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European CF Society Neonatal Screening Working Group (ECFS NSWG): Sabine Renner , Max Zeyda , Elke de Wachter , Luc Regal , Felix Votava, Andrea Holubova, Marianne Skov , Tessa Morgan, Paul Bregeaut , Loretta O'Grady, Ines Bucci , Stefano Pantano , Simonetta Simonetti, Domenica De Venuto, Donatello Salvatore, Nicola Perrotti, Mimma Caloiero, Giuseppe Castaldo, Antonella Tosco, Francesca Righetti, Giovanna Pisi, Fiorella Battistini, Antonio Angeloni, Giuseppe Cimino, Giovanni Fiocchi, Antonella Angiolillo, Michela Cassanello, Luisella Alberti, Laura E Claut, Raffaele Badolato, Enza Pavanello, Benedetta Fabrizzi, Elisabetta Bignamini, Anna Cardillo, Mariangela Lombardo, Letizia Cocciadiferro, Lisa Termini, Daniela Dolce, Vito Terlizzi, Anna Tamanini, Francesca Pauro, Giancarlo la Marca, Elina Aleksejeva, Dita Gaidule-Logina, Stoika Fustik, Violeta Anastasovska, Marelle Bouva, Alastair Reid, Jennifer Cundick, Emma Lundman, Egil Bakkeheim, Katarzyna Zybert, Mariusz Oltarzewski, Laura Vilarinho, Victoria Sherman, Elena Kondratyeva, Sarah Smith, Gordana Vilotijevic Dautovic, Maria Knapkova, Zuzana Mydlova, Rosa Mª López, Valle Velasco, Felicitas Díaz Flores, Cristóbal Colón Mejeras, Eva Sl Pedersen, Ugur Ozcelik, Bulent Karadag, Halyna Makukh, Moat Stuart. European CF Society Neonatal Screening Working Group (ECFS NSWG): INSA - Laura Vilarinho Background: The aim of this study was to record the current status of newborn bloodspot screening (NBS) for CF across Europe and assess performance. Methods: Survey of representatives of NBS for CF programmes across Europe. Performance was assessed through a framework developed in a previous exercise. Results: In 2022, we identified 22 national and 34 regional programmes in Europe. Barriers to establishing NBS included cost and political inertia. Performance was assessed from 2019 data reported by 21 national and 21 regional programmes. All programmes employed different protocols, with IRT-DNA the most common strategy. Six national and 11 regional programmes did not use DNA analysis. Conclusions: Integrating DNA analysis into the NBS protocol improves PPV, but at the expense of increased carrier and CFSPID recognition. Some programmes employ strategies to mitigate these outcomes. Programmes should constantly strive to improve performance but large datasets are needed to assess outcomes reliably. Highlights: In 2022, newborn bloodspot screening (NBS) for CF is undertaken in 30 European countries, 26 of them are national programmes; Some programmes are still not achieving ECFS standards. Compared to 2014, there is an improvement in sensitivity but a deterioration in achieving a sufficient PPV; There continues to be a wide variety of approaches, but the majority of national programmes are now using DNA analysis as a 2nd tier; This survey demonstrates areas of good practice, but there is considerable scope for improvement in the quality of NBS for CF across Europe; The framework of the 20 parameters to calculate the 8 key outcomes should be part of any annual report of a CF NBS programme, and thus improve future surveys. The survey was funded by the European CF Society info:eu-repo/semantics/publishedVersion

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder of adrenal steroidogenesis with a broad spectrum of clinical presentations, ranging from the severe classical salt-wasting (SW) and simple-virilizing (SV) form, to the mild nonclassical form. A large variety of CYP21A2 genotypes in correlation with phenotype have been described. DNA samples from a 14-day-old male newborn with clinical and laboratory signs of SW CAH and family members were subjected for molecular analysis of the nine most common point CYP21A2 mutations by ACRS/PCR method. Direct DNA sequencing of the whole CYP21A2 gene was performed to detect the second mutant allele in the patient. The in silico predicting analysis and the crystal structure analysis of the mutated CYP21A2 protein have been performed. Molecular analysis confirmed that the patient was compound heterozygote carrying p.Q318X mutation inherited from the mother and a novel c.1271_1279delGTGCCCGCG (p.G424_R426del) variant in exon 10 inherited from the father. The in silico predicting software tools classified the novel mutation as pathogenic. Crystal structure analysis showed that the three residues affected by the novel in-frame deletion form several hydrogen bonds that could lead to impaired stability and function of the CYP21A2 protein. These findings were concordant with the patient’s phenotype. The need of several molecular methods to elucidate the genotype in this patient has also been discussed. A novel 9 bp deletion in CYP21A2 gene with predicted pathogenic effect on the enzyme activity was detected in neonatal patient causing severe SW CAH.

