Adrijan Sarajlija

DNA polymerase-α regulates the activation of type I interferons through cytosolic RNA:DNA synthesis

Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations that disrupt nucleic acid metabolism can lead to autoinflammatory disorders. Here we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts the expression of POLA1, which encodes the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency resulted in increased production of type I interferons. This enzyme is necessary for the synthesis of RNA:DNA primers during DNA replication and, strikingly, we found that POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Together this work identifies POLA1 as a critical regulator of the type I interferon response.

Clinical characteristics of respiratory syncytial virus infection in neonates and young infants.

INTRODUCTION/AIM Infection with respiratory syncytial virus (RSV) occurs during the first year of life in 50% of children and 20%-40% of them have signs of lower respiratory tract infection (bronchiolitis or pneumonia). There is an increased risk for complicated course and death from RSV infection in premature infants, especially those with bronchopulmonary dysplasia (BPD) or congenital heart disease. The aim of our study was to analyze clinical characteristics of laboratory confirmed RSV infection in order to evaluate the need for preventive measures in neonates and young infants. METHODS The prospective study included children under age of 12 months admitted to our hospital in the period November 2008-March 2009 who were positive for RSV by enzyme immunoassay membrane test. The course of disease was assessed by clinical score and radiographic findings. RESULTS Infection with RSV was confirmed in 91 patients: 21 (23.0%) were under the age of 30 days, 37 (40.7%) were between 31-60 days, and 33 patients (36.3%) were older than 60 days (p > 0.05). The highest hospitalization rate was in January--33 patients (36.3%; p < 0.01). Disease severity score in these age groups (AG) were: 8.4 +/- 0.4 (AG 0-30 days); 9.0 +/- 0.3 (AG 31-60 days) and 8.3 +/- 0.3 (AG > 60 days), without statistically significant difference among the groups (p > 0.05). Clinical scores in patients with and without risk factors were 10.5 +/- 0.5 and 8.3 +/- 0.2, respectively (p < 0.01). Pathological radiographic findings were observed in 72 (79.1%) and complications (apnea, significant atelectasis, encephalopathy) occured in 15 (16.5%) patients. The average length of hospital stay in complicated and uncomplicated course of the disease was 9 days and 6 days, respectively (p < 0.01). Therapy in 85 (93.4%) patients included bronchodilators, while systemic glucocorticoids and oxygen therapy were used in 51 (56.0%) and 44 (48.4%) patients, respectively. Death occured in 2 (2.2%) patients, both from a high risk group (the patient with BPD and the other one with congenital heart disease and Down syndrome). CONCLUSION Infection with RSV in our settings showed marked seasonal characteristics with highest hospitalization rate in January. Although the course and outcome of the disease were favorable in the majority of our patients, the need for hospitalization and administration of therapy with possible side effects warrants that general measures for prevention of respiratory infections are followed especially in the first year of life. Severe disease and death are more probable in neonates and infants with risk factors. In these children passive immunisation with specific monoclonal antibody (e.g. palivizumab) during RSV season should be considered.

Epidemiology of Rett Syndrome in Serbia: Prevalence, Incidence and Survival

Background: Rett syndrome (RTT) is a severe neurodevelopmental disorder that represents the second most common cause of mental retardation in females. However, incidence and prevalence of RTT are scarcely reported. Methods: A retrospective study included all patients with RTT diagnosed between 1981 and 2012 in Serbia. Estimation of incidence and prevalence was calculated on the basis of vital statistics reported by Statistical Office of Republic of Serbia. Results: From 1981 to 2012, RTT has been diagnosed in 102 girls in Serbia. Incidence of RTT in Serbia is estimated at 0.586:10,000 female live births. We estimated the prevalence of RTT in population of females younger than 19 years at 1:8,439. Death occurred in 19 patients (18.63%), with pneumonia as the most common cause. The lethal outcome by the age of 12 years could be expected for 11% of patients. The mean age at diagnosis was 3.5 years and we have confirmed a significant trend towards earlier dianosis during studied period. Conclusions: Rett syndrome incidence in Serbia is in accordance with reports from other countries. Serbian RTT patients have increased risk for early death when compared to patients in more developed countries, most commonly due to pneumonia. There was significant trend towards early diagnosis of RTT in Serbia over recent decades.

