The clinical use of polygenic risk scores.

Polygenic risk scores (PRS) identify at-risk individuals for many common diseases. A discussion of strengths and limitations is carried out in this review. PRS complement traditional genetic testing and have shown utility in establishing a proper diagnosis and guiding primary and secondary prevention. Some individuals with high PRS have risks similar to those with monogenic predisposition. Limitations include potential misinterpretations, problems with application across ancestries, and limited usefulness in low-heritability traits. Despite its shortcomings PRS are predicted to play major roles in the future of personal medicine and genetic testing.

Clinical interpretation of cell-based non-invasive prenatal testing for monogenic disorders including repeat expansion disorders: potentials and pitfalls.

Introduction: Circulating fetal cells isolated from maternal blood can be used for prenatal testing, representing a safe alternative to invasive testing. The present study investigated the potential of cell-based noninvasive prenatal testing (NIPT) for diagnosing monogenic disorders dependent on the mode of inheritance. Methods: Maternal blood samples were collected from women opting for prenatal diagnostics for specific monogenic disorders (N = 7). Fetal trophoblasts were enriched and stained using magnetic activated cell sorting and isolated by fluorescens activated single-cell sorting. Individual cells were subject to whole genome amplification, and cells of fetal origin were identified by DNA-profiling using short tandem repeat markers. The amplified fetal DNA was input for genetic testing for autosomal dominant-, autosomal recessive-, X-linked and repeat expansion disorders by direct variant analysis and haplotyping. The cell-based NIPT results were compared with those of invasive testing. Results: In two cases at risk of skeletal dysplasia, caused by variants in the FGFR3 gene (autosomal dominant disorders), cell-based NIPT correctly stated an affected fetus, but allelic dropout of the normal alleles were observed in both cases. Cell-based NIPT gave an accurate result in two cases at risk of autosomal recessive disorders, where the parents carried either different diastrophic dysplasia causing variants in the SLC26A2 gene or the same cystic fibrosis disease-causing variant in the CFTR gene. Cell-based NIPT accurately identified an affected male fetus in a pregnancy at risk of Duchenne muscular dystrophy (DMD gene, X-linked recessive disorders). In two cases at risk of the myotonic dystrophy type 1 (DMPK gene, repeat expansion disorder), cell-based NIPT correctly detected an affected and an unaffected fetus, respectively. Discussion: Circulating fetal cells can be used to detect both maternally- and paternally inherited monogenic disorders irrespective of the type of variant, however, the risk of allelic dropout must be considered. We conclude that the clinical interpretation of the cell-based NIPT result thus varies depending on the disorders' mode of inheritance.

Multifocal ectopic purkinje-related premature contractions and related cardiomyopathy.

In the past 20 years, genetic variants in SCN5A encoding the cardiac voltage-gated sodium channel Nav1.5 have been linked to a range of inherited cardiac arrhythmias: variants resulting in loss-of-function of Nav1.5 have been linked to sick sinus syndrome, atrial stand still, atrial fibrillation (AF) impaired pulse generation, progressive and non-progressive conduction defects, the Brugada Syndrome (BrS), and sudden cardiac death. SCN5A variants causing increased sodium current during the plateau phase of the cardiac action potential is associated with Long QT Syndrome type 3 (LQTS3), Torsade de Pointes ventricular tachycardia and SCD. Recently, gain-of-function variants have been linked to complex electrical phenotypes, such as the Multifocal Ectopic Purkinje-related Premature Contractions (MEPPC) syndrome. MEPPC is a rare condition characterized by a high burden of premature atrial contractions (PACs) and/or premature ventricular contractions (PVCs) often accompanied by dilated cardiomyopathy (DCM). MEPPC is inherited in an autosomal dominant fashion with an almost complete penetrance. The onset is often in childhood. The link between SCN5A variants, MEPPC and DCM is currently not well understood, but amino acid substitutions resulting in gain-of-function of Nav1.5 or introduction of gating pore currents potentially play an important role. DCM patients with a MEPPC phenotype respond relatively poorly to standard heart failure medical therapy and catheter ablation as the PVCs originate from all parts of the fascicular Purkinje fiber network. Class 1c sodium channel inhibitors, notably flecainide, have a remarkable positive effect on the ectopic burden and the associated cardiomyopathy. This highlights the importance of genetic screening of DCM patients to identify patients with SCN5A variants associated with MEPPC. Here we review the MEPPC phenotype, MEPPC-SCN5A associated variants, and pathogenesis as well as treatment options.

