The Therapeutic Hypothermia in Treatment of Hyperammonemic Encephalopathy due to Urea Cycle Disorders and Organic Acidemias.

BACKGROUND: Hyperammonemic encephalopathy in newborns with urea cycle disorders and certain organic acidurias can cause severe brain injury, coma and death. Standard therapy includes protein restriction, nitrogen-scavenging drugs, prevention of catabolism and hemodialysis. Neuroprotective hypothermia as part of the treatment has been reported only 3 times. It has been suggested that mild systemic hypothermia can contribute to better neurological outcomes in hyperammonemic encephalopathy. However, the limited experience precludes accurate conclusions on safety and efficacy. METHODS: Whole body therapeutic hypothermia was included in the standard treatment of hyperammonemic encephalopathy in 4 neonates with urea cycle disorder or organic aciduria. RESULTS: Two patients survived the initial crisis. One patient has a developmental quotient of 0.8, while the other shows severe developmental delay. The cooling protocol had to be discontinued in 3 patients due to the otherwise untreatable complications (hypotension and hemorrhage). CONCLUSION: The efficacy and safety of therapeutic hypothermia in the treatment of neonatal hyperammonemic encephalopathy depend on various factors, requiring further evaluation.

[Lysosomal acid lipase deficiency in children: our experience and a novel possibility of enzyme replacement therapy]. / Manjak lizosomske kisele lipaze u djece: vlastita iskustva i nova mogucnost enzimskoga nadomjesnog lijecenja.

Lysosomal acid lipase deficiency is an autosomal recessive disorder with two distinct clinical phenotypes. Wolman disease is rapidly progressive with onset in early infancy. Complete enzyme deficiency results in massive accumulation of cholesterol esters and triglycerides in intestines, liver, spleen and other monocyte-macrophage system cells causing malabsorption, hepatosplenomegaly, liver failure and death in early infancy. Cholesterol ester storage disease may be diagnosed in childhood or later in life. It is characterized by chronic course and variable progression. Main features are variously expressed hepatopathy, including cirrhosis and liver failure, hypercholesterolemia and premature atherosclerosis. Characteristic is pathohistological finding of microvesicular steatosis and fibrosis and patognomonic are typical cholesterol ester crystals. Diagnosis is confirmed by enzyme assay and/or gene analysis. Until recently treatment was symptomatic. Ongoing clinical trials of enzyme replacement therapy have shown very promising results. We are presenting an infant with Wolman disease and two children with cholesterol ester storage disease with the aim to raise awareness about this disease and to start optimal care early.

Newborn screening in southeastern Europe.

The aim of our study was to assess the current state of newborn screening (NBS) in the region of southeastern Europe, as an example of a developing region, focusing also on future plans. Responses were obtained from 11 countries. Phenylketonuria screening was not introduced in four of 11 countries, while congenital hypothyroidism screening was not introduced in three of them; extended NBS programs were non-existent. The primary challenges were identified. Implementation of NBS to developing countries worldwide should be considered as a priority.

[Congenital hyperinsulinism--novel insights into etiology, diagnosis and treatment]. / Kongenitalni hiperinzulinizam- -novosti o nastanku, dijagnosticiranju i lijecenju bolesti.

Congenital hyperinsulinism (CHI) is a major cause of persistent hypoglycemia in the neonatal and early infancy periods. Althought the disease is relatively rare with incidence of about 1:25 000-50 000 live births, the importance of the disease should not be underestimated. Namely, prompt recognition and management of patients with CHI is essential, if permanent neurological impairment is to be avoided. CHI is caused by mutations in one of the 7 genes involved in the regulation of insulin secretion in pancreatic beta-cells. It is important to introduce specific medical therapy as soon as diagnosis is established. Severe, neonatal forms of CHI are often resistant to medications, thus they require surgical procedure. The preoperative genetic testing and scintigraphy are indicated to distinguish histological subtypes of the disease (focal vs. diffuse CHI). Patients with focal disease are usually cured after pancreatic resection, while diffuse disease has much worse prognosis. This manuscript offers novel insights into CHI and emphasizes the role of early diagnosis as crucial for succesful treatment that was recently enriched with novel options.

[Vitamin B12 deficiency in children--underestimated danger in the light of new knowledge]. / Manjak vitamina B12 u djece --podcijenjena opasnost u svjetlu novih spoznaja.

