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To proceed the clinical evaluation of DNA microarray for thalassemia gene detection.Peripheral blood samples of 166 thalassemia gene test subjects were collected and tested for thalassemia genes by microarray chip method and Gap-PCR method combined with PCR-reverse dot blot hybridization method according to double-blind control test. The specificity, sensitivity, positive predictive value, negative predictive value, and total coincidence rate of the microarray chip method were evaluated. When the two methods were inconsistent, multiplex ligation dependent probe amplification (MLPA) was used to verify the deletional α-thalassemia.Compared with Gap-PCR method, specificity, sensitivity, positive predictive value, negative predictive value, Youden index, and total coincidence rate of microarray chip method was 100% (70/70), 96.88% (93/96), 100% (93/93), 95.89% (70/73), 0.969, and 97.59% (162/166), respectively, while compared with PCR-reverse dot blot hybridization method was 100% (125/125), 100% (41/41), 100% (41/41), 100% (125/125), 1, and 100% (166/166), respectively.The microarray chip method for α-thalassemia gene detection shows the advantages of high specificity, sensitivity, and throughput.微阵列芯片法在α-地中海贫血检测中的方法学评价.对微阵列芯片法用于地中海贫血基因检测进行临床评价.收集166份地中海贫血基因受检者的外周血样本，按照双盲对照试验，分别应用微阵列芯片法和Gap-PCR法联合PCR-反向点杂交法对各样本进行地中海贫血基因检测，评价微阵列芯片法的特异性、灵敏度、阳性预测值、阴性预测值及总符合率。两种方法不一致时，对于缺失型α-地中海贫血采用多重连接依赖探针扩增（MLPA）法进行验证.微阵列芯片法与Gap-PCR法比较，特异性为100%（70/70），灵敏度为96.88%（93/96），阳性预测值为100%（93/93），阴性预测值为9589%（70/73），约登指数为0.969，总符合率为97.59%（162/166）；与PCR-反向点杂交法比较，其特异性为100%（125/125），灵敏度为100%（41/41），阳性预测值为100%（41/41），阴性预测值为100%（125/125），约登指数为1，总符合率为100%（166/166）.微阵列芯片法用于α-地中海贫血基因检测具有特异性高、灵敏度高、高通量等优点.

To perform dried blood spots thalassemia gene detection in patients with positive blood phenotypes by microarray technology, and evaluate its value in clinical detection.DNA samples were extracted from dried blood spots of 410 patients. Microarray technology was used to detect 3 deletion and 3 non-deletion types of α-thalassemia and 19 β-thalassemia point mutations which were common gene mutions in China.There were 357 positive cases in all the 410 tested samples with the positive rate 87.07%, among which 299 cases (72.93%) carried deletion or point mutations of α-thalassemia, 29 cases (7.07%) carried point mutations of β-thalassemia and 29 cases (7.07%) carried gene mutations of complex αβ-thalassemia syndrome. The mutations of α-thalassemia were involved with --The most common genetic mutations are --微阵列芯片法在地中海贫血患者基因突变检测中应用的研究.应用微阵列芯片技术对血液学表型分析阳性的患者干血斑进行地中海贫血基因检测，评价其在临床检测中的价值。.对410例患者的干血斑样本进行DNA提取，采用微阵列芯片技术对中国人常见的3种缺失型及3种非缺失型α-地中海贫血、19种β-地中海贫血点突变进行基因检测。.410例样本中，共检出阳性357例，总阳性率为87.07%。单纯α-地中海贫血基因缺失和点突变的样本有299例，占72.93%（299/410），其中177例为广西柳州地区α、β地中海贫血基因主要突变类型分别为

The aim of this study was to investigate a family with nonhomologous sequence recombination of

This study aimed to analyze the clinical phenotype of chromosome 9p deletion or duplication and its relationship with karyotype. A patient, female, aged 6 months, visited the hospital due to motor developmental delay. Karyotype analysis identified abnormalities of chromosome 9 short arm, and high-throughput sequencing found 9p24.3-9p23 deletion and 9p23-9p13.1 duplication. Her parents had a normal karyotype. Karyotype analysis combined with high-throughput sequencing is of great significance for improving the efficiency of etiological diagnosis in children with motor developmental delay or multiple congenital deformities and mental retardation.

OBJECTIVE: To study the gene mutation profile of primary carnitine deficiency (PCD) in neonates, and to provide a theoretical basis for early diagnosis and treatment, genetic counseling, and prenatal diagnosis of PCD. METHODS: Acylcarnitine profile analysis was performed by tandem mass spectrometry using 34 167 dry blood spots on filter paper. The SLC22A5 gene was sequenced and analyzed in neonates with free carnitine (C0) levels lower than 10 μmol/L as well as their parents. RESULTS: In the acylcarnitine profile analysis, a C0 level lower than 10 μmol/L was found in 10 neonates, but C0 level was not reduced in their mothers. The 10 neonates had 10 types of mutations at 20 different sites in the SLC22A5 gene, which included 4 previously unreported mutations:c.976C > T, c.919delG, c.517delC, and c.338G > A. Bioinformatics analysis showed that the four new mutations were associated with a risk of high pathogenicity. CONCLUSIONS: Tandem mass spectrometry combined with SLC22A5 gene sequencing may be useful for the early diagnosis of PCD. Identification of new mutations enriches the SLC22A5 gene mutation profile.