Molecular and phenotypic characteristics of seven novel mutations causing branched-chain organic acidurias

Specific mitochondrial enzymatic deficiencies in the catabolism of branched‐chain amino acids cause methylmalonic aciduria (MMA), propionic acidemia (PA) and maple syrup urine disease (MSUD). Disease‐causing mutations were identified in nine unrelated branched‐chain organic acidurias (BCOA) patients. We detected eight previously described mutations: p.Asn219Tyr, p.Arg369His p.Val553Glyfs*17 in MUT, p.Thr198Serfs*6 in MMAA, p.Ile144_Leu181del in PCCB, p.Gly288Valfs*11, p.Tyr438Asn in BCKDHA and p.Ala137Val in BCKDHB gene. Interestingly, we identified seven novel genetic variants: p.Leu549Pro, p.Glu564*, p.Leu641Pro in MUT, p.Tyr206Cys in PCCB, p.His194Arg, p.Val298Met in BCKDHA and p.Glu286_Met290del in BCKDHB gene. In silico and/or eukaryotic expression studies confirmed pathogenic effect of all novel genetic variants. Aberrant enzymes p.Leu549Pro MUT, p.Leu641Pro MUT and p.Tyr206Cys PCCB did not show residual activity in activity assays. In addition, activity of MUT enzymes was not rescued in the presence of vitamin B12 precursor in vitro which was in accordance with non‐responsiveness or partial responsiveness of patients to vitamin B12 therapy. Our study brings the first molecular genetic data and detailed phenotypic characteristics for MMA, PA and MSUD patients for Serbia and the whole South‐Eastern European region. Therefore, our study contributes to the better understanding of molecular landscape of BCOA in Europe and to general knowledge on genotype–phenotype correlation for these rare diseases.

Vitamin D Deficiency in Serbian Patients With Rett Syndrome

INTRODUCTION Rett syndrome (RTT) is a severe neurodevelopmental disorder. Bone manifestations of RTT include osteopenia and fractures. Studies addressing serum vitamin D levels in patients with RTT are scarce. GOALS The goals of this study were (1) to determine the prevalence of vitamin D deficiency in patients with RTT, (2) to compare serum vitamin D levels between patients with RTT and those with other neurological diseases, and (3) to explore the correlation between demographic and clinical characteristics of patients with RTT and vitamin D levels. METHODS Demographic and clinical characteristics included age, body mass index Z-score, mutation status, clinical severity score, presence of epilepsy, number of antiepileptic drugs, history of fractures, scoliosis, and ambulation ability. Laboratory parameters included serum 25-hydroxyvitamin D [25(OH)D], PTH, calcium, and alkaline phosphatase. RESULTS The study included 35 patients with RTT and 35 age-matched females with other neurological diseases. The median serum 25(OH)D concentration in the RTT group was 26.25 nmol/L, with values <75 nmol/L in all participants. Severe deficiency (<25 nmol/L) was detected in 17 of 35 (48.6%) patients. The median 25(OH)D concentration was significantly lower in patients with RTT than in control subjects. The risk for fracture by 12 years of age in patients with RTT was 35.3%. An inverse correlation of the 25(OH)D level to age and PTH level was detected. Patients receiving antiepileptic polytherapy had a 3.3 times greater chance for severe vitamin D deficiency than patients receiving monotherapy. CONCLUSION The prevalence of vitamin D deficiency in patients with RTT is higher than that in patients with other neurological diseases. The high risk for vitamin D deficiency should be accounted for in the strategy of antiepileptic treatment in RTT, especially when polytherapy is considered.

Genetic characterization of GSD I in Serbian population revealed unexpectedly high incidence of GSD Ib and 3 novel SLC37A4 variants: SKAKICet al.

