EDM1: a novel point mutation in cartilage oligomeric matrix protein gene in a Chinese family with multiple epiphyseal dysplasia / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Multiple epiphysis dysplasia (MED) is a common skeletal dysplasia with a significant locus heterogeneity. In the majority of clinically defined cases, mutations have been identified in the gene encoding cartilage algometric matrix protein (COMP).</p><p><b>METHODS</b>Five patients were included in the study. Linkage analysis and mutation analysis of the COMP gene were conducted in the patients and their family members.</p><p><b>RESULTS</b>We have identified a novel mutation in axon 14 of COMP gene in the family.</p><p><b>CONCLUSIONS</b>This mutation produced a severe MED phenotype with marked short stature, early onset osteoarthritis, and remarkable radiographic changes. Our results extended the range of disease-causing mutations in COMP gene and contributed more information about relationship between mutations and phenotype.</p>

Clinical characterization and mutation identification for multiple sulfatase deficiency patients in China / 中华儿科杂志

<p><b>OBJECTIVE</b>Multiple sulfatase deficiency is a rare autosomal recessively inherited lysosomal storage disorder characterized by the accumulation of sulfated lipids and acid mucopolysaccharides. The aim of this study was to explore the clinical manifestations, enzyme activities and SUMF1 gene mutations in two Chinese patients with multiple sulfatase deficiency.</p><p><b>METHOD</b>One boy and one girl from two families were studied. Both patients presented with mental retardation, mild coarse facial features, a neurodegenerative course of disease with loss of sensory and motor function after 2 years of age, ichthyosis and skeletal abnormalities (kyphosis or/and scoliosis). Clinical characteristics indicate multiple sulfatase deficiency.Sulfatases activities in blood leucocytes, plasma or cultured fibroblast of the patients were measured.Genomic DNAs were extracted from peripheral blood leukocytes from the patients and their parents. All SUMF1 gene exons and intron-exon boundaries were amplified by PCR and subjected for direct sequencing.</p><p><b>RESULT</b>In case 1, five sulfatases activities of blood leucocytes and four sulfatases of cultured skin-fibroblasts were analyzed.In case 2, three sulfatases activities of blood leucocytes were tested.Significantly decreased sulfatases activities confirmed the diagnosis of multiple sulfatase deficiency.On SUMF1 gene, c.793_794 insATG (p. P265X)/ c.1045C>T (p.R349W) in case 1 and c.451A>G (p.K151E)/ c.1046G>C (p.R349Q) in case 2 were detected, respectively. Three novel mutations c.793_794insAGT, c.1046G>C and c.451A>G were identified.</p><p><b>CONCLUSIONS</b>Multiple sulfatase deficiency usually results in multi-organ damage, especially neurologic, skeletal and skin.Sulfatases assay and SUMF1 gene analysis are necessary for the diagnosis. Two Chinese cases with multiple sulfatase deficiency were firstly reported. Three novel mutations were found.It should be considered that the mutation profile of SUMF1 gene in Chinese patients is different from other populations.</p>

Analysis of ITD characteristics in acute myeloid leukemia patients with FLT3-ITD positive / 中国实验血液学杂志

The aim of this study was to analyze the FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) allelic ratios (AR), number of ITD, ITD length and positions of ITD insertions in de novo acute myeloid leukemia (AML) patients with FLT3-ITD positive, and the relationship between mutant level and therapeutic efficacy. Genomic DNA was amplified by PCR, capillary electrophoresis was used to detect the ITD characteristics in 31 de novo AML patients, and DNA sequences analysis of FLT3-ITD(+) were performed in 13 patients. The results showed that the ratios of mutant to wild type FLT3 allele ranged from 0.01 to 2.8; 28 patients (90.32%) had a single ITD, the remaining 3 patients had more than one ITD; the ITD length ranged from 3 to 144 bp in all FLT3-ITD(+) patients. 13 sequence-analyzed patients, 4 patients were of pure duplications, and 2 patients had foreign bases inserted, and the other 7 patients were partial duplications. The ITD occurred in the regions from p.E573 to p.P606 of the FLT3 protein, with the majority clustered in a stretch between p.F590 and p.R595. The complete remission (CR) rate in AR < 0.5 patients (43.75%) were more prevalent as compared with AR ≥ 0.5 patients (16.67%) (p > 0.05). It is concluded that the ITD length and AR are vary widely. Some of the insertions are foreign bases, and all of the 13 sequences-analyzed ITD were concentrated on the juxtamembrane domain. The CR rate in patients of AR < 0.5 had no statistical significance compared with patients of AR ≥ 0.5.

