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TRIO gene; Macrocephaly; Phenotype Gen TRIO; Macrocefàlia; Fenotip Gen TRIO; Macrocefalia; Fenotipo The TRIO gene encodes a rho guanine exchange factor, the function of which is to exchange GDP to GTP, and hence to activate Rho GTPases, and has been described to impact neurodevelopment. Specific genotype-to-phenotype correlations have been established previously describing striking differentiating features seen in variants located in specific domains of the TRIO gene that are associated with opposite effects on RAC1 activity. Currently, 32 cases with a TRIO gene alteration have been published in the medical literature. Here, we report an additional 25, previously unreported individuals who possess heterozygous TRIO variants and we review the literature. In addition, functional studies were performed on the c.4394A > G (N1465S) and c.6244-2A > G TRIO variants to provide evidence for their pathogenicity. Variants reported by the current study include missense variants, truncating nonsense variants, and an intragenic deletion. Clinical features were previously described and included developmental delay, learning difficulties, microcephaly, macrocephaly, seizures, behavioral issues (aggression, stereotypies), skeletal problems including short, tapering fingers and scoliosis, dental problems (overcrowding/delayed eruption), and variable facial features. Here, we report clinical features that have not been described previously, including specific structural brain malformations such as abnormalities of the corpus callosum and ventriculomegaly, additional psychological and dental issues along with a more recognizable facial gestalt linked to the specific domains of the TRIO gene and the effect of the variant upon the function of the encoded protein. This current study further strengthens the genotype-to-phenotype correlation that was previously established and extends the range of phenotypes to include structural brain abnormalities, additional skeletal, dental, and psychiatric issues. This work was supported by grants from the Agence Nationale de la Recherche to Anne Debant (ANR-2019 TRIOTISM). Diana Baralle is supported by National Institute for Health Research (NIHR) (RP-2016-07-011) research professorship.

Despite major technical and genetic advances, more than half of the neurodevelopmental disorders (NDDs) cases remain undiagnosed. We explored the frequency of non-random XCI in the mothers of male patients and in affected females from a clinically heterogeneous cohort of unsolved NDD cases, negative at FRAXA, chromosomal microarray analysis and Trio Exome Sequencing. We hypothesize that an unbalanced XCI could unmask previously discarded genetic variants on the X chromosome connected both to XCI and NDD. A multiplex fluorescent-PCR-based assay was used to screen the XCI pattern after methylation sensitive HhaI digestion. Trio-based ES re-analysis was performed in families with skewed XCI occurrence. Linkage analysis and RT-PCR were used to further study the X-chromosome inactive allele. X-drop was used to define the chromosome deletion boundaries. We found a skewed XCI (>90%) in 16/186 mothers of affected NDD males (8.6%) and 12/90 female patients (13.3%), far beyond the expected XCI in normal population (3.6%, OR=4.10; OR=2.51). Reanalyzing ES and clinical data, we solved 7/28 cases (25%). These included variants in the KDM5C, PDZD4, PHF6, TAF1, OTUD5, and ZMYM3, and a genomic deletion spanning exons 3-4 of the ATRX gene. The identification of a skewed XCI is an easy assay that can help selecting a subgroup of patients for the re-evaluation of X-linked variants, improving the diagnostic yield in NDD patients, and allowing the identification of new X-linked disorders.

