Genetic testing in Poland and Ukraine: should comprehensive germline testing of BRCA1 and BRCA2 be recommended for women with breast and ovarian cancer?

PURPOSE: To characterize the spectrum of BRCA1 and BRCA2 pathogenic germline variants in women from south-west Poland and west Ukraine affected with breast or ovarian cancer. Testing in women at high risk of breast and ovarian cancer in these regions is currently mainly limited to founder mutations. METHODS: Unrelated women affected with breast and/or ovarian cancer from Poland (n = 337) and Ukraine (n = 123) were screened by targeted sequencing. Excluded from targeted sequencing were 34 Polish women who had previously been identified as carrying a founder mutation in BRCA1. No prior testing had been conducted among the Ukrainian women. Thus, this study screened BRCA1 and BRCA2 in the germline DNA of 426 women in total. RESULTS: We identified 31 and 18 women as carriers of pathogenic/likely pathogenic (P/LP) genetic variants in BRCA1 and BRCA2, respectively. We observed five BRCA1 and eight BRCA2 P/LP variants (13/337, 3.9%) in the Polish women. Combined with the 34/337 (10.1%) founder variants identified prior to this study, the overall P/LP variant frequency in the Polish women was thus 14% (47/337). Among the Ukrainian women, 16/123 (13%) women were identified as carrying a founder mutation and 20/123 (16.3%) were found to carry non-founder P/LP variants (10 in BRCA1 and 10 in BRCA2). CONCLUSIONS: These results indicate that genetic testing in women at high risk of breast and ovarian cancer in Poland and Ukraine should not be limited to founder mutations. Extended testing will enhance risk stratification and management for these women and their families.

Evaluation of a clinical pharmacogenetics model to predict methotrexate response in patients with rheumatoid arthritis.

Variability of response to treatment hinders successful management of rheumatoid arthritis (RA). Consequently, a clinical pharmacogenetics model for predicting response to methotrexate (CP-MTX) has been previously proposed that includes four clinical variables (disease activity, sex, the presence of rheumatoid factor and smoking status) and four SNPs (rs2236225, rs17602729, rs1127354, and rs2372536) in genes of the folate pathway. It showed good performance, but failed to attract attention, likely, in relation with lack of clear clinical benefit. Here, we have revised the value of the CP-MTX model directly addressing its clinical benefit by focusing on the expected benefit-cost of the predictions. In addition, our study included a much larger number of RA patients (n = 720) in MTX monotherapy than previous studies. Benefit of CP-MTX prediction was defined as the patients that would have received combination therapy as first treatment because they were correctly predicted as non-responders to MTX monotherapy. In contrast, cost of CP-MTX prediction was defined as the responder patients that were wrongly predicted as non-responders. Application of CP-MTX predictions to our patients showed a good benefit-cost relationship, with half of the 66.7% non-responders to MTX monotherapy rightly directed to alternative treatments (a benefit of 33.3%) at the cost of 8.5% wrongly predicted non-responders. These benefits-costs were consistent with reanalysis of the previously published studies. Therefore, predictions of CP-MTX showed a good benefit-cost relationship for informing MTX prescription.

FANCM and RECQL genetic variants and breast cancer susceptibility: relevance to South Poland and West Ukraine.

BACKGROUND: FANCM and RECQL have recently been reported as breast cancer susceptibility genes and it has been suggested that they should be included on gene panel tests for breast cancer predisposition. However, the clinical value of testing for mutations in RECQL and FANCM remains to be determined. In this study, we have characterised the spectrum of FANCM and RECQL mutations in women affected with breast or ovarian cancer from South-West Poland and West Ukraine. METHODS: We applied Hi-Plex, an amplicon-based enrichment method for targeted massively parallel sequencing, to screen the coding exons and proximal intron-exon junctions of FANCM and RECQL in germline DNA from unrelated women affected with breast cancer (n = 338) and ovarian cancer (n = 89) from Poland (n = 304) and Ukraine (n = 123). These women were at high-risk of carrying a genetic predisposition to breast and/or ovarian cancer due to a family history and/or early-onset disease. RESULTS: Among 427 women screened, we identified one carrier of the FANCM:c.1972C > T nonsense mutation (0.23%), and two carriers of the frameshift insertion FANCM:c.1491dup (0.47%). None of the variants we observed in RECQL were predicted to be loss-of-function mutations by standard variant effect prediction tools. CONCLUSIONS: Our study of the Polish and Ukrainian populations has identified a carrier frequency of truncating mutations in FANCM consistent with previous reports. Although initial reports suggesting that mutations in RECQL could be associated with increased breast cancer risk included women from Poland and identified the RECQL:c.1667_1667 + 3delAGTA mutation in 0.23-0.35% of breast cancer cases, we did not observe any carriers in our study cohort. Continued screening, both in research and diagnostic settings, will enable the accumulation of data that is needed to establish the clinical utility of including RECQL and FANCM on gene panel tests.

