Low frequency of Fabry disease in patients with common heart disease

PurposeTo test the hypothesis that undiagnosed patients with Fabry disease exist among patients affected by common heart disease.MethodsGlobotriaosylceramide in random whole urine using tandem mass spectroscopy, α-galactosidase A activity in dried blood spots, and next-generation sequencing of pooled or individual genomic DNA samples supplemented by Sanger sequencing.ResultsWe tested 2,256 consecutive patients: 852 women (median age 65 years (19–95)) and 1,404 men (median age 65 years (21–92)). The primary diagnoses were coronary artery disease (n = 994), arrhythmia (n = 607), cardiomyopathy (n = 138), and valvular disease (n = 568). Urinary globotriaosylceramide was elevated in 15% of patients and 15 males had low α-galactosidase A activity. GLA variants found included R118C (n = 2), D83N, and D313Y (n = 7); IVS6-22 C>T, IVS4-16 A>G, IVS2+990C>A, 5′UTR-10 C>T (n = 4), IVS1-581 C>T, IVS1-1238 G>A, 5′UTR-30 G>A, IVS2+590C>T, IVS0-12 G>A, IVS4+68A>G, IVS0-10 C>T, IVS2-81–77delCAGCC, IVS2-77delC. Although the pathogenicity of several of these missense mutations and complex intronic haplotypes has been controversial, none of the patients screened in this study were diagnosed definitively with Fabry disease.ConclusionThis population of patients with common heart disease did not contain a substantial number of patients with undiagnosed Fabry disease. GLA gene sequencing is superior to urinary globotriaosylceramide or α-galactosidase A activity in the screening for Fabry disease.     

CD38 gene polymorphisms and genetic predisposition to multiple myeloma

Single nucleotide polymorphisms (SNPs) of adhesion and signaling genes may influence the etiopathogenesis of multiple myeloma (MM). CD38 molecule and its ligand CD31 are expressed and interact in malignant plasma cells and MM microenvironment. In this study we evaluated allele frequencies and distribution of two potentially functional CD38 SNPs, intronic rs6449182 (184C>G) and missense rs1800561 (418C>T, Arg¹⁴⁰Trp) in 175 Caucasian patients with MM and 207 healthy blood donors. The carriers of variant G allele of the rs6449182 SNPs were found to have significantly elevated risk of MM as compared to non-carriers; odds ratio = 5.69 (95% confidence interval = 3.7–8.7), p < 0.0001. In contrast, rs1800561 SNP minor T allele was detected at very low and comparable frequencies in patients and controls groups. In conclusion, our data suggest that inherited genetic variation in CD38 gene may impact on the risk of MM development.     

Association of inhibin α gene promoter polymorphisms with risk of idiopathic primary ovarian insufficiency in Korean women

ObjectiveThe aim of this study was to investigate whether two polymorphisms in the promoter region of inhibin alpha (INHA) are associated with risk of idiopathic primary ovarian insufficiency (POI) in Korean women, which is a controversial topic.Study designWe genotyped the INHA polymorphisms c.-16C > T (rs35118453) and c.-124A > G (rs11893842) of 136 POI patients and 225 controls in Korean women by polymerase chain reaction and restriction fragment length polymorphism analysis. We then compared differences in genotype and allele frequencies (AF) of the polymorphisms between the two groups to determine odds ratios (OR) and 95% confidence intervals (CI) as measures of the strength of association between genotype and POI.ResultsThere were no significant differences in genotype or AF of the polymorphisms between the POI patients and controls. Haplotype analysis revealed that the T–G haplotype of the two variant alleles was more frequent in POI patients than in the controls (OR = 1.630, 95% CI = 1.081–2.457). Combination genotype analysis showed that the CT + TT/GG genotype frequency was higher in POI patients than in the controls (OR = 2.414, 95% CI = 1.190–4.895).ConclusionsWe provide evidence to suggest that when the two variant alleles are combined, the c.-16C > T and c.-124A > G polymorphisms are associated with increased POI risk in Korean women. We postulate that interactions between the INHA polymorphisms may affect POI risk.     