AimsThis study assesses the diagnostic utility of whole genome sequence analysis in a well-characterised research cohort of individuals referred with a clinical suspicion of Cornelia de Lange syndrome (CdLS) in whom prior genetic testing had not identified a causative variant.MethodsShort read, whole genome sequencing was performed in 195 individuals from 105 families, 108 of whom were affected. 100/108 of the affected individuals had prior relevant genetic testing with no pathogenic variant being identified. The study group comprised 42 trios (affected individuals with both unaffected parents), 61 singletons (unrelated affected individuals) and two families with more than one affected individual.Results32/105 (30.5%) unrelated probands had likely causative coding region disrupting variants. 4 loci were identified in >1 proband; NIPBL (10), ANKRD11 (6), EP300 (3), EHMT1 (2). Single alleles were detected in the remaining genes (EBF3, KMT2A, MED13L, NLGN3, NR2F1, PHIP, PUF60, SET, SETD5, SMC1A, TBL1XR1). Possibly causative variants in non-coding regions of NIPBL were identified in four individuals. Single de novo variants were identified in five genes not previously reported to be associated with any developmental disorder: ARID3A, PIK3C3, MCM7, MIS18BP1 and WDR18.ConclusionsClustering of de novo non-coding variants implicate a single uORF and a small region in intron 21 in NIPBL regulation. Causative variants in genes encoding chromatin-associated proteins, with no defined influence on cohesin function, appear to result in CdLS-like clinical features.

Background: Neuroblastoma tumorigenesis is a cascading process where several cytogenetic findings can be detected MYCN oncogene is a potent transcription factor that controls main cell functions. Several genetic methods can be applied in order to detect quantity of amplified MYCN oncogene. The purpose of this study is to improve the technique of determining the amplification of the MYCN oncogene in each evaluated tumor cell. Results: Standard G banded karyotype and fluorescence in-situ hybridization (FISH) on interphase nuclei using N-MYC amplification probe was performed in five patients with different clinical presentation of neuroblastoma. Both bone marrow and tumor tissue were analysed in four and in one patient only tumor tissue. Follow up study was performed in order to obtain additional prognostic information. Additional grading system was implemented to obtain MYCN amplification status. Significant amount of amplified units was detected in two patients with adverse outcome, which was not the case in other three patients who had minor or none amplification of MYCN. Furthermore, there were no cells with significant MYCN amplification in more than 30% of the cell surface in patient three and four that represented a good prognostic factor for their survival. Conclusion: Our study confirmed that patients with both chromosomal changes and significant MYCN amplification are characterized with aggressive clinical course. Accuracy in quantifying the amount of MYCN amplification is crucial in planning the therapeutic approach. FISH is proved to be rapid, sensitive, and reliable method for detection of MYCN oncogene amplification in routinely processed samples.

Individuals with autism spectrum disorder (ASD) exhibit an increased burden of de novo mutations (DNMs) in a broadening range of genes. While these studies have implicated hundreds of genes in ASD pathogenesis, which DNMs cause functional consequences in vivo remains unclear. We functionally test the effects of ASD missense DNMs using Drosophila through “humanization” rescue and overexpression-based strategies. We examine 79 ASD variants in 74 genes identified in the Simons Simplex Collection and find 38% of them to cause functional alterations. Moreover, we identify GLRA2 as the cause of a spectrum of neurodevelopmental phenotypes beyond ASD in 13 previously undiagnosed subjects. Functional characterization of variants in ASD candidate genes points to conserved neurobiological mechanisms and facilitates gene discovery for rare neurodevelopmental diseases.

Introduction. Diabetes is a chronic metabolic disease characterized with a rapid progression of prevalence in last 3 decades, especially in countries with low and middle income. Next three decades this number of diabetes in the world is expected to be doubled. Early diagnosis and appropriate management of diabetes is the primary way to prevent and delay cardiovascular complications. Patients and methods. In this retrospective study, we used the data from National electronic system e-health which was performed in late 2014, wich gives us nearly precise data, and we made statistical analysis for diabetes in last 5 years (2015-2019). Results. In 2015 we have registered 103480 patients with DM, in 2016 108130 patients, in 2017 114408, in 2018 119999 and in 2019 124450 patients with DM. 95% of patients are with T2DM and 4, 1% with T1DM. According the data from State statistical office for population of Republic of Macedonia, the prevalence of T2DM for the years 2015-2019 is as follows: 5,66% in 2015, 6.13% In 2016, 6.55% I 2017, 7,06% in 2018 and 7,2% in 2019. Conclusions. The number of registered patients with diabetes in last 5 years has grown up for 20970 or 20%, in the last 5 years the number of patients with type 2 diabetes has grown up for 18272 patients or 11%. The prevalence of T2DM has increased for 1.54%. Involvement of primary health care professionals has improved the early diagnosis of type 2 diabetes.

