Next-generation sequencing reveals novel variants and large deletion in FANCA gene in Polish family with Fanconi anemia.

BACKGROUND: Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. However, establishing its molecular diagnosis remains challenging. Chromosomal breakage analysis is the gold standard diagnostic test for this disease. Nevertheless, molecular analysis is always required for the identification of pathogenic alterations in the FA genes. RESULTS: We report here on a family with FA diagnosis in two siblings. Mitomycin C (MMC) test revealed high level of chromosome breaks and radial figures. In both children, array-Comparative Genomic Hybridization (aCGH) showed maternally inherited 16q24.3 deletion, including FANCA gene, and next generation sequencing (NGS) disclosed paternally inherited novel variants in the FANCA gene-Asn1113Tyr and Ser890Asn. A third sibling was shown to be a carrier of FANCA deletion only. CONCLUSIONS: Although genetic testing in FA patients often requires a multi-method approach including chromosome breakage test, aCGH, and NGS, every effort should be made to make it available for whole FA families. This is not only to confirm the clinical diagnosis of FA in affected individuals, but also to enable identification of carriers of FA gene(s) alterations, as it has implications for diagnostic and genetic counselling process.

Molecular Background and Disease Prevalence of Biotinidase Deficiency in a Polish Population-Data Based on the National Newborn Screening Programme.

Biotinidase deficiency (BD) is a rare autosomal recessive metabolic disease. Previously the disease was identified only by clinical signs and symptoms, and since recently, it has been included in newborn screening programs (NBS) worldwide, though not commonly. In Europe, BD prevalence varies highly among different countries, e.g., from 1:7 116 in Turkey to 1:75 842 in Switzerland. This paper aimed to present the molecular spectrum of BD (profound and partial forms) in Polish patients diagnosed within the national NBS of 1,071,463 newborns. The initial suspicion of BD was based on an abnormal biotinidase activity result determined in a dry blood spot (DBS) by colorimetric and by fluorimetric methods while biochemical verification was determined by serum biotinidase activity (as quantitative analysis). The final diagnosis of BD was established by serum enzyme activity and the BTD gene direct sequencing. The obtained results allowed for the estimation of disease prevalence (1:66,966 births, while 1:178,577 for profound and 1:107,146 for partial forms), and gave novel data on the molecular etiology of BD.

Tumor Necrosis Factor Receptor-Associated Periodic Syndrome (TRAPS) with a New Pathogenic Variant in TNFRSF1A Gene in a Family of the Adult Male with Renal AA Amyloidosis-Diagnostic and Therapeutic Challenge for Clinicians.

Tumor necrosis factor receptor-associated periodic syndrome (TRAPS) belongs to systemic autoinflammatory diseases (AIDs). Many of these syndromes are genetically conditioned and can be inherited. Diagnosis relies on clinical symptoms and should be confirmed by genetic testing. One of the most serious complications is AA amyloidosis. We present the diagnostic route of a 33-year-old male with AA amyloidosis and his children, leading to diagnosis of monogenic autoinflammatory syndrome, confirmed by genetic analysis. A novel variant of the in-frame insertion type in one allele of TNFRSF1A gene was found by whole exome sequencing and confirmed by Sanger sequencing, which allowed a diagnosis of TRAPS. Three-dimensional modeling was used to assess the structural changes introduced into TNFR1 molecule by the insertion. The analysis of the 3D model revealed that accommodation of the 4AA insert induces misalignment of three cysteine bridges (especially the C70-C96 bridge) in the extracellular domain, leading to putatively misfolded and improperly functioning TNFR1. Three of the patient's daughters inherited the same variant of the TNFRSF1A gene and presented TRAPS symptoms. TRAPS is a very rare disease, but in the presence of suggestive symptoms the genetic diagnostic workout should be undertaken. Early diagnosis followed by appropriate clinical management can prevent irreversible complications.

Clinical characteristics of rare CFTR mutations causing cystic fibrosis in Polish population.

