Implementation of Exome Sequencing in Prenatal Diagnosis and Impact on Genetic Counseling: The Polish Experience.

BACKGROUND: Despite advances in routine prenatal cytogenetic testing, most anomalous fetuses remain without a genetic diagnosis. Exome sequencing (ES) is a molecular technique that identifies sequence variants across protein-coding regions and is now increasingly used in clinical practice. Fetal phenotypes differ from postnatal and, therefore, prenatal ES interpretation requires a large amount of data deriving from prenatal testing. The aim of our study was to present initial results of the implementation of ES to prenatal diagnosis in Polish patients and to discuss its possible clinical impact on genetic counseling. METHODS: In this study we performed a retrospective review of all fetal samples referred to our laboratory for ES from cooperating centers between January 2017 and June 2021. RESULTS: During the study period 122 fetuses were subjected to ES at our institution. There were 52 abnormal ES results: 31 in the group of fetuses with a single organ system anomaly and 21 in the group of fetuses with multisystem anomalies. The difference between groups was not statistically significant. There were 57 different pathogenic or likely pathogenic variants reported in 33 different genes. The most common were missense variants. In 17 cases the molecular diagnosis had an actual clinical impact on subsequent pregnancies or other family members. CONCLUSIONS: Exome sequencing increases the detection rate in fetuses with structural anomalies and improves genetic counseling for both the affected couple and their relatives.

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.

Prenatal Diagnosis by Array Comparative Genomic Hybridization in Fetuses with Cardiac Abnormalities.

Congenital heart defects (CHDs) appear in 8-10 out of 1000 live born newborns and are one of the most common causes of deaths. In fetuses, the congenital heart defects are found even 3-5 times more often. Currently, microarray comparative genomic hybridization (array CGH) is recommended by worldwide scientific organizations as a first-line test in the prenatal diagnosis of fetuses with sonographic abnormalities, especially cardiac defects. We present the results of the application of array CGH in 484 cases with prenatally diagnosed congenital heart diseases by fetal ultrasound scanning (256 isolated CHD and 228 CHD coexisting with other malformations). We identified pathogenic aberrations and likely pathogenic genetic loci for CHD in 165 fetuses and 9 copy number variants (CNVs) of unknown clinical significance. Prenatal array-CGH is a useful method allowing the identification of all unbalanced aberrations (number and structure) with a much higher resolution than the currently applied traditional assessment techniques karyotype. Due to this ability, we identified the etiology of heart defects in 37% of cases.

The MED13L haploinsufficiency syndrome associated with de novo nonsense variant (P.GLN1981*).

The Mediator complex subunit 13-like is a part of the large Mediator complex. Recently, a large number of patients were diagnosed with mutations in this gene, which makes it one of the most frequent causes of syndromic intellectual disability. In this work, we report a patient with a novel de novo likely pathogenic variant c.5941C>T, p.(Gln1981*) in the MED13L gene with severe intellectual disability and facial dysmorphism. Uncommon findings like lack of speech, strabismus and self-destructive behaviour present in our patient allowed us to further define the phenotypic spectrum of mental retardation and distinctive facial features with or without cardiac defects syndrome.

Bi-allelic Pathogenic Variants in TUBGCP2 Cause Microcephaly and Lissencephaly Spectrum Disorders.

