Vesna Kušec

Two Novel CYP11B1 Gene Mutations in Patients from Two Croatian Families with 11 β -Hydroxylase Deficiency.

Steroid 11β-hydroxylase deficiency (11β-OHD) is the second most common cause of congenital adrenal hyperplasia. Mutations in the CYP11B1 gene, which encodes steroid 11β-hydroxylase, are responsible for this autosomal recessive disorder. Here, we describe the molecular genetics of two previously reported male siblings in whom diagnosis of 11β-OHD has been established based on their hormonal profiles displaying high levels of 11-deoxycortisol and hyperandrogenism. Both patients are compound heterozygous for a novel p.E67fs (c.199delG) mutation in exon 1 and a p.R448H (c.1343G>A) mutation in exon 8. We also report the biochemical and molecular genetics data of one new 11β-OHD patient. Sequencing of the CYP11B1 gene reveals that this patient is compound heterozygous for a novel, previously undescribed p.R141Q (c.422G>A) mutation in exon 3 and a p.T318R (c.953C>G) mutation in exon 5. All three patients are of Croatian (Slavic) origin and there is no self-reported consanguinity in these two families. Results of our investigation confirm that most of the CYP11B1 mutations are private. In order to elucidate the molecular basis for 11β-OHD in the Croatian/Slavic population, it is imperative to perform CYP11B1 genetic analysis in more patients from this region, since so far only four patients from three unrelated Croatian families have been analyzed.

Classical Congenital Adrenal Hyperplasia due to 21-Hydroxylase Deficiency in Croatia between 1995 and 2006

Aims: To evaluate the incidence, gender, symptoms and age at diagnosis among patients with classical congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency in Croatia. Methods: Data were collected retrospectively for all classical CAH patients born or electively aborted following prenatal diagnosis between 01.01.1995 and 31.12.2006 and were compared with the data of the previously conducted study evaluating CAH patients discovered between 1964 and 1984. Results: During a 12-year period, 34 classical CAH patients were born. There were 20 salt-wasting (SW) (12 females/8 males) and 14 simple virilizing (SV) patients (7 females/7 males). If 3 female fetuses, electively aborted, were added, that would be a total of 37 CAH patients. With 532,942 live births and 34 CAH patients born over this period, incidence of classical CAH was estimated to 1:15,574 or 1:14,403 if 3 electively aborted fetuses were included. The lower incidence of SW boys compared to SW girls (8:12) and similar number of SW and SV boys (8:7) indicate that substantial proportion of SW boys die unrecognized. Owing to better health care, diagnosis was established significantly earlier in SW and SV girls compared to the period of 1964–1984 (p < 0.003). During 1995–2006, none of the patients died following the diagnosis of CAH, and there was no erroneous sex assignment. Conclusion: Despite of improvement in health care, diagnosis of CAH in Croatia is still delayed and some of the patients go unrecognized or die. Therefore, we think that the results of our study support the need for the introduction of newborn screening.

Molecular genetic analysis in 93 patients and 193 family members with classical congenital adrenal hyperplasia due to 21-hydroxylase deficiency in Croatia.

Congenital adrenal hyperplasia owing to 21-hydroxylase deficiency is caused by mutation in the CYP21A2 gene. The frequency and spectrum of CYP21A2 mutations and genotype-phenotype correlations among different populations are variable. Aim of this study was to define mutation frequency and spectrum of CYP21A2 gene mutations in patients with classical 21-hydroxylase deficiency (21OHD) and their family members in Croatia and study genotype-phenotype correlation. Clinical features and mutations of CYP21A2 gene in 93 unrelated 21OHD patients and 193 family members were examined. In this cohort, 66 patients were affected with salt wasting (SW) form, and 27 were affected with simple virilizing (SV) form of the disease. Mutations were identified in both alleles (67% compound heterozygous and 33% homozygous) in 91 of 93 patients. Deletions and conversions were found in 18.8% and point mutations in 79.6% alleles. Mutations in 3 alleles (1.6%) remained unidentified (in one patient we found only one, while in other no mutations were found at all). The most common point mutations were Intron 2 splice mutation IVS2-13 A/C>G (35.5%) and p.R357W (16.7%). Genotypes were categorized into Groups 0, A, B and C according to the extent of enzyme impairment. Genotype-phenotype concordance was 100%, 85% and 75% for Groups 0, A and B, respectively. Since only classical 21OHD patients were studied, Group C comprised solely p.P31L mutation and had 73% patients with SV and 27% with SW phenotype. Intrafamilial phenotypic variability was found in two families. CYP21A2 genetic analysis in 193 family members showed that 126 parents were heterozygous carriers, 3 were newly discovered patients, 2 fathers were not biological parents, and mutations were not detected in 3. Among 59 siblings, 32 were heterozygous carriers, 15 carried normal alleles, and 12 were patients (4 newly diagnosed). Genotype-phenotype divergence observed in this study suggests caution in preconceptional counseling and prenatal diagnosis of CAH. High frequency of p.R357W mutation was found in Croatian patients with classical 21-OHD. Genotyping of family members discovered new patients and thus avoided pitfalls in genetic counseling when the parents were found to be affected.

