Thyroid function and dysfunction in preterm infants-Challenges in evaluation, diagnosis and therapy.

Thyroid hormone levels have a crucial role for optimal brain development from gestation through the first 2 postnatal years. However, thyroid hormones vary with gestational age, and their levels vary between term and preterm infants. Preterm newborns are prone to thyroid dysfunction which is now more frequently observed with the advances of neonatal care and improved survival of extremely premature infants. Thus, hypothyroxinaemia of prematurity associated with delayed TSH elevation is very common in low birth weight premature infants most likely due to the immaturity of the hypothalamic-pituitary thyroid axis. Furthermore, postnatal illness, medications and iodine status may contribute to the thyroid dysfunction or affect the interpretation of the thyroid function tests. Despite available guidelines, timing of screening and optimal treatment of thyroid dysfunction in premature infants remains controversial. Furthermore, it is unknown whether untreated thyroid dysfunction in premature babies affects neurodevelopmental outcome. In the vast majority of preterm infants, hypothyroxinaemia is transient; however, permanent hypothyroidism due to thyroid dysgenesis or enzyme defects might also occur. Therefore, careful monitoring of thyroid function and long-term follow-up is needed to assess an appropriate therapeutic approach. This article reviews thyroid physiology in preterm infants, the influences of gestation and other neonatal conditions on thyroid function tests, optimal timing of screening and possible predictors to differentiate transient hypothyroxinaemia from permanent hypothyroidism.

First insights into the genetics of 21-hydroxylase deficiency in the Roma population.

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.

Trends and cyclical variation in the incidence of childhood type 1 diabetes in 26 European centres in the 25 year period 1989-2013: a multicentre prospective registration study.

AIMS/HYPOTHESIS: Against a background of a near-universally increasing incidence of childhood type 1 diabetes, recent reports from some countries suggest a slowing in this increase. Occasional reports also describe cyclical variations in incidence, with periodicities of between 4 and 6 years. METHODS: Age/sex-standardised incidence rates for the 0- to 14-year-old age group are reported for 26 European centres (representing 22 countries) that have registered newly diagnosed individuals in geographically defined regions for up to 25 years during the period 1989-2013. Poisson regression was used to estimate rates of increase and test for cyclical patterns. Joinpoint regression software was used to fit segmented log-linear relationships to incidence trends. RESULTS: Significant increases in incidence were noted in all but two small centres, with a maximum rate of increase of 6.6% per annum in a Polish centre. Several centres in high-incidence countries showed reducing rates of increase in more recent years. Despite this, a pooled analysis across all centres revealed a 3.4% (95% CI 2.8%, 3.9%) per annum increase in incidence rate, although there was some suggestion of a reduced rate of increase in the 2004-2008 period. Rates of increase were similar in boys and girls in the 0- to 4-year-old age group (3.7% and 3.7% per annum, respectively) and in the 5- to 9-year-old age group (3.4% and 3.7% per annum, respectively), but were higher in boys than girls in the 10- to 14-year-old age group (3.3% and 2.6% per annum, respectively). Significant 4 year periodicity was detected in four centres, with three centres showing that the most recent peak in fitted rates occurred in 2012. CONCLUSIONS/INTERPRETATION: Despite reductions in the rate of increase in some high-risk countries, the pooled estimate across centres continues to show a 3.4% increase per annum in incidence rate, suggesting a doubling in incidence rate within approximately 20 years in Europe. Although four centres showed support for a cyclical pattern of incidence with a 4 year periodicity, no plausible explanation for this can be given.

Efficacy and safety of a fixed combination of insulin degludec/insulin aspart in children and adolescents with type 1 diabetes: A randomized trial.