INTRODUCTION: More than 2000 mutations have been identified since the discovery of the CFTR gene in 1989. However, only 346 mutations have been classified as cystic fibrosis (CF)-causing mutations. Due to the increasing number of mutations and poor correlation between the genotype and phenotype, there is an urgent need to determine the mutations that are pathogenic, nonpathogenic, or lead to variable symptoms. AIM: The aim of the study was to present the clinical characteristics of Polish patients with rare and novel CFTR mutations, with an attempt to determine the pathogenicity status of those variants. MATERIALS AND METHODS: The group included 13 patients born between September 2006 and May 2019, who underwent CF newborn screening and in whom two CFTR mutations, including at least one rare or a novel mutation, were identified. RESULTS: We identified 13 patients with mutations in both alleles of the CFTR gene, one of which was at least rare in Polish population (R289NfsX17, I618RfsX2, T682KfsX40, S1347PfsX13, W356X, E33X, dup.16,17A) or was a mutation of unknown clinical consequences (H199R, L468P, A1217E, Q359R, T1036I, W1282R). None of them were described in the CFTR2 database. In all examined patients, sweat tests were elevated. The diagnosed patients presented with a wide spectrum of clinical symptoms. Broad clinical characteristics and test results are presented. CONCLUSION: Pathogenic mutations are H199R, L468P, A1217E, Q359R, T1036I, W1282R, R289NfsX17, I618RfsX2, T682KfsX40, S1347PfsX13, W356X, E33X, dup.16,17A. Every patient with a mutation of unknown clinical consequences in one CFTR allele requires attentive follow-up.

Fitting the pieces of the puzzle together: a case report of the Dunnigan-type of familial partial lipodystrophy in the adolescent girl.

BACKGROUND: Familial partial lipodystrophy of the Dunnigan type (FPLD 2) is a rare autosomal dominant disorder caused by the mutations of the lamin A/C gene leading to the defective adipogenesis, premature death of adipocytes and lipotoxicity. FPLD 2 is characterized by a progressive loss of subcutaneous adipose tissue in the limbs and trunk, and accumulation of body fat in the face and neck with accompanying severe metabolic derangements including insulin resistance, glucose intolerance, diabetes, dyslipidemia, steatohepatitis. Clinical presentation of FPLD 2 can often lead to misdiagnosis with metabolic syndrome, type 2 diabetes or Cushing syndrome. CASE PRESENTATION: We report a case of a 14-year-old girl admitted to the Department of Paediatrics due to chronic hypertransaminasemia. On physical examination the girl appeared to have athletic posture. She demonstrated the absence of subcutaneous adipose tissue in the extremities, sparing the face, neck and gluteal area, pseudo-hypertrophy of calves, prominent peripheral veins of limbs, massive acanthosis nigricans around the neck, in axillary and inguinal regions and natural skin folds, hepatosplenomegaly. Laboratory results revealed hypertransaminasemia, elevated γ-glutamyltranspeptydase, and dyslipidemia, hyperinsulinaemia with insulin resistance, impaired glucose tolerance, and hyperuricemia. Diffuse steatoheptitis in the liver biopsy was stated. Clinical suspicion of FPLD 2 was confirmed genetically. The pathogenic mutation, R482W (p.Arg482Trp), responsible for the FPLD 2 phenotype was identified in one allele of the LMNA gene. CONCLUSIONS: Presented case highlights the importance of the holistic approach to a patient and the need of accomplished collaboration between paediatricians and geneticists. FPLD 2 should be considered in the differential diagnosis of diabetes, dyslipidemia, steatohepatitis, acanthosis nigricans and polycystic ovary syndrome.

Newborn screening for cystic fibrosis: Polish 4 years' experience with CFTR sequencing strategy.

Newborn screening for cystic fibrosis (NBS CF) in Poland was started in September 2006. Summary from 4 years' experience is presented in this study. The immunoreactive trypsin/DNA sequencing strategy was implemented. The group of 1,212,487 newborns were screened for cystic fibrosis during the programme. We identified a total of 221 CF cases during this period, including, 4 CF cases were reported to be omitted by NBS CF. Disease incidence in Poland based on the programme results was estimated as 1/4394 and carrier frequency as 1/33. The frequency of the F508del was similar (62%) to population data previously reported. This strategy allowed us to identify 29 affected infants with rare genotypes. The frequency of some mutations (eg, 2184insA, K710X) was assessed in Poland for the first time. Thus, sequencing assay seems to be accurate method for screening programme using blood spots in the Polish population.