Primary hypothyroidism and nipple hypoplasia in a girl with Wolcott–Rallison syndrome

Wolcott–Rallison syndrome (WRS), caused by mutation in the EIF2AK3 gene encoding the PERK enzyme, is the most common cause of permanent neonatal diabetes mellitus (PNDM) in consanguineous families and isolated populations. Besides PNDM, it also includes skeletal abnormalities, liver and renal dysfunction, and other inconsistently present features. We present two siblings, who are WRS patients, and are Albanians from Kosovo born to unrelated parents. The older sister presented with PNDM, exocrine pancreatic insufficiency, short stature, microcephaly, normocytic anemia, delay in speech development, skeletal abnormalities, primary hypothyroidism, and hypoplastic nipples. Sequencing of the EIF2AK3 gene identified a homozygous mutation R902X in exon 13. The younger brother was diagnosed with PNDM and died from hepatic failure suggesting that he has been suffering from WRS as well. Including one previously reported patient from Kosovo carrying the same homozygous mutation, there are three WRS patients from this very small, ethnically homogenous region suggesting founder effect in this population. Conclusion: We postulate that thyroid hypoplasia with primary subclinical hypothyroidism already reported in two WRS patients and nipple hypoplasia could also be the phenotypic reflection of the mutation of pleiotropic EIF2AK3 gene in secretory cells.

Five patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency (one with associated neuroblastoma) discovered in three generations of one family.

Background: Most patients with 21-hydroxylase deficiency (21-OHD) are compound heterozygous carriers. Their phenotype usually reflects a less severe allelic mutation, although discordance between the genotype and the phenotype has been observed. Case Report: We present 5 patients with congenital adrenal hyperplasia (CAH) due to 21-OHD belonging to the 3 generations of the same family (grandmother, parents and their 2 children). As each patient carries at least one mild mutation of the CYP21 gene, their genotypes correspond to nonclassical CAH. The propositus is the older brother, who is compound heterozygous with a mild and severe CYP21 mutation (P30L/R356W). In spite of one mild CYP21 mutation, he presented with the clinical picture of a simple virilizing form of 21-OHD and required glucocorticoid replacement therapy from the age of 4. Both probands’ parents are compound heterozygous carriers of different CYP21 gene mutations causing various degrees of enzymatic activity impairment, which explains the different genotypes and phenotypes in their offspring. The probands’ mother, besides the nonclassical 21-OHD, also had neuroblastoma of the adrenal gland. Conclusion: The potential discordance between the genotype and the phenotype in some patients with CAH is emphasized. The existence of a mild CYP21 mutation P30L in a compound heterozygous with CAH might be associated with progressive virilization requiring glucocorticoid therapy from early childhood. The occurrence of neuroblastoma with 21-OHD may support the hypothesis that an impairment in the synthesis and secretion of glucocorticoids may play role in the development and functioning of the adrenal medulla.

Diversity of bone cell activity as a histomorphometric feature of idiopathic osteoporosis in men

Although osteoporosis in men is an increasing health problem, studies on osteoporosis in males are still scarce. The aim of our study was to determine the characteristics of bone tissue and bone turnover in men with idiopathic osteoporosis. Transiliac crest bone samples were histomorphometrically analyzed after double tetracycline labeling in 32 men aged 37–65 years who were diagnosed with idiopathic osteoporosis by densitometry of the lumbar spine and hip. Bone volume, osteoid surface, osteoblast surface, eroded surface, osteoid thickness, trabecular thickness, trabecular number, trabecular separation, and mineral apposition rate (MAR) were determined in all trabecular bone specimens. Bone volume and structural parameters indicated trabecular bone loss in most patients. Cellular parameters and MAR indicated variations in bone cell actions. No age-related decrease in histomorphometric parameters was found. After the patients were grouped according to MAR values, osteoblast and eroded surfaces were found to be lower in the group with decreased MAR values and elevated in the group of patients with increased MAR parameter. Trabecular thickness was greater in patients with lower than normal MAR, due to reduced resorption and probably loss of very thin trabeculae. Our results suggest that idiopathic osteoporosis in man resembles many characteristics of postmenopausal osteoporosis in women resulting in impaired trabecular structure due to unbalanced cellular activity and bone turnover rate.