Association of nitric oxide synthase gene polymorphisms (−786T>C, 4a4b, 894G>T) with primary ovarian insufficiency in Korean women

ObjectivesThe aim of our study was to investigate whether the endothelial nitric oxide synthase (eNOS) gene polymorphisms ?786T>C, 4a4b, and 894G>T that affect nitric oxide (NO) generation confer a risk for primary ovarian insufficiency (POI) in Korean women.Study designWe genotyped 136 POI patients and 236 controls among Korean women for the three single nucleotide polymorphism sites with PCR-RFLP analysis. Differences in the eNOS ?786T>C (rs2070744), 4a4b (rs61722009), and 894G>T (rs1799983) genotype frequencies between patients and controls were compared, and odds ratios and 95% confidence intervals were determined as a measure of the strength of the association between the genotypes and POI.ResultsThe POI patients had significantly decreased frequencies of the eNOS 894GT and ?786TT/894GT genotypes (P = 0.025 and 0.027, respectively). However, the significant association between these eNOS polymorphisms and POI disappeared after adjustment for multiple comparisons (adjusted P = 0.075 and 0.081, respectively). The eNOS ?786T/894G haplotype is more frequent in patients than controls (P = 0.030), whereas the ?786T/894T haplotype was less frequent in patients (P = 0.031). The associations between ?786/894 haplotypes and POI were confirmed by permutation tests. We did not find associations between the eNOS ?786T>C or 4a4b polymorphisms and POI.ConclusionsOur data suggest that the eNOS ?786T/894T haplotype is associated with a decreased POI risk, and we postulate that the eNOS ?786T/894T haplotype may confer less risk on POI occurrence by reducing pathologically increased NO generation by eNOS in POI. Further study is warranted to elucidate the effect of the eNOS 894G>T polymorphism and POI occurrence.     

Długotrwała pancytopenia po chemioterapii jako objaw demaskujący chorobę Gauchera u pacjentki z rakiem płuca

The diagnosis of congenital metabolic disease can be very difficult and often extends in time. This applies particularly to metabolic diseases of milder phenotype, such as an adult form (type 1) of Gaucher disease caused by the inherited (autosomal recessive) deficiency of the lysosomal enzyme glucocerebrosidase.In this work, we present a case of 48-year-old Polish patient (living in Sweden) with lung cancer, who developed a prolonged pancytopenia complicated by sepsis after each cycle of chemotherapy. These symptoms led to initiation of hematological diagnostic work-up and the assumption that the complications are caused by Gaucher disease. Definitive diagnosis of Gaucher disease was confirmed by results of enzymatic analyses, which revealed reduced activity of glucocerebrosidase in peripheral blood lymphocytes to 0.44 μkat/kg protein (ref.: 2.1–3.8), increased activity of plasma chitotriosidase to 1241 nkat/L (ref.: <40), and elevated plasma concentrations of chemokine CCL18/PARC to 1228 μg/L (ref.: <100). Direct DNA sequencing of the GBA1 gene revealed the presence of heterozygous mutation c.604C>T (R163X) and c.1226A>G (N370S), confirming diagnosis of type 1 Gaucher disease in the patient. The presence of the mutation c.604C>T has never been previously reported in a Polish patient with Gaucher disease. Administration of enzyme replacement therapy with imiglucerase (Cerezyme™) led to a rapid improvement of peripheral blood counts and enabled further continuation of intensive chemotherapy for lung cancer.In conclusion, the authors would like to emphasize that knowledge of the symptoms and the principles of diagnosis of Gaucher disease among hematologists is very important for efficient diagnostics of patients affected by this rare disease.     

