The impact of renin–angiotensin system,angiotensin І converting enzyme (insertion/deletion), and angiotensin ІІ type 1 receptor (A1166C) polymorphisms on breast cancer survival in Iran

Introduction

Several proteins of renin–angiotensin system (RAS) have been implicated in the process of growth promotion or inhibition of breast tissue and cancer cells. This study aimed to investigate the association between angiotensin I converting enzyme (ACE) insertion/deletion (I/D) and angiotensin receptor-1 (AGTR1) A1166C polymorphisms and survival of 110 women with breast cancer.

Materials and methods

The I/D and A1166C polymorphisms were evaluated by using Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP) in 110 breast cancer patients who had been treated between 2007 and 2009. Genomic DNA was extracted from a Formalin-Fixed Paraffin-Embedded (FFPE) tissue of breast cancer sample blocks. All the potential clinical and pathological prognostic variables were analyzed to establish the impact of I/D and A1166C polymorphisms on disease-free and overall survival rates. Disease-free and overall survival rates were the primary endpoints of the study.

Results

The ACE (I/D) polymorphism was associated with 3-year disease-free survival. Disease-free survival in DD carriers was significantly increased compared to ID plus II carriers (HR = 4.75; 95% CI, 1.39–16.24; p = 0.013). No significant association was found between AGTR1 (A1166C) and 3-year disease-free survival (p = 0.233). Also, the ACE (I/D) and AGTR1 (A1166C) polymorphisms were not associated with breast cancer overall survival.

Conclusion

The ACE (I/D) polymorphism was associated with 3-year disease-free survival of the women with breast cancer. Besides, disease-free survival in DD carriers was significantly increased compared to ID plus II carriers.     

Involvement of 1,25D3-MARRS (membrane associated, rapid response steroid-binding), a novel vitamin D receptor, in growth inhibition of breast cancer cells

In addition to classical roles in calcium homeostasis and bone development, 1,25 dihydroxyvitamin D₃ [1,25(OH)₂D₃] inhibits the growth of several cancer types, including breast cancer. Although cellular effects of 1,25(OH)₂D₃ traditionally have been attributed to activation of a nuclear vitamin D receptor (VDR), a novel receptor for 1,25(OH)₂D₃ called 1,25D₃-MARRS (membrane-associated, rapid response steroid-binding) protein was identified recently. The purpose of this study was to determine if the level of 1,25D₃-MARRS expression modulates 1,25(OH)₂D₃ activity in breast cancer cells.Relative levels of 1,25D₃-MARRS protein in MCF-7, MDA MB 231, and MCF-10A cells were estimated by real-time RT-PCR and Western blotting. To determine if 1,25D₃-MARRS receptor was involved in the growth inhibitory effects of 1,25(OH)₂D₃ in MCF-7 cells, a ribozyme construct designed to knock down 1,25D₃-MARRS mRNA was stably transfected into MCF-7 cells. MCF-7 clones in which 1,25D₃-MARRS receptor expression was reduced showed increased sensitivity to 1,25(OH)₂D₃ ( IC₅₀ 56 ± 24 nM) compared to controls (319 ± 181 nM; P < 0.05). Reduction in 1,25D₃-MARRS receptor lengthened the doubling time in transfectants treated with 1,25(OH)₂D₃. Knockdown of 1,25D₃-MARRS receptor also increased the sensitivity of MCF-7 cells to the vitamin D analogs KH1060 and MC903, but not to unrelated agents (all-trans retinoic acid, paclitaxel, serum/glucose starvation, or the isoflavone, pomiferin). These results suggest that 1,25D₃-MARRS receptor expression interferes with the growth inhibitory activity of 1,25(OH)₂D₃ in breast cancer cells, possibly through the nuclear VDR. Further research should examine the potential for pharmacological or natural agents that modify 1,25D₃-MARRS expression or activity as anticancer agents.     

