Indra Sari Kusuma Harahap

Valproic acid increases SMN2 expression and modulates SF2/ASF and hnRNPA1 expression in SMA fibroblast cell lines

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder that is caused by loss of the survival motor neuron gene, SMN1. SMA treatment strategies have focused on production of the SMN protein from the almost identical gene, SMN2. Valproic acid (VPA) is a histone deacetylase inhibitor that can increase SMN levels in some SMA cells or SMA patients through activation of SMN2 transcription or splicing correction of SMN2 exon 7. It remains to be clarified what concentration of VPA is required and by what mechanisms the SMN production from SMN2 is elicited. We observed that in two fibroblast cell lines from Japanese SMA patients, more than 1mM of VPA increased SMN2 expression at both the transcript and protein levels. VPA increased not only full-length (FL) transcript level but also exon 7-excluding (Δ7) transcript level in the cell lines and did not change the ratio of FL/Δ7, suggesting that SMN2 transcription was mainly activated. We also found that VPA modulated splicing factor expression: VPA increased the expression of splicing factor 2/alternative splicing factor (SF2/ASF) and decreased the expression of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1). In conclusion, more than 1mM of VPA activated SMN2 transcription and modulated the expression of splicing factors in our SMA fibroblast cell lines.

A polymorphic mutation, c.-3279T > G, in the UGT1A1 promoter is a risk factor for neonatal jaundice in the Malay population.

The uridine diphosphoglucuronate-glucuronosyltransferase 1A1 (UGT1A1) gene encodes the enzyme responsible for bilirubin glucuronidation. To evaluate the contribution of UGT1A1 promoter mutations to neonatal jaundice, we determined the genotypes of c.-3279T>G, c.-3156G>A, and A(TA)7TAA in Malay infants with neonatal jaundice (patients) and in infants without neonatal jaundice (controls). In our population study, only c.-3279T>G was associated with neonatal jaundice. The genotype distributions between both groups were significantly different (p = 0.003): the frequency of homozygosity for c.-3279G was much higher in patients than those in controls. Allele frequency of c.-3279G was significantly higher in patients than those in controls (p = 0.006). We then investigated changes in transcriptional activity because of c.-3279T>G. Luciferase reporter assay in HepG2 cells demonstrated that transcriptional activity of the c.-3279G allele was significantly lower than that of the c.-3279T allele in both the absence and presence of bilirubin. Luciferase reporter assay in COS-7 cells elucidated that c.-3279T>G modified the synergistic effects of the nuclear factors associated with transcriptional machinery. In conclusion, the c.-3279T>G mutation in the UGT1A1 promoter is a genetic risk factor for neonatal jaundice.

Spinal Muscular Atrophy Patient Detection and Carrier Screening Using Dried Blood Spots on Filter Paper

AIM Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder. It is caused by mutations in the SMN1, and its clinical severity is modified by copy number variations of the SMN2. According to previous studies, deletion of SMN1 exon 7 is the most frequently observed in patients with SMA. Therefore, molecular analyses exploiting this genetic lesion could be beneficial in the diagnosis of SMA. Unfortunately, in many geographical regions, physicians do not have the latest molecular screening technologies at their immediate disposal. Thus, to overcome this issue, we developed an SMA-diagnosing system using dried blood spots (DBS) placed on filter paper to facilitate remote diagnosis. METHODS In this study, we validate the applicability of DBS on Flinders Technology Associates (FTA) filter paper for detecting SMN1 exon 7 deletions and copy number variations of SMN1 and SMN2. To detect exon 7 deletions in SMN1, polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis was conducted by using DNA extracted from the DBS on FTA filter paper that had been stored at room temperature for a period of up to 4 years. To determine the copy numbers of SMN1 and SMN2, we carried out SYBR green-based real-time PCR by using the same blood specimens. RESULTS The results obtained from the DBS on FTA filter paper were in complete concordance with those analyses using fresh blood specimens. This indicates that DBS on filter papers is a reliable method for SMA patient detection and carrier screenings. CONCLUSION The SMA-diagnosing system, combined with the mailing of DBS on filter paper, will be beneficial for patients suffering from neuromuscular disorders in areas with limited or no access to diagnostic facilities with molecular capabilities.

Diagnosis of spinal muscular atrophy via high-resolution melting analysis symmetric polymerase chain reaction without probe: a screening evaluation for SMN1 deletions and intragenic mutations.

AIM Spinal muscular atrophy (SMA) is a well-defined autosomal recessive neuromuscular disorder caused by mutations in the survival motor neuron 1 (SMN1) gene. The most frequently observed mutation is a deletion of exon 7, which has been documented in >95% of SMA patients. A novel technique for detecting mutations known as high-resolution melting analysis (HRMA) has rapidly become the tool of choice for screening pathogenic genetic variants. In the present study, we attempt to validate the applicability of HRMA to the detection of exon 7 deletions and other intragenic mutations in SMN1. RESULTS Three primer sets were adopted in our HRMA screening for deletion of SMN1 exon 7. In screening attempts utilizing two primer sets, the results of HRMA were not compatible with those obtained by polymerase chain reaction-restriction fragment length polymorphism. Therefore, we applied a modified protocol using revised primer sets, which resulted in an absolute compatibility of results between HRMA and polymerase chain reaction-restriction fragment length polymorphism. With regard to screenings for intragenic mutations in SMN1 exon 3, two primer sets were adopted for use in HRMA. In the initial HRMA screening using the first primer set, we failed to identify any intragenic mutations; however, when using a revised primer set, HRMA successfully detected the presence of a c.275G>C mutation. CONCLUSION HRMA is a simple but versatile tool to add to the existing arsenal of diagnostic techniques that could aid clinicians/researchers in diagnosing SMA. However, as we demonstrate in the present study, the design and selection of primers is of monumental importance in ensuring the successful application of HRMA to screening for pathogenic variants.