Polymorphisms in tumor necrosis factor-alpha (−863C>A, −857C>T and +488G>A) are associated with idiopathic recurrent pregnancy loss in Korean women

Polymorphisms in TNF-a have been reported as genetic risk factors for recurrent spontaneous abortion and TNF-α may be immunologically important. We therefore examined the contribution of several TNF-a mutations to this phenomenon. The study participants consisted of 388 patients with idiopathic recurrent pregnancy loss (RPL), which was diagnosed on the basis of at least two consecutive spontaneous abortions; control subjects were 224 healthy women with a history of successful pregnancies. Polymerase chain reaction-restriction fragment length polymorphism analysis was performed to determine the TNF-α −863C>A, −857C>T, and +488G>A genotypes. The TNF-α −863C>A variants correlated with increased risk of RPL (CA + AA; adjusted odds ratio [AOR], 2.142; 95% confidence interval [CI], 1.493–3.074). These data did not differ in a stratified analysis according to number of consecutive spontaneous abortions. In haplotype analysis, there were similar trends of data for combination analysis, but in patients with 3+ pregnancy losses, a stratified analysis revealed that this correlation did not increase directly with the number of pregnancy losses. The TNF-α −863C>A variant is a possible genetic risk factor for idiopathic RPL in Korean women.     

A Japanese child with asymptomatic elevation of serum creatine kinase shows PTRF-CAVIN mutation matching with congenital generalized lipodystrophy type 4

Congenital generalized lipodystrophy (CGL), characterized by generalized absence of adipose tissue, has heterogeneous causes. Recently, a novel type of CGL complicated by muscular dystrophy was categorized as CGL4 caused by PTRF-CAVIN deficiency. However, it is unknown whether CGL4 exhibits clinical abnormalities during the infantile period. Here, we describe the youngest Japanese case of CGL4—a Japanese girl with asymptomatic high serum creatine kinase (CK) levels at 3 months old. She was referred to our hospital at 5 months of age because of her elevated serum CK (2528 IU/L). Generalized absence of adipose tissue was first recognized at 2 years of age. Mutation analysis of genes known to be responsible for CGL1-3 failed to disclose any abnormalities. Instead, analysis of the PTRF-CAVIN gene encoding PTRF-CAVIN revealed compound heterozygous mutations, one allele contained an insertion (c.696_697insC) and the other allele harbored a novel nonsense mutation (c.512C>A). Our patient had low serum leptin and adiponectin levels and insulin resistance. Pathological studies on biopsied muscle disclosed mild dystrophic change and highly reduced expression of PTRF-CAVIN. It was concluded that our PTRF-CAVIN deficient patient showed not only CGL but also asymptomatic elevation of serum CK because of her mild muscle dystrophic change.     

Incidence and carrier frequency of Sandhoff disease in Saskatchewan determined using a novel substrate with detection by tandem mass spectrometry and molecular genetic analysis

Sandhoff disease is a rare progressive neurodegenerative genetic disorder with a high incidence among certain isolated communities and ethnic groups around the world. Previous reports have shown a high occurrence of Sandhoff disease in northern Saskatchewan. Newborn screening cards from northern Saskatchewan were retrospectively screened in order to investigate the incidence and determine the carrier frequency of Sandhoff disease in these communities. PCR-based screening was conducted for the c.115delG (p.(Val39fs)) variant in the HEXB gene that was previously found in 4 Sandhoff disease patients from this area. The carrier frequency for this allele was estimated to be ~ 1:27. MS/MS-based screening of hexosaminidase activity along with genetic sequencing allowed for the identification of additional variants based on low total hexosaminidase activity and high % hexosaminidase A activity relative to c.115delG carriers. In total 4 pathogenic variants were discovered in the population (c.115delG, c.619A>G, c.1601G>T, and c.1652G>A) of which two are previously unreported (c.1601G>T and c.1652G>A). The combined carrier frequency of these alleles in the study area was estimated at ~ 1:15. Based on the number of cases of Sandhoff disease from this area we estimate the incidence to be ~ 1:390 corresponding to a child being born with the disease every 1–2 years on average. The results from our study were then compared with variants in the HEXB gene from the genomes available from the 1000 Genomes project. A total of 19 HEXB variants were found in the 1092 genomes of which 5 are suspected of having a deleterious effect on hexosaminidase activity. The estimated carrier frequency of Sandhoff disease in Saskatchewan at 1:15 is more than 3 times higher than the carrier frequency in the global sample provided by the 1000 Genomes project at 1:57.     

