Shyh-Shin Chiou

The effect of hydroxyurea in spinal muscular atrophy cells and patients

BACKGROUND Spinal muscular atrophy (SMA) is a degenerative motor neuron disease caused by homozygous mutations of the survival motor neuron 1 (SMN1) gene. Effective treatment for SMA is unavailable at present. The aim of this study was to investigate the effect of hydroxyurea (HU) in SMA cells and patients. MATERIALS AND METHODS Fifteen SMA lymphoid and three fibroblast cell lines, 2 from SMA patients and 1 control, were treated with HU at different concentrations, and 33 patients (types II, III) randomized into three groups on different HU dosage, 20, 30, 40 mg/kg/day, were treated for 8 weeks and followed up for another drug-free 8 weeks. The effect of HU on SMN2 gene expression and clinical manifestations was evaluated. RESULTS After treatment, in vitro, full-length mRNA level and gems number increased significantly, and hnRNP A1 protein decreased. In vivo, there were slight increases in muscle strength scores at 4 weeks and full-length SMN mRNA at 8 weeks in 30 mg/kg/day subgroup. CONCLUSIONS Treating with HU enhanced SMN2 gene expression in SMA cells and showed slight trend towards improvement in some clinical outcome measures in SMA patients which suggests HU may be safe to use in SMA patients but larger randomized, placebo-controlled, double-blind trials are needed to further investigate its efficacy.

Diagnosis of thalassaemia by non‐isotope detection of α/β and ζ/α mRNA ratios

Summary. The α/β and ζ/α messenger RNA (mRNA) ratios in the thalassaemia syndromes were investigated by polymerase chain reaction (PCR) with silver staining of the PCR products. In this study we used the PCR to amplify cDNA copies of circulating erythroid cell mRNA in order to measure the relative amounts of α‐, β‐ and ζ‐globin contained within. Quantitation was performed by scanning the silver stain of specific globin cDNA bands. We found that there were significant differences of α/β‐mRNA and ζ/α‐mRNA in patients with Hb H disease and α‐thalassaemia‐1 compared to normal subjects. There was a marked increase in the α/β‐mRNA ratio but not in the ζ/α‐mRNA ratio in patients with β‐thalassaemia. In two β‐thalassaemia cases abnormal increases of ζ‐globin bands were noted and they were confirmed through DNA analysis to be combined with α‐thalassaemia‐1. This method provides a simple, rapid and non‐radioactive approach to detect thalassaemia syndromes, and can help to screen cases of β‐thalassaemia with α‐thalassaemia‐1.

Demethylation within the proximal promoter region of human estrogen receptor alpha gene correlates with its enhanced expression: Implications for female bias in lupus

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease primarily affecting women. Previous studies have indicated that sex hormone estrogens contribute to the female predilection of SLE. Estrogen regulates gene expression by translocating estrogen receptors (ER) α and β into the nucleus where they induce transcription by binding to estrogen response elements of target genes. We have previously observed that expression of ERα gene and protein in lupus patients is significantly higher than in healthy controls and that estradiol up-regulates calcineurin expression via over-expression of ERα gene in SLE. However, the pathogenesis of over-expression of ERα gene is unknown. Here we report that enhanced expression of ERα mRNA and protein in SLE and rheumatoid arthritis is associated with DNA demethylation within the proximal promoter region located between -232 and +81 base pair relative to transcription start site of human ERα gene (GenBank Accession no. AL356311.6). The frequency of DNA demethylation was comparable between male and female. These findings suggest that estrogen and demethylated ERα promoter associated up-regulated ERα genes are two critical factors in the gender biased development of autoimmune diseases besides genetic factor.

Molecular basis and haematological characterization of β-thalassaemia major in Taiwan, with a mutation of IVS-1 3' end TAG→GAG in a Chinese patient

Summary We studied 41 patients with β‐thalassaemia major and their parents by using a combination of polymerase chain reaction (PCR) amplification, slot‐blot hybridization of allele‐specific oligonucleotide (ASO), and direct genomic sequencing. Eight different point mutations were characterized. C to T substitution at nucleotide (nt) 654 of intervening sequences (IVS) 2, accounting for 46.3% of mutant β‐globin genes, is the most common mutation in Taiwan, followed by frameshift codons 41/42 with four nucleotides (TCTT) deletion for 31.7%, A to G substitution at position −28 of promotor area for 8.5%, A to T substitution at codon 17 for 6.1%, frameshift codons 27/28 (insertion of C) for 2.4%, G to T substitution at nucleotide 1 of IVS‐1 for 2.4%, frameshift codons 71/72 (insertion of A) and IVS‐1 3’end TAG→GAG for 1.2%. The former four mutations showed no obvious difference between two major ethnic groups in Taiwan. As to mutations in each individual of β‐thalassaemia major, the incidence of compound heterozygotes of two different mutations is much higher than homozygotes of single mutation, 78.0%v 22.0%. Compound heterozygotes of C to T substitution at nt 654 of IVS‐2 and frameshift codons 41/42 with four nucleotides deletion is the most common pattern of β‐thalassaemia mutations in each individual (41.5%). The results are somewhat different from other documented reports concerning the mutations of β‐thalassaemia in southern China. This is the first report of mutation of IVS‐1 3’end TAG→GAG which causes consensus change in Chinese people. Patients with heterozygotes of β° and –28 β+‐thalassaemia mutations would have a greater delay in initial transfusion in comparison to patients with homozygotes of both β°‐thalassaemia mutation, but their initial clinical manifestation might be aggravated when combined with a glucose‐6‐phosphate dehydrogenase (G‐6‐PD) deficiency and an insult such as exposure to infection and certain drugs.