Background 21-hydroxylase deficiency (21OHD) is an autosomal recessive disorder with an incidence of 1:10,000-1:20,000 and is the result of various mutations in the CYP21A2 gene. 21OHD has been described in many different populations, but it has not been studied in Roma individuals so far. The aim of the study was to analyse the genotype in Roma patients with 21OHD and the prevalence of the disease in the Roma population of North Macedonia. Methods Molecular analysis of the nine most frequent CYP21A2 mutations in all known Roma patients with CAH in North Macedonia, relatives and healthy individuals of Roma ancestry, using the PCR/ACRS method. Results Ten Roma patients with 21OHD were identified, of which nine had the salt-wasting and one had the simple virilizing form. Calculated incidence of 21OHD in the North Macedonian Roma population was 1:3375. Interestingly, 9/10 patients (90%) were homozygous for the In2G splicing mutation (293-13A/C > G). Standard therapy with hydrocortisone and fludrocortisone had been introduced according to the guidelines. In 16 healthy relatives investigated for CYP21A2 mutations, heterozygosity for the In2G mutation was detected in 13/32 (40.6%) alleles. In 100 healthy Roma individuals, none related to the analysed families, no CYP21A2 mutations were detected. Conclusion The Roma population in North Macedonia had a very high incidence of classic 21OHD. Almost all patients had the severe salt-wasting form and the In2G/In2G genotype.

Despite numerous studies in the field of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency, some clinical variability of the presentation and discrepancies in the genotype/phenotype correlation are still unexplained. Some, but not all, discordant phenotypes caused by mutations with known enzyme activity have been explained by in silico structural changes in the 21-hydroxylase protein. The incidence of P30L mutation varies in different populations and is most frequently found in several Central and Southeast European countries as well as Mexico. Patients carrying P30L mutation present predominantly as non-classical CAH; however, simple virilizing forms are found in up to 50% of patients. Taking into consideration the residual 21-hydroxulase activity present with P30L mutation this is unexpected. Different mechanisms for increased androgenization in patients carrying P30L mutation have been proposed including influence of different residues, accompanying promotor allele variability or mutations, and individual androgene sensitivity. Early diagnosis of patients who would present with SV is important in order to improve outcome. Outcome studies of CAH have confirmed the uniqueness of this mutation such as difficulties in phenotype classification, different fertility, growth, and psychologic issues in comparison with other genotypes. Additional studies of P30L mutation are warranted.

Background Steroid 21-hydroxylase deficiency is an autosomal recessive disorder, present in 90–95% of all cases with congenital adrenal hyperplasia (CAH). The classical simple virilizing (SV) form of the disease causes virilization of the external genitalia in newborn females and pseudo-precocious puberty in both sexes, due to reactive androgen overproduction. Case presentation We describe a 3.5-year-old girl presenting with pubarche, P2 according to Tanner, advanced bone age of 6 years and 10 months, and high serum levels of 17-hydroxyprogesterone (17-OHP). Molecular analysis of the nine most common pseudogene-derived CYP21A2 point mutations was performed in the patient and her family members using the polymerase chain reaction/amplification-created restriction site (PCR/ACRS) method. We detected the P30L/I172N genotype in the patient. She had inherited a mild P30L mutation from her mother and a severe I172N mutation from her father. Conclusions Although the CAH phenotype is determined by the allele that produces most of the enzyme activity and the mild non-classical (NC) phenotype should be expected, the mild P30L known to be more virilizing probably induced the classical SV phenotype in our patient. A continuous regimen of hydrocortisone at a recommended dose failed to decrease the 17-OHP sufficiently. Careful tapering of the dose did not help, and her pubic hair advanced to P3 according to Tanner. Individually tailored treatment is warranted in this patient.