Molecular analysis of mucopolysaccharidosis type VI in Poland, Belarus, Lithuania and Estonia.

Mucopolysaccharidosis VI (MPS VI) is a rare autosomal recessive disorder caused by a deficiency of N-acetylgalactosamine-4-sulfatase (ARSB). Over 130 ARSB gene mutations have been identified thus far and most mutations are unique to individual families. We aimed to analyze the spectrum of mutations in the ARSB gene responsible for the disorder in Poland, Belarus and Baltic States. Twenty one families with MPS VI patients, in whom diagnosis was confirmed biochemically and enzymatically, were studied. Direct sequencing of patient genomic DNA was used to identify ARSB mutations. In total, fourteen different disease-causing mutations were found. Three novel mutations included insertion c.375_376insT, a missense mutation c.499G>A (p.G167R) and deletion/insertion c.750_754delinsCCTGAAGTCAAG. We also report 11 previously described mutations (p.A33V, p.W57C, p.Q88X, p.T92K, p.Q97X, p.R152W, p.R160Q, p.R160X, p.Y210C, p.Y266S, p.G302R). The mutation p.R152W was present at a high prevalence of 50% (21/42) the mutated alleles in this group of patients. High prevalence of p.R152W mutation in Poland, Belarus and Baltic States indicates a possible founder effect and suggests that screening for this mutation may be appropriate in MPS VI patients from this region. Our study has also provided evidence to support genotype-phenotype correlation.

[Genetic risk markers of low bone mineral density in cystic fibrosis children]. / Genetyczne markery ryzyka wystapienia obnizonej gestosci mineralnej tkanki kostnej u dzieci z mukowiscydoza.

THE AIM OF THE STUDY: Verification of the hypothesis concerning the association between polymorphic variants of the following genes: COLIA1, VDR and CALCR considered to be risk factors of bone metabolism disturbances and decreased bone mineral density (BMD) in children with cystic fibrosis (CF). MATERIAL AND METHODS: Clinical evaluation of CF phenotype progression in 101 patients was assessed according to the Shwachman-Kulczycki score. In the project the best value of forced expiratory volume of one second (FEV1) from the six months before densitometric measurements was used. Evaluation of bone tissue condition parameters was always correlated with the analysis of calcium-phosphate metabolism and bone turnover parameters. Densitometric measurements of L1-L4 lumbar spine were made using Lunar DPX IQ 2898. Age and sex of examined persons were standardized in respect to clinical and biochemical parameters. Molecular analysis was performed in CF patients with the following genotypes: F508del/F508del--55 persons, F508del/m--37 persons, m/m--9 persons. In this project 102 persons formed the control group. Presence of polymorphisms in studied genes was compared with bone tissue parameters. RESULTS: Low bone mineral density (Z-score < -1 SD) was observed in 53.5% patients and in 26.7% of them BMD was below -2 SD. Patients with low BMD had worse BMI, FEV1 and more severe symptoms of CF. Allele T (Ball) in COLIA I and allele C (L447P) in CALCR were found to be more frequent in CF patients than in the control group. Allele C in CALCR gene was associated with reduced bone mass. No significant correlation was found between COLIA1 and VDR polymorphisms and BMD. CONCLUSIONS: Process of bone loss in CF patients starts in early childhood and recurrent respiratory infection, malnutrition and corticosteroid therapy are the main factors disturbing metabolic balance of bone tissue. There is a correlation between bone mass loss in CF patients and the appearance of defined gene alleles of bone metabolism. However, we have to emphasize that this type of study needs confirmation on larger groups of patients.

Modifier gene study of meconium ileus in cystic fibrosis: statistical considerations and gene mapping results.

