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.

Functional analysis of novel RUNX2 mutations identified in patients with cleidocranial dysplasia.

RUNX2 (Runt-related transcription factor 2) is a master regulator of osteoblast differentiation, cartilage and bone development. Pathogenic variants in RUNX2 have been linked to the Cleidocranial dysplasia (CCD), which is characterized by hypoplasia or aplasia of clavicles, delayed fontanelle closure, and dental anomalies. Here, we report 11 unrelated Polish patients with CCD caused by pathogenic alterations located in the Runt domain of RUNX2. In total, we identified eight different intragenic variants, including seven missense and one splicing mutation. Three of them are novel: c.407T>A p.(Leu136Gln), c.480C>G p.(Asn160Lys), c.659C>G p.(Thr220Arg), additional three were not functionally tested: c.391C>T p.(Arg131Cys), c.580+1G>T p.(Lys195_Arg229del), c.652A>G p.(Lys218Glu), and the remaining two: c.568C>T p.(Arg190Trp), c.673C>T p.(Arg225Trp) were previously reported and characterized. The performed transactivation and localization studies provide evidence of decreased transcriptional activity of RUNX2 due to mutations targeting the Runt domain and prove that impairment of nuclear localization signal (NLS) affects the subcellular localization of the protein. Presented data show that pathogenic variants discovered in our patients have a detrimental effect on RUNX2, triggering the CCD phenotype.

Two novel COL1A1 mutations in patients with osteogenesis imperfecta (OI) affect the stability of the collagen type I triple-helix.

Osteogenesis imperfecta (OI) is a bone dysplasia caused by mutations in the COL1A1 and COL1A2 genes. Although the condition has been intensely studied for over 25 years and recently over 800 novel mutations have been published, the relation between the location of mutations and clinical manifestation is poorly understood. Here we report missense mutations in COL1A1 of several OI patients. Two novel mutations were found in the D1 period. One caused a substitution of glycine 200 by valine at the N-terminus of D1 in OI type I/IV, lowering collagen stability by 50% at 34 degrees C. The other one was a substitution of valine 349 by phenylalanine at the C-terminus of D1 in OI type I, lowering collagen stability at 37.5 degrees C. Two other mutations, reported before, changed amino residues in D4. One was a lethal substitution changing glycine 866 to serine in genetically identical twins with OI type II. That mutated amino acid was near the border of D3 and D4. The second mutation changed glycine 1040 to serine located at the border of D4 and D0.4, in a proband manifesting OI type III, and lowered collagen stability at 39 degrees C (2 degrees C lower than normal). Our results confirm the hypothesis on a critical role of the D1 and D4 regions in stabilization of the collagen triple-helix. The defect in D1 seemed to produce a milder clinical type of OI, whereas the defect in the C-terminal end of collagen type caused the more severe or lethal types of OI.

[Characterization of marker chromosomes using molecular cytogenetic methods in patients with mental retardation and congenital malformations]. / Charakterystyka chromosomów markerowych metodami cytogenetyki molekularnej u pacjentów z cechami niepelnosprawnosci intelektualnej i wspólistnieniem wad rozwojowych.

INTRODUCTION: Until recently, great variety of marker chromosomes and difficulties with their identification have presented a problem for cytogenetic and clinical interpretation of the karyotype. At present, molecular cytogenetic methods of chromosome analysis enable precise characterization of such abnormalities providing knowledge necessary for estimation of their genetic risk. AIM: The aim of the study was molecular cytogenetic characterization of marker chromosomes recognized in three patients, an analysis of clinical features in relation to the abnormality and estimation of genetic risk of identified markers. PATIENTS AND METHODS: Karyotypes of three phenotypically abnormal patients were estimated in lymphocytes from peripheral blood by G banding analysis. Marker chromosomes were identified by fluorescence in situ hybridization (FISH), multiplex FISH, multicolor band and high resolution comparative genomic hybridization methods. RESULTS: Marker chromosomes were identified as inv dup(22)(pter->q11.2::q11.2->pter), der(8)(:p22->q11.2:), der(2l)(:pter->q21.3:) and der(19)(:p11->q13.1). All of them contained euchromatic sequences. First marker, an inverted duplication of chromosome 22q11.2 corresponding to tetrasomy of this chromosome region was recognized in a child with partial cat eye syndrome. Two further markers derived from chromosomes 8 and 21 were found in a child with mosaic karyotype and clinical features of trisomy 8p. In the third case additional chromosome material was derived from chromosome 19 and it was found in a patient with mild mental retardation and clinical features of ovary dysgenesis. Genetic risk of identified marker chromosomes except for mar(19) was estimated as high. CONCLUSIONS: Our results provide further evidence for diagnostic value of molecular cytogenetic methods. They also confirmed the general opinion of the high risk of phenotypic abnormalities in the carriers of marker chromosomes containing euchromatic sequences.