Genetic Variation in One-Carbon Metabolism and Changes in Metabolic Parameters in First-Episode Schizophrenia Patients.

Background: In this study, we aimed to investigate the effects of polymorphisms in genes encoding 1-carbon metabolism enzymes on differential development of metabolic parameters during 12 weeks of treatment with second-generation antipsychotics in first-episode schizophrenia patients. Methods: The following polymorphisms in 1-carbon metabolism genes were genotyped: MTHFR (C677T and A1298C), MTHFD1 (G1958A), MTRR (A66G), and BHMT (G742A). A broad panel of metabolic parameters including body mass index, waist circumference, total cholesterol low and high density lipoproteins, triglycerides, homocysteine, folate, and vitamin B12 was determined. Results: There was a significant effect of the interaction between the MTHFR C677T polymorphism and time on body mass index and waist circumference in the allelic and genotype analyses. Indeed, patients with the MTHFR 677CC genotype had higher increase in body mass index and waist circumference compared with other corresponding genotypes or the MTHFR 677T allele carriers (CT and TT genotypes). In addition, patients with the MTHFR 677TT genotype had higher waist circumference in all time points. Similarly, patients with the MTHFR 677TT genotype had higher body mass index in all time points, but this effect was not significant after correction for multiple testing. Conclusions: Our results indicate that the MTHFR C677T polymorphism may predict antipsychotic-induced weight gain. Effects of the MTHFR C677T polymorphism might be different in initial exposure to antipsychotics compared with long-term perspective.

[Rapid diagnosis of the most common fetal aneuploidies with the QF-PCR method--a study of 100 cases]. / Szybka diagnostyka najczestszych aneuploidii u plodu meaoda QF-PCR--analiza 100 przypadków.

UNLABELLED: The aim of the study was to assess whether commercial kit QF-PCR can be used as the only method for rapic prenatal dia gnosis of chromosomes 13, 18, 21, X and Y aneuploidies, omitting cell culture and complete cyt6genetik analysis of fetal chromosomes. MATERIAL AND METHODS: DNA from amniocytes (94 cases) and trophoblast cells (6 cases) was analyzed witt QF-PCR according to the manufacturer's protocol. The obtained products were separated using ABI 310 Genetic Analyzer and the resulting data were analyzed using GeneMarker software. RESULTS: The results of QF-PCR were obtained in 95 out of 100 cases (95%). Abnormalities were found in 28 casea (29.5%). All these results were confirmed in subsequent cytogenetic analysis. Normal results were obtained in 62 patients (70.5%). However in that group, we found three chromosomal aberrations other than those analyzed b3 QF-PCR. Additionally two abnormal and three normal karyotypes were found in patients with inconclusive QF-POF results. CONCLUSIONS: QF-PCR is a fast and reliable tool for chromosomal aneuploidy analysis and can be used as the only method without a full analysis of the karyotype, but only in cases of suspected fetal 13, 18, 21 trisomy or numerica aberrations of X chromosome. In other cases, fetal karyotype analysis from cells obtained after cell culture should be offered to the patient.

[The 2nd conference 'Rare diseases not only in the curriculum', Szczecin - Wroclaw, 26 and 30 May 2015].