Fraxinus excelsior seed extract FraxiPure™ limits weight gains and hyperglycemia in high-fat diet-induced obese mice

Purpose

The aim of this study was to determine whether a Fraxinus excelsior L. seed extract, FraxiPure™ (0.5% in the diet), limits weight gain and hyperglycemia in mice. In a previous report, we identified several secoiridoids in FraxiPure™, some of which activated peroxisome proliferator-activated receptor alpha (PPARα) in vitro and inhibited the differentiation of 3T3-L1 preadipocyte cells. In a separate study, FraxiPure™ reduced glycemia in healthy volunteers, following an oral glucose tolerance test. These findings suggest that FraxiPure™ has antiobesity and antihyperglycemia effects.

Materials and methods

FraxiPure™ was tested in mice that were fed a high-fat diet over 16 weeks and compared with low-fat and high-fat diet controls. Weight gain, omental and retroperitoneal fat, fasting blood glucose, and fasting blood insulin were measured.

Results

FraxiPure™ reduced gains in body weight by 32.30% (p < 0.05), omental fat by 17.92%, and retroperitoneal fat by 17.78%. FraxiPure™ also lowered fasting blood glucose levels by 76.52% (p < 0.001) and plasma insulin levels by 53.43% (p < 0.05) after 16 weeks. Moreover, FraxiPure™ lowered liver weight gains by 63.62% (p < 0.05) and the incidence of fatty livers by 66.67%.

Conclusions

Our novel results demonstrate the antiobesity effects of chronic administration of an F. excelsior seed extract and confirm its ability to regulate glycemia and insulinemia. In addition, this extract, which is rich in secoiridoid glucosides, protects against obesity-related liver steatosis.     

Protective effect of tetramethylpyrazine isolated from Ligusticum chuanxiong on nephropathy in rats with streptozotocin-induced diabetes

Purpose

This study was designed to investigate the protective effect of tetramethylpyrazine isolated from Ligusticum chuanxiong, a traditional Chinese medicine, on diabetic nephropathy in a rat model, and to explore the possible mechanism involved in a protective function.

Materials

Diabetes was induced in male Sprague-Dawley rats by a single intraperitoneal injection of 70 mg/kg of streptozotocin. One week later, 200 mg/kg/day of tetramethylpyrazine was administered intragastric gavage daily for 8 weeks. Renal functions and expression of vascular endothelial growth factor were examined at 4 and 8 weeks after tetramethylpyrazine administration.

Results

Blood glucose and renal function were significantly improved in the tetramethylpyrazine-treated group compared to the untreated diabetic rats. Diabetic nephropathy resulted in an increase in the expression of vascular endothelial growth factor, while tetramethylpyrazine administration greatly decreased the expression.

Conclusions

Our results suggest that administration of tetramethylpyrazine may reduce kidney damage caused by diabetes. This protective effect may be mediated, in part, by downregulated expression of vascular endothelial growth factor in the kidney.     

The sedimentation properties of ferritins. New insights and analysis of methods of nanoparticle preparation

Background

Ferritin exhibits complex behavior in the ultracentrifuge due to variability in iron core size among molecules. A comprehensive study was undertaken to develop procedures for obtaining more uniform cores and assessing their homogeneity.

Methods

Analytical ultracentrifugation was used to measure the mineral core size distributions obtained by adding iron under high- and low-flux conditions to horse spleen (apoHoSF) and human H-chain (apoHuHF) apoferritins.

