphyses or metaphyses including capital femoral, metacarpophalangeal, interphalangeal joints and metatarsals. Normal laboratory values were found for the erythrocyte sedimentation rate and C-reactive protein, rheumatoid factors, antinuclear antibodies etc. (2) The three different mutations of WISP3 gene were identified in three patients with PPD, including two small insert mutations (c.624_625insA, c.866_867insA), one was deletion mutation (c.729_735delGAGAAAA). The types of mutation of two alleles in three patients were c.624_625insA/c.729_735delGAGAAAA, c.624_625insA/c.866_867insA and c.866_867 insA/c.866_867insA, respectively. These mutations were found in exon 4 and exon 5 of WISP3 gene, accounting for 50%(3/6) respectively. All three different mutations were novel variations, and none of 3 novel variations was found in the 50 controls.</p><p><b>CONCLUSION</b>The characteristic evidences of PPD were non-inflammatory multiple enlarged joints (including hand and feet), limited movement, normal laboratory values such as rheumatoid factors. It is essential for making diagnosis to carefully examine the entire skeleton including spine. The characteristics of bone imaging are platyspondyly, widened epiphyses or metaphyses including large and small joints and narrow joint spaces. Three different novel variations of WISP3 gene were identified in three PPD patients, they are c.624_625insA, c.866_867insA and c.729_735delGAGAAAA. Each of novel mutations is insert or deletion mutation.</p>

Analysis of gene mutations in Chinese patients with methylmalonic acidemia and homocysteinemia / 中华儿科杂志

<p><b>OBJECTIVE</b>Methylmalonic acidemia complicated with homocysteinemia, cblC type, is the most common inborn error of cobalamin metabolism. The gene MMACHC (OMIM 277400) is located on chromosome 1p34.1 with four coding exons and a 5th non-coding exon. It encodes for a protein with 282 amino acid residues. So far, more than 40 mutations have been detected, in which 271dupA (R91KfsX14) is the hot spot of MMACHC gene. However, there have not been relevant reports in China. The present study aimed to identify the mutation types of MMACHC gene and analyze the genotype-phenotype correlations in Chinese patients.</p><p><b>METHOD</b>The diagnosis of this disease mainly depends on the measurement of C3 propionylcarnitine, C3/C0 (free carnitine) and C3/C2 (acetylcarnitine) in the blood by tandem mass spectrometry, the detection of methylmalonic acid in the urine by gas-chromatography mass spectrometry, the determination of total homocysteine in the serum, and the loading test of vitamin B12. The entire coding region of MMACHC gene was screened by polymerase chain reaction (PCR) combined with DNA direct sequencing in 28 Chinese patients. Genomic DNA was extracted using phenol-chloroform method from the peripheral blood leukocytes of each patient. PCR amplification products were checked by 1.8% agarose gel electrophoresis and were subsequently sequenced with both the forward and reverse primers. Mutational analyses were performed using normal human genomic MMACHC sequence as a reference (GenBank ID: 25974).</p><p><b>RESULT</b>In this study, ten mutations were identified in 27 of 28 Chinese patients. Most of them were located in exons 3 and 4 (91.3%). We detected four mutations reported, which were 609G>A (W203X), 217C>T (R73X), 271dupA (R91KfsX14), and 394C>T (R132X), and six novel mutations, which were 1A>G, 365A>T, 658_660delAAG, 301-3_327del 30, 567_568insT, and 625_626insT. The 609G>A (W203X) is the most common mutation, which was detected in 30 of 56 alleles (53.6%), including 10 homozygote mutations and 10 heterozygote mutations. In addition, three gene polymorphisms were detected, namely, -302T>G (rs3748643), -234A>G (rs3728644), and 321G>A (rs2275276). These mutations include missense mutations, nonsense mutations, duplication, deletions, and insertions.</p><p><b>CONCLUSION</b>In this study, we found a part of gene mutations spectrum in Chinese patients with methylmalonic acidemia and homocysteinemia, in which the 609G>A (W203X) may be the hotspot mutation of MMACHC gene. This would be helpful in the prenatal diagnosis and gene screening programs of methylmalonic acidemia and homocystinemia.</p>