Vitamin B12 (cobalamin) has two active forms, adenosylcobalamin and methylcobalamin which have a key role in two important metabolic pathways in humans and their deficiency is responsible for clinical problems. Cobalamin is essential during whole life, but its sufficient amount is extra important in fetal and neonatal period, when it is essential for normal child growth and development as well as for normal development of the central nervous system. Because of very complex transport and metabolism, its deficiency can be manifested in numerous congenital and acquired disorders. Vitamin B12 deficiency mostly has non-specific clinical features, it carries a great risk of permanent consequences, but most frequently it is easily curable if diagnosed on time. In Croatia cobalamin deficiency in children has been diagnosed too rarely. Accordingly, the aim of this paper is to point to the recently gained knowledge on cobalamin metabolism, present typical case reports and to provide guidelines for rapid and proper diagnostic and therapeutic approach.

Pallister Killian syndrome: unusual significant postnatal overgrowth in a girl with otherwise typical presentation.

Pallister Killian syndrome (PKS) is a rare genetic disorder caused by tetrasomy of the short arm of chromosome 12, revealed usually in mosaic distribution of an extra i (12) (p10) chromosome in fibroblasts. The syndrome presents with a recognizable pattern of findings including pigmentary skin changes, coarse face, high forehead, sparse anterior scalp hair, hypertelorism, seizures and progressive psychomotor developmental delay. It was first described independently by Pallister in 1977 and by Killian and Teschler-Nikola in 1981. We report a case of 21 month old girl with PKS and significant overgrowth. Cytogenetic analysis was performed using the GTG banding technique. The karyotype of cultured lymphocytes was normal. The karyotype from skin fibroblasts was established as mosaic tetrasomy of 12p 47,XX,+i (12) (p10)/46,XX. The origin of the extra marker chromosome was determinated by fluorescence in situ hybridization with chromosome 12 specific DNA probes confirming that supernumerary marker is chromosome i (12p) in 68% of cells. Despite the excessive postnatal growth we found low serum growth hormone levels and reduced response to pharmacological stimulation test. This is also the first report of a postnatal patient in our country.

Genotype-predicted tetrahydrobiopterin (BH4)-responsiveness and molecular genetics in Croatian patients with phenylalanine hydroxylase (PAH) deficiency.

Specific mutations in the gene encoding phenylalanine hydroxylase (PAH), located on chromosome 12q22-24.1, are linked to tetrahydrobiopterin (BH4; sapropterin)-responsive phenylketonuria (PKU). Diagnosis is usually done through the newborn screening for PKU, followed by a BH4 loading test. So far, more than 60 mutant alleles, presenting with a substantial residual PAH activity (average approximately 47%), were identified in more than 500 patients worldwide. We investigated the predictive value of BH4-responsive PAH mutations in Croatian population. From a group of 127 PKU patients, 62 were selected (based on the genotype) as potentially BH4-responsive and 39 loaded with BH4 (20 mg/kg). The overall frequency of BH4-responsiveness (>30% blood phenylalanine reduction within 24 h) was 36% (14 out of 39 patients with 23 different genotypes), significantly less than expected. The best responders were patients with mild hyperphenylalaninemia (4/4; 100%), followed by mild PKU (8/9; 89%), and classical PKU (2/26; 8%). The most common BH(4)-responsive genotypes were p.E390G/p.R408W and p.P281L/p.E390G. These genotypes correspond for approximately >30% residual PAH activity. The p.E390G mutation was 100% associated with BH4-responsiveness, regardless of the second allele (p.R408W, p.P281L, p.F55Lfs, p.L249P). With regard to the predicted relative PAH activity of recombinantly expressed mutant alleles, there was a significant (p<0.002) difference between BH4-responders and non-responders. In a general Croatian PKU population, disease-causing mutations were identified on 226 alleles (99%). There were 35 different mutations: 21 missense, 8 splice site, 3 nonsense, 2 single nucleotide deletions, and 1 in-frame deletion. Four mutations are reported for the first time: p.E76D, p.L333P, p.G346E, and IVS8-2A>G. Five mutations accounted for over two-thirds of investigated alleles: p.L48S, p.R261Q, p.P281L, p.E390G, and p.R408W. Thus, the Croatian PKU population seems to be more homogenous than some other Mediterranean or Central European populations. This study reveals the importance of a full genotype for the prediction of BH4-responsiveness. In contrast to previous assumption and with exception of the p.E390G mutation, single allele mutations are not reliable for the selection of potential PKU candidates for pharmacological therapy with BH4.

Hypophosphatasia: phenotypic variability and possible Croatian origin of the c.1402g>A mutation of TNSALP gene.