Expanded clinical and molecular spectrum of guanidinoacetate methyltransferase (GAMT) deficiency

Guanidinoacetate methyltransferase (GAMT) deficiency is a disorder of creatine biosynthesis, characterized by excessive amounts of guanidinoacetate in body fluids, deficiency of creatine in the brain, and presence of mutations in the GAMT gene. We present here 8 new patients with GAMT deficiency along with their clinical, biochemical and molecular data. The age at diagnosis of our patients ranges from 0 to 14 years. The age of onset of seizures usually ranges from infancy to 3 years. However, one of our patients developed seizures at age 5; progressing to myoclonic epilepsy at age 8 years and another patient has not developed seizures at age 17 years. Five novel mutations were identified: c.37ins26 (p.G13PfsX38), c.403G > T (p.D135Y), c.507_521dup15 (p.C169_S173dup), c.402C > G (p.Y134X) and c.610_611delAGinsGAA (p.R204EfsX63). Six patients had the c.327G > A (last nucleotide of exon 2) splice-site mutation which suggests that this is one of the most common mutations in the GAMT gene, second only to the known Portuguese founder mutation, c.59G > C (p.W20S). Our data suggests that the clinical presentation can be variable and the diagnosis may be overlooked due to unawareness of this disorder. Therefore, GAMT deficiency should be considered in the differential diagnosis of progressive myoclonic epilepsy as well as in unexplained developmental delay or regression with dystonia, even if the patient has no history of seizures. As more patients are reported, the prevalence of GAMT deficiency will become known and guidelines for prenatal diagnosis, newborn screening, presymptomatic testing and treatment, will need to be formulated.     

Ultrastructural examination of skin biopsies may assist in diagnosing mitochondrial cytopathy when muscle biopsies yield negative results

Ultrastructural evaluation of skin biopsies has been utilized for diagnosis of mitochondrial disease. This study investigates how frequently skin biopsies reveal mitochondrial abnormalities, correlates skin and muscle biopsy findings, and describes clinical diagnoses rendered following the evaluation. A retrospective review of surgical pathology reports from 1990 to 2015 identified skin biopsies examined by electron microscopy for suspected metabolic disease. A total of 630 biopsies were included from 615 patients. Of these patients, 178 also underwent a muscle biopsy. Of the 630 skin biopsies, 75 (12%) showed ultrastructural abnormalities and 34 (5%) specifically showed mitochondrial abnormalities including increased size (n = 27), reduced or abnormal cristae (n = 23), dense matrices (n = 20), and increased number (n = 8). Additional findings included lysosomal abnormalities (n = 13), lipid accumulation (n = 2) or glycogen accumulation (n = 1). Of the 34 patients with mitochondrial abnormalities on skin biopsy, 20 also had muscle biopsies performed and nine showed abnormalities suggestive of a mitochondrial disorder including absent cytochrome oxidase staining (n = 2), increased subsarcolemmal NADH, SDH, or cytochrome oxidase staining (n = 1), or ultrastructural findings including large mitochondrial size (n = 5), abnormal mitochondrial structure (n = 5), and increased mitochondrial number (n = 4). The most common presenting symptoms were intellectual disability (n = 13), seizures (n = 12), encephalopathy (n = 9), and gastrointestinal disturbances (n = 9). At last known follow-up, 12 patients had a definitive diagnosis of a mitochondrial disorder. One patient each had Complex I deficiency, Complex III deficiency, Charcot-Marie-Tooth disease, pyruvate dehydrogenase deficiency, and Phelan-McDermid syndrome. Our results suggest that skin biopsy sometimes yields diagnostic clues suggestive of a mitochondrial cytopathy in cases with a negative muscle biopsy.     

