[Cases Analysis of Hemoglobin H Disease Caused by HBA2:c.2T>C and HBA2:c.2delT Mutations].

OBJECTIVE: To investigate two cases of rare pathogenic genes, initiation codon mutations in HBA2 gene, combined with Southeast Asian deletion and their family members to understand the relationship of HBA2:c.2T>C and HBA2:c.2delT mutations with clinical phenotype. METHODS: The peripheral blood of family members was obtained for blood cell analysis and capillary electrophoresis hemoglobin analysis. Gap-PCR and reverse dot blotting (RDB) were used to detect common types of mutations in ɑ-thalassaemia gene. Sanger sequencing was used to analyze HBA1 and HBA2 gene sequence. RESULTS: Two proband genotypes were identified as --SEA/αα with HBA2:c.2T>C and --SEA/αα with HBA2:c.2delT. HBA2:c.2T>C/WT and HBA2:c.2delT/WT was detected in family members. They all presented with microcytic hypochromic anemia. CONCLUSION: When HBA2:c.2T>C and HBA2:c.2delT are heterozygous that can lead to static α-thalassemia phenotype, and when combined with mild α-thalassemia, they can lead to the clinical manifestations of hemoglobin H disease. This study provides a basis for genetic counseling.

[Methodological Evaluation of Microarray in the Detection of α-Thalassemia].

OBJECTIVE: To proceed the clinical evaluation of DNA microarray for thalassemia gene detection. METHODS: 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. RESULTS: 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. CONCLUSION: The microarray chip method for α-thalassemia gene detection shows the advantages of high specificity, sensitivity, and throughput.

[Application of DNA Microarray in Genetic Mutation Detection in Patients with Thalassemia].

OBJECTIVE: To perform dried blood spots thalassemia gene detection in patients with positive blood phenotypes by microarray technology, and evaluate its value in clinical detection. METHODS: 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 ß-thalassemia point mutations which were common gene mutions in China. RESULTS: 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 ß-thalassemia and 29 cases (7.07%) carried gene mutations of complex αß-thalassemia syndrome. The mutations of α-thalassemia were involved with --SEA heterozygous deletion (177 cases, 59.2%), αCS heterozygote (60 cases, 20.07%) and several other genotypes. The common mutations of ß- thalassemia were involved with ßCD41-42 heterozygote (10 cases, 34.48%) and ßCD17 heterozygote (9 cases, 31.03%). The mutations of complex αß-thalassemia syndrome were mainly involved with --SEA/αα+ßCD17/ßN (7 cases, 24.14%), αCSα/αα + ßCD41-42/ßN (3 cases, 10.34%) and -α4.2/αα + ßCD17/ßN (3 cases, 10.34%). CONCLUSION: The most common genetic mutations are --SEA for α-thalassemia and CD41-42 for ß-thalassemia in Liuzhou, Guangxi Zhuang Autonomous Region. A and ß-thalassemia can be detected at the same time by microarray chip technology in a high throughput manner.

Pedigree Analysis of Nonhomologous Sequence Recombination of HBA1 and HBA2 Genes.

The aim of this study was to investigate a family with nonhomologous sequence recombination of HBA1 and HBA2 genes and provide a favorable basis for genetic counseling and eugenics. Peripheral blood of family members was collected. Hematological parameters were determined by an automated cell counter and hemoglobin (Hb) analysis was performed using high performance liquid chromatography (HPLC). Villus samples were taken for prenatal diagnosis (PND). Gap-polymerase chain reaction (gap-PCR) and reverse dot-blot were used for thalassemia genotyping. DNA sequencing was used to analyze the gene sequence of HBA1 (α1-globin) and HBA2 (α2-globin). The nonhomologous sequence recombination allelic variant of HBA1 and HBA2 genes were identified, namely, a gene conversion on the HBA2 gene called α12 (HBA12). The α12 allele consists primarily of the HBA2 gene sequence except for a segment of the IVS-II in which HBA2-specific sequences have been replaced by HBA1-specific sequences. The following genotypes were observed: - -SEA/αα12 (Southeast Asian deletion), αα/αα12 and αQSα/αα12 (Hb Quong Sze or Hb QS; HBA2: c.377T>C), and all manifested as small cell hypochromic anemia. To find the α12 allele in the Chinese population and clarify the influence of the α12 allele and its common inheritance with abnormal Hb and α-thalassemia (α-thal) on α-globin gene expression can help guide clinical diagnosis and genetic counseling.

[Clinical and cytogenetic study in a child with de novo chromosome 9 abnormality].

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.

[Genetic diagnosis of 10 neonates with primary carnitine deficiency].

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.

Evaluation and comparison of three assays for molecular detection of spinal muscular atrophy.