Cystic fibrosis (CF) is a monogenic disease due to mutations in the CFTR gene. Yet, variability in CF disease presentation is presumed to be affected by modifier genes, such as those recently demonstrated for the pulmonary aspect. Here, we conduct a modifier gene study for meconium ileus (MI), an intestinal obstruction that occurs in 16-20% of CF newborns, providing linkage and association results from large family and case-control samples. Linkage analysis of modifier traits is different than linkage analysis of primary traits on which a sample was ascertained. Here, we articulate a source of confounding unique to modifier gene studies and provide an example of how one might overcome the confounding in the context of linkage studies. Our linkage analysis provided evidence of a MI locus on chromosome 12p13.3, which was segregating in up to 80% of MI families with at least one affected offspring (HLOD = 2.9). Fine mapping of the 12p13.3 region in a large case-control sample of pancreatic insufficient Canadian CF patients with and without MI pointed to the involvement of ADIPOR2 in MI (p = 0.002). This marker was substantially out of Hardy-Weinberg equilibrium in the cases only, and provided evidence of a cohort effect. The association with rs9300298 in the ADIPOR2 gene at the 12p13.3 locus was replicated in an independent sample of CF families. A protective locus, using the phenotype of no-MI, mapped to 4q13.3 (HLOD = 3.19), with substantial heterogeneity. A candidate gene in the region, SLC4A4, provided preliminary evidence of association (p = 0.002), warranting further follow-up studies. Our linkage approach was used to direct our fine-mapping studies, which uncovered two potential modifier genes worthy of follow-up.

Analysis of CFTR, SPINK1, PRSS1 and AAT mutations in children with acute or chronic pancreatitis.

OBJECTIVES: Defects of PRSS1, SPINK1, CFTR and AAT are considered causative or predisposing to pancreatitis. The aim of this study was to evaluate the impact of these defects into molecular pathology of chronic pancreatitis (CP) and acute recurrent pancreatitis (ARP). METHODS: Ninety-two children with CP or ARP, 55 family members and 50 controls were investigated. The subjects were screened for PRSS1 mutations: R122H, R122C, A16V, N29I; SPINK1 N34S variant; panel of 14 CFTR defects: INNOLiPA CFTR12, CFTRdele2,3 and IVS8-T variant or panel of 3 CFTR defects-F508del, CFTRdele2,3 and IVS8-T; AAT mutations: E264V, E342K. RESULTS: We identified 1 mutated allele in at least 1 of 4 genes in 31 of 92 patients and 12 of 50 controls (P = 0.157). Mutations in SPINK1 and PRSS1 were most frequent. PRSS1 mutations were identified mainly in CP patients (9.6% of CP vs 2.5% of ARP alleles, P = 0.094), whereas N34S SPINK1 mutation was present with comparable frequency in CP and ARP patients (7.7% vs 10.0%, P = 0.768). The frequency of mutations in CFTR alleles was similar to controls (4.9% vs 5%, P = 0.587). Overall frequency of AAT mutations was lower than in the controls. Family studies showed that defects in the examined genes did not always segregate with disease. CONCLUSIONS: PRSS1 defects seem to be causative for pancreatitis, whereas defects in SPINK1 are suggested to be associated with the disease. No association between CFTR mutations and pancreatitis was observed. The importance of AAT variants remains speculative.

[Prenatal diagnosis of cystic fibrosis in risk families in Poland--results of molecular analysis]. / Diagnostyka prenatalna mukowiscydozy w rodzinach ryzyka w Polsce -- wyniki badan molekularnych.

BACKGROUND: Cystic fibrosis is one of the most common genetic disorders in the Caucasian population, inherited as an autosomal recessive trait. Diagnosis of CF classifies the patient's family to the group of high genetic risk. In spite of the significant therapeutic advantages this disease is still the cause of preterm death of affected patients. One of the diagnostic tests that are offered to CF risk families is prenatal diagnostics of the disease. This kind of analysis may be performed in the first trimester of pregnancy and is based on the DNA analysis of the foetus. AIM: To sum up and analyse the results of CF prenatal diagnostic studies, in Poland, in the period 1990-2003. MATERIAL AND METHOD: In total 45 tests in 38 risk families have been carried out. In case of 7 families in formative results have been obtained due to application of the polymorphic markers analysis. In the remaining cases molecular analysis of foetal DNA focused on identification of specific CFTR gene mutations has been carried out. RESULTS: In 16 cases the CF genotype was identified. The disease was excluded in 29 foetuses. Some issues of genetic counselling in the context of the possibility of prenatal disease diagnosis have also been discussed.

