David Stejskal

QF-PCR-based prenatal detection of common aneuploidies in the Czech population: Five years of experience

We present the results from the largest clinical application of QF-PCR for antenatal rapid aneuploidy detection (RAD) in routine prenatal diagnosis in the Czech Republic. QF-PCR was performed in addition to karyotyping (dual testing) in two settings: the first was a single multiplex reaction testing only trisomy 21 and amelogenin X/Y alleles in the second trimester screened positive cases (T21 test), and the second setting consisted of two multiplexes (2M test) for common aneuploidies (13, 18, 21, X and Y) in cases with other RAD indications such as ultrasound findings, late booking or maternal anxiety. Dual testing was performed in 6349/12,778 (49.7%) of prenatal samples using either T21 or 2M test between 2002 and 2007. The clinical acceptability of our dual testing policy, methodological efficiency of RAD and residual risks of other chromosomal aberrations (CHAs) were evaluated. QF-PCR detected 92% (175/190) of significant CHAs. The 2M test identified 93.5% and the T21 test identified 87.5% of the significant CHAs with complete specificity. The residual risk of significant CHA was 1/231 in the 2M test and 1/565 in the T21 test. If RAD for all common aneuploidies is used as the sole prenatal diagnosis method, the odds of missing a CHA of any type are 1:90 and the odds of missing significant CHA with no ultrasound findings are 1:1513. If prenatal karyotyping were used as an additional procedure to RAD in cases only with ultrasound findings, 186/190 (97.8%) of the significant CHAs would be detected when 15.7% cases were karyotyped, according to our data. We consider RAD directed towards trisomy 21 alone (our T21 test) as an economically and clinically acceptable part of second trimester screening for Down syndrome. Both RAD tests allow fast alleviation of maternal anxiety with low residual risk when the test results are negative, and allow fast decision making if the results are positive. However, replacement of dual testing with only the RAD procedure in specific indications accepted in some countries (Great Britain) remains in the Czech Republic a theme for debate.

SNP array and phenotype correlation shows that FLI1 deletion per se is not responsible for thrombocytopenia development in Jacobsen syndrome

Jacobsen syndrome (JBS) is a rare chromosomal disorder caused by terminal deletion of the long arm of chromosome 11. We report on four prenatally diagnosed patients with JBS with variable prenatal and postnatal phenotypes and 11q deletions of varying sizes. Precise characterization of the deleted region in three patients was performed by SNP arrays. The severity of both the prenatal and postnatal phenotypes did not correlate with the size of the haploinsufficient region. Despite the large difference in the deletion size (nearly 6 Mb), both of the live‐born patients had similar phenotypes corresponding to JBS. However, one of the most prominent features of JBS, thrombocytopenia, was only present in the live‐born boy. The girl, who had a significantly longer deletion spanning all four genes suspected of being causative of JBS‐related thrombocytopenia (FLI1, ETS1, NFRKB, and JAM3), did not manifest a platelet phenotype. Therefore, our findings do not support the traditional view of deletion size correlation in JBS or the causative role of FLI1, ETS1, NFRKB, and JAM3 deletion per se for the development of disease‐related thrombocytopenia.

OmniPlex—a new QF-PCR assay for prenatal diagnosis of common aneuploidies based on evaluation of the heterozygosity of short tandem repeat loci in the Czech population

The aim of our study was to assess the utility of commonly used multiplex assays of short tandem repeat markers used for quantitative fluorescent polymerase chain reaction (QF‐PCR) for prenatal rapid aneuploidy detection (RAD) in routine prenatal diagnosis in the Czech population.

ISPD gene homozygous deletion identified by SNP array confirms prenatal manifestation of Walker-Warburg syndrome.

Walker-Warburg syndrome (WWS) is a rare form of autosomal recessive, congenital muscular dystrophy that is associated with brain and eye anomalies. Several genes encoding proteins involved in abnormal α-dystroglycan glycosylation have been implicated in the aetiology of WWS, most recently the ISPD gene. Typical WWS brain anomalies, such as cobblestone lissencephaly, hydrocephalus and cerebellar malformations, can be prenatally detected through routine ultrasound examinations. Here, we report two karyotypically normal foetuses with multiple brain anomalies that corresponded to WWS symptoms. Using a SNP-array examination on the amniotic fluid DNA, a homozygous microdeletion was identified at 7p21.2p21.1 within the ISPD gene. Published data and our findings led us to the conclusion that a homozygous segmental intragenic deletion of the ISPD gene causes the most severe phenotype of Walker-Warburg syndrome. Our results also clearly supports the use of chromosomal microarray analysis as a first-line diagnostic test in patients with a foetus with one or more major structural abnormalities identified on ultrasonographic examination.