The 2nd conference 'Rare diseases not only in the curriculum', that took place on 26th May, 2015 in Pomeranian Medical University in Szczecin and 30 May in Wroclaw Medical University. In accordance with the convention adopted at the first conference in 2014 in Szczecin participants of the meeting realized the idea expressed in the title of the conference by presenting issues of commonly known rare diseases and those that are not discussed in the course of medical studies. The active participants of the meeting were scientific workers medical schools in Szczecin, Wroclaw and Bialystok, medical students and PhD students. The significance of newborn screening in the early diagnosis of rare diseases in children, possibilities of supporting children with genetically conditioned rare diseases and their caregivers, application of guidelines of evidence-based medicine in the process of diagnosing non-routine patients and methods of physiotherapy of children with spinal muscular atrophy were discussed. Detailed issues of diagnosing and treatment of many rare diseases were also presented, among others Wilson disease, Alström syndrome, Cohen syndrome, Rubinstein-Taybi syndrome, Cornelia de Lange syndrome, Poland syndrome, Netherton syndrome, inborn aniridia and congenital arhinia - very rare defect requiring further scientific studies.

Effects of second-generation antipsychotics on selected markers of one-carbon metabolism and metabolic syndrome components in first-episode schizophrenia patients.

PURPOSE: Alterations in one-carbon metabolism (OCM) have been repeatedly reported in schizophrenia. However, there is a scarcity of studies addressing the effects of antipsychotics on selected OCM markers in schizophrenia and provided results are inconsistent. METHODS: We recruited 39 first-episode schizophrenia (FES) patients and determined serum profile of total homocysteine (tHcy), folate, vitamin B12, lipoproteins and glucose at baseline and after 12 weeks of treatment with second-generation antipsychotics (SGA) including olanzapine and risperidone in monotherapy. RESULTS: After 12 weeks of treatment, all patients had significantly higher body mass index (BMI), serum levels of total cholesterol (TC), low-density lipoproteins (LDL), triglycerides (TG) and tHcy together with significantly lower levels of folate and vitamin B12. The analysis of differences between SGA revealed the same biochemical alterations in patients treated with olanzapine as in the whole group, while those receiving risperidone had no statistically significant changes in serum folate, vitamin B12 and TG. There was a significantly higher increase in BMI and TC in patients treated with olanzapine in comparison with those treated with risperidone. Patients receiving olanzapine had a higher decrease in vitamin B12 than those assigned to the treatment with risperidone. Changes in folate, vitamin B12, tHcy and TC levels were significant only in males, even after Bonferroni correction. Multiple regression analysis revealed that changes in tHcy levels are associated with gender and baseline metabolic parameters (BMI, glucose, TC, LDL and HDL) but not with selected SGA. CONCLUSIONS: These results indicate that SGA may influence OCM, especially in first-episode schizophrenia (FES) males.

Elevated homocysteine level in first-episode schizophrenia patients--the relevance of family history of schizophrenia and lifetime diagnosis of cannabis abuse.

Accumulating evidence indicates that elevated homocysteine (Hcy) level occurs in first-episode schizophrenia (FES) patients. We included 56 FES patients and 53 healthy controls (HC). Plasma level of Hcy was significantly higher in FES patients than HC (p = 0.044). In addition, plasma levels of high-density lipoproteins (HDL) and folate were significantly lower in FES than in HC (p < 0.001). Positive family history of schizophrenia was associated with lower plasma HDL (p = 0.041) and vitamin B12 (p = 0.017), as well as higher level of Hcy (p = 0.017). Patients with FES, who abused cannabis, had higher levels of Hcy (p = 0.017), as well as lower levels of vitamin B12 (p = 0.017) and HDL (p = 0.041). Plasma Hcy negatively correlated with duration of untreated psychosis (r = -0.272, p = 0.042). There was a positive correlation between Hcy level and the severity of negative symptoms (r = 0.363, p = 0.006) and general psychopathology (r = 0.349, p = 0.008) assessed using Positive and Negative Syndrome Scale (PANSS). Vitamin B12 level was negatively associated with the severity of negative symptoms (r = -0.406, p = 0.002), while folate level negatively correlated with general psychopathology score (r = -0.365, p = 0.006) in PANSS. These results indicate that the severity of one-carbon metabolism alterations and HDL deficiency might be associated with family history of schizophrenia and cannabis abuse. Lower vitamin B12 and folate along with elevated Hcy may influence the severity of FES psychopathology.