A decade of change - lessons learned from prenatal diagnostics in Central Denmark region in 2008-2018.

INTRODUCTION: In 2011, it was decided to implement chromosomal microarray in prenatal testing in the Central Denmark Region, mainly due to the expected higher diagnostic yield. Chromosomal microarray was introduced gradually for an increasing number of pregnancies and without a transition period where both karyotyping and chromosomal microarray were performed: first malformations (2011), then large nuchal translucency (2013), then high risk at combined first trimester risk screening (2016) and finally for all indications (2018). This retrospective study summarizes 11 years of using chromosomal microarray in invasive prenatal testing and presents the effect on diagnostic yield and turnaround time. Furthermore, the concerns when introducing chromosomal microarray are presented and discussed. MATERIAL AND METHODS: Registry data from the Danish Fetal Medicine Database, the regional fetal medicine database, the Danish Cytogenetic Central Register and the local laboratory database at Department of Clinical Genetics were all combined, and a cohort of 147 158 singleton pregnancies with at least one ultrasound examination was established RESULTS: Of the 147 158 pregnancies, invasive sampling was performed (chorionic villi or amniocytes) in 8456, corresponding to an overall invasive rate of 5.8%. Between 2016 and 2018, 3.4% (95% confidence interval [CI] 2.8-4.2%; n = 86) of the invasive samples (n = 2533) had a disease causing copy number variant and 5.3% (95% CI 4.4-6.2%; n = 133) had trisomies and other aneuploidies. The turnaround time more than halved from 14 days to an average of 5.5 days for chorionic villus sampling. CONCLUSIONS: Chromosomal microarray identified 5.3% trisomies and 3.4% copy number variants, thereby increased the diagnostic yield by more than 64% compared with karyotype only and it also more than halved the turnaround time. Some preliminary concerns proved real, eg prenatal counseling complexity, but these have been resolved over time in a clinical path with expert consultations.

Genetic analysis identifies the SLC4A3 anion exchanger as a major gene for short QT syndrome.

BACKGROUND: A variant in the SLC4A3 anion exchanger has been identified as a novel cause of short QT syndrome (SQTS), but the clinical importance of SLC4A3 as a cause of SQTS or sudden cardiac death remains unknown. OBJECTIVE: The purpose of this study was to investigate the prevalence of potential disease-causing variants in SQTS patients using gene panels including SLC4A3. METHODS: In this multicenter study, genetic testing was performed in 34 index patients with SQTS. The pathogenicity of novel SLC4A3variants was validated in a zebrafish embryo heart model. RESULTS: Potentially disease-causing variants were identified in 9 (26%) patients and were mainly (15%) located in SLC4A3: 4 patients heterozygous for novel nonsynonymous SLC4A3 variants-p.Arg600Cys, p.Arg621Trp, p.Glu852Asp, and p.Arg952His-and 1 patient with the known p.Arg370His variant. In other SQTS genes, potentially disease-causing variants were less frequent (2× in KCNQ1, 1× in KCNJ2, and CACNA1C each). SLC4A3 variant carriers (n = 5) had a similar heart rate but shorter QT and J point to T wave peak intervals than did noncarriers (n = 29). Knockdown of slc4a3 in zebrafish resulted in shortened heart rate-corrected QT intervals (calculated using the Bazett formula) that could be rescued by overexpression of the native human SLC4A3-encoded protein (AE3), but neither by the mutated AE3 variants p.Arg600Cys, p.Arg621Trp, p.Glu852Asp nor by p.Arg952His, suggesting pathogenicity of these variants. Dysfunction in slc4a3/AE3 was associated with alkaline cytosol and shortened action potential of cardiomyocytes. CONCLUSION: In about a quarter of patients with SQTS, a potentially disease-causing variant can be identified. Nonsynonymous variants in SLC4A3 represent the most common cause of SQTS, underscoring the importance of including SLC4A3 in the genetic screening of patients with SQTS or sudden cardiac death.

