Paula Grigorescu-Sido

Alterations in Lipid and Carbohydrate Metabolism in Patients with Classic Congenital Adrenal Hyperplasia due to 21-Hydroxylase Deficiency

Background: Classic 21-hydroxylase deficiency (21HD) presents some traits of the metabolic syndrome. Aim: To characterize discrete alterations of lipid and carbohydrate metabolism in children and young adults with classic 21HD, which could predict early atherogenesis. Patients and Methods: Twenty-seven Caucasian patients with classic 21HD (4–31 years); 27 sex-, age- and BMI-matched controls. Clinical parameters, hormonal status and genotype were assessed in all patients. Lipid parameters, including relative (%) and absolute (mg/dl) small-dense low-density lipoproteins subfractions (sd-LDL) were measured in patients and controls. Oral glucose tolerance tests were performed in both groups. Results: sd-LDL (%) was significantly higher in patients than controls (39.7 ± 5.9 vs. 35.5 ± 5.7%; p = 0.008). The same applies for absolute sd-LDL (mg/dl) (42.6 ± 11.9 vs. 36.4 ± 7.5; p = 0.029). HDL-cholesterol was lower in patients (p = 0.032). Fasting glucose and insulin were significantly higher in patients. Similar differences were noticed for HOMA-IR (p = 0.001), IRI (p = 0.001) and HOMA-B (p = 0.002). IRI correlated directly and significantly with the total hydrocortisone dose and the duration of treatment. Fasting glucose correlated with absolute sd-LDL. No obvious differences were seen between clinical forms or genotype groups. Conclusions: Substitution therapy should be adapted particularly at young ages to prevent early atherogenesis and cardiovascular risk in later life.

Gaucher disease in Romanian patients: incidence of the most common mutations and phenotypic manifestations.

Gaucher disease (GD) is an inherited glycolipid storage disorder resulting from the deficiency of glucocerebrosidase. It is the most frequent lysosomal storage disease in Romania, accounting for 70% of all lysosomal disorders diagnosed since 1997 in this country. The prevalence of six common mutations (N370S, L444P, R463C, 84GG, recNciI and recTL) and their phenotypic impact were studied in 20 type 1 GD patients of non-Jewish origin. Mutation analysis identified 77.8% of the GD alleles. The N370S mutation had the highest prevalence (50%), followed by the L444P (22.2%) and the recNciI (5.6%) alleles. Mutations R463C, 84GG and recTL have not been found in our patients. Rare or novel mutations likely accounted for 22.2% of the disease-producing uncharacterised alleles. Our study indicates a high prevalence of type 1 among Romanian GD patients. Clinical phenotype and disease severity were evaluated according to the standardised severity score index. Genotype–phenotype correlations were similar to those reported for other Caucasian non-Jewish populations. The absence of neuronopathic disease in patients presenting at least one copy of the N370S allele was confirmed, but the relative mildness of N370S homozygotes was not a constant feature among our patients. The presence of the L444P or of uncharacterised sporadic mutations was always associated with severe clinical manifestations, even in compound heterozygotes with the N370S allele. A large degree of phenotypic variability was observed in patients displaying the same genotype. The particularities of genotype–phenotype correlations may suggest the impact of other genetic or non-genetic factors on the clinical picture.