Background Spinal muscular atrophy (SMA) is mainly caused by deletions in SMA-related genes. The objective of this study was to develop gene-dosage assays for diagnosing SMA. Methods A multiplex, quantitative PCR assay and a CNVplex assay were developed for determining the copy number of SMN1, SMN2, and NAIP. Reproducibility and specificity of the two assays were compared to a multiple ligation-dependent probe amplification (MLPA) assay. To evaluate reproducibility, 30 samples were analyzed three times using the three assays. A total of 317 samples were used to assess the specificity of the two assays. Results The multiplex quantitative PCR (qPCR) assay had higher reproducibility. Intra-assay CVs were 3.01%-8.52% and inter-assay CVs were 4.12%-6.24%. The CNVplex assay had ratios that were closer to expected (0.49-0.5 for one copy, 1.03-1.0 for two copies, and 1.50-1.50 for three copies). Diagnostic accuracy rates for the two assays were 100%. Conclusions The multiplex qPCR assay was a simple, rapid, and cost-effective method for routine SMA diagnosis and carrier screening. The CNVplex assay could be used to detect SMAs with complicated gene structures. The assays were reliable and could be used as alternative methods for clinical diagnosis of SMA.

This study aimed to identify the type of carnitine palmitoyl transferase 2 (CPT2) gene mutation in the child with carnitine palmitoyl transferase Ⅱ (CPT Ⅱ) deficiency and her parents and to provide the genetic counseling and prenatal diagnosis for the family members. As the proband, a 3-month-old female baby was admitted to the hospital due to fever which had lasted for 8 hours. Tandem mass spectrometric analysis for blood showed an elevated plasma level of acylcarnitine, which suggested CPT Ⅱ deficiency. The genomic DNA was extracted from peripheral blood of the patient and her parents. Five exon coding regions and some intron regions at the exon/intron boundaries of the CPT2 gene were analyzed by PCR and Sanger sequencing. Amniotic fluid was taken from the mother during the second trimester, and DNA was extracted to analyze the type of CPT2 gene mutation. Sanger sequencing results showed that two mutations were identified in the CPT2 gene of the proband:c.886C > T (p.R296X) and c.1148T > A (p.F383Y), which were inherited from the parents; the second child of the mother inherited the mutation of c.886C > T (p.R296X) and showed normal acylcarnitine spectrum and normal development after birth. It is concluded that the analysis of CPT2 gene mutations in the family suggested that the proband died of CPT Ⅱ deficiency and that the identification of the mutations was helpful in prenatal diagnosis in the second pregnancy.

X-linked ichthyosis (XLI) is a metabolic disease with steroid sulfatase deficiency and often occurs at birth or shortly after birth. The encoding gene of steroid sulfatase, STS, is located on the short arm of the X chromosome, and STS deletion or mutation can lead to the development of this disease. This study collected the data on the clinical phenotype from a family, and the proband, a boy aged 11 years with full-term vaginal delivery, had dry and rough skin and black-brown scaly patches, mainly in the abdomen and extensor aspect of extremities. Peripheral blood samples were collected from each family member and DNA was extracted. Multiplex ligation-dependent probe amplification (MLPA) was used to measure the copy number of STS on the X chromosome. Whole-genome microarray was used to determine the size of the segment with microdeletion in the X chromosome. MLPA was then used for prenatal diagnosis for the mother of the proband. The results revealed that the proband and another two male patients had hemizygotes in STS deletion. Gene microarray identified a rare deletion with a size of 1.6 Mb at Xp22.31 (chrX:6, 516, 735-8, 131, 442). Two female family members were found to be carriers. Prenatal diagnosis showed that the fetus carried by the proband's mother was a carrier of this microdeletion. This study showed STS gene deletion in this family of XLI, which causes the unique skin lesions of XLI. MLPA is a convenient and reliable technique for the molecular and prenatal diagnosis of XLI.