[Cystic fibrosis--a disease with many faces. The variable clinical picture versus the heterogeneity of molecular defect]. / Muskowidoza -- choroba o róznych obliczach. Zmienny obraz kliniczny a heterogennosc defektu molekularnego.

Cystic fibrosis (CF) is the most common genetic disorder in the Caucasian population with an autosomal recessive mode of inheritance. The disease is caused by mutations in the CFTR gene. So far, over 1300 of them have been identified. Primarily, the disease affects the epithelial cells of the airways, pancreas, intestines, gall bladder and sweat glands. However, cystic fibrosis is a clinically heterogeneous disorder. Especially the respiratory symptoms are significantly variable, even among patients with the same CFTR genotype. Therefore, there is an increasing interest in searching for the genetic modifiers of the clinical outcome in CF. This review presents the actual state of knowledge in terms of the potential genes-modifiers of the pulmonary form of cystic fibrosis.

[II. Pharmacogenetics--the future of modern pharmacology and genetics]. / II. Farmakogenetyka -- nadzieja wspólczesnej farmakologii i genetyki.

Pharmacogenetics is a scientific discipline connecting pharmacology and genetics. Its point of interest is an analysis of the variable, genetically determined patient's response to drugs. The basic discovery in this field was the elucidation of the heterogenic response of patients to the tuberculostatic drug -- isoniazid. The new era of searching for the molecular background of the variable drugs metabolism has begun after identification, in the 50's, of the polymorphic variants of NAT2 gene. It encodes for N-acetyltransferase 2, an isoniazid metabolizing enzyme. The key catalysts of the biotransformation reactions are cytochrome P450 isoenzymes. Identification of the polymorphic variants of genes encoding these enzymes enabled an explanation of the heterogenic drugs tolerance. The current knowledge shows that the genetic defects that are responsible for variable pharmacological response concern not only genes encoding the metabolising enzymes but also transporter proteins and drug receptors. Due to achievements of the molecular genetics and putting into practice the modern bioinformatic technics new pharmacological opportunities have been created. It becomes possible to predict the patient's individual reactions and needs, without the necessity of treatment by "trial and error". Pharmacogenetics will allow to implement personalized therapy leading to safer and more effective drugs usage. Identification of the individual metabolising paths will reduce patient's exposition to the side effects of many drugs. In spite of many financial and logistic limitations there are chances of introducing the pharmacogenetic analysis -- as an obligatory step -- to the phases II and III of clinical trials by 2020.

[I. Single nucleotide polymorphism in human genetic analyses]. / I. Polimorfizm pojedynczego nukleotydu w badaniach genetycznych czlowieka.

The new era of human genetic analyses has been began by finishing of The Human Genome Project. Discovery of the almost complete sequence of human DNA showed surprisingly small differences between the genetic materials of randomly chosen people. Genetists pay intensive attention to the very small part of nucleotide sequence - 0.1% - which contains polymorphic changes. The most frequent type of these changes is polymorphism of a single nucleotide (SNP). It makes up about 90% of all molecular differences in human DNA sequence. There are about 3 mln positions in DNA sequence containing SNPs. Polymorphic changes serve as genetic markers and they enable to map genes or to follow their inheritance. Changes of this kind seem also to be the possible cause of the remarkable variety of susceptibility to many common diseases e.g.: diabetes, cancer and cardiovascular disorders. SNPs are also the object of interest of intensively developing scientific domain - pharmacogenetics. Scientists working on this interdisciplinary field - connecting pharmacology and genetics - try to find out the reason of great variety of response to medicines and their side effects in case of patients belonging to the same therapeutical groups. Progress in this kind of research in near future will enable a significant improvement of pharmacological therapy, which will be based on matching the drug to genetically determined traits of patients.