Different laboratory and muscle biopsy findings in a family with an m.8851T > C mutation in the mitochondrial MTATP6 gene

We report the second known family with a very rare, maternally inherited missense m.8851T > C mutation in the mitochondrial MTATP6 gene. A failure to thrive, microcephaly, psychomotor retardation and hypotonia were present in a 3-year-old girl with a high mtDNA mutation load (87–97%). Ataxia and Leigh syndrome were subsequently documented in a neurological examination and brain MRI. A muscle biopsy demonstrated decreased ATP synthase and an accumulation of succinate dehydrogenase products, indicating mitochondrial myopathy. Her 36-year-old mother (68% blood heteroplasmy) developed peripheral neuropathy and muscle weakness at the age of 22 years. Our findings extend the clinical and laboratory phenotype associated with the m.8851T > C mutation.     

Aberrant NKG2D expression with IL-17 production of CD4+ T subsets in patients with type 2 diabetes

Type 2 diabetes (T2D) is a systemic inflammatory disease. Although the natural killer group 2, member D (NKG2D) receptor, was not expressed normally on CD4+ T cells, the aberrant expression was found in pathological conditions such as in auto-immune diseases. However, the involvement of NKG2D in pathogenesis of T2D is unclear. We hypothesize that there is an inflammatory CD4+ T cell subpopulation expressing NKG2D and producing interleukin (IL)-17 in T2D. NKG2D expression on CD4+ T cells and their subsets were analyzed by multi-color staining using flow cytometry. Lymphocytes were activated by phorbol-12-myristate-13-acetate (PMA) and ionomycin, and were stained for intracellular IL-17. To investigate the mechanism of IL-17 production, patients’ lymphocytes were stimulated using specific anti-T cell receptor (TCR) alone, anti-NKG2D alone or a combination of the two antibodies. CD4+ T cells and particularly, CD4 + CD28^null T subset of T2D patients were highly expressed NKG2D and more prevalent compared to non-diabetic individuals (ND) (P = 0.039 and P = 0.022, respectively). Significantly higher percentages of CD4 + CD28^nullNKG2D + T cells of patients produced IL-17 when compared to those of ND (P = 0.024) and were positively correlated with the level of glycated hemoglobin A1c (HbA1c) (R² = 0.386, P = 0.041). Additionally, this cell population could be stimulated by specific monoclonal anti-NKG2D to produce IL-17. In conclusion, CD4 + CD28^nullNKG2D+ T cells were expanded in T2D, especially in patients with poor glycemic control. NKG2D may be one of the surrogate co-stimulatory receptors leading to irregular inflammatory function producing IL-17. An IL-17 producing CD4 + CD28^nullNKG2D + T cells may potentially be involved in pathogenesis and drive severity of the disease with the glycemic dependence. This particular cell type could be targeted for prognostic or therapeutic purposes.     

Identification and characterization of a novel splice site mutation in the SERPING1 gene in a family with hereditary angioedema

Hereditary angioedema due to C1-inhibitor deficiency (HAE-C1INH) is a rare autosomal-dominant disease caused by mutations in SERPING1 gene. The main clinical feature of C1INH deficiency is the spontaneous edema of the subcutaneous and submucosal layers. More than 280 different mutations scattering the entire SERPING1 gene have been reported. We identified and characterized a new mutation in SERPING1 gene in a Spanish family with hereditary angioedema. The mutation (c.685 + 2 T > A) disrupts the donor splice site of intron 4 leading to the loss of exon 4 in mutant mRNA. We demonstrated that mutant mRNA is mostly degraded, probably by the surveillance pathway no-go mRNA decay. Bioinformatic analysis showed that the mutant protein, if produced, would be non-functional since the protein lacks a stretch of 45 amino acids affecting the functional RCL loop. Finally, we found a reduction of the wild-type mRNA expression in c.685 + 2 T > A carriers.     