Molecular genetic analysis of 10 Chinese patients with glycogen storage disease type III / 中华儿科杂志

<p><b>OBJECTIVE</b>Glycogen debranching enzyme (AGL) plays an important role in complete degradation of the glycogen, and has two independent catalytic activities, i.e., those of alpha-1, 4-glucanotransferase (EC 2.4. 1.25) and amylo-1,6-glucosidase (EC 3.2. 1.33). A deficiency in activities of AGL causes excessive accumulation of glycogen with short branched outer chains and results in glycogen storage disease type III (GSD III; MIM #232 400), an autosomal recessive inborn disorder of glycogen metabolism. The present study aimed to investigate the mutation of AGL in 10 Chinese patients with GSD III.</p><p><b>METHOD</b>Clinical and laboratory data of 10 patients with typical clinical manifestations of GSD III suggesting hypoglycemia, hyperlipidemia, increased creatine-phosphokinase and its isozyme were collected. The coding regions and their flanking introns of AGL gene of the 10 patients were amplified by PCR and analyzed by direct DNA sequencing. All the mutated alleles were confirmed by bidirectional DNA sequencing. The 3 novel splicing mutations were analyzed by restriction fragment length polymorphism (RFLP) in 50 healthy children (control). The 2 small deletions (c.408-411delTTTG, c.2717-2721delAGATC) were analyzed by fluorescent polymerase chain reaction and gene scan analysis to confirm the number of deleted bases.</p><p><b>RESULT</b>Thirteen different mutations were identified, including 4 splicing mutations (IVS6 + 1G > A, IVS6-1G > A, IVS14 + 1G > T, IVS26-2A > C), 5 nonsense mutations (R469X, R864X, S929X, R977X, Y1428X), 3 small deletions (c.408-411delTTTG, c.2717-2721delAGATC, c.2823delT) and 1 insert mutation (c.4234insT). Except for IVS14 + 1G > T, R864X, and R977X, the other 10 mutations are novel; 18 mutated alleles were identified in the 20 alleles (90%). IVS14 + 1G > T was the most frequently seen mutation, accounting for 5 of 20 (25%) alleles examined. None of homozygote and heterozygote of the 3 novel splicing mutations was found in the 50 healthy controls by RFLP analysis. With the fluorescent polymerase chain reaction and gene scan analysis, c.408411deTTTG mutation and c.2717-2721delAGATC mutation were confirmed to have 4 and 5 bases deletion respectively.</p><p><b>CONCLUSION</b>Thirteen mutations were identified in the 10 cases with GSD III, with 10 novel mutations. IVS14 + 1G > T was a relatively common mutation. This study revealed the heterozygosity of AGL gene in Chinese patients with GSD III.</p>

Gene mutation analyses in Chinese children with multiple carboxylase deficiency / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To confirm the diagnosis of multiple carboxylase deficiency (MCD) on the gene level and explore the mutations in Chinese children with MCD.</p><p><b>METHODS</b>Biotinidase (BT) and holocarboxylase synthetase (HLCS) genes were analyzed by PCR and direct sequencing for the 4 BT deficiency patients and 8 HLCS deficiency patients, respectively. The identified mutations in the parents of the patients and 50 normal controls were screened by PCR-restriction fragment length polymorphism and direct DNA sequencing.</p><p><b>RESULTS</b>Total detection rate of gene mutation is 100% in the 12 children with MCD. Six mutations were detected in the 4 children with BT deficiency, they were c. 98-104del7ins3, c. 1369G>A (V457M), c. 1157G>A(W386X), c. 1284C>A(Y428X), c. 1384delA and c. 1493_1494insT. The last four were novel mutations. Four mutations were found in the 8 children with HLCS deficiency. They were c. 126G>T (E42D), c. 1994G>C (R665P), c. 1088T>A (V363D) and c. 1522C>T (R508W). The last two were hot-spot mutations [75%(12/16)], and c. 1994G>C (R665P) was a novel mutation.</p><p><b>CONCLUSION</b>This study confirmed the diagnosis of 12 patients with MCD on the gene level. Six mutations were found in the BT gene and 4 in the HLCS gene, including 5 novel mutations. Two mutations of the HLCS gene are probably hot-spot mutations in Chinese children with HLCS deficiency.</p>

