The birth prevalence of lysosomal storage disorders in the Czech Republic: comparison with data in different populations

The aim of this retrospective study was to determine the prevalence of lysosomal storage disorders (LSDs) in the Czech Republic. The data on cases diagnosed between 1975 and 2008 were collected and analyzed. The overall prevalence of LSDs in the Czech population (12.25 per 100,000) is comparable to that reported for the countries with well-established and advanced diagnostics of LSDs such as the Netherlands (14 per 100,000), Australia (12.9 per 100,000) and Italy (12.1 per 100,000). Relatively higher prevalence of LSDs was reported in the north of Portugal (25 per 100,000). Thirty-four different LSDs were diagnosed in a total of 478 individuals. Gaucher disease was the most frequent LSD with a birth prevalence of 1.13 per 100,000 births. The most frequent LSD groups were lipidoses, mucopolysaccharidoses, and neuronal ceroid lipofuscinoses, with combined prevalences of 5.0, 3.72, and 2.29 per 100,000 live births, respectively. Glycoproteinoses (0.57 per 100,000 live births), glycogenosis type II (0.37), and mucolipidoses (0.31) rarely occur in the Czech population, and a range of other LSDs have not been detected at all over the past three decades. Knowledge of the birth prevalence and carrier frequency of particular disorders is important in genetic counselling for calculation of the risk for the disorder in the other members of affected families. Earlier diagnosis of these disorders will permit timely intervention and may also result in lowering of the number of newborns with LSDs

Determination of cystathionine beta-synthase activity in human plasma by LC-MS/MS: potential use in diagnosis of CBS deficiency

Cystathionine β-synthase (CBS) deficiency is usually confirmed by assaying the enzyme activity in cultured skin fibroblasts. We investigated whether CBS is present in human plasma and whether determination of its activity in plasma could be used for diagnostic purposes. We developed an assay to measure CBS activity in 20 μL of plasma using a stable isotope substrate - 2,3,3-2H serine. The activity was determined by measurement of the product of enzyme reaction, 3,3-2H-cystathionine, using LC-MS/MS. The median enzyme activity in control plasma samples was 404 nmol/h/L (range 66–1,066; n = 57). In pyridoxine nonresponsive CBS deficient patients, the median plasma activity was 0 nmol/ho/L (range 0–9; n = 26), while in pyridoxine responsive patients the median activity was 16 nmol/hour/L (range 0–358; n = 28); this overlapped with the enzyme activity from control subject. The presence of CBS in human plasma was confirmed by an in silico search of the proteome database, and was further evidenced by the activation of CBS by S-adenosyl-L-methionine and pyridoxal 5′-phosphate, and by configuration of the detected reaction product, 3,3-2H-cystathionine, which was in agreement with the previously observed CBS reaction mechanism. We hypothesize that the CBS enzyme in plasma originates from liver cells, as the plasma CBS activities in patients with elevated liver aminotransferase activities were more than 30-fold increased. In this study, we have demonstrated that CBS is present in human plasma and that its catalytic activity is detectable by LC-MS/MS. CBS assay in human plasma brings new possibilities in the diagnosis of pyridoxine nonresponsive CBS deficiency

Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells

The prevalent c.903+469T>C mutation in MTRR causes the cblE type of homocystinuria by strengthening an SRSF1 binding site in an ESE leading to activation of a pseudoexon. We hypothesized that other splicing regulatory elements (SREs) are also critical for MTRR pseudoexon inclusion. We demonstrate that the MTRR pseudoexon is on the verge of being recognized and is therefore vulnerable to several point mutations that disrupt a fine-tuned balance between the different SREs. Normally, pseudoexon inclusion is suppressed by a hnRNP A1 binding exonic splicing silencer (ESS). When the c.903+469T>C mutation is present two ESEs abrogate the activity of the ESS and promote pseudoexon inclusion. Blocking the 3′splice site or the ESEs by SSOs is effective in restoring normal splicing of minigenes and endogenous MTRR transcripts in patient cells. By employing an SSO complementary to both ESEs, we were able to rescue MTRR enzymatic activity in patient cells to approximately 50% of that in controls. We show that several point mutations, individually, can activate a pseudoexon, illustrating that this mechanism can occur more frequently than previously expected. Moreover, we demonstrate that SSO blocking of critical ESEs is a promising strategy to treat the increasing number of activated pseudoexon

