David Araújo-Vilar

Functional Consequences of Seven Novel Mutations in the CYP11B1 Gene: Four Mutations Associated with Nonclassic and Three Mutations Causing Classic 11β-Hydroxylase Deficiency

CONTEXT Steroid 11beta-hydroxylase (CYP11B1) deficiency (11OHD) is the second most common form of congenital adrenal hyperplasia (CAH). Cases of nonclassic 11OHD are rare compared with the incidence of nonclassic 21-hydroxylase deficiency. OBJECTIVE The aim of the study was to analyze the functional consequences of seven novel CYP11B1 mutations (p.M88I, p.W116G, p.P159L, p.A165D, p.K254_A259del, p.R366C, p.T401A) found in three patients with classic 11OHD, two patients with nonclassic 11OHD, and three heterozygous carriers for CYP11B1 mutations. METHODS We conducted functional studies employing a COS7 cell in vitro expression system comparing wild-type (WT) and mutant CYP11B1 activity. Mutants were examined in a computational three-dimensional model of the CYP11B1 protein. RESULTS All mutations (p.W116G, p.A165D, p.K254_A259del) found in patients with classic 11OHD have absent or very little 11beta-hydroxylase activity relative to WT. The mutations detected in patients with nonclassic 11OHD showed partial functional impairment, with one patient being homozygous (p.P159L; 25% of WT) and the other patient compound heterozygous for a novel mild p.M88I (40% of WT) and the known severe p.R383Q mutation. The two mutations detected in heterozygous carriers (p.R366C, p.T401A) also reduced CYP11B1 activity by 23 to 37%, respectively. CONCLUSION Functional analysis results allow for the classification of novel CYP11B1 mutations as causative for classic and nonclassic 11OHD, respectively. Four partially inactivating mutations are predicted to result in nonclassic 11OHD. These findings double the number of mild CYP11B1 mutations previously described as associated with mild 11OHD. Our data are important to predict phenotypic expression and provide important information for clinical and genetic counseling in 11OHD.

High variability in CYP21A2 mutated alleles in Spanish 21‐hydroxylase deficiency patients, six novel mutations and a founder effect

Objective To detect common as well as rare and novel CYP21A mutations in 21‐hydroxylase deficiency patients. To estimate the distribution of mutations and compare them with other European studies. To construct haplotypes linked to a recurrent novel mutation.

Site-dependent differences in both prelamin A and adipogenic genes in subcutaneous adipose tissue of patients with type 2 familial partial lipodystrophy

Background: Type 2 familial partial lipodystrophy (FPLD2) is characterised by loss of fat in the limbs and buttocks and results from mutations in the LMNA gene. Aim: To evaluate the role of several genes involved in adipogenesis in order to better understand the underlying mechanisms of regional loss of subcutaneous adipose tissue (scAT) in patients with FPLD2. Methods: In total, 7 patients with FPLD2 and 10 healthy control participants were studied. A minimal model was used to calculate the insulin sensitivity (IS). scAT was obtained from abdomen and thigh by biopsy. Relative gene expression was quantified by real-time reverse transcription PCR in a thermal cycler. Prelamin A western blot analysis was carried out on scAT and prelamin A nuclear localisation was determined using immunofluorescence. Adipocyte nuclei were examined by electron microscopy. Results: Patients with FPLD2 were found to have significantly lower IS. The expression of LMNA was similar in both groups. The expression of PPARG2, RB1, CCND3 and LPL in thigh but not in abdomen scAT was significantly reduced (67%, 25%, 38% and 66% respectively) in patients with FPLD2. Significantly higher levels of prelamin A were found in peripheral scAT of patients with FPLD2. Defects in the peripheral heterochromatin and a nuclear fibrous dense lamina were present in the adipocytes of patients with FPLD2. Conclusions: In FPLD2 participants, prelamin A accumulation in peripheral scAT is associated with a reduced expression of several genes involved in adipogenesis, which could perturb the balance between proliferation and differentiation in adipocytes, leading to less efficient tissue regeneration.

A novel phenotypic expression associated with a new mutation in LMNA gene, characterized by partial lipodystrophy, insulin resistance, aortic stenosis and hypertrophic cardiomyopathy

Background  Lipodystrophies are a heterogeneous group of diseases characterized by abnormal fat distribution. Familial partial lipodystrophy 2 (FPLD2) is due to mutations in the LMNA gene. Previous studies have suggested that LMNA mutations 5′ to the nuclear localization signal (NLS) are more likely to underlie laminopathies with cardiac or skeletal muscle involvement, while mutations 3′ to the NLS are more likely to underlie lipodystrophy and progeroid syndromes.

