Comparative Genomic Hybridization to Microarrays in Fetuses with High-Risk Prenatal Indications: Polish Experience with 7400 Pregnancies.

The aim of this study was to determine the suitability of the comparative genomic hybridization to microarray (aCGH) technique for prenatal diagnosis, but also to assess the frequency of chromosomal aberrations that may lead to fetal malformations but are not included in the diagnostic report. We present the results of the aCGH in a cohort of 7400 prenatal cases, indicated for invasive testing due to ultrasound abnormalities, high-risk for serum screening, thickened nuchal translucency, family history of genetic abnormalities or congenital abnormalities, and advanced maternal age (AMA). The overall chromosomal aberration detection rate was 27.2% (2010/7400), including 71.2% (1431/2010) of numerical aberrations and 28.8% (579/2010) of structural aberrations. Additionally, the detection rate of clinically significant copy number variants (CNVs) was 6.8% (505/7400) and 0.7% (57/7400) for variants of unknown clinical significance. The detection rate of clinically significant submicroscopic CNVs was 7.9% (334/4204) for fetuses with structural anomalies, 5.4% (18/336) in AMA, 3.1% (22/713) in the group of abnormal serum screening and 6.1% (131/2147) in other indications. Using the aCGH method, it was possible to assess the frequency of pathogenic chromosomal aberrations, of likely pathogenic and of uncertain clinical significance, in the groups of cases with different indications for an invasive test.

Mutations in the COL1A1 and COL1A2 genes associated with osteogenesis imperfecta (OI) types I or III.

Although over 85% of osteogenesis imperfecta (OI) cases are associated with mutations in the procollagen type I genes (COL1A1 or COL1A2), no hot spots for the mutations were associated with particular clinical phenotypes. Eight patients that were studied here, diagnosed with OI by clinical standards, are from the Polish population with no ethnic background indicated. Previously unpublished mutations were found in six out of those eight patients. Genotypes for polymorphisms (Sp1 - rs1800012 and PvuII - rs412777), linked to bone formation and metabolism were determined. Mutations were found in exons 2, 22, 50 and in introns 13 and 51 of the COL1A1 gene. In COL1A2, one mutation was identified in exon 22. Deletion type mutations in COL1A1 that resulted in OI type I had no effect on collagen type I secretion, nor on its intracellular accumulation. Also, a single base substitution in I13 (c.904-9 G>T) was associated with the OI type I. The OI type III was associated with a single base change in I51 of COL1A1, possibly causing an exon skipping. Also, a missense mutation in COL1A2 changing Gly→Cys in the central part of the triple helical domain of the collagen type I molecule caused OI type III. It affected secretion of the heterotrimeric form of procollagen type I. However, no intracellular accumulation of procollagen chains could be detected. Mutation in COL1A2 affected its incorporation into procollagen type I. The results obtained shall help in genetic counseling of OI patients and provide a rational support for making informed, life important decisions by them and their families.

Low-symptomatic skeletal muscle disease in patients with a cardiac disease - Diagnostic approach in skeletal muscle laminopathies.

Mild skeletal muscle symptoms might be accompanied with severe cardiac disease, sometimes indicating a serious inherited disorder. Very often it is a cardiologist who refers a patient with cardiomyopathy and/or cardiac arrhythmia and discrete muscle disease for neurological consultation, which helps to establish a proper diagnosis. Here we present three families in which a diagnosis of skeletal muscle laminopathy was made after careful examination of the members, who presented with cardiac arrhythmia and/or heart failure and a mild skeletal muscle disease, which together with positive family history allowed to direct the molecular diagnostics and then provide appropriate treatment and counseling.

Healthy Baby Born to a Robertsonian Translocation Carrier following Next-Generation Sequencing-Based Preimplantation Genetic Diagnosis: A Case Report.

Preimplantation genetic diagnosis (PGD) is well established method for treatment of genetic problems associated with infertility. Moreover, PGD with next-generation sequencing (NGS) provide new possibilities for diagnosis and new parameters for evaluation in, for example, aneuploidy screening. The aim of the study was to report the successful pregnancy outcome following PGD with NGS as the method for 24 chromosome aneuploidy screening in the case of Robertsonian translocation. Day 3 embryos screening for chromosomal aneuploidy was performed in two consecutive in vitro fertilization (IVF) cycles, first with fluorescent in situ hybridization (FISH), and then with NGS-based protocol. In each IVF attempt, three embryos were biopsied. Short duration of procedures enabled fresh embryo transfer without the need for vitrification. First IVF cycle with the embryo selected using PGD analysis with the FISH method ended with pregnancy loss in week 8. The second attempt with NGS-based aneuploidy screening led to exclusion of the following two embryos: one embryo with 22 monosomy and one with multiple aneuploidies. The transfer of the only euploid blastocyst resulted in the successful pregnancy outcome. The identification of the euploid embryo based on the NGS application was the first successful clinical application of NGS-based PGD in the case of the Robertsonian translocation carrier couple.