Noninvasive prenatal screening for cystic fibrosis using circulating trophoblasts: Detection of the 50 most common disease-causing variants.

OBJECTIVES: Cystic fibrosis (CF) is one of the most common severe autosomal recessive disorders. Prenatal or preconception CF screening is offered in some countries. A maternal blood sample in early pregnancy can provide circulating trophoblasts and offers a DNA source for genetic analysis of both the mother and the fetus. This study aimed to develop a cell-based noninvasive prenatal test (NIPT) to screen for the 50 most common CF variants. METHODS: Blood samples were collected from 30 pregnancies undergoing invasive diagnostics and circulating trophoblasts were harvested in 27. Cystic fibrosis testing was conducted using two different methods: by fragment length analysis and by our newly developed NGS-based CF analysis. RESULTS: In all 27 cases, cell-based NIPT provided a result using both methods in agreement with the invasive test result. CONCLUSION: This study shows that cell-based NIPT for CF screening provides a reliable result without the need for partner- and proband samples.

Spontaneous rescue of a FMR1 repeat expansion and review of deletions in the FMR1 non-coding region.

Fragile X syndrome (FXS) is caused by CGG-repeat expansion in the 5' UTR of FMR1 of >200 repeats. Rarely, FXS is caused by deletions; however, it is not clear whether deletions including only the non-coding region of FMR1 are pathogenic. We report a deletion in the 5' UTR of FMR1 in an unaffected male infant and review 12 reported deletions involving only the non-coding region of FMR1. Genetic testing was requested in a male infant born to a mother harbouring a FMR1 full mutation. The maternal grandmother carried a FMR1 premutation. FMR1 CGG repeats were analysed using repeat-primed PCR. Only a short PCR fragment was amplified and subsequent Sanger sequencing detected an 88 bp deletion in hemizygous form. The deletion included all CGG repeats and flanking sequences but no FMR1 exons. Linkage analysis using STR markers revealed that the deletion had occurred on the allele, which was expanded in the mother and the maternal grandmother. Reverse transcription and quantitative PCR showed normal FMR1 mRNA levels. Grønskov et al. reported a 157 bp deletion of all CGG repeats and flanking sequences in a female without FXS hemizygous for the FMR1 gene due to a deletion on the other X chromosome. Protein expression was unaffected by the deletion. The reported deletion comprises the deletion detected in the male infant. At almost 2 years of age he is unaffected. Based on these observations and the normal FMR1 mRNA level, we conclude that a spontaneous rescue of an FMR1 repeat expansion has occurred.

Genetic analysis of Charcot-Marie-Tooth disease in Denmark and the implementation of a next generation sequencing platform.

Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of hereditary polyneuropathies. Variants in more than 80 different genes have been associated with the disorder. In recent years, the introduction of next generation sequencing (NGS) techniques have completely changed the genetic diagnostic approach from the analysis of a handful of genes to the analysis of all genes associated with CMT in a single run. In this study we describe the CMT diagnostics in Denmark in 1992-2012, prior to the implementation of NGS, by combining laboratory- and national registry data. We investigate the effect of implementing a targeted NGS approach of 63 genes associated with CMT in the diagnostic laboratory setting. This was performed by analyzing a cohort of 195 samples from patients previously analyzed by Sanger sequencing and quantitative analysis for the common causes of CMT without reaching a molecular diagnosis. A total of 1442 CMT analyses were performed in Denmark in the period 1992-2012; a disease-causing variant was detected in 21.6% of the cases. Interestingly, the diagnosis was genetically confirmed in significantly more women than men; 25.9% compared to18.5%. In our study cohort, we found a 5.6% increase in the diagnostic yield with the introduction of a targeted NGS approach.