Lissencephaly comprises a spectrum of malformations of cortical development. This spectrum includes agyria, pachygyria, and subcortical band heterotopia; each represents anatomical malformations of brain cortical development caused by neuronal migration defects. The molecular etiologies of neuronal migration anomalies are highly enriched for genes encoding microtubules and microtubule-associated proteins, and this enrichment highlights the critical role for these genes in cortical growth and gyrification. Using exome sequencing and family based rare variant analyses, we identified a homozygous variant (c.997C>T [p.Arg333Cys]) in TUBGCP2, encoding gamma-tubulin complex protein 2 (GCP2), in two individuals from a consanguineous family; both individuals presented with microcephaly and developmental delay. GCP2 forms the multiprotein γ-tubulin ring complex (γ-TuRC) together with γ-tubulin and other GCPs to regulate the assembly of microtubules. By querying clinical exome sequencing cases and through GeneMatcher-facilitated collaborations, we found three additional families with bi-allelic variation and similarly affected phenotypes including a homozygous variant (c.1843G>C [p.Ala615Pro]) in two families and compound heterozygous variants consisting of one missense variant (c.889C>T [p.Arg297Cys]) and one splice variant (c.2025-2A>G) in another family. Brain imaging from all five affected individuals revealed varying degrees of cortical malformations including pachygyria and subcortical band heterotopia, presumably caused by disruption of neuronal migration. Our data demonstrate that pathogenic variants in TUBGCP2 cause an autosomal recessive neurodevelopmental trait consisting of a neuronal migration disorder, and our data implicate GCP2 as a core component of γ-TuRC in neuronal migrating cells.

Echocardiographic analysis of left ventricular function in term and preterm neonates at week 40 of postconceptional life.

BACKGROUND Both gestational and chronological age of the neonate may influence and impair the function of the delicate and immature myocardium. However, the transition from fetal to neonatal circulation in preterm neonates is poorly understood. AIMS This study aimed to compare left ventricular (LV) systolic and diastolic function between premature neonates at expected term and term neonates during the postnatal cardiovascular transitional period. METHODS Using echocardiography, we assessed systolic and diastolic function of the LV in 89 preterm neonates at week 40 of postconceptional age and 29 term neonates after closure of the patent ductus arteriosus (PDA) and on the 28th day of life. Based on M­mode images, we measured myocardial thickness and fractional shortening (FS%). Using pulsed­wave Doppler echocardiography, we estimated cardiac output, myocardial performance index (MPI), and LV diastolic function (E and A waves, E/A ratio). Systolic and diastolic function was also assessed by tissue Doppler imaging. RESULTS Compared with term neonates on the 28th day of life, preterm neonates had reduced myocardial thickness (P ≤0.04), FS% (P = 0.002), and cardiac output (P = 0.01). However, preterm neonates had a lower MPI than term neonates after PDA closure (P <0.001) and on the 28th day of life (P = 0.02). The E/A ratio and S' wave values were similar in preterm and term neonates (P >0.05). CONCLUSIONS Preterm neonates at 40 weeks of postconceptional age have preserved systolic and diastolic function of the LV.

Comprehensive genomic analysis of patients with disorders of cerebral cortical development.

Malformations of cortical development (MCDs) manifest with structural brain anomalies that lead to neurologic sequelae, including epilepsy, cerebral palsy, developmental delay, and intellectual disability. To investigate the underlying genetic architecture of patients with disorders of cerebral cortical development, a cohort of 54 patients demonstrating neuroradiologic signs of MCDs was investigated. Individual genomes were interrogated for single-nucleotide variants (SNV) and copy number variants (CNV) with whole-exome sequencing and chromosomal microarray studies. Variation affecting known MCDs-associated genes was found in 16/54 cases, including 11 patients with SNV, 2 patients with CNV, and 3 patients with both CNV and SNV, at distinct loci. Diagnostic pathogenic SNV and potentially damaging variants of unknown significance (VUS) were identified in two groups of seven individuals each. We demonstrated that de novo variants are important among patients with MCDs as they were identified in 10/16 individuals with a molecular diagnosis. Three patients showed changes in known MCDs genes  and a clinical phenotype beyond the usual characteristics observed, i.e., phenotypic expansion, for a particular known disease gene clinical entity. We also discovered 2 likely candidate genes, CDH4, and ASTN1, with human and animal studies supporting their roles in brain development, and 5 potential candidate genes. Our findings emphasize genetic heterogeneity of MCDs disorders and postulate potential novel candidate genes involved in cerebral cortical development.