Two novel EIF2AK3 mutations in a Chinese boy with Wolcott-Rallison syndrome / 中华儿科杂志

<p><b>OBJECTIVE</b>Wolcott-Rallison syndrome (WRS) is a rare autosomal recessive disorder characterized by the association of permanent neonatal or early-infancy insulin-dependent diabetes, multiple epiphyseal dysplasia and growth retardation, and other variable multisystem clinical manifestations. Here we describe a Chinese boy affected by WRS. Genetic testing of his EIF2AK3 gene was performed in order to elucidate molecular variations and subsequently to provide credible genetic counseling for prenatal diagnosis in his family.</p><p><b>METHOD</b>Based on analysis of a nine-year-old boy's clinical symptoms associated with biochemical examination and imaging, the diagnosis of WRS was therefore made. Genomic DNAs were extracted from peripheral blood leukocytes from the boy and his parents with their informed consent for genetic studies. All EIF2AK3 exons and intron-exon boundaries were amplified by Touch-down polymerase chain reaction (Touch-down PCR) and sequenced.</p><p><b>RESULT</b>Direct sequencing of PCR products revealed the presence of a heterozygous T insertion (c.1408_1409insT) in exon 8 of the EIF2AK3 gene leading to frameshifting and termination, and another heterozygous T to A exchange (c.1596T > A) in exon 9 of the EIF2AK3 gene resulting in nonsense C532X mutation.</p><p><b>CONCLUSION</b>Combining mutation screening of EIF2AK3 gene with clinical manifestations and effective examination may provide a reliable diagnostic method for patients. In this research, two novel mutations identified in the Chinese boy locate in the catalytic domain of the EIF2AK3 gene, disrupting the ability of autophosphorylation, leading to the truncated proteins that are unable to phosphorylate the natural substrate, which are responsible for the phenotype of Wolcott-Rallison syndrome.</p>

Mutation analysis of PTPN11 gene in Noonan syndrome / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the mutations in protein tyrosine phosphatase, nonreceptor-type 11 (PTPN11) gene in patients with Noonan syndrome (NS).</p><p><b>METHODS</b>Three sporadic patients with NS were studied. Genomic DNAs were extracted from peripheral blood leukocytes. All 15 coding exons and their flanking intronic boundaries of the PTPN11 gene were amplified by polymerase chain reaction and followed by direct sequencing. DNAs from parents were sequenced in the corresponding region when the mutation was detected in their affected child. The identified mutation was screened in 100 healthy individuals for exclusion of polymorphism by restriction endonuclease digestion of the PCR products. Protein conservation analysis was performed among 10 species using an online ClustalW tool.</p><p><b>RESULTS</b>Direct DNA sequence analysis identified a heterozygous 181G to A change in exon 3 of the PTPN11 gene in one patient, which resulted in the substitution of an aspartic acid residue by an asparagine at codon 61. The mutation was absent in his parents and 100 controls, and is located in a highly conserved amino acid site. No mutation in the coding region of PTPN11 gene was observed in the other two patients.</p><p><b>CONCLUSION</b>The p.D61N mutation was reported previously in Caucasians and is a de-novo mutation in this patient. Our study further confirmed that the p.D61N is a pathogenic mutation for NS and consistent with the clinical diagnosis. Additional genes may be involved in the other two patients with NS, indicating high genetic heterogeneity of this disease.</p>