Validation of the minisequencing method for detection of G1691A (Leiden) factor V mutation.

INTRODUCTION: The factor V (FV) plays an important role in the coagulation process and belongs to the group of factors that significantly increases the risk of thrombophilia. Our study presents a novel, rapid method for the detection of FV (R506Q) mutation using minisequencing approach. MATERIAL AND METHODS: Samples of peripheral blood were obtained from 300 females of the Lower Silesian population. Minisequencing, as one of the polymerase chain reaction (PCR)-based methods, was used for detection the of FV (R506Q) point mutations. The allele restriction mutation system PCR (ARMS-PCR) verifying method was applied. RESULTS: By using minisequencing reaction we examined the FV genotypes in the female group who experienced at least one unexplained spontaneous miscarriage. The results of the ARMS-PCR, as a verifying test, were fully consistent with the results of the minisequencing technique. DISCUSSION: One of the many factors which may cause thrombophilia is the FV gene mutation R506Q. A full validation of the minisequencing method was carried out in order to apply this method to clinical tests. The validation shows that the minisequencing technique is highly precise and may be used in routine diagnostics of the FV R506Q mutation.

Pro12Ala PPAR γ2 gene polymorphism in PCOS women: the role of compounds regulating satiety.

UNLABELLED: Five to ten percent of women of reproductive age suffer from polycystic ovary syndrome (PCOS). Leptin, NPY, galanin, cholecystokinin (CCK) are involved in the regulation of eating behavior. PPARγ are receptors that are probably involved in hyperandrogenism. This study was designed to assess associations between the Pro12Ala PPARγ2 gene polymorphism and satiety factors in PCOS. Fifty-four PCOS women and 51 healthy women were studied. Leptin, NPY, galanin, CCK levels, and genetic studies to detect Pro12Ala PPARγ2 gene polymorphism were assessed. The leptin levels in the PCOS women carrying Pro12Ala genotype were higher than in those with Pro12Pro and Ala12Ala. The PCOS women had higher leptin and NPY levels and lower galanin levels. Obese PCOS patients had lower CCK levels. CONCLUSIONS: In the PCOS women, a single Ala allele may have a protective role as far as hyperleptinemia is concerned. The PCOS women may reveal a disrupted central leptin/NPY feedback loop with some shifts in food intake.

Co-occurrence of severe Goltz-Gorlin syndrome and pentalogy of Cantrell - Case report and review of the literature.

Goltz-Gorlin syndrome is a highly variable disorder affecting many body parts of meso-ectodermal origin. Mutations in X-linked PORCN have been identified in almost all patients with a classical Goltz-Gorlin phenotype. The pentalogy of Cantrell is an infrequently described congenital disorder characterized by the combination of five anomalies: a midline supra-umbilical abdominal wall defect; absent or cleft lower part of the sternum; deficiency of the diaphragmatic pericardium; deficiency of the anterior diaphragm; and congenital heart anomalies. Etiology and pathogenesis are unknown. We report on an infant with findings fitting both Goltz-Gorlin syndrome (sparse hair; anophthalmia; clefting; bifid nose; irregular vermillion of both lips; asymmetrical limb malformations; caudal appendage; linear aplastic skin defects; unilateral hearing loss) and the pentalogy of Cantrell (absent lower sternum; anterior diaphragmatic hernia; ectopia cordis; omphalocele). The clinical diagnosis Goltz-Gorlin syndrome was confirmed molecularly by a point mutation in PORCN (c.727C>T). The presence of molecularly confirmed Goltz-Gorlin syndrome and pentalogy of Cantrell in a single patient has been reported twice before. The present patient confirms that the pentalogy of Cantrell can be caused in some patients by a PORCN mutation. It remains at present uncertain whether this can be explained by the type or localization of the mutation within PORCN, or whether the co-occurrence of the two entities is additionally determined by mutations or polymorphisms in other genes, environmental factors, and/or epigenetic influences.