Three new 46,XX male patients: A clinical, cytogenetic and molecular analysis

BACKGROUND XX males range phenotypically from completely masculinised individuals to true hermaphrodites and include a subset of SRY negative patients. The correlation between genotype (SRY+/-) and phenotype is still unclear. AIM To report three new patients with this rare condition, one of whom was diagnosed prenatally and another was SRY negative, and to verify in our patients whether the presence of SRY results in a more masculinised phenotype. PATIENTS AND METHODS We present two phenotypically normal XX male patients (10 and 13.5 years) and one 3.1 years old XX male with ambiguous external male genitalia Prader IV. The patients were diagnosed by clinical, hormonal, sonographic, genetic and histological criteria. RESULTS Basal hormonal status was normal for phenotype but an excessive response to GnRH testing was noticed in the second patient together with insufficient hCG stimulation in all three patients. Pelvic ultrasound displayed male structures without Müllerian ducts; testicular biopsy, performed only in the intersex patient, showed Sertoli and Leydig cell hypoplasia. Chromosome analysis confirmed 46,XX karyotype. FISH analysis and molecular analysis by PCR were positive for Yp fragments/SRY gene on Xp in two patients and negative in the patient with ambiguous external genitalia. CONCLUSIONS In our observation Y chromosome-specific material containing the SRY gene translocated to the X chromosome results in a completely masculinised phenotype. In the intersex patient, incomplete masculinisation without SRY suggests a mutation of one or more downstream non-Y testis-determining genes.

Mutational Analysis and Genotype-Phenotype Correlation in Patients with Classic 21-Hydroxylase Deficiency from Transylvania (North-West Romania)

The regional incidence of 21-hydroxylase deficiency, its mutational spectrum and the correlation of genotype and phenotype has been studied by European, American and Latin-American groups. However, little information is known about the molecular background of the disease in patients from Central-Eastern Europe. The present study aimed to genotype a group of patients from Transylvania, the north-western part of Romania, in order to gain some insight into the molecular pattern and the genotype-phenotype correlation of congenital adrenal hyperplasia (CAH) in this region. We genotyped 17 patients with classic 21-hydroxylase deficiency and, whenever available, their parents in order to verify mutational segregation. The patients came from 13 unrelated families. DNA was prepared from peripheral blood leucocytes and four gene fragments were amplified by PCR. The 21-hydroxylase gene (CYP21B) was completely sequenced and analyzed for point mutations or deletions. Percentage distribution of mutations was as follows: 12G--34.6%, deletions and large conversions (del)--19.2%, P30L--15.4%, 1172N--15.4%, P30L+I2G+del8bp (triple mutation)--11.5%, and R356W--3.8%. Mutational percentage distribution compared to other Latin populations is higher for I2G and I172N and lower for deletions, while the P30L mutation was found at a higher rate than in any other analyzed population. Some differences may arise from the low patient number and from ethnic particularities. The incidence of compound heterozygotes in our group was 76.5%. The genotype seemed to correlate fairly well to phenotype, with a general concordance rate of 82.35%. Clear divergence was found in two patients with the simple virilizing form, exhibiting a homozygous status for I2G and del, respectively. This study offers the first information about the molecular pathology of CAH in Romania and should help to improve management and clinical outcome for patients with CAH in Transylvania. Hopefully, it might also be the first step towards exact and accurate prenatal diagnosis in our country.

Modelling long-term evolution of chitotriosidase in non-neuronopathic Gaucher disease

Abstract Chitotriosidase, an enzyme secreted by activated macrophages, is widely used as a biomarker for therapeutic monitoring and patient follow-up in Gaucher disease (GD), a lysosomal disorder caused by an inherited deficiency of glucocerebrosidase. We analyzed the long-term evolution of chitotriosidase aiming to establish an accurate model that describes the influence of enzyme replacement therapy (ERT) and the impact of several covariates. A total of 55 patients with non-neuronopathic (type 1) GD were followed for almost 17 years (during a maximum of 7.57 and 8.96 years, before and after the onset of ERT, respectively). Plasma chitotriosidase activity, measured yearly before the onset of ERT and at 6-month intervals after the initiation of ERT, was analyzed as a function of several covariates (age at diagnosis and at ERT initiation, nature of the most frequent genotypes, spleen status and the occurrence of bone complications). The evolution of chitotriosidase was approximated by a sigmoidal function, which allows the calculation of predicted values, based on several parameters inferred from our data. Splenectomy and the occurrence of bone complications significantly delayed the decline in chitotriosidase activity and induced higher mean residual values after long-term (4–9 years) ERT. Likewise, patients who started ERT infusions under 15 years of age had significantly higher mean residual chitotriosidase activities. The influence of other covariates did not reach statistical significance. In conclusion, we propose a novel model describing the evolution of chitotriosidase, allowing more accurate treatment adjustments, according to the variations of this biomarker.