FMS-like tyrosine kinase 3 gene mutation in acute myeloid leukemia detected by denaturing PAGE and its clinical significance / 中国实验血液学杂志

The aim of this study was to analyze the frequency of flt3 length mutation (flt3-LM) in de novo acute myeloid leukemia patients and the relationship between flt3-LM and chromosome alterations, FAB subgroups, as well as efficiency of therapy. Genomic DNA was amplified by PCR; 2% agarose gel or 8% denaturing PAGE were used to detect the length mutation of flt3 gene in 99 de novo acute myeloid leukemia patients; karyotyping in 72 AML patients was performed by G banding technique. The results showed that the flt3-LM was detected in 20.2% (20/99) patients by agarose gel electrophoresis, and in 29.9% (29/99) by denaturing PAGE. The flt3-LM was not detected in M(0) (only one patient was available), but flt3-LM occurrence in AML subtypes was as follow: in M(2) (9/30), M(3) (6/27), M(4) (4/14), M(5) (7/19), M(6) (3/8) respectively. flt3-LM in patients with normal karyotypes (39.13%) was more prevalent as compared with patients of abnormal karyotype (24.49%), but there was no statistical difference (p > 0.05). The complete remission (CR) rate in flt3-LM positive patients (36.36%) was lower than that in flt3-LM negative patients (62.75%) in the 73 patients (p < 0.05) whose karyotypic detection was performed. The distributions of flt3-LM were observed in 8 out of 40 CR patients, 8 out of 21 PR patients, and 6 out of 12 NR patients. It is concluded that the denaturing PAGE is more sensitive and reliable to detect the flt3-LM. The flt3 mutation represents a common genetic abnormality in AML patients, and the flt3-LM is associated with lower CR rate.

Genetic analysis and serum level of cartilage oligomeric matrix protein in patients with pseudoachondroplasia / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Pseudoachondroplasia (PSACH) is an autosomal-dominant osteochondrodysplasia due to mutations in the gene encoding cartilage oligomeric matrix protein (COMP). Clinical diagnosis of PSACH is based primarily on family history, physical examination, and radiographic evaluation. There is evidence that decreased serum COMP concentration may serve as a diagnostic marker in PSACH. Here, we investigated the role of this gene and the serum COMP concentration in Chinese patients with PSACH.</p><p><b>METHODS</b>A family with three patients and a sporadic case were recruited. Genomic and phenotypic data were recorded. The diagnosis of PSACH was made on the base of clinical evaluation. The genomic DNA was extracted from peripheral blood leukocytes. The 8-19 exons and flanking intron-exon boundary sequences of COMP were amplified by polymerase chain reaction (PCR) and screened for mutation by direct DNA sequencing. Serum COMP concentrations of 4 patients and age-compatible control group of 20 unrelated healthy subjects were analyzed on the basis of an ELISA Kit for human cartilage oligomeric matrix protein.</p><p><b>RESULTS</b>A deletion (c.1447-1455del) was identified in exon 13 in the sporadic case. The mean serum COMP concentrations of four patients (3.12+/-2.28) were significantly lower than those of control group (10.86+/-2.21, P<0.05). There was no overlap in the distribution of serum COMP concentration between PSACH patients and controls.</p><p><b>CONCLUSIONS</b>Mutations in COMP gene are responsible for the PSACH. Serum COMP concentration may be suggested as an additional diagnostic marker to aid clinical findings in suspected cases of PSACH.</p>