Results

More uniform core sizes are obtained with the homopolymer human H-chain ferritin than with the heteropolymer horse spleen HoSF protein in which subpopulations of HoSF molecules with varying iron content are observed. A binomial probability distribution of H- and L-subunits among protein shells qualitatively accounts for the observed subpopulations. The addition of Fe²⁺ to apoHuHF produces iron core particle size diameters from 3.8 ± 0.3 to 6.2 ± 0.3 nm. Diameters from 3.4 ± 0.6 to 6.5 ± 0.6 nm are obtained with natural HoSF after sucrose gradient fractionation. The change in the sedimentation coefficient as iron accumulates in ferritin suggests that the protein shell contracts ∼ 10% to a more compact structure, a finding consistent with published electron micrographs. The physicochemical parameters for apoHoSF (15%/85% H/L subunits) are M = 484,120 g/mol, ν? = 0.735 mL/g, s_20,w = 17.0 S and D_20,w = 3.21 × 10⁻⁷ cm²/s; and for apoHuHF M = 506,266 g/mol, ν? = 0.724 mL/g, s_20,w = 18.3 S and D_20,w = 3.18 × 10⁻⁷ cm²/s.

Significance

The methods presented here should prove useful in the synthesis of size controlled nanoparticles of other minerals.     

Serum steroid profiling by isotopic dilution-liquid chromatography-mass spectrometry: comparison with current immunoassays and reference intervals in healthy adults

Background

The simultaneous, rapid and reliable measurement of a wide steroid panel is a powerful tool to unravel physiological and pathological hormone status. Clinical laboratories are currently dominated by high-throughput immunoassays, but these methods lack specificity due to cross-reactivity and matrix interferences. We developed and validated an isotopic dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method for the simultaneous measurement of cortisol, corticosterone, 11deoxycortisol, androstenedione, deoxycorticosterone (DOC), testosterone, 17OHprogesterone, dehydroepiandrosterone (DHEA) and progesterone in serum, and compared it to routine immunoassays employed in our laboratory. We also established adult reference intervals in 416 healthy subjects.

Methods

0.9 ml of serum were spiked with labelled internal standards (IS) and extracted on C18 cartridges. Eluate was injected into a two-dimensional LC-system, purified in a perfusion column and separated on a C8 column during a 21 min gradient run. Analytes were revealed by atmospheric pressure chemical ionization (APCI) followed by multiple reaction monitoring (MRM) analysis.

Results

Of the four immunoassays compared with the ID-LC-MS/MS method, only the results of ElecsysE170 for cortisol, testosterone in males and progesterone > 1 ng/ml were in agreement with ID-LC-MS/MS. ElecsysE170 for testosterone in females and progesterone < 1 ng/ml, Immulite2000 for androstenedione, DSL-9000 for DHEA and 17OHP Bridge for 17OHprogesterone, respectively, showed poor agreement. Reference intervals and steroid age and fertility related fluctuations were established.

Conclusion

Our ID-LC-MS/MS method proved to be reliable and sensitive in revealing steroid circulating concentrations in adults and in highlighting the limits of routine immunoassays at low concentrations.     

Pharmacological mechanism responsible for the Atractylodes japonica-induced distal colonic contraction in rats

Background and aim

Atractylodes japonica Koidz (Compositae) has been commonly used to treat the gastrointestinal (GI) disorders in Korean traditional medicine, but its pharmacological roles in the regulation of GI motility have not been clarified yet.

Methods

Atractylodes japonica was sequentially partitioned with MeOH, n-hexane, CHCl₃, EtOAc and n-BuOH saturated with H₂O, and the effects of Atractylodes japonica extracts on the spontaneous contractility of GI muscle strips prepared from rats were measured.

Results

Among five different fractionations, EtOAc extracts of Atractylodes japonica (AJEA) dose-dependently increased the low frequency contraction of distal colon longitudinal muscles (DCLM), and the ED₅₀ values were revealed to be 1.71 × 10⁻⁹ g/ml. Among GI tracts, a prominent contractile response to AJEA was observed only in the DCLM. The contractile patterns produced by AJEA remarkably differed from those caused by acetylcholine and 5-HT. 4-DAMP and methoctramine at 0.5 μM significantly blocked the AJEA (1.0 μg/ml)-induced contraction of DCLM, but ondansetron, GR113808 and methysergide at 1.0 μM in combination did not change the AJEA-induced DCLM contractions. Acetylethylcholine mustard (5.0 μM) significantly diminished the AJEA-induced DCLM contractions, whereas p-chlorophenyl alanine (1.0 μM) did not affect the stimulatory effects of AJEA on the DCLM contractions.