Unexplained cholestasis in adults and adolescents: diagnostic benefit of genetic examination.

OBJECTIVES: A few adult and adolescent patients with even severe cholestatic liver disease remain unexplained after standard diagnostic work-up. We studied the value of genetic examination in such patients and developed a panel of eight genes with known cholestatic associations. MATERIALS AND METHODS: Thirty-three patients with unexplained cholestasis despite a thorough clinical work-up were examined for sequence variations in the coding regions of the ABCB4, ABCB11, ABCC2, ABCG5, ATP8B1, JAG1, NOTCH2, and UGT1A1 genes and the promoter region of UGT1A1 by massive parallel sequencing of DNA extracted from whole blood. Hepatologists and clinical geneticists evaluated the causal potential of genetic variants. RESULTS: In 9/33 patients (27%), we identified genetic variants as a certain causal factor and in further 9/33 (27%) variants as a possible contributing factor. In most cases, a detailed family history was necessary to establish the importance of genetic variants. Genetic causes were identified in 6/13 women (46%) with intrahepatic cholestasis during pregnancy and persisting abnormal biochemistry after delivery. CONCLUSIONS: Our study suggests that a small number of well-known genetic variants are involved in at least 27-54% of patients with unexplained cholestasis. An expanded panel will likely explain more cases. This motivates genetic testing of these patients. Genetic testing, however, cannot stand alone but should be combined with a clinical genetic work-up in collaboration between hepatologists and clinical geneticists.

On the road to replacing invasive testing with cell-based NIPT: Five clinical cases with aneuploidies, microduplication, unbalanced structural rearrangement, or mosaicism.

OBJECTIVE: Trophoblastic fetal cells harvested from maternal blood have the capacity to be used for copy number analyses in a cell-based non-invasive prenatal test (cbNIPT). Potentially, this will result in increased resolution for detection of subchromosomal aberrations due to high quality DNA not intermixed with maternal DNA. We present 5 selected clinical cases from first trimester pregnancies where cbNIPT was used to demonstrate a wide range of clinically relevant aberrations. METHOD: Blood samples were collected from high risk pregnancies in gestational week 12 + 1 to 12 + 5. Fetal trophoblast cells were enriched and stained using fetal cell specific antibodies. The enriched cell fraction was scanned, and fetal cells were picked using a capillary-based cell picking instrument. Subsequently, whole genome amplification (WGA) was performed on fetal cells, and the DNA was analyzed blindly by array comparative genomic hybridization (aCGH). RESULTS: We present 5 cases where non-invasive cell-based prenatal test results are compared with aCGH results on chorionic villus samples (CVS), demonstrating aneuploidies including mosaicism, unbalanced translocations, subchromosomal deletions, or duplications. CONCLUSION: Aneuploidy and subchromosomal aberrations can be detected using fetal cells harvested from maternal blood. The method has the future potential of being offered as a cell-based NIPT with large high genomic resolution.

Cell-free DNA in pregnancy with choriocarcinoma and coexistent live fetus: A case report.

BACKGROUND: This case report describes the use of analysis of cell-free DNA in the blood of a patient with a pregnancy with one live fetus and a choriocarcinoma diagnosed at 22 weeks of gestation. RESULTS: The result of the analysis of 16 microsatellite loci on 14 chromosomes in the cell-free DNA in plasma was consistent with the result of the analysis of a tumor biopsy indicating biparental diploid origin of the genome. The DNA markers were discordant with the markers of the placenta indicating two separate conceptions. CONCLUSION: Our results indicate that analysis of cell-free DNA in plasma allows determination of the origin of a choriocarcinoma without tissue biopsy, even in the presence of a co-existent pregnancy.