Micromastia or breast hypoplasia is described as underdevelopment of a woman’s mammary tissue. We present the case of a 15-year-old girl with unilateral micromastia, with familial predisposition. Ultrasound, hormonal, dysmorphic, cardiologic, genetic examinations and testing were performed. No mutation in the whole- exome sequencing was found, nor novel mutation. Some of these cases have been reported to be related to breаst cancer so further follow-up is mandatory. Therapy consists of surgical reconstruction of the affected breast. This is a rare condition and it requires a multidisciplinary approach., Микромастија или хипоплазија на дојки е опишана како недоразвиеност на ткивото на дојката кај жените. Опишавме случај на 15-годишно девојче со унилатерална микромастија со фамилијарна предиспозиција. Беа направени ехосонографски, хормонални, дисморфични, кардиолошки и генетски испитувања и тестови. Не беше пронајдена мутација на целокупниот секвенциониран ексом, ниту пак нова мутација. Некои од овие случаи се прикажани како да се поврзани со канцер на дојката и затоа се потребни понатамошни задолжителни следења. Терапијата се состои од хируршка реконструкција на афектираната дојка. Ова претставува ретка состојба, но бара мултидисциплинарен пристап.

USP9X is an X-chromosome gene that escapes X-inactivation. Loss or compromised function of USP9X leads to neurodevelopmental disorders in males and females. While males are impacted primarily by hemizygous partial loss-of-function missense variants, in females de novo heterozygous complete loss-of-function mutations predominate, and give rise to the clinically recognisable USP9X-female syndrome. Here we provide evidence of the contribution of USP9X missense and small in-frame deletion variants in USP9X-female syndrome also. We scrutinise the pathogenicity of eleven such variants, ten of which were novel. Combined application of variant prediction algorithms, protein structure modelling, and assessment under clinically relevant guidelines universally support their pathogenicity. The core phenotype of this cohort overlapped with previous descriptions of USP9X-female syndrome, but exposed heightened variability. Aggregate phenotypic information of 35 currently known females with predicted pathogenic variation in USP9X reaffirms the clinically recognisable USP9X-female syndrome, and highlights major differences when compared to USP9X-male associated neurodevelopmental disorders.

Genetics in Macedonia-Following the international trends.

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.

Hearing impairment is the most common sensory disorder, which occurs in 1 of 1000 newborns. It is caused by heterogeneous conditions with more than a half due to genetic etiology. Although hundreds of genes are implicated in hearing process and have been found to be associated with nonsyndromic hearing loss, pathogenic variants in GJB2 gene have been considered as the main cause of deafness among nonsyndromic hearing loss (NSHL) population worldwide. Pathogenic variants in MT-RNR1 or mtDNA12SrRNA gene were also implicated predominantly in postlingual progresive deafness. The aim of this study was to analyze the implication of GJB2 and MT-RNR1 genes in the molecular etiology of deafness among 130 NSHL patients in the Republic of Macedonia. The presence of the del (GJB6-D13S1830) was also analysed. We performed SSCP and/or sequence analysis of GJB2 and identified sequence variants in 62 out of 130 patients (47.7%); (51 homozygous or compound heterozygous and 11 with only one variant allele). We found 8 different allelic variants, the most prevalent being c.35delG (65.49%), and p.W24*(23.01%), followed by other less frequent alleles (p.V27I, p.V37I, p. P175T and cd. delE120 or delGAG at 360). In addition, two polymorphic substitutions in the GJB2 gene with no clinical significance (p.V153I and p.R127H) were detected. No del(GJB6-D13S1830) was found. SNaPshot analysis was used to screen for the five most frequent allelic variants in the MT-RNR1 gene. Two MT-RNR1 mutations (A827G and T961G) were detected in three patients where only one GJB2 pathogenic variant was found. A new MT-RNR1 gene variant G1303A was also detected. In conclusion, MT-RNR1 mutations were not a significant contributor to the etiology of deafness in Macedonia, although could be considered as a modifier gene affecting the expression of deafness in patients carrying one GJB2 variant. On the other hand, the high percenttage of GJB2 pathogenic variants identified among NSHL cases indicates the necessity of molecular newborn screening for the two most common GJB2 variants (c.35delG and p.W24*) in the Republic of Macedonia.