[The importance of pharmacogenetic tests in evaluation of the effectiveness of methotrexate treatment in rheumatoid arthritis (part 1)]. / Znaczenie badan farmakogenetycznych w efektywnosci leczenia metotreksatem chorych na reumatoidalne zapalenie stawów (czesc 1).

Methotrexate (MTX) is still the gold standard in the treatment of rheumatoid arthritis and is used worldwide in more than 0.5 million patients with RA. Much hope is currently associated with the individualization of therapy provided to RA patients. The search is underway for biochemical and clinical markers that would be useful in predicting good response to MTX therapy. Along with clinical factors, genetic predisposition may also be helpful. Polymorphism of genes participating in MTX metabolism may affect the drug's efficacy and the rate of adverse effects. Pharmacogenetic studies may contribute to more effective individualization of therapy for RA patients. There are many potential enzymes and transport proteins present in polymorphic forms, which are involved in transport of MTX into the cell, its metabolism and elimination from the cell. There is a chance that in the future through individualized therapy we will be able to customize therapy (type of drug, dose, route of administration) to the molecular subtype of the disease and the genotype of the patient. Patients with a specific type of polymorphism would require more frequent monitoring of the efficacy and safety of treatment. Note, however, that genetic predisposition is only one of the factors contributing to differences in pharmacotherapy in individual patients.

[The importance of pharmacogenetic tests in evaluation of the effectiveness of methotrexate treatment in rheumatoid arthritis (part 2)]. / Znaczenie badan farmakogenetycznych w efektywnosci leczenia metotreksatem chorych na reumatoidalne zapalenie stawów (czesc 2).

Much hope is currently associated with individualization of therapy provided to rheumatoid arthritis (RA) patients. The search is underway for biochemical and clinical markers that would be useful in prediction of a good response to methotrexate (MTX) therapy. Along with clinical factors, genetic predisposition may also be helpful. Polymorphism of genes participating in MTX metabolism may affect the drug's efficacy and the rate of adverse effects. Pharmacogenetic studies may contribute to more effective individualization of therapy for RA patients. The purpose of the study was to determine the significance of gene polymorphisms MTHFR C677T and A1298C for efficacy of MTX therapy in RA patients. Patients possessing determined polymorphisms should be particularly carefully evaluated because of a higher risk of occurrence of adverse effects.

[The role of 1691G>A (Leiden) mutation in Factor V gene, 20210G>A in prothrombin gene and 677C>T in MTHFR gene in etiology of early pregnancy loss]. / Ocena roli mutacji 1691G>A (Leiden) w genie czynnika V, 20210G>A w genie protrombiny oraz 677C>T w genie MTHFR w etiologii wczesnych poronien samoistnych.

OBJECTIVES: The objective of this study was to investigate the prevalence of common hereditary risk factors for thrombophilia (mutations 1691G>A, 20210G>A and 677C>T variant in factor V Leiden (FV), prothrombin (FII) and MTHFR gene, respectively)--in a cohort of women with early pregnancy loss. MATERIAL AND METHODS: Frequency of mutations in FV, FII and MTHFR was assessed by PCR-RFLP or minisequencing in a cohort of 313 women with a history of at least two miscarriages and the control group consisting of 200 women without obstetric complications. RESULTS: Compared with controls, neither FV mutation (3.2% vs 3%; p=0.45) nor the MTHFR 677TT variant (8.4% vs 11.1%; p=0.58) was more prevalent in the patients. Mutation in FII gene was more frequent in the patients (3.5% vs 0.5%; p=0.03) when compared with controls, however, the frequency of this mutation in controls was lower than estimated frequency in the population.

Pro12Ala PPAR gamma2 gene polymorphism in women with polycystic ovary syndrome.