Neonatal screening in Macedonia detects congenital hypothyroidism (CH) with an incidence of 1 in 1,585, and more than 50% of cases exhibit a normally located gland-in-situ (GIS). Monogenic mutations causing dyshormonogenesis may underlie GIS CH; additionally, a small proportion of thyroid hypoplasia has a monogenic cause, such as TSHR and PAX8 defects. The genetic architecture of Macedonian CH cases has not previously been studied. We recruited screening-detected, non-syndromic GIS CH or thyroid hypoplasia cases (n = 40) exhibiting a spectrum of biochemical thyroid dysfunction ranging from severe permanent to mild transient CH and including 11 familial cases. Cases were born at term, with birth weight >3,000 g, and thyroid morphologies included goiter (n = 11), thyroid hypoplasia (n = 6), and apparently normal-sized thyroid. A comprehensive, phenotype-driven, Sanger sequencing approach was used to identify genetic mutations underlying CH, by sequentially screening known dyshormonogenesis-associated genes and TSHR in GIS cases and TSHR and PAX8 in cases with thyroid hypoplasia. Potentially pathogenic variants were identified in 14 cases, of which four were definitively causative; we also detected digenic variants in three cases. Seventeen variants (nine novel) were identified in TPO (n = 4), TG (n = 3), TSHR (n = 4), DUOX2 (n = 4), and PAX8 (n = 2). No mutations were detected in DUOXA2, NIS, IYD, and SLC26A7. The relatively low mutation frequency suggests that factors other than recognized monogenic causes (oligogenic variants, environmental factors, or novel genes) may contribute to GIS CH in this region. Future non–hypothesis-driven, next-generation sequencing studies are required to confirm these findings.

Introduction Chronic periodontitis is as an inflammatory disease of the supporting tissue of the teeth caused by periopatogens microorganisams. New concept about etiology of periodontal disease suggests that herpes viruses may play an important role in the pathogenesis of periodontal disease. The aim of this study was to analyze the presence of herpes simplex virus type 1and 2 (HSV-1, HSV-2) cytomegalovirus (HCMV) and Epstein-Barr virus (EBV), in supra-gingival dental plaque .Materials and Methods The study comprised a total of 89 patients who were divided in two groups: patients who were diagnosed moderate stage of periodontal disease and patients who were diagnosed with advanced stage of periodontal disease. Clinical examinations included determination of plaque index, gingival index, sulcus bleeding index, probing depth, and clinical attachment level. Supra-gingival dental plaque samples were taken with sterile coton ball scrub on tooth enamel. Molecular detection of HSV1, HSV2 , EBV and CMV was analyzed using polymerase chain reaction (PCR).Results Molecular analysis of HSV-1 ,HSV-2, EBV and CMV in supra-gingival dental plaque in patients with chronic periodontal disease (a total of 89) showed the presence of EBV in 12,40% of patients , HSV-1 was found in 11,20% of patients, in 7,90% of patients was detected HSV-2, CMV was established in 9,00% of patients. In 3,40% patients in supra-gingival dental plaque was detected combination of viruses HSV1 and CMV, in 3,40% of patient combination of HSV1 and EBV ; and in 2,20% of patient combination ofHSV2,EBV was observed; in 2,20% of patients was found combination of 3 viruses HSV1; HSV2; EBV and in 49,50% patients we found negative finding. Patients with moderate stage of periodontal disease have a 0,78 times(OR = 0,78 / 0,33-1,84/)no significantly lower risk of probability of finding viruses in the supra-gingival plaque compared to patients who had advanced stage of periodontal disease. In the shown cross-culture of the presence of viruses in supra-gingival plaque in patients with moderate and advanced stage of periodontal disease for Fisher, s Exact Test = 5,19 and p >0.05 (p = 0.809 / Monte Carlo sig. (0.799-0.819)there is no significant difference.Conclusion We can associate the presence of herpes simplex viruses in supra-gingival dental plaque with periodontal disease. Our findings suggest that there is no significant difference in presence of viruses in supra-gingival plaque in patients with moderate and sereve stage of periodontal disease.