Conclusion

The present results suggest that AJEA may specifically act on the DCLM among GI smooth muscles, and AJEA-induced DCLM contraction is likely mediated, at least, by activation of ChAT and acetylcholinergic muscarinic receptors.     

Integration of molecular and physiological models to explain time of anthesis in wheat

Background and Aims

A model to predict anthesis time of a wheat plant from environmental and genetic information requires integration of current concepts in physiological and molecular biology. This paper describes the structure of an integrated model and quantifies its response mechanisms.

Methods

Literature was reviewed to formulate the components of the model. Detailed re-analysis of physiological observations are utilized from a previous publication by the second two authors. In this approach measurements of leaf number and leaf and primordia appearance of near isogenic lines of spring and winter wheat grown for different durations in different temperature and photoperiod conditions are used to quantify mechanisms and parameters to predict time of anthesis.

Key Results

The model predicts the time of anthesis from the length of sequential phases: 1, embryo development; 2, dormant; 3, imbibed/emerging; 4, vegetative; 5, early reproductive; 6, pseudo-stem extension; and 7, ear development. Phase 4 ends with vernalization saturation (VS), Phase 5 with terminal spikelet (TS) and Phase 6 with flag leaf ligule appearance (FL). The durations of Phases 4 and 5 are linked to the expression of Vrn genes and are calculated in relation to change in Haun stage (HS) to account for the effects of temperature per se. Vrn1 must be expressed to sufficient levels for VS to occur. Vrn1 expression occurs at a base rate of 0·08/HS in winter ‘Batten’ and 0·17/HS in spring ‘Batten’ during Phases 1, 3 and 4. Low temperatures promote expression of Vrn1 and accelerate progress toward VS. Our hypothesis is that a repressor, Vrn4, must first be downregulated for this to occur. Rates of Vrn4 downregulation and Vrn1 upregulation have the same exponential response to temperature, but Vrn4 is quickly upregulated again at high temperatures, meaning short exposure to low temperature has no impact on the time of VS. VS occurs when Vrn1 reaches a relative expression of 0·76 and Vrn3 expression begins. However, Vrn2 represses Vrn3 expression so Vrn1 must be further upregulated to repress Vrn2 and enable Vrn3 expression. As a result, the target for Vrn1 to trigger VS was 0·76 in 8-h photoperiods (Pp) and increased at 0·026/HS under 16-h Pp as levels of Vrn2 increased. This provides a mechanism to model short-day vernalization. Vrn3 is expressed in Phase 5 (following VS), and apparent rates of Vrn3 expression increased from 0·15/HS at 8-h Pp to 0·33/HS at 16-h Pp. The final number of leaves is calculated as a function of the HS at which TS occurred (TS^HS): 2·86 + 1·1 × TS^HS. The duration of Phase 6 is then dependent on the number of leaves left to emerge and how quickly they emerge.

Conclusions

The analysis integrates molecular biology and crop physiology concepts into a model framework that links different developmental genes to quantitative predictions of wheat anthesis time in different field situations.     

Leaf press juice from Bryophyllum pinnatum (Lamarck) Oken induces myometrial relaxation

Aims

The use of preparations from Bryophyllum pinnatum (Lamarck) Oken (Kalanchoe pinnata (Lamarck) Persoon) in tocolysis is supported by clinical evidence. We studied here the effect of B. pinnatum leaf press juice and its chemical fractions on the response of human myometrial strips. No data are available if the influence on myometrial strips of the juice differs from that of its components in the chemical fractions, in order to increase the pharmacological effect.