Evaluation of neopterin as a biomarker for the monitoring of Gaucher disease patients

Objectives: Biomarker research is an important area of investigation in Gaucher disease, caused by an inherited deficiency of a lysosomal enzyme, glucocerebrosidase. We evaluated the usefulness of neopterin, as a novel biomarker reflecting chronic inflammation and immune system activation in Gaucher disease and analysed its evolution in response to enzyme replacement therapy (ERT). Methods: Circulating plasma neopterin levels in 31 patients with non-neuronopathic Gaucher disease were measured before and after the onset of ERT and were compared with those of 18 healthy controls. Plasma chitotriosidase activity was also monitored, as a reference biomarker, against which we evaluated the evolution of neopterin. Results: Neopterin levels were significantly increased in treatment-naïve patients (mean 11.90 ± 5.82 nM) compared with controls (6.63 ± 5.59 nM, Mann–Whitney U test P = 0.001), but returned to normal levels (6.92 ± 4.66 nM) following ERT. Investigating the diagnostic value of neopterin by receiver operating characteristic analysis, we found a cut-off value of 7.613 nM that corresponds to an area under the curve of 0.780 and indicates a good discrimination capacity, with a sensitivity of 0.774 and a specificity of 0.778. Discussion: Our results suggest that measurement of circulating neopterin may be considered as a novel test for the confirmation of diagnosis and monitoring of the efficacy of therapeutic intervention in Gaucher disease. Plasma neopterin levels reflect the global accumulation and activation of Gaucher cells and the extent of chronic immune activation in this disorder. Conclusion: Neopterin may be an alternative storage cell biomarker in Gaucher disease, especially in chitotriosidase-deficient patients.

Baseline characteristics and outcome in Romanian patients with Gaucher disease type 1

BACKGROUND/AIM To present clinical and genetic characteristics of all Romanian patients with Gaucher disease type 1, in whom specific diagnosis has been confirmed by enzymatic and molecular methods and to analyze their outcome with and without enzymatic replacement therapy (ERT). PATIENTS, METHODS There are fifty patients (F/M - 1.63/1) with Gaucher disease type 1. Clinical status, haemoglobin, thrombocytes, hepatic/splenic volume, bone mineral density and severity score were assessed at baseline and every six months thereafter. Thirty-nine patients (78%) received imiglucerase (44.4+/-13.6 U/kg/2 weeks) for 3.1+/-1.4 years. RESULTS Based on general prevalence data, our group represents 22.7% of the expected total number of patients with Gaucher disease type 1 in Romania. Mean age was 15.5 years at clinical onset and 28.9 years at confirmation of diagnosis. The genotype N370S/L444P was frequent in our group (35.9% of alleles). Anaemia, thrombocytopenia, splenomegaly and bone disease were present at 38%, 70%, 100% and 84%, respectively. Mean values for haemoglobin, thrombocytes, hepatic volume and chitotriosidase normalized after 0.5, 1.5, 2.5 and 3 years of ERT, respectively. Splenomegaly regressed from 14.4 x N (normal) to 3.06 x N over four years of treatment. Bone disease was ameliorated under ERT, yet bone mineral density worsened in patients treated with 30 U/kg/2 weeks. CONCLUSIONS The genotype N370S/L444P is frequent in our patients, in line with the severe phenotypes. ERT improved haematological parameters and visceromegaly, without a clear benefit for bone mineral density. To attain therapeutic goals, an early treatment start with optimal dosage is mandatory.