BACKGROUND: Insulin degludec/insulin aspart (IDegAsp) is a fixed soluble co-formulation of basal and bolus insulin. OBJECTIVE: To evaluate efficacy and safety of IDegAsp in pediatric patients with type 1 diabetes (T1D). SUBJECTS: Children and adolescents (aged 1 to <18 years) with T1D. METHODS: A 16-week, phase 3b, treat-to-target, parallel-group, open-label, non-inferiority trial was conducted at 63 sites in 14 countries from October 2013 to November 2014. Patients were randomized 1:1 (age stratified: 1-<6 years; 6-<12 years; 12-<18 years) to IDegAsp once daily (OD) plus insulin aspart (IAsp) for remaining meals (IDegAsp + IAsp), or IDet OD or twice daily plus mealtime IAsp (IDet + IAsp). The primary end-point was HbA1c change from baseline at week 16. RESULTS: A total of 362 participants were randomized to IDegAsp + IAsp (n = 182) or IDet + IAsp (n = 180). HbA1c decreased from baseline to week 16 by 0.3% in both groups (estimated treatment difference: -0.04%-points [-0.23; 0.15]95%CI (-0.45 mmol/mol [-2.51; 1.60]95%CI ), confirming non-inferiority. There were no significant differences between treatment groups in fasting or self-measured plasma glucose. Confirmed hypoglycemia rates did not significantly differ between groups. There was a significant reduction in basal and total insulin dose with IDegAsp + IAsp vs IDet + IAsp (post hoc analysis). Mean number of injections/day was 3.6 and 4.9 with IDegAsp + IAsp and IDet + IAsp, respectively (post hoc analysis). A non-significant higher rate of severe hypoglycemia was observed with IDegAsp + IAsp vs IDet + IAsp. The most frequent adverse events in both groups were hypoglycemia, headache, and nasopharyngitis. CONCLUSIONS: IDegAsp + IAsp was non-inferior to IDet + IAsp regarding HbA1c, had similar hypoglycemia rates and required fewer injections.

Targets and teamwork: Understanding differences in pediatric diabetes centers treatment outcomes.

OBJECTIVE: The reason for center differences in metabolic control of childhood diabetes is still unknown. We sought to determine to what extent the targets, expectations, and goals that diabetes care professionals have for their patients is a determinant of center differences in metabolic outcomes. RESEARCH DESIGN AND METHODS: Children, under the age of 11 with type 1 diabetes and their parents treated at the study centers participated. Clinical, medical, and demographic data were obtained, along with blood sample for centralized assay. Parents and all members of the diabetes care team completed questionnaires on treatment targets for hemoglobin A1c (HbA1c) and recommended frequency of blood glucose monitoring. RESULTS: Totally 1113 (53% male) children (mean age 8.0 ± 2.1 years) from 18 centers in 17 countries, along with parents and 113 health-care professionals, participated. There were substantial differences in mean HbA1c between centers ranging from 7.3 ± 0.8% (53 mmol/mol ± 8.7) to 8.9 ± 1.1% (74 mmol/mol ± 12.0). Centers with lower mean HbA1c had (1) parents who reported lower targets for their children, (2) health-care professionals that reported lower targets and more frequent testing, and (3) teams with less disagreement about recommended targets. Multiple regression analysis indicated that teams reporting higher HbA1c targets and more target disagreement had parents reporting higher treatment targets. This seemed to partially account for center differences in Hb1Ac. CONCLUSIONS: The diabetes care teams' cohesiveness and perspectives on treatment targets, expectations, and recommendations have an influence on parental targets, contributing to the differences in pediatric diabetes center outcomes.

Optic glioma and precocious puberty in a girl with neurofibromatosis type 1 carrying an R681X mutation of NF1: case report and review of the literature.

BACKGROUND: Neurofibromatosis type 1 (NF1) is a common autosomal dominant genetic disorder with an extremely variable phenotype. In childhood NF1 can be associated with optic glioma and central precocious puberty; the latter is more common when the optic chiasm is affected. The mutational spectrum of the NF1 gene is wide and complex; R681X is a rare severe mutation of the NF1 gene known to cause truncation of neurofibromin, with only ten reported cases in the literature so far. CASE PRESENTATION: We describe a girl with NF1 associated with early central precocious puberty appearing at 2.5 years of age and optic glioma affecting the optic chiasm as seen on magnetic resonance imaging (MRI). Genetic analysis confirmed the presence of R681X. Therapy with a gonadotropin-releasing hormone agonist was instituted with good response to therapy. The lesions on MRI were stable and no significant vision impairment was present during the 6 years of follow-up. CONCLUSION: Of the ten reported cases of NF1 due to R681X, one has presented with optic glioma and none with precocious puberty. Thus, to our knowledge, this is the first reported case of this mutation presenting with precocious puberty. We believe that this is a contribution to the few reports on the phenotype of this mutation and to the future elucidation of genotype-phenotype correlations of this disease.

Clinical practice: experience with newborn screening for congenital hypothyroidism in the Republic of Macedonia - a multiethnic country.

UNLABELLED: 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.

Metabolic outcomes in young children with type 1 diabetes differ between treatment centers: the Hvidoere Study in Young Children 2009.