Gene mapping of autosomal dominant retinitis pigmentosa in a Chinese family / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The autosomal dominant form of retinitis pigmentosa (ADRP) can be caused by mutations in 14 genes and further loci remains to be identified. This study was intended to identify mutations in a Chinese pedigree with ADRP.</p><p><b>METHODS</b>A large Chinese family with retinitis pigmentosa was collected. The genetic analysis of the family suggested an autosomal dominant pattern. Microsatellite (STR) markers tightly linked to genes known to be responsible for ADRP were selected for linkage analysis. Exons along with adjacent splice junctions of PRPF31 were amplified by polymerase chain reaction (PCR) and screened by direct sequencing.</p><p><b>RESULTS</b>The caused gene of ADRP was mapped to 19q13.4 between markers D19S572 and D19S877, with a maximum LOD score of 3.01 at marker D19S418 (recombination fraction = 0).</p><p><b>CONCLUSION</b>The affected gene linked to the 19q13.4 in a Chinese family with ADRP, which is different from other mutations at the same loci in other Chinese families.</p>

Gene mutation analysis of a collodion baby / 中华儿科杂志

<p><b>OBJECTIVE</b>To determine the mutations pattern of the genes of a collodion baby.</p><p><b>METHODS</b>Collodion baby is a genetic heterogeneous disease caused by mutations of several genes. Since the most common mutations were observed in TGM1 gene, this gene was chosen for mutation screening. The screening was carried out by PCR and direct sequencing. The allele specific primers were designed for a missense mutation and allele-specific (AS) PCR was carried out in 50 normal individuals for population study.</p><p><b>RESULTS</b>Three novel alterations were detected in TGM1 gene of the proband, a missense mutation c.463C > T (p.Arg155Trp) in exon 3, a nonsense mutation c.578G > A (p.Trp193X) in exon 4, and a single nucleotide deletion (c.694delG) also in exon 4 of TGM1 gene. This infant's father was heterozygote of c.694delG mutation, while his mother carried the two mutations (c.463C > T and c.578G > A) on the same chromosome. The missense mutation was not detected in his father and in any of the control individuals by AS-PCR.</p><p><b>CONCLUSION</b>Three novel mutations were identified in TGM1 gene in a Chinese collodion baby. A double mutation (c.463C > T and c.578G > A) located on the maternal allele while the c.694delG deletion on the paternal allele.</p>

Detection of microdeletion in Williams syndrome by multiplex ligation-dependent probe amplification / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a method of multiplex ligation-dependent probe amplification (MLPA) for clinical screening of Williams syndrome (WS) and for routine use in WS diagnosis.</p><p><b>METHODS</b>Probes for MLPA were designed according to the frequent deletion regions, and used to screen the two patients suspected with Williams syndrome, and the density of the bands were analyzed with software. Linkage analysis using polymorphic markers was performed to confirm the positive result of MLPA.</p><p><b>RESULTS</b>The MLPA data indicated that the two children had possible microdeletions in the WS critical region. The deletions were confirmed and both were maternal origin by polymorphism analysis.</p><p><b>CONCLUSION</b>MLPA is a quick and convenient method for detecting deletion or duplication mutations. It can provide reliable and helpful information for clinical diagnose of Williams syndrome.</p>

Mutation analysis of the PAH gene in patients with phenylketonuria in Gansu province / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To characterize the mutations of the phenylalanine hydroxylase (PAH) gene in patients with phenylketonuria in Gansu province.</p><p><b>METHODS</b>Mutations of the PAH gene were detected in exons 3, 5, 6, 7, 11 and 12 with flaking introns of PAH gene by PCR and DNA sequencing.</p><p><b>RESULTS</b>Mutations were identified in 45/58 alleles (detection rate: 96.4%), in total of 18 variants. Among them IVS12+5G>C was a novel mutation. The most frequent mutations were R243Q (22.7%), V399V (12.1%), EX6-96A>G (5.2%), R413P (5.2%) and IVS4-1G>A (5.2%), followed by Y356X (3.4%), R111X (3.4%) and INS7+2T>A (3.4%).</p><p><b>CONCLUSION</b>The mutations of the phenylalanine hydroxylase gene in patients with phenylketonuria in Gansu province were similar to that in other areas of China, with obvious difference in mutation rate of some mutations.</p>