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.

[CPT2 gene mutation analysis and prenatal diagnosis in a family with carnitine palmitoyltransferase II deficiency].

This study aimed to identify the type of carnitine palmitoyltransferase 2 (CPT2) gene mutation in the child with carnitine palmitoyltransferase 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.

[Pancytopenia and metabolic decompensation in a neonate].

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.

[Identification of gene mutation and prenatal diagnosis in a family with X-linked ichthyosis].

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.

[An analysis of clinical characteristics and gene mutation in two patients with medium- and short-chain acyl-CoA dehydrogenase deficiency].

Medium- and short-chain acyl-CoA dehydrogenase deficiency is a disorder of fatty acid ß-oxidation. Gene mutation prevents medium- and short-chain fatty acids from entry into mitochondria for oxidation, which leads to multiple organ dysfunction. In this study, serum acylcarnitines and the organic acid profile in urea were analyzed in two children whose clinical symptoms were hypoglycemia and metabolic acidosis. Moreover, gene mutations in the two children and their parents were evaluated. One of the patients was a 3-day-old male who was admitted to the hospital due to neonatal asphyxia, sucking weakness, and sleepiness. The serum acylcarnitine profile showed increases in medium-chain acylcarnitines (C6-C10), particularly in C8, which showed a concentration of 3.52 µmol/L (reference value: 0.02-0.2 µmol/L). The analysis of organic acids in urea gave a normal result. Sanger sequencing revealed a reported c.580A>G (p.Asn194Asp) homozygous mutation at exon 7 of the ACADM gene. The other patient was a 3-month-old female who was admitted to the hospital due to cough and recurrent fever for around 10 days. The serum acylcarnitine profile showed an increase in serum C4 level, which was 1.66 µmol/L (reference value: 0.06-0.6 µmol/L). The analysis of organic acids in urea showed an increase in the level of ethyl malonic acid, which was 55.9 (reference value: 0-6.2). Sanger sequencing revealed a reported c.625G>A (p.Gly209Ser) homozygous mutation in the ACADS gene. This study indicates that screening tests for genetic metabolic diseases are recommended for children who have unexplained metabolic acidosis and hypoglycemia. Genetic analyses of the ACADM and ACADS genes are helpful for the diagnosis of medium- and short-chain acyl-CoA dehydrogenase deficiency.

Molecular characterization and copy number of SMN1, SMN2 and NAIP in Chinese patients with spinal muscular atrophy and unrelated healthy controls.

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 < 0.001) in people with and without SMA. CONCLUSION: The copy numbers and gene structures of SMA-related genes were different in Chinese SMA patients and healthy controls.

Evidence of recent natural selection on the Southeast Asian deletion (--(SEA)) causing α-thalassemia in South China.

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.

Reliable detection of paternal SNPs within deletion breakpoints for non-invasive prenatal exclusion of homozygous α-thalassemia in maternal plasma.

Reliable detection of large deletions from cell-free fetal DNA (cffDNA) in maternal plasma is challenging, especially when both parents have the same deletion owing to a lack of specific markers for fetal genotyping. In order to evaluate the efficacy of a non-invasive prenatal diagnosis (NIPD) test to exclude α-thalassemia major that uses SNPs linked to the normal paternal α-globin allele, we established a novel protocol to reliably detect paternal SNPs within the (--(SEA)) breakpoints and performed evaluation of the diagnostic potential of the protocol in a total of 67 pregnancies, in whom plasma samples were collected prior to invasive obstetrics procedures in southern China. A group of nine SNPs identified within the deletion breakpoints were scanned to select the informative SNPs in each of the 67 couples DNA by multiplex PCR based mini-sequencing technique. The paternally inherited SNP allele from cffDNA was detected by allele specific real-time PCR. A protocol for reliable detection of paternal SNPs within the (--(SEA)) breakpoints was established and evaluation of the diagnostic potential of the protocol was performed in a total of 67 pregnancies. In 97% of the couples one or more different SNPs within the deletion breakpoint occurred between paternal and maternal alleles. Homozygosity for the (--(SEA)) deletion was accurately excluded in 33 out of 67 (49.3%, 95% CI, 25.4-78.6%) pregnancies through the implementation of the protocol. Protocol was completely concordant with the traditional reference methods, except for two cases that exhibited uncertain results due to sample hemolysis. This method could be used as a routine NIPD test to exclude gross fetal deletions in α-thalassemia major, and could further be employed to test for other diseases due to gene deletion.

Molecular characterization of SMN copy number derived from carrier screening and from core families with SMA in a Chinese population.

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.

Development and evaluation of a reverse dot blot assay for the simultaneous detection of six common Chinese G6PD mutations and one polymorphism.

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.