Methodology

In vitro study to test the effect of repeated addition of B. pinnatum leaf press juice (BPJ) and its chemical components in several dilutions (undiluted, 1-10%) on myometrium strips hang up in a myograph chamber. Chemical analysis is including HPLC, MPLC with Sephadex LH-20 and TLC.

Results

All test solutions are inhibiting contractility by reducing the amplitude and the area under the curve (AUC) of the contractions. Undiluted BPJ and its undiluted chemical fraction 4 are reducing most effective these two parameters: the amplitude was at 78% of the baseline (95% CI (77-89); p < 0.05) at the second addition of the BPJ and at 70% (95% CI (50-90); p < 0.05) of the first addition of fraction 4; the AUC was at 82% (95% CI (69-95); p < 0.05) of the baseline at the first addition of the press juice and at 51% (95% CI (27-74); p < 0.05) at the first addition of fraction 4. The BPJ decreased amplitude and AUC significantly faster and increased frequency significantly faster than the control. Fractions could be tentatively assigned to bufadienolids, flavonoids and cinnamic acids. Fraction 4, accounted for flavonoids, increased the frequency of the contractions most effectively: 557% of the baseline (95% CI (316-797); p < 0.05) at the first addition.

Conclusion

Leaf juice of B. pinnatum and its flavonoid fraction are most effective in relaxing myometrial strips by inducing frequency.     

Differential voltage-sensitivity of D2-like dopamine receptors

Agonist potency at some neurotransmitter receptors has been shown to be regulated by transmembrane voltage, a mechanism which has been suggested to play a crucial role in the regulation of neurotransmitter release by autoreceptors and in synaptic plasticity. We have recently described the voltage-sensitivity of the dopamine D_2L receptor and we now extend our studies to include the other members of the D₂-like receptor subfamily; the D_2S, D₃, and D₄ dopamine receptors. Electrophysiological recordings were performed on Xenopus oocytes coexpressing human dopamine D_2S, D₃, or D₄ receptors with G protein-coupled potassium (GIRK) channels. Comparison of concentration-response relationships at −80 mV and at 0 mV for dopamine-mediated GIRK activation revealed significant rightward shifts for both D_2S and D₄ upon depolarization. In contrast, the concentration-response relationships for D₃-mediated GIRK activation were not appreciably different at the two voltages. Our findings provide new insight into the functional differences of these closely related receptors.     

Analysis of DNA repair gene polymorphisms in glioblastoma

Background

Glioblastoma is the most common and aggressive primary brain tumor in adults. Despite several factors such as ionizing radiation exposure or rare genetic syndromes have been associated with the development of glioblastoma, no underlying cause has been identified for the majority of cases. We thus aimed to investigate the role of DNA repair polymorphisms in modulating glioblastoma risk.

Methods

Genotypic and allelic frequencies of seven common polymorphisms in DNA repair genes involved in nucleotide excision repair (ERCC1 rs11615, ERCC2 rs13181, ERCC6 rs4253079), base excision repair (APEX1 rs1130409, XRCC1 rs25487), double-strand break repair (XRCC3 rs861539) and mismatch repair (MLH1 rs1800734) pathways were analyzed in 115 glioblastoma patients and 200 healthy controls. Haplotype analysis was also performed for ERCC1 rs11615 and ERCC2 rs13181 polymorphisms, located on the same chromosomal region (19q13.32).

Results

Our results indicated that carriers of the ERCC2 Gln/Gln genotype were associated with a lower glioblastoma risk (OR = 0.32, 95% CI 0.12–0.89; P = 0.028), whereas carriers of the MLH1 AA genotype were associated with an increased risk of glioblastoma (OR = 3.14, 95% CI 1.09–9.06; P = 0.034). Furthermore, the haplotype containing the C allele of ERCC2 rs13181 polymorphism and the T allele of ERCC1 rs11615 polymorphism was significantly associated with a protective effect of developing glioblastoma (OR = 0.34, 95% CI 0.16–0.71; P = 0.004).