Background Diagnostic re-evaluation is important for all patients with congenital hypothyroidism (CH) for determining the etiology and identifying transient CH cases. Our study is a first thyroxine therapy withdrawal study conducted in Macedonian CH patients for a diagnostic re-evaluation. We aimed to evaluate the etiology of CH, the prevalence of transient CH and identify predictive factors for distinguishing between permanent (PCH) and transient CH (TCH). Materials and methods Patients with CH aged >3 years underwent a trial of treatment withdrawal for 4 weeks period. Thyroid function testing (TFT), ultrasound and Technetium-99m pertechnetate thyroid scan were performed thereafter. TCH was defined when TFT remained within normal limits for at least 6-month follow-up. PCH was diagnosed when TFT was abnormal and classified according the imaging findings. Results 42 (55%) patients had PCH and 34 (45.0%) patients had TCH. Thyroid agenesia was the most prevalent form in the PCH group. Patients with TCH had lower initial thyroid-stimulating hormone (TSH) values (P P = 0.0023) and lower mean doses of levothyroxine during treatment period (P Conclusion TCH presents a significant portion of patients with CH. Initial TSH value and levothyroxine dose during treatment period has a predictive role in differentiating TCH from PCH. Earlier re-evaluation, between 2 and 3 years age might be considered in some patients requiring low doses of levothyroxine.

Background The simple virilizing (SV) form of congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder usually caused by steroid 21-hydroxylase deficiency due to I172N missense mutation at the CYP21A2 gene. Clinical presentation encompasses virilization of external genitalia in newborn females and pseudoprecocious puberty in both sexes, due to reactive androgen overproduction. The aim of this study was to present two sisters with an SV form of CAH and distinctive genotype, detected and treated since childhood with a poor compliance and poor metabolic control hindering the fertility. Case presentation We retrospectively reviewed the clinical, biochemical, and molecular data of two sisters with CAH a 46,XX karyotype when they reached an age of 35 and 38 years, respectively, and were attempting conception for several years. They had been diagnosed with SV form of CAH at the age of 7 and 9 years, respectively, by the standard clinical and biochemical procedures, presenting with severe virilization due to androgen excess. Follow-up was performed through standard methods of measurement of 17-OHP, testosterone, and ACTH. Clitoroplasty with vaginoplasty was performed at the age of 18 in the older sister. Using PCR/ACRS, we performed molecular analysis of the nine most common point CYP21A2 mutations in the patients and family members. The P30L/II72N genotype was observed in both sisters. They had inadequate metabolic control due to noncompliance until decision to conceive. IVF was performed three times in the older sister without success. Sufficient follicles were harvested and fertilized; however, the embryos were lost 3–5 days after implantations. The younger sister is preparing for IVF. She underwent follicle harvesting and the embryos were frozen awaiting appropriate hormonal balance for embryo transfer. The I172N mutation in the heterozygote state was observed in their other two sisters, whose fertility was unaffected. Conclusions Despite significant improvements over the last years in achieving fertility in female patients with SV CAH, it is highly dependent upon the severity of virilization and the metabolic control. The role of P30L mutation in infertility and unsuccessfully assisted reproduction remains to be elucidated.

Iodine deficiency is associated with goiter and impaired brain function leading to cretinism. An increased frequency of thyroid-stimulating hormone (TSH) measurements above 5 mlU/L on newborn screening points toward an impaired iodine status of the population.A 13-year retrospective analysis was performed in 228,266 newborns participating in the national thyroid newborn screening program. The TSH concentration was measured in dry blood spots collected by heel stick on filter paper, 48 hours after birth, using fluoroimmunometric DELFIA method.Out of 236,378 live-born infants, 228,266 (96.6%) have been screened for TSH, of which 198,213 (86.8%) were retrospectively evaluated for TSH levels above 5 mlU/L. Neonates with congenital hypothyroidism, prematurity, and low birth weight were excluded from the evaluation, as well as the inadequately sampled neonates (13.2%). A national prevalence of 3.08% newborns (n=6,105) with TSH5 mIU/L was found. Higher percentages were noted in two regions of the country, indicating possible mild iodine deficiency in these regions and shifting the overall average to above 3%.Our results indicate overall iodine sufficiency in the Macedonian population. Additional assessment of the iodine intake in the regions with suspected mild iodine deficiency is needed to prevent suboptimal cognitive and psychomotor outcomes.Nedostatak joda je povezan sa strumom i kompromitovanom funkcijom mozga sa konsekutivnim kretenizmom. Povecćana učestalost nivoa tireostimulišućeg hormona (TSH) iznad 5 mU/L dobijena na skriningu novorolenih govori o poremecenom statusu joda u stanovništva.Uralena je retrospektivna analiza kod 228,266 no- vorolenčadi koja su tokom proteklih 13 godina bila obuhvaćena nacionalnim neonatalnim tiroidnim skrining programom. Koncentracija TSH je merena u suvim kapima krvi pokupljene iz pete novorolencadi na filter hartiji, 48časova nakon rolenja, koristeći fluoroimunometrijsku DELFIA metodu.Od 236,378 živorodenih, 228,266 (96,6%) testi- rani su za TSH, od kojih su 198,213 (86,8%) retrospek-tivno analizirani za nivo TSH iznad 5 mU/L. Novorolencčad sa urolenom hipotireozom, preranim rolenjem i niskom porolajnom tezinom, kao i novorolencad sa neadekvatno sakupljenim uzorcima krvi (13,2%) isključena su iz pro - cene. Dobijena je nacionalna prevalencija od 3,08% novorolenčadi (n=6,106) sa TSH5 mU/L. Veći procenat od prosečnog je zabeležen u dva regiona u zemlji, ukazujući namoguć blagi nedostatak joda u ovim regionima.Naži rezultati ukazuju na sveukupnu dovoljnost joda kod makedonskog stanovništva. Dodatna procena unosa joda u regionima sa sumnjom na blagi nedostatak joda je potrebna da bi se omogucio normalan kognitivni i psihomotorni razvoj kod dece.