Evaluation of left ventricular function in preterm infants with bronchopulmonary dysplasia using various echocardiographic techniques.

AIM: Echocardiographic evaluation of left ventricular function in preterm infants with and without bronchopulmonary dysplasia. METHODS: In 82 preterm infants (32 in no-bronchopulmonary-dysplasia group, 35 in mild-bronchopulmonary-dysplasia group, and 15 in severe-bronchopulmonary-dysplasia group), echocardiography was performed on the first day of life, at 28 days of life, and at 36 weeks postconceptional age. RESULTS: The mean E/A ratio at 36 PCA was 0.94±0.31 and 0.73±0.12 in the mild- and severe-bronchopulmonary-dysplasia groups, respectively (P=.037). The mean E'-wave velocity was 5.62±1.61 cm/s vs 4.32±1.11 cm/s at 1 day of life (P=.006) and 6.40±1.39 cm/s vs 5.34±1.37 cm/s at 28 days of life (P=.030) in the no-bronchopulmonary-dysplasia and mild-bronchopulmonary-dysplasia groups, respectively. This measure tended to be lower in the severe-bronchopulmonary-dysplasia group compared to the no-bronchopulmonary-dysplasia group (5.25±1.29 cm/s at 28 days of life; P=.081). The E/E' ratio differed between the no-bronchopulmonary-dysplasia (7.21±1.85) and mild-bronchopulmonary-dysplasia groups (9.03±2.56; P=.019) at 1 day of life. The left ventricle myocardial performance index decreased between 1 day of life and 36 postconceptional age in infants without bronchopulmonary dysplasia and those with mild bronchopulmonary dysplasia, but not in those with severe bronchopulmonary dysplasia. CONCLUSION: E/A and E/E' ratios are the most sensitive indicators of impaired left ventricle diastolic function in preterm infants with bronchopulmonary dysplasia.

Echocardiographic evaluation of right ventricular function in preterm infants with bronchopulmonary dysplasia.

AIM: To evaluate right ventricular function in preterm infants with and without bronchopulmonary dysplasia. METHODS: Eighty-nine preterm infants (<32 weeks) were divided into three groups: (1) no-bronchopulmonary dysplasia (n=32); (2) mild-bronchopulmonary dysplasia (n=35); (3) severe-bronchopulmonary dysplasia (n=15). Right ventricular echocardiographic parameters included the following: (1) pulsed-wave Doppler through the tricuspid valve (E/A ratio), pulmonary artery acceleration time, right ventricular ejection time, right ventricular velocity-time integral; (2) tissue Doppler measurements of myocardial velocities and atrioventricular conduction times; (3) pulsed-wave Doppler and tissue Doppler evaluation of myocardial performance index and E/E' ratio; and (4) M-mode detection of right ventricular end-diastolic wall diameter. RESULTS: The severe-bronchopulmonary dysplasia group had higher mean right ventricular myocardial performance index (on the 28th day of life by pulsed-wave Doppler) than the no-bronchopulmonary dysplasia (P=.014) or mild-bronchopulmonary dysplasia (P=.031) groups; no differences were found between no-bronchopulmonary dysplasia and mild-bronchopulmonary dysplasia groups (P=.919). A reduction in right ventricular myocardial performance index at later time points was observed in all three groups (P<.05). We found no differences between preterm infants with differing bronchopulmonary dysplasia severity in other right ventricular echocardiographic parameters. CONCLUSION: Right ventricular myocardial performance index measured by pulsed-wave Doppler indicates impaired right ventricular function in preterm infants with severe bronchopulmonary dysplasia.

Myocardial performance index (Tei index) in term and preterm neonates during the neonatal period.