High frequency of BRCA1/2 germline mutations in consecutive ovarian cancer patients in Poland.

BACKGROUND: We estimated the prevalence of BRCA1/2 germline mutations in consecutive ovarian cancers and correlated the mutation status with clinicopathological features. METHODS: 151 consecutive primary ovarian cancer patients were screened for BRCA1/2 germline mutations. RESULTS: We identified BRCA1/2 germline mutations in 21 (13.9%) patients. Seventeen (81%) of carriers have BRCA1 and four (19%) have BRCA2 mutation. BRCA1/2 carriers have a distinctly longer overall survival than sporadic cases (log-rank, p=0.014). CONCLUSIONS: The relatively high proportion of BRCA1/2 carriers among unselected ovarian cancer patients indicates the necessity of searching for recurrent BRCA mutations in each case of ovarian carcinoma. This routine screen should be widened to include denaturing high performance liquid chromatography (DHPLC) analysis of both exons 11 of BRCA1 and BRCA2 genes in women with positive family history.

Frontometaphyseal dysplasia: mutations in FLNA and phenotypic diversity.

Frontometaphyseal dysplasia is an X-linked trait primarily characterized by a skeletal dysplasia comprising hyperostosis of the skull and modeling anomalies of the tubular bones. Extraskeletal features include tracheobronchial, cardiac, and urological malformations. A proportion of individuals have missense mutations or small deletions in the X-linked gene, FLNA. We report here our experience with comprehensive screening of the FLNA gene in a group of 23 unrelated probands (11 familial instances, 12 simplex cases; total affected individuals 32) with FMD. We found missense mutations leading to substitutions in the actin-binding domain and within filamin repeats 9, 10, 14, 16, 22, and 23 of filamin A in 13/23 (57%) of individuals in this cohort. Some mutations present with a male phenotype that is characterized by a severe skeletal dysplasia, cardiac, and genitourinary malformations that leads to perinatal death. Although no phenotypic feature consistently discriminates between females with FMD who are heterozygous for FLNA mutations and those in whom no FLNA mutation can be identified, there is a difference in the degree of skewing of X-inactivation between these two groups. This observation suggests that locus heterogeneity may exist for this disorder.

HER2 Amplification Has no Prognostic Value in Sporadic and Hereditary Ovarian Tumours.

Whereas HER2 amplification is a well-known phenomenon in breast tumours, its frequency and clinical importance in ovarian cancer have not been established. The aim of the study was to compare the frequency of HER2 amplification in hereditary (BRCA-positive) and sporadic (BRCA-negative) ovarian tumours and to estimate the association of this gene alteration on clinical outcome in ovarian cancer patients. We analysed HER2 amplification in 53 ovarian tumours: 20 from mutation carriers (18 in BRCA1 and 2 in BRCA2 gene) and 33 from non-carriers. Fluorescence in situ hybridization for HER2 was performed on 'touch' slides from frozen tumour samples or formalin-fixed, paraffin-embedded tissue. Our results indicate that high amplification (HER2: centromere ratio>5) is an infrequent phenomenon in ovarian tumours (6/53 cases). It occurs in both hereditary (4/20) and sporadic (2/33) tumours and no difference in the frequency of HER2 amplification exists between these groups. There is no significant difference in the clinical outcome of patients with HER2 amplified and non-amplified tumours (p = 0.3). Our results suggest a different biological role of HER2 amplification in ovarian and breast cancer.

Prevalence and clinical correlations of BRCA1/BRCA2 unclassified variant carriers among unselected primary ovarian cancer cases - preliminary report.