BACKGROUND: Periodontal disease is an inflammatory-destructive condition of the supporting tissues of the teeth. Microorganisms found in the dental plaque were considered to be the primary local etiologic factor responsible for the periodontal destruction. It is also evident that herpes simplex viruses may have an impact in the etiopathogenesis of periodontal disease. AIM: This study has been made with the aim to analyse the prevalence of herpes simplex virus type 1 (HSV-1) in the dental plaque (supra- and subgingival) of patients with the chronic periodontal disease. MATERIAL AND METHODS: The study comprised a total of 89 patients with chronic periodontal disease divided into two groups (patients with moderate and severe periodontitis). Supragingival dental plaque samples were taken with sterile cotton (supragingival), and subgingival dental plaque samples were taken with paper absorbents. Samples were subjected to extraction of DNA and further analysis with multiplex PCR for the presence of herpes viral DNA. RESULTS: HSV-1 virus was detected In 24.7% of all patients included in the study. HSV-1 was detected in 22.2% of patients with the moderate stage of the disease, of which in all (100%) in the supragingival plaque samples and only 16.7% in subgingival plaque samples. In two patients HSV-1 was concomitantly detected in supra and subgingival plaque samples. In patients with advanced stage of the disease, the HSV-1 virus was detected in 28.6% patients. In two of the patients, HSV-1 was concomitantly detected in supra and subgingival plaque samples. Statistically, a significant difference was found in HSV-1 positive patients with a moderate stage of disease, between the presence of the virus in subgingival (100%) and subgingival (16.7%) dental plaque samples, p < 0.05. CONCLUSION: Herpes simplex viruses type 1 are present in supragingival and subgingival dental plaque.

Background Testicular adrenal rest tumors (TARTs) are found in 30–94% of adult males with congenital adrenal hyperplasia (CAH). We sought to explore TART appearance through yearly ultrasound examination of testes in young boys with CAH, and its association with metabolic control and genetic mutations. Methods Twenty-five boys with 21-hydroxylase deficiency in the age group 4–18 years diagnosed during the period 2001–2016 were included in the study. ACTH, 17-hydroxyprogesterone, androstenedione and testosterone were measured at 4-month intervals. Growth and BMI were assessed at the time of evaluation. PCR/ACRS method was used for CYP21A2 gene analysis. Testicular ultrasound examination was performed yearly. Results TARTs were detected by ultrasound in 8 children at the age of 6–16 years (13.2 years average). Five had salt-wasting form, two had simple virilizing form and one had non-classic form of CAH. Significant differences in the17OHP and androstenedione levels were detected between the boys, adherent and non-adherent to therapy. Inadequate metabolic control was not different in boys with and without TART (11/17 and 5/8 respectively). No significant difference was detected in the distribution of genetic mutations or adherence to therapy between patients with and without TARTs. One patient had a mutation not reported thus far in TART and another developed leukemia. Conclusion TART is not rare in young boys with CAH, irrespective of the specific mutation or metabolic control. Ultrasound screening helps timely diagnosis and adjustment of therapy.