Glycogen storage disease (GSD) type I is inborn metabolic disease characterized by accumulation of glycogen in multiple organs. We analyzed 38 patients with clinical suspicion of GSD I using Sanger and next‐generation sequencing (NGS). We identified 28 GSD Ib and 5 GSD Ia patients. In 5 patients, GSD III, VI, IX, cholesteryl‐ester storage disease and Shwachman‐Diamond syndrome diagnoses were set using NGS. Incidences for GSD Ia and GSD Ib were estimated at 1:172 746 and 1:60 461 live‐births, respectively. Two variants were identified in G6PC gene: c.247C>T (p.Arg83Cys) and c.518T>C (p.Leu173Pro). In SLC37A4 gene, 6 variants were detected. Three previously reported variants c.81T>A (p.Asn27Lys), c.162C>A (p.Ser54Arg) and c.1042_1043delCT (p.Leu348Valfs*53) accounted for 87% of all analyzed alleles. Computational, transcription studies and/or clinical presentation in patients confirmed pathogenic effect of 3 novel variants: c.248G>A (p.Gly83Glu), c.404G>A (p.Gly135Asp) and c.785G>A (p.Ser263Glyfs*33 or p.Gly262Asp). In the cohort, hepatomegaly, hypoglycemia and failure to thrive were the most frequent presenting signs of GSD Ia, while hepatomegaly and recurrent bacterial infections were clinical hallmarks of GSD Ib. All GSD Ib patients developed neutropenia while 20.6% developed inflammatory bowel disease. Our study revealed the highest worldwide incidence of GSD Ib. Furthermore, description of 3 novel variants will facilitate medical genetic practice.

Late-Presenting Congenital Diaphragmatic Hernia in a Child with Tmem70 Deficiency.

Transmembrane protein 70 (TMEM70) deficiency has been delineated as a distinct mitochondrial disease less than a decade ago (Cízková et al. 2008). Hallmarks of the disease include hypotonia, developmental delay, hypertrophic cardiomyopathy (HCM), facial dysmorphism, microcephaly, hypospadia, lactic acidosis and 3-methylglutaconic aciduria (3MGA). Although there is no curative treatment, proper management of hyperammonemic metabolic crises seems to be crucial for the prognosis of these patients. Characteristic facial features with high forehead, downslanting palpebral fissures, curved eyebrows, prominent nasal bridge, low-set ears, thin lips, microretrognathia and ptosis have been described in two-thirds of patients. The disease is panethnic but particularly common in the Roma population with prevalent c.317-2 A > G mutation (Spiegel et al. 2011, Magner et al. 2015). Congenital malformations in patients with TMEM70 deficiency include heart defects (36%) with valve defects and aortic coarctation, hypospadia (50% of affected boys), umbilical and inguinal hernias (approximately one-third of cases), syndactyly between the second and third finger, congenital cataract, and agenesis or hypoplasia of corpus callosum (Spiegel et al. 2011, Magner et al. 2015). Congenital diaphragmatic hernia (CDH) has been described only in one patient with this disease, so far (Catteruccia et al. 2014). We report herein the second case of unique association of TMEM70 deficiency and CDH. Severe lactic acidosis and HCM were noted in a boy of Roma ethnicity in the neonatal period. Microcephaly, hypotonia and dysmorphic facial features (synophris, curved eyebrows, anteverted nostrils, low set ears, microretrognathia), but no hypospadia, were observed later in infancy. Chest radiograph made at the age of one year did not show presence of diaphragmatic hernia. At the age of 3.5 years, the boy was admitted to hospital due to vomiting and somnolence. Physical exam revealed tachycardia, tachypnoea and decreased breathing sounds on the left side of the chest. Chest radiograph showed hydroaeric levels at the left thoracic side (Fig. 1A). Upper gastrointestinal series with small bowel follow-through showed normally positioned esophagus and stomach, whereas duodenum ascended towards the left part of the chest cavity, which was filled with intestinal loops (Fig. 1B). Posterolateral left Bochdalek hernia with defect diameter of 5 cm and hernial sac protruding to the chest cavity was visualized during surgical intervention. The whole jejunum and ileum, including the ileocecal valve, was herniated. After repositioning of the abdominal organs, plastic of diaphragm and Ladd procedure followed. Twelve months later, the boy succumbed to severe metabolic crisis with lactic acidemia but absent 3MGA. Unfortunately, ammonia level was not measured at the time. Post-mortem mutation analysis of TMEM70 in the patient’s DNA sample documented homozygous mutation c.3172 A > G. Associated congenital anomalies are found in 8.6% of patients with late presenting CDH, cardiac anomalies being the most frequent (Bagłaj 2004). In dual-hit hypothesis of CDH evolution, a primary bilateral lung hypoplasia results from combined genetic and external factors, while the second hit affects only the left lung by pressure of intrathoracic bowels (Keijzer et al. 2000). Although the lung hypoplasia was described in one patient with TMEM70 (Spiegel et al. 2011), there was no proof of hypoplastic lungs in our patient or in the patient reported by Catterucia et al. (2014). Hypoplastic lungs may cause the persistent pulmonary hypertension in newborn (PPHN), which is frequently observed in children with TMEM70 (>50%). However, this finding has probably no relation to the reported CDH (Catteruccia et al. 2014). The second case, reported herein, further supports the association between TMEM70 deficiency and CDH, which might help to diagnose this rare but treatable disorder.