The objective of this study was to determine the prevalence of BRCA1 and BRCA2 gene mutations in unselected ovarian cancer patients, and to analyse clinical and pathological features of ovarian cancer unclassified variant mutation carriers in comparison with BRCA1 pathogenic mutation carriers and sporadic cases. A consecutive sample of 205 women with primary ovarian cancer was screened for mutations in the BRCA1 and BRCA2 genes using a direct test for small deletions and insertions, conformational sensitive gel electrophoresis and direct sequencing. Data regarding medical and familial history were collected using questionnaires. Clinical and pathological data were extracted from medical records. Unclassified variants and polymorphic mutations accounted for 8% (n = 16) and 6% (n = 13) of all cases, respectively. BRCA1 pathogenic mutations were found in 18 (9%) patients. None were found in BRCA2. The mean age of onset for BRCA1-associated tumours was 43.1 years (standard deviation (SD: 7.3) whereas in the patients with an unclassified variant, polymorphism, or no detectable gene changes, the mean age of onset ranged from 49.5-56.4 years. The most significant predictors for pathogenic or unclassified variant changes in BRCA1 in ovarian cancer patients were a younger age of onset and a history of hyperthyroidism and infertility. Except for infertility and hyperthyroidism, unclassified variant-linked ovarian tumours share features with sporadic tumours rather than with BRCA1 pathogenic mutations.

Analysis of candidate genes for genotypic diagnosis in the long QT syndrome.

Patients with the long QT syndrome (LQTS) suffer from cardiac arrhythmias that can lead to abrupt loss of consciousness and sudden death, already in young individuals. Thus, an early diagnosis of LQTS is essential for patients and their family members. So far, six genes (KCNQ1, HERG, SCN5A, ANK2, KCNE1, KCNE2) have been demonstrated to be involved in the development of LQTS. Since this syndrome is genetically heterogeneous and large-sized families are often not available for linkage analysis, alternative tools are required for a genetic diagnosis. To investigate genes with numerous exons, like KCNQ1, HERG, SCN5A and ANK2, segregation analysis of a Polish Romano-Ward family with eight members was performed as a reliable method faster than linkage analysis or direct sequencing. To test these four LQT loci, an appropriate selection of microsatellite markers covering different chromosomal regions was applied. Furthermore, two small genes KCNE1 and KCNE2 (at the LQT5 and LQT6 loci), and the SGK1 gene (encoding a kinase regulating KCNE1 and SCN5A channels) were sequenced. All six LQT loci and the SGK1 gene were excluded by these analyses, thus a different pathogenic mechanism of LQT syndromes can be presumed.

Loss of heterozygosity at chromosomes 3p and 17p in primary non-small cell lung cancer.

BACKGROUND: Loss of heterozygosity (LOH) of selected regions at chromosomes 3p and 17p in non-small cell lung cancer (NSCLC) and the association of these abnormalities with major clinical parameters and prognosis were studied. MATERIALS AND METHODS: The study group included 92 consecutive primary NSCLC tumours and four microsatellite markers from chromosome 3p and three markers from 17p were analyzed. RESULTS: LOH of at least one locus was found in 83% of all analyzed tumours. Most frequently deletion (58%) was found at locus D3S1481 (3p14.2). Sequence deletions of D17S520 (17p12) and TP53 (17p13.1) occurred in 52% of tumours. LOH occurrence at 3p and 17p was more frequent in squamous cell carcinomas compared to adenocarcinomas (89% vs. 75%), but this difference was not significant. CONCLUSION: No significant association was found between LOH on any analyzed loci and tumour stage (TNM) and grade (G). There was no correlation between LOH and survival.

Left ventricular size, mass and function in relation to angiotensin-converting enzyme gene and angiotensin-II type 1 receptor gene polymorphisms in patients with coronary artery disease.

The objective of the present study was to analyse the potential synergistic influence of the insertion/deletion polymorphism of the angiotensin-converting enzyme gene (I/D ACE) and the A1166C polymorphism of the angiotensin-II type 1 receptor gene polymorphisms (A1166C AT1R) on the left ventricular size and performance. Three hundred sixty and one consecutive, Caucasian patients with angiographically confirmed coronary artery disease (CAD) were enrolled into the study. Left ventricular diameter, mass and function were evaluated by echocardiography. Screening for the I/D ACE and A1166C AT1R genotypes was performed by polymerase chain reaction of genomic DNA, followed by restriction enzyme digestion and agarose gel electrophoresis. The I/D ACE and A1166C AT1R genotypes separately were not significantly associated with the left ventricular size and function parameters in CAD patients. However, trends towards decreased left ventricular ejection fraction (LVEF) as well as increased left ventricular end-diastolic diameter (LVEDD) and left ventricular mass index (LVMI) were observed when patients with genotype DD+CC/AC and DD+CC were compared to patients homozygous only in one locus (DD or CC). Significant increase in LVEDD and LVMI was observed only in patients with a history of anterior myocardial infarction with combined genotype DD+CC/AC or DD+CC. This study does not support the role of the ACE I/D and AT1R A1166C polymorphisms in the determination of the left ventricular size and performance in patients with significant coronary atherosclerosis. However, it indicates that the influence of polymorphisms may be present in specific patient populations.