A Chinese girl with fibrodysplasia ossificans progressiva caused by a de novo mutation R206H in ACVR1 gene / 中华儿科杂志

<p><b>OBJECTIVE</b>Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant inherited disease caused by mutations of ACVR1 gene and can be inherited from either mother or father. FOP is characterized by the presence of malformations of the big toes and of progressive extra-skeletal ossification. Direct sequence analyses of genomic DNA have demonstrated that there is an identical single nucleotide substitution (c617G-->A, R206H) in the glycine-serine (GS) activation domain of ACVR1 gene, responsible for all affected individuals reported so far. We report a Chinese girl with typical FOP characteristics, in whom the same mutation in ACVR1 was identified.</p><p><b>METHODS</b>Clinical diagnosis was based on physical examination, radiological findings, and biochemical tests. For mutation detection, peripheral blood was obtained with informed consent from the patient and the parents. Genomic DNA was extracted from peripheral blood using standard method. Exon 4 of ACVR1 was amplified by polymerase chain reaction (PCR), and the PCR products were subjected to automatic DNA sequencing.</p><p><b>RESULTS</b>The affected girl is 3-year-old and showed typical clinical manifestations of FOP. She had malformations of the halluces at birth and subsequently progressive extra-skeletal ossification developed at the age of 8 - 9 months. Then, she gradually developed stiffness of the knee joint and neck but remained ambulant. Radiographic changes were observable, e.g., the extra-skeletal ossification was found at cervical spine. Her mother has congenital malformations of the halluces, but had no postnatal progressive extra-skeletal ossification. Her father and other family members are normal. With direct sequencing of the PCR products, a G to A substitution at c617 of ACVR1 (R206H) was detected in the patient only but not in her parents. Paternity analysis suggested that it is a de novo mutation.</p><p><b>CONCLUSION</b>This is the first case reported in a Chinese patient with FOP in the mainland of China, which was confirmed by direct sequencing. Although sporadic cases of FOP have been reported in diverse geographic and ethnic group, the mutations of ACVR1 c617 (R206H) are identical up to now. The presence of mutation hot spot facilitates molecular diagnosis in clinical practice. Genetic detection is important for FOP patients to avoid misdiagnosis and further damages, including those from medical intervention.</p>

The power of linkage analysis on PAH gene in prenatal gene diagnosis is improved with three additional short tandem repeat markers / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To increase the success rate of prenatal diagnosis for classical phenylketonuria(PKU).</p><p><b>METHODS</b>Three new short tandem repeat (STR) markers (PAH26, PAH32 and PAH9) within and surrounding phenylalanine hydroxylase(PAH) gene were selected for amplified fragment length polymorphism. The allele frequencies and polymorphism information contests (PIC) were determined in Chinese population.</p><p><b>RESULTS</b>The PIC of these three new STR markers was 0.518 (PAH26), 0.413 (PAH32) and 0.362 (PAH9) respectively. There was linkage disequilibrium between PAH9 marker and PAH-STR marker (TCTA)n in the intron 3 of PAH gene. The linkage phase of the mutant genes and the markers was established using the combination of PAH-STR, PAH26 and PAH32 in 95% families. Prenatal diagnosis was performed successfully with these markers in four cases.</p><p><b>CONCLUSION</b>By selecting or combining the three STR markers, the mutant genes could be distinguished from the normal allele in up to 95% of families with classical PKU.</p>

Erectile dysfunction in Fragile X patients / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To report the clinical experience during collecting sperm samples in the Fragile X syndrome (FXS) male patients.</p><p><b>METHODS</b>Two different polymerase chain reaction (PCR) based methods were used for the molecular diagnosis of FXS. Sperm collection was done mostly according to the laboratory manual of the World Health Organization.</p><p><b>RESULTS</b>We failed to collect sperm samples from five Fragile X subjects aged 18-60 years as a result of an unexpected erectile dysfunction (ED). Multiple examinations of the same subject at different times, and of different subjects from different provinces examined by different physicians, showed the same result consistently in all the five subjects.</p><p><b>CONCLUSION</b>Erectile reflex is an instinctive response in all healthy males. The absence of erection can be caused by hormonal, physical or neuronal malfunction. As hormonal profiles were reported to be generally normal in Fragile X men, we propose that an unknown physical factor or the neuronal circuit, or both, underlying the erection is compromised. The finding of this symptom in Fragile X patients may help better understand the clinical spectrum and pathogeneses of the disease.</p>