Carnitine palmitoyltransferase 1A (CPT1A) P479L prevalence in live newborns in Yukon,Northwest Territories,and Nunavut

Carnitine palmitoyltransferase 1A (CPT1A), encoded by the gene CPT1A, is the hepatic isoform of CPT1 and is a major regulatory point in long-chain fatty acid oxidation. CPT1A deficiency confers risk for hypoketotic hypoglycaemia, hepatic encephalopathy, seizures, and sudden unexpected death in infancy (SUDI). It remains controversial whether the CPT1A gene variant, c.1436C>T (p.P479L), identified in Inuit, First Nations, and Alaska Native infants, causes susceptibility to decompensation, in particular during times of fever and intercurrent illness. Although newborn screening for the P479L variant occurs in some jurisdictions, background knowledge about the presence of the variant in Canadian Aboriginal populations is lacking. In an effort to understand the population implications of the variant in northern Canada, overall frequencies of the variant were assessed. Further studies are underway to determine associated risk. Ethics approval was obtained from university REBs, local research institutes, and with consultation with territorial Aboriginal groups. Newborn screening blood spots from all infants born in 2006 in the three territories were genotyped for the p.P479L variant. p.P479L (c.1436C>T) allele frequencies in the three territories were 0.02, 0.08, and 0.77 in Yukon (n = 325), Northwest Territories (n = 564), and Nunavut (n = 695), respectively. Homozygosity rates were 0%, 3%, and 64%. Aboriginal status was available only in NWT, with allele frequencies of 0.04, 0.44, 0.00, and 0.01 for First Nations, Inuvialuit/Inuit, Métis, and non-Aboriginal populations. Although individual blood spots were not identified for Aboriginal ethnicity in Nunavut infants, ~ 90% of infants in Nunavut are born to Inuit women. The allele frequency and rate of homozygosity for the CPT1A P479L variant were high in Inuit and Inuvialuit who reside in northern coastal regions. The variant is present at a low frequency in First Nations populations, who reside in areas less coastal than the Inuit or Inuvialuit in the two western territories. The significance of the population and geographic distribution remains unclear, but the high population frequencies of the variant suggest a historically low penetrance for adverse outcomes. Further evidence is needed to determine if there is an increased risk for infant mortality and morbidity and whether newborn screening will be indicated on a population basis.     

Influence of thymidylate synthase gene polymorphisms on total plasma homocysteine concentrations

BackgroundThymidylate synthase (TS) is a key enzyme that regulates the production of nucleotide synthesis by catalyzing the conversion of deoxyuridylate to thymidylate. Three functional polymorphisms in the TS gene have been identified including: (i) the thymidylate synthase enhancer region (TSER) tandem repeat polymorphism and (ii) the G to C single nucleotide polymorphism (G/C SNP) both of which occur in the 5′untranslated region (UTR) of the TS gene; and (iii) the 6 base pair deletion at base pair 1494 (TS1494del6) located in the 3′UTR.PurposeThe purpose of this research was to investigate the relationship between TS polymorphisms and total plasma homocysteine (tHcy) levels.MethodsThe study population consisted of 396 healthy male and female volunteers from Kingston, Ontario and Halifax, Nova Scotia, Canada between 2006 and 2008. The effect of each TS polymorphism on tHcy concentrations was investigated and further analyses were conducted on categorization of polymorphisms based on 5′ or 3′UTR. The combined effect of TS polymorphisms on tHcy concentration was also investigated, in addition to interactions between polymorphisms in TS and MTHFR 677C>T and interactions between TS polymorphisms and serum folate and vitamin B₁₂ status.ResultsAn association between TS 5′polymorphisms and tHcy concentration was observed (p = 0.05). The combined effect of the TS polymorphisms was also found to be associated with tHcy concentration (p = 0.05). Additionally, an antagonistic interaction was observed between TS 5′polymorphism and MTHFR 677C>T on tHcy concentrations (p = 0.04).ConclusionsThe findings of this research provide evidence of an association between TS polymorphisms and tHcy concentrations.     