[Cytogenetic-molecular analysis of balanced chromosomal rearrangements in nine patients with intellectual disability, dysmorphic features and congenital abnormalities]. / Cytogenetyczno-molekularna charakterystyka zrównowazonych rearranzacji chromosomowych u dziewieciu pacjentów z cechami uposledzenia umyslowego, dysmorfii oraz wadami rozwojowymi.

INTRODUCTION: In about 6% of individuals with intellectual disability, dysmorphic features and congenital anomalies, an abnormal, apparently balanced karyotype is found. These abnormalities may result from abnormal expression of genes at the breakpoints, presence of a submicroscopic deletion, or other unbalanced chromosome aberrations. In such cases, the detailed analysis of breakpoints of balanced chromosome rearrangements may help with identification of genes responsible for patient's clinical features. AIM OF WORK: Was the explanation of causes of abnormal phenotype in the carriers with abnormal but balanced karyotype. MATERIAL AND METHODS: Cytogenetic-molecular analysis performed in nine patients with mental retardation, dysmorphic features and congenital anomalies. Studies with subtelomeric probes, high resolution comparative genomic hybridization (HR-CGH) and fluorescence in situ hybridization (FISH) with region-specific BAC clones were performed. RESULTS: Seventeen chromosome breakpoint regions were narrowed to 200-400 kb. In one case, an 0.5-Mb submicroscopic deletion associated with more complex rearrangement has been found. Mapping of the breakpoints and information obtained from the UCSC Human Genome Browser data base enabled identification of 46 genes in these regions. Twelve genes, that may have been disrupted as a result of the patients' chromosomal rearrangement, were found. At four different breakpoints the identified genes (NRCAM, NPTX1, NMT1, MAPT, HDAC5 and MEF2C) may be due to a position effect. CONCLUSIONS: The results confirm earlier suggestions concerning reasons of abnormal phenotype in the patients with balanced chromosome rearrangements and present the value of detailed analysis of the genome in such cases.

Molecular analysis of a constitutional complex genome rearrangement with 11 breakpoints involving chromosomes 3, 11, 12, and 21 and a approximately 0.5-Mb submicroscopic deletion in a patient with mild mental retardation.

Complex chromosome rearrangements (CCRs) are extremely rare but often associated with mental retardation, congenital anomalies, or recurrent spontaneous abortions. We report a de novo apparently balanced CCR involving chromosomes 3 and 12 and a two-way translocation between chromosomes 11 and 21 in a woman with mild intellectual disability, obesity, coarse facies, and apparent synophrys without other distinctive dysmorphia or congenital anomalies. Molecular analysis of breakpoints using fluorescence in situ hybridization (FISH) with region-specific BAC clones revealed a more complex character for the CCR. The rearrangement is a result of nine breaks and involves reciprocal translocation of terminal chromosome fragments 3p24.1-->pter and 12q23.1-->qter, insertion of four fragments of the long arm of chromosome 12: q14.1-->q21?, q21?-->q22, q22-->q23.1, and q23.1-->q23.1 and a region 3p22.3-->p24.1 into chromosome 3q26.31. In addition, we detected a approximately 0.5-Mb submicroscopic deletion at 3q26.31. The deletion involves the chromosome region that has been previously associated with Cornelia de Lange syndrome (CdLS) in which a novel gene NAALADL2 has been mapped recently. Other potential genes responsible for intellectual deficiency disrupted as a result of patient's chromosomal rearrangement map at 12q14.1 (TAFA2), 12q23.1 (METAP2), and 11p14.1 (BDNF).