Two maternal duplications involving the CDKN1C gene are associated with contrasting growth phenotypes.

BACKGROUND: The overgrowth-associated Beckwith-Wiedemann syndrome (BWS) and the undergrowth-associated Silver-Russell syndrome (SRS) are characterized by heterogeneous molecular defects affecting a large imprinted gene cluster at chromosome 11p15.5-p15.4. While maternal and paternal duplications of the entire cluster consistently result in SRS and BWS, respectively, the phenotypes associated with smaller duplications are difficult to predict due to the complexity of imprinting regulation. Here, we describe two cases with novel inherited partial duplications of the centromeric domain on chromosome 11p15 associated with contrasting growth phenotypes. FINDINGS: In a male patient affected by intrauterine growth restriction and postnatal short stature, we identified an in cis maternally inherited duplication of 0.88 Mb including the CDKN1C gene that was significantly up-regulated. The duplication did not include the long non-coding RNA KCNQ1OT1 nor the imprinting control region of the centromeric domain (KCNQ1OT1:TSS-DMR or ICR2) in which methylation was normal. In the mother, also referring a growth restriction phenotype in her infancy, the duplication was de novo and present on her paternal chromosome. A different in cis maternal duplication, 1.13 Mb long and including the abovementioned duplication, was observed in a child affected by Tetralogy of Fallot but with normal growth. In this case, the rearrangement also included most of the KCNQ1OT1 gene and resulted in ICR2 loss of methylation (LOM). In this second family, the mother carried the duplication on her paternal chromosome and showed a normal growth phenotype as well. CONCLUSIONS: We report two novel in cis microduplications encompassing part of the centromeric domain of the 11p15.5-p15.4 imprinted gene cluster and both including the growth inhibitor CDKN1C gene. Likely, as a consequence of the differential involvement of the regulatory KCNQ1OT1 RNA and ICR2, the smaller duplication is associated with growth restriction on both maternal and paternal transmissions, while the larger duplication, although it includes the smaller one, does not result in any growth anomaly. Our study provides further insights into the phenotypes associated with imprinted gene alterations and highlights the importance of carefully evaluating the affected genes and regulatory elements for accurate genetic counselling of the 11p15 chromosomal rearrangements.

Diagnostic performance of quantitative fluorescence PCR analysis in high-risk pregnancies after combined first-trimester screening.

INTRODUCTION: We aimed to determine the diagnostic efficiency of quantitative fluorescence polymerase chain reaction (QF-PCR) in a clinical setting where most of the analyses are performed on chorion villus samples from high-risk pregnancies as determined by combined first-trimester screening. METHODS: A retrospective study on QF-PCR data from all pregnancies in the Central and North Denmark Regions over a four-year period (n = 2,550) with invasive prenatal testing carried out due to a high risk of carrying a foetus with Down's syndrome. Results of QF-PCR were compared with those obtained by karyotyping. Other supplementary data were obtained from the Danish Foetal Medicine Database and the Danish Cytogenetic Central Register. RESULTS: QF-PCR for common aneuploidies is fast, has a low failure rate, and is associated with high positive and negative predictive values (PPV, NPV) (> 99.8%) for all analysed abnormal karyotypes except for mosaicism for trisomy 13 (PPV = 20%) and sex chromosome mosaic cases (PPV = 40%; NPV = 99.7%)). In 25 (1%) cases, clinically significant chromosome abnormalities other than chromosomes 13, 18, 21, X, and Y were identified by karyotyping. CONCLUSION: QF-PCR is a rapid and accurate diagnostic method to detect common aneuploidies in high-risk pregnancies. However, the rapid test cannot stand alone as several clinically significant abnormal karyotypes would be overlooked. FUNDING: not relevant. TRIAL REGISTRATION: not relevant.