The effect of 17 years of increased salt iodization on the prevalence and nature of goiter in Croatian schoolchildren

Abstract Background High goiter prevalence caused by iodine deficiency (medium content 5.6 mg potassium iodide [KI]/kg of salt, median urine iodine concentration [UIC] 68 μg/L) in Croatia was observed in 1991 and 1995 when salt was iodized with 10 mg KI/kg. A new regulation introduced in 1996, specified 25 mg KI/kg of salt resulting in an increase of median UIC to 248 μg/L. Afterwards, goiter prevalence was only assessed in two small studies. Methods In this study, we investigated the prevalence and etiology of goiter in 3594 schoolchildren 17 years after an increase in salt iodization in Croatia. Thyroid size was determined by palpation in 1777 girls and 1817 boys aged 10–18 years. In goitrous children, a thyroid ultrasound and thyroid-stimulating hormone, free thyroxine (fT4), free triiodothyronine (fT3), thyroid peroxidase (TPO) and thyroglobulin (TG) antibody measurements were performed. Results Goiter was found in 32 children (0.89% vs. 2.8% in 1991, p<0.00001 and 27% in 1995, p<0.00001), simple goiter (SG) in 18/32 (56%) goitrous children vs. 126/152 (82.8%) in 1991 p<0.00001, autoimmune thyroiditis (AT) in 13/32 (40.6%) vs. 19/152 (12.5%) in 1991 p<0.0009, nodules in four: two cysts, toxic adenoma and carcinoma (in 1991 two adenomas and one cyst), Graves’ disease was not found (four in 1991). Subclinical hypothyroidism was found in three children. Thyroid disease was diagnosed in four of 32 children before the investigation. Increased iodine supply decreased goiter prevalence and SG/AT ratio in goitrous patients. Conclusions As thyroid abnormalities were found in 0.89% of children and some required treatment, thyroid examination is important in apparently healthy children regardless of sufficient iodization.

Impaired regulation of calcium excretion in kidney transplant recipients.

ZusammenfassungStörungen des Mineralstoffwechsels und Hyperkalzämie sind bei Nierentransplantierten häufig. Der Quotient der Calcium/Kreatinin (Ca/Kr) Clearance wurde als Kriterium zur Unterscheidung von verschiedenen Erkrankungen des Calciumstoffwechsels verwendet. Wir untersuchten 91 Nierentransplantierte (davon 53 Männer) im Alter von 23–70 Jahren mit einer Kr. Clearance von >60 ml/min. Folgende mit dem Mineralstoffwechsel im Zusammenhang stehende Parameter wurden im Serum gemessen: iPTH (Parathormon), gesamte alkalische Phosphatase (tALP), Telopeptid (ein Marker des Knochenabbaus, CTX), 25-OH-Vitamin D3, gesamtes und ionisiertes Calcium, anorganisches Phosphat, Kreatinin. Kr und Ca wurden auch im Harn, ebenso wie der Quotient der Ca/Kr Clearance, bestimmt. Die Patienten wurden entsprechend ihres Ca/Kr Clearance Quotienten in folgende 3 Gruppen geteilt: 1) <0,01: Erkrankungen, die durch gestörten Ca-sensitiven Rezeptor verursacht sind (N = 30); 2) 0,01–0,02: Normale (N = 45); und 3) >0,02: Hyperparathyreoidismus (N = 16). In der Gruppe 1 hatten 7 Patienten eine Hypercalzämie und 4 Patienten eine Hyperkalzämie mit erhöhten PTH Werten. Eine Verschlechterung der renalen Ca Ausscheidung scheint bei Nierentransplantierten auch bei guter Nierenfunktion auftreten zu können. Unverhältnismäßig geringe Calciurie und gestörte Sensitivität des Ca-sensitiven Rezeptors könnten pathogenetische Faktoren für die Hypercalcaämie bei Nierentransplantierten sein.SummaryDisorders of mineral metabolism and hypercalcemia are frequent in kidney transplant recipients. Calcium to creatinine (Ca/Cr) clearance ratio was used as a criterion to distinguish between different calcium metabolism disorders. The study comprised 91 (53 men, 38 women) kidney recipients aged 23–70 years, with creatinine clearance (CrCl) >60 ml/min. The following parameters related to mineral metabolism were measured in serum: iPTH, total alkaline phosphatase (tALP), telopeptide (bone degradation marker, CTX), 25(OH)D3, total and ionized calcium, Ca++ , Pi, creatinine (Cr). Creatinine and Ca were also determined in urine, as well as Ca/Cr clearance ratio. According to the Ca/Cr clearance ratio, patients were divided into three groups as follows: <0.01 (found in disorders caused by reduced calcium-sensing receptor sensitivity, N = 30), 0.01–0.02 (normal value, N = 45), and >0.02 (found in hyperparathyroidism, N = 16). In the group of patients with Ca/Cr clearance ratio <0.01, seven patients had hypercalcemia, and four patients had hypercalcemia and elevated iPTH. It seems that impairment of renal calcium excretion may occur in kidney transplant recipients with good kidney function. Inappropriately low calciuria and impaired sensitivity of calcium-sensing receptor may be pathogenetic factors causing hypercalcemia in kidney transplant recipients.