Lower cutoff levels in screening programs have led to an increase in the proportion of detected cases with transient hypothyroidism, leading to increase of the overall incidence of primary congenital hypothyroidism (CH) in several countries. We have performed retrospective evaluation on the data from 251,008 (96.72%) neonates screened for thyroid-stimulating hormone (TSH) level in dried blood spot specimens taken 48 hours after birth, between 2002 and 2015, using DELFIA method. A TSH value of 15 mIU/L was used as the cutoff point until 2010 and 10 mIU/L thereafter. Primary CH was detected in 127 newborns (1/1976) of which 81.1% had permanent and 18.9% had transient CH. The incidence of primary CH was increased from 1/2489 until to 2010 to 1/1585 thereafter (p=0.131). However, the incidence of permanent CH was slightly increased (p=0.922), while the transient CH incidence had 8-fold increasing after lowering the TSH cutoff level (p

Summary Background: Steroid 21-hydroxylase deficiency is present in 90-95% of all cases with congenital adrenal hyperplasia (CAH), an autosomal recessive disorder. It can present as the severe classical salt wasting (SW) or simple virilising (SV) form, or the milder, nonclassical form. Nine pseudo- gene-derived point mutations account for about 80% of all defects in the CYP21A2 gene coding the 21-hydroxylase enzyme. Methods: We have studied nine CYP21A2 point mutations in 61 Macedonian and 24 Serbian patients with different clinical presentations of CAH, using the PCR/ACRS method. Results: Six different mutations were detected in 71.3% of alleles of the Macedonian patients. The most prevalent mutation was IVS2. Mutations were detected in 85.4% of the SW, 83.4% SV and 47.7% LO alleles. In the Macedonian patients the most common genotype was IVS2/IVS2. Five different mutations were detected in 64.6% of alleles of the Serbian patients. The most preva- lent was P30L. Mutations were present in 83.3% SW, 80% SV and 50% of the LO alleles. In the Serbian patients, the P30L/P30L genotype was the most frequent. Conclusions: Specific CYP21A2 mutations are involved in different clinical forms of CAH. High frequency of P30L was found in both populations. Also, high prevalence of the mild P30L mutation was found in both the Macedonian and Serbian classical SV patients. Our findings support the role of the P30L mutation in pronounced virilisation. An unusual finding is the low frequency of V281L in the Macedonian non-classical patients and its absence in the ones from Serbia.

Background:Congenital hypothyroidism (CH) is a common and preventable cause of intellectual disability for which early diagnosis is difficult without newborn screening. Genetic and environmental factors, race, ethnicity, sex, and pregnancy outcomes were noted as risk factors. In the study we aimed to determine the incidence of CH among different ethnic groups in the capital of Macedoina – a multiethnic city.Methods:A 14-year retrospective cohort analysis was performed on 121,507 newborns in the capital of Macedonia, Skopje, screened for whole-blood thyroid-stimulating hormone (TSH), in dry blood spots collected 48–72 h after birth, during the period 2002–2015. A TSH value of 15 mIU/L was used as cutoff point until 2010 and 10 mIU/L thereafter.Results:Primary CH was detected in 46 newborns (female to male ratio 1.3) with overall incidence of 3.8/10,000 (1/2641). The incidence of primary CH was significantly increased after lowering the TSH cutoff value (p=0.038), primarily due to detected neonates with transient CH for this period. Ethnic differences in the incidence of primary CH were detected. CH incidence among Roma neonates (6.7/10,000) was significantly higher (pConclusions:Increased incidence of CH in Roma newborns was detected as compared to other ethnicities in the capital of Macedonia. Further analysis of factors in direct interrelationship with the increased CH incidence in Roma newborns, as well as elucidation of impact of the CH incidence in this ethnicity on the overall incidence in Skopje, is warranted.

BACKGROUND The essential role of thyroid hormones for normal brain development during a critical period of life is well established. Neonatal screening programs for congenital hypothyroidism (CH) enable early detection of patients and attainment of euthyroid status as quickly as possible. Adequate and accurate monitoring of thyroid function is necessary. In the study we aimed to determine the periodicity of thyroid function testing in the first year of life (3-month intervals versus