[Epilepsy in three children with Wolf-Hirschhorn syndrome]. / Padaczka u trojga dzieci z zespolem Wolfa-Hirschhorna.

OBJECTIVES: Wolf-Hirschhorn syndrome (4p detetion) belongs to the group of disorders caused by chromosomal aberrations, associated with frequent occurrence of epilepsy. To illustrate phenotype - genotype association, the study presents 3 children with this syndrome and epilepsy. MATERIALS AND METHODS: The diagnosis of Wolf-Hirschhorn syndrome was established in 2 patients during neonatal period and in the third child, the 5 month of life. In the majority of patients characteristic phenotype and associated malformations were detected, and the low birth weight and fronto-temporal diameter of the head as well. The structural neuroimaging revealed the diffuse decrease of brain volume without neurodevelopmental malformations. The earliest manifestation of epilepsy was observed in the 4 month old child with 4p. microdeletion as a status epilepticus. In remaining two children with 4p deletion, generalised tonic-clonic seizures were observed for the first time in the 7th month and 7th year, respectively. The standard EEG was performed in infants, while in the 7 year old child a-one-hour videoEEG was recorded. RESULTS: In older children diazepam and clona-zepam were effective to abort seizures, patients became seizure free on carbamazepin and phenobarbital. In the youngest child, status epilepticus being resistant to benzodiazepins, was interrupted with difficulties. Despite generalised type of seizures, EEG revealed focal changes of bioelectrical activity of the brain in two children. In the 7 month old child there were high voltage slow waves in the parieto-temporo-occipital region, while in the 7 year old child videoEEG demonstrated inter-hemispheric asymmetry and asynchrony, and the presence of epileptic grapho-elements, such as spikes and the spike-slow wave complexes as well. CONCLUSION: Epilepsy appeared in children with Wolf-Hirschhorn syndrome with deletion and microdeletion. Status epilepticus in this syndrome may be resistant to benzodiazepins.

[Thanatophoric dysplasia: three patients hospitalized in PAIP in 1994-2000]. / Karlowatosc tanatoforyczna--prezentacja trzech pacjentów hospitalizowanych w PAIP w latach 1994-2000.

BACKGROUND: Thanatophoric dwarfism is a lethal bone dysplasia causing severe disturbance in body proportions, shortening and deformation of the long bones and maldevelopment of the chest leading to severe respiratory failure and early death. The disease is caused usually by de novo mutation in the gene of fibroblast growth factor receptor 3 (FGFR3). Inheritance is autosomal dominant. The most common mutation C742T leads to substitution of arginine by cysteine in 248 position of polypeptide (R248C). GOAL: Presentation of clinical picture, radiological findings and molecular diagnostics in three patients with TD hospitalized in PAIP in 1994-2000. PATIENTS: Three patients with TD were hospitalized in PAIP between 1994 and 2000. They were admitted in the 1st, 2nd, 19th day of life. Two patients were referred with diagnosis of achondroplasia. One newborn was born after uncomplicated pregnancy with cesarean section due to large head circumference found on prenatal USG. Two other newborns were born preterm (34 week of gestation), vaginally. One pregnancy was complicated by polyhydramnios. All patients required oxygen therapy, two were artificially ventilated (21 and 16 days). Three newborns died due to respiratory failure, average length of life--29 days. METHODS AND RESULTS: The diagnosis was established based on clinical presentation (abnormal proportions, shortening and deformation of the extremities, maldevelopment of the chest, large cranium) and radiological presentation (typical vertebral bodies, long bones shaped as telephone receiver). In two cases molecular analysis was performed, which excluded achondroplasia, in one of those patients molecular studies directly confirmed presence of the most common mutation leading to TD (R248C).