The MHC2TA 1614 C&gt;G gene polymorphism is associated with risk of developing acute coronary syndrome

BackgroundInflammation plays an essential role in the development and progression of atherosclerotic lesions. The major histocompatibility complex class II trans-activator (MHC2TA) is considered an important molecule in the inflammatory process regulation. The aim of the present study was to evaluate the role of MHC2TA gene polymorphisms as susceptibility markers for acute coronary syndrome (ACS).MethodsThree polymorphisms (−168 A>G, 1614 C>G, and 2536 G>A) of the MHC2TA gene were analyzed by 5′ exonuclease TaqMan genotyping assays in a group of 297 patients with ACS and 283 healthy controls. Haplotypes were constructed after linkage disequilibrium analysis.ResultsThe 1614 C allele and CC genotype were associated with risk of developing ACS (PC = 0.014, OR = 1.37 and PC = 0.006, OR = 1.90, respectively). Based on Hosmer–Lemeshow Goodness of Fit test, the recessive model was selected to estimate risk between ACS patients and controls adjusted by cardiovascular risk factors using a multiple logistic analysis. In this case, the OR adjusted was 1.78 for the 1614 CC genotype (P = 0.023). The analysis of linkage disequilibrium showed one risk haplotype (ACG) and one protective haplotype (AGG) for developing ACS (P = 0.02, OR = 1.5 and P = 0.04, OR = 0.72, respectively).ConclusionThe results suggest that MHC2TA 1614 gene polymorphism could be involved in the risk of developing ACS.     

Mitochondrial DNA deletion and sarcopenia

PurposeAccumulation of mitochondrial DNA deletions and the resultant impaired oxidative phosphorylation may play a pathogenic role in the mediation of age-related sarcopenia.MethodsTwenty four participants of the New Mexico Aging Process Study were classified as normal lean (n = 15) or sarcopenic (n = 9) based on body composition determined by Dual Energy x-ray Absorptiometry. Complex I and Complex IV activities were measured in the skeletal muscle samples obtained from gastrocnemius muscle. A two-stage nested polymerase chain reaction strategy was used to identify the mitochondrial DNA deletions in the entire mitochondrial genome in the skeletal muscle samples.ResultsAlthough Complex I activity was not significantly different (5.5 ± 0.9 vs. 4.6 ± 0.7 mU/mg protein, P > 0.05), Complex IV activity was higher in sarcopenic subjects (1.4 ± 0.3 vs. 1.0 ± 0.1 mU/mg protein, P < 0.05). Mitochondrial DNA deletions were mostly located in the region of Complex I and spanned from nicotinamide adenine dinucleotide dehydrogenase 1 to nicotinamide adenine dinucleotide dehydrogenase 6. Deletions in the 8,577–10,407 bp and 10,233–11,249 bp regions were associated with a significant decrease in Complex I activity (P < 0.05 and P = 0.02, respectively). Total cumulative deletion, defined as the sum of individual length of deletions in a subject, was comparable in subjects with and without sarcopenia (1760 ± 726 vs. 1782 ± 888 bp, P > 0.05). The magnitude of mitochondrial DNA deletion, however, correlated positively with lean body mass (r = 0.43, P < 0.05).ConclusionThus, mitochondrial DNA deletions are common in elderly subjects and are negatively related to Complex I activity. The positive association between mitochondrial DNA deletions and lean body mass needs to be confirmed by studies in a larger study population.     

Relevance of HLA-G,HLA-E and IL-10 expression in lip carcinogenesis

HLA-G, HLA-E and IL-10 are molecules which can provide tumor immunosuppression as well as the capacity of evasion to the immune system host. This study set out to evaluate HLA-G, HLA-E and IL-10 expression in lip squamous cell carcinoma (LSCC) and in a potentially malignant disorder (actinic cheilitis – AC), correlating the expression of these proteins with the degree of epithelial dysplasia. Immunohistochemistry was undertaken to identify HLA-G, HLA-E and IL-10 in samples from patients with LSCC (n = 20), AC (n = 30) and healthy lip mucosa (control) (n = 10). A semiquantitative scoring system was used for analysis. Differences between the groups were evaluated using the Pearson Chi-Squared test. The percentage of LSCC samples showing high immunoreactivity (IRS > 2) for HLA-G, HLA-E and IL-10 (neoplastic/epithelial cells) and HLA-E (stroma/connective tissue) was significantly higher that of the control (P < 0.05). A tendency for a progressive increase in the proteins analyzed was observed from the control to AC and to LSCC. The degree of dysplasia in the AC samples was not significantly associated with the proteins evaluated (P > 0.05). The high expression of HLA-G, HLA-E and IL-10 in AC and LSCC reflects the capacity that these pathologies have for evasion and progression.     