Prematurely aged children: molecular alterations leading to Hutchinson-Gilford progeria and Werner syndromes.

Ageing is thought to be a polygenic and stochastic process in which multiple mechanisms operate at the same time. At the level of the individual organism ageing is associated with a progressive deterioration of health and quality of life, sharing common features such as: alopecia and grey hair, loss of audition, macular degeneration, neurodegeneration, cardiovascular diseases, osteoporosis, cataract formation, type-2 diabetes, lipodystrophies; a generally increased susceptibility to infection, autoimmune disorders and diseases such as cancer; and an impaired ability to cope with stress. Recent studies of mechanisms involved in the ageing process are contributing to the identification of genes involved in longevity. Monogenic heritable disorders causing premature ageing, and animal models have contributed to the understanding of some of the characteristic organism-level features associated with human ageing. Werner syndrome and Hutchinson-Gilford progeria syndrome are the best characterized human disorders. Werner syndrome patients have a median life expectancy of 47 years with clinical conditions from the second decade of life. Hutchinson-Gilford progeria syndrome patients die at a median age of 11-13 years with clinical conditions appearing soon after birth. In both syndromes, alterations in specific genes have been identified, with mutations in the WRN and LMNA genes respectively being the most closely associated with each syndrome. Results from molecular studies strongly suggest an increase in DNA damage and cell senescence as the underlying mechanism of pathological premature ageing in these two human syndromes. The same general mechanism has also been observed in human cells undergoing the normal ageing process. In the present article the molecular mechanisms currently proposed for explaining these two syndromes, which may also partly explain the normal ageing process, are reviewed.

A new seipin-associated neurodegenerative syndrome

Background Seipin/BSCL2 mutations can cause type 2 congenital generalised lipodystrophy (BSCL) or dominant motor neurone diseases. Type 2 BSCL is frequently associated with some degree of intellectual impairment, but not to fatal neurodegeneration. In order to unveil the aetiology and pathogenetic mechanisms of a new neurodegenerative syndrome associated with a novel BSCL2 mutation, six children, four of them showing the BSCL features, were studied. Methods Mutational and splicing analyses of BSCL2 were performed. The brain of two of these children was examined postmortem. Relative expression of BSCL2 transcripts was analysed by real-time reverse transcription-polymerase chain reaction (RT-PCR) in different tissues of the index case and controls. Overexpressed mutated seipin in HeLa cells was analysed by immunofluorescence and western blotting. Results Two patients carried a novel homozygous c.985C>T mutation, which appeared in the other four patients in compound heterozygosity. Splicing analysis showed that the c.985C>T mutation causes an aberrant splicing site leading to skipping of exon 7. Expression of exon 7-skipping transcripts was very high with respect to that of the non-skipped transcripts in all the analysed tissues of the index case. Neuropathological studies showed severe neurone loss, astrogliosis and intranuclear ubiquitin(+) aggregates in neurones from multiple cortical regions and in the caudate nucleus. Conclusions Our results suggest that exon 7 skipping in the BSCL2 gene due to the c.985C>T mutation is responsible for a novel early onset, fatal neurodegenerative syndrome involving cerebral cortex and basal ganglia.

Influence of moderate physical exercise on insulin-mediated and non—insulin-mediated glucose uptake in healthy subjects

To establish the relative importance of insulin sensitivity and glucose effectiveness during exercise using Bergmans minimal model, 12 nontrained healthy subjects were studied at rest and during 95 minutes of moderate exercise (50% maximum oxygen consumption [VO2max]). Each subject underwent two frequently sampled intravenous glucose tolerance tests (FSIGTs) for 90 minutes, at rest (FSIGTr) and during exercise (FSIGTe). Plasma glucose, insulin, and C-peptide were determined. Insulin sensitivity (S(I)), glucose effectiveness at basal insulin (S(G)), insulin action [X(t)], and first-phase (phi1) and second-phase (phi2) beta-cell responsiveness to glucose were estimated using both minimal models of glucose disposal (MMg) and insulin kinetics (MMi). Glucose effectiveness at zero insulin (GEZI), glucose tolerance index (K(G)), and the area under the insulin curve (AUC(0-90)) were also calculated. Intravenous glucose tolerance improved significantly during physical exercise. During exercise, S(I) (FSIGTr v FSIGTe: 8.5 +/- 1.0 v 25.5 +/- 7.2 x 10(-5) x min(-1) [pmol x L(-1)]-1, P < .01), S(G) (0.195 +/- 0.03 v 0.283 +/- 0.03 x 10(-1) x min(-1), P < .05), and GEZI (0.190 +/- 0.03 v 0.269 +/- 0.04 x 10(-1) x min(-1), P < .05) increased; however, no changes in phi1 and phi2 were found. Despite a significant decrease in the insulin response to glucose (AUC0-90, 21,000 +/- 2,008 v 14,340 +/- 2,596 pmol x L(-1) x min, P < .01), insulin action [X(t)] was significantly higher during the FSIGTe. These results show that physical exercise improves mainly insulin sensitivity, and to a lesser degree, glucose effectiveness. During exercise, the insulin response to glucose was lower than at rest, but beta-cell responsiveness to glucose did not change.

