Multiplex ligation-dependent probe amplification assay for diagnosis of congenital adrenal hyperplasia

Mutations in the CYP21A2 gene encoding the 21-hydroxylase enzyme account for >90% of congenital adrenal hyperplasia (CAH) cases. Approximately 20% of mutant alleles carrying large deletion/duplication have also been reported. Herein, we describe the use of the multiplex ligation-dependent probe amplification (MLPA) method for convenient and rapid detection of deletions/duplications in the CYP21A2 gene. We used MLPA to analyze the gene dose of CYP21A2 MLPA in 13 Korean patients who previously underwent direct sequencing for the molecular diagnosis of CAH. The MLPA assays identified 5 patients with CYP21A2 deletions; all 5 patients carried a single mutant allele peak in sequence analysis. These results demonstrate the diagnostic usefulness of MLPA to detect CYP21A2 deletions/duplications for diagnosis of CAH.     

Multiplex ligation-dependent probe amplification (MLPA) assay for the detection of mitochondrial DNA deletion in chronic progressive external ophthalmoplegia (CPEO)

Chronic progressive external ophthalmoplegia (CPEO) is a mitochondrial myopathy commonly caused by deleterious changes in the mitochondrial DNA (mtDNA). We describe a 45-year-old man who was referred to us for investigation of progressive ptosis. We performed a multiplex ligation-dependent probe amplification (MLPA) assay of mtDNA from muscle tissue and peripheral blood leukocytes, and followed up with gap-polymerase chain reaction (PCR) and direct sequence analysis. Results showed a deletion of a 4,407 bp segment in the mtDNA region, ranging from nucleotide position 8,577 in the MT-ATP6 gene to nucleotide position 12,983 in the MT-ND5 gene. To the best of our knowledge, this is the first report of a CPEO patient with a large novel deletion of mtDNA genetically confirmed by MLPA assay. MLPA can be a feasible platform for clinical laboratories to detect large deletion mutations in the mtDNA for suspected cases.     

Gender-related difference in left ventricular diastolic elastance during exercise in patients with diabetes mellitus

Background Because the ratio of mitral inflow and annular velocity to stroke volume has been reported as an index of diastolic elastance (Ed), the hypothesis tested in the present study was that Ed during exercise would be more abnormal in female than in male patients with type 2 diabetes. Methods and Results Ed was measured at rest and during graded supine bicycle exercise (25W, 3-min increments) in 53 patients (27 males, mean age 53+/-14 years) with type 2 diabetes and 53 age- and gender-matched controls. The patients with diabetes were divided into 2 groups by gender. Ed was not significantly different at rest between men and women, but was significantly higher during exercise in women than in men (25 W, 0.15+/-0.04 vs 0.20+/-0.07, p=0.009; 50 W, 0.16+/-0.05 vs 0.21+/-0.08, p=0.0175). Conclusion Left ventricular (LV) diastolic elastance is abnormal during exercise, but not at rest, in patients with diabetes without overt heart disease. Female gender was associated with increased LV stiffness during exercise among patients with type 2 diabetes. (Circ J 2008; 72: 1443 - 1448).     

Multiplex ligation-dependent probe amplification screening of isolated increased HbF levels revealed three cases of novel rearrangements/deletions in the β-globin gene cluster

Investigations of naturally occurring mutations, such as the deletional thalassaemias and hereditary persistence of fetal haemoglobins (HPFHs), have brought many insights into human globin switching, but limited data have been reported so far. We selected 15 individuals with elevated fetal haemoglobin (HbF) levels (>5%) from a previous screening of 27 006 Korean individuals and analysed dosage changes of the globin gene cluster using multiplex ligation-dependent probe amplification (MLPA). Dosage changes detected by the MLPA probes were followed up with gap-polymerase chain reaction and sequence analysis. Three subjects were found to have deletions in the globin gene cluster, including a β-thalassaemia due to deletion of HBB (β-globin gene), an HPFH due to deletions of HBD (δ-globin gene) and HBB , and an HPFH due to a novel HBG2–HBG1 fusion gene consisting of exons 1 and 2 of HBG2 (^Gγ-globin gene) and exon 3 of HBG1 (^Aγ-globin gene). The case with the HBG2–HBG1 fusion suggested the existence of another mechanism for the reactivation of HBG2 and HBG1 . The IVS2 of HBG2 and HBG1 might play a role in HbF regulation, and combinations of specific polymorphisms could influence the reactivation of these genes in adults.     

