Identification of New Copy Number Variation and the Evaluation of a CNV Detection Tool for NGS Panel Data in Polish Familial Hypercholesterolemia Patients.

Familial hypercholesterolemia (FH) is an inherited, autosomal dominant metabolic disorder mostly associated with disease-causing variant in LDLR, APOB or PCSK9. Although the dominant changes are small-scale missense, frameshift and splicing variants, approximately 10% of molecularly defined FH cases are due to copy number variations (CNVs). The first-line strategy is to identify possible pathogenic SNVs (single nucleotide variants) using multiple PCR, Sanger sequencing, or with more comprehensive approaches, such as NGS (next-generation sequencing), WES (whole-exome sequencing) or WGS (whole-genome sequencing). The gold standard for CNV detection in genetic diagnostics are MLPA (multiplex ligation-dependent amplification) or aCGH (array-based comparative genome hybridization). However, faster and simpler analyses are needed. Therefore, it has been proposed that NGS data can be searched to analyze CNV variants. The aim of the study was to identify novel CNV changes in FH patients without detected pathogenic SNVs using targeted sequencing and evaluation of CNV calling tool (DECoN) working on gene panel NGS data; the study also assesses its suitability as a screening step in genetic diagnostics. A group of 136 adult and child patients were recruited for the present study. The inclusion criteria comprised at least "possible FH" according to the Simon Broome diagnostic criteria in children and the DLCN (Dutch Lipid Clinical Network) criteria in adults. NGS analysis revealed potentially pathogenic SNVs in 57 patients. Thirty selected patients without a positive finding from NGS were subjected to MLPA analysis; ten of these revealed possibly pathogenic CNVs. Nine patients were found to harbor exons 4−8 duplication, two harbored exons 6−8 deletion and one demonstrated exon 9−10 deletion in LDLR. To test the DECoN program, the whole study group was referred for bioinformatic analysis. The DECoN program detected duplication of exons 4−8 in the LDLR gene in two patients, whose genetic analysis was stopped after the NGS step. The integration of the two methods proved to be particularly valuable in a five-year-old girl presenting with extreme hypercholesterolemia, with both a pathogenic missense variant (c.1747C>T) and exons 9−10 deletion in LDLR. This is the first report of a heterozygous deletion of exons 9 and 10 co-occurring with SNV. Our results suggest that the NGS-based approach has the potential to identify large-scale variation in the LDLR gene and could be further applied to extend CNV screening in other FH-related genes. Nevertheless, the outcomes from the bioinformatic approach still need to be confirmed by MLPA; hence, the latter remains the reference method for assessing CNV in FH patients.

Identification of New Genetic Determinants in Pediatric Patients with Familial Hypercholesterolemia Using a Custom NGS Panel.

The most common form of inherited lipid disorders is familial hypercholesterolemia (FH). It is characterized primarily by high concentrations of the clinical triad of low-density lipoprotein cholesterol, tendon xanthomas and premature CVD. The well-known genetic background are mutations in LDLR, APOB and PCSK9 gene. Causative mutations can be found in 60−80% of definite FH patients and 20−30% of those with possible FH. Their occurrence could be attributed to the activity of minor candidate genes, whose causal mechanism has not been fully discovered. The aim of the conducted study was to identify disease-causing mutations in FH-related and candidate genes in pediatric patients from Poland using next generation sequencing (NGS). An NGS custom panel was designed to cover 21 causative and candidate genes linked to primary dyslipidemia. Recruitment was performed using Simon Broome diagnostic criteria. Targeted next generation sequencing was performed on a MiniSeq sequencer (Illumina, San Diego, CA, USA) using a 2 × 150 bp paired-end read module. Sequencing data analysis revealed pathogenic and possibly pathogenic variants in 33 out of 57 studied children. The affected genes were LDLR, APOB, ABCG5 and LPL. A novel pathogenic 7bp frameshift deletion c.373_379delCAGTTCG in the exon 4 of the LDLR gene was found. Our findings are the first to identify the c.373_379delCAGTTCG mutation in the LDLR gene. Furthermore, the double heterozygous carrier of frameshift insertion c.2416dupG in the LDLR gene and missense variant c.10708C>T in the APOB gene was identified. The c.2416dupG variant was defined as pathogenic, as confirmed by its cosegregation with hypercholesterolemia in the proband's family. Although the APOB c.10708C>T variant was previously detected in hypercholesterolemic patients, our data seem to demonstrate no clinical impact. Two missense variants in the LPL gene associated with elevated triglyceride plasma level (c.106G>A and c.953A>G) were also identified. The custom NGS panel proved to be an effective research tool for identifying new causative aberrations in a genetically heterogeneous disease as familial hypercholesterolemia (FH). Our findings expand the spectrum of variants associated with the FH loci and will be of value in genetic counseling among patients with the disease.