A novel mutation in the EDAR gene causes severe autosomal recessive hypohidrotic ectodermal dysplasia.

We report on a 2-year-old girl presenting with a severe form of hypohidrotic ectodermal dysplasia (HED). The patient presented with hypotrichosis, anodontia, hypohidrosis, frontal bossing, prominent lips and ears, dry, pale skin, and dermatitis. The patient had chronic rhinitis with malodorous nasal discharge. The girl was the second born child of first-cousin immigrants from Northern Iraq. A novel homozygous mutation (c.84delC) in the EDAR gene was identified. This mutation most likely causes a frameshift in the protein product (p.S29fs*74). This results in abolition of all ectodysplasin-mediated NF-kB signalling. This complete loss-of-function mutation likely accounts for the severe clinical abnormalities in ectodermal structures in the described patient.

Comparison of recombinant human haptocorrin expressed in human embryonic kidney cells and native haptocorrin.

Haptocorrin (HC) is a circulating corrinoid binding protein with unclear function. In contrast to transcobalamin, the other transport protein in blood, HC is heavily glycosylated and binds a variety of cobalamin (Cbl) analogues. HC is present not only in blood but also in various secretions like milk, tears and saliva. No recombinant form of HC has been described so far. We report the expression of recombinant human HC (rhHC) in human embryonic kidney cells. We purified the protein with a yield of 6 mg (90 nmol) per litre of cell culture supernatant. The isolated rhHC behaved as native HC concerning its spectral properties and ability to recognize both Cbl and its baseless analogue cobinamide. Similar to native HC isolated from blood, rhHC bound to the asialoglycoprotein receptor only after removal of terminal sialic acid residues by treatment with neuraminidase. Interestingly, rhHC, that compared to native HC contains four excessive amino acids (…LVPR) at the C-terminus, showed subtle changes in the binding kinetics of Cbl, cobinamide and the fluorescent Cbl conjugate CBC. The recombinant protein has properties very similar to native HC and although showing slightly different ligand binding kinetics, rhHC is valuable for further biochemical and structural studies.

The expression level of the chromatin-associated HMGB1 protein influences growth, stress tolerance, and transcriptome in Arabidopsis.

High mobility group (HMG) proteins of the HMGB family are small and relatively abundant chromatin-associated proteins. As architectural factors, the HMGB proteins are involved in the regulation of transcription and other DNA-dependent processes. We have examined Arabidopsis mutant plants lacking the HMGB1 protein, which is a typical representative of the plant HMGB family. In addition, our analyses included transgenic plants overexpressing HMGB1 and mutant plants that were transformed with the HMGB1 genomic region (complementation plants), as well as control plants. Both the absence and overexpression of HMGB1 caused shorter primary roots and affected the sensitivity towards the genotoxic agent methyl methanesulfonate. The overexpression of HMGB1 decreased the seed germination rate in the presence of elevated concentrations of NaCl. The complementation plants that expressed HMGB1 at wild-type levels did not show phenotypic differences compared to the control plants. Transcript profiling by microarray hybridization revealed that a remarkably large number of genes were differentially expressed (up- and down-regulated) in plants lacking HMGB1 compared to control plants. Among the down-regulated genes, the gene ontology category of stress-responsive genes was overrepresented. Neither microscopic analyses nor micrococcal nuclease digestion experiments revealed notable differences in overall chromatin structure, when comparing chromatin from HMGB1-deficient and control plants. Collectively, our results show that despite the presence of several other HMGB proteins, the lack and overexpression of HMGB1 affect certain aspects of plant growth and stress tolerance and it has a marked impact on the transcriptome, suggesting that HMGB1 has (partially) specialized functions in Arabidopsis.