Diagnosis of rare inherited neuromuscular disorders is sometimes delayed due to variations in time of onset, different clinical appearance and limited diagnostic possibilities. The management of patients starts with neurological examination, followed by specific laboratory tests and neurophysiologic assessment. In the era of molecular medicine, molecular biology tools are useful in avoiding some of the invasive investigations such as muscle biopsy. We present a boy with a mild form of metabolic myopathy due to carnitine palmitoyltransferase 2 deficiency diagnosed upon timed functional assessment. A child had delayed developmental milestones, associated with fatigue and muscle pain during exercising and longer walks. There were no episodes of myoglobinuiria during exercise or during febrile illnesses. Neurological examination reveled proximal muscle weakness. Serum creatine kinase (CK) and serum lactate were above normal limits. Serum acylcarnitine profile was normal. Short timed functional tests such as 10 meters walk/run test showed normal results. Nord Star Ambulatory Assessment showed difficulties in balance and jumping. Diagnosis of myopathy was suspected after performance of 6-minute walk test, when the passed distance was 327 meters with slowing and fatigue. EMG and echocardiography were within normal range. Diagnosis was established by sequencing of the CPT II gene which revealed c.338C>T (p.Ser113Leu) mutation in homozygous form as characteristic CPT II deficiency profile., Дијагнозата на ретките невромускулни заболувања е понекогаш пролонгирана заради различното вре- ме на почеток на симптомите, различната клиничка слика и ограничените дијагностички можности. Обработката на пациентот започнува со невролошки преглед, по што следат специфични лаборато- риски тестови и неврофизиолошки испитувања. Во ера на молекуларната медицина, генетската анали- за овозможува да се избегнат некои од инвазивните испитувања, како на пример мускулната биопсија. Прикажуваме момче со блага форма на метаболичка миопатија со карнитин палмитоил трансфераза 2 (CPT II) дефицит, која е дијагностицирана врз основа на временска функционална проценка. Детето имало забавен моторен развој, со замор и мускулна болка во тек на вежбање или подолго одење. Нема- ло епизоди на миоглобинурија во тек на вежбање или во тек на фебрилни болести. Невролошкиот пре- глед покажа проксимална мускулна слабост. Серумската креатин киназа и серумското ниво на лакта- ти беа над нормалните граници. Серумскиот профил на ацил карнитини беше нормален. Кратките функционални тестови како 10 метри одење/трчање покажаа уредни резултати. Nord Star Ambulatory Assessment – тестирањето покажа потешкотии во рамнотежата и при скокање. Сомнение за вродена миопатија се постави по изведување на подолготраен функционален тест – 6-минутниот тест на одење кога поминатата дистанца беше 327 метри со успорување и замор. Електроневромиографијата и ехо- кардиографијата кај детето покажаа нормални наоди. Дијагнозата беше потврдена со секвенциони- рање на CPT II генот со регистрирање на c.338C>T (p.Ser113Leu) мутација во хомозиготна форма која е карактеристична за CPT II дефицит.