Estradiol biodisponible (non-SHBG-bound E2). Dosage ou calcul en relation avec la densité minérale osseuse lombaire (L1–L4) chez l'homme âgé

With age, decreased levels of bioavailable T and E₁ + E₂ are mainly the result of the increase of SHBG levels. Several studies have shown the association of low levels < 11 pg/ml (40 pM) of bioavailable estradiol with vertebral fractures and osteoporosis in older men. They used assayed E₂ Bio or calculated non-SHBG-bound E₂ calculated using the equation derived from the law of mass action for the model: two binding proteins (SHBG, Albumin) and two ligands (T, E₂) (FE₂c_II Södergard) while serum SHBG, albumin, T and E₂ are determined experimentally in every sample. The aim of the study is to evaluate the performance of E₂ Bio and FE₂c_II associated with vertebral osteoporosis on 62 men (median age: 64 years). As expected, SHBG increases markedly with age (P = 0.003) and Ttot increases with SHBG (P = 0.0003). DMO and T score are positively correlated (P = 0.086–0.048) with E₂ Bio in men with E₂ Bio < 40 pM and are not correlated (P = 0.70–0.48) in men with E₂ Bio > 40 pM. E₂ Bio (P < 0.003) is inversely associated with SHBG levels but FE₂c_II is not correlated with SHBG (P > 0.45) and with DMO or T score adjusted for body weight (P = 0.48). Considering the lack of sensitivity of calculated FE₂c_II to estimate the "non-SHBG-bound E₂", the bioavailable estradiol assay by ammonium sulfate precipitation is the preferred method for detecting elderly men in the lowest quartile for E₂ Bio and at risk for developing osteoporosis later in life.     

Testostérone libre ou biodisponible : dosages ou calculs. Comparaison critique de différents modes d'approche

The aim of the study is to find the most reliable practical approach to the estimation of free or bioavailable serum testosterone. We compare assayed values of bioavailable testosterone (T Bio), Free testosterone DPC (Free T DPC), total testosterone (Ttot) and calculated Free Androgen Index FAI = [Ttot/SHBG] × 100, calculated Free Testosterone using the equation derived from the law of mass action for the model: two binding proteins (SHBG, Albumin) and two ligands (T,E₂) (FTc_II Södergaard) or one ligand (T) (Ftc_I Kaufman and Fiers). Serum SHBG, Albumin, E₂ are determined exprimentally in every sample. The bioavailable (or non-SHBG bound) T correlates well with Free T by ultrafiltration (r = 0.96; P < 0.01), is easy to perform, reliable and sensitive if a particular care is ensured in the purification of the tracer. Assayed women values of T Bio agreed well with calculated values for FTc _II or FTc _I (r = 0.93; P < 0.01) using the laws of mass action. In contrast, the ratios of FTc _II/T Bio and FTc _I/T Bio (which should be constant if indexes reflect T Bio) remain negatively SHBG dependent for women and positively SHBG dependent for men confirming that the assumptions of the model are too simplified and the association constants Ka values too approximative. Calculated FTc is an acceptable substitute for an estimation of bioavailable T if we presume women with standard SHBG binding conditions and sera free of significant amounts of substances or steroids that could occupy the binding sites in the SHBG moiety and invalidate the calculation. Although showing a good correlation (r = 0.89; P < 0.01) with T Bio, FAI is not a useful index: the FAI/T Bio ratio is negatively correlated (r = –0.86 P < 0.001) with SHBG and overestimate strongly the SHBG binding capacity contribution for a reliable quantification of the non-SHBG bound T. The Free T DPC is inaccurate, not sufficiently sensitive, not free of proteins effects and less correlated with T Bio (r = 0.49; P < 0.05) than Ttot (r = 0.64; P < 0.01) for women!