BACKGROUND: The myocardial performance index (MPI) is a noninvasive method to measure global systolic and diastolic myocardial function. In both term and premature neonates, changes in the systolic and diastolic function of the left ventricle (LV) and right ventricle (RV) reflect the degree of neonatal myocardial immaturity and the co-existence of foetal circulation. AIM: To assess MPI (or Tei indices) of both ventricles in term and preterm newborns, and to observe MPI trends throughout the neonatal period. METHODS: Heart ultrasound imaging was performed on the first day of life (DOL), after patent ductus arteriosus (PDA) closure, and on the 28th DOL, in 29 term and 29 preterm newborns. RVMPI and LVMPI were measured within the preterm group at 40 weeks of post-conception age (PCA). RESULTS: A statistically significant reduction in RVMPI was observed in both term and preterm newborns. In term newborns, the RVMPI value on the first DOL was 0.42 ± 14, dropping to 0.29 ± 0.09 after PDA closure, and finally reaching 0.22 ± 0.09 on the 28th DOL. The respective RVMPI values for the preterm newborns were 0.44 ± 0.15, 0.30 ± 0.12, and 0.21 ± 0.08. Little variability in the mean values of LVMPI was observed in both groups throughout the neonatal period. The LVMPI for term neonates in successive measurements was 0.37 ± 0.10, 0.39 ± 0.07, and 0.37 ± 0.11, respectively, and for the preterm neonates it was 0.37 ± 0.10, 0.35 ± 0.09, and 0.36 ± 0.10, respectively. The MPI values from preterm newborns taken at 40 weeks PCA (RVMPI = 0.28 ± 0.09; LVMPI = 0.37 ± 0.05) were comparable to those measured in term newborns after PDA closure. CONCLUSIONS: Observed postnatal changes in RVMPI correspond to changes in ventricular function, reflecting the haemodynamic changes of the transitional circulation. The relatively small postnatal changes in LVMPI in term and preterm newborns may reflect an immature myocardium. The RVMPI and LVMPI values at 40 weeks PCA in preterm newborns correlate best with MPI values in term newborns just after PDA closure.

[Familial congenital diaphragmatic hernia with anencephly-exencephaly and spina bifida]. / Rodzinne wystepowanie wrodzonej przepukliny przeponowej z wada dystroficzna i bezczaszkowiem.

Congenital Diaphragmatic Hernia (CDH) occurs with an estimated incidence of 1 to 2500 live births. Even though the exact etiology is still unknown, more and more often current research points out genetic factors as the possible cause of the defect. According to the latest data and the own experience 50-60% of CDH cases are isolated. The rest forms a group of CDH complicated by an additional anatomic defect or a genetic syndrome caused by a mutation of a single gene or the whole chromosome. We have presented a case study of a 32 years-old multigravida para 3, who has been referred to the Reference Centre of Prenatal Cardiology in 30 weeks of gestation due to the diagnosis of acrania with exencephaly, spina bifida and suspicion for CDH in a fetus. Although the patient's first child died due to CDH, the patient neither before nor during the pregnancy was getting a folic acid supplementation. Moreover, she has not agreed on a further cytogenetic testing or an advanced consultation with a clinical geneticist. The child died after delivery in a local hospital. The case was described to indicate the problem that the CDH diagnostic procedure is still missing a molecular genetic analysis especially in the cases of recurrent CDH. By saying that in the cases of CDH we should always strive to complete the molecular testing having in mind that by discovering pathogenesis and genes responsible for the formation of CDH we not only might improve the therapeutic methods but also find a way to prevent its development.

The usefulness of array comparative genomic hybridization in clinical diagnostics of intellectual disability in children.