SPAST mutation spectrum and familial occurrence among Czech patients with pure hereditary spastic paraplegia

The SPAST gene has a major role in hereditary spastic paraplegias (HSPs). This is the first report mapping characteristics of the SPAST gene in a large cohort of Czech HSP patients. All 17 coding exons of the SPAST gene were Sanger sequenced in 327 patients from 263 independent families with suspected uncomplicated HSP. The selected 126 independent patients, without mutation in the SPAST gene after Sanger sequencing, were subsequently tested by Multiplex Ligation-dependent Probe Amplification (MLPA) assay for large deletions or copy number variations affecting the SPAST gene. Among the 263 independent patients, 35 different, small mutations in 44 patients were found. Twenty-one mutations are novel with the majority of frameshift mutations. Seven mutations were found in more than one family. The age at onset ranged between preschool childhood and the fifth decade with inter- and intra-familiar differences. SPAST small mutations were detected in 16.7% (44/263) of independent tested patients. Mutations in the SPAST gene were found more frequently in familial cases (with affected relatives). Mutation were found in 31.9% (29/91 familial tested) in the familial patient group, whereas in the sporadic patient group, mutations were found in only 4.7% of cases (5/106 sporadic cases). Among SPAST-positive patients, 65.9% (29/44) were familial but only 11.4% (5/44) were sporadic. MLPA testing revealed four large deletions in four independent patients, all in familial-positive cases. Mutations in the SPAST gene are 5.8 × more frequent in familial than in sporadic cases. Large deletions were found only in familial patients. Diagnostic testing of the SPAST gene is useful only in positive family history patients not in sporadic cases.

Exponentially weighted moving average chart as a suitable tool for nuchal translucency quality review

This study aimed to design an exponentially weighted moving average (EWMA) chart for the quality review of nuchal translucency (NT) and to assess its performance compared with the methods currently in use: retrospective distribution‐based methods and the cumulative sum (CUSUM) chart.

Preimplantation genetic diagnosis of X-linked Charcot-Marie-Tooth disease by indirect linkage analysis

OBJECTIVE:To present methodical approach of preimplantation genetic diagnosis (PGD) as an option for an unaffected pregnancy in reproductive-age couples who have a genetic risk of the X-linked dominant peripheral neuropathy Charcot-Marie-Tooth type 1 disease. PATIENTS AND METHODS:We performed PGD of X-linked Charcot-Marie-Tooth type 1 disease using haplotyping/indirect linkage analysis, when during analysis we reach to exclude embryos that carry a high-risk haplotype linked to the causal mutation p.Leu9Phe in the GJB1 gene. RESULTS:Within the PGD cycle, we examined 4 blastomeres biopsied from cleavage-stage embryos and recommended 3 embryos for transfer. Two embryos were implanted into the uterus; however, it resulted in a singleton pregnancy with a male descendant. Three years later, the couple returned again with spontaneous gravidity. A chorionic biopsy examination of this gravidity ascertained the female sex and a pericentric inversion of chromosome 5 in 70% of the cultivated foetal cells. CONCLUSION:Using indirect linkage analysis, PGD may help to identify genetic X-linked defects within embryos during screening, thereby circumventing the potential problems with abortion.

Importance of the integrated test in the Down’s syndrome screening algorithm

Objective In the Czech Republic, over 97% of all pregnant women undergo some type of antenatal screening for Down’s syndrome. In about 95% of cases with a confirmed fetal chromosomal abnormality, the pregnancy is terminated. The most commonly used test is the first trimester combined test. We investigated the impact of implementing an integrated sequential test to improve the detection of Down’s syndrome pregnancies. Methods Data on the incidence of congenital defects, number of births, and affected pregnancies terminated are recorded in the National Registry of Congenital Anomalies. Anonymous data on cases of Down’s syndrome diagnosed antenatally or postnatally between 2010 and 2015 in one of the large antenatal care centers were analyzed. Results There were 600 diagnoses of Down’s syndrome (5.7 per 1000 births), 90% of which were made antenatally. Of antenatally detected cases, 80% were indicated for diagnostic procedure by multimarker screening results. In the multimarker screen positive group, 75% cases were first trimester positive and 25% second trimester positive (most of these had positive integrated test results). Among Down’s syndrome cases indicated for antenatal diagnosis by multimarker screening results 6.25% (n = 26) were first trimester negative, and became positive after integration with the second trimester screening results. Conclusions Results from five major Czech antenatal centers confirm that an integrated sequential test would detect 80–85% of Down’s syndrome fetuses in the first trimester and at least an extra 5–10% of Down’s syndrome pregnancies in the second trimester of pregnancy. These are important data that should be considered in implementing the national antenatal screening program.