Mojca Zerjav Tansek

COL2A1-related skeletal dysplasias with predominant metaphyseal involvement

Skeletal dysplasias induced by mutations in the collagen 2 gene (the so‐called “type 2 collagenopathies”) form a wide spectrum in severity and are distinguished by subtle clinical and radiographic differential signs. The unifying features are predominant involvement of the vertebral bodies and the epiphyses of the long bones (“spondylo‐epiphyseal” pattern). A mild degree of metaphyseal dysplasia can be seen in the so‐called Strudwick variant of spondyloepimetaphyseal dysplasia and is generally mild or absent in other forms.

Comparison of tandem mass spectrometry and amino acid analyzer for phenylalanine and tyrosine monitoring—Implications for clinical management of patients with hyperphenylalaninemia

OBJECTIVES Regular and accurate monitoring of blood phenylalanine (Phe) and tyrosine (Tyr) levels is prerequisite for a successful management of patients with hyperphenylalaninemia (HPA). We aimed to compare the tandem mass spectrometry (MS/MS) and the amino acid analyzer (AAA) as methods to measure blood Phe and Tyr levels and Phe/Tyr ratio. METHODS Venous blood samples were collected for the AAA analysis, using Pinnacle PCX (Pickering Laboratories), with HPLC Series 1200 (Agilent). Capillary blood was spotted directly on filter paper (Whatman 903) for the MS/MS analysis, using 3200 QTrap AB SCIEX and Perkin Elmer Series 200 HPLC system. The Bland-Altman test was used to compare agreement between the methods and Pearson correlation coefficient to assess the association between the methods. RESULTS 207 pairs of measurements were performed. The Phe levels (range 0-2500μM) obtained by the MS/MS were on average 26.1% (SD 13.9%) lower compared to those obtained by the AAA. The Tyr levels by the MS/MS were on average 15.5% (SD 20.6%) lower. The Phe/Tyr ratio by the MS/MS was on average 10.6% (SD 15.9%) lower. The Pearson correlation coefficients for Phe (range 0-2500μM), Tyr and the Phe/Tyr ratio were 0.984 (p<0.001), 0.841 (p<0.001) and 0.987 (p<0.001) respectively. CONCLUSIONS When monitoring blood Phe and Tyr levels in patients with HPA, clinicians need to be informed about the method used. Due to the considerable inter-assay variability, a single method is preferable for long-term follow-up of patients. When using MS/MS, on average 26% lower blood Phe levels were obtained as compared to the AAA. The guidelines and recommendations on HPA management should take into consideration the differences in laboratory methods.

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.

Fifty years of phenylketonuria newborn screening — A great success for many, but what about the rest?

Guthries landmark discovery and the subsequent implementation of the first newborn screening programs for phenylketonuria (PKU) and other inherited errors of metabolism (IEM) could be - in a 50 year retrospective - easily considered among the greatest advances in medicine. They have not just improved the quality of hundreds of thousands of lives, but also transformed our understanding and approach to PKU and IEM in general. However, according to the available albeit very scarce data, many countries and regions seem not to share the benefits of the last 50 years of development. Many of them have not yet introduced the newborn screening for PKU or face significant problems in its implementation. In addition, the issue seems to be underrated by the relevant professional forums. Action to improve the current situation should urgently be taken.

Assessment of tetrahydrobiopterin (BH4)-responsiveness and spontaneous phenylalanine reduction in a phenylalanine hydroxylase deficiency population

A BH(4) loading test was performed in 36 patients from 34 unrelated families. The patients had 29 different genotypes, and previous data on only eight of them were found in the BIOPKU database. Thirteen patients were classified as classic PKU (35.1%), 14 as mild PKU (37.8%) and 9 as MHP (27.0%). Blood Phe levels were shown to reach a plateau after three full days of increased natural protein ingestion. Measuring the 24-hour blood Phe levels (T(-24), T(-16), T(0)) on the fourth day of increased protein ingestion before BH(4) administration showed that within 24h Phe on average increased by 2.4% in MHP patients, decreased by 2.7% in mild PKU patients and increased by 9.7% in classic PKU patients (NS for all comparisons); Phe only slightly decreased in responders by 0.2% but increased in non-responders by 7.8% (P>0.05). Altogether, 16 of 36 (44.4%) patients represented by 12 of 29 (41.4%) different genotypes were proven to be BH(4) responders, and four (10.8%) were slow-responders. Responders were 6/9 (66.7%) MHP patients, 10/14 (71.4%) mild PKU patients and 0/13 classic PKU patients. Twenty of the 29 (68.9%) genotypes harbored at least one mutation with a known PRA of 10% or more but only 11 (55%) of them were BH(4)-responsive. Spontaneous reduction of blood Phe levels within 24h on the fourth day of natural protein loading was observed only in mild PKU patients and was shown not to be an important part of the BH(4)-response. 73.3% of genotypes containing at least one allele with a PRA of at least 30% were found to be BH(4) responsive; a PRA of at least 15.5% was needed for the responder genotype in our population.

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

BackgroundWe aimed to assess the current state of PKU screening and management in the region of southeastern Europe.MethodsA 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.ResultsResponses 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.ConclusionsThe 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.

