A Novel Hemoglobin Variant Hb Liaobu [α107(G14)Val→Leu, HBA2: c.322G>C] Detected by Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry.

We here describe a novel hemoglobin (Hb) variant, Hb Liaobu [α107(G14)Val→Leu, HBA2: c.322G>C], in a Chinese family. The structurally abnormal α chain variant could not be detected using capillary electrophoresis (CE) and was subsequently characterized by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS), and further confirmed by reversed phase high performance liquid chromatography (HPLC). Sanger sequencing revealed a novel base mutation on the α2-globin gene and RNA analysis by reverse transcription polymerase chain reaction (RT-PCR) showed the presence of an abnormal HBA transcript. The isopropanol stability test indicated the stable state of this structural Hb variant. In conclusion, a new Hb variant, Hb Liaobu, was discovered and characterized. It was proven to be a nonpathogenic variant. Our study resolved the confusion in the clinical diagnosis of individuals with this novel Hb variant in this family.

C1orf194 deficiency leads to incomplete early embryonic lethality and dominant intermediate Charcot-Marie-Tooth disease in a knockout mouse model.

Charcot-Marie-Tooth (CMT) disease is the most common inherited peripheral neuropathy and shows clinical and genetic heterogeneity. Mutations in C1orf194 encoding a Ca2+ regulator in neurons and Schwann cells have been reported previously by us to cause CMT disease. In here, we further investigated the function and pathogenic mechanism of C1or194 by generating C1orf194 knockout (KO) mice. Homozygous mutants of C1orf194 mice exhibited incomplete embryonic lethality, characterized by differentiation abnormalities and stillbirth on embryonic days 7.5-15.5. Heterozygous and surviving homozygous C1orf194 KO mice developed motor and sensory defects at the age of 4 months. Electrophysiologic recordings showed decreased compound muscle action potential and motor nerve conduction velocity in the sciatic nerve of C1orf194-deficient mice as a pathologic feature of dominant intermediate-type CMT. Transmission electron microscopy analysis revealed demyelination and axonal atrophy in the sciatic nerve as well as swelling and loss of mitochondrial matrix and other abnormalities in axons and Schwann cells. A histopathologic examination showed a loss of motor neurons in the anterior horn of the spinal cord and muscle atrophy. Shorter internodal length between nodes of Ranvier and Schmidt-Lanterman incisures was detected in the sciatic nerve of affected animals. These results indicate that C1orf194 KO mice can serve as an animal model of CMT with a severe dominant intermediate CMT phenotype that can be used to investigate the molecular mechanisms of the disease and evaluate the efficacy of therapeutic strategies.

Anterograde Viral Tracer Herpes Simplex Virus 1 Strain H129 Transports Primarily as Capsids in Cortical Neuron Axons.

The features of herpes simplex virus 1 (HSV-1) strain 129 (H129), including natural neurotropism and anterograde transneuronal trafficking, make it a potential tool for anterograde neural circuitry tracing. Recently anterograde polysynaptic and monosynaptic tracers were developed from H129 and have been applied for the identification of novel connections and functions of different neural circuitries. However, how H129 viral particles are transported in neurons, especially those of the central nervous system, remains unclear. In this study, we constructed recombinant H129 variants with mCherry-labeled capsids and/or green fluorescent protein (GFP)-labeled envelopes and infected the cortical neurons to study axonal transport of H129 viral particles. We found that different types of viral particles were unevenly distributed in the nucleus, cytoplasm of the cell body, and axon. Most H129 progeny particles were unenveloped capsids and were transported as capsids rather than virions in the axon. Notably, capsids acquired envelopes at axonal varicosities and terminals where the sites forming synapses are connected with other neurons. Moreover, viral capsids moved more frequently in the anterograde direction in axons, with an average velocity of 0.62 ± 0.18 µm/s and maximal velocity of 1.80 ± 0.15 µm/s. We also provided evidence that axonal transport of capsids requires the kinesin-1 molecular motor. These findings support that H129-derived tracers map the neural circuit anterogradely and possibly transsynaptically. These data will guide future modifications and improvements of H129-based anterograde viral tracers.IMPORTANCE Anterograde transneuronal tracers derived from herpes simplex virus 1 (HSV-1) strain 129 (H129) are important tools for mapping neural circuit anatomic and functional connections. It is, therefore, critical to elucidate the transport pattern of H129 within neurons and between neurons. We constructed recombinant H129 variants with genetically encoded fluorescence-labeled capsid protein and/or glycoprotein to visualize viral particle movement in neurons. Both electron microscopy and light microscopy data show that H129 capsids and envelopes move separately, and notably, capsids are enveloped at axonal varicosity and terminals, which are the sites forming synapses to connect with other neurons. Superresolution microscopy-based colocalization analysis and inhibition of H129 particle movement by inhibitors of molecular motors support that kinesin-1 contributes to the anterograde transport of capsids. These results shed light into the mechanisms for anterograde transport of H129-derived tracer in axons and transmission between neurons via synapses, explaining the anterograde labeling of neural circuits by H129-derived tracers.