Molecular screening of the TSH receptor (TSHR) and thyroid peroxidase (TPO) genes in Korean patients with nonsyndromic congenital hypothyroidism

Objective To investigate thyroid‐stimulating hormone receptor (TSHR) and thyroid peroxidase (TPO) mutations in Korean patients with primary congenital hypothyroidism (CH). Context Congenital hypothyroidism is a common genetic disorder in which the majority of mutations occur in the TSHR and TPO genes. Design We examined the frequencies of TSHR and TPO mutations among Korean patients with primary CH. Furthermore, we explored the relationships between imaging findings and mutation status. Patients A total of 193 paediatric patients with nonsyndromic CH were enrolled in the present study. Measurements Patients with decreased ^99mTc uptake were screened for TSHR mutations using Sanger sequencing, and those with increased uptake were screened for TPO mutations. The relationships between scintigraphic and ultrasonographic findings and mutation status were analysed. Results Thirteen (16·5%) of 79 patients with decreased ^99mTc uptake were found to harbour TSHR mutations including G132R, G245S, R450H, R519C and F525S. The R450H mutation was present in 13 (72·2%) of 18 disease alleles. Seven (10·3%) of 68 patients with increased ^99mTc uptake harboured TPO mutations including R189Q, K439E, G493S, C808LfsX72, A863T, R875Hfs and P883S. The TSHR and TPO mutations were observed only in patients with normal to slightly enlarged thyroid glands. Conclusions This study identified underlying TSHR and TPO mutations in Korean patients with CH and revealed a possible relationship between imaging findings and mutation status. In addition, the low rate of mutation positivity suggests significant genetic heterogeneity of CH in the Korean population.     

Left ventricular remodeling can be predicted with left ventricular volume response during dobutamine echocardiography after acute myocardial infarction

OBJECTIVES: This study was performed to evaluate the significance of left ventricular (LV) volume response during dobutamine stress echocardiography (DSE) in the prediction of LV volume change during follow-up (F/U) in patients with acute myocardial infarction (AMI). METHODS: Forty-five patients with AMI (male 39, age 57+/-10 y, anterior myocardial infarction [MI] 29) underwent DSE 6+/-4 d after AMI. Revascularization of the infarct-related artery was performed if severe stenosis was present. A F/U echocardiography was performed 7.5+/-3.4 mo after DSE. The LV end-diastolic volume (EDV) and end-systolic volume (ESV) using the modified Simpson's method were measured at baseline echocardiography, low-dose (10 microg x kg(-1) x min(-1)) DSE, and F/U echocardiography. RESULTS: Patients were divided into 2 groups; Group I (n = 21) with an abnormal response (<10% decrease) in LVEDV during low-dose DSE; Group II (n = 24) with a normal response (> or =10% decrease) in LVEDV during low-dose DSE. At F/U echocardiography, the (%) change of LVEDV was significantly different between the 2 groups (-2.0+/-16.7 versus - 22.6+/-24.7%, p<0.01). Using multivariate analysis, the response of LVEDV (%) at low-dose DSE was the only significant independent predictor of the change of LVEDV (%) during F/U (y = 0.85 x - 0.03, r = 0.63, p<0.001). CONCLUSIONS: The response of LVEDV during DSE can be used as a predictor for the LV volume change after AMI.     