Clinical and genetic characteristics in a group of 45 patients with Turner syndrome (monocentric study)

Introduction Recent years have seen a shift in perspective on Turner syndrome, as it is no longer considered a significant disability due to therapeutic advances. The delay of diagnosis and the underdiagnosis are common in Turner syndrome, especially because of the great phenotypic variability and lack of firm diagnostic criteria. Aim Our first aim was to assess the clinical and the cytogenetic characteristics and growth rate in growth hormone (GH)-treated patients as compared to those with spontaneous growth. The second aim was to analyze the Y chromosomal sequences. Materials and methods We analyzed 45 patients diagnosed with Turner syndrome in Genetic Pathology Centre of Cluj Emergency Children’s Hospital. We carried out a study of the clinical features, the correlations between the karyotype and the phenotype, and we also made a research of Y chromosome sequences. Results The average age at diagnosis was 8.9±5.4 years. A significant association was observed between the number of external phenotypical abnormalities and internal malformations (r=0.45), particularly the cardiovascular ones (r=0.44). Patients treated with GH showed improvement in growth rate, with final stature significantly better than in untreated patients; benefits following treatment were greater if diagnosis was made before the age of 5 years. Thirteen percent of patients experienced spontaneous and complete puberty, whereas 30% experienced incomplete puberty. Patients with the 45,X genotype had a greater stature deficit and a higher incidence of cardiac malformations, compared with patients with 45,X/46,XX mosaic karyotype. Y chromosome sequences were found in only one patient, who subsequently underwent gonadectomy. Conclusion The importance of this study resides, to the best of our knowledge, in the fact that the largest group of patients in Romania was analyzed and assessed. To draw firm conclusions on the most valuable clinical indicators for Turner syndrome diagnosis in clinical practice, studies on large groups of patients should be conducted.

The c.301_302delAG PROP1 gene mutation in Romanian patients with multiple pituitary hormone deficiency.

Abstract Objective: To establish the frequency of the c.301_302 delAG mutation of the PROP1 gene in Romanian patients with multiple pituitary hormone deficiency (MPHD). Subjects and methods: Somatic assessment, hormonal test, bone age, magnetic resonance imaging of the pituitary gland, and molecular diagnosis were performed in 26 patients with MPHD (7 patients with familial form of MPHD and 19 patients with sporadic form of MPHD). Results: The c.301_302delAG mutation was detected in the homozygous state in 10 patients belonging to 5 unrelated families (7 patients with familial history of MPHD and 3 patients with sporadic form of MPHD). Those 10 patients presented variable pituitary hormone deficiency and pituitary morphology. Conclusions: The c.301_302delAG homozygous genotype had a high frequency of 38% (10/26), reaching 100% (7/7) in group with familial cases of MPHD and 16% (3/19) in group with sporadic forms of MPHD.

GJB2 and GJB6 genes mutations in children with non-syndromic hearing loss

Abstract Introduction. At the moment there is not enough data in Romania about the incidence of the main genetic mutations which can cause hearing loss. Objective. The current research aims to determine on a representative sample the prevalence of two mutations of genes GJB2 -c.35delG and p.W24X- and two mutations of genes GJB6 -del(GJB6-D13S1830), del(GJB6-D13S1854) respectively - in patients with congenital nonsyndromic sensorineural hearing loss (CNSHL). Methods: The sample group included 179 children with CNSHL. The evaluation consist in: a.Clinical, laboratory and imagistic examination; b.ENT exam and audiological evaluation. c.Two methods (semi-nested PCR technique followed by RFLP, validated with ARMS-PCR analysis) for detection of c.35delG and pW24X mutations; d.PCR-multiplex technique for detecting del(GJB6-D13S1830) and del (GJB6-D13S1854). Results: The audiological diagnosis was: profound hearing loss in 116 patients (64.8%), severe hearing loss in 29 children (16.2%) and moderate hearing loss in 34 patients (representing 19% of the trial patients). The prevalence for the three mutations was: 27.3 % for c.35delG, 3.6 % for p.W24X and 0.28% for del(GJB6-D13S1830). The detection of the three mutations (two on GJB2 gene and one on GJB 6 gene) has allowed to establish the genetic cause for deafness in 45 patients, representing 25.14% of the sample group. Our study is reporting the first case in Romania with a mutation of gene GJB6. Mutation del(GJB6-D13S1854) lacked in all 179 patients. Conclusion: The prevalence data obtained in the current research are comparable to data communicated by studies from other European countries.