Hypophosphatasia is a metabolic bone disease characterized by bone and teeth hypomineralization due to defective function of tissue-nonspecific alkaline phosphatase (TNSALP). The disorder is caused by various mutations in the TNSALP gene localized on short arm of chromosome 1. Infantile hypophosphatasia is a severe form of the disease inherited as an autosomal recessive trait which presents before age of six months and often has fatal outcome. We report a patient with typical clinical course for infantile hypophosphatasia who was homozygous for the c.1402G>A mutation. The same mutation has been previously associated with a more severe perinatal form also in a Croatian family what indicates a possible common ancestral origin and phenotypic variability potential of c.1402G>A mutation of TNSALP gene.

Molecular basis and clinical presentation of classic galactosemia in a Croatian population.

BACKGROUND: Classic galactosemia is an autosomal recessive disorder of galactose metabolism caused by severely decreased activity of galactose-1-phosphate uridylyltransferase (GALT) due to pathogenic mutations in the GALT gene. To date more than 330 mutations have been described, with p.Q188R and p.K285N being the most common in Caucasian populations. Although acute manifestations can be fully avoided by a galactose-restricted diet, chronic complications, such as neurological ones, cannot be prevented in a significant number of patients despite compliance with the dietary treatment. METHODS: A cohort of 16 galactosemic Croatian patients, including one pair of siblings, was studied. Molecular characterization was performed by direct sequence analysis of the GALT gene. RESULTS: Sixteen patients were analyzed and only four different mutations were detected. As expected, p.Q188R and p.K285N were common, accounting for 40% and 37% of unrelated alleles, respectively. The third mutation accounting for 20% of mutant alleles was p.R123X causing a premature stop codon, is thus considered to be severe, which is in accordance with the phenotype presented by the homozygous patient described here. The fourth mutation p.E271D was found in a single allele. More than half of our patients manifested some chronic neurological complications. CONCLUSIONS: This is the first report on mutational and phenotypic spectra of classic galactosemia in Croatia that expands the knowledge on the mutational map of the GALT gene across Europe and reveals the genetic homogeneity of the Croatian population.

Hyperinsulinism-hyperammonemia syndrome: a de novo mutation of the GLUD1 gene in twins and a review of the literature.

Hyperinsulinism-hyperammonemia (HI/HA) syndrome is a rare autosomal dominant disease characterized by recurrent hypoglycemia and persistent mild elevation of plasma ammonia. HI/HA syndrome is one of the more common forms of congenital hyperinsulinism (CHI), caused by activating mutations within the GLUD1 gene that encodes the mitochondrial enzyme glutamate dehydrogenase (GDH). We report here on monozygotic twin girls presented with fasting- and protein-induced hypoglycemia and mild persistent hyperammonemia. Genetic analysis revealed that both girls were heterozygous for a novel missense mutation within exon 11 [c.1499A>T, p.(R443W)] of the GLUD1 gene. Despite early treatment with diazoxide and a low protein diet, they both developed non-hypoglycemic seizures in early childhood followed by cognitive impairment. In addition to their clinical course, a review of the literature on HI/HA syndrome is provided.

Phenylketonuria screening and management in southeastern Europe - survey results from 11 countries.

BACKGROUND: We aimed to assess the current state of PKU screening and management in the region of southeastern Europe. METHODS: A survey was performed involving all identified professionals responsible for the PKU management in the 11 countries from South-Eastern region of Europe (Albania, Bulgaria, Bosnia and Herzegovina, Croatia, Kosovo, Macedonia, Moldova, Montenegro, Romania, Serbia, Slovenia). The questionnaire was designed to assess the characteristics regarding PKU management in three main areas: nation-wide characteristics, PKU screening, and characteristics of the PKU management in the responding centre. It consisted of 56 questions. The distribution and collection of the questionnaires (via e-mail) was taking place from December 2013 to March 2014. RESULTS: Responses from participants from 11 countries were included; the countries cumulative population is approx. 52.5 mio. PKU screening was not yet introduced in 4 of 11 countries. Reported PKU incidences ranged from 1/7325 to 1/39338 (and were not known for 5 countries). National PKU guidelines existed in 5 of 11 countries and 7 of 11 countries had PKU registry (registries included 40 to 194 patients). The number of PKU centers in each country varied from 1 to 6. Routine genetic diagnostics was reported in 4 of 11 countries. Most commonly used laboratory method to assess phenylalanine levels was fluorometric. Tetrahydrobiopterine was used in only 2 of 11 countries. Most frequently, pediatricians were caring for the patients. Dietitian was a member of PKU team in only 4 of 11 countries, while regular psychological assessments were performed in 6 of 11 countries. Patient's PKU society existed in 7 of 11 countries. CONCLUSIONS: The region of southeastern Europe was facing certain important challenges of PKU screening and management. Neonatal PKU screening should be introduced throughout the region. Furthermore, PKU management was falling behind internationally established standards-of-care in many aspects.