OBJECTIVE: To investigate whether center differences in glycemic control are present in prepubertal children <11 yr with type 1 diabetes mellitus. RESEARCH DESIGN AND METHODS: This cross-sectional study involved 18 pediatric centers worldwide. All children, <11 y with a diabetes duration ≥12 months were invited to participate. Case Record Forms included information on clinical characteristics, insulin regimens, diabetic ketoacidosis (DKA), severe hypoglycemia, language difficulties, and comorbidities. Hemoglobin A1c (HbA1c) was measured centrally by liquid chromatography (DCCT aligned, range: 4.4-6.3%; IFFC: 25-45 mmol/mol). RESULTS: A total of 1133 children participated (mean age: 8.0 ± 2.1 y; females: 47.5%, mean diabetes duration: 3.8 ± 2.1 y). HbA1c (overall mean: 8.0 ± 1.0%; range: 7.3-8.9%) and severe hypoglycemia frequency (mean 21.7 events per 100 patient-years), but not DKA, differed significantly between centers (p < 0.001 resp. p = 0.179). Language difficulties showed a negative relationship with HbA1c (8.3 ± 1.2% vs. 8.0 ± 1.0%; p = 0.036). Frequency of blood glucose monitoring demonstrated a significant but weak association with HbA1c (r = -0.17; p < 0.0001). Although significant different HbA1c levels were obtained with diverse insulin regimens (range: 7.3-8.5%; p < 0.001), center differences remained after adjusting for insulin regimen (p < 0.001). Differences between insulin regimens were no longer significant after adjusting for center effect (p = 0.199). CONCLUSIONS: Center differences in metabolic outcomes are present in children <11 yr, irrespective of diabetes duration, age, or gender. The incidence of severe hypoglycemia is lower than in adolescents despite achieving better glycemic control. Insulin regimens show a significant relationship with HbA1c but do not explain center differences. Each center's effectiveness in using specific treatment strategies remains the key factor for outcome.

Mutational Spectrum and Genotype-phenotype Correlations in Neurofibromatosis Type 1 Patients from North Macedonia: Identification of Ten Novel NF1 Pathogenic Variants

Background: Neurofibromatosis type 1 (NF1) is an autosomal dominant neurocutaneous disorder, characterized by multiple café-au-lait macules, axillary and inguinal freckling, tumors of the nervous system, and iris hamartomas. More than 3,100 different pathogenic variants have been reported in the NF1 gene, including missense, nonsense, frameshift, in-frame, splicing, and large deletions. Aims: To determine the NF1 mutational spectrum in patients with NF1 from the Republic of North Macedonia. Study Design: A cohort study. Methods: Molecular analyses included reverse transcription and cDNA sequencing of the NF1 gene and next-generation sequencing using the TruSight Cancer panel, along with the multiple ligation probe amplification method to detect single nucleotide variants and copy number variations. Direct DNA sequencing was also used for the family member analysis. Results: Our 9-year study of patients suspected of having NF1 in the Republic of North Macedonia encompassed molecular characterization of 30 cases of the disease. We identified 28 unique pathogenic NF1 variants (NM_001042492.3), of which ten were novel: c.208delA; c.341_364del; c.1480_1481delTT; c.2325+1G>C; c.2495_2496dupAC; c.2533_2541del; c.4517delC; c.5844C>G; c.6971delA; c.7605_7606delGAinsAT. In addition to the variant spectrum analysis, our research revealed two positive genotype-phenotype correlations. One between the clinical manifestation of cognitive impairment and gross deletions in the NF1 gene, and the other between cognitive impairment and truncating variants located in the RAS-GAP functional domain. Conclusion: This is the first study of NF1 patients in the Republic of North Macedonia, and it contributes ten novel variants to the global spectrum of pathogenic NF1 variants. It also corroborates the crucial importance of NF1 genetic testing for a prompt and precise diagnosis, particularly in younger patients.

Prevalence of congenital hypothyroidism in North Macedonia: data from a newborn screening program conducted for twenty years.