Genotype-phenotype correlation in 44 Czech, Slovak, Croatian and Serbian patients with mucopolysaccharidosis type II

Mucopolysaccharidosis type II (Hunter syndrome, MPS II, OMIM 309900) is an X‐linked lysosomal storage disorder caused by deficiency of iduronate‐2‐sulfatase (IDS). We analyzed clinical and laboratory data from 44 Slavic patients with this disease. In total, 21 Czech, 7 Slovak, 9 Croatian and 7 Serbian patients (43 M/1 F) were included in the study (median age 11.0 years, range 1.2–43 years). Birth prevalence ranged from 1:69,223 (Serbia) to 1:192,626 (Czech Rep.). In the majority of patients (71%), the disease manifested in infancy. Cognitive functions were normal in 10 patients. Four, six and 24 patients had mild, moderate, and severe developmental delay, respectively, typically subsequent to developmental regression (59%). Residual enzyme activity showed no predictive value, and estimation of glycosaminoglycans (GAGs) had only limited importance for prognosis. Mutation analysis performed in 36 families led to the identification of 12 novel mutations, eight of which were small deletions/insertions. Large deletions/rearrangements and all but one small deletion/insertion led to a severe phenotype. This genotype–phenotype correlation was also identified in six cases with recurrent missense mutations. Based on patient genotype, the severity of the disease may be predicted with high probability in approximately half of MPS II patients.

Tetrahydrobiopterin deficiency among Serbian patients presenting with hyperphenylalaninemia

Abstract Hyperphenylalaninemia (HPA) [phenylketonuria (PKU) and tetrahydrobiopterin (BH4) deficiencies] is rare inborn metabolic disease characterized by elevated phenylalanine level in body fluids. In Serbia, 62 HPA patients have been identified through newborn screening since 1983. However, pterin pattern analysis is not performed. We present a patient initially diagnosed and treated as classical PKU. At 3 years of age, during infection with H1N1 influenza A virus, the patient first developed a neurologic crisis with encephalopathy and dystonic movements. We suspected that the patient is the first case of BH4 deficiency identified in Serbia. Genetic analyses showed that the patient does not have disease-causing variants of the PAH gene and carries a p.Asp136Val mutation in homozygous state in the PTS gene. For patients with treatable rare diseases, like PKU and BH4 deficiencies, correct diagnosis is crucial for the implementation of optimal treatment. If biochemical tests needed for differential diagnosis are not available, our experience emphasizes the necessity of immediate genetic testing after newborn screening.

Appendiceal involvement in a patient with Gaucher disease

Almost any anatomical compartment may be involved in Gaucher disease (GD). Abdominal lymphadenopathy occurred during enzyme replacement therapy in more than a dozen children with GD so far. A fourteen-year-old boy from Serbia developed clinical signs of acute appendicitis six years after the onset of GD type 3 related abdominal lymphadenopathy. Ultrasound examination showed diffuse thickening of the intestinal wall in the ileocoecal region with periappendicular infiltration. An appendectomy was performed four months after conservative treatment with antibiotics. Histopathology revealed macrophages with cytological characteristics of Gaucher cells densely crammed in mesoappendiceal adipose tissue. Also the multifocal replacement of subserosal tissue by Gaucher cells and their infiltration to a variable depth of muscularis propria of the appendix were verified. Frank infiltration of the vermiform appendix with Gaucher cells represents a novel observation in a wide spectrum of manifestations reported in GD. A possible causative relationship of this infiltration with appendicitis is considered.