Generalized epilepsy with febrile seizures plus (GEFS+) spectrum: Clinical manifestations and SCN1A mutations in Indonesian patients

Generalized epilepsy with febrile seizures plus (GEFS+) is a childhood genetic epilepsy syndrome. GEFS+ includes a wide spectrum of clinical manifestations, and SCN1A mutations have frequently been reported among the GEFS+-related gene abnormalities. In this study, to clarify the distributions of the clinical subtypes, we analyzed 34 families with GEFS+ in Indonesia using the hospital records of the patients and questionnaires for the family members. The number of patients with febrile seizures plus (FS+), FS+ and afebrile generalized/partial seizures, borderline severe myoclonic epilepsy in infancy (SMEB) and severe myoclonic epilepsy in infancy (SMEI) were 9, 11, 7, and 7, respectively. Most patients had a family history of febrile seizures. Next, we performed molecular analyses to clarify the contributions of SCN1A mutations to the development of the GEFS+ subtypes. Only 3 of 34 probands showed SCN1A mutations. These mutations were two missense mutations, p.V1612I and p.C1756G, in two patients with SMEI and SMEB, and one silent mutation, p.G1762G, in a patient with FS+ and afebrile partial seizures. In conclusion, the majority of GEFS+ patients in Indonesia were not associated with SCN1A mutations. To detect the GEFS+-causing mutations, we must search and analyze other genes in these patients.

Acetaminophen administration in a patient with Gilbert's syndrome

The patient was an 8‐year‐old Japanese girl with Gilberts syndrome (GS). Based on the DNA analysis, she was homozygous for a T‐to‐G transversion at nucleotide position 1456 in the UGT1A1 gene, leading to the substitution of aspartate for tyrosine at position 486 of the UGT1A1 enzyme. Because this mutation is located in an exon common to UGT1A genes, all the UGT1A enzymes may be affected. It is well‐known that UGT1A1, UGT1A6 and UGT1A9 enzymes glucuronidate acetaminophen. To evaluate acetaminophen tolerance in the patient, serum acetaminophen levels were determined after oral administration of acetaminophen (15 mg/kg). The maximum serum acetaminophen level reached (12.8 µg/mL) was far below the toxic level. The finding suggested that the usual therapeutic dose of acetaminophen is safe for the GS patient. The combination of mutation analysis in UGT1A1 and acetaminophen loading test may be useful to avoid adverse effect in GS patients.

Herbal Medicine Containing Licorice May Be Contraindicated for a Patient with an HSD11B2 Mutation

Licorice ingestion, as well as mutations in the HSD11B2 gene, inhibits 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) enzyme activity, causing the syndrome of apparent mineral corticoid excess (AME). However, the combined effect of licorice ingestion and an HSD11B2 mutation has never been reported, until now. In this study, we demonstrated that licorice ingestion can produce overt hypertension in an individual without medical history of hypertension who is heterozygous for wild-type and mutant HSD11B2 genes. Our patient was a 51-year-old female with serious hypertension who had been taking herbal medicine containing licorice for more than one year. She was clinically diagnosed as having licorice intoxication, because she did not present with hypertension after ceasing the herbal medicine. Molecular analysis showed that she carried a missense mutation, c.40C>T, in HSD11B2. In conclusion, licorice ingestion is an environmental risk factor for hypertension or AME state in patients with a mutation in HSD11B2. Carrying a mutation in HSD11B2 is, conversely, a genetic risk factor for licorice-induced hypertension or AME state. Herbal medicine containing licorice may, therefore, be contraindicated in patients with an HSD11B2 mutation.

Paramyotonia congenita: From clinical diagnosis to in silico protein modeling analysis

Background:  Paramyotonia congenita (PMC) is an autosomal dominant disorder characterized by cold‐ or exercise‐induced myotonia. PMC is caused by a mutation in SCN4A which encodes the α‐subunit of the skeletal muscle sodium channel.

A Study of valproic acid for patients with spinal muscular atrophy

Valproic acid (VPA) is expected to become an effective therapeutic agent for spinal muscular atrophy (SMA) because of its histone deacetylase inhibitor effect.

Valproic Acid Treatment for Spinal Muscular Atrophy Patient with Abnormal Electroencephalogram Findings

Background: Valproic Acid (VPA), an anticonvulsant, is a histone deacetylase inhibitor that can increase the Survival Motor Neuron (SMN) level in Spinal Muscular Atrophy (SMA) patients and anticipated to be an effective therapeutic agent for SMA.Patient description: VPA was administered for SMA to a 6-year-old female who showed non-symptomatic abnormal electroencephalogram findings, as well as SMA signs and symptoms. Although the VPA concentration of our patient remained in a low range, electroencephalogram abnormalities disappeared within 10 months after starting administration. On the other hand, clinical signs and symptoms of SMA, respiratory function values, and modified Hammersmith Functional Motor Scale results showed no remarkable changes. We also analyzed survival motor neuron (SMN) transcripts and splicing factor transcripts, such as splicing factor 2/alternative splicing factor (SF2/ASF) and heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), using quantitative real-time PCR assays performed before and after VPA treatment. The amounts of the FL-SMN, total-SMN, ASF/SF2, and hnRNPA1 transcripts showed an increasing tendency, whereas that of Δ7-SMN was decreased.Conclusion: Our findings indicate a discrepancy between SMN transcript response, and improvements in SMA symptoms and signs following VPA administration in some SMA patients.