Clinical Features of Familial Hypercholesterolemia in Children and Adults in EAS-FHSC Regional Center for Rare Diseases in Poland.

BACKGROUND: Familial hypercholesterolemia (FH) is a genetic autosomal co-dominant metabolic disorder leading to elevated circulating concentrations of low-density lipoprotein cholesterol (LDL-C). Early development of atherosclerotic cardiovascular disease (ASCVD) is common in affected patients. We aimed to evaluate the characteristics and differences in the diagnosis and therapy of FH children and adults. Methods: All consecutive patients who were diagnosed with FH, both phenotypically and with genetic tests, were included in this analysis. All patients are a part of the European Atherosclerosis Society FH-Study Collaboration (FHSC) regional center for rare diseases at the Polish Mother's Memorial Hospital Research Institute (PMMHRI) in Lodz, Poland. Results: Of 103 patients with FH, there were 16 children (15.5%) at mean age of 9 ± 3 years and 87 adults aged 41 ± 16; 59% were female. Children presented higher mean levels of total cholesterol, LDL-C, and high-density lipoprotein cholesterol (HDL-C) measured at the baseline visit (TC 313 vs. 259 mg/dL (8.0 vs. 6.6 mmol/L), p = 0.04; LDL 247 vs. 192 mg/dL (6.3 vs. 4.9 mmol/L), p = 0.02, HDL 53 vs. 48 mg/dL (1.3 vs. 1.2 mmol/L), p = 0.009). Overall, 70% of adult patients and 56% of children were prescribed statins (rosuvastatin or atorvastatin) on admission. Combination therapy (dual or triple) was administered for 24% of adult patients. Furthermore, 13.6% of adult patients and 19% of children reported side effects of statin therapy; most of them complained of muscle pain. Only 50% of adult patients on combination therapy achieved their treatment goals. None of children achieved the treatment goal. CONCLUSIONS: Despite a younger age of FH diagnosis, children presented with higher mean levels of LDL-C than adults. There are still urgent unmet needs concerning effective lipid-lowering therapy in FH patients, especially the need for greater use of combination therapy, which may allow LDL-C targets to be met in most of the patients.

Helicobacter pylori Infection in Children with Phenylketonuria Does Not Depend on Metabolic Control and Is Not More Frequent Than in Healthy Subjects-A Cross-Sectional Study.

In a small preliminary study, phenylketonuria and poor metabolic control were suggested as risk factors for Helicobacter pylori infection in children as detected with an antigen stool test. We aimed to determine Helicobacter pylori prevalence in an adequately sized group of individuals with phenylketonuria and healthy subjects using the standard gold test (urea breath test). Further, we correlated Helicobacter pylori infection with metabolic control. The study comprised 103 individuals with phenylketonuria and 103 healthy subjects on whom a 13C urea breath test was performed. Blood phenylalanine levels in the preceding year were analysed. The infection rate did not differ between individuals with phenylketonuria and healthy subjects (10.7% vs 15.5%; p = 0.41). The frequency of testing and phenylalanine concentrations of Helicobacter pylori-positive and Helicobacter pylori-negative patients with phenylketonuria did not differ (p = 0.92 and p = 0.54, respectively). No associations were detected for body mass index or metabolic control. Forward stepwise regression models revealed that age (p = 0.0009-0.0016) was the only independent correlate of Helicobacter pylori infection with a relatively low fraction of the variability of the condition being explained (adjR2 = 0.0721-0.0754; model p = 0.020-0.023). In conclusion, Helicobacter pylori infection in phenylketonuria is not more frequent than in the general population. Moreover, it does not depend on metabolic control.