Conclusions

These results pointed out that MLH1 rs1800734 and ERCC2 rs13181 polymorphisms might constitute glioblastoma susceptibility factors, and also suggested that the chromosomal region 19q could be important in glioblastoma pathogenesis.     

A novel bifunctional peptidic aspartic protease inhibitor inhibits chitinase A from Serratia marcescens: Kinetic analysis of inhibition and binding affinity

Background

Chitinase inhibitors have chemotherapeutic potential as fungicides, pesticides and antiasthmatics. The majority of chitinase inhibitors reported are natural products like argifin, argifin linear fragments, argadin, allosamidin and disulfide-cyclized peptides. Here, we report a novel peptidic inhibitor API (Aspartic Protease Inhibitor), isolated from Bacillus licheniformis that inhibits chitinase A (ChiA) from Serratia marcescens.

Methods

The binding affinity of API with ChiA and type of inhibition was determined by the inhibition kinetics assays. Fluorescence and CD spectroscopic analysis and chemical modification of API with different affinity reagents elucidated the mechanism of binding of API with ChiA.

Results and conclusions

The peptide has an amino acid sequence N-Ile¹-Cys²-Glu³-Ala⁴-Glu⁵-His⁶-Lys⁷-Trp⁸-Gly⁹-Asp¹⁰-Tyr¹¹-Leu¹²-Asp¹³-C. The ChiA–API kinetic interactions reveal noncompetitive, irreversible and tight binding nature of API with I₅₀ = 600 nM and K_i = 510 nM in the presence of chromogenic substrate p-nitrophenyl-N,N′-diacetyl-β-chitobioside[p-NP-(GlcNAc)₂]. The inhibition progress curves show a two-step slow tight binding inhibition mechanism with the rate constant k₅ = 8.7 ± 1 × 10^− 3 s^− 1 and k₆ = 7.3 ± 0.6 × 10^− 5 s^− 1. CD-spectra and tryptophanyl fluorescence analysis of ChiA incubated with increasing API concentrations confirms conformational changes in enzyme structure which may be due to irreversible denaturation of enzyme upon binding of API. Chemical modifications by WRK abolished the anti-chitinase activity of API and revealed the involvement of carboxyl groups in the enzyme inactivation. Abolished isoindole fluorescence of OPTA-labeled ChiA demonstrates the irreversible denaturation of ChiA upon incubation with API for prolonged time and distortion of active site of the enzyme.

General significance

The data provide useful information that could lead to the generation of drug-like, natural product-based chitinase inhibitors.     

Polymorphisms of the TLR4 gene and risk of gastric cancer

Objective

Toll-like receptor 4 (TLR4) is an important lipo-polysaccharide (LPS) receptor in gastric epithelial cell signaling transduction and plays critical roles in the development and progression of gastric cancer (GC). We investigated the effects of TLR4 gene polymorphisms and gene–environmental interactions on the risk of GC in Northeastern China.

Methods

We genotyped two single-nucleotide polymorphisms (SNPs) in TLR4 (rs10116253 and rs1927911) in 217 GC patients and 294 cancer-free controls using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Odds ratio (OR) and 95% confidence intervals (CIs) were estimated by unconditional logistic-regression models.

Results

Individuals carrying CC genotype of rs10116253 and TT genotype of rs1927911 had a significantly decreased risk of GC (adjusted OR = 0.33, 95% CI 0.18–0.60, P < 0.001 and adjusted OR = 0.37, 95% CI 0.21–0.67, P = 0.001 respectively), compared with TT genotype of rs10116253 and CC genotype of rs1927911. In addition, the SNP effects were additive to the effects of some known environmental factors without any interaction between them in the susceptibility to GC.

Conclusion

Our data suggested that TLR4 gene polymorphisms may be associated with a decreased risk of GC in Chinese population. And these SNPs and their combined effects with environmental factors may be associated with the risk of GC.     