The molecular basis of phenylalanine hydroxylase deficiency in Croatia.

We present the results of a comprehensive analysis of mutations, polymorphisms and haplotypes in the phenylalanine hydroxylase (PAH) gene in 39 Croatian families with phenylketonuria (PKU). A total of 21 disease-causing mutations was identified on 78 out of 79 independent chromosomes. The commonest mutation, R408W on haplotype 2 was found with a relative frequency of 37 %. P281L accounted for 11 %, R261Q and E390G each for 9 % of mutant chromosomes. There were three novel mutations: L249P (c.746T>C) in exon 7, IVS8+2T>C (c.912T>C) in intron 8, and F402L (c.1206T>G) in exon 12 of the PAH gene. Two known PKU mutations were found in cis on the same chromosome in one family, highlighting the need to perform full mutation scanning in recessive disease genes for molecular diagnosis even if two known mutations have been identified in a patient. This is the first comprehensive report on PKU mutations in southeastern Europe, adding to the growing bulk of molecular data for population genetic investigations.

[Glutaric aciduria type 1: an example of the importance of early detection of so-called cerebral organic aciduria]. / Glutaricka acidurija tipa 1: primjer vaznosti ranog prepoznavanja tzv. cerebralnih organskih acidurija.

Glutaric aciduria type 1 (GA 1) is a preventable cause of acute brain damage in early childhood, leading to a severe dystonic-dyskinetic disorder. Typically between 6 and 18 months of age, a non-specific illness such as respiratory or gastrointestinal infection or immunization leads to encephalopathic crisis, usually resulting in degeneration of the putamen and caudate. GA 1 is an autosomal recessive disease of catabolism of amino acids lysine, hydroxylysine and tryptophane leading to accumulation of glutaric acid, 3-hydroxyglutaric acid and glutaconic acid. Recognition of this biochemical disorder before the brain has been injured is essential to the outcome. Diagnosis depends upon the recognition of relatively non-specific physical findings such as hypotonia, tremor, irritability and macrocephaly, and on urinary organic acids analysis. The diagnosis may also be suggested by characteristic findings of neuroimaging. Specific management includes pharmacological doses of 1-carnitine and dietary protein restriction. Metabolic decompensation must be treated vigorously to avoid permanent brain damage. With this case report the authors want to contribute to the early recognition of GA1, to the prevention of the related brain damage, and to increase awareness of the existence of so-called cerebral organic acidurias.

S-adenosylhomocysteine hydrolase deficiency in a human: a genetic disorder of methionine metabolism.

We report studies of a Croatian boy, a proven case of human S-adenosylhomocysteine (AdoHcy) hydrolase deficiency. Psychomotor development was slow until his fifth month; thereafter, virtually absent until treatment was started. He had marked hypotonia with elevated serum creatine kinase and transaminases, prolonged prothrombin time and low albumin. Electron microscopy of muscle showed numerous abnormal myelin figures; liver biopsy showed mild hepatitis with sparse rough endoplasmic reticulum. Brain MRI at 12.7 months revealed white matter atrophy and abnormally slow myelination. Hypermethioninemia was present in the initial metabolic study at age 8 months, and persisted (up to 784 microM) without tyrosine elevation. Plasma total homocysteine was very slightly elevated for an infant to 14.5-15.9 microM. In plasma, S-adenosylmethionine was 30-fold and AdoHcy 150-fold elevated. Activity of AdoHcy hydrolase was approximately equal to 3% of control in liver and was 5-10% of the control values in red blood cells and cultured fibroblasts. We found no evidence of a soluble inhibitor of the enzyme in extracts of the patient's cultured fibroblasts. Additional pretreatment abnormalities in plasma included low concentrations of phosphatidylcholine and choline, with elevations of guanidinoacetate, betaine, dimethylglycine, and cystathionine. Leukocyte DNA was hypermethylated. Gene analysis revealed two mutations in exon 4: a maternally derived stop codon, and a paternally derived missense mutation. We discuss reasons for biochemical abnormalities and pathophysiological aspects of AdoHcy hydrolase deficiency.