[Effect of genetic modifiers on the clinical severity of ß-thalassemia].

ß-thalassemia (ß-thal) is a fatal and disabling inherited blood disorder with diverse phenotypes. The same or similar genotype of ß-thal can manifest variable clinical severities. It is the hotspot and emphasis in the field of hematopathy and genetic diseases to explore genetic modifiers that influence the phenotype of ß-thal. This review illustrates the deteriorating and amelioratig modifiers from two aspects: genotypes of α-globin and quantitative trait locus of fetal hemoglobin (Hb F). Variations of transcription factors which reactive the γ-globin gene expression and ß-globin cluster cis-acting elements were introduced emphatically. Finally, clinical applications and future development prospects of ß-thal genetic modifiers are introduced by examples.

[Congenital ectrodactyly caused by chromosome 10q24.31 duplication and its pathogenetic analysis].

In order to investigate the genetic variations and the clinical manifestations of a range of congenital ectrodactyly family and to summarize the split hand/foot malformation (SHFM) types and their related pathogenic genes, we conducted phenotypic analyses of patient's limbs by physical and X-ray examination. The haplotypes were analyzed by using the extracted genes from peripheral blood on D10S1709, D10S192, D10S597, D10S1693 and D10S587 loci, and the mutation duplication loci were confirmed by Array-CGH detection. The pathogenic factors and inheritance pattern of SHFM were analyzed based on family investigation and gene analysis. Results demonstrate the proband's phenotype is typically of a congenital SHFM which is manifested by missing bilateral index and middle fingers, short bilateral thumbs, deformed left ring finger with webbing of the skin missing at the middle finger; bilateral big toe with the second and the third toe missing, fourth and fifth toe fusion leading to a deformed toe separated from the first toe by the middle of the foot. The haplotype analyses show that there is a repeat of at least 610 kb in chromosome 10q24.31-10q24.32 region. Array-CGH analysis shows 10q24.31 (102 832 650-103 511 083) ×3. Our results demonstrate that the pathogenic gene variation of ectrodactyly in this family is due to duplication of 10q24.31 (102 832 650~103 511 083). The haplotype 165-251-289-219-102 can be used as a disease marker for detecting 10q24.31~10q24.32 allele for SHFM.

[A comprehensive repository of mutation data and a clinical assistant decision system for hemoglobinopathy in the Chinese population].

Personal genomic information benefits from accumulated big data and its application is no longer limited to scientific research. Presently, it is undergoing the transformation to daily medical practice. Systematic arrangement, archiving and rational utilization of disease-related genomic information is an important foundation of future precision medicine. Hemoglobinopathy is prevalent in southern China, but its molecular pathological basis has racial specificity. To facilitate clinical diagnosis and genetic screening of hemoglobinopathy in southern China, we established the LOVD gene data management system for the variation and phenotype spectrum of hemoglobinopathy. Then we designed an integrated and efficient on-line auxiliary accurate diagnosis and risk assessment system in order to assist clinicians to make comprehensive diagnosis and genetic counseling in a short time based on cloud standardized annotated library of specific hemoglobinopathy variants and diagnostic repository. The methodology and experience of improving the clinical decision-making efficiency of diseases with big data and artificial intelligence technology can be used as an example in the clinical and preventive application of other diseases.