INTRODUCTION: The pathogenesis of PCOS has not been definitively determined and includes a number of genes linked with steroidogenesis, regulation of gonadotropin secretion, actions of insulin, obesity as well as chronic inflammatory processes. Some authors indicate that PPARgamma play a role in insulin sensitivity and are probably involved in hyperandrogenism in PCOS. The aim of the study was to assess the frequency of the Pro12Ala and Pro115Gln PPARgamma2 gene polymorphisms in women with PCOS. SUBJECTS AND METHODS: 54 PCOS women and 51 healthy women were recruited. Genetic studies to detect Pro12Ala and Pro115Gln PPARgamma2 gene polymorphism were performed. RESULTS: In the whole studied group the Pro115Gln polymorphism of the PPARgamma2 gene was not found. The frequency of the Pro12Ala polymorphism was estimated at 26.47% in the controls and at 23.15% in the PCOS patients. Women from the control and PCOS groups with BMI > or = 30 had statistically higher occurrence of the Ala allele than women with BMI <30 (38.80% versus 12.50% and 38.23% versus 18.75%). CONCLUSIONS: The frequency of the Pro12Ala polymorphism observed in the sample of women from the Lower Silesian population was significantly higher than in the majority of European populations.

Occurrence of Esophageal Atresia With Tracheoesophageal Fistula in Siblings From Three-Generation Family Affected by Variable Expressivity MYCN Mutation: A Case Report.

The MYCN oncogene encodes a transcription factor belonging to the MYC family. It is primarily expressed in normal developing embryos and is thought to be critical in brain and other neural development. Loss-of-function variants resulting in haploinsufficiency of MYCN, which encodes a protein with a basic helix-loop-helix domain causes Feingold syndrome (OMIM 164280, ORPHA 391641). We present an occurrence of esophageal atresia (EA) with tracheoesophageal fistula in siblings from a three-generation family affected by variable expressivity of MYCN mutation p.(Ser90GlnfsTer176) as a diagnostic effect of searching the cause of familial esophageal atresia using NGS-based whole-exome sequencing (WES). All of our affected patients showed microcephaly and toe syndactyly, which were frequently reported in the literature. Just one patient exhibited clinodactyly. None of the patients exhibited brachymesophalangy or hypoplastic thumbs. The latest report noted that patients with EA and Feingold syndrome were also those with the more complex and severe phenotype. However, following a thorough review of the present literature, the same association was not found, which is also confirmed by the case we described. The variable phenotypic expression of the patients we described and the data from the literature guide a careful differential diagnosis of Feingold syndrome even in cases of poorly expressed and non-specific symptoms.

Further Delineation of Phenotype and Genotype of Primary Microcephaly Syndrome with Cortical Malformations Associated with Mutations in the WDR62 Gene.

Type 2 congenital microcephaly (MCPH2) is a brain development disorder characterized by primary microcephaly with or without brain malformations. MCPH2 is caused by mutations in the WDR62 gene. We present three new patients with MCPH2 and compound heterozygous mutations in the WDR62 gene. In all the cases, the parents were healthy and unrelated. All children were clinically diagnosed with congenital microcephaly and retardation of motor and speech development. Sequencing results in the presented patients revealed five new variants in the WDR62 gene (c.4273C>T, c.1711_1712insTA, c.1777_1778delGA, c.1642+2T>G, c.194T>A) and one previously described in the German population (c.2864_2867delACAG). In two of the presented cases, variants in the SMAD4, DKC1, and ATRX genes were also found with unknown effects on the course of the disease. Moreover, in the article we collected and compared the most common clinical symptoms, dysmorphic features, and changes in radiographic examinations of the brain observed in 120 patients with recessive primary microcephaly type 2 caused by mutations in the WDR62 gene.

Exome Sequencing Reveals Novel Variants and Expands the Genetic Landscape for Congenital Microcephaly.

Congenital microcephaly causes smaller than average head circumference relative to age, sex and ethnicity and is most usually associated with a variety of neurodevelopmental disorders. The underlying etiology is highly heterogeneous and can be either environmental or genetic. Disruption of any one of multiple biological processes, such as those underlying neurogenesis, cell cycle and division, DNA repair or transcription regulation, can result in microcephaly. This etiological heterogeneity manifests in a clinical variability and presents a major diagnostic and therapeutic challenge, leaving an unacceptably large proportion of over half of microcephaly patients without molecular diagnosis. To elucidate the clinical and genetic landscapes of congenital microcephaly, we sequenced the exomes of 191 clinically diagnosed patients with microcephaly as one of the features. We established a molecular basis for microcephaly in 71 patients (37%), and detected novel variants in five high confidence candidate genes previously unassociated with this condition. We report a large number of patients with mutations in tubulin-related genes in our cohort as well as higher incidence of pathogenic mutations in MCPH genes. Our study expands the phenotypic and genetic landscape of microcephaly, facilitating differential clinical diagnoses for disorders associated with most commonly disrupted genes in our cohort.