BACKGROUND: Congenital hypothyroidism (CH) is a common endocrine disorder that can be treated if timely detected by newborn screening, optimizing the developmental outcome in affected children. In the present study, we analyze the data of the national newborn thyroid screening program in North Macedonia collected over twenty years, including the CH prevalence as well as its geographical and ethnic variations. METHODS: The thyroid-stimulating hormone (TSH) was measured on a filter paper blood spot sample using the DELFIA fluoroimmunometric assay. A TSH value of 15 mIU/L whole blood was used as the cutoff point until 2010 and 10 mIU/L thereafter. RESULTS: Out of 377,508 screened live births, a total of 226 newborns with primary CH were detected, providing an overall prevalence of 6.0 per 10,000. Lowering the TSH cutoff led to an apparently increased prevalence of the transient CH, from 0.2 to 2.4 per 10,000 live births (p < 0.0001) with an impact on the overall prevalence of primary CH (from 4.0 to 7.1 per 10,000, p=0.0001). Taking ethnicity into account, the significantly highest primary CH prevalence of 11.3 per 10,000 live births was observed among the Roma neonates, with a predominance of permanent CH (75.5%). There were also regional differences in the prevalence of primary CH. The highest primary CH prevalence of 11.7 per 10,000 live births was observed in the Vardar region, together with the highest regional prevalence of the transient CH (3.2 per 10,000). The highest prevalence of permanent CH was observed in the Pelagonia region (6.6 per 10,000) where the largest percentage of the Roma population lives. CONCLUSIONS: The overall CH prevalence is high in North Macedonia, with substantial ethnic and geographical variations. Further analysis to elucidate the causes for the significant variations in the CH prevalence including environmental factors is warranted.

Unique concurrent appearance of two rare conditions in a young girl: central precocious puberty due to hypothalamic hamartoma and uncommon type of diabetes.

Hypothalamic hamartomas (HH) are rare congenital nonneoplastic lesions of the tuber cinereum, which usually present as precocious puberty of central origin in young girls and respond well to treatment with long acting gonadotropin releasing hormone (GnRH) analogs. No association of this condition with diabetes mellitus of any form has been reported so far. On the other hand, diabetes mellitus in children and adolescents, when it is not autoimmune type 1 diabetes, is difficult to classify. We present a girl with early onset of central precocious puberty at the age of 8 months, due to hypothalamic hamartoma. Treatment with depot of a GnRH analog for a period of 9 years and 8 months was successful, and her puberty continued 6 months after the discontinuation of triptorelin. At the age of 9 years 6 months, the girl presented with diabetes. She was negative for islet, GAD and IA2 antibodies and her insulinemia and C-peptide remained within normal limits during the 2 years of follow-up. Her metabolic control is excellent with a combination of metformin and a low-dose of mixed insulin. To our knowledge, this is the first description of the simultaneous appearance of these two endocrinological conditions.

Characteristics of In2G Variant in Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency.

Substantial research has been performed during the last decades on the clinical and genetic variability of congenital adrenal hyperplasia (CAH) and its most common form, 21-hydroxylase deficiency (21OHD). CAH is one of the most prevalent autosomal recessive diseases in humans, and it can be divided into classic-further subdivided into salt wasting (SW) and simple virilizing (SV)-and non-classic (NC) forms. Pathogenic variants of CYP21A2 gene, encoding the 21-hydroxylase enzyme, have been reported with variable prevalence in different populations. NM_000500.9:c.293-13C/A>G (In2G) variant represents the most common CYP21A2 gene changes related to the classic 21OHD form. However, the phenotype of In2G carriers is variable depending on the variant homozygous/heterozygous status and combination with other CYP21A2 pathogenic variants. In addition, identical genotypes, harboring the homozygous In2G variant, can present with variable phenotypes including the SW and SV or rarely NC form of the disease. Here, we analyze and present the clinical aspects, genotype/phenotype correlations, and other characteristics related to the CYP21A2 In2G variant.

A novel 9 bp deletion (c.1271_1279delGTGCCCGCG) in exon 10 of CYP21A2 gene causing severe congenital adrenal hyperplasia.

BACKGROUND: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Current Status of Newborn Screening in Southeastern Europe.

Significant part of Southeastern Europe (with a population of 76 million) has newborn screening (NBS) programs non-harmonized with developed European countries. Initial survey was conducted in 2013/2014 among 11 countries from the region (Albania, Bulgaria, Bosnia and Herzegovina (BIH), Croatia, Kosovo, Macedonia, Moldova, Montenegro, Romania, Serbia, and Slovenia) to assess the main characteristics of their NBS programs and their future plans. Their cumulative population at that time was ~52,5 million. At that time, none of the countries had an expanded NBS program, while phenylketonuria screening was not introduced in four and congenital hypothyroidism in three of 11 countries. We repeated the survey in 2020 inviting the same 11 countries, adding Cyprus, Greece, Hungary, and Malta (due to their geographical position in the wider region). The aims were to assess the current state, to evaluate the change in the period, and to identify the main obstacles impacting the implementation of expanded NBS and/or reaching a wider population. Responses were collected from 12 countries (BIH-Federation of BIH, BIH-Republic of Srpska, Bulgaria, Croatia, Greece, Hungary, Kosovo, North Macedonia, Malta, Montenegro, Romania, Serbia, Slovenia) with a population of 68.5 million. The results of the survey showed that the regional situation regarding NBS only modestly improved in this period. All of the surveyed countries except Kosovo screened for at least congenital hypothyroidism, while phenylketonuria was not screened in four of 12 countries. Croatia and Slovenia implemented an expanded NBS program using tandem mass spectrometry from the time of last survey. In conclusion, the current status of NBS programs in Southeastern Europe is very variable and is still underdeveloped (or even non-existent) in some of the countries. We suggest establishing an international task-force to assist with implementation and harmonization of basic NBS services where needed.