A research on TGFBI gene mutations in Chinese families with corneal dystrophies / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify what kind of TGFBI gene mutation happening to Chinese patients with corneal dystrophies.</p><p><b>METHODS</b>Three Chinese families with stromal corneal dystrophies and one Chinese family with Thiel-Behnke corneal dystrophies were studied, of whom three were Han race and another was Mongolia race in China. All members of families were examined clinically and their genomic DNAs were extracted from blood leukocytes. Thirteen exons in TGFBI gene were amplified by polymerase chain reaction (PCR) and directly sequenced for molecular analysis.</p><p><b>RESULTS</b>Mutations in TGFBI gene were detected from all the patients with corneal dystrophy, but not found in normal subjects of families. The mutation R555W was found and identified from the family with granular corneal dystrophy; R555Q from the family with Thiel-Behnke corneal dystrophy; and R124H from the other two families with Avellino corneal dystrophy.</p><p><b>CONCLUSION</b>The above study results show that the amino acids R124 and R555, if their genetic codes result from the mutations, play an important role in the pathogenesis of autosomal dominant corneal dystrophy of Chinese patients, and the molecular genetic analysis can improve the accuracy of diagnosing corneal dystrophy. In China, the mutation R555Q found in the family with Thiel-Behnke corneal dystrophy is reported for the first time.</p>

Postnatal and prenatal diagnosis of mucopolysaccharidosis type II (Hunter syndrome) / 中华儿科杂志

<p><b>OBJECTIVE</b>Mucopolysaccharidosis type II (MPS II, Hunter syndrome, OMIM 309900) is an X-linked recessive lysosomal storage disease resulting from a deficiency of iduronte-2-sulphate sulphatase (IDS). The present study aimed to establish an enzyme assay method for IDS activity for carrying out postnatal and prenatal diagnosis of MPS II by means of IDS activity assay on plasma, uncultured chorionic villi (CV) and cultured amniotic fluid cells (AF cell) using a new synthesized substrate.</p><p><b>METHODS</b>A fluorigenic substrate (4-methylumbelliferyl-alpha-iduronate-2-sulphate, MU-alpha-Idu-2S) was used for the assay of IDS activity. IDS activity in plasma was determined for diagnosis of the proband. Prenatal diagnosis in 10 pregnancies at risk was carried out according to IDS activity on uncultured CV at 11th week or on cultured AF cell at 18th week of gestation. At the same time, IDS activity was also determined in the maternal plasmas to observe the change of IDS activity in pregnancy. The fetal sex determination was performed by PCR amplification of the ZFX/ZFY genes.</p><p><b>RESULT</b>The IDS activity in plasma of normal controls and obligate heterozygotes were 240.2 - 668.2 nmol/(4 hxml) and 88.7 - 547.9 nmol/(4 hxml), respectively, while the enzyme activities in plasmas were in the range of 0.3 - 18.6 nmol/(4 hxml) in affected male. The IDS activities were 37.2 - 54.9 nmol/(4 hxmg protein) and 21.4 - 74.4 nmol/(4 hxmg protein) in CV and cultured AF cells respectively. Out of 50 suspected cases, 46 were diagnosed as having MPS II and 4 were excluded. Prenatal diagnosis was performed on 10 pregnancies at risk. Four of 5 male fetuses [IDS activity were 4.7, 1.8, 7.0 nmol/(4hxmg protein) in CV, 0.6 nmol/(4 hxmg protein) in AF cell] were diagnosed as having MPS II and the other 5 fetuses were normal females [IDS activity were: 48.7, 5.9, 25.2 nmol/(4 hxmg protein) in CV, 55.2, 40.9 nmol/(4 hxmg protein) in AF cell]. Increased IDS activity was observed in plasma of the pregnant women with unaffected fetuses, while the IDS activity decreased in pregnancies with affected fetuses. IDS activity of one female fetus was very low [5.9 nmol/(4 hxmg protein)], but the IDS activity in maternal plasmas increased, this fetus was a normal female.</p><p><b>CONCLUSIONS</b>The method using a synthesized fluorigenic 4-methylumbelliferyl-substrate was a sensitive, rapid and convenient assay of IDS activity and was reliable for early prenatal diagnosis. Determination of fetal sex would be helpful in excluding the female fetus with low IDS activity from being considered as an affected male fetus. It would be further helpful if IDS activity in maternal plasma was taken into account.</p>