Diagnosis, treatment and gene mutation analysis in children with holocarboxylase synthetas deficiency / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To report the clinical diagnosis, treatment and follow-up of children with holocarboxylase synthetas(HCS) deficiency and explore the gene mutation spectrum of the disease.</p><p><b>METHODS</b>Eleven children with HCS deficiency were enrolled. Mass spectrometry analysis and biotinidase activity determination were used for diagnosis of HCS deficiency. HCS gene mutations were analyzed by PCR directed sequencing methods. Ten patients received oral biotin treatment (10-40 mg/d). Clinical effects of biotin treatment were observed.</p><p><b>RESULTS</b>All 11 cases developed apathetic, lethargy and metabolic acidosis at different degrees, and 10 cases presented with skin lesions. The average blood 3-hydroxyisovaleryl-carnitine concentrations and urinary 3-methylcrontonylglycine and methylcitrate concentrations increased significantly. The biotinidase activity increased, being higher over 30% of the normal reference value. Four mutations in HCS gene were identified, and they were c.1522C>T (R508W), c.1088T>A (V363D), c.126G>T (E42D) and c.1994G>C (R665P) (a new variant) and the frequency was 50%, 29%, 7% and 14% respectively. The symptoms disappeared in 10 cases 1-2 weeks after biotin treatment, and blood and urinary abnormal metabolites were gradually reduced to normal 2-6 months after treatment.</p><p><b>CONCLUSIONS</b>HCS deficiency is characterized by nervous system damage, skin lesions and metabolic acidosis. Mass spectrometry analysis, biotinidase activity determination and gene mutation analysis may be helpful in the definite diagnosis of this disorder. The effect of early biotin treatment is satisfactory. The mutations R508W and V363D might be hot-spots in Chinese children with HCS deficiency.</p>

Gene mutation analysis in patients with propionic acidemia / 中华儿科杂志

<p><b>OBJECTIVE</b>Propionic acidemia is a common organic acidemia, caused by deficiency of propionyl-CoA carboxylase (PCC), which catalyzes the carboxylation of propionyl-CoA to D-methylmalonyl-CoA. PCC is a dodecameric enzyme of alpha-PCC and beta-PCC subunits, nuclearly encoded by genes PCCA and PCCB, respectively. Mutation in either gene cause propionic acidemia, the PCCA gene is located on chromosome 13q32 with 24 exons and the PCCB gene is located on chromosome 3q13.2-q22 with 15 exons. In this study, we analyzed gene mutations of 11 PCCA and PCCB deficient patients from China and to explore the possible mutation spectrum.</p><p><b>METHODS</b>All 39 exons of PCCA and PCCB genes in 11 unrelated Chinese PA patients were analyzed by polymerase chain reaction (PCR) and direct sequencing. Genomic DNA was extracted using phenol-chloroform method from the peripheral blood leukocytes of each patient. PCR amplification products were checked by 1.8% agarose gel electrophoresis and were subsequently sequenced with ABI 3700 Automated DNA Sequencer.</p><p><b>RESULTS</b>The authors identified 13 PA mutations, 8 affecting the PCCA gene, 5 affecting the PCCB gene, including 10 novel mutations and 3 previously reported mutations. Three missense mutations (1079T > G, 1102G > C and 1850T > C), one splicing mutation (716-2A > G) and one short deletion (1863delA) were found in alpha-PCC subunit while three missense mutations (484G > A, 601G > A and 1253C > T) and two short insertion-deletions (167-179del13ins1, 560-561delCCinsT) were found in beta-PCC subunit. The 167-179del13ins1 change was identified in two homozygous PA patients, with allelic frequency of 40% in beta-PCC subunit deficiencies.</p><p><b>CONCLUSION</b>Thirteen mutations were found in 11 Chinese PA patients including ten novel mutations. No mutation is predominant in Chinese PCCA and PCCB deficient patients.</p>