Both a reduced acute insulin response to glucose and lower glucose effectiveness are responsible for the worsening of intravenous glucose tolerance in healthy subjects independently of the degree of obesity

The effects of the acute insulin response to glucose (AIRg), insulin sensitivity (SI), and glucose effectiveness at zero insulin (GEZI) on intravenous glucose tolerance were studied in 94 non elderly healthy subjects with a wide range of body mass index (BMI). Conrads coefficient of glucose assimilation (KG) was calculated between 10 and 19 minutes of an intravenous glucose tolerance test. Both SI and GEZI were estimated using Bergmans minimal model. AIRg was calculated as the area under the insulin curve above basal between 0 and 10 minutes, and the suprabasal insulin effect was determined by the product of SI x AIRg. Stepwise multiple regression showed that the combined effect of SI x AIRg and GEZI explained 67% of the KG index variance. Division of the sample into tertiles according to KG shows that subjects with the lowest KG (KG < 1.32 min[-1]) had the lowest AIRg (2,832 +/- 1,362 v 6,510 +/- 4,410 [pmol x L(-1)] min, P = .0005), the lowest GEZI (0.092 +/- 0.06 v 0.179 +/- 0.09 min(-1), P = .0004), and the lowest SI x AIRg (0.014 +/- 0.008 v 0.022 +/- 0.01 min(-1), P = .00001), and were the oldest (41 +/- 10 v 31 +/- 10 years, P = .002) compared with subjects with the highest KG (KG > 1.8 min[-1]). However, no differences in SI (4.86 +/- 4.6 v 6.5 +/- 3.7 min(-1) [pmol x L(-1)],(-1) NS) or BMI (29.6 +/- 5.0 v 26.6 +/- 5.9 kg x m(-2), NS) were observed. These results did not vary when lean and obese subjects were analyzed separately. Age correlated significantly only with SI x AIRg. In conclusion, although the main factors that determine intravenous glucose tolerance are the suprabasal insulin effect and GEZI, worsening of the KG index depends on inadequate insulin secretion for the degree of insulin sensitivity and lower non-insulin-mediated glucose uptake. Age seems to be another factor in the worsening of intravenous glucose tolerance through a lower suprabasal insulin effect.

Non-insulin-mediated glucose uptake in several insulin-resistant states in the postabsortive period

The aim of our work was to study non-insulin-mediated glucose uptake (NIMGU), in the postabsorptive state, in several pathologies characterized by peripheral insulin resistance, namely, obesity (n = 10), NIDDM (n = 7), acromegaly (n = 7) and Cushings disease (n = 6). These groups were compared with a group of 16 healthy subjects. To estimate peripheral insulin sensitivity (SI) and glucose effectiveness (SG), we used the minimal model of glucose metabolism. Although all of these pathologies showed severe insulin resistance (control: 6.44 +/- 2.63, obesity: 2.84 +/- 1.57, NIDDM: 1.71 +/- 0.77, acromegaly: 1.88 +/- 1.23, Cushings disease: 1.87 +/- 0.66 x 10(-4) min-1 (microU/ml)-1, P < 0.01), fasting insulin-mediated glucose uptake (IMGU) did not differ significantly among the five groups, because reactive hyperinsulinaemia was present in all of these states. The contribution of NIMGU to whole-body glucose uptake did not differ significantly among the five groups (control: 77 +/- 8%; obesity: 77 +/- 9%; acromegaly: 82 +/- 8%; Cushings disease: 83 +/- 8%; NIDDM: 84 +/- 7%). In conclusion, our data show that, in the postabsorptive period, non-insulin mediated glucose uptake is a major determinant of glucose disposal and is similar in the different pathologies studied; on the other hand, although absolute rates of basal insulin-mediated glucose uptake are reduced in insulin-resistant states, they did not achieve statistical value compared with control subjects because of compensatory hyperinsulinaemia.

Reduced adipogenic gene expression in fibroblasts from a patient with type 2 congenital generalized lipodystrophy

Diabet. Med. 27, 1178–1187 (2010)