The Impact of the COVID-19 Pandemic on the Perception of Health and Treatment-Related Issues among Patients with Phenylketonuria in Poland-The Results of a National Online Survey.

There is agreement that the pandemic has affected the healthcare system and behaviour of patients. This study aims to identify problems encountered by patients with phenylketonuria (PKU) and their parents/caregivers during the six-week pandemic lockdown in Poland (15 March to 30 April 2020). To determine the factors that influenced health and treatment-related issues, as well as the respondents' perception of the impact of the pandemic, study participants were asked to complete a non-validated online questionnaire comprising 31 questions (including 27 single-choice, two multiple-choice and two open-ended ones). A total of 571 patients or their parents completed the questionnaire, with 9.5% of respondents not performing any blood phenylalanine (Phe) test in the analysed period, 21.3% declaring a blood Phe increase, and 15.3% a decrease. Increased problems in contacting the doctor or dietitian were reported by 26.1% of subjects, whereas 39.3% of them felt restricted access to dietary products. Most (63.4%) participants were satisfied with remote contact with their PKU clinic. Better compliance was associated with higher odds of acceptance of remote contact and of reporting fewer problems with contacting the doctor, and with lower odds of missing Phe testing. Self-reported high stress was associated with higher odds of reporting the limited availability of low-Phe products and Phe-free formulas, as well as with increased Phe concentrations and non-PKU-related health problems. These patients also had poor dietary compliance and experienced more problems in contacting specialists. Health and treatment-related problems experienced during the pandemic lockdown were related to a higher intensity of stress in patient's family and worse therapy compliance before the pandemic. Previous experience of remote visits resulted in a better perception of this method of contact. It seems that this form of communication should be popularized and improved to increase therapy effectiveness in case of different limitations in the future. Special attention should be paid to vulnerable patients who may be at extra risk when the provision of standard care is affected.

Carriership of the rs113883650/rs2287120 haplotype of the SLC7A5 (LAT1) gene increases the risk of obesity in infants with phenylketonuria.

PURPOSE: Phenylketonuria (PKU) can be effectively treated with the use of a low-phenylalanine diet. However, some patients become overweight despite proper dietary treatment. We hypothesized that this phenomenon could be explained by the presence of specific variants within the genes involved in phenylalanine transport or in the phenylalanine transamination/oxygenation pathway. METHODS: We selected a clinically homogenous group of 100 infants with PKU and assessed their growth patterns in the context of dietary phenylalanine tolerance. Next, within the sample, we performed exome sequencing and assessed a potential relationship between the observed phenotypical variability and the presence of structural variants in a priori selected genes of interest. RESULTS: We detected a highly significant association between overweight and carriership of the rs113883650/rs2287120 haplotype of the SLC7A5 (LAT1) gene, which encodes the main transmembrane transporter of large neutral amino acids and of thyroid hormones. CONCLUSIONS: Our findings suggest a pharmacogenetic effect of the relatively common rs113883650/rs2287120 haplotype of the SLC7A5 gene. This can have practical implications for patients with PKU, since treatment protocols need to be reassessed to better prevent overweight in the carriers of the above variant.

"Apple does not fall far from the tree" - subclinical atherosclerosis in children with familial hypercholesterolemia.