Diagnosis, treatment and long-term following up of 223 patients with hyperphenylalaninemia detected by neonatal screening programs / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the incidence of hyperphenylalaninemia (HPA) caused by different etiologic factors in China and the relationship between the phenylalanine and mental development of patients with HPAs who were diagnosed by neonatal screening and early treated.</p><p><b>METHODS</b>Two hundred and twenty-three patients with HPA detected by neonatal screening programs were refered to us at the age of (41 +/- 27) days after birth. The differential diagnosis was performed by BH(4) (20 mg/kg) loading test, urinary pterin analysis and dihydropteridine reductase (DHPR) activity determination respectively. The control of phenylalanine (Phe) metabolism, growth and mental development were evaluated in all treated patients. Related gene mutation analysis was performed in some patients</p><p><b>RESULTS</b>One hundred and twenty-nine of 223 patients (57.8%) were diagnosed as phenylalanine hydroxylase deficiency (PAHD), 64 patients (28.7%) as BH(4) responsive PAHD, 30 patients (13.5%) as 6-pyruvoyl tetrahydropterin synthase deficiency (PTSD). One hundred and forty-nine patients were followed at age of 4 m - 2 y in our clinic. The 136 of 149 patients were treated according to different etiology at the age of 1.6 m (0.5 - 3.5 m) after birth. Thirteen patients were followed up without the need for treatment. All patients had normal growth development. One hundred and eight (79.4%) of 136 treated patients had normal mental development. The negative correlation (r = -0.439, P < 0.01) between IQ and average Phe levels were observed in 58 patients. Twenty-eight patients were able to go to primary school or even university. Nine kinds of PTS gene mutations were found in 9 cases with PTSD, among which 286G-->A and 259C-->T were most commonly seen, accounting for 45%. Seven kinds of PAH gene mutations were found in 13 cases with BH(4) responsive PAHD with the R241C (43.8%) mutation being the most frequent one.</p><p><b>CONCLUSION</b>The differential diagnosis should be quickly made in all HPA patients detected by neonatal screening. Near 80% patients early treated had normal mental development. The good control of blood Phe level is a key factor for mental development.</p>

Screening for tetrahydrobiopterin metabolic disorders and related gene analysis among the patients with motor disturbance and mental retardation / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the incidence of various enzyme deficiency in tetrahydrobiopterin (BH4) metabolism and the related gene mutation among the patients with motor disturbance and mental retardation.</p><p><b>METHODS</b>One hundred patients with unknown motor disturbance and mental retardation were referred to this study. All patients were performed by phenylalanine (Phe) and BH4 loading test, urinary pterin analysis and dihydropteridine reductase (DHPR) activity. Some patients received the dopa treatment for diagnosis of dopa-responsive dystonia (DRD). The analysis of GTP cyclohydrolase 1 gene (GCH1) mutation for DRD patients and the analysis of 6-pyruvoyl tetrahydropterin synthase (PTS) gene mutations for PTS deficient patients were done under the consent from their parents.</p><p><b>RESULTS</b>Seventy of 100 patients had normal basic blood Phe levels, six (6%) patients were diagnosed as DRD. Thirty patients had hyperphenylalaninemia (HPA), eight (8%) were diagnosed as PTS deficiency and 22(22%) were diagnosed as phenylalanine hydroxylase (PAH) deficiency. All patients had normal DHPR activity. The mutation IVS5+3insT of GCH1 was found in 2 patients with DRD. Seven kinds of PTS mutations were found in 8 patients with PTS deficiency, and 75% of the mutations were 259C-->T,286G-->A and 155A-->G.</p><p><b>CONCLUSION</b>Some patients with unknown motor disturbance and mental retardation may suffer from BH4 metabolism related diseases. Theses patients are necessary to be screened for such kind of diseases in order to confirm the diagnosis.</p>