Association between the Pl(A) platelet glycoprotein GPIIIa polymorphism and extent of coronary artery disease.

BACKGROUND: The Pl(A2) allele of the gene encoding for GPIIIa subunit of the platelet membrane receptor glycoprotein (GP) IIb/IIIa has been suggested as a significant risk factor for thrombotic complications of coronary artery disease (CAD). The aim of the current investigation was to investigate the association between Pl(A) GPIIIa polymorphism and the extent of angiographically confirmed CAD in patients from the north region of Poland. METHODS: The study was performed in 397 male Caucasian patients. All subjects had significant coronary artery stenosis confirmed by elective coronary angiography. Screening for the Pl(A) GPIIIa genotypes was performed by polymerase chain reaction of genomic DNA, followed by NciI digestion and agarose gel electrophoresis. RESULTS: The genotype distribution of the Pl(A) GPIIIa polymorphism in our study group was Pl(A1/A1)-75%, Pl(A1/A2)-24% and Pl(A2/A2)-1% with Pl(A1) and Pl(A2) allele frequencies of 0.87 and 0.13, respectively. The prevalence of the homozygous Pl(A1/A1) genotype among subjects with multiple-vessel CAD (two or three vessels with at least 50% stenosis) was significantly higher than in patients with single-vessel disease; the odds ratio of Pl(A2/A2) or Pl(A1/A2) patients for having multiple-vessel CAD was 0.46 (95% CI 0.27-0.77, P<0.01). The mean CAD score for Pl(A1/A1) patients was significantly higher in comparison to Pl(A2/A2) and Pl(A1/A2) patients (7.58+/-2.20 and 6.98+/-2.37, respectively, P<0.05). CONCLUSIONS: Our results suggest, that the Pl(A1/A1) genotype of Pl(A) GPIIIa polymorphism is associated with more severe CAD in male Caucasian patients from the north region of Poland.

Association of the ScaI atrial natriuretic peptide gene polymorphism with nonfatal myocardial infarction and extent of coronary artery disease.

BACKGROUND: There is growing evidence from recent studies that atrial natriuretic peptide (ANP) plays an important part in coronary blood flow regulation and in atherosclerosis. Transition T2238-->C in the atrial natriuretic peptide (ANP) precursor gene, which leads potentially to the translation of ANP with 2 additional arginines, has been suggested to be associated with salt-sensitive hypertension. According to our knowledge, this study is the first to look for the potential association of the ScaI ANP gene polymorphism with the history of nonfatal myocardial infarction and the extent of coronary artery disease (CAD). METHODS: The study was performed in 847 consecutive, white patients (719 men and 128 women) with significant coronary artery stenosis confirmed by means of elective coronary angiography (at least 1 coronary artery with > or =50% lumen narrowing). Screening for the T2238-->C substitution was performed by means of polymerase chain reaction of genomic DNA, followed by ScaI digestion and agarose gel electrophoresis. RESULTS: We found a significant association of the A2A2 ScaI ANP genotype with a higher incidence of positive history of nonfatal myocardial infarction (odds ratio 1.85, 95% CI 1.33-2.58) and multiple-vessel CAD (odds ratio 1.45, 95% CI 1.02-2.06). The ScaI ANP genotype distribution did not differ with age, sex, body mass index, plasma lipids, hypertension, diabetes mellitus, and family history of CAD in studied groups. CONCLUSIONS: Our results suggest that the ScaI ANP polymorphism may be associated with nonfatal myocardial infarction and the extent of CAD. However, the precise mechanism of this association remains to be determined.

[X-linked agammaglobulinemia (XLA) associated with agranulocytosis--case report]. / Agammaglobulinemia sprzezona z chromosomem X z towarzyszaca agranulocytoza--opis przypadku.

In this case study authors presented the clinical characteristics of X-linked agammaglobulinemia (XLA) associated with agranulocytosis diagnosed in a 2-year-old boy. Affected child lacked circulating mature B cells, presented low levels of serum immunoglobulins, but did not suffer from recurrent bacterial infections. XLA is a primary immunodeficiency caused by a defective tyrosine kinase (Btk) in B cells. Our patient and his mother have a mutation in the BTK gene, described as W281X. During therapy with intravenous gammaglobulin, the boy has not experienced agranulocytosis. It is important to consider a primary immunodeficiency diagnosis when a child presents agranulocytosis or neutropenia and a recurrent infectious disease.