Genetic heterogeneity for familial recurrent hydatidiform mole / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To determine the parental origin of the genome in the molar pregnancies of two familes with familial recurrent hydatidiform mole (FRHM) and to investigate whether the gene responsible for FRHM is likely to be located within the 19q13.4 region in these familes.</p><p><b>METHODS</b>The features of complete hydatidiform mole (CHM) were confirmed by pathological examination. DNA of CHM was prepared from sections of formalin-fixed paraffin-embedded blocks of molar tissue following laser capture microdissection. The polymerace chain reaction was used to amplify microsatellite polymorphisms in DNA from the patients, their husbands and the captured molar tissue. Parental contributions to the molar tissue were determined using ABI 310 GeneScan software. Genotyping and haplotype analysis of the candidate region on 19q13.4 was performed for members of both families using 25 microsatellite markers.</p><p><b>RESULTS</b>One CHM from each family was identified as a biparental complete hydatidiform mole. All patients were heterozygous for most of the markers in the chromosome region of interest. In addition the two affected sisters in one of the families had different genotypes for the 19q13.4 region, suggesting that mutations in a different locus might be responsible for the disorder in this family.</p><p><b>CONCLUSION</b>The location of the gene responsible for FRHM is unlikely to be located in the 19q13.4 chromosomal region in these two families suggesting that FRHM shows genetic heterogeneity.</p>

Mutation analysis of glycogen debrancher enzyme gene in five Chinese patients with glycogen storage disease type III / 中华儿科杂志