Molecular Characterization of the Promoter of ATP7B in Wilson Disease / 实用儿科临床杂志

Objective To detect molecular characterization of the promoter and 5′UTR region of ATP7B gene in Chinese children with Wilson disease (WD) and explore the distribution of polymorphisms and mutations in different ethnicity.Methods One hundred and ten patients with WD and 90 healthy children were enrolled into the study and analyzed by polymerase chain reaction-single strand configuration polymorphism (PCR-SSCP) and DNA sequence analysis.Results 1.Five polymorphisms were identified as follows, -1294T→G,-105C→G,-116C→T ,-132delGCCGC and -75A→C(transcription start site as +1). The former three ones had never been reported before. The fourth one had not been reported either in China. 2.The polymorphism -132delGCCGC and -75A→C both exhibited with allelic frequency at above 70%, which was much higher than other races. The -132delGCCGC polymorphism shared almost complete linkage disequilibrium with the -75A→C polymorphism (in 98% patients) and their regularity was 96.9%.3. Almost all the polymorphisms distributed in flanking sequence of EXON 1 in Chinese. Race and geological distribution may be dominant factors of the variability of promoter and 5′UTR region of ATP7B gene.Conclusions Three novel polymorphisms and a linkage disequilibrium with the -132delGCCGC and -75A→C were identified in Chinese with WD. It also suggests that the mutation in the promoter of ATP7B is uncommon in Chinese patients.

Prenatal diagnosis of glycogen storage disease Ia by screening for hot spot mutations in combination with the 1176 nucleotide polymorphism linkage analysis / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To develop and evaluate a simple, fast and accurate prenatal diagnosis method for glycogen storage disease Ia (GSD Ia) in Chinese.</p><p><b>METHODS</b>This study involved 3 unrelated families. Genomic DNA samples were extracted from the blood of three GSD Ia patients and their parents, from the amniocytes of 3 fetuses and the blood of 2 newborns. By the way of restriction enzyme analysis, the screening for 727G-->T and R83H mutations of glucose-6-phosphatase gene was carried out in conjunction with 1176 nucleotide polymorphism linkage analysis so as to make the gene and prenatal diagnosis of 3 GSD Ia families. Direct DNA sequencing of the corresponding PCR products was used to confirm the unveiled mutations and 1176 nucleotide polymorphism.</p><p><b>RESULTS</b>Three probands were homozygotes for the 727G-->T mutation and their parents were heterozygotes for this mutation. The fetuses of family 1 and 3 were heterozygotes for the 727G-->T mutation, whereas the fetus of family 2 did not carry this mutation. The 1176 nucleotide polymorphisms of 3 fetuses were different from those of the corresponding probands. The prenatal diagnoses of family 1 and 2 were confirmed by the postnatal biochemical and molecular studies.</p><p><b>CONCLUSION</b>These findings suggest that the screening for 727G-->T and R83H mutations in conjunction with the 1176 polymorphism linkage analysis be a simple, fast and accurate method for gene and prenatal diagnosis of GSD Ia in Chinese.</p>

Study on mutations of exon 12 of the PAH gene in 127 phenylketonuria patients / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To obtain the mutation spectrum of exon 12 of the phenylalanine hydroxylase(PAH) gene.</p><p><b>METHODS</b>The samples from 127 patients with phenylketonuria(PKU) were studied by polymerase chain reaction(PCR)-single strand conformation polymorphism(SSCP) PCR-denaturing gradient gel electrophoresis(DGGE) and direct DNA sequencing.</p><p><b>RESULTS</b>Four heterozygous mutations including R413P S411X R408W R408Q in 10 of 127 patients were identified by DNA sequencing. The frequencies of R413P S411X R408W R408Q mutation were 2.76%, 0.39%, 0.39% and 0.39%, respectively. The S411X mutation in Chinese was first reported. Only 2 cases with R413P mutation were detected by SSCP. DGGE analysis showed that 10 cases displayed 3 kinds of abnormal electrophoretic bands. There were no obvious differences in the frequency of R413P mutation between northern and southern Chinese with PKU, and between classical PKU and hyperphenylalaninemia.</p><p><b>CONCLUSION</b>DGGE is more sensitive than SSCP in screening the mutations of exon 12 of the PAH gene. DGGE combined with DNA sequencing can be used to define all the mutations of exon 12 of the PAH gene. These results indicate the similarity in the frequency of R413P mutation between northern and southern patients.</p>

Study on Mutation in Exon 8 of ATP7B Gene in Chinese Patients with Wilson Disease / 实用儿科临床杂志

G,the noval insertion mutation of c.2298_2299insC is identified in Chinese patients.