A case report of short-chain acyl-CoA dehydrogenase deficiency (SCADD)

Background Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is a rare inherited mitochondrial fatty acid oxidation disorder associated with variations in the ACADS (Acyl-CoA dehydrogenase, C-2 to C-3 short chain) gene. SCADD has highly variable biochemical, genetic and clinical characteristics. Phenotypes vary from fatal metabolic decompensation to asymptomatic individuals. Subject and methods A Romani boy presented at 3 days after birth with hypoglycaemia, hypotonia and respiratory pauses with brief generalized seizures. Afterwards the failure to thrive and developmental delay were present. Organic acids analysis with gas chromatography-mass spectrometry (GS/MS) in urine and acylcarnitines analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS) in dried blood spot were measured. Deoxyribonucleic acid (DNA) was isolated from blood and polymerase chain reactions (PCRs) were performed for all exons. Sequence analysis of all exons and flanking intron sequences of ACADS gene was performed. Results Organic acids analysis revealed increased concentration of ethylmalonic acid. Acylcarnitines analysis showed increase of butyrylcarnitine, C4-carnitine. C4-carnitine was 3.5 times above the reference range (<0.68 µmol/L). Confirmation analysis for organic acids and acylcarnitine profile was performed on the second independent sample and showed the same pattern of increased metabolites. Sequence analysis revealed 3-bp deletion at position 310-312 in homozygous state (c.310_312delGAG). Mutation was previously described as pathogenic in heterozygous state, while it is in homozygous state in our patient. Conclusions In our case clinical features of a patient, biochemical parameters and genetic data were consistent and showed definitely SCAD deficiency.

Long-term BH4 (sapropterin) treatment of children with hyperphenylalaninemia - effect on median Phe/Tyr ratios.

Abstract Background: Phenylalanine hydroxylase deficiency causes various degrees of hyperphenylalaninemia (HPA). Tetrahydrobiopterin (BH4; sapropterin) reduces phenylalanine (Phe) levels in responders, enabling relaxation of dietary therapy. We aimed to assess long-term effects of BH4 treatment in HPA patients. Methods: Nine pre-pubertal BH4 responsive children were treated with BH4 for at least 2 years. The median dietary tolerance to Phe and levels of blood Phe, tyrosine (Tyr), zinc, selenium and vitamin B12 and anthropometric measurements, in the 2 years periods before and after the introduction of BH4 treatment were analyzed and compared. Adverse effects of BH4 were assessed. Results: The daily Phe tolerance had tripled, from pretreatment median value of 620 mg (IQR 400–700 mg) to 2000 (IQR 1000–2000 mg) after 2 years of follow up (p<0.001). The median blood Phe levels during the 2 years period before introducing BH4 did not change significantly during the 2 years on therapy (from 200 μmol/L; IQR 191–302 to 190 μmol/L; IQR 135–285 μmol/L), but the median blood Phe/Tyr ratio had lowered significantly from pre-treatment value 4.7 to 2.4 during the 2 years on therapy (p=0.01). Median zinc, selenium, vitamin B12 levels and anthropometric measurements did not change while on BH4 therapy (p=NS). No adverse effects were noticed. Conclusions: BH4 therapy enabled patients much higher dietary Phe intakes, with no noticeable adverse effects. Median blood Phe and Tyr levels, median zinc, selenium, vitamin B12 levels and anthropometric measurements did not change significantly on BH4 therapy, but median Phe/Tyr ratios had lowered.

Next generation sequencing as a follow-up test in an expanded newborn screening programme.

OBJECTIVES Contrary to many western European countries, most south-eastern European countries do not have an expanded newborn screening (NBS) program using tandem mass spectrometry. This study would represent one of the first expanded NBS studies in south-eastern Europe and will enable the estimation of the incidences of IEM in Slovenia. We proposed an expanded NBS approach including next-generation sequencing (NGS) as a confirmational analysis. DESIGN & METHODS We conducted a pilot study of expanded NBS for selected inborn errors of metabolism (IEM) in Slovenia including 10,048 NBS cards. We used an approach including tandem mass spectrometry followed by second tier tests including NGS. Based on the NBS results, 85 children were evaluated at a metabolic follow-up; 80 of them were analyzed using NGS. RESULTS Altogether, glutaric acidemia type 1 was confirmed in one patient who was a compound heterozygote for two known causative GCDH variants. A patient with suspected very long-chain acyl-CoA dehydrogenase deficiency had negative metabolic follow-up tests, but had two heterozygous ACADVL variants; one known disease-causing variant and one indel, namely c.205-8_205-7delinsGC, that is predicted to be causative. Nine participants had elevated metabolites characteristic of 3-methylcrotonyl-CoA carboxylase deficiency, 2 of them had known causative homozygous variants in MCCC1. The other seven were heterozygous; two had a novel genetic variant c.149_151dupCCA (p.Thr50dup). Cumulative incidences of IEM in Slovenia were similar to other European countries. CONCLUSIONS NGS proved to be valuable in explaining the abnormal metabolite concentrations in NBS as it enabled the differentiation between affected patients and mere heterozygotes, and it improved the turnaround time of genetic analysis. The results of this study will be instrumental in the routine implementation of expanded NBS in Slovenia.

The IVS8-2A>G (c.913-2A>G) mutation and the PAH deficiency populations of Central Europe

The recent report by Sterl et al provides a very interesting account on the genetic characteristics of the Austrian PAH deficiency population, not just rounding the genetic data on the Central European populations but also positioning Austria as their very integral part, with evidently strong links to Eastern Europe (Sterl et al 2012). However, the IVS8-2A>G (c.913-2A>G) mutation—one of the presumably five novel mutations in the Austrian PAH deficiency population where it is found in 3/294 (1 %) of the alleles— has been previously reported in the Croat population (its frequency is not shown) (Karacic et al 2009). It is also found in the Slovene population where it represents 4/214 (2 %) of the alleles (Groselj et al 2012). The simultaneous presence of the IVS8-2A>G (c.9132A>G) mutation in the three populations is not surprising considering the close geographical proximity and also the common historical roots of the three populations in the Austro-Hungarian Empire, in what has been traditionally known as Mitteleuropa. Conflict of interest None.