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.
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.
In the past 20 years, genetic variants in
SCN5A
encoding the cardiac voltage-gated sodium channel Na
v
1.5 have been linked to a range of inherited cardiac arrhythmias: variants resulting in ...loss-of-function of Na
v
1.5 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 Na
v
1.5 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.
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.
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.
Abstract
Background and Aims
Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most frequent, inherited, cystic kidney disease. It is characterized by formation and growth of renal cysts ...obstructing normal tubules leading to a decline in renal function and eventually end stage renal disease (ESRD). The disease is mainly caused by mutations in either PKD1 or PKD2. Both the involved genes and the type of mutation have been shown to provide significant prognostic information in relation to the risk of early progression to ESRD. However, even within families, progression of disease varies widely between individuals with the same disease causing mutation. This variation might be explained by other genetic components. Previous studies have suggested genetic components affect the progression of Chronic Kidney Disease (CKD) and ESRD.
To explain the variation in disease progression, not accounted for by the disease causing gene, 15 Single Nucleotide Variants (SNPs) associated to CKD and/or ESRD were chosen for further analysis. In addition, 3 SNPs within CFTR was included, since variants within this gene is known to affect the progression of ADPKD.
Method
A cohort of 120 Danish individuals suffering from ADPKD was sequenced using targeted Next Generation Sequencing (NGS). The targeted NGS panel included PKD1, PKD2, rs13538, rs12460876, rs17319721, rs10109414, rs653178, rs267734, rs1260326, rs347685, rs11959928, rs6420094, rs7805747, rs4744712, rs626277, rs881858, rs12917707, rs213950, rs213965, rs1042180. If no causative variant was identified using the NGS panel, Multiplex Ligation-dependent Probe Amplification was performed to identify larger deletions and duplications. In addition, clinical information was obtained to divide patients into two established or predicted progression groups; fast or slow.
Results
Based on clinical information we identified 39 patients with established or predicted fast progression and 81 with slow progression. The distribution of SNPs in the fast progression group was compared to the distribution within a European population. Out of 18 SNPs, one was significantly different distributed among the fast progressive ADPKD patients, rs13538 (p=0.01); however, in contrast to expected the frequency of the SNP was lower in the fast progression group compared to the European population. To examine if the shift could be associated to a specific disease causing mutation type, the distribution of each SNP within different mutation types were analysed. The mutations were divided into three groups; PKD1 truncating, PKD1 nontruncating and PKD2. A significant difference was observed in all three groups.
Conclusion
A single SNP associated to progression of CKD was observed to have a different distribution in the fast progressive ADPKD patients. The distribution differed for all analysed mutation types, thus no specific mutation type or gene could explain the shift in distribution alone. Hence, none of the analysed SNPs seem to influence the rate of progression in the analysed cohort of ADPKD patients.
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.
Key points
What's already known about this topic?
Many pregnant women are positive towards prenatal screening for Cystic fibrosis (CF).
The only current pregnancy marker is echogenic bowel in the second trimester, but this test has low sensitivity and specificity.
What does this study add?
Prenatal screening for CF can be done using circulating trophoblasts isolated from maternal blood early in pregnancy
Circulating trophoblasts can provide screening for CF as a simple single‐visit setup without the need for a paternal sample.
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.
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.
What's already known about this topic?
Enriched fetal cells from maternal blood can be used for performing whole genome amplification, followed by array CGH.
What does this study add?
cbNIPT can detect aneuploidy, microduplication, unbalanced structural rearrangements, and mosaic cases.
cbNIPT can identify subchromosomal aberrations >10 Mb
ABSTRACT Background Genomic disorders caused by copy number variations (CNVs) are prevalent in patients with kidney disease; however, their contribution to chronic kidney failure (KF) of undetermined ...aetiology (uKF) is unclear. We screened patients with uKF aged 50 years or younger to establish the prevalence of causative CNVs. Methods We enrolled patients with an onset of KF ≤50 years from suspected undetermined aetiology for initial review of medical records to exclude patients with clear-cut clinical or histopathological kidney diagnoses or patients with already established genetic kidney diseases. Next, we performed single nucleotide polymorphism (SNP) array–based CNV screening. All the detected CNVs were systematically classified and evaluated as possible causes of the patient's kidney disease. Patients with CNVs not explaining the kidney phenotype were additionally screened for causal variants in 540 genes using whole-genome sequencing. Results We enrolled 172 patients, of whom 123 underwent SNP-array. Pathogenic CNVs corresponding to known genomic disorders were identified in 12 patients (9.8%). The identified genomic disorders provided a causative kidney diagnosis in three patients, all of whom had reached KF by age 18 years. The remaining nine patients had CNVs with unclear kidney disease causality. Subsequently, whole-genome sequencing provided a causative genetic diagnosis in an additional four patients, including two diagnostic sequence variants unrelated to the detected CNVs. Conclusions Genomic disorders were prevalent in this cohort with uKF, and causative CNVs were identified in 5 of 123 patients. Further studies combining the analysis of CNVs and sequence variants are needed to clarify the causal role of genomic disorders in kidney disease.
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