A 9-day-old male patient was admitted to the hospital because of cough, anhelation, feeding difficulty and lethargy. The diagnostic examinations indicated pulmonary infection, severe metabolic acidosis, hyperglycemia, hyperammonemia and pancytopenia in the patient. Blood and urine screening and isovaleryl-CoA dehydrogenase (IVD) gene detection for inherited metabolic diseases were performed to clarify the etiology. Tandem mass spectrometric screening for blood showed an elevated isovalerylcarnitine (C5) level. The organic acid analysis of urine by gas chromatography-mass spectrometry showed significantly increased levels in isovaleryl glycine and 3-hydroxyisovaleric acid. Homozygous mutations (c.1208A > G, p.Tyr403Cys) in the IVD gene were identified in the patient. His parents were heterozygous carriers. After the treatment with low-leucine diets and L-carnitine for 3 days, the patient showed a significant improvement in symptoms, but he died one week later. It is concluded that the neonates with pneumonia and metabolic decompensation of unknown etiology should be screened for genetic metabolic disease.

Screening for carriers of spinal muscular atrophy (SMA) is necessary for effective clinical/prenatal diagnosis and genetic counseling. However, a population-based study of SMA prevalence in mainland China has not yet been conducted. In this study, the copy number of survival motor neuron (SMN) genes was determined in 1712 newborn cord blood samples collected from southern China and from 25 core families, which included 26 SMA patients and 44 parents, to identify SMA carriers. The results presented 13 groups with different SMN1/SMN2 ratios among 1712 newborn individuals, which corresponded to 1535 subjects with two copies of SMN1, 119 with three copies of SMN1, 17 with four copies of SMN1, and 41 with a heterozygous deletion of SMN1 exon 7. Simultaneously, two ‘2+0' genotypes and two point mutations were found among the 44 obligate carriers in the core families, including a novel SMN1 splice-site mutation that was identified in the junction between intron 6 and exon 7 (c. 835–1G>A). These results indicated that the carrier frequency is 1/42 in the general Chinese population and that duplicated SMN1 alleles and de novo deletion mutations are present in a small number of SMA carriers. In addition, we developed and validated a new alternative screening method using a reverse dot blot assay for rapid genotyping of deletional SMA. Our research elucidated the genetic load and SMN gene variants that are present in the Chinese population, and could serve as the basis for a nationwide program of genetic counseling and clinical/prenatal diagnosis to prevent SMA in China.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited disorder worldwide including southern China. We developed and validated a reverse dot blot (RDB) assay for the rapid and simultaneous genotyping of six mutations (c.95A>G, c.871G>A, c.1004C>T, c.1024C>T, c.1376G>T and c.1388G>A), that were common mutations in the Chinese G6PD deficiency population, and one polymorphism (c.1311C>T). Reliable genotyping of wild-type and mutant genomic DNA samples was achieved by means of a test strip onto which allele-specific oligonucleotide probe lines are fixed in parallel. This method involves a multiplex PCR amplification of three fragments in the G6PD target sequence and a manual hybridization/detection protocol. The entire procedure starting from blood sampling to the identification of mutations requires less than 6 h. The diagnostic reliability of this reverse dot blot assay was evaluated on 207 pre-typed samples by using direct DNA sequence analysis in a blind study. The reverse dot blot typing was in complete concordance with the reference method. The reverse dot blot assay was proved to be a simple, rapid, highly accurate, and cost-effective method to identify common G6PD mutations in Chinese population.

Background Spinal muscular atrophy (SMA) is caused by SMN1 dysfunction, and the copy number of SMN2 and NAIP can modify the phenotype of SMA. The aim of this study was to analyze the copy numbers and gene structures of SMA-related genes in Chinese SMA patients and unrelated healthy controls. Methods Forty-two Chinese SMA patients and two hundred and twelve unrelated healthy Chinese individuals were enrolled in our study. The copy numbers and gene structures of SMA-related genes were measured by MLPA assay. Results We identified a homozygous deletion of SMN1 in exons 7 and 8 in 37 of 42 patients (88.1%); the other 5 SMA patients (11.9%) had a single copy of SMN1 exon 8. The proportions of the 212 unrelated healthy controls with different copy numbers for the normal SMN1 gene were 1 copy in 4 individuals (1.9%), 2 copies in 203 (95.7%) and 3 copies in 5 (2.4%). Three hybrid SMN genes and five genes that lack partial sequences were found in SMA patients and healthy controls. Distributions of copy numbers for normal SMN2 and NAIP were significantly different (P

Background The Southeast Asian deletion (--SEA) is the most commonly observed mutation among diverse α-thalassemia alleles in Southeast Asia and South China. It is generally argued that mutation --SEA, like other variants causing hemoglobin disorders, is associated with protection against malaria that is endemic in these regions. However, little evidence has been provided to support this claim. Results We first examined the genetic imprint of recent positive selection on the --SEA allele and flanking sequences in the human α-globin cluster, covering a genomic region spanning ~410 kb, by genotyping 28 SNPs in a Chinese population consisting of 76 --SEA heterozygotes and 138 normal individuals. The pattern of linkage disequilibrium (LD) and the long-range haplotype test revealed a signature of positive selection. The network of inferred haplotypes suggested a single origin of the --SEA allele. Conclusions Thus, our data support the hypothesis that the --SEA allele has been subjected to recent balancing selection, triggered by malaria.