Mutations in C1orf194, encoding a calcium regulator, cause dominant Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease is a hereditary motor and sensory neuropathy exhibiting great clinical and genetic heterogeneity. Here, the identification of two heterozygous missense mutations in the C1orf194 gene at 1p21.2-p13.2 with Charcot-Marie-Tooth disease are reported. Specifically, the p.I122N mutation was the cause of an intermediate form of Charcot-Marie-Tooth disease, and the p.K28I missense mutation predominately led to the demyelinating form. Functional studies demonstrated that the p.K28I variant significantly reduced expression of the protein, but the p.I122N variant increased. In addition, the p.I122N mutant protein exhibited the aggregation in neuroblastoma cell lines and the patient's peroneal nerve. Either gain-of-function or partial loss-of-function mutations to C1ORF194 can specify different causal mechanisms responsible for Charcot-Marie-Tooth disease with a wide range of clinical severity. Moreover, a knock-in mouse model confirmed that the C1orf194 missense mutation p.I121N led to impairments in motor and neuromuscular functions, and aberrant myelination and axonal phenotypes. The loss of normal C1ORF194 protein altered intracellular Ca2+ homeostasis and upregulated Ca2+ handling regulatory proteins. These findings describe a novel protein with vital functions in peripheral nervous systems and broaden the causes of Charcot-Marie-Tooth disease, which open new avenues for the diagnosis and treatment of related neuropathies.

Analysis of variants in upstream open reading frames of human globin-related genes.

ß-thalassemia is an autosomal recessive monogenic disease that is caused by defects in the production of ß-like globin chains. Activation of γ-globin gene and the increase in fetal hemoglobin expression have been demonstrated as one of the most important factors to ameliorate the clinical outcome of ß-thalassemia patients. In this study, 202 genes or miRNAs associated with human hemoglobin gene expression from 1802 ß-thalassemia patients were analyzed with target capture and next generation sequencing strategies in terms of functional variants that might affect hemoglobin gene expression. The subsequent bioinformatics analysis included assessments of sequence quality, the variants within the target regions and the 5'UTR with potential effects on upstream open reading frames (uORFs). Among the 41 variants in 5'UTR potentially affecting the uORFs identified in the study, two variants (chr19: 41859418 G > A and chr1:153606541 C > T) were experimentally validated with dual-luciferase assays to be capable of significantly down-regulating the expression of TGFB1 and CHTOP gene, respectively. The present study demonstrated a system suitable for evaluating the importance of variants in 5'UTRs affecting uORFs in 202 human genes associated with hemoglobin expression. Research with this approach could provide potential targets that may contribute to the clinical phenotypes and provide biomarkers for precise diagnosis of ß-thalassemia.

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.

Development of a capillary zone electrophoresis method for rapid determination of human globin chains in α and ß-thalassemia subjects.

Thalassemia is an inherited autosomal recessive blood disorder characterized by the underproduction of globin chains as a consequence of globin gene defects, resulting in malfunctioning red blood cells and oxygen transport. Analysis of globin chains is an important aspect of thalassemia research. In this study we developed a capillary zone electrophoresis (CZE) method for human globin determination in the diagnosis of thalassemia and hemoglobin variants. To demonstrate the utility of this approach, α/ß area ratios were determined for samples from 310 thalassemia patients and healthy controls. The separation was performed on uncoated capillary with simple preparation. Distinct globin peaks were resolved in 17 min, and coefficients of variation (CV) for migration time and areas ranged from 0.37%-1.69% and 0.46%-6.71%, respectively. Receiver operating characteristic (ROC) curve analysis of the α/ß area ratios gave 100% sensitivity and specificity for indicating ß-TI/TM, and 100% sensitivity and 97.4% specificity for Hb H disease. Hemoglobin G-Honolulu (Hb G-Honolulu) and Hb Westmead (Hb WS) were successfully detected using this CZE method. This automated methodology is simple, rapid and cost-effective for the fast determination of human globin chains, which could be an important diagnostic tool in the field of hemoglobinopathies.