Survival of motor neuron protein downregulates miR-9 expression in patients with spinal muscular atrophy

Spinal muscular atrophy (SMA) is a lethal hereditary disease caused by homozygous absence of the survival of the motor neuron (SMN) 1 gene (SMN1), and it is the leading genetic cause of infant mortality. The severity of SMA is directly correlated with SMN protein levels in affected patients; however, the cellular regulatory mechanisms for SMN protein expression are not completely understood. In this study, we investigated the regulatory effects between SMN expression and miR‐9a, a downstream noncoding small RNA. Using an inducible SMN short hairpin RNA interference (shRNAi) system in NSC 34 and human skin fibroblast cells, cellular miR‐9 levels and SMN protein repression were time‐dependently upregulated. Conversely, cellular miR‐9 levels decreased when HeLa cells were transfected with SMN protein fused with green fluorescent protein. In SMA‐like mice spinal cords and human primary skin fibroblasts isolated from patients with different degrees of SMA, human SMN exhibited a disease severity‐dependent decrease, whereas cellular miR‐9 levels increased. These results clearly suggested that cellular SMN proteins regulated miR‐9 expression and that miR‐9 expression was related to SMA severity. Thus, miR‐9 may be a marker for SMA prognosis.

Rapid identification of the copy number of α-globin genes by capillary electrophoresis analysis.

OBJECTIVES The current study aimed at the rapid identification of the copy number of α-globin genes for the diagnosis of α-thalassemia. DESIGN AND METHODS To identify the copy number of α-globin genes in α-thalassemia, we developed a novel method using a multiplex polymerase chain reaction (PCR) in combination with the CE analysis. RESULTS The proposed method provides a rapid detection of the common α-globin gene deletions. Sixty-six patients with α-thalassemia and 46 normal controls were included in the present study. The obtained results showed good correlation with those obtained by gap PCR. Moreover, a low amount of maternal cell contamination in the fetus specimen for the prenatal diagnosis of hemoglobin Barts hydrops fetalis as well as the rare multiplicated α-globin genes can be identified using this method. CONCLUSION This method provides a convenient and efficient tool for the rapid identification of the copy number of α-globin genes in α-thalassemia and the individuals with α-globin gene multiplication.

Whole-exome sequencing for the genetic diagnosis of congenital red blood cell membrane disorders in Taiwan

PURPOSE Congenital hemolytic anemia caused by red blood cell (RBC) membrane defects is a heterogeneous group of disorders. The present study aimed to search the causative gene mutations in patients with RBC membrane disorders in Taiwan. MATERIALS AND METHODS Next-generation sequencing approach using whole-exome sequencing (WES) was performed. Sanger sequencing was performed for confirmation of variants detected in WES in patients and their family members. RESULTS Five causative variants, including two ANK1, two SPTA and one SPTB variants, were detected in four patients. All these variants, except one SPTA1 variant c.83G > A (p.R28H), are novel variants. Their pedigree analysis showed one de novo SPTA1 mutation c.83G > A (p.R28H) combined with αLELY, one de novo ANK1 mutation c.1034C > A (p.A345E), one autosomal dominant combined SPTA1 c.4604A > C (p.Q1535P) and SPTB c.6203 T > C (p.L2068P) mutations and one autosomal dominant ANK1 c.4462C > T (p.R1488X) mutation. CONCLUSIONS Our data demonstrated that WES is an efficient tool for determining genetic etiologies of RBC membrane disorders and can facilitate accurate diagnosis and genetic counseling. Additional studies should be conducted on larger cohorts to investigate the distribution of gene mutations in patients with RBC membrane disorders in Taiwan.

Recurrent spinal primitive neuroectodermal tumor with brain and bone metastases: A case report

Rationale: Primary spinal primitive neuroectodermal tumor (PNET) is relatively rare in all age groups, and the prognosis in most cases of spinal PNETs appears to be poor, with a median patient survival of 1 to 2 years. We present a case with recurrent spinal PNET with brain and bone metastases that was successfully treated by multimodality treatment. Patient concerns: A 14-year-old teenage girl had suffered from progressive left upper back pain with bilateral lower legs weakness and numbness for 1 year. After treatment, left neck mass was noted 3 years later. Diagnoses: Initially, magnetic resonance imaging (MRI) showed neurogenic tumor involving intradural extramedullary space of T5-T10. Pathology report showed PNET (World Health Organization grade IV) featuring lobules of neoplastic cells with round regular nuclei, high nucleus-to-cytoplasm ratio, and fibrillary cytoplasm. At the time of tumor recurrence, chest MRI then showed recurrent tumor at T2-T3 level of the epidural space with right neural foramina invasion. Brain MRI showed extensive bilateral calvarial metastases and leptomeningeal metastases in the right frontoparietal regions. Bone scan showed multiple bone metastases. Interventions: T-spine tumor removal and adjuvant radiotherapy (RT) to T-spine tumor bed were performed in the initial treatment. After clinical tumor recurrence, tumor removal was done again. She then received chemotherapy followed by whole brain irradiation with hippocampal sparing with 35 gray in 20 fractions. Outcomes: After treatment, follow-up images showed that the disease was under control. There was no neurological sequela. She has survived more than 7 years from diagnosis and more than 4 years from recurrence to date. Lessons: Multimodality treatments including operation, RT, and chemotherapy should be considered in the initial treatment planning, and salvage chemotherapy was useful in this case.