Functional relevance of the cannabinoid receptor 2 — heme oxygenase pathway: A novel target for the attenuation of portal hypertension

Aims

In liver cirrhosis, inflammation triggers portal hypertension. Kupffer cells (KC) produce vasoconstrictors upon activation by bacterial constituents. Here, we hypothesize that the anti-inflammatory action of the cannabinoid receptor 2 (CB₂) agonists JWH-133 and GP 1a attenuate portal hypertension.

Main methods

In vivo measurements of portal pressures and non-recirculating liver perfusions were performed in rats 4 weeks after bile duct ligation (BDL). Zymosan (150 μg/ml, isolated liver perfusion) or LPS (4 mg/kg b.w., in vivo) was infused to activate the KC in the absence or presence of JWH-133 (10 mg/kg b.w.), GP 1a (2.5 mg/kg b.w.) or ZnPP IX (1 μM). Isolated KC were treated with Zymosan (0.5 mg/ml) in addition to JWH-133 (5 μM). The thromboxane (TX) B₂ levels in the perfusate and KC media were determined by ELISA. Heme oxygenase-1 (HO-1) and CB₂ were analyzed by Western blot or confocal microscopy.

Key findings

JWH-133 or GP 1a pre-treatment attenuated portal pressures following KC activation in all experimental settings. In parallel, HO-1 expression increased with JWH-133 pre-treatment. However, the inhibition of HO-1 enhanced portal hypertension, indicating the functional role of this novel pathway. In isolated KC, the expression of CB₂ and HO-1 increased with Zymosan, LPS and JWH-133 treatment while TXB₂ production following KC activation was attenuated by JWH-133 pre-treatment.

Significance

JWH-133 or GP 1a treatment attenuates portal hypertension. HO-1 induction by JWH-133 plays a functional role. Therefore, the administration of JWH-133 or GP 1a represents a promising new treatment option for portal hypertension triggered by microbiological products.     

A rational,non-radioactive strategy for the molecular diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency

Context

Molecular diagnosis of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD) has not been straightforward.

Objective

To conduct a comprehensive genetic analysis by Multiplex Ligation dependent Probe Amplification (MLPA) and evaluate its reliability for the molecular CAH-21OHD diagnosis.

Patients and methods

We studied 99 patients from 90 families with salt-wasting (SW; n = 32), simple-virilizing (SV; n = 29), and non-classical (NC; n = 29) CAH-21OHD. Molecular analysis was sequentially performed by detecting the most frequent point mutations by allele-specific oligonucleotide polymerase chain reaction (ASO-PCR), large rearrangements by MLPA, and rare mutations by direct sequencing. Parental segregation was evaluated.

Results

ASO-PCR detected microconversions in 164 alleles (91.1%). MLPA identified CYP21A1P large conversions to CYP21A2 in 7 of the remaining 16 (43.7%), 30-kb deletions including the 3′-end of CYP21A1P, C4B, and the 5′-end of CYP21A2 in 3 of the 16 (18.7%), and a complete CYP21A2 deletion in one (6.3%). Five alleles (2.7%) required direct sequencing; three mutations located in the CYP21A2 gene and two derived from CYP21A1P were found. No parental segregation was observed in patients with the c.329_336del and/or the CL6 cluster mutations. These cases were not diagnosed by ASO-PCR, but MLPA detected deletions in the promoter region of the CYP21A2 gene, explaining the genotype/phenotype dissociation.

Conclusion

Using the proposed algorithm, all alleles were elucidated. False-positive results in MLPA occurred when mutations or polymorphisms were located close to the probe-binding regions. These difficulties were overcome by the association of MLPA with ASO-PCR and paternal segregation. Using these approaches, we can successfully use MLPA in a cost-effective laboratory routine for the molecular diagnosis of CAH-21OHD.