<p><b>OBJECTIVE</b>Type III glycogen storage disease (GSD-III, McKusick 232400), is a rare autosomal recessive disorder, also known as Cori's or Forbe's disease. The affected enzyme is amylo-1,6-glucosidase, 4-alpha-glucanotransferase (glycogen debrancher enzyme, GDE or amylogluco-sidase, AGL), which is responsible for the debranching of the glycogen molecule during catabolism. The AGL gene is located on chromosome 1p21 and contains 35 exons translated in a monomeric protein product. The clinical manifestations of GSD-III are represented by hepatomegaly, recurrent hypoglycemia, seizures, growth failure, dysmorphism, hyperlipidemia, raised transaminases and creatine kinase concentrations and, in a number of subjects, myopathy and cardiomyopathy. The hepatocellular adenoma, hepatocellular carcinoma, diabetes mellitus and liver fibrosis remain rare events. The diagnosis of debrancher deficiency was established by laboratory tests, electromyography (EMG), and muscle and liver biopsy.</p><p><b>METHODS</b>We studied six GSD-III families after patients or parental consent and the clinical characteristics were documented. Analysis of 33 exons and part exon-intron boundaries of the AGL gene in patients and their parents were carried out by PCR and direct DNA sequencing.</p><p><b>RESULTS</b>The clinical features included hepatomegaly, splenomegaly, recurrent hypoglycemia, hyperlipidemia, growth failure, raised transaminases and acidosis. Administration of epinephrine 2 hours after a carbohydrate meal could provoke normal rise of blood glucose in the affected individuals, but could not evoke any response after overnight fasting. Administration of raw-corn-starch could maintain normoglycemia and improve the disease condition. Mutation analysis for patient 1 was normal. Patient 2 had a compound heterozygote: a C-to-T transition at nucleotide 1294 (come from father, 1294C > T, L 298 L) in exon 8 and a G-to-T transition at nucleotide 4747 (from mother, 4747G > T, E1450X) in exon 34. Patient 3 had a compound heterozygote: a C-to-T transition at nucleotide 1294 (from father, 1294C > T, L 298 L) in exon 8 and a G-to-A transition at nucleotide -10 (from mother, -10G > A) in exon 3. Patient 4 was a homozygote: an insertion of a nucleotide CT into position +65 in exon 35 (4664 ins CT). Patient 5 had a compound heterozygote: a 8 bp deletion at nucleotide 2341 (from father, 2341delGCCATAGA, frameshift mutation) in exon 16 and a G-to-A transition at nucleotide 1559 (from mother, 1559G > A, R 387 Q) in exon 10. Patient 6 had a compound heterozygote: a T-to-G transition at nucleotide 1686 (from mother, 1686T > G, Y429 X) in exon 12 and a G-to-A transition at nucleotide 3742 (from father, 3742G > A, G 1115 R) in exon 26.</p><p><b>CONCLUSION</b>GSD-III patients have variable phenotypic characteristics. Administration of raw-corn-starch can effectively improve the disease outcome. We identified 8 new mutations on AGL gene through nucleotide sequence analysis.</p>

Mutation-function analysis in the lipoprotein lipase gene of Chinese patients with hypertriglyceridemic type 2 diabetes / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the role of lipoprotein lipase (LPL) gene on Chinese patients with hypertriglyceridemic type 2 diabetes.</p><p><b>METHODS</b>Three subject groups, including hypertriglyceridemic group, normalipidemic type 2 diabetes group and healthy controls, were recruited and screened for sequence changes in LPL gene with PCR, SSCP, restriction analysis and direct DNA sequencing. LPL mass and activity in post-heparin plasma and in in vitro expression were investigated. Comparative modeling was performed via Swiss-PDB Viewer to provide the potential 2-D structures of wildtype and mutant proteins.</p><p><b>RESULTS</b>Four missense mutations, Ala71Thr, Val18Ile, Gly188Glu and Glu242Lys, were identified in patients with hypertriglyceridemic type 2 diabetes, and not in both normalipidemic diabetes and the control subjects. The four missense mutations were located in the highly conserved amino acid sites, which are involved in highly conserved exon 3, 5, or 6 regions. They led to reduced LPL mass and enzyme activities in both post-heparin plasma and in vitro expression. The modeled structures displayed the differences to a great extent between the mutant and wide-type molecules.</p><p><b>CONCLUSION</b>These results indicated that the 4 missense mutations lead to LPL deficiency and subsequent hypertriglyceridemia. The LPL deficiency predispose a progressive diabetic pathway to those affected individuals. LPL gene is one of susceptibility gene for hypertriglyceridemic type 2 diabetes.</p>

Identification of a novel mutation of C1 inhibitor gene in a Chinese family with hereditary angioedema / 中国医学科学院学报

<p><b>OBJECTIVE</b>To identify the mutation of C1 inhibitor (C1 INH) gene in a Chinese family with hereditary angioedema (HAE).</p><p><b>METHODS</b>Polymerase chain reaction and direct sequencing were used to identify the mutation type. The sequencing results were compared with the normal sequences in GenBank to find the mutation. In order to exclude the polymorphism, 30 normal volunteers were analyzed.</p><p><b>RESULTS</b>One novel mutation (17839 del C) was detected in 5 patients with HAE. The mutation was not found in controls.</p><p><b>CONCLUSION</b>The mutation of C1 INH gene (17839 del C) is identified in the family. Molecular diagnosis can be made by detecting the mutation.</p>