INTRODUCTION: Intellectual disability (ID)/Developmental delay (DD), which occurs in 1-3% of the population, accounts for a large number of cases regularly seen in genetics clinics. Currently, Array Comparative Genomic Hybridization (array CGH) is recommended by the International Standards for Cytogenomic Arrays (ISCA) Consortium as a first line test in the diagnostics of ID/DD, replacing G-banded chromosome analysis. THE AIM: Application of array CGH in clinical diagnostics of developmental delay/ intellectual disability in children. MATERIAL AND METHODS: We present the results of 8x60K oligonucleotide array application that was successfully implemented in a cohort of 112 patients with the clinical diagnosis of intellectual disability and accompanying dysmorphic features and/or congenital malformations. RESULTS: We have identified 37 copy number variants (CNVs) with the size ranging from 40 kb to numerical chromosomal aberrations, including unbalanced translocations and chromosome Y disomy, receiving an overall diagnostic yield of 33%. Known pathogenic changes were identified in 21.4% of the cases. Among patients with pathogenic CNVs identified by array CGH, 41.7% had a previously normal karyotype analysis. CONCLUSIONS: Our studies provide more insights into the benefits derived by using chromosomal microarray analysis and demonstrate the usefulness of array CGH as a first-tier clinical setting test in patients with intellectual disability.

Doppler tissue imaging assessment of myocardial velocities and atrioventricular time intervals in term newborn infants during the neonatal period.

BACKGROUND: In both term and premature neonates, changes in the systolic and diastolic function of the left ventricle (LV) and right ventricle (RV) reflect the degree of neonatal myocardial immaturity and the co-existence of foetal circulation as well as the presence of concurrent diseases. AIM: To evaluate the changes in values of systolic and diastolic LV and RV function using pulse tissue Doppler imaging (TDI) in 20 healthy term newborn infants from birth to the 28th day of life. METHODS: Ventricular peak myocardial velocities were recorded during early diastole (Em wave), atrial contraction (Am wave), and systole (Sm wave). TDI derived atrioventricular (AV) intervals were measured as the period from atrial contraction (Am) to isovolumic contraction (IV), from Am to ventricular systole (Sm), from Sm to the following Am, and from IV to the following Am. The first measurements were taken as soon as possible after birth, the second on the third day, and the final one on the 28th day of life. RESULTS: The diastolic myocardial velocities recorded in the RV were higher than those in the LV. Statistically significant differences were observed for time intervals in the RV: Am-IV and Am-Sm (day 1-3), p < 0.02; IV-Am (day 1-28), p < 0.005; Sm-Am (day 1-28), p < 0.01. Statistically significant differences for time intervals were also evident in the LV: Am-IV (day 1-28), p < 0.05; and for Sm-Am (day 1-28), p < 0.01. Mean isovolumetric contraction time (ICT) and isovolumetric relaxation time (IRT) intervals remained stable for all measurements recorded in the RV. However, a statistically significant difference was evident for both ICT and IRT intervals in the LV between days 1 and 28 of life (p < 0.01). CONCLUSIONS: 1. Cardiac TDI is feasible in the neonate. 2. In neonates, the diastolic and systolic function recorded in the RV was better than that in the LV. This may reflect the 'persistent' foetal status of this ventricle in the first day of life. 3. The differences observed in conduction times also reflect the haemodynamic changes which occur in the circulatory system of the neonate in the first month of life. 4. Further investigation of a larger population of neonates throughout the whole neonatal period is indicated.

[Right ventricular hypertrophic cardiomyopathy in the fetus --may it be caused by chronic oral tocolysis with fenoterol?]. / Kardiomiopatia przerostowa prawej komory u plodu--czy przewlekla doustna tokoliza fenoterolem moze byc jej przyczyna?

The use of fenoterol in the treatment of preterm labor is associated with the risk of many complications in the mother and the fetus. We present a case of a multipara treated with oral fenoterol due to threatening preterm labor 14 weeks. At 35 weeks of gestation the fetus was diagnosed with hypertrophic cardiomyopathy with severe impairment of the right ventricle. The only factor that might have caused such a state of the fetal circulatory system was fenoterol, used from 21 weeks of gestation. After the withdrawal of the fenoterol the fetal right ventricular function improved gradually. However fetal cardiac hypertrophy persisted until the birth at 39 weeks of gestation. Concentric hypertrophy of the right ventricular wall and interventricular septum were confirmed in the newborn.