Technique of PCR-ACRS for the detection of CYP21 gene mutations / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a rapid method of detecting CYP21 gene mutations.</p><p><b>METHODS</b>Fifty Chinese patients with 21-hydroxylase deficiency and some of their families were investigated. Blood samples were obtained for extraction of peripheral blood lymphocytes. A search for restriction sites discriminating between the morbid and the normal in CYP21 gene was made by the computer program DNAssist. PCR-based amplication-created restriction site(PCR-ACRS) was performed at I172N and R356W which are not natural recognition sequence. In addition, I172N and R356W were analysed in five families which conform to the applicability of PCR-ACRS.</p><p><b>RESULTS</b>In 50 identified 21-hydroxylase deficient Chinese patients, 21 were found to have I172 N (3 were homozygote, 18 were heterozygote); 8 were found to have R356W, all of them were heterozygote. By analysing the families, the findings were consistent with the characteristics of autosomal recessive genetic deficiency.</p><p><b>CONCLUSION</b>Analysis of CYP21 gene point mutations using PCR-ACRS is relatively simple, accurate and feasible.</p>

CYP21 gene point mutations study in 21-hydroxylase deficiency patients / 中华儿科杂志

<p><b>OBJECTIVE</b>The major cause of congenital adrenal hyperplasia (CAH) is 21-hydroxylase deficiency, which accounts for 90% - 95% of all cases in most populations. This study was conducted to characterize the molecular basis of the 21-hydroxylase deficiency and to obtain the spectrum of the CYP21 gene mutations in a group of Chinese patients, and analyze the relationship of genotype and phenotype.</p><p><b>METHODS</b>To detect the distribution of gene mutations in Chinese population samples from 52 patients with 21-hydroxylase deficiency from 51 families were collected, including two parents samples in 30 patients and one parents sample in 10 patients. Blood samples were obtained for extraction of peripheral blood lymphocytes. Polymerase chain reaction (PCR) followed by nesed PCR were used to study the 21-hydroxylase gene (CYP21) mutations. The primary PCR amplified two overlapping CYP21-specific DNA fragments, The product of the nested PCR which used products from the primary PCR was analysed by restriction fragment length polymorphism (RFLP) or amplification-created restriction site (ACRS). All patients were studied by 6 mutations, including P30L, I2g (intron 2 nt 656 c/a-->g splice mutation), E3Delta8nt (exon 3 codon111-codon113 8 bp deletion), I172N, V281L and Q318X.</p><p><b>RESULTS</b>Through analysis of 52 patients with 21-hydroxylase deficiency, in 5 patients no mutations were detected, in 17 patients only one mutated allele could be characterized, two different mutations were identified in 21 patients, three mutations were detected in 2 patients. Totally, in 73% of alleles the genotypes could be detected. The most common mutation was I2g, which present on 31% affected alleles, then followed by I172N, Q318X, V281L, P30L, E3Delta8nt, accounting for 23%, 14%, 9%, 3%, 2% of all identified mutations respectively, which included multiple mutations accounting for 6%. The most frequent molecular defects of the salt-wasting form were the I2g (45.7%), Q318X (26%). Of the simple virilizing form, the dominant mutations were I172N (40.7%) and I2g (18.5%).</p><p><b>CONCLUSION</b>Six different mutations were examined in this study, and the detected mutations accounted for 73% affected alleles, in which I2g and I172N were the most common mutations (accounting for 54%). Correlation between genotypes and phenotypes was compatible with the reported data. Two rounds of PCR followed by RFLP or ACRS analysis may provide important information for genetic counseling and for prenatal diagnosis.</p>

Analysis of haplotype-based haplotype relative risk and transmission disequilibrium test in childhood absence epilepsy / 中华儿科杂志