Phenotypic expansion in Zhu-Tokita-Takenouchi-Kim syndrome caused by de novo variants in the SON gene.

BACKGROUND: The genetic etiology of intellectual and psychomotor disability without a defined spectrum of dysmorphic features is usually monogenic. As no diagnostic criteria for such diseases are established, the clinical diagnosis becomes to be a challenge. The object of our paper is to present two patients with non-specific clinical symptoms for whom whole-exome-sequencing identified the new SON mutations and thus allowed for establishing the diagnosis of Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome. In both patients, the same symptoms including hypotonia, developmental and speech delay, feeding difficulties as well as frequent infections of the respiratory tract and internal ear were observed. However, both cases presented also with exceptional symptoms such as in case 1 ventriculomegaly and asymmetry of ventricles, hypoplastic left heart syndrome (HLHS), intellectual disability, intestinal malrotation, gastroparesis, and duodenal atresia and in the case 2 febrile seizures and reduced IgA levels. We will be presenting the patients and comparing them to 30 previously described cases. METHODS: Whole-exome sequencing (WES) was performed on the probands' DNA and paired-end sequenced (2x100 bp) on HiSeq 1500. Variants considered as disease-causing were validated in the proband and studied in all available family members by amplicon deep sequencing performed using Nextera XT Kit and sequenced on HiSeq 1500. RESULTS: We have identified two new variants in SON gene. In case 1 it has been a heterozygous frameshift variant p.(Ala1340GlnfsTer26), while in case 2 it has been a heterozygous frameshift variant, p.(Asp1640GlyfsTer7). Both variants are described for the first time and up to now, are not mentioned in any database. CONCLUSION: As there are no precise criteria established for the clinical diagnosis of ZTTK, an identification of SON gene mutation by whole-exome-sequencing is the best method that allows for a diagnosis of this syndrome.

Multiplex ligation-dependent probe amplification as a screening test in children with autism spectrum disorders.

BACKGROUND: Autism spectrum disorders (ASDs) are a heterogeneous group of neurodevelopmental disorders, characterized by the presence of various symptoms related to deficits in communication and social interactions as well as stereotyped and repetitive behavior. Increasing evidence indicates the contribution of genetic factors in the etiology of ASDs. Genetic diagnosis in ASDs is based on identifying chromosome aberrations, microaberrations and point mutations in specific genes. One of the diagnostic tools is multiplex ligase-dependent probe amplification (MLPA) with a set of probes dedicated to ASDs (SALSA MLPA P343 Autism-1; MRC-Holland BV, Amsterdam, the Netherlands) targeting the genes located in the regions 15q11-q13, 16p11 and the SHANK3 gene in the 22q13 region. OBJECTIVES: Our study included 240 patients referred to the clinical genetics unit because of ASDs and/or developmental delay and/or an intellectual disability. Before genetic testing, the patients underwent a comprehensive medical work-up. MATERIAL AND METHODS: Multiplex ligase-dependent probe amplification was performed in 256 DNA samples from 240 probands and 16 family members using the SALSA MLPA P343 Autism-1 probe mix (MRC-Holland BV) according to the manufacturer's protocol. RESULTS: We obtained 234 normal results and 22 abnormal results (15 probands and 7 abnormal results for probands' parents or siblings). We diagnosed 1 16p11 microdeletion syndrome and 1 16p11 microduplication syndrome. We also found 3 deletions and 1 duplication in 15q13 region including 2 or 3 genes and 9 single probe alterations in the regions examined (1 duplication and 7 deletions). CONCLUSIONS: Due to the low costs, MLPA test may be a good tool for the genetic screening of ASD patients.