Vascular presentation of cystathionine beta-synthase deficiency in adulthood

Several recent studies describing a solely vascular presentation of cystathionine beta-synthase (CBS) deficiency in adulthood prompted us to analyze the frequency of patients manifesting with vascular complications in the Czech Republic. Between 1980 and 2009, a total of 20 Czech patients with CBS deficiency have been diagnosed yielding an incidence of 1:311,000. These patients were divided into three groups based on symptoms leading to diagnosis: those with vascular complications, with connective tissue manifestation and with neurological presentation. A vascular event such as a clinical feature leading to diagnosis of homocystinuria was present in five patients, while two of them had no other symptoms typical for CBS deficiency at the time of diagnosis. All patients with the vascular manifestation were diagnosed only during the past decade. The median age of diagnosis was 29 years in the vascular, 11.5 years in the connective tissue and 4.5 years in the neurological group. The ratio of pyridoxine responsive to nonresponsive patients was higher in the vascular (4 of 5 patients) and connective tissue groups (6 of 7 patients) than in the neurological group (2 of 8 patients). Mutation c.833T>C (p.I278T) was frequent in patients with vascular (6/10 alleles) and connective tissue presentation (8/14 alleles), while it was not present in patients with neurological involvement (0/16 alleles). During the last decade, we have observed patients with homocystinuria diagnosed solely due to vascular events; this milder form of homocystinuria usually manifests at greater ages, has a high ratio of pyridoxine responsiveness/nonresponsiveness, and the mutation c.833T>C (p.I278T) is often present

Recommendations for reporting results of diagnostic genetic testing (biochemical, cytogenetic and molecular genetic)

Genetic test results can have considerable importance for patients, their parents and more remote family members. Clinical therapy and surveillance, reproductive decisions and genetic diagnostics in family members, including prenatal diagnosis, are based on these results. The genetic test report should therefore provide a clear, concise, accurate, fully interpretative and authoritative answer to the clinical question. The need for harmonizing reporting practice of genetic tests has been recognised by the External Quality Assessment (EQA), providers and laboratories. The ESHG Genetic Services Quality Committee has produced reporting guidelines for the genetic disciplines (biochemical, cytogenetic and molecular genetic). These guidelines give assistance on report content, including the interpretation of results. Selected examples of genetic test reports for all three disciplines are provided in an annexe.</p

Postauthorization safety study of betaine anhydrous

Patient registries for rare diseases enable systematic data collection and can also be used to facilitate postauthorization safety studies (PASS) for orphan drugs. This study evaluates the PASS for betaine anhydrous (Cystadane), conducted as public private partnership (PPP) between the European network and registry for homocystinurias and methylation defects and the marketing authorization holder (MAH). Data were prospectively collected, 2013–2016, in a noninterventional, international, multicenter, registry study. Putative adverse and severe adverse events were reported to the MAH's pharmacovigilance. In total, 130 individuals with vitamin B6 nonresponsive (N = 54) and partially responsive (N = 7) cystathionine beta-synthase (CBS) deficiency, as well as 5,10-methylenetetrahydrofolate reductase (MTHFR; N = 21) deficiency and cobalamin C (N = 48) disease were included. Median (range) duration of treatment with betaine anhydrous was 6.8 (0–9.8) years. The prescribed betaine dose exceeded the recommended maximum (6 g/day) in 49% of individuals older than 10 years because of continued dose adaptation to weight; however, with disease-specific differences (minimum: 31% in B6 nonresponsive CBS deficiency, maximum: 67% in MTHFR deficiency). Despite dose escalation no new or potential risk was identified. Combined disease-specific treatment decreased mean ± SD total plasma homocysteine concentrations from 203 ± 116 to 81 ± 51 μmol/L (p < 0.0001), except in MTHFR deficiency. Recommendations for betaine anhydrous dosage were revised for individuals ≥ 10 years. PPPs between MAH and international scientific consortia can be considered a reliable model for implementing a PASS, reutilizing well-established structures and avoiding data duplication and fragmentation