[The possibility of cardiovascular system evaluation in fetuses at 11.0 to 13.6 weeks of gestation in a reference perinatal cardiology centre]. / Mozliwosc oceny plodowego ukladu krazenia pomiedzy 11.0 a 13.6 tygodniem ciazy w referencyjnym osrodku kardiologii perinatalnej.

OBJECTIVE: To assess the possibility of foetal circulatory system evaluation between 11+0 to 13+6 weeks of gestation in a reference perinatal cardiology centre. MATERIAL AND METHODS: A prospective study was conducted between January 2004 and June 2008 in patients admitted to the reference perinatal cardiology centre for a foetal echocardiographic examination. 1170 foetuses were examined: 31 in 2004, 167 in 2005, 310 in 2006, 406 in 2007 and 262 in the first half of 2008. All foetuses had early echocardiography performed during 11-13+6 weeks of gestation, following the Foetal Medicine Foundation guidelines. The examination included: position and size of the heart, 4-chamber view and outflow tracts evaluation. The flow through ductus venosus, atrio-ventricular and arterial valves were assessed with colour and pulsed Doppler. 982 patients with confirmed normal heart anatomy in the second trimester were further analysed. RESULTS: Mean maternal age was 30 years. 237 (20%) patients were >35 years old. Mean CRL (crown rump length) was 65 mm. 4-chamber view was obtained in 933 foetuses (95%) and outflow tracts in 813 (83%) cases. Diagnostic flow through the tricuspid valve was detected in 894 (91%) foetuses. CONCLUSIONS: Early foetal echocardiography is available in a reference perinatal cardiology centre since 11th week of gestation, in most cases with transabdominal probe. Possibility of the foetal heart anatomy evaluation increases with gestational age. Only patients from the high risk group (i.e. previous child with a major cardiac defect, diabetes mellitus, monochorionic pregnancy) and foetuses with increased NT should be examined. In every case, a control echocardiographic examination in the second trimester must be performed.

[The possibility of evaluation of the myocardial performance index in fetuses at 11,0 to 13,6 week of gestation]. / Moiliwosci oceny skurczowo-rozkurczowej funkcji miesnia sercowego u plodów miedzy 11,0 a 13,6 tygodniem ciazy.

OBJECTIVES: To assess myocardial performance index (Tei index) for left and right ventricle in fetuses at 11.0 to 13.6 week of gestation. MATERIAL AND METHODS: Flow velocity waveforms of mitral, tricuspid, aortic and pulmonary valves were obtained. The Tei index for the left ventricle (Tei LV) was calculated in 55, and for the right ventricle (Tei RV) in 27 fetuses. RESULTS: The value of Tei LV ranged from 0.28 to 0.59, mean 0.41 +/- 0.08, and for Tei RV from 0.23 to 0.56, mean 0.37 +/- 0.11. There was no statistically important correlation either between Tei index and both ventricles or gestational age and fetal heart rate. The possibility to measure Tei index increased with the progress of the pregnancy. There remains a significant correlation between Tei LV and Tei RV values. CONCLUSIONS: Tei index may be useful for the assessment of fetal myocardial performance in the first trimester of pregnancy. Tei index is independent of gestational age and fetal heart rate. It is easier to measure the Tei index for the left ventricle than for the right one.

Ethical and legal aspects in teaching students of medicine.

Due to the rapid advances in medical technology, medical students are now being faced with increasingly complex and unparalleled ethical and practical dilemmas during their training. The new and future challenges of high-tech medicine demand improvements in current medical education, not only by meeting the needs of students through humanized training programs, but also by involving them in finding solutions to the ethical and legal quandaries they encounter. Today's students of medical universities must acquire knowledge and understanding of the ethical and legal issues relevant to the practice of medicine, and we have to do everything possible to introduce these students to the current discussions on more or less controversial ethical and legal topics. Although final answers may not be found, the very discussion, argumentation, and awakening of students' interest should become an essential part of the core curriculum of every doctor.