Дијагнозата на ретките невромускулни заболувања е понекогаш пролонгирана заради различното вре- ме на почеток на симптомите, различната клиничка слика и ограничените дијагностички можности. Обработката на пациентот започнува со невролошки преглед, по што следат специфични лаборато- риски тестови и неврофизиолошки испитувања. Во ера на молекуларната медицина, генетската анали- за овозможува да се избегнат некои од инвазивните испитувања, како на пример мускулната биопсија. Прикажуваме момче со блага форма на метаболичка миопатија со карнитин палмитоил трансфераза 2 (CPT II) дефицит, која е дијагностицирана врз основа на временска функционална проценка. Детето имало забавен моторен развој, со замор и мускулна болка во тек на вежбање или подолго одење. Нема- ло епизоди на миоглобинурија во тек на вежбање или во тек на фебрилни болести. Невролошкиот пре- глед покажа проксимална мускулна слабост. Серумската креатин киназа и серумското ниво на лакта- ти беа над нормалните граници. Серумскиот профил на ацил карнитини беше нормален. Кратките функционални тестови како 10 метри одење/трчање покажаа уредни резултати. Nord Star Ambulatory Assessment  – тестирањето покажа потешкотии во рамнотежата и при скокање. Сомнение за вродена миопатија се постави по изведување на подолготраен функционален тест – 6-минутниот тест на одење кога поминатата  дистанца беше 327 метри со успорување и замор. Електроневромиографијата и ехо- кардиографијата кај детето покажаа нормални наоди. Дијагнозата беше потврдена со секвенциони- рање на CPT II генот со регистрирање на c.338C>T (p.Ser113Leu) мутација  во хомозиготна форма која е карактеристична за CPT II дефицит., Diagnosis of rare inherited neuromuscular disorders is sometimes delayed due to variations in time of onset, different clinical appearance and limited diagnostic possibilities. The management of patients  starts  with neurological examination, followed by  specific laboratory tests  and neurophysiologic assessment. In the  era of molecular medicine, molecular biology tools are useful in avoiding some of the invasive investigations such as muscle biopsy. We present a boy with a mild form of metabolic myopathy due to carnitine  palmitoyltransferase  2 deficiency diagnosed upon timed functional assessment. A child had delayed developmental milestones, associated with fatigue and muscle pain during exercising and longer walks. There were no episodes of myoglobinuiria during exercise or during febrile illnesses. Neurological examination reveled proximal muscle weakness. Serum creatine kinase (CK) and serum lactate were above normal limits. Serum acylcarnitine profile was normal. Short timed functional tests such as 10 meters  walk/run test  showed normal results. Nord Star Ambulatory Assessment showed difficulties in balance and jumping. Diagnosis of myopathy was suspected after performance of 6-minute walk test, when the passed distance was 327 meters with slowing and fatigue. EMG and echocardiography were within normal range. Diagnosis was established by sequencing  of the CPT II gene which revealed  c.338C>T (p.Ser113Leu) mutation in homozygous form as characteristic CPT II deficiency profile.

Summary Background: In the past decades, the obesity epidemic in children of all ages has been an important research field for detecting the metabolic causes and consequences of obe- sity, the major focus being on insulin and adipocytokine lev- els. Metabolic work-up in obese children is recommended in the age group as young as 2-6 years. There is evidence that birth weight can be a factor causing obesity later in life accompanied by metabolic complications. Methods: Insulin, leptin, and adiponectin levels were ana- lyzed in 269 obese children and 60 controls, as well as 110 newborn children with different birth weight and different length of gestation, using standard methods. Results: In 53.6% of the obese children, complications of obesity such as diabetes mellitus, obesity, hyperlipidemia, heart attack or stroke were found in family members. The peak insulinemia on OGTT was significantly higher in the pubertal compared to the prepubertal group (110.5± 75.9 μU/mL versus 72.2±62.7 μU/mL) (p Conclusion: Obese children have high insulinemia in all ages, reaching its peak towards puberty. The leptin and adiponectin levels might be indicators of the metabolic syn- drome. Our findings in newborns might influence the nutritional approach in the future in order to prevent complications of obesity.