To evaluate the thyroid screening program and to estimate the prevalence of congenital hypothyroidism (CH) among newborns in the Republic of Macedonia, we measured thyroid-stimulating hormone (TSH) levels in dried blood spot specimens using the DELFIA fluoroimmunoassay, over a period of 12 years. The TSH cutoff level was 10 mU/L blood. A total of 215,077 newborns were screened (94.76 %). Out of 254 recalled newborns (a recall rate of 0.15 %), 83 newborns with CH were detected, yielding a CH prevalence at screening of 1/2,591 (female to male ratio, 1.86:1). Of the CH cases, 47/107,754 (56.6 %) neonates were Macedonian, 29/70,330 (34.9 %) were Albanian, and 7/15,055 (8.4 %) were Roma. The thyroid gland was undetectable on ultrasound in 43 (51.8 %) newborns with CH, thyroid hypoplasia was confirmed in 8 (9.6 %), while 29 (34.9 %) had a normal thyroid gland. In three newborns (3.6 %), agenesis of one lobe was confirmed. Therapy with levothyroxin was initiated on average 11.7 days after birth. Conclusion: The national thyroid newborn screening program in Macedonia has been successful and effective, providing timely diagnosis and treatment of children with congenital hypothyroidism.

Deficiency of 21-hydroxylase is present in 90-95% cases of congenital adrenal hyperplasia (CAH), an autosomal recessive disorder. Eleven common pseudogene-derived mutations account for approximately 95% of all affected CYP21A2 alleles in all three clinical forms of the disease.To analyse the detected heterozygotes during the molecular analysis of eleven CYP21A2 common pseudogene-derived point mutations in Macedonian CAH patients and their relatives, using the PCR-ACRS protocol.We performed direct molecular detection of CYP21A2 mutations: p.P30L, IVS2-655 C/A→G, G110Δ8nt, p.I172N, p.I236N, p.V237E, p.M239K, p.F306+t, p.V281L, p.Q318X and p.R356W, in 51 CAH Macedonian patients and their 70 healthy relatives (parents and siblings), using the differential PCR-ACRS protocol.Six of the analysed mutations were detected in 29.4% (15/51) of the patients, in the heterozygous state, with the following distribution: IVS2-655 C/A→G (13.7%), p.P30L (11.8%), p.Q318X (9.8%), p.I172N (3.9%), p.R356W (3.9%) and p.V281L (1.96%). Six cases (6/15) were compound heterozygotes and nine (9/15) were simple heterozygotes. Genotype-phenotype correlation was observed in all of our detected compound heterozygote patients. Their clinical presentation was correlated with the less severely mutated allele. Four of the analysed mutations (p.P30L 20%, IVS2-655 C/A→G 12.9%, p.Q318X 7.1% and p.R356W 2.9%) appeared in thirty healthy relatives (42.9%), in the heterozygous state.Distribution of the analysed CYP21A2 mutations among CAH patients and their relatives is comparable to studies from other populations. As expected, high carrier frequency of alleles causing 21-hydroxylase deficiency was observed in relatives of CAH patients.

UNLABELLED Steroid 21-hydroxylase deficiency is a most frequent cause of congenital adrenal hyperplasia (CAH), due to mutations in the CYP21A2 gene. Approximately 75% of patients with classical form of CAH have severe impairment of 21-hydroxylase activity. METHODS We have performed direct molecular diagnosis of the nine common CYP21A2 point mutations in 24 Macedonian CAH patients from 20 unrelated families, using differential PCR and ACRS. RESULTS Five of the analysed mutations were detected in 23 patients: 15 patients were homozygous for one mutation, four patients were compound heterozygotes and four patients were heterozygotes. The most common was IVS2-13A/C mutation found in 60.4% of the alleles, followed by Q318X (22.9%), R356W (4.2%), V281L (2.1%) and P30L (2.1%). The concordance of genotype to phenotype in the patients was 83.3% with complete concordance in the genotypes predicting the SW and SV phenotype. CONCLUSION The distribution of the detected mutations in the Macedonian CAH patients was similar with those described in other European populations. The genotype-phenotype correlation observed in our patients strengthens the fact that the genotype cannot be completely predictive of phenotype.

Objective: Thyroid ectopy is a rare condition resulting from abnormal embryologic development and migration of the gland. Sublingual is the most common thyroid ectopy; all other ectopic thyroid locations occur very rare. There are no reports in the literature that describe the clinical course of patients with congenital hypothyroidism due to thyroid ectopy. Methods and Results: We present a child with congenital hypothyroidism detected on neonatal screening which had a subclinical course during follow-up. Scintigraphy revealed submental thyroid ectopy, a rare ectopic location and no orthotopic thyroid gland. Conclusion: Our case is unique because of the rare ectopic thyroid location but also of the unexpected clinical course; however, further thyroid monitoring is required for the therapy adjustment and detection of any changes in the ectopic tissue.