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.

DNA hypermethylation and X chromosome inactivation are major determinants of phenotypic variation in women heterozygous for G6PD mutations.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked incompletely dominant enzyme deficiency that results from G6PD gene mutations. Women heterozygous for G6PD mutations exhibit variation in the loss of enzyme activity but the cause of this phenotypic variation is unclear. We determined DNA methylation and X-inactivation patterns in 71 G6PD-deficient female heterozygotes and 68 G6PD non-deficient controls with the same missense mutations (G6PD Canton c.1376G>T or Kaiping c.1388G>A) to correlate determinants with variable phenotypes. Specific CpG methylations within the G6PD promoter were significantly higher in G6PD-deficient heterozygotes than in controls. Preferential X-inactivation of the G6PD wild-type allele was determined in heterozygotes. The incidence of preferential X-inactivation was 86.2% in the deficient heterozygote group and 31.7% in the non-deficient heterozygote group. A significant negative correlation was observed between X-inactivation ratios of the wild-type allele and G6PD/6-phosphogluconate dehydrogenase (6PGD) ratios in heterozygous G6PD Canton (r=-0.657, p<0.001) or Kaiping (r=-0.668, p<0.001). Multivariate logistic regression indicated that heterozygotes with hypermethylation of specific CpG sites in the G6PD promoter and preferential X-inactivation of the wild-type allele were at risk of enzyme deficiency.

[Analysis of clinical phenotypes of compound heterozygotes of Hb J-Bangkok and ß-thalassemia].

OBJECTIVE: To analyze hematological characteristics of compound heterozygotes of Hb J-Bangkok and ß-thalassemia, and to explore the influence of Hb J-Bangkok on the phenotype of ß-thalassemia. METHODS: Peripheral blood samples from a patient carrying Hb J-Bangkok and a ß-thalassemia mutation, her family members and three sporadic Hb J-Bangkok carriers were collected. RBC analysis and hemoglobin electrophoresis were performed. Genotypes of α- and ß-globin genes were analyzed. RESULTS: The father of the proband and the three sporadic cases were single carriers of Hb J-Bangkok. All of them were asymptomatic and have normal hematological parameters except for an abnormal hemoglobin band detected on hemoglobin electrophoresis. The proband was a compound heterozygote for Hb J-Bangkok and ß-thalassemia mutation IVS-Ⅱ-654. She presented typical ß-thalassemia trait, featuring hypochromic microcytic anemia and increased Hb A2 level. An abnormal hemoglobin band was also detected. CONCLUSION: Carriers of Hb J-Bangkok alone are asymptomatic. Co-existence of Hb J-Bangkok and ß-thalassemia may not aggravate the phenotype. Therefore, couples with one carrying Hb J-Bangkok and another carrying a ß-thalassemia mutation do not require prenatal diagnosis.

A novel COL7A1 gene mutation causing pretibial epidermolysis bullosa: report of a Chinese family with intra-familial phenotypical diversity.

Pretibial epidermolysis bullosa (PEB) is an extremely rare subtype of dominant dystrophic epidermolysis bullosa (DDEB) caused by mutation of the COL7A1 gene. More than 730 mutations have been identified in patients with DDEB, but only five mutations have been found to be related to PEB. In this study, a novel heterozygous nucleotide G>T transition at position 6101 in exon 73 of COL7A1 was detected, which resulted in a glycine to valine substitution (G2034V) in the triple-helical domain of type-VII collagen. This is the first report to show that one mutation caused a broad range of severity of disease in one family with PEB. These data suggest that c.6101G>T may influence the phenotype of PEB. They also contribute to the expanding database on COL7A1 mutations.

A novel fusion gene and a common α(0)-thalassemia deletion cause hemoglobin H disease in a Chinese family.