BACKGROUND: Familial hypercholesterolemia (FH) increases the risk of atherosclerosis in children and adults. Atherosclerotic cardiovascular disease in young patients FH is usually subclinical but recognition of children with more pronounced changes is crucial for adjusting effective management. Aim of this research was to use ultrasonography with two-dimensional speckle tracking (2DST) and tonometry to evaluate atherosclerotic changes in patients with FH (parents and their offspring). METHODS: Applanation tonometry and carotid arteries sonography with evaluation of the intima-media complex thickness (IMCT) and application of the 2DST were performed in 20 families with FH (20 parents and 29 children). The same size control group (age and sex matched) was included. Results were compared between peers and between generations together with the correlation analysis. RESULTS: Adults with FH, in comparison with healthy peers, presented significantly more atherosclerotic plaques (9 vs. 2, p = 0.0230), had significantly thicker IMC (0.84 ± 0.19 vs. 0.56 ± 0.06 mm, p < 0.0001) and had stiffer arterial wall (for stain: 6.25 ± 2.3 vs. 8.15 ± 2.46, p = 0.0103). In children from both groups there were no atherosclerotic plaques and IMCT did not differ significantly (0.42 ± 0.07 vs. 0.39 ± 0.04, p = 0.1722). However, children with FH had significantly stiffer arterial wall according to 2DST (for strain: 9.22 ± 3.4 vs. 11.93 ± 3.11, p = 0.0057) and tonometry (for the pulse wave velocity: 4.5 ± 0.64 vs.3.96 ± 0.62, p = 0.0047). These parameters correlated with atherosclerosis surrogates in their parents (p < 0.001) but were not significantly affected by presence of presumed pathogenic gene variant. CONCLUSIONS: Children with FH presented subclinical atherosclerosis manifested as decreased arterial wall elasticity. Degree of stiffening was associated with advancement of atherosclerosis in their parents but did not present significant association with gene variants. Sonography with application of 2DST seems to be a good candidate for comprehensive evaluation of atherosclerosis in families with FH.

Phenylketonuria patients' and their parents' knowledge and attitudes to the daily diet - multi-centre study.

BACKGROUND: The aim of the study was to assess both patients' and their parents' knowledge of phenylketonuria (PKU) treatment and compliance with PKU diet. METHODS: The study included 173 PKU patients aged 10-19 and 110 parents of PKU children who were enrolled in the study on the basis of questionnaire data. The study also included 45 patients aged ≥20. RESULTS: Our study demonstrated that only 45% (n = 74) of PKU patients knew daily Phe intake recommendations, 27% of patients (n = 41) knew the Phe content in a minimum of three out of four researched food products. Patients' knowledge concerning Phe intake (p = 0.0181) and the knowledge of selected food products (p = 0.041819) improved with age. We did not establish such a correlation in the group of PKU children's parents. Approximately 31% of patients and 22% of parents reported helplessness, which increased with the child's age, associated with the necessity to adhere to the diet; 30% of patients reported feeling ashamed of the fact that they could not eat all food products. Regardless of age, children were more likely than parents to report helplessness (p = 0.032005). Among patients, 41.40% declared that they would wish to select products unassisted but their parents did not permit them to do so. The question of whether parents teach children self-reliance in meal preparation was answered affirmatively by 98% of parents and only 81% of children (p = 0.0001). CONCLUSION: Our data demonstrated that parents' and children's knowledge concerning treatment recommendations and food products does not have a direct impact on attitude to the PKU diet. Limiting children's independence in meal selection, growing helplessness in the face of dietary adherence and shame resulting from the necessity to follow a different diet observed in PKU families are responsible for shaping and perpetuating a consistently negative attitude to the diet. The care of PKU paediatric patients requires consistent, long-term family and individual therapy which may counteract the effects of learned helplessness. In regard to the educational effort, a good parent-child relationship as well as the teaching of behaviours motivating patients to comply with the diet are of great importance.

Hen's egg as a source of valuable biologically active substances.

The aim of this article is to show current knowledge concerning valuable substances biologically active present in hen eggs and underline important nutritive role of hen eggs. Hen egg is a good source of nutrients such as proteins, vitamins (A, B2, B6, B12, D, E, K), minerals and lipids. The significant part of lipids is a group of unsaturated phospholipids, which are components of cell membranes, act protectively on the cardiovascular system and contribute to a decrease of cholesterol level and blood pressure. Therefore, the consumption of unsaturated phospholipids is recommended especially in patients suffering from diseases of the cardiovascular system. Another important substance is egg cystatin, which has a wide spectrum of biological functions, for example the ability to stimulate cell growth, inhibit inflammatory processes and has antibacterial and antiviral properties. Other substance presented in the egg white which helps fight bacteria is lysozyme. It is used in medicine as an aid in antibiotic therapy and analgesic in the course of infection, as well as in tumor malignancies. Among the components contained in the egg yolk there is also immunoglobulin Y which due to its therapeutic importance deserves special attention. Its use offers the possibility of replacing chemotherapeutic agents in the treatment of bacterial infections of digestive system, as well as an opportunity for the development of medicine associated with passive immunization of patients. The egg is a rich source of retinol which gradual depletion in the organism causes many eye pathologies. A very important and useful part of the egg, used in medicine is a shell and its membranes, due to the high collagen content relevant in the treatment of connective tissue diseases.