Neonatal Screening in Europe Revisited: An ISNS Perspective on the Current State and Developments Since 2010.

Neonatal screening (NBS) was initiated in Europe during the 1960s with the screening for phenylketonuria. The panel of screened disorders ("conditions") then gradually expanded, with a boost in the late 1990s with the introduction of tandem mass spectrometry (MS/MS), making it possible to screen for 40-50 conditions using a single blood spot. The most recent additions to screening programmes (screening for cystic fibrosis, severe combined immunodeficiency and spinal muscular atrophy) were assisted by or realised through the introduction of molecular technologies. For this survey, we collected data from 51 European countries. We report the developments between 2010 and 2020 and highlight the achievements reached with the progress made in this period. We also identify areas where further progress can be made, mainly by exchanging knowledge and learning from experiences in neighbouring countries. Between 2010 and 2020, most NBS programmes in geographical Europe matured considerably, both in terms of methodology (modernised) and with regard to the panel of conditions screened (expanded). These developments indicate that more collaboration in Europe through European organisations is gaining momentum. We can only accomplish the timely detection of newborn infants potentially suffering from one of the many rare diseases and take appropriate action by working together.

Disease progression and search for monogenic diabetes among children with new onset type 1 diabetes negative for ICA, GAD- and IA-2 Antibodies.

BACKGROUND: To investigate disease progression the first 12 months after diagnosis in children with type 1 diabetes negative (AAB negative) for pancreatic autoantibodies [islet cell autoantibodies(ICA), glutamic acid decarboxylase antibodies (GADA) and insulinoma-associated antigen-2 antibodies (IA-2A)]. Furthermore the study aimed at determining whether mutations in KCNJ11, ABCC8, HNF1A, HNF4A or INS are common in AAB negative diabetes. MATERIALS AND METHODS: In 261 newly diagnosed children with type 1 diabetes, we measured residual ß-cell function, ICA, GADA, and IA-2A at 1, 6 and 12 months after diagnosis. The genes KCNJ11, ABCC8, HNF1A, HNF4A and INS were sequenced in subjects AAB negative at diagnosis. We expressed recombinant K-ATP channels in Xenopus oocytes to analyse the functional effects of an ABCC8 mutation. RESULTS: Twenty-four patients (9.1%) tested AAB negative after one month. Patients, who were AAB-negative throughout the 12-month period, had higher residual ß-cell function (P = 0.002), lower blood glucose (P = 0.004), received less insulin (P = 0.05) and had lower HbA1c (P = 0.02) 12 months after diagnosis. One patient had a heterozygous mutation leading to the substitution of arginine at residue 1530 of SUR1 (ABCC8) by cysteine. Functional analyses of recombinant K-ATP channels showed that R1530C markedly reduced the sensitivity of the K-ATP channel to inhibition by MgATP. Morover, the channel was highly sensitive to sulphonylureas. However, there was no effect of sulfonylurea treatment after four weeks on 1.0-1.2 mg/kg/24 h glibenclamide. CONCLUSION: GAD, IA-2A, and ICA negative children with new onset type 1 diabetes have slower disease progression as assessed by residual beta-cell function and improved glycemic control 12 months after diagnosis. One out of 24 had a mutation in ABCC8, suggesting that screening of ABCC8 should be considered in patients with AAB negative type 1 diabetes.

Genotype-phenotype correlation in CAH patients with severe CYP21A2 point mutations in the Republic of Macedonia.

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.

Clinical outcomes and characteristics of P30L mutations in congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

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.

Genetics of Gland-in-situ or Hypoplastic Congenital Hypothyroidism in Macedonia.

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.

The impact of CYP21A2 (P30L/I172N) genotype on female fertility in one family.

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.