It has been well documented that hypercholesterolemia represents both a common and a dominant, although non-obligatory, risk factor in the progression of atherosclerosis. Research was conducted upon experimentally induced hyperlipidemic animals by means of a custom-tailored atherogenic diet. Cell susceptibility to nonenzyme-induced oxidative stress appears to be influenced by membrane fatty acid composition. This study was undertaken to determine whether differences in lipid peroxidation in steady-state and induced lipid peroxidation is a result of a different fatty acid supplementation. Adult Wistar strain rats of male gender were exposed to an atherogenic diet for a period of 160 days, before randomization into 6 dietary groups with different intragastral oil supplementation. Lipid peroxidation products were measured in 2.5% (w/v) of fresh liver homogenates (Tris-HCl, pH 7.4), by the assay of a thiobarbituric acid reactive substances (TBARS) formation using the procedures described by Okhawa (1979), including modifications (1989) in three different experimental conditions: steady-state (which corresponds to concentration of lipid peroxides in vivo ), spontaneous and metal-stimulated lipid peroxidation. Results were expressed as nmol TBARS per g of liver homogenate, calculated from the absorbency at 532 nm, using TEP as an external standard. This study shows that prolonged atherogenic dietary treatment causes moderate hypercholesterolemia and enhanced hypertriglyceridemia (+34.1% and +114.8, p0.001, respectively). Despite the lowering effects of the lipoprotein profiles, resulting from a fatty acid supplementation, at the end of each supplementation period, omega-6 fatty acids (soybean and corn oil) revealed an enhancement in the production of lipid peroxides (TBARS formation) measured in steady-state levels (+22.9%, p0.05 and +22.6%, p0.05, respectively). When liver homogenates were exposed to Fe2+ and ascorbic acid-induced oxidative stress, lipid peroxidation (LPO) was enhanced in the group treated with soybean oil (omega-6) and fish oil (omega-3; +48.4 %, p0.001 and +44.1%, p0.001, respectively), but not in the group receiving corn oil. The achieved results support the hypothesis that the process of lipid peroxidation is not always in correlation with the number of double bonds in fatty acids esterified in phospholipid molecules. Consequently, it can be concluded that supplementation of unsaturated fatty acids, in the therapy of cardiovascular diseases, should include the administration of antioxidants, in order to prevent fatty acid decomposition in the case of oxidative insult.

The enzyme glucose-6-phosphate dehydrogenase (G6P-DH, EC.1.1.1.49) catalyzes the oxidation of glucose-6-phosphate in 6-phosphogluconat which is indispensable in the defence of erythrocytes from oxidative insult. The aim of this study was to examine the influence of commonly used drugs in our medical practice, amoxiclav (amoxicillin-clavulanate combination) and lidaprim (trimethoprim-sulfamethrole combination) respectively, upon the erythrocyte G6P-DH activity in experimental rats. In addition, the effect of the toxic drug 1-chloro-2,4-dinitrobenzen (CDNB) on the activity of G6P-DH was examined. The experiment was conducted in fresh blood haemolysates of white laboratory rats, Wistar type, of both genders (n=80). The enzyme activity was determined by "Boehringer-Mannheim" diagnostic assay kits (Kornberg et al., 1955). However, the measured enzyme activity in the control group of rats was found to be a statistically insignificant difference between the genders (140.2 +/- 21.2 mU/10(9)Er in male rats, 144.3 +/- 20.6 mU/10(9) in the female group). Hence, the established enzyme activity does not differ from the activity of the same enzyme in healthy human subjects. The administered dose of lidaprim did not affect the activity of G6P-DH in the treated group of rats, thus attaining levels similar to the control group. By contrast, amoxiclav administration provoked a significant reduction in enzyme activity of 13.6% in male and 19.5% in female rats (p0.001), while the treatment with CDNB significantly increased the activity of the latter to 49.7% in male and 30.1% in female rats (p0.001) in comparison with the control ones. Testing of haemolitical potential is strongly recommended prior to the use of new drugs, particularly in the Mediterranean region, were this enzymopathy is found to be frequent bearing in mind that there is an established list of drugs which affect the G6P-DH activity in the erythrocytes. The above-mentioned method may be used in experimental animal models allowing for administration of a wider selection of drugs in this type of research.