Phenylketonuria patients' and their parents' acceptance of the disease: multi-centre study.

PURPOSE: Phenylketonuria (PKU) still poses a therapeutic challenge for patients and medical professionals. The aim of the study was to assess both patients' and their parents' acceptance of the disease. METHODS: The study included 218 PKU patients and 178 parents of PKU children who were enrolled in the study on the basis of questionnaire data. RESULTS: Regarding attitude towards the disease, our study demonstrated that 63 (28.9 %) PKU patients did not accept the disease. Patients who found accepting the disease difficult, more frequently perceived themselves as inferior/different in comparison with their peers. In total, 36 % of patients did not want their friends to be aware of their condition, while only 18 % of parents believed that their children's peers should not know about their disease. In total, 42 % of parents wanted to talk to other parents of PKU children and only 13 % to a doctor. Only 20 % of patients saw the need to discuss their condition with a doctor. In total, 8 % of children, regardless of age, and 14 % of parents preferred to talk to a psychologist. CONCLUSION: Our data demonstrated that disease acceptance played an essential role in patients' social integration. The study also indicated the need to overcome communication barriers between patients and their healthy peers and for patients to find the courage to be open about the disease. The importance of support groups for PKU families and the significance of strict cooperation between patients and their families with PKU treatment teams were also revealed.

Molecular genetics of PKU in Poland and potential impact of mutations on BH4 responsiveness.

Tetrahydrobiopterin (BH4) has been recently approved as a treatment of patients with phenylketonuria. However, as a confirmation of BH4-responsiveness, it might require a very expensive trial treatment with BH4 or prolonged BH4-loading procedures. The selection of patients eligible for BH4-therapy by means of genotyping of the PAH gene mutations may be recommended as a complementary approach. A population-wide genotyping study was carried out in 1286 Polish phenyloketonuria-patients. The aim was to estimate the BH4 demand and to cover prospectively the treatment by a National Health Fund. A total of 95 types of mutations were identified. Genetic variants corresponding with probable BH4-responsiveness were found in 28.2% of cases. However, patients with mild or classical phenylketonuria who require continuous treatment accounted for 11.4% of the studied population only. Analysis of the published data shows similar percentage of the "BH4-responsive" variants of a PAH gene in patients from other countries of Eastern Europe. Therefore, it can be concluded, that the proportion of phenylketonuria-patients who could benefit from the use of BH4 reaches approximately 10% in the entire region.

Impact of lipophilic antioxidants and level of antibodies against oxidized low-density lipoprotein in Polish children with phenylketonuria.

The treatment of phenylketonuria (PKU) patients constitutes a phenylalanine (Phe) intake restriction in their diet, which is achieved by adding a special Phe-free amino acid mixture to the diet. It has been reported that this diet could have some micronutrient deficiency. Several authors have also reported an increased oxidative stress or impaired antioxidant status in human and experimental PKU. Our project assessed the concentrations of retinol, alpha-tocopherol, coenzyme Q10, and anti-oxidized low-density lipoprotein (ox-LDL) antibodies in PKU children's plasma. It was found that retinol concentration in PKU children remains within the norm despite a low intake. The lower plasma alpha-tocopherol concentration in PKU children compared with normal children was associated with the lower level of antibodies against ox-LDL. This raises the question whether higher than observed circulatory alpha-tocopherol is indeed beneficial to lower plasma ox-LDL levels. Further studies are needed to explain the genetic factor in PKU patients (e.g., CD36/FAT polymorphism gene). The open clinical question is whether daily supplementation of alpha-tocopherol changes the PKU patients' level of antibodies against ox-LDL.