<p><b>OBJECTIVE</b>Childhood absence epilepsy (CAE), a common form of idiopathic generalized epilepsy, accounts for 8% - 15% of all childhood epilepsies. A positive family history of epilepsy, a hereditary factor being one of the pathogeneses, is found in 15% - 44% of children with absence seizures. The phenotype of CAE is specific (including seizure forms and EEG), therefore it is suitable for genetic study. The purpose of this study was to confirm the linkage of childhood absence epilepsy to chromosome 8q24 in China.</p><p><b>METHODS</b>Twenty-nine trios families (a patient and his/her parents) as patient group and 10 normal trios families as control group were investigated for chromosome 8q24 by haplotype analysis with 5 microsatellite DNA markers (D8S554, D8S534, D8S1100, D8S1783, D8S1753). Genomic DNA was isolated from 4 ml human peripheral blood by using the conventional procedure, and then was treated using the PCR method. PCR products were analyzed by gene scan. Statistical methodology included haplotype-based haplotype relative risk (HHRR) and transmission disequilibrium test (TDT).</p><p><b>RESULTS</b>In this study, the polymorphism information content (PIC) of 5 microsatellite DNA markers were: 0.519, 0.828, 0.528, 0.654 and 0.772. HHRR showed D8S554(4) (chi(2) = 5.939, P < 0.05), D8S1100(3) (chi(2) = 5.081, P < 0.05), D8S1783(6) (chi(2) = 4.308, P < 0.05). TDT showed D8S554(4) (chi(2) = 4.46, P < 0.05), D8S1783(6) (chi(2) = 4, P < 0.05). In order to exclude false association results, the authors analyzed every family in detail. Four trios families transmitted allele D8S1783(6) to their offspring, and the same allele hasn't been found in controls. The further work showed that locus D8S1783 had transmission disequilibrium with CAE, the other two loci were a false association.</p><p><b>CONCLUSION</b>(1) Childhood absence epilepsy in the Chinese population may be linked to chromosome 8q24, the CAE gene is transmitted disequilibrium on locus D8S1783. Combined with other research results, we suppose that CAE gene may be in the ECA1 area on chromosome 8q24. (2) The CAE gene perhaps has a genetic heterogeneity in the population of different areas and different races. (3) HHRR and TDT seem to be the best statistical methods to do linkage disequilibrium study in the trios family.</p>

Genotype and phenotype correlation in Chinese patients with Wilson's Disease / 中华儿科杂志

<p><b>OBJECTIVE</b>To determine distribution and mutation pattern of type P ATP7B gene mutation and to explore genotype and phenotype correlation in patients with Wilson's disease (WD).</p><p><b>METHODS</b>Sixty patients with WD from 57 no kinship families, 37 male and 23 female, were enrolled in this study. The age of onset ranged from 4.6 - 39 years, < or = 16 years in 55 patients. Some exons of ATP7B gene mutation were analyzed in patients with WD by using biochemical methods, polymerase chain reaction-single strand configuration polymorphism (PCR-SSCP), restriction fragment and DNA sequence analysis. Totally 778 coding regions were identified with restriction enzyme Msp I. The activity of Cu-ATPase was assessed by measuring inorganic phosphorus in 3 patients with known genotype.</p><p><b>RESULTS</b>Fifty-two of 60 patients (86%) had presented with hepatic manifestations, 30 of them had only hepatic manifestations, 12/52 patients had hepatic and neurological manifestations at the same time; 10/52 patients had hepatic and other symptom; 7/60 patients had only neurological symptom, one patient had no symptom. Eleven mutations were detected by DNA sequencing, including five missense mutations (R778L, V1140A,G943S, V1106I and V1216M), one deletion (1384del17) and five polymorphisms (IVS4-5T/C, A2495G, C2310G, IVS18 + 6C/T and IVS20 + 5A/G) were identified. R778L mutation was identified 52/114 alleles (45.6%). R778L occurred in 38/52 patients with hepatic manifestation (73%), homozygosis of R778L was demonstrated in 14 patients and heterozygosity of R778L in 24 patients. V1106I mutation was 1.7%, G943S, V1140A, and V1216M was 0.86% respectively in this study. Two patients with delayed onset of neurological symptoms occurred V1106I mutation of ATP7B. Cu-ATPase activity of 3 patients with known mutation (R778L/V1106I, R778L/V1216M and R778L/R778L) declined by 44.55%, 88.23% and 69.49%, respectively, compared with normal control.</p><p><b>CONCLUSION</b>The 1384del17bp and V1106I are two novel mutations found in patients with WD. R778L was common mutation of ATP7B gene with frequency of 45.6% in this study. The mutation in exon 8 of WD gene may play an important role in pathogenesis of WD in Chinese. Carriage of R778L mutation seems to be correlated with hepatic manifestation.</p>