Genetic recombination has been implicated as a mechanism that drives mutagenesis in the human globin gene clusters, either as a result of unequal crossover or gene conversion. In this paper, a novel fusion gene was identified in a Chinese girl with hemoglobin H disease. The proband's father was a compound heterozygote for the common -α(4.2) deletion and this fusion gene, and her mother was heterozygous for the common --(SEA) deletion (--(SEA)/αα). Both her parents had a hypochromic and microcytic red cell phenotype and a normal hemoglobin level. Molecular studies revealed a compound heterozygote for the --(SEA) deletion and this novel fusion gene and the patient had the clinical features of classic hemoglobin H disease. Sequence analysis revealed that the mutant gene was the result of a fusion between the α2 and ψα1 genes. The recombination began at exon 3 of α2 gene, crossing with exon 3 of the ψα1 gene. With this recombination, the conservative 3'UTR of the α2 gene was changed, and an extensive transcript with a new signal 1048bp 3' to the terminating codon was found. The abnormal transcripts of the fusion gene read through the intergenic sequence.

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.

Rapid determination of human globin chains using reversed-phase high-performance liquid chromatography.

Reversed-phase high-performance liquid chromatography (RP-HPLC) of human globin chains is an important tool for detecting thalassemias and hemoglobin variants. The challenges of this method that limit its clinical application are a long analytical time and complex sample preparation. The aim of this study was to establish a simple, rapid and high-resolution RP-HPLC method for the separation of globin chains in human blood. Red blood cells from newborns and adults were diluted in deionized water and injected directly onto a micro-jupiter C18 reversed-phase column (250 mm × 4.6 mm) with UV detection at 280 nm. Under the conditions of varying pH or the HPLC gradient, the globin chains (pre-ß, ß, δ, α, (G)γ and (A)γ) were denatured and separated from the heme groups in 12 min with a retention time coefficient of variation (CV) ranging from 0.11 to 1.29% and a peak area CV between 0.32% and 4.86%. Significant differences (P<0.05) among three groups (normal, Hb H and ß thalassemia) were found in the area ratio of α/pre-ß+ß applying the rapid elution procedure, while P≥0.05 was obtained between the normal and α thalassemia silent/trait group. Based on the ANOVA results, receiver operating characteristic (ROC) curve analysis of the δ/ß and α/pre-ß+ß area ratios showed a sensitivity of 100.0%, and a specificity of 100.0% for indicating ß thalassemia carriers, and a sensitivity of 96.6% and a specificity of 89.6% for the prediction of hemoglobin H (Hb H) disease. The proposed cut-off was 0.026 of δ/ß for ß thalassemia carriers and 0.626 of α/pre-ß+ß for Hb H disease. In addition, abnormal hemoglobin hemoglobin E (Hb E) and Hb Westmead (Hb WS) were successfully identified using this RP-HPLC method. Our experience in developing this RP-HPLC method for the rapid separation of human globin chains could be of use for similar work.

A quantitative assay to detect α-thalassemia deletions and triplications using multiplex nested real-time quantitative polymerase chain reaction.

Increasing evidence indicates that copy number variants (CNVs) have great relevance to common human diseases. In α-thalassemia, clinical phenotypes are related to genotypes, specifically copy number changes in the human α-globin gene cluster. Assays are available for high-throughput screening of unknown CNVs genome-wide and also for targeted CNV genotyping at loci associated with genetic disorders. Here we describe a universal quantitative approach based on nested real-time quantitative polymerase chain reaction for accurate determination of copy numbers at multiple particular gene loci. We used the α-globin gene as a model system, obtaining the reproducibility and sensitivity to analyze different gene copies and testing 95 DNA samples with 16 different known genotypes. Our results showed that this approach has high sensitivity and low standard deviations for correctly genotyping DNA samples containing different copy numbers of the α1 and α2 globin genes. Our method is rapid, simple, and reliable, and it could be used to simultaneously screen for α-thalassemia deletions or triplications. Moreover, it has potential as a versatile technology for the rapid genotyping of known CNVs in a targeted region.