Is albuminuria an indicator of myocardial dysfunction in diabetic patients without overt heart disease? A study with Doppler strain and strain rate imaging

The aim of this study was to address whether albuminuria could predict myocardial dysfunction in diabetic patients without overt heart disease. We studied 67 patients with normal left ventricular (LV) ejection fraction and no evidence of LV hypertrophy or coronary artery disease (47 patients with type 2 diabetes mellitus and hypertension and 20 patients with hypertension only). Diabetes patients were divided into 3 groups based on albuminuria status: group II = no albuminuria (n = 20, <30 mg/d), group III = microalbuminuria (n = 13, 30-300 mg/d), and group IV = macroalbuminuria (n = 14, >300 mg/d). Twenty patients with hypertension only served as a control group (group I). Conventional 2-dimensional and Doppler echocardiography was done. Peak strain, peak systolic strain rate (SR), and peak diastolic SR of 6 LV segments in the apical views were measured and averaged in each patient. Conventional 2-dimensional parameters such as LV ejection fraction; left atrium volume index; LV mass; deceleration time; and mitral early peak, mitral late peak, myocardial early peak diastolic, and myocardial peak systolic velocities were not different among the 4 groups. However, peak strains were significantly lower in group III (P = .002) and group IV (P < .001) than in group I; and the absolute value of peak systolic SR was lower in group III (P = .033) and group IV (P < .001) than in group I. Furthermore, the value of peak diastolic SR was lower in group IV than in group I (P = .014). In diabetic patients with albuminuria, Doppler strain and SR imaging detected subclinical LV systolic and diastolic dysfunction; and albuminuria was associated with myocardial dysfunction in diabetic patients without overt heart disease.     

Genetic polymorphisms of NAT2 and CYP2E1 associated with antituberculosis drug-induced hepatotoxicity in Korean patients with pulmonary tuberculosis

Antituberculosis drug-induced hepatitis attributed to isoniazid (INH) is one of the most prevalent drug-induced liver injuries. INH is metabolized by hepatic N-acetyltransferase (NAT) and cytochrome P450 2E1 (CYP2E1) to form hepatotoxins. The aim of this study was to evaluate whether polymorphisms of the NAT2 and/or CYP2E1 genes were associated with antituberculosis drug-induced hepatotoxicity in Korean patients. A total of 132 patients with tuberculosis who received antituberculosis treatment were followed prospectively. Their NAT2 and CYP2E1 genotypes were determined using polymerase chain reaction (PCR) with or without sequencing. Eighteen (13.6%) patients developed antituberculosis drug-induced hepatotoxicity. Regarding NAT2, slow acetylators had a higher incidence of hepatotoxicity than rapid acetylators (36.8% vs. 9.7%, P=0.005) and there was a 3.8-fold risk of hepatotoxicity for the slow acetylators compared to the rapid acetylators. For the CYP2E1 gene, the RsaI polymorphism in the 5' untranslated region, and a polymorphic repetitive sequence at the CYP2E1 5'-flaking region were analyzed; there was no significant association between any CYP2E1 genotype and antituberculosis drug-induced hepatotoxicity. In conclusion, slow acetylator status of NAT2 was a significant susceptibility risk factor for antituberculosis drug-induced hepatotoxicity; NAT2 genotyping may be a useful tool for predicting antituberculosis drug-induced hepatotoxicity.     

Sequestering carbon dioxide into complex structures of naturally occurring gas hydrates

Large amounts of CH4 in the form of solid hydrates are stored on continental margins and in permafrost regions. If these CH4 hydrates could be converted into CO2 hydrates, they would serve double duty as CH4 sources and CO2 storage sites. We explore here the swapping phenomenon occurring in structure I (sI) and structure II (sII) CH4 hydrate deposits through spectroscopic analyses and its potential application to CO2 sequestration at the preliminary phase. The present 85% CH4 recovery rate in sI CH4 hydrate achieved by the direct use of binary N2+CO2 guests is surprising when compared with the rate of 64% for a pure CO2 guest attained in the previous approach. The direct use of a mixture of N2+CO2 eliminates the requirement of a CO2 separation/purification process. In addition, the simultaneously occurring dual mechanism of CO2 sequestration and CH4 recovery is expected to provide the physicochemical background required for developing a promising large-scale approach with economic feasibility. In the case of sII CH4 hydrates, we observe a spontaneous structure transition of sII to sI during the replacement and a cage-specific distribution of guest molecules. A significant change of the lattice dimension caused by structure transformation induces a relative number of small cage sites to reduce, resulting in the considerable increase of CH4 recovery rate. The mutually interactive pattern of targeted guest-cage conjugates possesses important implications for the diverse hydrate-based inclusion phenomena as illustrated in the swapping process between CO2 stream and complex CH4 hydrate structure.