Background Killian-Pallister syndrome (KPS) is a rare form of chromosomal mosaicism and is defined by the existence of an extra chromosome 12 in some cell lines in one individual. The degree of mosaicism varies among tissues and dictates the clinical presentation of the syndrome. The clinical features of Killian-Pallister syndrome include mental retardation, typical facial dysmorphism and pigmentation defects. Case presentation We present a rare case of Killian-Pallister syndrome with severe form of the disease associated with isolated growth hormone deficiency and low-rate mosaicism on buccal smear. The absence of a marker chromosome 12p in lymphocyte cultures and the low degree of mosaicism lead to frequent misdiagnosis of this condition. Conclusions The selection of tissue sampling is crucial in establishing the diagnosis of Killian-Pallister syndrome. Fluorescent in situ hybridisation on buccal smear remains the golden standard as a screening method if a suspicion of the syndrome exists.

Трудот е презентиран и печатен на симпозиум посветен на ендокринолошките проблеми и метаболниот профил кај деца со различна гестациска возраст и големина на раѓањето, барајќи асоцијација помеѓу трофиката и метаболниот профил.

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.

We report on a patient with a contiguous interstitial germline deletion of chromosome 10q23, encompassing BMPR1A and PTEN, with clinical manifestations of juvenile polyposis and minor symptoms of Cowden syndrome (CS) and Bannayan–Riley–Ruvalcaba syndrome (BRRS). The patient presented dysmorphic features as well as developmental delay at the age of 5 months. Multiple polyps along all parts of the colon were diagnosed at the age of 3 years, following an episode of a severe abdominal pain and intestinal bleeding. The high-resolution comparative genomic hybridisation revealed a 3.7-Mb deletion within the 10q23 chromosomal region: 86,329,859–90,035,024. The genotyping with four polymorphic microsatellite markers confirmed a de novo 10q deletion on the allele with a paternal origin, encompassing both PTEN and BMPR1A genes. The karyotype analysis additionally identified a balanced translocation involving chromosomes 5q and 7q, and an inversion at chromosome 2, i.e. 46,XY,t(5;7)(q13.3-q36), inv(2)(p25q34). Although many genetic defects were detected, it is most likely that the 10q23 deletion is primarily the cause for the serious phenotypic manifestations. The current clinical findings and deletion of BMPR1A indicate a diagnosis of severe juvenile polyposis, but the existing macrocephaly and PTEN deletion also point to either CS or BRRS, which cannot be ruled out at the moment because of their clinical manifestation later in life and the de novo character of the deletion. The deletion detected in our patient narrows the genetic region deleted in all reported cases with juvenile polyposis by 0.04 Mb from the telomeric side, mapping it to the region chr10:88.5–90.03Mb (GRCh37/hg19), with an overall length of 1.53 Mb.

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.

The lowest incidence of childhood type 1 diabetes in Europe has been reported from the Republic of Macedonia. To assess the possible genetic contribution we analyzed the distribution of HLA-DR-DQ haplotypes among 163 diabetic children and 239 healthy controls. Similar disease associations were found as in other Caucasian populations. HLA-(DR3)-DQA1*05-DQB1*02 was the most common disease associated haplotype, but several DRB1*04-DQB1*0302 haplotypes were also found increased among patients. DRB1*0402 was the most common DR4 allele among them. The high frequency of protective (DR11/12)-DQA1*05-DQB1*0301 and (DR14)-DQB1*0503 haplotypes as well as of neutral (DR1/10)-DQB1*0501 and (DR16)-DQB1*0502 haplotypes were characteristic for the background population. Although a relatively low frequency of predisposing and a high frequency of protective haplotypes was detected, the haplotype frequency distribution did not markedly differ from that reported from other Eastern Mediterranean populations and these differences cannot be the sole explanation for the low disease incidence in Macedonia.